**2. Material and methods**

#### **2.1. Study area**

**Keywords:** polychlorinated biphenyls, pollution, fish, parasites, water reservoir

The ability of persistent organic pollutants (POPs) to spread through long distances and remain in the environment has resulted in their presence worldwide. They have a tendency to accumulate within the food chain and, as a result, pose a high risk to human and animal health. The risks caused by industrial POPs are best illustrated by polychlorinated biphenyls (PCBs), extensively used in a wide variety of applications from the early 20th century. PCBs became ubiquitous contaminants of various biotic and abiotic environments worldwide due to their

PCBs are highly persistent compounds in the environment, especially in aquatic sediments, which act as a stable reservoir from which PCBs can continue to be released over a long period of time, because of their low solubility in water and low volatility [3]. The involvement of PCBs in the food chain occurs through the incorporation of suspended particles in phytoplankton and zooplankton, at the base of the food chain. Bottom feeders and other aquatic organisms ingest, accumulate and pass PCBs upward in the food chain [4, 5]. Fish near the top of the aquatic food web have a relatively long life span and concentrate high amounts of PCBs [6]. The concentration of poorly metabolized chemicals accumulated in fish can thus reflect the

Biomonitoring is a vital and rapidly growing field that uses several biological groups, such as phytoplankton, macrophytes, invertebrates and fish, as bioindicators [9]. This ecological methodology is increasingly used to assess the level of pollution in aquatic environments [10– 13]. Numerous studies have also documented the close relationship between aquatic pollution and parasitism [e.g. 13, 14]. Certain parasitic organisms have the ability to concentrate high quantities of pollutants in their tissues and organs and thus can provide information about the chemical state of the environment [15, 16]. These studies have mainly focused on intestinal parasites, mostly acanthocephalans, as indicators of heavy-metal pollution [14, 17, 18], but data on organic pollutants, including PCBs and their bioaccumulation in parasitic organisms, are

The Zemplínska šírava water reservoir (eastern Slovakia; Fig. 1) is one of the most PCBcontaminated sites in Europe [19, 20]. Large amounts of PCB compounds have been released to the reservoir from a chemical factory in the nearby town of Strážske without treatment (decontamination) or preventive measures, leading to heavy contamination of soil, superficial and underground water and subsequently food chains in this area [21]. The PCBs in the reservoir and the inflowing Šíravský canal thus belong to the so-called old environmental burdens, and their amount varies from tens to hundreds of milligrams per kilogram of sediment dry weight. At least 40, 000 tons of PCB-contaminated sediments are assumed to be

massive and uncontrolled use in industry and agriculture [1, 2].

4 Emerging Pollutants in the Environment - Current and Further Implications

degree of pollution in an aquatic system [7, 8].

Zemplínska šírava

**1. Introduction**

still scarce.

The Zemplínska šírava reservoir (48°47′32″N; 22°0′42″E), one of the largest artificial waterstorage sites in Slovakia, is located in the eastern part of the country (Fig. 1).

**Figure 1.** Location of the sampling sites in the Zemplínska šírava reservoir.

It was built between 1961 and 1965 for regulating flood overflows from the Laborec River and partly for irrigation purposes in the area. The reservoir is 11 km long, ca. 3.5 km wide and covers an area of 33 km². PCB compounds from the chemical factory in Strážske were released into the Strážsky (effluent) canal to the Laborec River and from there by the Šíravský (influent) canal to the Zemplínska šírava reservoir (Fig. 1). The reservoir has abundant ichthyofauna, represented predominantly by dense populations of common carp, *Cyprinus carpio* L.; fresh‐ water bream, *Abramis brama* L.; pike-perch, *Sander lucioperca* L.; northern pike, *Esox lucius* L.; European perch, *Perca fluviatilis* L.; Wels catfish, *Silurus glanis* L. and goldfish, *Carassius auratus* L. The PCBs in fish were monitored twice, first in 2004 and then in 2009, at eight sites in the reservoir (Fig. 1).

#### **2.2. Fish and parasite collection**

A total of 50 fish of nine species belonging to five families (Anguillidae, Cyprinidae, Esocidae, Percidae and Siluridae) were collected. The distribution and concentrations of PCBs were determined in predatory (*P. fluviatilis*, *E. lucius*, *S. lucioperca* and *S. glanis*) and non-predatory (*C. carpio*; the European eel, *Anguilla anguilla*; *Ab. brama*; *C. auratus* and the roach, *Rutilus rutilus*) species. The fishes were transported to the laboratory alive, weighed, measured and divided into feeding guilds [24, 25] (Table 1).


*N*, number of fish examined.

\* Trophic level was assessed as in [25].

**Table 1.** Number of fish and tissue/organ samples examined, food items and trophic levels. *Abbreviations*: Musc, muscle; Liv, liver; Kidn, kidney; Hroe, hard roe; AT, adipose tissue (Data from [26, 27]).

Only muscle tissues (19 samples) were collected in 2004. A more detailed study in 2009 focused on the tissue-specific distribution of PCBs to assess the temporal variations of PCBs in the reservoir. Samples of the liver (hepatopancreas in cyprinids), kidney, adipose tissue, hard roe, bone and brain were collected from 31 fish belonging to seven species [26].

Only perch were infected with parasites and so received special attention. Twelve of 16 perch in a single catch were small, and therefore the tissue/organ samples from these fish were pooled by the weight of the fish: up to 10 g (*n*=7) and 14–30 g (*n*=5). The four remaining fish weighed 60–130 g and were analysed separately. A total of 24 samples of muscles, livers, kidneys, adipose tissues and brains were examined for the presence of PCBs (Table 1). Screening of the digestive tracts for parasites using a stereomicroscope found only one helminth species, the acanthocephalan *Acanthocephalus lucii*. The two largest perch (>120 g) contained 50 and 19 acanthocephalans. The remaining fish were either free of acanthocephalans or their infections were low, mostly 1–2 acanthocephalans per fish in the 10–30 g group. Perch weighing 60 g contained a maximum of five parasites. Perch harbouring such low parasite burdens were considered as uninfected for the comparison of PCB accumulation in infected versus unin‐ fected perch. The parasites were washed in saline, frozen and subsequently examined for PCBs. A total of 95 tissue/organ samples of all fish species (Table 1) and two samples of acanthoce‐ phalan parasites were analysed spectrophotometrically [27]. The scientific and common names of the fish match those in the FishBase database [25].

#### **2.3. Analytical procedure**

represented predominantly by dense populations of common carp, *Cyprinus carpio* L.; fresh‐ water bream, *Abramis brama* L.; pike-perch, *Sander lucioperca* L.; northern pike, *Esox lucius* L.; European perch, *Perca fluviatilis* L.; Wels catfish, *Silurus glanis* L. and goldfish, *Carassius auratus* L. The PCBs in fish were monitored twice, first in 2004 and then in 2009, at eight sites

A total of 50 fish of nine species belonging to five families (Anguillidae, Cyprinidae, Esocidae, Percidae and Siluridae) were collected. The distribution and concentrations of PCBs were determined in predatory (*P. fluviatilis*, *E. lucius*, *S. lucioperca* and *S. glanis*) and non-predatory (*C. carpio*; the European eel, *Anguilla anguilla*; *Ab. brama*; *C. auratus* and the roach, *Rutilus rutilus*) species. The fishes were transported to the laboratory alive, weighed, measured and

**Trophic**

Northern pike 1 2 Nekton 3.8–4.5 2 2 2 1 1 1 2 Pike-perch 2 3 Nekton 4.3 3 3 3 − 3 − 2 Wels catfish 0 3 Nekton 4.3–4.4 2 2 2 1 1 1 2

Common carp 2 1 Zoobenthos 2.1–3.1 1 1 1 − 1 1 1

Freshwater bream 5 1 Zoobenthos 2.9–3.1 1 1 − 1 1 1 1 Goldfish 1 5 Zoobenthos 2.0 5 5 5 1 − 2 5

**Table 1.** Number of fish and tissue/organ samples examined, food items and trophic levels. *Abbreviations*: Musc,

Only muscle tissues (19 samples) were collected in 2004. A more detailed study in 2009 focused on the tissue-specific distribution of PCBs to assess the temporal variations of PCBs in the

**level\* Number of samples**

3.2–4.4 6 5 5 − 3 − 5

2.3–3.4 1 − − − − − −

2004 2009 Musc Liv Kidn Hroe AT Bone Brain

in the reservoir (Fig. 1).

*Predators*

*Non*-*predators*

European perch 0 16

Roach 1 0

*N*, number of fish examined.

\* Trophic level was assessed as in [25].

**2.2. Fish and parasite collection**

divided into feeding guilds [24, 25] (Table 1).

6 Emerging Pollutants in the Environment - Current and Further Implications

European eel 7 0 Zoobenthos 2.3–3.5

**item**

Zoobenthos, nekton

Zoobenthos, plants

muscle; Liv, liver; Kidn, kidney; Hroe, hard roe; AT, adipose tissue (Data from [26, 27]).

**Fish species** *<sup>N</sup>* **Major food**

The extraction and clean-up of the samples followed the methods described by Himberg et al. and Fisher and Ballschmiter [28, 29], with slight modifications. Briefly, the fish and parasite samples were homogenized in anhydrous sodium sulfate and extracted with a mixture of petroleum ether (90%) and acetone (10%) using a separation funnel. The extracts were concentrated in a rotary evaporator and then purified using a Florisil chromatographic column according to STN EN 12393-2 (2001). The final extracts were analysed on a 6890 gas chroma‐ tograph (Hewlett-Packard) equipped with and electron capture detector. The HP-5 capillary column was 30 m in length, with an i.d. of 0.25. and having a film thickness of 0.25 µm. The chromatograph was operated at an injector temperature of 250°C and a detector temperature of 300°C and used helium as the carrier gas at a flow rate of 1.4 ml min–1. The following oven temperature program was used: start temperature 80°C for 1 min, 80–180°C at 30°C min–1 and maintained for 1 min, 185–205 °C at 6°C min–1 and maintained for 15 min and 205–290 °C at 20°C min–1 and maintained for 7.5 min. Individual PCB congeners were identified by their comparison with the retention times of known standards and qualified by comparing the peak areas to the appropriate peaks in the standard mixture (PCB Mix C-SCA-06). The extracts were injected under a splitless mode. The recovery rates of the PCBs in the spiked samples were 80– 95%, whereas the detection limits were 1 µg kg–1 based on wet weight. Six PCB indicator congeners – PCB 28, 52, 101, 138, 153 and 180 – were analysed. All PCB concentrations in the biological samples are given in mg kg–1 lipid weight (lipid wt).

#### **2.4. Statistical analysis**

The data obtained did not meet the requirements for parametric statistical tests (normality). So they were analysed non-parametrically or were log-transformed [ln (*x* + 1)] to satisfy the assumptions (normal distribution and homogeneity of variances) of analyses of variance (ANOVAs). Differences in PCB load between years and between fish tissues/organs and parasites were determined by *t*-tests. The effects of trophic strategy (predatory and nonpredatory fish) and tissue/organ type on PCB levels were tested with two-way ANOVAs. Main-effect ANOVAs were used instead of factorial ANOVAs because no significant interac‐ tions were confirmed between the factors. The data were consequently divided into two groups, predatory and non-predatory fish, and were processed separately. The concentrations are expressed as mean ± SD (standard deviation). Differences in mean concentrations (mg kg– 1 lipid wt) of the congeners and total PCBs in the fish tissues/organs (muscle, liver, kidney, brain, adipose tissue and bone for predatory/non-predatory fish) were tested with a nonparametric Kruskal-Wallis ANOVA with post-hoc multiple comparison. The analyses were performed in Statistica for Windows, version 9.0 [30].
