Changes of erythrocyte-metric parameters in

Changes of erythrocyte-metric parameters in Pelophylax ridibundus (Amphibia: Anura: Ranidae) inhabiting water bodies with different types of anthropogenic pollution in Southern Bulgaria Zhivko Zhelev, Georgi Popgeorgiev, Ivan Ivanov & Peter Boyadzhiev

Environmental Science and Pollution Research ISSN 0944-1344 Environ Sci Pollut Res DOI 10.1007/s11356-017-9364-z

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Author's personal copy Environ Sci Pollut Res DOI 10.1007/s11356-017-9364-z

RESEARCH ARTICLE

Changes of erythrocyte-metric parameters in Pelophylax ridibundus (Amphibia: Anura: Ranidae) inhabiting water bodies with different types of anthropogenic pollution in Southern Bulgaria Zhivko Zhelev 1 & Georgi Popgeorgiev 2 & Ivan Ivanov 2 & Peter Boyadzhiev 3

Received: 26 February 2017 / Accepted: 23 May 2017 # Springer-Verlag Berlin Heidelberg 2017

Abstract The article presents the basic erythrocyte-metric parameters: cell length (EL) and width (EW), EL/EW, erythrocyte size (ES), nucleus length (NL) and width (NW), NL/ NW, nucleus size (NS) and nucleocytoplasmic ratio (NS/ES) in the wild populations of marsh frogs Pelophylax ridibundus from five water bodies in Southern Bulgaria (two rivers and three reservoirs) with different degrees and types of anthropogenic pollution (less disrupted water basins, domestic sewage pollution and heavy metal pollution). The changes in erythrocyte-metric parameters depend on concentrations and types of toxicant and, to a lesser extent, on the type of water basin. We found that when P. ridibundus populations live in conditions of domestic sewage pollution, EL, EW and ES increase in comparison with the control samples, with regard to an elongated elliptical cell shape. Simultaneously, NL, NW and NS did not undergo any significant changes when compared with the control samples. The nuclei had elliptical shape. In the populations from the water basins with heavy metal pollution, EL, EW, ES, NL, NW and NS decreased. The cells and nuclei had a circular shape. NS/ES decreased when compared with the control sample, regardless of the type of toxicants. Responsible editor: Philippe Garrigues * Zhivko Zhelev [email protected]

1

Faculty of Biology, Department of Human Anatomy and Physiology, University of Plovdiv Paisii Hilendarski, 24 Tzar Asen, 4000 Plovdiv, Bulgaria

2

Bulgarian Society for the Protection of Birds (BSPB/Bird Life Bulgaria), 27-A Petko Todorov, 4000 Plovdiv, Bulgaria

3

Faculty of Biology, Department of Zoology, University of Plovdiv Paisii Hilendarski, 24 Tzar Asen, Plovdiv, Bulgaria

Keywords Erythrocyte-metric parameters . Pelophylax ridibundus . Domestic sewage pollution . Heavy metal pollution

Introduction Anthropogenic pollution is currently one of the global environmental problems. The study of adaptive variability of components of the natural biota in anthropogenically transformed areas allows clarification of the mechanisms for survival of organisms and their adaptation to new conditions of existence. Tailless amphibians (Order Anura) have fully developed blood-circulatory and immune systems (Маnning and Horton 1982). Blood, being highly differentiated and an inner reactive medium of the organism, reflects all changes in its functional state. Therefore, dependencies between the changes of basic blood cell parameters and the environmental factors could be analysed and used as a potential biomarker for the type and level of pollution (Davis et al. 2008). The blood parameters in anurans are sensitive to various toxicants which makes them very suitable as biomarkers (Cabagna et al. 2005; Teixeira et al. 2012; Carvalho et al. 2016; Medina et al. 2016). Haematologic parameters are important for assessing the environmental and health risks of exposure to potentially toxic chemicals and for developing measures that serve as early warning signals in polluted areas (Salinas et al. 2015; Pollo et al. 2016). These biomarkers represent the first level, in which the initial interaction between pollutants and organisms occurs (Cajaraville et al. 2000). Erythrocytes transport gases, and they are the largest cell group in the blood of vertebrate animals. According to Wintrobe (1933), the erythrocyte size reflects the position of a species on the evolutionary scale: in lower vertebrates and those with not so successful evolutionary past, e.g.

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cyclostomes, elasmobranches and urodeles have large erythrocytes, while in higher vertebrates (mammals), these cells are smaller and do not posses nucleus. Tailless amphibians have nucleated erythrocytes. The size of blood cells in anurans has been relatively poorly studied. Research work in this field is predominantly targeted at finding the reference ranges for different species and also ascertaining age-related changes in the size and morphology of blood cells (Atatür et al. 1999; Aliko et al. 2005; Arserim and Mermer 2008; Gül et al. 2011; Mahapatra et al. 2012; Arikan and Çiçek 2014; Meesawat et al. 2016). Recent phylogenetic studies have confirmed the species specificity of erythrocyte sizes in anurans and supported the opinion that species with larger erythrocytes usually have larger nuclei (Wei et al. 2015). Erythrocyte sizes in anurans may vary according to their age class (juvenile-adult), or according to ploidy, the size of erythrocytes being usually higher in hybrid species (Cal et al. 2005; Grenat et al. 2009; Bondarieva et al. 2012). However, there are some studies that have reported changes in the shape and size of red blood cells in different anuran species under the influence of environmental factors such as temperature (Haden 1940; Harris 1963), changes in climate (Sinha 1983), and altitude (Ruiz et al. 1989; Baraquet et al. 2013). The changes in erythrocytemetric parameters in amphibians that inhabit anthropogenically transformed environments have been poorly studied. Filling this gap in the research field is becoming increasingly necessary due to the global anthropogenic pollution and its negative effects on biocenosis of ecosystems. Currently, a decline in amphibian populations has been observed in all regions worldwide. Along with infectious diseases, climate changes and higher ultraviolet radiation, the anthropogenic pollution is one of the main reasons (Whittaker et al. 2013). However, some anuran species can easily adapt to anthropogenically polluted environments and retain the abundance of their population. In Eurasia, such anuran species is the marsh frog Pelophylax ridibundus (Pallas, 1771). This makes the species useful for uncovering the mechanisms of survival and it also has properties that can contribute to assessing the anthropogenic risk and environmental damage. In recent years, the number of studies, regarding the successful use of haematological indices of the genus Pelophylax (Fitzinger, 1843), has increased, in particular P. ridibundus—a species that is very dependent on the water basin in which it lives (they rarely move away and usually spend their lives close to their breeding place), which makes them very useful for biomonitoring (Romanova and Egorikhina 2006; Falfushinska et al. 2008; Vershinin and Vershinina 2013; Zhelev et al. 2013). European green frogs Pelophylax esculentus complex (Linnaeus, 1758) consists of two parental species: P. ridibundus and the pool frog Pelophylax lessonae (Camerano, 1882) and their hybrid—the edible frog P. kl. esculentus (Linnaeus, 1758). Among the species of the green

frog group, P. ridibundus is widespread in Bulgaria. P. kl esculentus occurs only along the Danube River and the territory of Northeast Bulgaria, while P. lessonae, despite its presumable presence, has not been reliably documented in this country (Stojanov et al. 2011; Tzankov and Popgeorgiev 2015; Natchev et al. 2016). There are few studies about changes in the size of blood cells in anurans that live in anthropogenically polluted environments (Drobot et al. 2008; Omelykovets and Berezyuk 2009; Mineeva and Mineev 2010). More research works have found changes in the morphology of erythrocytes in amphibians that inhabit anthropogenically transformed environments, compared to animals inhabiting background territories; nevertheless, there are no strong views about the relation of these changes to certain toxicants. In our previous study (Zhelev et al. 2006), we reported the finding of statistically reliable changes in erythrocyte-metric parameters in populations of P. ridibundus that live in conditions of anthropogenic pollution. Thus, when compared with the control group from a relatively clean habitat, in a region with chemical industry, we found that erythrocytes were elongated, having an ellipsoidal shape with a bigger cell size; the nuclei had smaller parameters and were ellipsoidal. In P. ridibundus populations that live in a region with energy industry, we found ellipsoidal erythrocytes having a statistically reliable decrease in the parameters erythrocyte cell length (EL), erythrocyte cell width (EW), nucleus length (NL) and nucleus width (NW). Finding a link between changes in physiological parameters of anurans and specific toxicants in conditions of natural (in the field), anthropogenically polluted habitats is a challenge for researchers, because environmental factors and those of anthropogenic origin in ecosystems involve diverse, complex and often synergetic relationships. This has determined the direction of the present research. It is an integral part of our extensive research work carried out with P. ridibundus populations that inhabit anthropogenically polluted biotopes in Southern Bulgaria, during the period 2009–2012. It has monitored the changes in haematological and morphophysiological parameters. The findings from this research showed that P. ridibundus from habitats with anthropogenic pollution live in conditions of strong stress. There were changes in basic haematologic parameters (high levels of erythrocytes, leukocytes and haemoglobin concentration) in anurans in response to environmental stress, indicating respiratory and/or ion-regulatory disturbances that imply an increase in energy consumption to restore homeostasis (Zhelev et al. 2013). Changes in major organs of metabolism were also found—increasing relative weights of the liver, spleen and kidney, indication for an accelerated metabolism and an active output of toxins (Zhelev et al. 2014, 2015). The results of these analyses indicate adaptive responses in amphibians’ organisms to the toxicants present in the environment. They caused stimulation of the haematopoietic organs and led to

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increasing number of blood cells that transport gases and provide immune protection. Activation of haemoglobin synthesis leads to a more intense haemoglobin saturation of red blood cells. It was logically necessary to find the answer to the question whether the erythrocyte sizes undergo any changes in amphibians living in anthropogenic stress conditions. The present study was extended, and apart from the rivers Sazliyka and Topolnitsa (Zhelev et al. 2013, 2014, 2015), three more water basins of static type in Southern Bulgaria were included. The aim of this study was to investigate the status of some basic erythrocyte-metric parameters in wild populations of P. ridibundus, living in flowing and static water bodies with different degrees and types of anthropogenic pollution in Southern Bulgaria. In the course of the work, three assumptions were investigated: & & &

If there are any differences in the values of erythrocytemetric parameters of P. ridibundus inhabiting water bodies with different levels of anthropogenic pollution; How the type of toxicant influences erythrocyte-metric parameters; If there are any differences in the values of erythrocytemetric parameters under the same physicochemical water characteristics in basins of different types (flowing and still).

2011 (Zhelev et al. 2016). From the still water basins, one sample from each water basin was studied, respectively, 3.1 (the reservoir Vacha, in the vicinity of the village of Mihalkovo, 41.8542° N, 24.4227° E, 530 m a.s.l.), 4.1 (the reservoir Rozov Kladenets near the village of Obruchishte, 42.1383° N, 25.9180° E, 230 m a.s.l.) and 5.1 (the reservoir Studen Kladenets from its Btail^, 41.6267° N, 25.3743° E, 540 m a.s.l.)—Fig. 1. Note *: Since the distance between the microhabitat 2.2 and reservoir Topolnitsa is no more than 2 km and because of the potential exchange of animals between the two basins, we believe that the microhabitat 2.2 can be considered in the current study neither as a reservoir of the flowing type nor as such of static type; therefore, in the commentary below, we consider the microhabitat 2.1 as typical for the river Topolnitsa and it is more than 30 km in a straight line away from the reservoir with the same name. This paper uses data from our research conducted on the rivers Sazliyka and Topolnitsa, in different seasons—apart from spring, the changes of erythrocyte-metric parameters in P. ridibundus populations were followed in the summer and autumn of 2011. The findings of this research are presented in Zhelev et al. (2016). The present work presents the data concerning P. ridibundus populations, which inhabit four microhabitats on these two rivers, in spring only—the time when the research work was done in each of the other water basins from the present study: three water bodies of static type and a new microhabitat on the river Topolnitsa; these data are provided only for the purposes of comparison.

Material and methods Sampling area

Data from physicochemical analysis of the water ecosystems

The sample specimens were gathered April of 2011 in the reproductive period of this species, in five water bodies (indicated by 1 to 5) in Southern Bulgaria; two of them are flowing—(1) the river Sazliyka (145.4 km total length from the spring to the estuary) and (2) the river Topolnitsa (154.8 km total length from the spring to the estuary); the remaining three basins are still—(3) the reservoir Vacha (49.7 km2), (4) the reservoir Rozov Kladenets (3.6 km2) and (5) the reservoir Studen Kladenets (29 km2). Two microhabitats, 30 km away from one another, were studied for the river Sazliyka: 1.1 (in the south of the village of Rakitnitsa, 42.3352° N, 25.5149° E, 200 m a.s.l.) and 1.2 (below the town of Radnevo and the influx of the river Blatnitsa, 42.2804° N, 25.9219° E, 113 m a.s.l.). Two microhabitats, 30 km apart, were studied for the river Topolnitsa: 2.1 (below the village of Chavdar, 42.6406° N, 24.0693° E, 530 m a.s.l.) and 2.2* (near the village of Poibrene, below the location where the river Medetska enters the reservoir Topolnitsa, 42.4968° N, 23.9975° E, 511 m a.s.l.). In microhabitats 1.1, 1.2 and 2.2, the research work continued in the summer and autumn of

The river Sazliyka is polluted mostly with domestic and industrial wastewaters from the towns Stara Zagora and Nova Zagora through its feeders: the rivers Bedechka and Blatnitsa. Part of its water is used for industrial purposes; it is diverted to TPS Brikell and then it enters the reservoir Rozov Kladenets. The pollutants of the river Topolnitsa are from Aurubis JSC, Bulgaria (former Pirdop)—copper smelter and refinery, the Asarel Medet JSC, Bulgaria—a copper extracting and processing factory, and the landfill of Chelopech Mining JSC, Bulgaria. The major source of pollution in the reservoir Studen Kladenets is the Lead and Zinc Plant Kardzhali JSC (Fig. 1). Monitoring and control of the surface water in Bulgaria were performed by the National System for Environmental Monitoring (NSEM). The present study uses the data from physicochemical monitoring water analysis done in the laboratory of the Basin Directorate for Water Management-East Aegean Region-Plovdiv, Ministry of the Environment and Waters (http://www.bg-ibr.org). The analyses were done for each water basin (each microhabitats) simultaneously with

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Fig. 1 Geographical location of the water ecosystems studied. The map has been prepared by the help of the Geographic information system (GIS). Water bodies with their microhabitats: 1.1 the river Sazliyka below the village of Rakitnitsa, 1.2 the river Sazliyka below the town of

Radnevo, 2.1 the river Topolnitsa below the village of Chavdar, 2.2 the river Topolnitsa below the village of Poibrene, 3.1 the Vacha reservoir, 4.1 the Rozov Kladenets reservoir, 5.1 the Studen Kladenets reservoir

frogs’ catching (April, 2011 in the reproductive period of this species)—Table 1. Table 1 presents the average annual data from 2011, so the exact condition of each water body is to be clarified. The microhabitats 1.1 and 3.1 are less disrupted (in our work, they are considered as Bconventional controls^ for the flowing types—1.1 and still types—3.1 of water basin), the other microhabitats are anthropogenically polluted: 1.2 and 4.1 with domestic sewage pollution; 2.1, 2.2 and 5.1 with heavy metals. To facilitate commenting on the work, each sample from the population inhabiting the corresponding microhabitat was marked with its number.

There is sexual dimorphism in P. ridibundus—females are bigger than males. Furthermore, the sizes of adults in both sexes are highly variable (males reached more than 70.0 mm, females—over 100.0 mm). The body size influences erythrocyte sizes (Frýdlová et al. 2012). To eliminate the influence of body size, frogs’ catching was selective in our study—only adults, sexually mature frogs from both sexes and equal in size were caught: 15 individuals: 8 males and 7 females from each microhabitat with SVL > 60.0 mm, not separated into age classes—3+, 4+, etc. (see Bannikov et al. 1977; Grenat et al. 2009). Manipulations were done in line with the ethical standards for research work with live animals, 1 day after the capture of the animals in laboratory conditions. To reduce the stress, animals were transported in buckets with water, as soon as possible, from the place of capture to the laboratory. The alive animals were anesthetized with ether. The blood (0.20 ml) was drawn through cardiac ventricular puncture, with small heparinized needles (20 mm length) via heparinized haematocrit capillaries. We prepared dry blood smears and stained them by the Giemmsa-Romanowski method (Рavlov et al. 1980). The erythrocyte-metric parameters were determined with an Olympus stereo microscope (SZX16, resolution 900 line pair/mm, Germany). Forty randomly selected erythrocytes (600 cells from each population) from each individual were evaluated with an ocular micrometer (MOB-1–15×). Four

Capturing frogs and experimental analyses This study was carried out with the marsh frog P. ridibundus, determined on the base of its morphological characteristics, namely, P. ridibundus differentiated from P. kl. esculentus in metatarsal tubercle size (see Biserkov et al. 2007) and bioacoustics identification (see Tzankov and Popgeorgiev 2015 and Natchev et al. 2016). According to clause number 42, clause number 41 and appendix 2 to the clause number 41 of the Bulgarian Law on Biological Diversity, which capture permits for P. ridibundus, are not required for the aims of scientific research. The animals were caught, at night, in the water, by using an electric torch.

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Recent data on the water bodies at the time of the study: physicochemical analysis—surface water sample

Parameters

SI units

Regulation no. 7/08.08.1986

Microhabitats: flowing rivers (*) and still reservoirs (**)

Categories

A—average annual data from 2011; B—data from April 2011

I

pH

pH units

Temperature

°C

Insoluble substances Electro-conductivity

mg/dm3 μS/cm

Dissolved oxygen

mgO2/dm3

Oxygenation

%

Biological oxygen demand 5 days (BOD5) Chemical oxygen demand (COD) Ammonium Nitrate (N-NH4) Nitrogen Nitrate (N-NO3) Nitrogen Nitrite (N-NO2) Orthophosphates (Р-PO4)

mgO2/dm3

II

III

70

100

mg/dm3

0.1

2

5

mg/dm3

5

10

20

mg/dm3

0.002

0.04

0.06

mg/dm3

0.2

1

2

Total Nitrogen

mg/dm3

1

5

10

Total Phosphorus (as P) Sulphates (SO42−)

mg/dm3

0.4

2

3

mg/dm3

200

300

400

Iron—total (Fe)

mg/dm3

SKOS—0.1

Manganese (Mn)

mg/dm

SKOS—0.05

Copper (Cu)

mg/dm3

SKOS—0.022

Lead and its compounds (Pb) Nickel and its compounds (Ni) Zinc (Zn)

mg/dm3

SKOS—0.0072

mg/dm3

SKOS—0.02

mg/dm

SKOS—1.0

Cadmium (Cd)

mg/dm3

SKOS—0.005

3

1.1*

6.5–8.5 6.0–9.0 6.0–9.0 A B To 3° middle of the season A B 30 50 100 A B 700 1300 1600 A B 6 4 2 A B 75 40 20 A B 5 15 25 A B

mgO2/dm3 25

3

A; B

A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B

8.06 8.0 15.1 12.7 6.1 5.3 891 659 7.88 8.2 82.2 86.3 1.8 3.0 6.7 5.7 0.079 0.035 1.2 1.4 0.012 0.011 0.316 0.025 1.8 1.5 0.303 0.05 and < compare mean values of the parameters

8.08 ± 0.05 (7.99–8.17) 2.1 < 2.2 < 5.1

2.1*

Heavy metal pollution

Microhabitats: flowing rivers (*) and still reservoirs (**)

9.67 ± 0.05 (9.56–9.77) 1.1 = 3.1 5.65 ± 0.03 (5.58–5.71) 1.1 > 3.1 1.73 ± 0.01 (1.71–1.74) 1.1 < 3.1 43.39 ± 0.44 (42.53–44.24) 1.1 > 3.1c 0.17 ± 0.002 (0.16–0.17) 1.1 > 3.1

5.1**

0.14 ± 0.001 (0.14–0.15)

35.58 ± 0.35 (34.89–36.27)

1.59 ± 0.01 (1.58–1.61)

5.31 ± 0.03 (5.26–5.36)

8.43 ± 0.05 (8.34–8.53)

9.78 ± 0.05 (9.68–9.89) 1.2 = 4.1 5.64 ± 0.03 (5.59–5.70) 1.2 > 4.1 1.74 ± 0.01 (1.73–1.76) 1.2 < 4.1 43.73 ± 0.40 (42.94–44.51) 1.2 = 4.1 0.16 ± 0.002 (0.15–0.16) 1.2 = 4.1

1.2*

1.1*

3.1**

Domestic sewage pollution

Less disrupted

Microhabitats: flowing rivers (*) and still reservoirs (**)

NL (μm) ANOVA

Parameters

NL (μm) ANOVA NW (μm) ANOVA NL/NW ANOVA t NS (μm2) ANOVA NS/ES (μm2) ANOVA t

Parameters

(1.1 = 1.2 = 4.1) > 5.1 > 2.2 > 2.1 (1.2 = 4.1) > 3.1 > 5.1 > 2.2 > 2.1 1.2 > 5.1 > 2.2 > 2.1 4.1 > 5.1 > 2.2 > 2.1 F6, 4193 = 252.757, P = 0.001 (1.1 = 1.2) > 4.1 > 5. 1 > (2.1 = 2.2) 1.2 > 4.1 > 3.1 > 5. 1 > (2.1 = 2.2) 1.2 > 5. 1 > (2.1 = 2.2) 4.1 > 5. 1 > (2.1 = 2.2) F6, 4193 = 54.115, P < 0.001 4.1 > (1.1 = 1.2) > (2.1 = 2.2 = 5.1) (3.1 = 4.1) > 1.2 > (2.1 = 2.2 = 5.1) 1.2 > (2.1 = 2.2 = 5.1) 4.1 > (2.1 = 2.2 = 5.1) F6, 4193 = 145.099, P < 0.001 (1.1 = 1.2 = 4.1) > 5.1 > 2.2 > 2.1 (1.2 = 4.1) > 3.1 > 5.1 > 2.2 > 2.1 1.2 > 5.1 > 2.2 > 2.1 4.1 > 5.1 > 2.2 > 2.1 F6, 4193 = 158.168, P < 0.001 1.1 > (1.2 = 1.4) > (2.2 = 5.1) > 2.1 (1.2 = 4.1) > 3.1 > (2.2 = 5.1) > 2.1 1.2 > (2.2 = 5.1) > 2.1 4.1 > (2 = 5.1) > 2.1 F6, 4193 = 38.608, P < 0.001

ANOVA

0.16 ± 0.001 (0.15–0.16)

42.71 ± 0.34 (42.04–43.39)

1.79 ± 0.01 (1.77–1.81)

5.53 ± 0.03 (5.47–5.58)

9.79 ± 0.05 (9.70–9.88)

4.1**

Table 3 Descriptive statistics and ANOVA: nuclei measurements (means ± SEM and their confidence—95%) in the peripheral blood of amphibians of the populations of Pelophylax ridibundus from the investigated water bodies from Southern Bulgaria

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Author's personal copy Environ Sci Pollut Res Fig. 2 Photomicrographs of erythrocytes of Pelophylax ridibundus populations from the investigated water bodies with their microhabitats (*rivers; **reservoirs) in Southern Bulgaria: (1.1*, 3.1**)—less disrupted, (1.2*, 4.1**)— domestic sewage polluted, and (2.1*, 5.1**)—heavy metal polluted water basins. Scale lines = 10 μm

decreased (it is the same in the rivers and reservoirs)—the nuclei got circular shapes. NS: In the populations, living in conditions of domestic sewage pollution, there were practically no significant changes in comparison with the animals from the less disrupted groups; the only difference was found in microhabitat 4.1, where NS slightly increased compared with those from 3.1. NS decreased in the populations from the heavy metal polluted habitats. NS/ES: The populations from the microhabitats with domestic sewage pollution had lower NS/ES values than those from the less disrupted group in the river (1.1), but they were higher than these from the other relatively clean microhabitat (closed type—3.1). NS/ES decreased in the populations from the microhabitats with heavy metal pollution, and NS/ES had higher values in water basins of closed type. Multivariate statistics—PCA The sum of the first three variables (PC1 33.09%, PC2 18.57% and PC3 14.48%) includes 66.14% of the variation for the environmental factors and erythrocyte-metric parameters of P. ridibundus from the six water basins (1.1, 1.2, 2.1,

3.1, 4.1 and 5.1): the eigenvalue (eigenvectors) of the data was fixed at ≥1 (10.257, 5.758 and 4.489, respectively). Factor loadings of the first three components (PC1, PC2 and PC3) indicated a link between changes of the tested erythrocyte-metric parameters and physicochemical data in the polluted water basins. All the nine erythrocyte-metric parameters tested (factor loadings 0.5) with this axis. Two physicochemical parameters: dissolved oxygen and oxygenation (factor loadings >0.5) showed the most strongly negative correlation with factor 1. The parameters pH, temperature and all heavy metals (factor loadings