FLUCTUATING ASYMMETRY IN PELOPHYLAX RIDIBUNDUS ( AMPHIBIA : RANIDAE ) AS A RESPONSE TO ANTHROPOGENIC POLLUTION IN SOUTH BULGARIA

The aim of this study was to investigate the integral indicator for developmental stability, the fluctuating asymmetry (FA), in the marsh frog Pelophylax ridibundus populations that inhabit biotopes of different types (running rivers and still, dam lakes), when exposed to different types of anthropogenic pollution (domestic sewage pollution and heavy metal pollution) in south Bulgaria. A total of 920 P. ridibundus individuals were used for FA analyses over three years (2009-2011). Fluctuating asymmetry was defined by 10 morphological traits, using the index frequency of asymmetric manifestation of an individual (FAMI). In closed water basins, regardless of the nature of toxicants, the FA values in P. ridibundus populations were statistically lower than those in river populations. The FA values were constantly the highest under conditions of sustained anthropogenic pollution, with high concentrations of toxicants in rivers with domestic sewage pollution and heavy-metal pollution. The results provide better opportunities to use FA in P. ridibundus populations for bioindication and biomonitoring, and for parallel and independent analyses of the physicochemical assessment of the environmental condition.


INTRODUCTION
Developmental stability is an important aspect of homeostasis.Morphogenetic homeostasis is the ability of organisms to form a genetically determined phenotype without ontogenetic disturbances (Leary et al., 1985;Zakharov and Graham, 1992;Klingenberg, 2003).In nature, "ideal forms" are rarely considered a priority.However, bilateral structures in bilaterally symmetrical organisms offer an ideal of perfect symmetry, against which deviations can be compared (Palmer and Strobek, 1986;Zakharov, 1987).Consequently, they provide a very convenient method for assessing deviations from the norm, and for studying the factors that might influence such deviations.Subtle deviations from symmetry are most commonly described using frequency distributions of right-left.Van Valen (1962, 1978) distinguishes three types of asymmetry: fluctuating asymmetry (FA), directional asymmetry and antisymmetry.
Asymmetry is a matter of great interest and worth studying, particularly fluctuating asymmetry, which is believed to be the indicator that effectively defines environmental stress (extreme temperatures, audiogenic stress, toxins), reflects the instability of ontogenesis (Parsons, 1990; Markovski, 1993; Palmer, 1994; Polak,  2003; Vasil , ev and Vasil , eva, 2009; Amaral et.al., 2012)  and could be used for assessing environmental influence on the organisms (Zakharov, 1992(Zakharov, , 2001)).FA has a role in determining the effects of some unusual
Over the past 10 years, there has been much research into the manifestations of fluctuating asymmetry in populations of tailless amphibians (order Anura) living in conditions of environmental stress, namely, urbanization and anthropogenic pollution.Meristic traits such as color marks (stripes and spots) on the back side of the body and limbs are mostly used as diagnostic markers because they are more sensitive to environmental changes than metric ones (Zakharov, 1987;Lajus, 2001).Additionally, the method does not require the killing of animals.
pollution; what these studies have in common is the fact that whatever the test-subject is, the values of the developmental stability indicator (FA) are higher in animals living in anthropogenically transformed habitats compared to those from background territories (Ustyuzhanina and Streltsov, 2001b;Zhigileva and Burakova, 2005;Peskova and Zhukova, 2009;Zhelev and Peskova 2010a;Peskova et al., 2011;Zhelev et al., 2014b).There are also studies showing the degree of asymmetry in meristic traits is not always directly proportional to the degree of anthropogenic disturbance of the environment (Lada et al., 2012).
In Bulgaria, P. ridibundus is a widely spread species, found throughout the country; P. kl. esculentus can be found only in the north, along the Danube River (south of Mt.Stara Planina no single match has been documented in the literature), while P. lessonae has not been reliably documented (Biserkov et al., 2007;Stojanov et al., 2011).Pelophylax ridibundus is a species tolerant to the anthropogenic load of the environment (Vershinin, 2007), able to dwell under conditions of high levels of pollution in a water body, even in wastewater from industries where other amphibian species are unable to survive (Misyura and Sporadets, 2005;Zhelev, 2007;Zhelev et al., 2006Zhelev et al., , 2013b)).
As an independent bioindication method, FA illustrates not only the extent of damage to the body; it also presents the opportunity for an independent analysis parallel to physicochemical analysis (which reflects the status of a water body at the time of sampling, but does not always catch "the burst effusions", especially those caused by industry, even at high doses of toxicants) and data assessment of the ecological condition of a biotope (Zakharov et al., 2000a(Zakharov et al., , 2000b)).At present, it is not clear whether the FA value indicators in P. ridibundus populations are different in polluted water basins with toxicants of a different type, or if there are differences in the FA value indicators in P. ridibundus populations that inhabit different type of water basins (still and flowing) with one and the same anthropogenic toxic pollutants.
The aim of this study was to find the values of the integral indicator for developmental stability (FA) in P. ridibundus populations inhabiting water basins of flowing type (rivers) and still type (dam lakes), with different levels and type of anthropogenic pollution (domestic sewage pollution, heavy metal pollution) over a three-year period (2009)(2010)(2011).Based on the values of the integral indicator for developmental stability, an evaluation can be given, which is parallel to the data from physicochemical analyses of the ecological conditions of each of the biotopes that were studied during the period of our research work.
The paper uses data from our research conducted on the rivers Sazliyka and Topolnitsa, as shown in our previous publications (Zhelev et al., 2012(Zhelev et al., , 2013a)).This data is provided only for comparison in this work.The present study is an integral part of our extensive research work carried out with P. ridibundus populations, which inhabit anthropogenically polluted biotopes in the Southern Bulgaria, in the period 2009-2012.The purpose of the present study is to affirm the usage of P. ridibundus as test subjects for bioindication and biomonitoring; it is a natural continuation of a series of previous investigations, concerning hematology (Zhelev et al., 2013b(Zhelev et al., , 2014a)), morphophysiology (Zhelev et al., 2014c), sex differences, age/size structure populations (Zhelev et al., 2014d).
The river Sazliyka (145.4 km total length from the spring to the estuary) is a left tributary of the Maritsa River that flows through the Thracian field and the mining complex (coal mining) "Maritsa East".Part of its water is used for industrial purposes; it is diverted to TPS "Brikell" and then it enters the dam lake "Rozov Kladenets" (3.6 km 2 , 200 m altitude).The river Topolnitsa is also a left tributary of the Maritsa River (154.8 km total length from the spring to the estuary).It flows through the mountain Sredna Gora and the Pazardzhik field.The dam lake "Topolnitsa" is on it (9.6 km 2 , 470 m altitude).The copper complex "Aurubis" and "Assarel-Medet" Mining and Processing Complex are in the river valley.
The dam lake "Studen Kladenets" (29 km 2 , 580 m altitude) is on the river Arda near the town of Kardzhali.The major source of pollution in the area, namely "Lead and Zinc Plant" JSC Kardzhali, is in the northern part.
The dam lake "Vacha" (49.7 km 2 ; 550 m altitude) is situated at the river of the same name in the western Rhodopi Mountain, on the road Krichim-Devin.
On the river Sazliyka, 4 micro-biotopes were studied (1.1 -south of the village Rakitnitsa, 1.2below the town Stara Zagora and the confluence of the river Bedechka; 1.3 -below the town of Radnevo and the confluence of the river Blatnitsa; 1.4 -below the village of Lyubenovo at the diverting part of the river to the dam lake "Rozov Kladenets").Two micro-biotopes were studied on the river Topolnitsa (2.1 -below the bridge on the road Panagyurishte-Pirdop; 2.2 -below the village Chavdar).Each micro-biotope described is at a distance of minimum 15-20 km from the previous one, so that exchange of animals between them is unlikely.Only one sample was examined from the still water basins as follows: "Vacha" Dam Lake -3.1 in the region of village of Mihalkovo; 4.1 -"Rozov Kladenets" Dam Lakeat the village Obruchishte; 5.1 -"Topolnitsa" Dam Lake -at the village Poibrene; "Studen Kladenets" Dam Lake -6.1 -at the "tail".
(the type of toxicants were recorded in the polluted water basins).As a basis for assessing the ecological status of water basins studied, we used the data from the physicochemical analyses of the water in each one of them for the period 2009-2011 (average annual values), and these analyses were done in the laboratories of Basin Water Management, in the Eastern Aegean Sea Region, town of Plovdiv (http://www.bg-ibr.org).The data is presented in the relevant yearbooks on the state of the environment (water) in the Republic of Bulgaria and is posted on the website EEA (http:// eea.goverment.bg)(Tables 1, 2, 3).
The average annual data in Tables 1, 2 and 3 are compared with the acceptable standards of the country for I (clean), II (slightly polluted), and III (mod-erately polluted) water categories, under Ordinance № 7 of 8.08.1986, about indicators and standards for water categorization in Bulgaria (State Gazette 96th of 12.12.1986).
Biotopes 1.1 at the river Sazliyka and 3.1 ("Vacha" Dam Lake) are relatively clean.In our work, they served as control biotopes for flowing and still type water basins.
The data from the tables reflects the sustainable presence of toxicants in biotopes 1.2, 1.3, 1.4 at Sazliyka River and the dam lake connected to it "Rozov Kladenets" (4.1).The pollution is of the domestic type (nitrite nitrogen, phosphates, sulfates, biological oxygen demand in five days -BOD 5 and insoluble solids).At the river Topolnitsa, in the two biotopes (2.1, 2.2), as -above the admissible concentration limit for category II; ** -above the admissible concentration limit for category III; ˝ -above SKOS; -no measurements done The digits in column "№" correspond to the same digits in Table 1.
well as in the dam lake situated on it (5.1), the pollution is from heavy metals (copper, iron, manganese, lead, arsenic).The dam lake "Studen Kladenets" (6.1) is also polluted with heavy metals.

Subject of study and methods of analysis
The marsh frog P. ridibundus is the subject of the study; they were distinguished from P. kl. esculentus, basing on the ratio between the length of first finger of the hind leg (Digitus primus, D.p.) and the size of metatarsal tubercle (Callus internus, C.int.: 2009 -305, 2010 -317, and 2011 -298).After having been analyzed, the animals were returned back into the wild.
The marsh frog P. ridibundus is listed in enclosure number 4 to the Bulgarian law for biological diversity (Biodiversity Act -Prom.SG 77, August 9 th 2002).According to clause number 42, clause number 41 and appendix 2 for clause number 41 of the same law capture permits for the P. ridibundus had not been issued when it was used for scientific research.
The animals were caught from the water along the banks in the evening time with an electric torch.One km-long and 4m-wide strips of the river bank were walked through, along the river below the respective town or village, or along the aquatory of each dam lake - above the admissible concentration limit for category II; ** -above the admissible concentration limit for category III; ˝ -above SKOS; -no measurements done.The digits in column "№" correspond to the same digits in Table 1. at sites so-called the "dam tail", according to Sutherland (2000).The analyses were done with live animals, after which they were returned back to their natural habitat.
Their age was determined on the basis of their body size.All examined animals were adults (L >60.0 mm) and sexually mature (Bannikov et al., 1977).
The Ethics Board for Experimental Animals, Faculty of Biology at Plovdiv University, approved the animal handing and methodology.All experiments were conducted in accordance with national and international guidelines of the European Parliament and the Council for the protection of animals used for scientific purposes (Directive 2010/63/EU).

Morphological analysis
As a method for assessing the developmental stability, we used fluctuating asymmetry in 10 morphological traits suggested by Chubinishvili (1997): 1 -number of stripes on the dorsal side of the thigh (femur), 2 -number of spots on the dorsal side of the thigh (femur), 3 -number of stripes on the dorsal side of the left shank (crus), 4 -number of spots on the dorsal side of the right shank (crus), 5 -number of stripes on the left foot (pes), 6 -number of spots on the right foot (pes), 7 -number of stripes and spots on the back (dorsum), 8 -number of white spots on the ventral side of the second finger of the hind leg, 9 -number of white spots on the ventral side of the third finger of the hind leg, 10 -number of white spots on the ventral side of the fourth finger of the hind leg, as for each individual we recognized the number of asymmetrical traits by the degree of their expression on the left and right side of their body (Fig. 2) The level of asymmetric manifestation for each of the ten traits was recorded for each individual; it may vary from 0 (no asymmetry) to 1 (all the traits are asymmetric).It is possible for some of the traits not to express asymmetry, but only in very rare cases it is possible for all 10 traits to be bilateral (Zakharov, 2000a(Zakharov, , 2000b)).
The fluctuating asymmetry was defined by the index frequency of asymmetric manifestation of an individual (FAMI).FAMI = (Σ d L-R / N) was calculated as the ratio of the sum of the number of individuals expressing asymmetric traits towards the total number of individuals (Markowski, 1993;Palmer, 1994;Zakharov et al., 2000a).The grade rating for the status of the populations (respectively for the corresponding biotope) achieved on the basis of the FAMI values, defined by a special scale for P. ridibundus, in the southern part of the area, where Bulgaria is (Peskova and Zhukova, 2007) -Table 4. The values of the FA indicators in the northern and central part of the area containing the species are slightly lower and they are recorded in accordance with the scale proposed by Zakharov еt al. (2000a).(1.1 -below the village of Rakitnitsa, 1.2 -below the town of Stara Zagora, 1.3 -below the town of Radnevo, 1.4 -below the village of Lyubenovo); the Topolnitsa River: (2.1 -below the bridge on the road Panagyurishte-Pirdop, 2.2 -below the village of Chavdar); 3.1 -the "Vacha" Dam Lake; 4.1 -the "Rozov Kladenets" Dam Lake; 5.1 -the "Topolnitsa" Dam Lake; 6.1 -the "Studen Kladenets" Dam Lake; n -numbers of individuals.(2.1 -below the bridge on the road Panagyurishte -Pirdop, 2.2 -below the village of Chavdar); 3.1 -"Vacha" Dam Lake; 4.1 -"Rozov Kladenets" Dam Lake; 5.1 -"Topolnitsa" Dam Lake; 6.1 -"Studen Kladenets" Dam Lake.* (P<0.05),** (P<0.01)*** (P<0.001),ns (P>0.05).For the entire three-year period of research, the FAMI values in the populations from the two control water basins (1.1, 3.1) were significantly lower than those in the populations from the polluted biotopes (regardless of the type of pollution and the type of water basin) -Table 5. Throughout each year of research, there were no considerable fluctuations in the values of the indicator for FA in the control water basins and they remained significantly lower in comparison with those from the polluted biotopes of running and still types (Fig. 3, Table 6).

In the polluted biotopes (rivers and dam lakes): 1.2, 1.3, 1.4, 4.1 (domestic sewage pollution) and 2.1, 2.2, 5.1, 6.1 (heavy metal pollution)
For the whole three-year period of research, the FAMI values in the populations from the biotopes 1.2 and 1.3 were significantly higher than those in the populations inhabiting the biotopes with heavy metals -2.1, 5.1, 6.1 (Table 5).There was no difference between FAMI of 1.3 and 2.2 populations.A closer analysis of the comparisons for each year of the study shows no significant difference in the values of the indicator for developmental stability in the populations from the two biotopes along the upper course of Sazliyka River and those in the populations from the two biotopes at Topolnitsa River (Fig. 3, Table 6).
The FAMI value for the population from the biotope in the middle of the course of the Sazliyka River (1.4) was significantly lower than those for the populations at Topolnitsa River throughout the period of the study, and at the same time significantly higher than those for the populations in the two dam lakes polluted with heavy metals (5.1 and 6.1).These types of differences were observed in each year, except 2010, when the differences in the values of FAMI from the populations in biotopes 2.1 and 2.2 were not statistically reliable.
Comparisons between the population from biotope 4.1 and those living in the two dam lakes polluted with heavy metals showed significantly higher FAMI values than the population from biotope 5.1 and no differences with that from biotope 6.1 (Table 5) throughout the three-year period of the study.For each year, the differences in the values of the indicator for FA in the populations from biotopes 4.1, 5.1 and 6.1 did not change considerably and they were either within the statistical error or unreliable (Fig. 3, Table 6).

In the controls: river (1.1) with a dam lake (3.1)
For the whole three-year period of research, the FAMI values for the P. ridibundus population, inhabiting the closed type of water basin "Vacha" Dam Lake, were significantly lower than those in the population that lives in the control biotope at Sazliyka River -Table 5.Each year, the differences in the FAMI values remained unchanged (Fig. 3, Table 6).

DISCUSSION
This study shows that the highest levels of FA were found in P. ridibundus populations under conditions of permanent anthropogenic pollution, in rivers, at high concentrations of toxicants, regardless of their nature.Despite the higher maximums of FAMI in the biotopes with domestic sewage pollution in the beginning of the investigation period, the average FAMI values from biotopes with different nature of toxicants became equalized over time.
The grade rating by Peskova and Zhukova's scale ( 2007) is 5 -the maximum for each of the four biotopes from the two rivers (1.2, 1.3, 2.1, 2.2), with the highest FAMI values for the whole period of study.According to the findings, P. ridibundus populations appear to be in crisis and there are many serious disorders of their genetic homeostasis in each of these biotopes.Similar high levels of FA (FAMI = 0.65-0.87)were found in the populations of P. ridibundus in the northern and central parts of the area in conditions of different types of anthropogenic pollution: the Voronezh River -metallurgic industry in the region of the town Novolipetsk (Nikashin, 2005), the Hadazhka River -a region of a pig farm in the West Caucasus (Peskova and Zhukova, 2007), the Sviyaga River, near the town of Ulyanovsk, pollution from chemical industry (Spirina, 2009).
For the whole three-year period of investigation, as well as for each year, the population from the biotope in the lower course of the river Sazliyka (1.4), with less domestic sewage pollution, had a significantly lower FAMI value than the populations from the biotopes at the upper (1.2) and middle (1.3) courses of the river; it is also lower than these in the two biotopes at Topolnitsa River (2.1, 2.2).However, the FAMI values in population 1.4 were also significantly higher than these in the populations living in the three water basins of closed type 4.1 (domestic sewage pollution), 5.1 and 6.1 (heavy metal pollution).According to the scale of Peskova and Zhukova (2007) the biotope 1.4 was evaluated with the grade 4, while water basins 4.1, 5.1 аnd 6.1 -with 3. The values of the indicator for FA in biotope 1.4 showed that, regardless of the status of P. ridibundus population (which is decreasing), here the life conditions were better than in the biotopes from the upper and middle parts of Sazliyka River.This is probably due to the lower levels of anthropogenic pollution and the enhanced self-purification processes occurring in the lower part of the river (Zhelev et al., 2013b).It is interesting to note that, with the same type and comparable levels of toxicant with biotope 4.1, the FAMI values in the population, inhabiting the river (1.4), are higher than those in the population from the water basin of closed type "Rozov Kladenets" Dam Lake.In our opinion, this means that at lower levels of anthropogenic pollution, the values of the integral indicator for developmental stability are influenced to a greater extent by the type of water basin, rather than by the type of toxicants.In support of this opinion, the FAMI values were higher in the population from biotope 1.4 than those living in the two dam lakes with heavy metal pollution (5.1 and 6.1).Levels of FA (FAMI = 0.55-0.60)corresponding to 3 and 4 (FAMI = 0.60-0.70)grades by the scale of Zakharov et al. (2000а), Peskova and Zhukova (2007) were found in Р. ridibundus populations living in Oka River -pesticide pollution (Ustyuzhanina and Streltsov, 2001a), in the region of the outgoing-cleaning facilities of TPP-1 and TPP-2 of the Voronezh reservoir (Hitsova et al., 2004), a bovine animal farm (Peskova, 2007) at the Maritza River, in the region of the chemical plant "Neochim" in southern Bulgaria (Zhelev and Peskova, 2010b), at Tsna River pollution from industrial enterprises of the town Kotovsk (Lada et. al., 2012).A possible explanation for the changes we found in the FAMI values in P. ridibundus populations from anthropogenically polluted water basins of flowing and still types in southern Bulgaria could be associated with the acidity of the medium.Over the three years of research in the two biotopes at Topolnitsa River (2.1, 2.1), pH values were lower than MAC (maximum admissible concentrations) and they typified the medium in them as acidic (especially in biotope 2.1).Heavy metals remain dissolved and more difficult to precipitate in such medium.Over the three years of the study, pH values in the two biotopes of closed type (5.1, 6.1) were higher than MAC and they typified the medium as alkaline.The process of heavy metal deposition in bottom sediments increases in an alkaline medium, and it is also known that P. ridibundus populations stand in the shallow coastal parts of water basins of closed type (Biserkov et al., 2007;Stojanov et al., 2011).This could be one of the reasons for the higher values of FAMI in P. ridibundus populations from the biotopes at Topolnitsa River in comparison with the populations from the two dam lakes (with the same type of toxicants).It is harder to explain the changes in the FAMI values in the populations living in biotopes of flowing and still type, with domestic sewage pollution, under similar parameters of anthropogenic pollution in them (biotopes 1.4, 4.1).As noted above, the FAMI value in the population from "Rozov Kladenets" Dam Lake is lower than population 1.4 but here the larger volume of /water mass probably causes changes (greater dilution).In support of this reasoning is the fact that in the populations from the two control water basins, the FAMI values were lower in the populations that inhabit the water basin of closed type "Vacha" Dam Lake and they were evaluated with grade 1, while the grade for biotope 1.1 was 2.
The present investigation reaffirms the opportunities for assessing the developmental stability of P. ridibundus populations that live in conditions of anthropogenic pollution through the method of FA.It supplements the data of FAMI values in P. ridibundus populations from rivers and dam lakes polluted with heavy metals and domestic sewage pollution.The results provide better opportunities to use FA in P. ridibundus populations for bioindication and biomonitoring, and for parallel and independent analyses on the physicochemical analyses assessment of the environmental condition.

Fig. 2 .
Fig. 2. Diagram of morphological features for assessing the developmental stability in Pelophylax ridibundus. 1 -number of stripes on the dorsal side of the thigh (femur), 2 -number of spots on the dorsal side of the thigh (femur), 3 -number of stripes on the dorsal side of the left shank (crus), 4 -number of spots on the dorsal side of the right shank (crus), 5 -number of stripes on the left foot (pes), 6 -number of spots on the right foot (pes), 7number of stripes and spots on the back (dorsum), 8 -number of white spots on the ventral side of the second finger of the hind leg, 9 -number of white spots on the ventral side of the third finger of the hind leg, 10 -number of white spots on the ventral side of the fourth finger of the hind leg.

Table 1 .
Indicators and standards for determining the quality of flowing surface water in the Republic of Bulgaria under Ordinance № 7 of 08.08.1986.

Table 2 .
Recent data on the biotopes (flowing water: rivers) at the time of the study (physicochemical analysis -surface water sample).

Table 3 .
Recent data on the biotopes (still water: dam lakes) at the time of the study (physicochemical analysis -surface water sample).

Table 4 .
Rating scale for the deviations in the status of Pelophylax ridibundus from the conventional standard.