Biomarkers For Pollution in Caged Mussels From Three Reservoirs in Bulgaria A Pilot Study

Heliyon 8 (2022) e09069
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Heliyon
journal homepage: www.cell.com/heliyon
Research article
Biomarkers for pollution in caged mussels from three reservoirs in Bulgaria:

A pilot study
Elenka Georgieva a, Laszlo Antal b, Stela Stoyanova a, Desislava Aranudova c, Iliana Velcheva d,
Ilia Iliev e, Tonka Vasileva e, Veselin Bivolarski e, Vesela Mitkovska c, Tsenka Chassovnikarova c, f,
Borislava Todorova d, Ifeanyi Emmanuel Uzochukwu b, g, Kriszti an Nyeste b, *, 1,
d, 1
Vesela Yancheva
a
Department of Developmental Biology, Faculty of Biology, Plovdiv University, Plovdiv, Bulgaria
b
Department of Hydrobiology, Faculty of Science and Technology, University of Debrecen, Debrecen, Hungary
c
Department of Zoology, Faculty of Biology, Plovdiv University, Plovdiv, Bulgaria
d
Department of Ecology and Environmental Conservation, Faculty of Biology, Plovdiv University, Plovdiv, Bulgaria
e
Department of Biochemistry and Microbiology, Faculty of Biology, Plovdiv University, Plovdiv, Bulgaria
f
Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria
g
Pal Juhasz-Nagy Doctoral School of Biology and Environmental Sciences, University of Debrecen, Debrecen, Hungary
H I G H L I G H T S
The effects of water pollution in caged mussels from three large dam reservoirs in Bulgaria were assessed.
A cocktail of different inorganic and organic toxicants was measured both in waters and mussels for the first time.
Different biomarker responses (cellular to individual) were also followed in gills and digestive glands of the transplants.
Correlation trends between the pollution levels and the applied biological tools were established.
A R T I C L E I N F O A B S T R A C T
Keywords: The mussel-watch concept was firstly proposed in 1975, which was later adopted by several international
Water monitoring programs worldwide. However, for the very first time, a field experiment with caged mussels was
Pollution performed in three reservoirs in Bulgaria to follow the harmful effects of sub-chronic pollution (30 days) of
Transplants
metals, trace, and macro-elements, as well as some organic toxicants, such as polybrominated diphenyl ethers and
Caged mussels
chlorinated paraffins. Therefore, we studied the biometric indices, histochemical lesions in the gills, biochemical
Biomarkers
changes in the digestive glands (antioxidant defense enzymes, such as catalase, glutathione reductase, and
glutathione peroxidase; metabolic enzymes, such as lactate dehydrogenase, alanine aminotransferase, and
aspartate aminotransferase, and the neurotransmitter cholinesterase), in addition to the DNA damage in the
Chinese pond mussel, Sinanodonta woodiana (Lea, 1834) in Kardzhali, Studen Kladenets and Zhrebchevo reser-
voirs in Bulgaria. Significant correlation trends between the pollution levels, which we reported before, and the
biomarker responses were established in the current paper. Overall, we found that both tested organs were
susceptible to pollution-induced oxidative stress. The different alterations in the selected biomarkers in the caged
mussels compared to the reference group were linked to the different kinds and levels of water pollution in the
reservoirs, and also to the simultaneously conducted bioaccumulation studies.
* Corresponding author.
E-mail address: [email protected] (K. Nyeste).
1
These authors contributed equally.
https://doi.org/10.1016/j.heliyon.2022.e09069
Received 15 November 2021; Received in revised form 11 January 2022; Accepted 3 March 2022
2405-8440/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
E. Georgieva et al. Heliyon 8 (2022) e09069
1. Introduction studied thoroughly to understand the extent of the damage, which is a

vital aspect of ecotoxicological research [33].
Pollution with metals and toxic elements has been a worldwide The results of the current field experiment are part of a pilot study оn
phenomenon for decades and affects various aquatic environments [1, 2, bioaccumulation of selected priority substances in water, as well as
3, 4, 5, 6, 7]. Metals and metalloids, such as arsenic, aluminum, and transplanted moss and mussels. The hypothesis, which was tested in the
mercury are considered to be among the most dangerous pollutants, present work was that the long-term pollution waters of three large
because of their persistence in the environment, nonbiodegradability, reservoirs in Bulgaria would alter selected biomarkers in caged mussels
toxicity character, bioaccumulation, and even biomagnification [8]. after a 30 days’ exposure period. Hence, we aimed to develop an inte-
According to Müller et al. and Zhang et al., polybrominated diphenyl grated methodology for the assessment of metals, trace and macro-
ethers (PBDEs) are a group of chemicals that have been marketed since elements, polybrominated diphenyl ethers, and short-chain chlorinated
the 1960s [9, 10]. They have been commonly applied as flame re- paraffins using transplants in Kardzhali, Student Kladenets and Zhreb-
tardants in various commercial products, such as electronic equipment, chevo reservoirs, located in Bulgaria, which have been subjected to
furniture, plastic materials, polyurethane foams, textile fabrics, etc. anthropogenic stress for several decades. In addition, we aimed to apply a
PBDEs belong to a class of hydrophobic, poorly degradable organic multi-biomarker approach on the Chinese pond mussel, Sinanodonta
pollutants, which are persistent and, are prone to adsorb onto particu- woodiana (Lea, 1834), which included biometric measurements, histo-
late matter, bioaccumulate in the fatty tissues and biomagnify through chemical, biochemical, and genotoxic assays to study the possible link
the food web [11]. Accordingly, the production and application of between pollutant levels and biological parameters. To our knowledge,
technical penta-, octa- and deca-BDE mixtures has been prohibited in this is the very first study, which was carried out on transplanted mussels
the EU [12] and partially restricted in America. Novak et al. stated that with the selected reservoirs, bivalve species, pollutants, and biological
despite the bans, PBDEs will continue to be discharged in nature from effect tools.
the available large depots of PBDE-containing products for many years
to come, and hence, will remain an environmental issue in the fore- 2. Materials and methods
seeable future [13].
Among the high production volume commercial organics, chlorinated 2.1. Test organism
paraffins (CPs) are of growing environmental concern. As explained by
Feo et al. they have been commonly used as metal flame retardants, fat According to Klimova et al., while mollusks of the genus Mytilus are
liquors of leather, plasticizers and working fluids [14]. According to Zhou considered the most sensitive test organisms in the biomonitoring of
et al., the synthesized products are conventionally classified as marine ecosystems, a model similar in characteristics has not yet been
short-chain (SCCPs, C10–13), medium-chain (MCCPs, C14–17) or proposed for freshwater ecosystems [34]. In this regard, the identifica-
long-chain CPs (LCCPs, C 18), and hazard evaluation and chemical tion of a freshwater bivalve bioindicator with suitable parameters as the
regulation of CPs have focused on the chain length categories, which has Mytilus species will play a crucial role for future biomonitoring and
thus taken most CP formulations into account [15]. In particular, SCCPs ecotoxicological studies of inland waters [35]. There are several studies
have been under global regulation as persistent organic pollutants (POPs) [36, 37, 38], which suggest that zebra mussel, Dreissena polymorpha
since 2018 [16], and MCCPs and LCCPs have been on the EU priority list (Pallas, 1771) could be a substitute for marine bivalves, but its relatively
of chemicals, considering their impact on the environment [17]. smaller size could be a problem in terms of multiple analyses.
Bivalve mollusks, especially mussels, are considered to be good bio- In the present study, we selected as a test organism the Chinese pond
indicator organisms of water pollution. First of all, it is so because of their mussel Sinanodonta woodiana (Lea, 1834) (the Eastern Asiatic freshwater
broad distribution, sedentary life mode and high abundance, and sec- clam or swan-mussel). Overall, the reasons we chose this particular
ondly, because of their filtering feeding mode, ability to accumulate high bivalve were because it is a natural biofilter and has a wide distribution,
concentrations of substances in their organs, and high tolerance to abiotic but also a relatively big size, which is essential in terms of performing
changes [18]. Mussels also attract the attention regarding the assessment several different analyses [39, 40]. However, in terms of its quick range
of human health risks associated with water deterioration [19]. In expansion, we also carefully selected a test organism, which occurs
addition, bivalves have been used as bioindicators of pollutants naturally, in both the study and reference sites. The Chinese pond mussel
throughout the United States and also around the globe [20, 21]. Until is a species, with a native range that expands into East Asia from the
today, mussels have been widely applied in many regional environmental Russian Far East (Amur Basin) to Indo-China, Malaysia, and Taiwan [41,
monitoring programs, such as the U.S. Mussel Watch Project, Assessment 42]. According to Demayo et al., Soroka et al., and Lopes-Lima et al., this
and Control of Pollution in the Mediterranean region (MEDPOL), and mussel has spread broadly across many areas, e.g., Costa Rica, Europe,
OSPAR's Coordinated Environmental Monitoring Program (CEMP) [22]. Hispaniola, Indonesia, Philippines, and the USA [43, 44, 45]. Therefore,
Also, resident and transplanted mussels have been used to assess patterns the Chinese pond mussel can be classified as an invasive species in
in the process of bioaccumulation in the Californian waters for over four Europe and its presence could have adverse effects on other indigenous
decades, including some of the earliest performed work, using the Unionid species in the same habitat. In Europe, the Chinese pond mussel
“Mussel Watch” approach [23]. Thus, in the “Mussel Watch” monitoring was firstly seen in Romania in 1979. Currently, the Chinese pond mussel
programs, resident and/or caged mussels can be used as bioindicators of is known to exist in 16 European countries, including Bulgaria and
chemical pollutants [24]. Recently, Avio et al., Catarino et al., d'Ericco Hungary, and also in the study sites of the presently reported research
et al., Manfra et al., and Railo et al. used caged mussels in specific areas to [46, 47]. Contrary to the European countries and the USA, the pop-
investigate pollution effects related to different anthropogenic activity, ulations of the Chinese pond mussel are also perceived as an important
such as plastic pollution, wastewater discharge, wreck removal, gas protein source to local communities, which has a great economic value in
production and dredging activities [25, 26, 27, 28, 29]. Furthermore, Indonesia [48]. Moreover, this mussel is also a traditionally edible spe-
according to Schøyen et al., mussel caging is particularly valuable when cies in its native range, including China as explained by Chen et al. [49].
the indigenous mussels are absent in the study sites [30].
According to Turja et al. and Larsson et al., responses to environ-
mental stress are usually measured using multiple biomarkers of different 2.2. Study sites
biological functions and from different stages of biological organization
[31, 32]. Thus, an integrated biomarker approach employs a set of Three large dam reservoirs in Bulgaria were selected as study sites –
biochemical, histological, genotoxic, and physiological features [31, 32], Kardzhali (41.638475 N, 25.304432 E), Studen Kladenets (41.622244 N,
which represent the kind, level, and state of alterations that need to be 25.441933 E), and Zhrebchevo (42.585571 N, 25.885592 E) reservoirs.
2
Figure 1. Map of South-Eastern Europe and the localities of the study sites: Kardzhali (K), Studen Kladenets (SK) and Zhrebchevo (Z) reservoirs, and reference site in
Plovdiv (P) in Bulgaria. Geocoordinates of the localities: K – 41.638475 N, 25.304432 E; SK – 41.622244 N, 25.441933 E; Z – 42.585571 N, 25.885592 E; P –
42.164785 N, 24.756515 E.
They have been subjected to long-term anthropogenic stress, which also the changes of zooplankton structure in Zhrebchevo Reservoir as a result
differs in its type and level (Figure 1). of water pollution and hydro-technical constructions were described as
Kardzhali is an artificial water reservoir, formed by damming the early as 1981 by Naidenow [62].
waters of the Arda River in the Rhodope Mountains. It is among the first
reservoirs in Bulgaria where fish were reared in net cages and currently 2.3. Field experiment
there are 7 net cage farms [50]. In addition, Kardzhali Reservoir has also
been intensively used for power production and cage farming for over 30 Mussels were hand-collected from one of the fish ponds (dig-out) of
years, with one of the largest sturgeon farms in Europe [51]. Arda River is the Institute of Fisheries and Aquaculture (42.143611 N, 24.816111 E) in
a large Bulgarian river, passing through other Balkan countries, such as Plovdiv, Bulgaria (reference site) where they exist naturally a few days
Greece and Turkey, which subsequently flows into the Aegean Sea. There before field deployment. The mussels were selected from one size class
used to be anthropogenic sources of metal pollution along its valley on (weight: 154 5.5 g; shell length: 11 3.5 cm). The Institute of Fisheries
the territory of Bulgaria; the lead-zinc processing plant and ore mines are and Aquaculture rears fish under strict and controlled conditions, and
now closed, but they have caused permanent water deterioration [52]. there is no known or published data on anthropogenic pressure in the
However, there is relatively scarce recent data on the pollution levels of area (www.ira-plovdiv.bg). Plovdiv is situated in south-central Bulgaria,
this dam reservoir and their effects on different bioindicator species [53]. standing on the two banks of the Maritsa River. Plovdiv was аlso the
Studen Kladenets Reservoir is the third largest dam in Bulgaria and it cultural capital of Europe in 2019 and it is the second-largest city after
has been significantly impacted by the activity of the former lead-zinc ore Sofia. Even though the city is rapidly growing, the Institute of Fisheries
processing plant “Kardzhali” and the very few published and old data and Aquaculture is far from the city center, located in an uninhabited
were on metal accumulation and its effects on some freshwater fish area surrounded by fields, which creates perfect conditions for fish
species [54, 55]. Furthermore, until now, according to Bachvarov and farming.
Velcheva [56], in the area of Studen Kladenets Reservoir, studies have No mussels from elsewhere were transplanted in the reference site
been mainly conducted related to the metal content in different fish or- and no mussels from other locations were used as transplants in the three
gans. There is almost no data about the impact of anthropogenic pollu- studied reservoirs.
tion on the various biomarkers in bioindicators, such as fish or mussels The mussels were transported to the laboratory at Plovdiv University
[57, 58]. in Bulgaria on the same day. They were placed in a 100 L water tank filled
Zhrebchevo Reservoir is located along Tundzha River (in the drainage with dechlorinated water, mixed with water from the reference site
area of Maritsa River, Aegean Sea basin) and it is mainly polluted due to (50:50) and fitted with air pumps. Conductivity, dissolved oxygen, pH
the intense agricultural activity in the region, but there is also some data and temperature were measured to be relatively constant, thus we veri-
on metal pollution [59, 60]. The reservoir is used for power generation, fied that they did not influence the tested biomarkers. Shortly after that,
irrigation, aquaculture, recreation, and sport fishing [61]. In addition, the mussels (n ¼ 30) were transported to the reservoirs in clean plastic
3
containers filled with oxygenated water and put randomly in stainless- 2.4.3. Histochemical technique
steel cages (30 15 10 cm) at a depth of 2 m in each of the studied The histochemical analysis was carried out with a cryostat (Leica, CM
reservoirs (n ¼ 10 in each reservoir) [63]. The cages' mesh size was 25 1520, Germany) to cut the samples. Multiple mussel gill sections (6 μm)
mm allowing water circulation and preventing mussels from falling out of of each specimen were prepared according to a standard PAS method-
the cages as explained by Kazour and Amara [64]. The mussels were not ology [73] and observed with a light microscope (Leica DM 2000 LED,
artificially fed. The cages were collected after 30 days of exposure. No set Germany), attached with a camera in a blinded manner. The gill histo-
of animals was sampled at time 0, only at day 30. There were funding chemical lesions of all the specimens, including the reference group, were
limitations and therefore, we used to control from the reference site for appraised individually and semi-quantitatively by using the grading
comparison. In addition, we thought that at time 0 we would not find any system of Bernet, which was adopted for this study [74]. The positive
disturbances in the studied biomarkers, which normally take a longer PAS reaction was presented in purple-magenta staining. The evaluation
time to manifest. The mussels’ total soft tissues were cut out at the of the histochemical changes was performed and presented as an average
selected study sites for further biomarker analyses according to the value. Each grade represents specific histochemical characteristics and
adapted for mussels EMERGE protocol of Rosseland et al. [65], but the was classified as follows: (0) – negative reaction of the histochemical
sex was not determined for the current study as we were not interested in staining; (1) – very weak positive reaction of the histochemical staining;
the sex-specific differences in regards to the toxicological responses in the (2) – weak positive reaction of the histochemical staining; (3) – moderate
present study. The transplant experiment was performed once in positive reaction of the histochemical staining; (4) – strong positive re-
June–July 2019. action of the histochemical staining.
2.4. Laboratory methodology 2.4.4. Biochemical analyses

All the chemicals, which were used in the biochemical assays were of
2.4.1. Bioaccumulation analyses analytical grade. They were purchased from Sigma Aldrich-Merck (Ger-
Concentrations of 17 elements, which included metals, trace and many). The biochemical analyses were measured at 25 C with a spec-
macro-elements (Al, As, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Na, Ni, P, trophotometer (Beckman Coulter, DU 800, USA).
Pb, Zn), as well as organic toxicants, such as PBDEs (PBDE 28, PBDE 47, The digestive glands were first thawed fast on ice and then, manually
PBDE 99, PBDE 100, PBDE 153, PBDE 154) and SCCPs were determined homogenized, using a Potter Elvehjem homogenizer fitted with a Teflon
simultaneously in surface water samples and the same set of transplanted pestle (Thomas Scientific, USA) in chilled phosphate buffer (pH 7.4, 50
mussel samples (whole mussel soft tissues) at day 30. The methods of mM, 300 mM NaCl). The homogenates were centrifuged at 4 C for 15 min
ICP-AES and ICP-MS in addition to the method of gas chromatography- at 9000 rpm in a cooling centrifuge (MPW 351 R, Poland). The supernatant
mass spectrometry (GC-MS) (Thermo Scientific, USA) were applied for fractions were aliquoted, transferred in new Eppendorf tubes, and stored at
the bioaccumulation analyses [63, 66]. Metal pollution index (MPI) was ‒80 C for further analyses of both, antioxidant and metabolic enzymes.
assessed to compare the total content of metals and trace elements, The catalase (CAT EC 1.11.1.6) activity was measured by the decrease
excluding the macro-elements for the mussels from the reference and in absorbance at 240 nm by H2O2 decomposition according to Aebi and
sampling sites (Al, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Zn). The MPI Beutler [75, 76].
formula is the following [67, 68] [Eq. (1)]: The glutathione reductase activity (GR, E.C. 1.8.1.7) was determined
by monitoring the glutathione-dependent oxidation of NADPH at 340 nm
MPI ¼ (C1 C2 C3 …Cn)1/n, (1) [15].
where Cn is the mean concentration of the element n in the analyzed The glutathione peroxidase (GPx, E.C. 1.11.1.9) was measured using
tissue (mg kg1 wet weight). the method described by Wendel [77].
The results on bioaccumulation and bioaccumulation factors in the The cholinesterase (ChE, E.C. 3.1.1.8) activity was determined by the
transplanted mussels from Kardzhali, Studen Kladenets, and Zhrebchevo decrease in absorbance at 405 nm according to Burtis and Ashwood [78].
reservoirs were previously calculated and reported as well [63, 66]. The lactate dehydrogenase (LDH, E.C. 1.1.1.27) activity was
Therefore, the main focus of the present research was the biomarker measured by measuring the amount of pyruvate consumed due to NADH
responses of the Chinese pond mussel associated with the toxicant levels oxidation at 340 nm (backward reaction) according to Vassault [79].
in the waters, which are presented below. The alanine aminotransferase (ALAT, E.C. 2.6.1.2) and aspartate
aminotransferase (ASAT, E.C. 2.6.1.1) activities were determined by the
2.4.2. Biometric measurements method of Reitman and Frankel [80] as described by Bergmeyer et al.,
The biometric calculations were carried out as described by Gasmi using commercially available kits (Merck, Germany) [81].
et al. [69]. Thus, the whole weight (ww) of each mussel was measured The total protein levels were detected by the method described by
and reported in g with an analytical scale (Kern, Germany) after Bradford [82] with Coomassie Brilliant Blue G-250 using bovine serum
cleaning the shell from epibionts and other debris. The mussels were albumin as standard. The absorbance was set at 595 nm and expressed as
then thoroughly opened to separate the flesh from the shell with a milligram protein per milliliter homogenate.
stainless scalpel. The intervalvular liquid was drained, and thus the soft All the assays were performed in triplicates and the enzyme activities
tissues were dried on absorbent paper and wet-weighed. The shells were were presented in international units per milligram of protein (U/mg
measured with calipers in mm to calculate the studied indices following protein).
Marques [70]; Kagley et al. [71] and Galvao et al. [72] [Eqs. (2), (3),
(4), (5), and (6)]: 2.4.5. Comet assay
Hemolymph was collected from the adductor muscle of each mussel,
CI total ¼ soft tissue ww/total ww x 100 (2) and the samples were centrifuged for 10 min at 1000 rpm (MPW 351 R,
Poland) before the comet assay analysis. The alkaline version was con-
CI 2 ¼ soft tissue ww/shell weight ww x 100 (3)
ducted, following the procedure of Singh et al. [83], which we slightly
CI 3 (state index) ¼ soft tissue ww/shell length x 100 (4) modified. The preparation of the agarose layers on the microscope slides
was performed according to Kolarevic et al. [84]. Nucleoids forming
CI 4 (shell component index) ¼ shell ww/(shell ww þ meat ww) x 100 (5) comet-like shapes were observed at a magnification of 400 after
staining with SYBR Green I (1: 10.000 dilution) with a Leica DM1000
CI 5 (condition factor) ¼ soft tissue ww/shell length3 x 100 (6)
LED epifluorescence microscope, equipped with an I3 filter (Leica, Ger-
many) and a camera. Fifty nucleoids per individual were scored, using
4
the Comet Assay IV Computer Software (Perceptive Instruments, UK). elements and organic pollutants in the caged mussels, which were
The DNA damage was evaluated by the comet parameter Tail intensity determined simultaneously and published separately in our previous
(TI%), which reflects the percentage of DNA in the comet tail. manuscripts [63, 66] (Table 1). Our results showed that Studen Kladenets
was highly polluted compared to the other two, which were also
2.5. Integrated biomarker response (IBR) calculation impacted, but to a lesser degree. Оne probable reason for the pollution of
the reservoir is that it is the final precipitator of the various emitters
Integrative indices are widely used to summarizing biomarker re- along Arda River, including the existing alloy factory and four sites for
sponses and simplifying their interpretation in biomonitoring programs discharge from terminated mine sections.
[28, 29]. Beliaeff and Burgeot [85] created the integrated biomarker The MPI values of mussels from the reference site, Zhrebchevo,
response (IBR) especially for biomonitoring purposes with mussels. The Kardzhali, and Studen Kladenets, and reservoirs were 0.95; 1.58; 1.65,
basis of the calculation is described here briefly. For each biomarker: and 1.72, respectively. According to the MPI values of mussels, the
amount of bioaccumulated metals was the lowest in the case of the
(1) Calculation of mean and standard deviation for each sampling reference site, and the highest was in the mussels from Studen Kladenets,
site. respectively. At the same time, the mean concentrations of BDEs were
(2) Standardisation of data for each sampling site [Eq. (7)]: highest in the mussels from Studen Kladanets too, and those of SCCPs
were the highest in the mussels from Zhrebchevo (Table 1).
Y ¼ (X – m)/s (7)
where Y ¼ the standardized value of the biomarker; X ¼ mean value of a

biomarker from each sampling site, m ¼ mean of the biomarker calcu- 3.2. Biomarkers
lated for all the sampling sites, and s ¼ standard deviation of m.
No lethal outcome was observed throughout the exposures in any of
(3) Then we computation of Z value [Eq. (8)], the tested reservoirs.
Z ¼ –Y or Z ¼ Y (8) 3.2.1. Biometric indices

in the case of a biological effect corresponding, respectively, to inhibition The results on calculated biological measurements are presented in
or activation. Table 2. After one month of caging, we observed deviations in the bio-
metric values of reference and transplanted mussels. There were no sig-
(4) Finally, the score (S) was computed as adding the absolute value nificant differences among CI, CI 2, CI 4 of mussels from the different
of minimum value (min) for all sampling sites for each biomarker sampling sites (CI: ANOVA, F ¼ 0.6378, p ¼ 0.6062; CI 2: ANOVA, F ¼
to Z value [Eq. (9)]: 2.583, p ¼ 0.1063; CI 4: ANOVA, F ¼ 2.713, p ¼ 0.0959; CI 5 ANOVA, F
¼ 1.074, p ¼ 0.1764) (Table 2). At the same time, the CI 3 of mussels
S ¼ Z þ |min| (9) from Studen Kladanets Reservoir was significantly lower compared to the
reference and the other sampling sites (ANOVA, F ¼ 7.656, p < 0.01)
where S 0.
(Table 2). We consider that such results can indicate that the individuals
All the biomarkers were computed this way. Calculation of star plot
exposed to pollution put their energy in detoxification processes, instead
areas by multiplication of the obtained value of each biomarker (Si), with
of other processes associated with metabolism, growth, etc. On one hand,
the value of the next biomarker, arranged as a set, dividing each calcu-
some authors [88, 89, 90] explained that this period (1 month) is
lation by 2 and summing up all values [86]. The corresponding IBR value
potentially too short to register changes in the biometric indices, which
is [Eq. (10)]:
was not the scenario in our case. On the other hand, our results are in line
IBR ¼ (((S1 S2)/2) þ ((S2 S3)/2) þ … ((Sn-1 Sn)/2) þ((Sn S1)/2))) with Lacroix et al. [90] that the period was long enough for histo-
(10) chemical, biochemical, and genotoxic alterations in the transplanted
mussels.
2.6. Data processing There are various approaches in the scientific literature [91, 92, 93,
94, 95] for calculating the status indices in bivalve mollusks; they can be
Past 3.03 [87] and GraphPad Prism 7 for Windows (USA) were used for based on the ratio of weight and length of the shell, with some formulas,
the statistical evaluation of the obtained data. The normality of data was using wet weight and others - dry weight, respectively. Furthermore,
tested by the Shapiro-Wilk test. The homogeneity of variances was tested some authors showed that the fluctuations in the index values can be
with Levene's test. The results were analyzed for the significance of dif- affected by water pollution [96, 97], which we also confirmed in our
ferences among the mussels from the reference and the exposed groups by study. We agree with Galvao et al. [72] according to whom the studied
one-way analysis of variance (ANOVA), followed by Tukey's test (means indices are not limited only to aquaculture, but are also commonly
comparison). In addition, Spearman's non-parametric correlation test was applied as biological tools in studies on environmental pollution, as well
used to check for significant relationships between the MPI and biomarker as to assess the relationship between toxic substances and mussels’
responses of mussels from the reference and sampling sites. Principal health.
component analysis (PCA) was also used to assess the differences in mussels
from the studied sampling sites in Bulgaria, based on the biometric mea- 3.2.2. Histochemical observations
surements; PAS-reaction in the gills; CAT, GPx, GR, ChE, ASAT, ALAT, LDH The results on histochemical analyses showed a moderately positive
activities in the digestive glands, and DNA damage in the hemocytes. The PAS reaction in the gills of the reference group. In contrast, in the case of
significance was set at p < 0.05. The results from all the performed tested reservoirs, we observed a general tendency towards reducing the
biomarker analyses were presented as mean standard deviation (SD). amount of glycogen in the mussels' gills. Furthermore, in the mussels'
gills from Kardzhali and Student Kladenets reservoirs, a weak positive
3. Results and discussion reaction of the histochemical staining was revealed, which was also
expressed in discrete pink-violet colors. Regarding the mussels from
3.1. Bioaccumulation analyses Zhrebchevo Reservoir, we found a moderately positive PAS reaction with
similar intensity to the reference group (Table 2). This in turn also
Оverall, in the present study we could link all the observed changes in showed a similar level of glycogen accumulation in the mussels’ gill cells
the studied biomarkers with the results on bioaccumulation of different (Figure 2).
5
Table 1. Concentrations (mean mg L1 in water and mg kg1 in mussels and their Table 2. Average results ( standard deviation) of biometric measurements;
relative standard deviation (RSD%)) of studied elements and organic pollutants PAS-reaction in the gills; oxidative stress related enzymes' activities (catalase –
in water and mussel samples from different sampling sites in Bulgaria. CAT, glutathione peroxidase – GPx, and glutathione reductase – GR) and meta-
bolic related enzymes’ activities (cholinesterase – ChE, aspartate aminotrans-
Reference site Sampling sites
ferase – ASAT, alanine aminotransferase – ALAT, and lactate dehydrogenase –
Zhrebchevo Kardzhali Studen Kladenets LDH) in the digestive glands (U/mg protein); and DNA damage (percentage of
Water DNA in the tail of the comet (tail intensity – TI%)) in the hemocytes; and the
Macro elements (mg L¡1) integrated biomarker response (IBR) values of mussels from different sampling
Ca n.a. n.a. n.a. n.a. sites in Bulgaria.
K n.a. n.a. n.a. n.a. Reference site Sampling sites
Mg 10.7 (3.3%) 12.1 (2.6%) 2.6 (5.9%) 3.6 (6.4%) Zhrebchevo Kardzhali Studen
Na 14.9 (2.6%) 11.1 (2.7%) 4.8 (3.6%) 7.6 (2.1%) Kladenets
P 0.13 (3.5%) <0.01 0.11 (7.6%) 0.04 (8.2%) Biometric indices
Trace elements (μg L¡1) CI total 25.57 1.50a 22.78 2.04a 25.57 24.29
Al 130 (3.5%) 50 (11.4%) 110 (7.6%) 30 (13.3%) 2.10a 3.31a
As 1.7 (8.2%) <1 1.2 (9.4%) 5.2 (4.9%) CI 2 62.10 39.49 1.57a 51.17 51.95
10.99a 8.28a 6.84a
Cd <0.1 <0.1 <0.1 0.32 (7.9%)
CI 3 419.63 330.43 329.13 255.94
Co 0.28 (5.1%) <0.01 <0.01 0.26 (5.4%)
24.72a 13.15a 58.60a 38.17b
Cr 0.38 (5.3%) 0.13 (4.2%) 0.18 (4.1%) 0.06 (6.9%)
CI 4 61.97 4.15a 71.70 0.81a 66.35 65.94
Cu 6.4 (4.2%) 0.3 (7.3%) 1.7 (6.2%) 1.8 (5.7%) 3.73a 2.99a
Fe 0.27 (9.8%) <0.01 <0.01 <0.01 CI 5 0.0030 0.0027 0.0025 0.0024
Hg <0.05 <0.05 <0.05 <0.05 0.0003a 0.0001a 0.0003a 0.0004a
Mn 0.049 (4.3%) 0.005 (5.3%) 0.008 (5.2%) 0.039 (3.3%) Histochemical alterations in gills
Ni 1.0 (3.1%) 0.4 (2.5%) 0.4 (7.5%) 0.6 (3.4%) Intensity of PAS- þþ þ þ/- þ/-
Pb 2.1 (4.5%) 0.3 (5.9%) 0.6 (5.0%) 17.7 (4.6%) reaction
Zn <1 <1 <1 19.9 (4.8%) Oxidative stress related enzymes in digestive gland
Organic compounds (μg L¡1) CAT 25.79 17.74 2.52a 34.23 7.59c 104.75
2.99a,c 5.06b
BDE 28 <0.004 <0.004 0.023 (26.0%) 0.032 (28.1%)
GPx 0.68 0.19a 0.74 0.18a 0.93 1.27 0.29b
BDE 47 0.005 (0.1%) 0.005 (0.1%) 0.012 (0.1%) 0.005 (0.1%)
0.23a,b
BDE 99 0.012 (0.1%) <0.004 0.017 (0.1%) 0.018 (0.1%)
GR 0.31 0.11a 0.49 0.09a 0.86 0.28b 1.10 0.25b
BDE 100 0.009 (0.1%) <0.004 <0.004 <0.004
Metabolic related enzymes in digestive gland
BDE 153 0.014 (0.1%) 0.014 (0.1%) 0.012 (0.1%) 0.018 (0.1%) ChE 31.82 3.73a 30.39 2.46b 5.98 2.76d 14.52 3.18c
BDE 154 <0.004 <0.004 0.010 (0.1%) <0.004 a b
ASAT 84.35 2.97 72.19 3.00 24.98 65.18 2.79c
SCCPs 0.58 (20.7%) 3.9 (20.5%) 0.86 (19.8%) 1.2 (20.0%) 2.54d
Mussels ALAT 47.07 3.64a 46.12 2.70a 34.41 35.32 2.85b
Macro elements (mg kg¡1) 1.72b
Ca 89 (3.1%) 196 (2.3%) 145 (2.1%) 185 (2.4%) LDH 147.28 52.28 6.70b 140.81 125.43
16.22a 8.76a,c 9.16c
K 211 (4.2%) 201 (4.7%) 229 (4.0%) 234 (5.5%)
DNA damage in haemocytes
Mg 268 (3.8%) 659 (2.6%) 454 (2.8%) 450 (3.1%)
Tail inensity, TI 5.54 0.92a 13.23 1.31b 21.84 2.60c 25.79 1.58d
Na 331 (3.6%) 514 (3.0%) 780 (3.0%) 436 (3.1%)
%
P 0.43 (6.4%) 1.20 (5.7%) 0.93 (6.0%) 1.07 (5.1%)
Integrated biomarker response (IBR)
Trace elements (mg kg¡1)
IBR 0 0.50 7.51 4.38
Al 14.4 (11.1%) 36.9 (6.9%) 36.6 (6.2%) 35.7 (5.7%)
a,b,c
As 0.37 (6.4%) 1.15 (8.8%) 0.80 (7.5%) 0.63 (9.3%) The values with different letters in the same row are significantly different
(Tukey's test, p < 0.05).
Cd 0.09 (10%) 0.19 (13%) 0.17 (6.0%) 0.38 (5.6%)
CI total ¼ weight of soft tissue (g)/total weight (g) x 100. CI 2 ¼ weight of soft
Co 0.14 (6.0%) 0.27 (7.4%) 0.20 (7.1%) 0.21 (5.8%)
tissue (g)/shell weight (g) x 100. CI 3 (state index) ¼ weight of soft tissue (g)/
Cr 0.15 (3.4%) 0.19 (8.6%) 0.22 (3.2%) 0.14 (11.3%) shell length (cm) x 100. CI 4 (shell component index) ¼ shell weight (g)/(shell
Cu 5.32 (4.9%) 25.35 (4.3%) 63.03 (3.4%) 31.43 (3.9%) weight (g) þ meat weight (g)) x 100. CI 5 (condition factor) ¼ weight of soft
Fe 170 (6.4%) 294 (3.4%) 212 (3.4%) 196 (5.0%) tissue (g)/shell length^3 (mm) x 100.
Hg 0.004 (16%) 0.009 (12%) 0.007 (12%) 0.009 (11%)
Mn 1.34 (7.5%) 1.88 (5.0%) 2.54 (4.0%) 1.98 (5.6%)
Based on the obtained results, we can summarize that in the gills of
Ni 0.14 (5.0%) 0.4 (2.5%) 0.37 (6.5%) 0.6 (3.4%)
the tested individuals from Kardzhali and Studen Kladenets reservoirs
Pb 1.6 (6.8%) 1.9 (3.6%) 1.7 (5.7%) 4.0 (7.9%)
there was a stronger decrease in the amount of glycogen compared to
Zn 55.1 (3.3%) 15.70 (3.8%) 11.47 (3.6%) 32.59 (4.7%)
Zhrebchevo Reservoir. This is a possible indication of chemical stress in
Organic compounds (mg kg¡1)
the mussels' organisms as a result of the polluted waters with metals,
BDE 28 0.005 (0.1%) <0.003 <0.003 0.005 (0.1%)
toxic elements, and organic compounds [63, 66]. The different degrees of
BDE 47 0.005 (0.1%) 0.005 (0.1%) 0.012 (0.1%) 0.005 (0.1%)
intensity of PAS reaction in the caged mussels (Table 2) also showed a
BDE 99 0.013 (0.1%) <0.003 0.015 (0.1%) 0.010 (0.1%)
different degree of glycogen depletion in the mussels’ cells. This could be
BDE 100 0.007 (0.1%) <0.003 <0.003 <0.003 explained by varying stages of glycolysis processes, associated with
BDE 153 0.014 (0.1%) <0.003 0.016 (0.1%) 0.014 (0.1%) depletion of energy reserves, as a result of induced chemical stress.
BDE 154 <0.003 <0.003 <0.003 0.010 (0.1%) Our opinion is in line with Au [98] that the use of histological and
SCCPs 7.4 (29.7%) 0.22 (31.8%) 0.56 (30.4%) 6.1 (29.5%) histochemical biomarkers to determine the effects of environmental
BDE: brominate diphenyl ethers; SCCPs: short-chain chlorinated paraffins. pollution is perceived as an extremely important approach, as they
6
Figure 2. Intensity of PAS-reaction in the gills of mussels: A – reference site, B – Zhrebchevo; C – Kardzhali; D – Studen Kladenets reservoirs.
specifically reflect the health status of organisms. Moreover, we agree Biomphalaria glabrata (Say, 1818). Lastly, we confirmed the results of Da
with Pathan et al. [99] who indicated that histochemical studies help to Silva Souza et al. [106] that the caged mussels from Kardzhali, Studen
demonstrate the localization of proteins, lipids, and glycogen at the Kladenets, and Zhrebchevo reservoirs most likely consumed their energy
cellular level, with the main advantage of applying such methods, being reserves in an attempt to reestablish the organism integrity, however, we
in the analysis of biological phenomena in “individual cells”. did not observe lethal outcome as the reported authors did.
Overall, a similar result to ours was observed by Bakry et al. [100]
who explained that a reduction in the glycogen content in tissues leads to 3.2.3. Biochemical responses
an increase in the glucose level, thus providing the organism enough Data of all examined enzyme activities were normally distributed
energy to detoxify, breakdown and excrete toxicants. The authors re- (Shapiro-Wilk test, p > 0.05) and their variances were homogeneous
ported a decrease in the levels of glycogen and an increase in the levels of (Levene's test, p > 0.05).
glucose in hemolymph from the freshwater snail Bulinus truncates
(Audouin, 1827) exposed to pesticides. We also confirmed the findings of 3.2.4. Oxidative stress-related enzymes
Ansaldo et al. [101] of the potential use of glycogen level as a biomarker Oxidative stress can take place in mussels under a series of environ-
of chemical stress in caged mussels for the aim of environmental moni- mental adverse conditions, including water pollution, which we showed
toring and risk assessment in natural water bodies or under laboratory in our study. In general, the biomarkers of oxidative stress can be split
conditions. We agree that under conditions of oxidative stress, caused by into two main groups as explained by Uchendu [107]: biomarkers for free
water pollution the body responds by including defense mechanisms, radicals in the biological systems and antioxidant defense factors. The
such as increasing the antioxidant defense mechanisms. However, in the biomarkers used in the present study belong to the group of antioxidant
case of excessive free radical formation, antioxidant mechanisms can be defense factors. The analyses revealed changes in the enzymatic activity
completely compromised. Our opinion is in line with Bickler and Buck of the following antioxidant enzymes - catalase (CAT), glutathione
[102] that another important response to oxidative stress in the regula- peroxidase (GPx), and glutathione reductase (GR) in the digestive glands
tion of the glycolysis process, which is an easily available energy of caged mussels from the tested reservoirs (Table 2).
resource. Hence, the main function of body fat is associated with lipid, Overall, the analyses showed that generally there were no significant
glycogen, and protein storage [103]. In addition, we confirmed that the differences between the oxidative stress-related enzymatic activities in
biotransformation of toxic compounds requires the use of energetic re- mussels from Zhrebchevo Reservoir and those in mussels from the
serves, which are needed for other vital processes as explained by K€ ohler reference site. However, oxidative stress-related enzymatic activities in
et al. [104] and Ansaldo et al. [101]. Furthermore, our findings are in line mussels from Studen Kladenets were significantly higher than those from
with Triebskorn and K€ ohler [105] who reported depletion of lipids and Zhrebchevo Reservoir and the reference site, respectively. We consider
glycogen in the hepatopancreas of the mollusk Deroceras reticulatum (O. that these phenomena indicated that Studen Kladenets Reservoir was the
F. Müller, 1774), with K€ ohler et al. [104] who found a decrease in the most polluted water basin according to the changes in enzymatic activ-
number of lipids in the hepatopancreas of the isopod Porcellio scaber ities, and thus Zhrebchevo Reservoir was the least polluted water body
(Latreille, 1804), and also with Ansaldo et al. [101] who observed that (the biochemical values did not differ significantly from the reference
the exposure to toxicants resulted in glycogen depletion in the gastropod site). Our bioaccumulation analyses also confirmed these results [63, 66].
7
The CAT activity in mussels from Studen Kladenets and Kardzhali oxidative stress, which includes enzymes, such as CAT, GPx, and GR. Due
reservoirs was significantly higher compared to the reference group to funding limitations, the activity of other key enzymes, such as GST,
(ANOVA, F ¼ 324.3, p < 0.001) (Table 2). At the same time, there were SOD, and lipid peroxidation was not measured. Hence, further studies in
no significant differences between the oxidative stress-related enzymatic this particular area are highly recommended, which will also provide
activities in mussels from Zhrebchevo Reservoir and mussels from the information if the protective defense mechanisms in transplanted mus-
reference site (p > 0.05) (Table 2). sels were impaired due to exceeded ROS generation, resulting in lipid
We agree with Regoli et al. [108] that when organisms are under peroxidation. This is in agreement with Lacroix et al. who proved that
stress conditions, antioxidant enzymes, including the CAT activity could oxidative pressure subsequently leads to oxidative damage (lipid perox-
be increased to eliminate the excessively produced reactive oxygen idation) in cases when the antioxidant mechanisms are not sufficient to
species (ROS), more precisely the higher rates of H2O2 production and maintain a balance between the generation and neutralization of ROS
prevent further cellular damage. Moreover, our findings are in agreement [90].
with Duarte et al. [109], Freitas et al. [110], and Velez et al. [111] who
assessed the impact of metals on the CAT activity in different mussel 3.2.5. Metabolic related enzymes
species. These results endorsed the capacity of mussel's cells to detoxify According to Dong et al. [122], metabolism is the basic process of
superoxide (O 2 ), which is the precursor of most of the other ROS through material circulation and energy exchange in a living entity. Metabolic
the antioxidant enzymatic system. enzymes perform different cellular functions vital for survival and ho-
The activity of GPx in mussels from Studen Kladenets Reservoir was meostasis, including proteolysis and digestion, cellular respiration, en-
significantly higher compared to the reference site and Zhrebchevo ergy storage, transcription, and response to the environment. These
Reservoir (ANOVA, F ¼ 7.066, p < 0.01) (Table 2). life-sustaining pathways are essential for the growth and maintenance
The GPx catalyzes the degradation of hydrogen peroxide (H2O2), of cellular integrity. Metabolic enzymes encompass a wide range of
which originates from the dismutation of superoxide radical by the su- different classes, including carboxylases, dehydrogenases, lipoxygenases,
peroxide dismutase (SOD) activity. As explained by Lesser [112] it also oxidoreductases, kinases, lyases, transferases, and more.
catalyzes the degradation of lipid hydroperoxides in alcohols. Our results Generally, the metabolic-related enzymes’ activities from the sam-
for the elevated levels of GPx are in line with Orbea and Cajaraville [113] pling sites were significantly lower than those of the reference site, which
who found an increased GPx activity in invertebrate species from highly indicated disturbances in the metabolism linked with polluted waters.
polluted sites compared to the respective reference sites, and also with The activity of LDH in caged mussels from Zhrebchevo and Studen
Cong et al. who reported an increased GPx activity in bivalves exposed to Kladenets reservoirs was significantly lower than that of the mussels from
metals under laboratory conditions [114]. the reference site (ANOVA, F ¼ 81.69, p < 0.001). The levels of LDH in
The GR activity in the mussels from Studen Kladenets and Kardzhali the specimens from Kardzhali Reservoir were similar to the reference site
reservoirs was significantly higher than that of the reference and (Table 2).
Zhrebchevo sites (ANOVA, F ¼ 15.680, p < 0.001) (Table 2). Our find- LDH plays a key role in the cellular metabolic activity under the in-
ings are coherent with Lacroix et al. [90] whose data suggested a higher fluence of oxidative stress, especially after exposure to metals [123]. LDH
GSH production, which could subsequently enhance ROS scavenging and as a metabolic enzyme is also involved in cellular respiration and the
phase-II biotransformation. Furthermore, we agree with Ferreira et al. production of the high-energy compound adenosine triphosphate (ATP)
who found in their study on transplanted oysters from an estuary in Brazil from glucose. Furthermore, LDH is responsible for the regeneration of
that the increased GR activity could be explained with a possible increase nicotinamide adenine dinucleotide (NADþ) necessary in the metabolism
in the cellular GSH supply [115]. of glucose and for the subsequent production of ATP from NADH for the
As explained by Regoli and Guiliani, and Ferreira et al., GR is an continuation of glycolysis. Therefore, the LDH activity is a measure of the
NADPH-dependent enzyme, involved in the reduction of oxidized anaerobic capacity and status of the cell as explained in detail by Gagnon
glutathione (GSSG) to GSH, previously needed by antioxidant and and Holdway [124].
biotransformation enzymes (e.g., GPx and GST) for ROS and chemical The activity of ASAT differed significantly among the sampling sites
detoxification [115, 116]. (ANOVA, F ¼ 412.80, p < 0.001). At the same time, the levels of ASAT in
We agree that the biochemical changes observed in the caged mussels mussels from the tested reservoirs were lower than the reference site. In
are expected to be energetically costly and therefore, lead to increased addition, the activity of ALAT in mussels from Studen Kladenets and
energy consumption as shown by Sokolova et al. [117]. We also consider Kardzhali reservoirs was significantly lower than that from the reference
that the energy needed for the normal function of the body will be put site (ANOVA, F ¼ 29.20, p < 0.001) (Table 2).
into detoxification processes and our results on the biometric measure- ASAT and ALAT are sensitive criteria for hepatotoxicity and can be
ments and histochemical analyses confirmed this suggestion. Similarly to assessed in a shorter time [125, 126]. According to Narvia and Ranta-
Lacroix et al. [90], we could hypothesize that there was probably acti- maki [127], transaminases are among the crucial enzymes in amino acid
vation of aerobic metabolism in the caged mussels to fulfill the energy metabolism, which in aquatic organisms is known to be affected by
demand, associated with an increase in the ROS generation and lipid exposure to organic pollutants. In addition, ASAT and ALAT not only
peroxidation due to water pollution. Moreover, ROS could be generated function as link enzymes between the protein and carbohydrate meta-
directly by toxicants, but also indirectly by increased aerobic metabolism bolism but also serve as indicators of the altered physiological condition
to sustain energetic costs of metabolic responses to pollutants, since the under chemical stress (Table 2).
electron transport chain is a major site of ROS production [116]. We From the obtained results we could consider that the decrease in the
consider that there was perhaps activation of aerobic metabolism in the enzymatic activities is a result of tissue lesions in the particular organ
digestive glands of transplanted mussels to fulfill the energy demand associated with degenerative processes. Therefore, we agree with
associated with water pollution and a ROS metabolism activation, lead- Almeida et al. [128] who consider that changes in the LDH, ASAT, and
ing to an increased ROS generation and probably lipid peroxidation. Our ALAT activity can be used as biomarkers for tissue damage and further
results correspond with those of Murphy and Sokolova et al. [117, 118]. determination and confirmation of the presence of histological lesions.
Regarding metal and organic (PAHs, PCBs) pollution of waters, it is Thus, we suggest that such analyses on the digestive gland of trans-
well documented that it promotes the generation of reactive oxygen planted mussels in Kardzhali, Studen Kladenets, and Zhrebchevo reser-
species (ROS) [115, 119, 120]. In addition, according to Wang et al. voirs should be also assessed in the research to come.
[121], it can trigger defense mechanisms or cause oxidative cellular From the conducted analyses on the changes in the ChE activity, we
damage to macromolecules, such as lipid peroxidation. In the present found that there were significant differences among the sampling sites
study we were interested in the response of transplanted mussels to (ANOVA, F ¼ 83.57, p < 0.001) (Table 2) with the lowest levels
8
measured in mussels from Kardzhali and Studen Kladenets reservoirs We proved that the monitoring of genotoxicity in freshwater, indig-
compared to the reference site (Table 2). enous and caged mussels, using the comet assay of hemocytes is a rather
The ChE activity has traditionally been applied as a specific indicator sensitive method as stated by other authors [146, 147, 148]. Our study
of exposure to some pesticides [96, 97], initially for organophosphorus also confirmed that the Chinese pond mussel is a good bioindicator
and carbamate insecticides, but it also responds to a large variety of species for in situ genotoxicity assessment. The latter has been demon-
chemicals, such as metals and toxic elements, hydrocarbons, detergents, strated already through the method of comet assay in native populations
etc. Thus, it has been also suggested as an indicator of general health from polluted waters [149, 150, 151]. The same is also valid for trans-
[129]. We agree with Bocquene et al. [130, 131] and Galgani and planted mussels [148].
Bocquene [132] that the inhibition of ChE activity can be used as an The significant differences among the TI% in the hemocytes of caged
indicator of stress under the influence of environmental toxicants. In mussels from the different sampling sites in our study confirmed the high
addition, our results confirmed that in bivalves the neurotoxic impacts of sensitivity of the tested freshwater mussel in detecting genotoxicity
different in nature pollutants were proven by the decrease of ChE activity induced by waterborne pollutants, such as metals, toxic elements, and
due to its high affinity for many neurotoxic compounds, including organic compounds.
metal(loid)s and organic chemicals [133, 134, 135, 136]. Our results revealed elevated levels of DNA damage in the mussels’
The action of metals, toxic elements, and organic pollutants, which hemocytes from all the studied reservoirs. This confirms the genotoxic
are contained in the waters of Kardzhali, Student Kladenets and effect, caused by the polluted waters [63, 66]. DNA damage detected in
Zhrebchevo reservoirs led to disturbances in the biochemical processes the mussels sampled from Studen Kladenets Reservoir was the highest
that occurred in the caged mussels. In recent years, the measurement of (Table 2, Figure 3) and can be attributed to the former lead-zinc ore
enzymatic activity in various organs of bivalves has been used as a processing plant “Kardzhali”. This corresponds to the results for bio-
reliable biomarker to identify the negative effects of a polluted envi- accumulation of metals and toxic elements, as well as organic priority
ronment. Our findings are in line with Sevgiler et al. [137] who iden- substances: six polybrominated diphenyl ethers (PBDEs) congeners and
tified changes in the enzymatic levels as one of the most sensitive short-chain chlorinated paraffins (SCCPs) in caged mussels from the
biomarkers for the negative effects of different anthropogenic toxicants. sampling sites [63]. The highest values for Cd, Pb, Zn, as well as for
Furthermore, we agree with Rogers et al. [138] that the damage to vital PBDEs and SCCPs, were measured in the mussels from Studen Kladenets
organs or tissues in bivalves is ecologically relevant because it reflects Reservoir. Cd, Pb, and Zn caused genotoxic responses – DNA and/or
the negative impact on important physiological functions, such as chromosomal fragmentations, and DNA strand breakage in aquatic
toxicant transformations and digestion, detoxification, growth, energy freshwater organisms as previously described [151, 152, 153]. Moreover,
substrate storage, ionic regulation, and respiration. Thus, we consider metals cause DNA damage in living cells because they form strong co-
that it is crucial to determine the harmful effects by considering the valent bonds with DNA [154]. This results in the formation of DNA ad-
alterations in the activity of complex enzymes and to study the mech- ducts, which prevent replication. The genotoxic effect of Cd is indirect
anisms of their action, as well as to try to imply different ways to reduce and is due to the oxidative stress and inactivation of several DNA repair
possible adverse effects. enzymes [153]. The genotoxic effects of Pb in vitro and in vivo are widely
known, but the biochemical and molecular mechanisms of Pb impact are
3.2.6. DNA damage still unclear. Inhibition of DNA repair or production of free radicals was
There were significant differences among the DNA tail intensity reported as indirect mechanism of Pb genotoxicity [155]. It is also known
percentage in the hemocytes of mussels from the different sampling sites that the brominated compounds cause double-strand breaks, as well as
(ANOVA, F ¼ 277.1, p < 0.001) (Table 2). Furthermore, a significant DNA damage mediated through reactive oxygen species (ROS) [156].
increase of DNA migration in the hemocytes’ nucleoids of caged mussels The comet assay revealed that all the assessed PBDEs exerted genotoxic
was observed in the three investigated reservoirs and the individuals effects in HepG2 cells according to Pereira et al. [157], and BDE-209
from Studen Kladenets Reservoir showed the most intensive migration induced DNA damages in in vivo exposed freshwater bivalve zebra
(Figure 3). mussel, Dreissena polymorpha under laboratory conditions [158]. Ac-
In more recent ecotoxicological studies the DNA damage is mostly cording to Wang et al. [121], the metabolism of SCCPs can cause the
evaluated by the use of DNA comets [139, 140, 141]. Furthermore, the generation of ROS. The deduction of oxidative stress destroys cellular
individual cell's DNA damage is detected by the use of comet assay components, causing DNA damage and genetic mutations, which may
(single-cell gel electrophoresis) for quick evaluation of genotoxicity in underlie the toxic effect mechanisms of SCCPs [121]. In our study,
aquatic organisms [142, 143, 144] caused by pollution. As a result, we oxidative stress-related enzymatic activities in mussels from Studen
agree that the method can be used for early detection of exposure to Kladenets Reservoir were significantly higher than those from Zhreb-
genotoxic agents like primary DNA damage (single or double-strand chevo Reservoir and the reference site. The SCCPs (6.12 μg kg1) in the
breaks, alkali labile sites, and crosslinks) [140, 141, 145]. mussels from Studen Kladenets Reservoir were 27 times higher than
Figure 3. Comet cells from the mussels' haemocytes: A – reference site; B – Studen Kladenets Reservoir (magnification 400 ).
9
those measured in the Zhrebchevo Reservoir and 10 times higher than

Table 3. Correlation coefficients between metal pollution index (MPI) and those in the Kardzhali Reservoir [63]. Therefore, we consider that the
biomarker responses of mussels from different sampling sites in Bulgaria, sig-
highest level of DNA damage in Studen Kladenets Reservoir can be
nificant at p < 0.05 (N ¼ 20).
attributed to pollutants, such as metals and toxic elements, PBDEs, and
Biometric indices/ Spearman's rang SCCPs accumulated in the soft tissues of caged mussels.
biomarker responses correlation coefficient
The genotoxicity detected at the other two reservoirs could be
CI total n.s.
attributed mainly to the measured metals concentrations, with the
CI 2 n.s. highest levels for Cr, Cu, Ni in the mussels from Kardzhali Reservoir, and
CI 3 -0.824 for Al, As, Co, Fe, Mn in the mussels from Zhrebchevo Reservoir [63]. A
CI 4 n.s. similar finding was observed by Khan et al. (Khan et al., 2019) who re-
CI 5 -0.592 ported that the higher concentrations of metals (Cd, Cr, Cu, Fe, Mn, Ni,
CAT 0.745 Pb, Zn) in the soft tissues of freshwater mussels showed significantly
GPx 0.718 higher DNA damage from metal polluted waters as compared to the
GR 0.904 reference site.
ChE -0.710 The observed DNA damage in transplants from all three reservoirs can
ASAT -0.760 also be further explained by the effect of synergism between the different
ALAT -0.772 toxicants. The various combinations of pesticides and metals act syner-
LDH n.s. gistically and result in a more severe toxic effect compared to a single
Tail intensity 0.953 molecule alone [159]. The effect of synergism can also explain the sta-
tistically significant DNA damage in the mussels from Zhrebchevo
n.s.: Non-significant.
Reservoir, mainly polluted due to intense agricultural activity, as well as
CI total ¼ weight of soft tissue (g)/total weight (g) x 100, CI 2 ¼ weight of soft
with the presence of metal pollution reported before [59, 60].
tissue (g)/shell weight (g) x 100, CI 3 (state index) ¼ weight of soft tissue (g)/
shell length (cm) x 100, CI 4 (shell component index) ¼ shell weight (g)/(shell The DNA damage due to genotoxic pollution was proved by the comet
weight (g) þ meat weight (g)) x 100, CI 5 (condition factor) ¼ weight of soft assay technique on hemocytes in transplants in other studies during the
tissue (g)/shell length^3 (mm) x 100. last decade [84, 148, 149]. The highest values of TI% (28.8 1.6 for Unio
ASAT – aspartate aminotransferase, ALAT – alanine aminotransferase, CAT – sp. and 21.9 1.5 for S. woodiana) in specimens collected from different
catalase, ChE – cholinesterase, GPx – glutathione peroxidase, GR – glutathione sites under pollution pressure in Danube River [84] were close to those
reductase, and LDH – lactate dehydrogenase in the digestive glands of caged for Studen Kladenets Reservoir (25.79 1.58) and Kardzhali Reservoir
mussels (U/mg protein). (21.84 2.60) in our study. Slightly lower levels of DNA damage of the
Tail intensity (TI%) – average results of DNA damage (percentage of DNA in the other polluted sites along the Danube River were obtained in the study of
tail of the comet) in the haemocytes of caged mussels.
Maric et al. [148] – the highest level of TI% recorded in hemocytes of
Figure 4. Principal component analysis of biometric

measurements; PAS-reaction in the gills; oxidative
stress related enzymes' activities (catalase – CAT,
glutathione peroxidase – GPx, and glutathione
reductase – GR) and metabolic related enzymes' ac-
tivities (cholinesterase – ChE, aspartate aminotrans-
ferase – ASAT, alanine aminotransferase – ALAT, and
lactate dehydrogenase – LDH) in the digestive glands
(U/mg protein); and DNA damage (percentage of DNA
in the tail of the comet (tail intensity – TI%)) in the
hemocytes of mussels from different sampling sites in
Bulgaria.
10
Chinese pond mussel specimens was 18.42 0.72, while the lowest was pilot study are essential for improving the assessment and monitoring
10.95 0.58. programs of water pollution effects. In addition, these data suggest that
Our results confirmed the comet assay as a sensitive biomarker for the the approach with transplanted mussels should be applied regularly in
detection of DNA damage in bivalves from polluted waters. Based on the ecotoxicological studies in Bulgaria and respectively on the Balkans,
obtained results, we can summarize that the significantly higher levels of along with classical bioaccumulation analyses of water/sediments and
DNA damage measured in the hemocytes of caged mussels from Studen biota in polluted reservoirs. We recommend that a battery of biomarkers,
Kladenets, Kardzhali, and Zhrebchevo reservoirs compared to the refer- which include biometric measurements, histochemical, biochemical, and
ence site revealed the genotoxic potential of the waters of the three genotoxic assays are also integrated because it is essential to use easily
reservoirs, which we associated with the presence of metals, toxic ele- applicable and representative biological tools for determining the toxi-
ments and organic toxicants, such as PBDEs and SCCPs. cant impact on bivalves in freshwater ecosystems. Lastly, we recommend
that further investigation is performed to study in detail the combined
3.2.7. Relationships between pollutant levels and biomarker responses exposure to inorganic and organic pollutants, and their effects on the
The results of correlation analysis, which was performed to study the Chinese pond mussel, which may reveal antagonistic or synergistic
possible relationships between the MPI and the different biomarker re- interactions.
sponses, such as biometric indices, enzymatic responses, and tail in-
tensity (DNA damage), were summarized in Table 3. Declarations
In the case of biometric indices, only the state index (CI 3) and the
condition factor (CI 5) showed a significant (p < 0.05) negative corre- Author contribution statement
lation with the MPI of caged mussels. According to these results, with the
increasing values of MPI, the state index (CI 3) and condition factor (CI 5) Elenka Georgieva: Conceived and designed the experiments;
decreased significantly. Therefore, the mussels with higher concentra- Contributed reagents, materials, analysis tools or data; Wrote the paper.
tions of metals had lower biometric indices. o Antal & Ifeanyi Emmanuel Uzochukwu: Analyzed and inter-
Laszl
Between the MPI and the oxidative stress-related enzymatic activities preted the data; Wrote the paper.
(CAT, GPx, GR) there were significant (p < 0.05) positive correlations. At Stela Stoyanova & Vesela Yancheva: Conceived and designed the
the same time, the metabolic-related enzyme activities (except for LDH) experiments; Performed the experiments; Contributed reagents, mate-
showed a significant decrease (p < 0.05) with the increasing values of rials, analysis tools or data; Wrote the paper.
MPI. These results indicated that the mussels with higher values of MPI Desislava Aranudova, Iliana Velcheva, Ilia Iliev, Tonka Vasileva,
increased the activities of oxidative stress-related enzymes. On the other Veselin Bivolarski, Vesela Mitkovska, Tsenka Chassovnikarova & Bor-
hand, the metabolic-related enzymes (ASAT, ALAT, ChE) showed a sig- islava Todorova: Performed the experiments.
nificant negative correlation with MPI. This means that mussels with Krisztian Nyeste: Analyzed and interpreted the data; Wrote the paper.
higher values of MPI had lower activities of metabolic-related enzymes.
Furthermore, it is also important to highlight that between the MPI
Funding statement
and TI% a significant (p < 0.05) positive correlation (Table 3) was
established. This result confirmed that the mussels with higher MPI had
This work was supported by the Live water, air and health with
notable DNA damage.
transplants - LIFE (FP19-BF-013), NPD – Plovdiv University “Paisii
The IBR values also showed that overall, the pollution status based on
Hilendarski”, the National Research, Development and Innovation Fund
the investigated biomarkers was the lowest in the case of reference site
of Hungary (TKP2021-NKTA-32) and the New National Excellence Pro-
(Table 2). Zhrebchevo Reservoir had a relatively low IBR value, which
gram of the Ministry of Human Capacities (ÚNKP-21-4).
indicates a lower pollution status of this sampling site. At the same time,
Kardzhali, and Studen Kladanets reservoirs were more impacted, which
supports the presently reported results. Data availability statement
The PCA showed clear separations among all investigated biomarkers
of mussels from the different sampling sites in Bulgaria (Figure 4). The Data included in article/supplementary material/referenced in
first component (PCA 1) contributed 58.56% of the total variance, while article.
the second (PCA 2) contributed 21.72% of the total variance (Figure 4).
Based on the biomarker pattern, the groups were completely separated Declaration of interests statement
from the other groups, which supported the results of the regression
analyses. The authors declare no conflict of interest.
In a similar way, Ferreira et al. [115] studied the possible relation-
ships between metal levels and biochemical biomarkers in oysters
Additional information
(Crassostrea gasar Deshayes, 1830). Ferreira et al. [119] also investigated
the relationship between organic toxicants (polycyclic aromatic hydro-
No additional information is available for this paper.
carbons, PAHs, polychlorinated biphenyls, PCBs, and linear alkylben-
zenes, LABs) and molecular, and biochemical markers in transplanted
oysters. Moreover, our results are in agreement with Capolupo et al. Acknowledgements
[160] who followed the correlation trends between CAT activity, DNA
damage, and caffeine concentrations in the Mediterranean mussel We sincerely thank Assoc. Prof. Gana Gecheva for fulfilling a wish to
(Mytillus galloprovincialis Lamark, 1819), which were dose-dependent. work on caged mussels despite all challenges in the field.
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Biomarkers For Pollution in Caged Mussels From Three Reservoirs in Bulgaria A Pilot Study

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Biomarkers for pollution in caged mussels from three reservoirs in Bulgaria:

1. Introduction studied thoroughly to understand the extent of the damage, which is a

2.4. Laboratory methodology 2.4.4. Biochemical analyses

where Y ¼ the standardized value of the biomarker; X ¼ mean value of a

Z ¼ –Y or Z ¼ Y (8) 3.2.1. Biometric indices

those measured in the Zhrebchevo Reservoir and 10 times higher than

Figure 4. Principal component analysis of biometric

[1] P. Chapman, Heavy metal—music, notscience, Environ. Sci. Technol. 41 (2007) 6.

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