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Biomarkers For Pollution in Caged Mussels From Three Reservoirs in Bulgaria A Pilot Study
Biomarkers For Pollution in Caged Mussels From Three Reservoirs in Bulgaria A Pilot Study
Heliyon
journal homepage: www.cell.com/heliyon
Research article
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
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E. Georgieva et al. Heliyon 8 (2022) e09069
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
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E. Georgieva et al. Heliyon 8 (2022) e09069
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.
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E. Georgieva et al. Heliyon 8 (2022) e09069
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)
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E. Georgieva et al. Heliyon 8 (2022) e09069
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
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E. Georgieva et al. Heliyon 8 (2022) e09069
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
E. Georgieva et al. Heliyon 8 (2022) e09069
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
E. Georgieva et al. Heliyon 8 (2022) e09069
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
E. Georgieva et al. Heliyon 8 (2022) e09069
10
E. Georgieva et al. Heliyon 8 (2022) e09069
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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