Assessment of the Health Status of Whitefish (Coregonus lavaretus Linnaeus, 1758) and the Quality of Its Habitat in Lake Sevan (Armenia) Using a Multi-Biomarker Approach

Abstract

Lake Sevan is a freshwater reservoir in the Caucasus region. Since the first half of the 20th century, the lake has undergone significant changes caused by human activity and anthropogenic pressure. To identify the current ecological state of two bays—Lchashen and Lichk—located in different parts of the lake, a study was conducted in 2022–2023 using a multi-biomarker approach. For this purpose, biomarkers for assessing the health status of fish and the quality of their living conditions were used such as the activity of acetylcholinesterase (AChE) in the brain, glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), and the concentration of reduced glutathione (RGS) and malondialdehyde (MDA) in the liver and gills of the whitefish (Coregonus lavaretus Linnaeus, 1758). In addition, hydrochemical and ichthyological analyses were conducted. This study demonstrated seasonal dynamics for all biomarkers. Comparative analysis of biomarkers and hydrochemical and ichthyological data showed that the whitefish in Lchashen Bay is in worse health, and its living conditions there are less favorable than in Lichk Bay.

1. Introduction

Anthropogenic pollution of aquatic ecosystems globally is one of the pressing environmental problems of modern society. Pollution of water bodies disrupts the functioning of their ecosystems and related ecosystem services. In particular, wastewater from the food processing industry and municipal and agricultural wastewater cause an additional flow of easily oxidized organic matter and biogenic elements into water bodies, which leads to an increase in their trophicity and saprobity. This, in turn, can cause a decrease in the concentration of dissolved oxygen leading to the creation of hypoxia or anoxia and deterioration of the living conditions of aquatic organisms. The discharge of industrial wastewater containing heavy metals and inorganic and organic toxic compounds cause pathological disorders in the body of aquatic animals and their death. All this leads to a change in the structure of aquatic communities and ecosystems [1,2,3,4,5,6].

Lake Sevan, one of the largest freshwater high-mountain lakes in the world, is the largest freshwater reservoir in the Caucasus region. It consists of two parts, Small and Big Sevan, which were formed in the late Miocene and then significantly transformed in the Pleistocene and Holocene [7]. The ecosystem of Lake Sevan is a complex object of great natural and socio-economic significance representing a strategic source of drinking water, providing water for recreation, irrigation, hydropower generation, and fish resources [8,9].

Since the first half of the 20th century, the lake has experienced significant changes caused by human activities: declining water levels, loss of biodiversity, habitat degradation, intensive nutrient input from the catchment area, and overfishing. As a result, since the mid-20th century, the oligotrophic lake has turned into a mesotrophic one [8].

Bays of Lake Sevan are one of the main habitats of different fishes. Young whitefish (Coregonus lavareus) mainly feed in the bays of the lake, the Lichk and Lchashen Bays also serve as spawning grounds for whitefish. The whitefish was introduced into the lake in the late 1920s and 1930s and became the main commercial species of the lake by the 1960 [10,11]. While they have been the main pelagic consumer of zooplankton [12], the whitefish have large feeding plasticity and can feed on any available prey [13].

However, the bays are subject to anthropogenic input associated with industrial activity and municipal and agricultural wastewater [14], and the whitefish (Coregonus lavaretus) belonging to the Salmonidae family is sensitive to changes in the water quality. For example, the feeding of Atlantic salmon may be reduced to as low as 60% at an oxygen level around 40% saturation [15]. Changes in the biochemical parameters of fish may be early signs of deteriorated feeding conditions long before the irreversible changes are shown at the population level [16]. For example, the activity of acetylcholinesterase (AChE) is a widely used method for monitoring pollution, mainly due to its high sensitivity to anticholinergic chemicals such as organophosphate pesticides and carbamates [17]. AChE has the ability to stoichiometrically bind organophosphorus (OP) and carbamate compounds (Cs), which leads to irreversible inhibition of ChE activity, thereby causing disruption of the normal functioning of the organism, ultimately leading to its death [17,18]. Moreover, there are data of AChE activity inhibition via cyanotoxins [19]. In this connection, a decrease in cholinesterase activity in animal tissues is a specific and long-term biomarker of fish being poisoned with these compounds [20]. On the other hand, it has been shown that the activity of AChE in the fish brain can increase during acute and chronic stress, induced by different factors [21].

At the molecular biological level, the damaging effect of xenobiotics is caused by excessive production of reactive oxygen species (ROS) in living organisms. Under normal conditions, ROS are produced in small quantities as a by-product of metabolism. ROS are strong oxidizers and extremely active compounds that destroy submolecular cellular structures and functional molecules. At the same time, the intensity of lipid peroxidation (LPO) and oxidative damage to DNA and proteins increases and the activity of the antioxidant system (AOS) is changed [22]. Increased accumulation of lipid peroxidation products, in particular malondialdehyde (MDA), is one of the signs indicating oxidative stress and the presence of active oxygen species in the cell. The cellular antioxidant defense system neutralizes the damaging effects of ROS. Important elements of AOS are the enzymes catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GSH), glutathione peroxidase (GPO), and glutathione transferase (GST).

Hence, the aim of this work is to evaluate the health of whitefish and its habitat conditions in the two bays of Lake Sevan (Lichk and Lchashen) via biochemical methods (biomarkers of oxidative stress, AChE, and LPO) using a multi-biomarker approach. In parallel, ichthyological investigations were conducted to evaluate the changes in the Condition Factor (CF) of fish which is indicative of the fish feeding state in the bays.

2. Materials and Methods

2.1. Study Area and Fish Sampling

The studies were conducted on Lake Sevan (Armenia), located at an altitude of about 1900 m above sea level and consisting of two morphometrically different parts: Small Sevan (SS) in the northwest and Big Sevan (BS) in the southeast, connected by a narrow strait (Figure 1). Samples were collected in May, August, and October of 2022 and 2023 years at two stations located along the eastern coast of the lake at a distance of 30 m from the shore: one in the northern part of Small Sevan in Lchashen Bay (40°30′45.0″ N 44°57′25.3″ E) and the other in the southern part of Big Sevan in Lichk Bay (40°11′12.4″ N 45°14′39.3″ E). Both bays are ecosystems with rich biodiversity due to their shallow depth and unique hydrological regime. Due to the relatively shallow depth compared to other parts of the lake and the direct influence of runoff from the drainage areas, the most intensive processing of nutrients and energy transfer occurs in these bays. The Lichk Bay receives the input of two rivers, while in Lchashen Bay, the water is mostly stagnant due to the absence of inflowing rivers. Nevertheless, both bays are under the influence of anthropogenic loads associated with industrial activity and domestic and agricultural wastewater [14].

Whitefish (Coregonus lavaretus Linnaeus, 1758) were caught using gill nets (length 100 m, width 3 and 5 m, mesh size 45 × 45 mm), which were set in the previous evening at 19:00–20:00 and removed around 06:00–07:00 the following morning. The total number of whitefish studied was 120 individuals—10 specimens in each season in each bay.

2.2. Ichthyological Analysis

The fish were taken out of the nets, and the length to the tips of the caudal fins and the weight of whole fish were measured as quickly as possible. The fish were killed via cervical transection, dissected, and sexed. Biometric and morphological analyses were carried out using standard ichthyological methods [23,24,25,26]. Age structure was determined through counting annual rings on a scale [24,26]. Size structure was identified through measuring the total length. The total body weight (W) was recorded. The sex structure—sex and the level of development of gonads (according to a 6-grade system) were identified [24]. Food components were investigated according to a guide for the investigation of fish feeding [27,28].

CF was calculated according to the following formula [27,28]:

CF = (whole body weight, g/total length³, cm) × 100

2.3. Biochemical Analysis

Immediately after biometric analyses, fish were placed on ice and dissected: the brain, liver, and gills were removed, weighed, washed in a chilled 0.1 M phosphate buffer with pH 7.5, and dried on filter paper. The samples were placed in a sealed bag and stored at a temperature of no more than −86 °C until biochemical analysis was performed. Each tissue sample was homogenized in ice-cold phosphate buffer pH 7.5 at a 1:5 ratio (w/v) for enzyme activity assays (1 g tissue in 5 mL buffer) and a 1:1 ratio for reduced glutathione (GSH) assay (1 g tissue in 1 mL buffer) using an Ultra-Turrax T10 Basic homogenizer (IKA). Homogenates were centrifuged at 12,000× g for 10 min at 4 °C in a Hettich Mikro 22 R (Germany) centrifuge. After centrifugation, the lipid phase was removed, and supernatants were collected for further analysis. The brain was used for the determination of acetylcholinesterase activity (AChE), and the liver and gill were used for the determination of glutathione S-transferase (GST) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, reduced glutathione (GSH) concentrations, and lipoperoxidation (LOP). Biochemical assays were carried out using an MRC Spectro UV-18 (Israel) spectrophotometer. Each sample was measured in duplicate. Protein contents were measured using the method of Bradford (1976) [29] with bovine serum albumin as a standard.

The AChE activity was determined following the procedure of Ellman (1961) [30]. The enzyme activities were assayed via the addition of a substrate of acetylthiocholine iodide (Sigma-Aldrich^®, Darmstadt, Germany) (AThCh; 4.3 × 10⁻⁴ M final concentration) and dithiobisnitrobenzoic acid (Sigma-Aldrich^®, Germany) (DTNB; 7.1 × 10⁻⁵ M final concentration) mixture. After 20 min of incubation, the reaction was stopped with the addition of prostigmine (Sigma-Aldrich^®, Germany) to each of the tubes except the blank control, and the enzyme activities were determined. Samples were measured at 412 nm. The activity of AChE was calculated as nmol/µg protein/min.

The contents of MDA as a measure of LOP intensity were assayed via color reaction with 2-thiobarbituric acid [31]. Trichloroacetic acid (Component-Reaktiv, Moscow, Russia) solution at 30%, 0.5 M HCl (Component-Reaktiv, Russia) solution, and 0.75% 2-thiobarbituric acid (Sigma-Aldrich^®, Germany) solution were successively added to tissue homogenate. The mixture was stirred vigorously and heated for 20 min in a bath of boiling water. After cooling, samples were separated via centrifuging. An ethanol solution of ionol (Sigma-Aldrich^®, Germany) was added to the supernatant (final concentration 5 mM) in order to prevent lipid peroxidation in the sample. Samples were measured at 532 nm. A molar extinction coefficient of 1.56 × 10⁵ M⁻¹ cm⁻¹ was used for calculations. The content of MDA was calculated as pmol/µg protein.

The CAT activity was measured by following the rate of 0.3% H₂O₂ decomposition, which can be determined directly by the decrease in absorbance at 410 nm, using a molar extinction coefficient of 22.2 × 10³ M⁻¹ cm⁻¹. Change in absorbance per minute was measured and calculated as pmol/µg protein/min [32].

The SOD activity was measured by the inhibition of nitroblue tetrazolium reduction in reaction with phenazine methosulfate and NADH under basic conditions [33]. Measurement was performed in 0.15 M phosphate buffer pH 7.8 at 20 °C. The reaction mixture contained 0.00033 M EDTA (Sigma-Aldrich^®, Germany), 0.0006 M phenazine methosulfate (Sigma-Aldrich^®, Germany), 0.00135 M nitroblue tetrazolium (Sigma-Aldrich^®, Germany), 0.078 M NADH (Sigma-Aldrich^®, Germany), and 0.3 mL sample aliquot. SOD activity determination was performed at 540 nm and calculated as the increase in concentration (ΔE × 10^–6) of nitroformazan produced per µg protein in the sample per 1 min of reaction time.

The GSH concentrations were detected in reaction with 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB). Trichloroacetic acid (30%) was added to homogenate aliquot, mixed thoroughly, and placed on ice for 1 h. The resulting precipitate was separated via centrifugation at 10,000× g for 10 min at 4 °C. Each sample cuvette contained 0.1 M phosphate buffer pH 7.5, 0.001 M DTNB (Sigma-Aldrich^®, Germany), and 0.5 mL supernatant fraction. The colored complex was registered at 412 nm. GSH content was calculated using a molar extinction coefficient of 13.6 × 10³ M⁻¹ cm⁻¹ and calculated as pmol/µg protein [34].

The GST activity was determined by monitoring the conjugation of GSH with 1-chloro-2,4-dinitrobenzene used as a substrate [35]. The reaction mixture was composed of 0.1 M phosphate buffer pH 7.5, 0.1 M GSH, 0.05 M 1-chloro-2,4-dinitrobenzene (Sigma-Aldrich^®, Germany), and 0.1 mL of the sample aliquot. Absorbance was measured at 340 nm, 20 °C. The increase in absorbance was recorded for 3 min. Enzyme activity was measured using a molar extinction coefficient of 9.6 × 10³ M⁻¹ cm⁻¹ and calculated as the concentration of 1-cloro-2,4-dinitrobenzene produced per mg protein in the sample per minute of reaction time (nmol/µg protein/min).

All reagent work solutions for analysis were prepared in the laboratory and commercial kits were not used.

2.4. Hydrochemical and Hydrophysical Analysis

Simultaneously with the catch of fish, water samples were taken in the same places using a Molchanov bathometer (Zawod Gidrometpribor, Russia) from 5 to 10 m depth of the lake. Water samples were collected for physical–chemical analysis using 1 L clean polyethylene bottles (ten bottles from each bay) after being rinsed three times with a few milliliters of lake water sample.

Dissolved oxygen (DO) concentration, biochemical oxygen demand (BOD₅), permanganate oxidation, nutrients (nitrates, nitrites, ammonium ions, and phosphates), metals (K, Na, Ca, Mg, total Fe, Cu, Zn, Pb, and Cd) concentrations, and pH were determined. Nitrate ion was analyzed via an ion chromatograph with a photoelectrocolorimetric detector Metrohm 940 Professional IC Vario (Switzerland); nitrites, ammonium, and phosphate ions were measured via the photoelectrocolorimetric method using a Specord 210 PLUS Analytic Jena spectrophotometer (Germany). BOD₅ was measured via the electrochemical method with a portable dissolved oxygen meter H19146 (Germany). The concentrations of metals (K, Na, Ca, Mg, total Fe, Cu, Zn, Pb, and Cd) in water were measured on a 7900 ICP-MS Agilent inductively coupled plasma mass spectrometer (US).

2.5. Statistical Analysis

The data are presented as the means with the standard errors (mean ± SEM). The data were statistically processed using the Statistica 8.0 software package. The differences in the levels of AChE, antioxidants, and MDA and the length and weight of the whitefish among the season sampling points were examined using one-way ANOVA analysis with Tukey post hoc tests or Student’s t-test between bays at a p = 0.05 significance level.

3. Results

3.1. Hydrochemical and Hydrophysical Analyses

Hydrochemical and hydrophysical parameters of the water taken from the Lichk and Lchashen Bays are presented in

Table 1. Hydrochemical and hydrophysical parameters of the water in Lichk and Lchashen Bays.

Bay	Temperature (°C)	pH	Dissolved Oxygen (Mg L−1)	BOD5 (mg L−1)	Permanganate Oxidation (mg O L−1)	Ammonium Ion (mg N L−1)	Nitrate Ion (mg N L−1)	Nitrite Ion (mg N L−1)	Phosphate Ion (mg P L−1)
May, 2022
Lchashen	22	8.4	6.8	3.9	6	0.1	0.24	0.007	0.06
Lichk	18	8.4	8.3	3.5	5.7	0.07	1.1	0.012	0.09
August 2022
Lchashen	20	8.5	2.4	4.5	6.8	0.27	0.18	0.005	0.09
Lichk	23	8.4	6.1	3.9	6.2	0.1	0.16	0.004	0.08
October, 2022
Lchashen	16.7	8.4	5.1	4.2	6.2	0.21	0.23	0.008	0.07
Lichk	19.7	8.4	6.9	3.6	5.9	0.08	0.19	0.005	0.09
May, 2023
Lchashen	21	8.3	6.5	3.8	5.9	0.13	0.2	0.006	0.08
Lichk	19	8.4	8.3	3.2	5.3	0.08	1.14	0.01	0.06
August, 2023
Lchashen	23	8.5	3.7	4.6	6.4	0.18	0.17	0.004	0.1
Lichk	22	8.3	6.4	3.7	5.8	0.11	0.16	0.007	0.07
October, 2023
Lchashen	17	8.5	5.8	4	6	0.17	0.19	0.007	0.09
Lichk	19	8.4	6.9	3.3	5.4	0.1	0.17	0.006	0.06
National surface water quality criteria *	-	6.5–8.5	≥6	3.0	5	0.39	9.0	0.02	3.5

Notes: * National surface water quality criteria adopted by the Ministry of Environment of Armenia (http://env.am/en/environment/environmental-monitoring, accessed 10 July 2024).

and

Table 2. Concentrations (mg L⁻¹) of select elements present in the water samples of Lichk and Lchashen Bays.

Bay	K+	Na+	Ca2+	Mg2+	Fe Total	Cu2+	Zn2+	Pb2+	Cd2+
May, 2022
Lchashen	13	62	26	45	0.02	<dL	<dL	<dL	<dL
Lichk	7.6	40	29	18	0.21	<dL	<dL	<dL	<dL
August, 2022
Lchashen	31.1	61	26.1	44	0.02	<dL	<dL	<dL	<dL
Lichk	19.4	56	27	34	0.09	<dL	<dL	<dL	<dL
October, 2022
Lchashen	12.8	58	27	44.3	0.01	<dL	<dL	<dL	<dL
Lichk	14	47	27.2	39	0.02	<dL	<dL	<dL	<dL
May, 2023
Lchashen	14	59	27	46	0.04	<dL	<dL	<dL	<dL
Lichk	7.3	41	28	23	0.19	<dL	<dL	<dL	<dL
August, 2023
Lchashen	26	60	27.1	46	0.03	<dL	<dL	<dL	<dL
Lichk	17	54	27	38	0.07	<dL	<dL	<dL	<dL
October, 2023
Lchashen	13.1	56	26.7	48	0.02	<dL	<dL	<dL	<dL
Lichk	12.6	49	27	41	0.02	<dL	<dL	<dL	<dL
“average” quality *	4 × B	4 × B	200	100	0.5	0.05	0.2	0.025	B + 0.002
“good” quality *	2 × B	2 × B	100	50	2 × B	B + 0.02
“excellent” quality *	B	B	B	B	B	B

Notes: * Armenia surface water quality guidelines. B—background value of a given element in a local environment; dL—detection limit: 0.002 mg L⁻¹.

.

The water temperature in both bays varied in the range of 16.7–23 °C, i.e., during the entire observation period the seasonal temperature range was no more than 6.3 °C, which corresponds to the usual seasonal temperature values and their variability in these parts of Lake Sevan (Table 1). In May, the water temperature in the Lchaschen Bay was always slightly higher, and in October, it was lower than in the Lichk Bay. In August, the temperature in both bays was almost the same and the highest of the season, and in October, the water temperature was minimal. Water pH values varied little during the entire observation period, equaling 8.3–8.5, and were within the norms for the previous period of 2017–2022 (http://env.am/en/environment/environmental-monitoring, accessed 10 July 2024). No pronounced seasonal variability was revealed.

The DO content varied over the observation period in a wide range from 2.4 to 8.3 mg L⁻¹ (Table 1). In both bays, it was highest in May, then decreased to a minimum in August, and increased again in October, but did not reach the May values. It should be noted that in Lchashen Bay, the values of this indicator were always lower than in Lichk Bay. At the same time, in Lchashen Bay in May, the dissolved oxygen content corresponded to the national standard level, while in August and October, it was 1.62–2.5- and 1.03–1.18-times lower, respectively, depending on the year. Note that in Lichk Bay, the content of DO for all months was above the national standard level. The reduced concentrations of DO in Lchashen Bay are most likely associated with the increased content of easily oxidized organic matter, the oxidation of which consumes oxygen.

The organic matter content measured as BOD₅ and permanganate oxidation in both bays was high during the observation period, varying in the range from 3.2 to 6.8 mg L⁻¹ (Table 1). It was highest in August compared to May and October. It should be noted that the organic content in Lchashen Bay was always higher than in Lichk Bay. At the same time, the BOD₅ and permanganate oxidation values in both bays exceeded the national water quality standards (by 1.13–1.5 and 1.2–1.4 times, respectively).

The content of nitrates and phosphates in both bays varied in the range of 0.004–1.14 mg L⁻¹ (Table 1). The concentration of nitrates and nitrites was highest in May, then decreased in August and increased again in October but did not reach the May values. In contrast, the ammonium and phosphate content were higher in August than in May and October. According to the data, the nitrate and phosphate values in Lchashen Bay were always higher than those in Lichk Bay, which was due to the increase in the concentration of ammonium ions and additionally indicates some obvious deterioration in water quality. The nitrate, nitrite, and phosphate content in both bays corresponded to the national standard level (Table 1).

The concentrations of all studied metal ions (K, Na, Ca, Mg, total Fe, Cu, Zn, Pb, and Cd) varied throughout the observation period in the range of 0.02–62 and were much lower than the “average” water quality indicator, mostly corresponding to the “good” and even “excellent” water quality criterion (Table 2) according to the national surface water quality criteria adopted by the Ministry of Environment of Armenia (http://env.am/en/environment/environmental-monitoring, accessed 10 July 2024).

3.2. Ichthyological Data

Biometric parameters of the lake’s whitefish in the study seasons are presented in

Table 3. Biometric indicators of whitefish from Lichk and Lchaschen Bays of Lake Sevan.

	Age	N	Length, cm	Weight, g	Condition Factor
Lichk Bay
May, 2022	1 + 2+	10 0	28.61 ± 0.346 a	224.6 ± 7.45 a	0.96 ± 0.01 a
August, 2022	1 + 2+	4 6	31.52 ± 0.59 b	318.3 ± 20.7 b	1.01 ± 0.05 a,b
October, 2022	1 + 2+	7 3	30.2 ± 0.65 b,c *	291.7 ± 21.1 b *	1.05 ± 0.02 a,b,c
May, 2023	1 + 2+	9 1	29.05 ± 0.42 a	255.3 ± 12.3 a *	1.04 ± 0.03 a,b,c *
August, 2023	1 + 2+	9 1	31.71 ± 0.43 b,c,d	358.3 ± 14.3 a,b,c *	1.12 ± 0.04 b,c *
October, 2023	1 + 2+	0 10	33.18 ± 0.55 b,c,d *	385.8 ± 11.1 b,c *	1.06 ± 0.02 a,b,c
Lchashen Bay
May, 2022	1 + 2+	10 0	28.4 ± 0.247 a	224.9 ± 3.59 a	0.98 ± 0.01 a
August, 2022	1 + 2+	2 8	32.11 ± 0.367 b	344.6 ± 11.7 b	1.04 ± 0.03 a,b
October, 2022	1 + 2+	10 0	28.4 ± 0.296 a *	223.3 ± 5.37 a *	0.98 ± 0.02 a,b
May, 2023	1 + 2+	10 0	28.65 ± 0.224 a	224.6 ± 6.32 a *	0.95 ± 0.02 a,b,c *
August, 2023	1 + 2+	9 1	30.88 ± 0.262 c	290.5 ± 8.9 c *	0.99 ± 0.02 a,b,c *
October, 2023	1 + 2+	6 4	31.99 ± 0.38 b,c *	329.4 ± 20.2 b,c *	1.00 ± 0.04 a,b,c

Note: a,b,c,d values marked with the same letters in each column are not significantly different (ANOVA and LSD test, p = 0.05). * Means for the same date differ significantly between stations (Student’s t-test, p = 0.05).

.

3.3. Biochemical Studies

Comparative analysis of the data showed that the values of all studied biochemical parameters of fish during the observation period of 2022–2023 were consistently and statistically significantly higher in fish from Lchashen Bay than in fish from Lichk Bay (Student’s t-test, p = 0.05). In both bays, fish showed similar seasonal dynamics for each indicator, and seasonal differences within each year, with some exceptions, were statistically significant, while interannual differences for each month were mostly absent or minimal (ANOVA Tukey test, p = 0.05). In the liver, the values of AOS indicators were slightly higher than in the gills.

AChE activity. The value of this indicator in the brain of whitefish from Lchashen Bay was approximately 2-times higher than that of fish from Lichk Bay and varied within the range of 0.3543–0.5215 and 0.1692–0.2546 nmol/μg protein/min, respectively (

Table 4. Values of acetylcholinesterase (AChE) in the brain of whitefish specimens.

Month, Year	Mean Values of Parameters ± S.E.
	AChE
	nmol per 1 μg Protein per 1 min
	Lichk Bay	Lchashen Bay
May, 2022	0.2546 ± 0.010 a	0.5117 ± 0.004 a *
August, 2022	0.1692 ± 0.008 b	0.3623 ± 0.012 b,d *
October, 2022	0.1934 ± 0.009 b,c	0.3973 ± 0.007 c *
May, 2023	0.2389 ± 0.010 a,d	0.5215 ± 0.004 a *
August, 2023	0.1807 ± 0.004 b,c	0.3543 ± 0.009 b *
October, 2023	0.2081 ± 0.005 c,d	0.3903 ± 0.0025 c,d *

Note: a,b,c,d values marked with the same letters in each column are not significantly different (ANOVA and LSD test, p = 0.05). * Means for the same date differ significantly between stations (Student’s t-test, p = 0.05). The same note for Table 5 and Table 6.

). The maximum seasonal activity was observed in May; in August, it decreased by 1.4–1.5 times, and in October, it increased again but did not reach the maximum value, remaining 1.3-times lower than the May values. The differences between the May (maximum) and August (minimum) values were statistically significant, and the October values, which occupied an intermediate position, did not statistically differ depending on the year from either the May or August values.

MDA content. The values of the indicator in the liver and gills of fish from Lchashen Bay were statistically significantly higher than that of the fish from Lichk Bay by 17–26% and 60–79%, respectively (

Table 5. Values of lipid peroxidation and antioxidant system in the liver of whitefish specimens.

Month, Year	Mean Values of Parameters ±S.E.
	MDA	GSH	GST	Catalase	SOD
	pmol per 1 μg Protein		nmol per 1 μg Protein per 1 min		ΔE × 10–6 per 1 μg Protein per 1 min
Lichk Bay
May, 2022	0.5213 ± 0.026 a,c	8.21 ± 0.27 a	2.27 ± 0.18 a	24.1± 0.35 a	7.6 ± 0.18 a
August, 2022	0.6140 ± 0.016 b	12.9 ± 0.15 b	1.15 ± 0.03 b,c	29.7 ± 0.17 b	16.2 ± 0.13 b
October, 2022	0.5835 ± 0.017 a,b	11.1 ± 0.16 c	1.54 ± 0.03 c	27.4 ± 0.13 c	13.8 ± 0.08 c
May, 2023	0.5102 ± 0.013 c	8.27 ± 0.16 a	2.21 ± 0.15 a	23.7 ± 0.17 a	8.2 ± 0.13 d
August, 2023	0.6320 ± 0.009 b	13.3 ± 0.13 b	1.11 ± 0.02 b	29.3 ± 0.127 b	15.7 ± 0.13 b
October, 2023	0.5792 ± 0.003 a,b	12.8 ± 0.095 b	1.27 ± 0.05 b,c	28.4 ± 0.14 d	14.9 ± 0.02 e
Lchashen Bay
May, 2022	0.6147 ± 0.020 a *	9.72 ± 0.21 a *	3.67 ± 0.26a *	36.3 ± 0.27 a	14.9 ± 0.24 a
August, 2022	0.7420 ± 0.012 b *	14.8 ± 0.1 b *	2.73 ± 0.12 b *	41.3 ± 0.14 b	28.3 ± 0.12 b
October, 2022	0.6912 ± 0.007 c *	13.3 ± 0.18 c *	2.97 ± 0.103 b *	39.7 ± 0.053 c	26.9 ± 0.12 c,e
May, 2023	0.6412 ± 0.005 a,d *	9.77 ± 0.045 a *	3.73 ± 0.05 a *	34.9 ± 0.02 d	14.2 ± 0.046 d
August, 2023	0.7390 ± 0.008 b *	14.1 ± 0.04 d *	2.67 ± 0.096 b *	40.7 ± 0.072 e	27.1 ± 0.11 e
October, 2023	0.6827 ± 0.007 c,d *	13.5 ± 0.09 c *	2.91 ± 0.13 b *	39.1 ± 0.02 f	26.3 ± 0.2 c

Note: a,b,c,d values marked with the same letters in each column are not significantly different (ANOVA and LSD test, p = 0.05).

and

Table 6. Values of lipid peroxidation and antioxidant system in the gill of whitefish specimens.

Month, Year	Mean Values of Parameters ±S.E.
	MDA	GSH	GST	Catalase	SOD
	pmol per 1 μg Protein		nmol per 1 μg Protein per 1 min		ΔE × 10–6 per 1 μg Protein per 1 min
Lichk Bay
May, 2022	0.3217 ± 0.026 a	5.26 ± 0.24 a	1.97± 0.13 a	17.3 ± 0.35 a	6.9 ± 0.16 a
August, 2022	0.3671 ± 0.014 a	8.90 ± 0.15 b	1.09± 0.04 b	21.8 ± 0.19 b	14.8 ± 0.11 b
October, 2022	0.3583 ± 0.008 a	7.40 ± 0.16 c	1.37 ± 0.07 b	19.5 ± 0.08 c	12.9 ± 0.11 c
May, 2023	0.3172 ± 0.016 a	5.71 ± 0.10 a	1.81 ± 0.14 a	16.7 ± 0.13 a	7.6 ± 0.12 d
August, 2023	0.3431 ± 0.010 a	8.31 ± 0.13 b,d	1.16 ± 0.014 b	23.1 ± 0.18 d	13.9 ± 0.13 e
October, 2023	0.3479 ± 0.011 a	7.80 ± 0.095 c,d	1.31 ± 0.04 b	21.2 ± 0.14 b	12.3 ± 0.12 f
Lchashen Bay
May, 2022	0.5134 ± 0.017 a	7.12 ± 0.12 a	2.73 ± 0.08 a	24.8 ± 0.16 a	11.9 ± 0.24 a
August, 2022	0.6052 ± 0.006 b	13.1 ± 0.11 b	1.89 ± 0.10 b,c	32.3 ± 0.15 b	26.1 ± 0.11 b
October, 2022	0.5943 ± 0.006 b	11.9 ± 0.07 c	2.18 ± 0.10 c	29.4 ± 0.13 c	23.2 ± 0.11 c
May, 2023	0.5371 ± 0.004 a	7.79 ± 0.04 d	2.87 ± 0.07 a	25.9 ± 0.02 d	12.8 ± 0.13 d
August, 2023	0.6149 ± 0.007 b	12.67 ± 0.16 e	1.71 ± 0.11 b	34.2 ± 0.33 e	27.4 ± 0.09 e
October, 2023	0.5818 ± 0.007 b	11.43 ± 0.08 f	2.03 ± 0.10 b,c	30.7 ± 0.16 f	24.9 ± 0.12 f

Note: a,b,c,d values marked with the same letters in each column are not significantly different (ANOVA and LSD test, p = 0.05).

).

In the liver, they varied in the range of 0.6147–0.7420 and 0.5102–0.6320 pmol/μg protein, respectively (Table 5). In May, the minimum seasonal values were observed; in August, they increased by 15–24%, and in October, they decreased again but did not reach the minimum, remaining 6–14% higher than the May values. The differences between the minimum (May) and maximum (August) values of the indicator were statistically significant, and the October values, which occupied an intermediate position, either statistically significantly differed from the other two months or did not.

In the gills, the indicator varied in the range of 0.5134–0.6149 and 0.3172–0.3671 pmol/μg protein, respectively (Table 6). For fish from Lchashen Bay, minimum seasonal values were observed in May; in August, they increased by 8–18%, reaching maximum values and remained at this level in October. The differences between the May (minimum) and August (maximum) values were statistically significant. In fish from Lichk Bay, the trend of seasonal dynamics was similar to that in fish from Lchashen Bay, but there were no statistically significant seasonal differences in the indicator within each year. Interannual differences in the same months in fish from both bays were mostly absent or statistically significant, but minimal.

GSH content. The value of the indicator in the liver and gills of fish from Lchashen Bay were statistically significantly higher than in the fish from Lichk Bay by 5–20% and 60–79% (Table 5 and Table 6).

In the liver, it varied in the range of 9.72–14.8 and 8.21–13.3 pmol/μg protein, respectively (Table 5). Seasonal minimum values were observed in May; in August, they increased by 44–61%, and in October, they decreased again but did not reach the minimum, remaining 35–55% higher than May values. Interannual differences in the same months were mostly absent or minimal.

In the gills they varied in the range of 7.12–12.67 and 5.26–8.90 pmol/μg protein (Table 6). The minimum values were noted in May; in August, they increased by 46–84%, and in October, they decreased again but did not reach the minimum, remaining 37–67% higher than the May values. There were no interannual differences in the same months in fishes from Lichk Bay, but in fishes from Lchashen Bay, they were minimal but statistically significant.

GST activity. The values of the indicator in the liver and gills of fish from Lchashen Bay were statistically significantly higher than in fishes from Lichk Bay by 62–193% and by 39–73% (Table 5 and Table 6).

In the liver, they varied in the range of 2.73–3.73 and 1.11–2.27 nmol/μg protein/min, respectively (Table 5). The maximum values were observed in May; in August, they decreased by 28–50%, while in October, they increased again but did not reach the maximum, remaining 19–43% lower than the May values. The differences between the maximum (May) and August minimum (August)) values were statistically significant, but the October values, which occupied an intermediate position, did not differ from the August ones. Interannual differences in the same months were either absent or minimal.

In the gills, they varied in the range of 1.71–2.87 and 1.09–1.97 nmol/μg protein/min, respectively (Table 6). In May, the maximum values were noted; in August, they decreased by 31–45%, and in October, they increased again but did not reach the maximum, remaining 13–27% higher than the May values.

CAT activity. The values of the indicator in the liver and gills of fish from Lchashen Bay were statistically significantly higher than in fishes from Lichk Bay by 38–51% and 45–55% (Table 5 and Table 6).

In the liver, they varied in the range of 34.9–41.3 and 23.7–29.7 nmol/μg protein/min, respectively (Table 5). The minimum values were observed in May; in August, they were maximum, increasing by 14–24%, and in October, they decreased again but did not reach the May minimum, remaining 9–20% higher. Interannual differences in the same months were either absent or minimal.

In the gills, they varied in the range of 24.8–34.2 and 16.7–23.1 nmol/μg protein/min (Table 6). In May, the minimum values were noted; in August, they were maximum, increasing by 26–38%, and in October, they decreased again but did not reach the May minimum, remaining 9–20% higher. Interannual differences in the same months in fish from Lichk Bay were absent, and for the fishes from Lchashen Bay, they were minimal but statistically significant.

SOD activity. The values of the indicator in the liver and gills of fish from Lchashen Bay were statistically significantly higher than in the liver of fishes from Lichk Bay by 73–96% and 68–124% (Table 5 and Table 6).

In the liver, they varied in the range of 14.2–28.3 and 7.6–16.2 ΔE × 10^–6/μg protein/min, respectively (Table 5). The minimum values were observed in May; in August, they were maximum, increasing by 90–113%, and in October, they decreased again but did not reach the May minimum, remaining 81–85% higher. Interannual differences in the same months were mostly absent or minimal.

In the gills, this indicator varied in the range of 11.9–27.4 and 6.9–14.8 ΔE × 10^–6/μg protein/min (Table 6). The minimum values were observed in May; in August, they were maximum, increasing by 83–119%, and in October, they decreased again but did not reach the May minimum, remaining 62–95% higher. The interannual differences in the same months were minimal but statistically significant.

4. Discussion

In this study, the brain AChE activity of the whitefish taken out from both bays of Lake Sevan varied within the range of 0.1692–0.5215 nmol/μg protein/min. This is in good agreement with previously published data, showing that the enzyme activity in the same whitefish, crucian carp (Carassius auratus gibelio (Bloch, 1782), and khramicarp (Capoeta sevangi De Filippi, 1865) from Lake Sevan averages 0.548, 5.01, and 4.25 nmol/μg protein/min, respectively [36]. Similar values were obtained for roach (Rutilus rutilus Linnaeus, 1758) and bream (Abramis brama Linnaeus, 1758) of the Rybinsk Reservoir, equal to 0.280–0.603 [37] and 0.15–0.20 nmol/μg protein/min [38], respectively, depending on the season. In bream (A. brama) from the Danube River in Serbia [39] and carp (Cyprinus carpio Linnaeus, 1758) from three reservoirs in Tunisia [40], the brain AChE activity was equal to 0.05 and 0.125–0.195, respectively. In capoeta (Capoeta umbla Heckel, 1843) from the Pülümür River (Pülümür Stream, Turkey), it varies within the range of 0.04–0.54 nmol/μg protein/min [41]. In the ten-spotted live-bearer fish (Cnesterodon decemmaculatus, Jenyns 1842) from the Lujan River basin (Argentina), the enzyme activity varied within the range of 0.113–0.285 nmol/μg protein/min throughout the year [42]. In the Mediterranean rainbow wrasse (Coris julis Linnaeus, 1758), the brain AChE activity depending on habitat varied between 0.048 and 0.95 nmol/μg protein/min [43].

The data on the AOS values in the liver and gills of whitefish are generally consistent with the results obtained earlier by different authors on other fish species. Thus, in the liver of bream (A. brama L.) of the Rybinsk Reservoir, the values of MDA, GSH, GST, CAT, and SOD activity depending on the fishing location varied within the range of 0.311–0.601 and 7.99–11.95 pmol/µg protein, 1.03–3.57, 25.4–37.6 nmol/µg protein/min, and 11.0–34.5 ΔE × 10^–6/μg protein/min, respectively [44]. In another study, GST and CAT activities in the liver of bream (A. brama) from the Danube River, depending on the catch location, averaged 0.2–0.3 and 12–14 nmol/µg protein/min, respectively [39]. In carp (C. carpio) from three reservoirs in Tunisia, GST activities ranged from 0.3 to 0.8 nmol/µg protein/min [40].

Our study revealed two main trends in the status of the studied biochemical parameters in whitefish in Lake Sevan. First, all of the parameters demonstrated stable seasonal dynamics, repeating without noticeable changes during two years of observations. Secondly, the values of all parameters were higher in whitefish from Lchashen Bay than in fish from Lichk Bay.

It is well known that seasonal changes in the metabolism of living organisms are adaptive in nature and are aimed at ensuring their normal functioning under periodically changing external natural conditions during the annual cycle. The main drivers of the rhythm of seasonal biological cycles in mid-latitudes are photoperiod and temperature. Moreover, the first serves as a trigger that launches the sequence and direction of biochemical, physiological, structural–morphological, and behavioral reactions of the body, and the temperature allows for the operational regulation of these processes, accelerating or slowing them down [37,44,45]. In general, when there is a longer night phase of the day and lower temperatures in the autumn–winter season, the intensity of morphological and functional processes is reduced, and in the spring–summer, when the duration of the light phase of the day and average daily temperatures are increased, it is increased. An additional factor influencing the metabolism and seasonal dynamics of the internal processes of the body is spawning.

The seasonal dynamics of brain AChE activity in the whitefish from Lake Sevan generally corresponds to the identified patterns. The highest enzyme activity over a two-year period (2022–2023) was observed in May; in August, it decreased and then increased slightly again but did not reach the May values. Such natural dynamics of AChE activity, in our opinion, may be due to the action of two factors. In May and August, it reflects the seasonal cyclicity of the photoperiod, and the observed increase in activity in October is caused by the beginning of fish preparation for spawning, which for whitefish in Lake Sevan occurs in late November-early December. Note that the similar seasonal dynamics of AChE activity were previously found in the brain of the European sardine (Sardina pilchardus Walbaum, 1792) [46].

Correspondence of the fish brain AChE activity to natural seasonal rhythms has also been shown for perch (P. fluviatilis Linnaeus, 1758), roach (R. rutilus Linnaeus, 1758), and bream (A. brama Linnaeus, 1758) in the Rybinsk Reservoir (Russia) [37,38], capoeta (C. umbla Heckel, 1843) from the Pülümür River (Pülümür Stream, Turkey) [41], and ten-spotted live-bearer fish (C. decemmaculatus) from the Lujan River basin (Argentina) [42]. In winter, the AChE activity in the brain is the lowest. In spring, when the water temperature is still close to 0 °C, but the light phase of the day is already increased, it also begins to increase. Subsequently, the activity increases with an increase in the average water temperature, reaching a maximum in the summer months. In fish, with a one-time spawning (whitefish, bream, roach, perch, capoeta, etc.), an additional increase in enzyme activity is observed in the prespawning–spawning period.

Changes in enzyme activity during the prespawning and spawning period compared to other seasonal stages of the life of fish and other aquatic organisms are associated with hormonal changes and stress states that they experience at this time. An increase in AChE activity has been experimentally demonstrated during the stress response induced by hormones (by adrenaline and dexamethasone) and by “handling” in the fish brain [21], as well as in sea urchin coelomocytes upon exposure to cold [47].

Seasonal dynamics of AOS indicators both in the liver and in the gills of whitefish from Lake Sevan were generally similar and also cyclical. Moreover, changes in different indicators over a two-year period (2022–2023) had a multidirectional nature. Thus, in May, the content of MDA and GSH, SOD, and CAT activity in both organs of whitefish showed the lowest seasonal values, while GST activity was the highest. In August, the values of the first four indicators have reached a seasonal maximum, and the last one decreased to a seasonal minimum. In October, the changes in indicators were in the opposite direction, but they did not reach the extreme values observed in May. Previously, the seasonal dynamics of AOS indicators was studied in bream (A. brama Linneaus 1758.) from the Rybinsk Reservoir [44]. It was found that all these AOS indicators demonstrate minimum values in the winter months and maximum values in spring or summer. It should be noted that the MDA content in both whitefish and bream had the lowest interseasonal variability, which amounted to 15 and 29%, respectively, and was not always statistically significant. That is, during the seasonal cycle, the fish organism managed to maintain the content of lipid peroxidation products (LPOs) at a relatively constant level, and, consequently, the content of ROS also remained constant due to the active work of the antioxidant defense (AOD) system. The intensification of the work of the AOD system is indicated by a wider range of seasonal changes in the values of its components: from 30% and higher for GSH and up to more than 2 times for SOD.

In our study, throughout the entire observation period, the values of all the studied biochemical markers of whitefish from Lchashen Bay were higher than those from Lichk Bay. These differences may be due to both internal biological and external environmental factors. Comparative analysis of biometric indicators of whitefish in the bays across the seasons showed consistently lower weight, length, or CF in the fish (1+ and 2+ years old) of Lchashen Bay starting from the October 2022 (Table 3). These values may primarily indicate worsened feeding conditions in Lchashen Bay, compared to the Lichk, caused by lesser food availability due to different anthropogenic pressures, e.g., organic pollution. The DO content was lower, while biological (BOD) and chemical (COD) oxygen demand, as well as the concentration of ammonium in the water throughout the year, were significantly higher in Lchashen Bay than in Lichk Bay. At the same time, the nitrate content was higher in Lichk bay. The remaining indicators, as well as the concentrations of the main elements, including metals, differ to a lesser extent and are significantly below the level of national water quality standards (http://env.am/en/environment/environmental-monitoring, accessed 10 July 2024). The most pronounced differences were observed for the DO indicator, the values of which in Lchashen Bay in August and October were below the national standard level (≥6 mg L⁻¹), decreasing especially sharply in August (2.7–3.4 mg L⁻¹), which is even below the critical level (4 mg L⁻¹) necessary for the normal existence of the fish. The reason for the decrease in the DO level in Lchashen Bay in August and partly in October may be the higher level of organic matter, which is oxidized using dissolved oxygen. This assumption is supported by the increased values of BOD, COD, and ammonium concentration in the water in Lchashen Bay compared to Lichk Bay. As Lchashen Bay is characterized by water stagnation due to the absence of inflowing rivers, pollution from neighboring lands may apparently have greater impact on the water chemistry and the biota inhabiting the bay. It can be assumed that Lchashen Bay is more susceptible to anthropogenic impact than Lichk Bay.

Hence, the elevated levels of brain AChE activity and AOS indices in the liver and gills of the whitefish from Lchashen Bay found in our case are due to hypoxia. In Lichk Bay, the oxygen regime of fish has corresponded to normal conditions and the values of all biochemical indicators were lower than those of fish from Lchashen Bay. The effect of hypoxia on the activity of brain AChE was previously demonstrated in rats. It has been established that after exposure to hypoxia in the prenatal period, an increased level of activity of the membrane-bound form of AChE is observed in the brains of animals throughout their life [48].

It is also known that the decrease in the DO level in water and hypoxia results in the development of AOS in fish [49,50,51,52]. In our study, the whitefish from Lchashen Bay had an increased MDA level in both organs, indicating an increase in the intensity of ROS formation. At the same time, the values of the AOD system indicators were also increased, indicating its activation. Normally, the intensity of ROS formation and neutralization in the cell, recorded via the MDA content, is balanced, and their content is at a stationary level [49,53]. With short-term exposure to a stress factor, acute oxidative stress is developed when the balance is shifted towards the formation of ROS. However, activation of the AOD system quickly returns their level to the normal values. With long-term action of the stress factor, chronic oxidative stress develops, and a return to the initial level of ROS content requires more intensive work of the AOD system for a long time. However, in some cases, the ROS content does not return to the initial level, despite the high activity of the AOD system, but is established at a new, higher level, called quasi-stationary. Based on the data we received, we can say that this is exactly the condition observed in whitefish from Lchashen Bay. It should be noted that such a state is less stable than a stationary one, and under the influence of additional stress factors, for example, elevated temperatures or increased anthropogenic load, a depletion of the AOD system and an uncontrolled increase in the content of ROS in the cell may occur. This can result in the development of numerous pathological processes in the body [50,52].

5. Conclusions

The AChE activity in the brain and AOS indicators in the liver and gills of whitefish from Lchashen and Lichk Bays of Lake Sevan demonstrate a stable seasonal cyclicity during the studied period 2022–2023. Comparative analysis of biochemical parameters showed that the whitefish in Lchashen Bay are in a worse condition, and their habitat conditions are less favorable than in Lichk Bay. The most probable reason for this is a low DO value and the increased level of organic pollution, which is confirmed by the data from hydrochemical analysis. The concentration of main elements and metals in waters of bays do not play a significant role in the fish biochemistry responses. The ichthyological data also demonstrated worse feeding condition for whitefish in Lchashen Bay compared to Lichk Bay. It can be assumed that in the future, additional anthropogenic load on Lchashen Bay can lead to an even greater deterioration in the health of fish and pronounced pathological disorders in their organisms.