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UDC 57.044:577.3:639.3:612.062
INFLUENCE OF CHLORAMINE-T ON OXIDATIVE STRESS BIOMARKERS IN THE MUSCLE TISSUE OF GRAYLING (THYMALLUS THYMALLUS)
H. Tkachenko, J. Grudniewska
ВЛИЯНИЕ ХЛОРАМИНА-Т НА СОДЕРЖАНИЕ БИОМАРКЕРОВ ОКИСЛИТЕЛЬНОГО СТРЕССА В МЫШЕЧНОЙ ТКАНИ ХАРИУСА
(TTHYMALLUS THYMALLUS)
Г. М. Ткаченко, Й. Грудневская
Chloramine-T is a widely used disinfectant for the treatment of gill diseases of fish in freshwater, and more recently attention has turned to its use in seawater. However, despite the wide use of chloramine-T, few studies have examined its toxicity to fish. Therefore, the aim of the present study was to examine the effects of exposure to chloramine-T on the muscle tissue of grayling (Thymallus thymallus Linck) using oxidative stress biomarkers (levels of 2-thiobarbituric acid reactive substances and oxidatively modified protein products) and total antioxidant capacity to observe its toxic effects. Our results showed that chloramine-T bathing markedly decrease lipid peroxidation with non-significant decrease of aldehydic and ketonic derivatives of oxidative proteins. However, reduced lipid peroxidation results in decrease of total antioxidant capacity. Moreover, decreased lipid peroxidation level causes decrease of aldehydic and ketonic derivatives of oxidatively modified proteins. These parameters could be effectively used as potential biomarkers of chloramine-T toxicity to the fish in the warning signal for pharmaceutical exposure to aquatic organisms. Our studies indicated that chloramine-T in dose 9 mg per L could at least partly attenuate oxidative stress and can be used for prophylactic treatment of grayling. However, more detailed studies on using these specific biomarkers to monitor the disinfectant treatment in aquaculture are needed.
chloramine-T, disinfection, grayling Thymallus thymallus, muscle tissue, lipid peroxidation, oxidatively modified proteins, total antioxidant capacity
Хлорамин-Т широко используется как дезинфицирующее средство для лечения жаберных заболеваний пресноводных рыб, а в последнее время стали обращать внимание также на его использование и в морской воде. Тем не менее, несмотря на широкое использование хлорамина-Т, существует мало исследований его токсичности для рыб. Поэтому цель нашего исследования состояла в изучении последствий воздействия хлорамина-Т на мышечную ткань хариуса (Thymallus thymallus) с использованием биомаркеров окислительного стресса (содержание реактивных соединений, реагирующих с 2-тиобарбитуровой кислотой, альдегидные и кетоновые производные окислительно модифицированных белков) и общую антиоксидантную активность. Наши результаты показали, что
дезинфекция хлорамином-Т существенно снижает перекисное окисление липидов с незначительным снижением содержания альдегидных и кетоновых производных окислительно модифицированных белков. Однако снижение перекисного окисления липидов сопровождается уменьшением общей антиоксидантной активности мышечной ткани. Эти параметры могут быть эффективно использованны в качестве потенциальных биомаркеров токсичности хлорамина-Т в аквакультуре. Наши исследования показали, что хлорамин-Т в дозе 9 мг на литр может, по крайней мере, частично ослабить окислительный стресс и быть использован для профилактической дезинфекции хариуса. Тем не менее нужны более подробные исследования по использованию этих конкретных биомаркеров для мониторинга дезинфицирующих мероприятий в аквакультуре.
хлорамин-Т, дезинфекция, хариус Thymallus thymallus, мышечная ткань, перекисное окисление липидов, окислительно модифицированные белки, общая антиоксидантная активность
Organic chlorine compounds (N-chloro compounds), which contain the =N-Cl group, show microbicidal activity. Examples of such compounds, are chloramine-T, dichloramine-T, halazone, halane, dichloroisocyanuric acid, sodium and potassium dichloroisocyanurates and trichloroisocyanuric acid. All appear to hydrolyze in water to produce an imino (=NH) group [1,2].
Their action is claimed to be slower than that of the hypochlorites, although this can be increased under acidic conditions [3]. Experiments where equal weights of disinfectants were used suggested that the greater penetrating power of monochloramine compensated for its limited disinfection activity. Studies of LeChevallier et al. (1988) showed that monochloramine was as effective as free chlorine for inactivation of biofilm bacteria [3].
Chloramine-T, as an anti-microbial agent, has had widespread use in a broad range of practices, including medical, dental, veterinary, food processing, and agricultural. As a disinfectant, it is used to disinfect surfaces and instruments. Chloramine-T has a low degree of cytotoxicity and has been used in direct contact with tissues. It is easy to use and effective against many bacteria (both Gram-negative and -positive), viruses (enveloped and naked), fungi, algae, yeast, and parasites [4].
The mode of action of chloramine-T is thought to be through oxidative processes, quickly destroying cell material or disrupting essential cellular processes. Microorganisms do not develop resistances to chloramine-T as often happens with antibiotics. In addition, the chloramine-T ion is highly stable and remains active over an extended period of time. Because chloramine-T is effective at low concentrations (200
INTRODUCTION
Chlorarnine T (sodiurn-p-toluene-sulphonchloramide)
to 300 ppm [710 to 1070 |iM]), it is an effective disinfectant without causing tissue cytotoxicity [4]. It may be used as a disinfectant for both skin and for wounds [4].
Chloramine-T is effective for the control of proliferative gill disease and bacterial gill disease, and flexibacteriosis. Bacterial gill disease is caused by a variety of Gram-negative bacteria (myxobacteria, aeromonads, and pseudomonads [4]. The disease is highly contagious among cultured salmonids and can lead to substantial fish losses. An approved therapeutant to control bacterial gill disease is needed to enable the production of salmonids for restoration of fish stocks and for sport and commercial fisheries [4]. As a therapeutic agent, it is used as an effective treatment of bacterial gill disease in freshwater or marine aquaria, garden ponds, or other aquatic systems at concentrations ranging from 6.5 to 10.0 mg/L [23.1 to 35.5 |iM] [5, 6] and as a preventative, prophylactic, and disinfectant treatment in many fresh water hatcheries [4, 6, 7].
Chloramine-T is a widely used disinfectant for the treatment of gill diseases of fish in freshwater, and more recently attention has turned to its use in seawater. However, despite the wide use of chloramine-T, few studies have examined its toxicity to fish [1]. Therefore, the aim of the present study was to examine the effects of exposure to chloramine-T on the muscle tissue of grayling (Thymallus thymallus Linck) using oxidative stress biomarkers (levels of 2-thiobarbituric acid reactive substances and oxidatively modified protein products) and total antioxidant capacity to observe the its toxic effects. The endpoints obtained from this study will be useful to monitor the effects of disinfectant bathing with chloramine-T for this species of fish.
MATERIALS AND METHODS Fish. Twenty clinically healthy grayling (Thymallus thymallus) were used in the experiments. The study was carried out in a Department of Salmonid Research, Inland Fisheries Institute near the village of Zukowo, Poland. Experiments were performed at a water temperature of 16±2°C and the pH was 7.5. The dissolved oxygen level was about 12 ppm with additional oxygen supply. All biochemical assays were carried out at Department of Zoology and Animal Physiology, Institute of Biology and Environmental Protection, Pomeranian University (Slupsk, Poland).
The fish were divided into two groups and held in 250-L square tanks (70 fish per tank) supplied with the same water as during the acclimation period (2 days). On alternate days, the water supply to each tank was stopped. In the disinfectant exposure, grayling (n=10) were exposed to chloramine-T in final concentration 9 mg per L. Control group of grayling (n=10) were handled in the same way as chloramine-T exposed groups. Fish were bathed for 20 min and repeated three times every 3 days. Two days after the last bathing fish were sampled. Fish were not anesthetized before tissue sampling.
Muscle tissue isolation. Muscle tissue was removed from grayling after decapitation. One grayling was used for each homogenate preparation. Briefly, muscle tissue were excised, weighted and washed in ice-cold buffer. The minced tissue was rinsed clear of blood with cold isolation buffer and homogenized in a glass Potter-Elvehjem homogenising vessel with a motor-driven Teflon pestle on ice. The isolation buffer contained 100 mM tris-HCl; pH of 7.2 was adjusted with HCl.
Analytical methods. All enzymatic assays were carried out at 25±0.5°C using a Specol 11 spectrophotometer (Carl Zeiss Jena, Germany). The enzymatic reactions were started by adding the homogenate suspension. The specific assay conditions are presented subsequently. Each sample was analyzed in triplicate. The protein concentration in each sample was determined according to Bradford (1976) using bovine serum albumin as a standard [8].
TBARS assay for lipid peroxidation. Lipid peroxidation level was determined by quantifying the concentration of 2-thiobarbituric acid reactive substances (TBARS), expressed as |imol of malondialdehyde (MDA) per mg of protein, according to Kamyshnikov (2004) [9]. The TBARS level was expressed in nmol MDA per mg protein by using 1.56105 mM-1 cm-1 as molar extinction coefficient. Carbonyl derivatives of oxidatively modified protein (OMP) assay. The rate of protein oxidative destruction was estimated from the reaction of the resultant carbonyl derivatives of amino acid reaction with DNFH as described by Levine et al. (1990) [10] and as modified by Dubinina et al. (1995) [11]. The carbonyl content was calculated from the absorbance measurement at 370 nm and 430 nm and an absorption coefficient 22,000 M-1xm-1. Carbonyl groups were determined spectrophotometrically from the difference in absorbance at 370 nm (aldehydic derivatives, OMP370) and 430 nm (ketonic derivatives, OMP430) and expressed in nmol per mg of tissue protein. Total antioxidant capacity (TAC) assay. The TAC level in the sample was estimated spectrophotometrically at 532 nm following the method with Tween 80 oxidation [12]. TAC was expressed in %.
Statistical analysis. The mean ± S.E.M. values was calculated for each group to determine the significance of inter group difference. All variables were tested for normal distribution using the Kolmogorov-Smirnov and Lilliefors test (p>0.05). Significance of differences between the oxidative stress biomarkers level (significance level, p<0.05) was examined using Kruskal-Wallis one-way analysis of variance by ranks test. Correlations between parameters at the set significance level were evaluated using Spearman's correlation analysis [13]. All statistical calculation was performed on separate data from each individual with STATISTICA 10.0.
RESULTS
Influence of chloramine-T on lipid peroxidation biomarker, measured as 2-thiobarbituric acid reactive substances in the muscle tissue of grayling are presented in Fig. 1 A. Significantly lower TBARS level (by 39%, p=0.004) in grayling disinfected by chloramine-T compared to control group was observed (Fig. 1 A).
Aldehydic and ketonic derivatives of oxidatively modified proteins in the muscle tissue of grayling disinfected by chloramine-T were non-significantly lower compared to controls (Fig. 1B). Significant decrease of TAC level (by 13%, p=0.026) in the muscle tissue of grayling as a consequence of bathing with chloramine-T were found (Fig. 1C).
А
В
C
Fig. 1. Influence of chloramine-T on lipid peroxidation biomarker, measured as 2-
thiobarbituric acid reactive substances (А), as well as aldehydic and ketonic derivatives of oxidatively modifiedproteins (B) and total antioxidant capacity (C) in the muscle tissue of grayling (Thymallus thymallus). * Data are represented as mean ± S.E.M.
the significant difference was shown as p<0.05 when compared control and chloramine-T exposed groups Рис. 1. Влияние хлорамина-Т на содержание биомаркера перекисного окисления
липидов (реактивные соединения, которые реагируют с 2-тиобарбитуровой кислотой) (А), содержание альдегидных и кетоновых производных окислительно модифицированных белков (В), общую антиоксидантную активность (С) в
мышечной ткани хариуса (Thymallus thymallus) Данные представлены как среднее ± S.E.M. (стандартная ошибка среднего). * статистически достоверные изменения (р<0,05) между средними в контрольной группе рыб и группе после дезинфекции хлорамином-Т
Several correlations between checked parameters were found. Muscle TBARS level correlated positively with aldehydic (r=0.854, p=0.002) and ketonic derivatives of oxidatively modified proteins (r=0.852, p=0.002) (Fig. 2).
Fig. 2. Correlations between TBARS, aldehydic and ketonic derivatives of oxidatively modified proteins content in the muscle tissue of grayling disinfected by
chloramine-T
Рис. 2. Корреляционные зависимости между биомаркерами перекисного окисления липидов, альдегидными и кетоновыми производными окислительно модифицированных белков в мышечной ткани хариуса обработанного
хлорамином-Т
DISCUSSION
Our results showed that chloramine-T bathing markedly decrease lipid peroxidation with non-significant decrease of aldehydic and ketonic derivatives of oxidative proteins (Figs 1А and 1В). However, reduced lipid peroxidation results in decrease of total antioxidant capacity (Fig. 1С). Moreover, decreased lipid peroxidation level causes decrease of aldehydic (r=0.854, p=0.002) and ketonic derivatives of oxidatively modified proteins (r=0.852, p=0.002) (Fig. 2).
Chloramine T was found to increase freshwater bathing efficacy and reduced amoeba survival [14]. Recent studies also suggest that chloramine-T in seawater is as effective in seawater as in fresh water [15, 16]. Concentrations of between 8.5 and 12 mg per L have been demonstrated to be successful for the control of bacterial gill diseases in hatcheries [5]. However, Powell et al. (1994) suggest that juvenile rainbow trout exposed to 10 and 20 mg chloramine-T per L showed significant predisposition to an erosive dermatitis of the caudal fin which appeared to be caused by opportunistic pathogens of the genus Pseudomonas spp. and Flavobacter spp. [17]. They recommend
that a prophylactic dose of chloramine-T must be less than 10 mg per L. Also repeated exposures of rainbow trout to chloramine T resulted in decreased growth rates [17].
In our previous study [18], chloramine-T bathing markedly decrease aldehydic and ketonic derivatives of oxidative protein, and aminotransferases activity only in rainbow trout liver, and their elevation is a compensatory mechanism to impaired metabolism. No significant changes were found in oxidative stress biomarkers between control and chloramine-treated brown trout. For grayling, chloramine-T exposure caused significantly elevation in the levels of severe oxidative stress biomarkers. Increased aldehydic and ketonic derivatives of oxidative protein could modify lactate and pyruvate levels, aminotransferases and lactate dehydrogenase activities, principally causing increased enzymes activity due to oxidative stress in the liver of chloramine-exposed fish [18].
Accumulating evidence has shown that chloramine-T causes oxidative stress by inducing the generation of reactive oxygen species (ROS) [19-21].The data suggest that HOCl and monochloramine can increase endothelial permeability by causing very rapid cytoskeletal shortening and cell retraction, possibly as a result of the oxidation of intracellular sulfhydryls [19]. Sakuma et al. (2009) assessed the influence of monochloramine on the conversion of xanthine dehydrogenase into xanthine oxidase in rat liver in vitro. When incubated with the partially purified cytosolic fraction from rat liver, monochloramine (2.5-20 microM) dose-dependently enhanced xanthine oxidase activity concomitant with a decrease in xanthine dehydrogenase activity, implying that monochloramine can convert xanthine dehydrogenase into the ROS producing form xanthine oxidase. It was found that monochloramine could increase ROS generation in the cytoplasm of rat primary hepatocyte cultures, and that this increase might be reversed by an xanthine oxidase inhibitor, allopurinol. These results suggest that monochloramine has the potential to convert xanthine dehydrogenase into xanthine oxidase in the liver, which in turn may induce the ROS generation in this region [21].
There is a strong link between chronic inflammation and the incidence of many cancers, which may be associated with the ability of HOCl and related oxidants such as N-chloramines to damage DNA [20]. Stanley et al. (2010) examined the ability of HOCl and various N-chloramines to form chlorinated base products on nucleosides, nucleotides, DNA, and in cellular systems. Experiments were performed with N-chloramines formed on Na-acetyl-histidine (His-C), Na-acetyl-lysine (Lys-C), glycine (Gly-C), taurine (Tau-C), and ammonia (Mono-C). Treatment of DNA and related materials with HOCl and Na-acetyl-histidine resulted in the formation of 5-chloro-2'-deoxycytidine, 8-chloro-2'-deoxyadenosine and 8-chloro-2'-deoxyguanosine. Cellular RNA was also a target for HOCl and His-C, with evidence for the formation of 5-chloro-cytidine. HOCl and the model N-chloramine, His-C, are able to chlorinate cellular genetic material, which may play a role in the development of various inflammatory cancers [20].
To estimate a Chloramine-T margin of safety, defined as the highest dosing regimen above the proposed maximum therapeutic regimen at which no adverse effects are observed, Bowker et al. (2011) conducted seven experiments with fry, fingerling, and juvenile rainbow trout that examined mortality and an eighth experiment that examined mortality, gross pathology, and histopathology after Chloramine-T exposure at different concentration [22]. Across experiments, 92% of all mortalities occurred within 20 h of the first exposure to Chloramine-T. The histopathological changes of
most concern were associated with gill tissues, but these were evident only in moribund fish exposed to doses of 60 mg/L or higher. Based on analysis of the survival data, the margin-of-safety estimates were approximately 100 mg/L for rainbow trout fry, at least 60 mg/L for fingerlings, and 50-60 mg/L for juveniles. Tissue responses to ChloramineT at these concentrations were minor and did not warrant decreasing these estimates [22].
Powell and Harris (2004) examined the acute (within 12 h) toxicity of freshwater- and seawater-acclimated Atlantic salmon Salmo salar smolts under aerated (100% air saturation with O2) and oxygen supersaturation conditions (200% air saturation with O2) [1]. Chloramine-T was more acutely toxic to salmon in seawater than to those in freshwater, and oxygen supersaturation enhanced the toxicity in both sea- and freshwater. Freshwater Atlantic salmon appear to be as sensitive as rainbow trout and more sensitive than channel catfish Ictalurus punctatus to chloramine-T toxicity. Seawater-acclimated salmon, however, appear to be more sensitive to chloramine-T than are trout and catfish. The primary mechanism of toxicity in both seawater and freshwater appears to be extensive oxidative necrosis of the gill filament and lamellar epithelium, causing acute osmoregulatory dysfunction [1].
In summary, chloramine-T has a profound influence on the levels of oxidative stress biomarkers in the muscle tissue of grayling. Chloramine-T markedly affects lipid peroxidation and total antioxidant capacity. These parameters could be effectively used as potential biomarkers of chloramine-T toxicity to the fish in the warning signal for pharmaceutical exposure to aquatic organisms. Our studies indicated that chloramine-T in dose 9 mg per L could at least partly attenuate oxidative stress and can be used for prophylactic treatment of grayling. However, more detailed studies on using of these specific biomarkers to monitor the disinfectant treatment in aquaculture are needed.
This work was supported by grant of the Pomeranian University for Young Scientists.
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ИНФОРМАЦИЯ ОБ АВТОРАХ
Ткаченко Галина Михайловна - Институт биологии и охраны среды, Поморская Академия, г. Слупск, Польша; кафедра зоологии и физиологии животных; кандидат биологических наук, докторант; E-mail: tkachenko@apsl.edu.pl
Tkachenko Halyna Mikhailovna - Institute of Biology and Environmental Protection, Pomeranian University, Slupsk, Poland; Department of Zoology and Animal Physiology; Candidate of Biological Sciences, Assistant Professor; E-mail: tkachenko@apsl.edu.pl
Грудневская Йоанна - Институт пресноводного рыбного хозяйства, Жуково, Польша; отдел исследований лососевых рыб; кандидат биологических наук, докторант; E-mail: jgrudniewska@infish.com.pl
Grudniewska Joanna - Inland Fisheries Institute, Zukowo, Poland; Department of Salmonid Research; Candidate of Biological Sciences, Assistant Professor; E-mail: jgrudniewska@i nfish.com.pl
