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Effects of various propolis concentrations on biochemical and hematological parameters of rainbow trout (Oncorhynchus mykiss)
ARTICLE IN PRESS Ecotoxicology and Environmental Safety 72 (2009) 1994–1998
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Effects of various propolis concentrations on biochemical and hematological parameters of rainbow trout (Oncorhynchus mykiss) Zeliha Selamoglu Talas , Mehmet Fuat Gulhan Department of Biology, Faculty of Arts and Science, Nigde University, Nigde, Turkey
a r t i c l e in f o
a b s t r a c t
Article history: Received 20 October 2008 Received in revised form 10 April 2009 Accepted 13 April 2009 Available online 9 May 2009
Biochemical and hematological parameters in blood of rainbow trout treated to various concentrations of propolis for 96 h were determined. Total leukocyte count and granulocytes values increased (po0.05) in 0.02 and 0.03 g/L propolis groups. There was a decrease in agranulocytes (po0.05) erythrocytes, hemoglobin and hematocrit values for fish exposed to 0.02 and 0.03 g/L propolis. MCV and MCH values (po0.05) were significantly increased; 0.02 and 0.03 g/L propolis caused an increase (po0.05) in the levels of glucose, blood urea nitrogen, triglyceride, total cholesterol, lactate dehydrogenase, amylase and gamma glutamyltransferase. There was a decrease in the levels of aspartate aminotransferase and alkaline phosphatase. Hematological and biochemical protective effects of 0.01 g/L propolis were investigated. Dose-dependent effects of propolis on blood of fish can be favorable, opening new perspectives of investigation on their biological properties and utilization. & 2009 Elsevier Inc. All rights reserved.
Keywords: Biochemical parameters Blood Hematological parameters Oncorhynchus mykiss Propolis
1. Introduction Propolis is a resinous material collected by bees from bud and exudates of plants, which is mixed with products of their salivary glands and wax. Its color varies from green, red to dark brown. Propolis has been used in folk medicine since ancient times. Propolis has attracted researchers’ interest in the last decades because of several biological and pharmacological properties, such as immunomodulatory, antitumor, antimicrobial, anti-inflammatory, and antioxidant, among others (Bankova et al., 2000; Kanbur et al., 2009). As produced by the bees, propolis is a strongly adhesive, resinous substance used to seal holes in their hives, smooth out the internal walls and protect the entrance against intruders. Although a common source of the resin is Populus balsamifera L. (and other Populus species), the precise composition of raw propolis varies with the source. In general, it is composed of 50% resin and vegetable balsam, 30% wax, 10% essential and aromatic oils, 5% pollen and 5% various other substances, including organic debris. Raw propolis is processed using water washing and solubilizing in 95% ethanol to remove the wax and organic debris, creating propolis tincture, ‘‘propolis balsam’’, or ethanol extract of propolis (Burdock, 1998). Besides, propolis-containing products have been intensely marketed by the pharmaceutical industry and health-food stores (Banskota et al., 2001a, b). The ethnopharmacological approach, combined with chemical and biological methods, may provide useful
Corresponding author. Fax: +90 388 2250180.
E-mail address: [email protected] (Z.S. Talas). 0147-6513/$ - see front matter & 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2009.04.011
pharmacological leads. Several researchers have reported the antitumoral property of propolis both in vivo and in vitro (Banskota et al., 2001a, b; Lee et al., 2005). Propolis has been used since ancient times as a medicine because of its biological properties as an antimicrobial, antifungal, antiprotozoan and antiviral agent (Alberto et al., 2005). Important sources of propolis include poplars, alders and birches, chestnut, ash and various Pinus and Salix species. Variations in the bees-wax content of raw propolis also affect the chemical composition. The main chemical classes found in propolis are flavonoids, phenolics and various aromatic compounds. However, propolis contains many of the B-complex vitamins, important minerals and trace elements. But its bioflavonoid content is now receiving attention. Bioflavonoids are antioxidant molecules that play very important roles in the scavenging of free radicals, which are produced in degenerative heart diseases, atherosclerosis, aging and effects of toxic substances (Kolankaya et al., 2002). Biochemical and hematological parameters of fish are determined as an index of their health status as well (Blaxhall and Daisley, 1973). Fish in particular are commonly used to estimate the influence of environmental compounds due to the sensitivity of their biochemical and hematological parameters under such conditions (Lopes et al., 2001). As a sign of stress, the use of hematological and biochemical methods provides valuable knowledge about physiological reactions occurring against changing environmental conditions, especially understanding the physiological, biochemical and hematological changes occurring at various concentrations of compounds, to predict the possible level of threat to life.
ARTICLE IN PRESS Z.S. Talas, M.F. Gulhan / Ecotoxicology and Environmental Safety 72 (2009) 1994–1998
In this study, we have tested for the first time the possible use of propolis using the rainbow trout, one of the most popular cultural fish in Turkey. To observe the effects of propolis, which is a natural product obtain from plants, rainbow trout, which is one of the most important fish in Turkey and human diet was studied. This study opens a new perspective on the investigation of propolis biological properties, mainly with respect to the hematological and biochemical parameters in blood of rainbow trout. This study was aimed at the investigation of effects and useful concentration of natural products like propolis, which has biologically benefits exists in natural environmental conditions, on living organisms that live in natural area.
1995
2.4. Hematological analysis Blood samples were collected prior to anesthetizing of fish to prevent hemolysis (McKnight, 1966). The samples were transferred to glass tubes and hemotimetric parameters were immediately determined. Red blood cell counting was done after 1:200 dilution into Hayem solution. Leukocyte counting was done in blood samples after proper dilution into Turck solution (De Wilde and Houston, 1961; Blaxhall and Daisley, 1973; Blaxhall, 1981). Hemoglobin (HGB) concentration was determined according to the cyano-methemoglobin procedure (Kit 525-A; Sigma Chemical, St. Louis, MO, USA; Blaxhall and Daisley, 1973). Nonclotted blood (20 ml aliquots) was diluted with 1 ml of Drabkin solution and left to stand for 10 min at room temperature. The absorbance was read at 540 nm and the amount of hemoglobin was calculated against a hemoglobin standard (Azizoglu and Cengizler, 1996). Hematocrit was determined as in Jewet et al., (1991) and Wilhelm Filho et al. (1992). Nonclotted blood was transferred to a microhematocrit capillary, centrifuged at 14,000g for 5 min and read against a standard chart.
2. Materials and methods
2.5. Biochemical assay
2.1. Experimental section
After treatments and propolis exposure 2 ml of blood was withdrawn from caudal vein. Blood samples were transferred to glass tubes, kept in cooled bath and promptly analyzed. The blood was centrifuged at 3000g, at 4 1C for 5 min. Alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyltransferase (GGT), lactate dehydrogenase (LDH) and amilase activities were assayed; glucose, total protein, creatinine, blood urea nitrogen (BUN), triglycerides, total cholesterol, chloride, sodium, potassium, total calcium, phosphates and ferric levels in plasma were determined. All such analyses were done (Olympus Optical Corp., Shizuoka-ken, Japan) using commercially available kits (Roche).
The rainbow trout (Oncorhynchus mykiss) were purchased from Camardı, Ecemis fish farm (Nigde, Turkey). Fish were fed for 15 days in a 8 5 1.5 m3 stock pond to be acclimatized. After the adaptation period, 8 fish were transferred to 200 L water tank filled with natural spring water. Fish used in this study had an average weight of 245.5175.22 g and length of 29.7573.81 cm. Physical and chemical properties of water are depicted in Table 1.
2.2. Preparation of propolis extractive solution 2.6. Statistical analysis Propolis extraction methods may influence its activity, since different solvents solubilize and extract different compounds. The most common extracts used in biological assays are ethanol, methanol and water. Its chemical composition is very complex; more than 300 components have already been identified, and its composition is dependent on the local flora. Moreover, propolis composition is highly variable, creating a problem the medical use and standardization. In the present work, propolis was collected from a farm at village Kocaavsar in Balikesir, Turkey. Propolis was dissolved to 30% in ethanol, protected from light and moderately shaken for 1 day at room temperature. Afterward, the extracts were filtered twice, dried and stored in sealed bottles at 4 1C until use. Three different concentrations (0.01, 0.02 and 0.03 g/L) of the dry propolis extracts were achieved in double distilled water.
2.3. Experimental design In the study, there were totally 32 rainbow trout in 0.01 g/L-propolis-exposed group, 0.02 g/L-propolis-treated group, 0.03 g/L-propolis-treated groups and control group (the average weights and lengths of rainbow trout were 240–250 g and 29.7573.81 cm, respectively). Thirty-two rainbow trout were divided into four groups, each consisting of eight animals. Each rainbow trout was weighed just before the start of the study; 0.01 g/L propolis was treated to the animals in group I for 96 h and not fed for 12 h before; 0.02 g/L propolis was treated to the animals in group II for 96 h and not fed for 12 h before; 0.03 g/L propolis was treated to the animals in group III for 96 h and not fed for 12 h before. Fish in group IV were used as a control and were killed 96 h later. Fish experiments were performed in accordance with the guidelines for fish research from the National Institute of Health and approved by the Committee of Fish Research at Nigde University, Nigde, Turkey.
Table 1 Some parameters of the water used in the experiment. Parameter (mg/L)
Before treatment
After treatment
Dissolved oxygen Chemical oxygen demand Suspended solids Calcium Sodium Chloride Total nitrogen Hardness (CaCO3) Temperature (1C) pH
7.870.2 15.170.1 36.871.2 126.071.6 22.470.8 16.071.5 6.070.2 174.373.1 11.571.0 7.870.1
7.670.1 16.270.2 40.171.8 114.171.1 19.770.8 18.071.4 6.870.3 168.272.9 1271 7.870.1
Hematological and biochemical data were analyzed with SPSS 9.0 for Windows using one-way analyses of variance (ANOVA). Differences between means were determined using Duncan’s multiple range test in which the significance level was defined as po0.05.
3. Results In order to determine the effects of propolis extracts, we used three concentrations. The effects on the fish, observed through biochemical parameters, are summarized in Table 2. Fish exposed to 0.01 g/L did not change (p40.05) the levels of glucose, total protein, creatinine, BUN, triglycerides, total cholesterol ALT, AST, LDH, ALP, amilase, and GGT as compared with the control group (Table 2). Fish exposed to 0.02 and 0.03 g/L presented significant increase (po0.05) in glucose, BUN, triglycerides, total cholesterol levels and LDH, amilase and GGT activities as compared with the control group. However there was no significant increase (p40.05) in the level of total protein and creatinine. There was significant decrease in AST and ALP as compared with the control group (Table 2). But no significant decrease in the levels of ALT was observed as compared with the control group (p40.05). It is seen (Table 3) that changes in the electrolytes in rainbow trout blood with three different concentrations of propolis administration, 0.01, 0.02 and 0.03 g/L, are no statistically significant changes (p40.05) in the levels of chloride, sodium, total calcium and phosphates as compared with the control group (Table 3). However, with 0.02 and 0.03 g/L administration there was a statistically significant decrease (po0.05) in the level of potassium as compared with the control and 0.01 g/L (Table 3). There was a statistically significant increase in the level of ferric as compared with the control and 0.01 g/L (p40.05) (Table 3). The effects of propolis on the hematological parameters are summarized in Table 4. Significant increases (po0.05) in total leukocyte (WBC), granulocyte, MCV and MCH values in fish exposed to 0.02 and 0.03 g/L propolis and significant decreases (po0.05) in agranulocyte, RBC, HGB and HCT values were observed in contrast with MCHC (Table 4).
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Z.S. Talas, M.F. Gulhan / Ecotoxicology and Environmental Safety 72 (2009) 1994–1998
Table 2 Changes in the biochemical parameters in rainbow trout blood with different concentrations of propolis. Biochemical parameters
Control
0.01 g/L propolis
0.02 g/L propolis
0.03 g/L propolis
Glucose (mg dL1) Total protein (g dL1) Creatinine (mg dL1) BUN (mg dL1) Triglycerides (mg dL1) Total cholesterol (mg dL1) ALT (IU l1) AST (IU l1) LDH (IU l1) ALP (IU l1) Amilase (IU l1) GGT (IU l1)
68.570.5 2.570.1 0.170.0 4.970.2 380.2736.2 180.3711.5 30.378.4 536.5743.1 784.2740.2 54.175.9 287.5714.2 5.270.9
66.170.6 2.670.08 0.170.0 5.170.2 387.1723.1 184.4713.1 26.573.3 470.6736.5 802.1719.1 53.374.2 292.8713.2 6.070.8
74.270.8 2.770.1 0.170.0 6.770.1 458.1721.4 221.4716.5 21.673.7 332.6721.2 1070.2760.4 40.573.8 454.1724.1 8.671.2
80.371.1 2.770.1 0.270.0 7.170.3 487.7717.2 244.7715.4 28.773.0 312.5730.5 1281.7778.5 34.274.1 591.7740.6 9.270.6
Data are mean values 7sd (n ¼ 8). Denotes statistically significant groups (po0.05).
Table 3 Changes in the electrolytes in rainbow trout blood with different concentrations of propolis. Electrolytes
Control
0.01 g/L propolis
0.02 g/L propolis
0.03 g/L propolis
Chloride (mmol l1) Sodium (mmol l1) Potassium (mmol l1) Total calcium (mmol l1) Phosphates (mmol l1) Ferric (mmol l1)
117.672.6 151.672.5 3.870.6 11.870.4 20.371.5 73.374.3
123.571.9 148.671.7 2.270.5 10.970.3 19.870.6 84.276.1
122.672.2 147.671.3 1.270.3 10.170.3 21.671.8 110.279.1
119.571.5 146.571.2 1.970.3 10.270.4 18.970.9 111.7711.8
Data are mean values 7sd (n ¼ 8). Denotes statistically significant groups (po0.05).
Table 4 Changes in the hematological parameters in rainbow trout blood with different concentrations of propolis. Hematological parameters 3
Control 3
Total leukocyte—WBC (m /10 ) Granulocytes (%) Agranulocytes (%) Erytrocyte—RBC (mm3/106) Hemoglobin—HGB (g/dL) Hematocrit—HCT (%) Erythrocyte indexes MCV (m3) MCH (mg) MCHC (%)
0.01 g/L propolis
0.02 g/L propolis
7.670.1 75.470.4 24.670.3 1.570.1 8.570.2 35.671.5
7.270.2 77.570.4 22.570.4 1.470.1 8.370.1 33.971.2
8.770.1 81.170.7 18.970.7 1.270.1 7.170.2 27.670.4
238.972.3 57.071.4 23.870.7
235.472.1 57.671.7 24.470.8
240.071.9 61.771.8 25.770.6
0.03 g/L propolis 9.570.7 82.670.8 17.470.8 1.070.1 6.970.2 26.170.6 263.072.7 70.472.4 26.470.7
Data are mean values 7sd (n ¼ 8). Denotes statistically significant groups (po0.05).
In the present study, the protective effect doses of propolis were compared to control group in hematological and biochemical parameters. The data show that propolis has an important effect on biochemical and hematological parameters at 0.01 g/L levels, whereas 0.02 and 0.03 g/L concentrations appear to be unfavorable for blood tissue of rainbow trouts.
4. Discussion Fish is considered as a valuable source of protein in the human diet. The significance of long-chain polyunsaturated fatty acids has gained attention because of their prevention of human
cardiovascular diseases (Shahidi and Botta, 1994). Investigations were carried out on the rainbow trout. Due to rapid growth and easy accommodation to environment conditions, rainbow trout are the common consumed and economical important fish. In recent years, there has been a great deal of studies carried out on propolis metabolism but not on dose studies of propolis and its effects on biochemical and hematological parameters of aquatic animals. Thus, this study aimed to discuss the doses of this bee product on biochemical and hematological parameters in blood of rainbow trout. Hematological and biochemical data showed that propolis at various concentrations exerted a certain influence on some of the blood indices studied. Propolis compounds may enter aquatic
ARTICLE IN PRESS Z.S. Talas, M.F. Gulhan / Ecotoxicology and Environmental Safety 72 (2009) 1994–1998
environments from agricultural and rural fields by rain water and have long-term effects in fish. These effects appear in population or ecosystems as physiological and biochemical reactions. To understand the effects of propolis on living organisms, metric, physiological, hematological and biochemical parameters should be examined (Lenhardt, 1992; Strmack and Braunbeck, 2000). As shown in various studies, propolis has several biological and pharmacological properties, such as those of antimicrobial, antiinflammatory, antioxidant, antifungal, antiprotozoan and antiviral agents. (Bankova et al., 2000; Kanbur et al., 2009). When these features are taken into account, it is thought that propolis might harm people and animals when they are exposed to amounts higher than needed for the normal diet (Burdock, 1998; Jingtao, 1999). Due to the cytotoxic effect of high concentration of propolis it may have a role in the inhibition of erythropoiesis in hematopoietic organisms. Administration of propolis at different doses resulted in evaluation of levels of glucose, total protein, creatinine, BUN, triglyceride, total cholesterol, LDH, amilase, GGT, and ferric. The elevation in their levels in fish exposed to 0.02 and 0.03 g/L propolis may be considered as one of the markers of renal and liver dysfunctions, and changes of lipid metabolism (Dobrowolski et al., 1991); 0.01 g/L propolis caused a significant (po0.05) decrease in most of these levels, therefore appeared to have potential to maintain liver and renal function, and avoid hypercatabolism at high concentration (0.02 and 0.03 g/L) in propolis-treated animals. As propolis possesses antioxidant properties, it is also likely to behave as an antioxidant. It is known that exogen agents can change hematological parameters, such as erythrocyte number, HGB amount, hematocrit value and total leukocytes. We emphasize that effective dose of propolis to inhibit changes in hematological and biochemical parameters exists at high concentration. With 0.02 and 0.03 g/L propolis while group levels of total leukocyte count and granulocytes, MCV, MCH and MCHC increased, erytrocyte count, agranulocytes, HGB and hematocrit levels decreased. Decreases in erythrocyte, HGB and hematocrit levels at high doses (0.02 and 0.03 g/L) of propolis may be an indicator of anemia with inhibition of erythropoesis in the hemopoetic organism (Kleinrok et al., 1978). In addition, increases in MCV, MCH and MHCH values indicated that the anemia was of a macrocytic type. However, high leukocyte values depend on stimulatory effects of cytotoxic agents on the immune system (Ivanov et al., 1973; Kleinrok et al., 1978; Sforcin, 2007). The usefulness of immunostimulants has been demonstrated in modern aquaculture and fish farmers use a wide range of immunostimulants that may need to be purified (vitamins, chitin, glucans, etc.) or not (microorganisms, animal and plant extracts, subproducts of other industries, etc, Sakai, 1999; Alberto et al., 2005). According to results of our study, 0.01 g/L propolis can act as an immunostimulant. Ample stimulation of the immune system by 0.02 and 0.03 g/L doses of propolis can increase the total leukocytes value. In conclusion, it can be said that propolis is important at various concentrations for the rainbow trout and may significantly influence certain physiological functions, especially hematopoiesis. Hematological and biochemical parameters of rainbow trout treated at various doses were investigated, and the effects of 0.01 g/L propolis were outlined, evidencing their preservation role on hematological and biochemical parameters. However, there is still a need for further studies regarding the use of propolis extracts or its constituents and environmental distribution as natural antioxidant, possible food supplement and natural protective agent.
1997
5. Conclusions Most recent studies have shown that natural preventive compounds have gained popularity day by day as some of the widely used synthetic pharmaceuticals and therapeutics might have some undesirable effects. One can think that certain natural food ingredients would be better and safer than synthetic ones. Many of these compounds, such as plant phenolics, often exhibit antioxidant activities; therefore the addition of these compounds into food products may be helpful to health the of consumers and also to the stabilization of food products. Due to the presence of some of these effective compounds such as flavonoids (flavones and flavanones), phenolic acids and their esters in propolis and propolis extract, if the positive physiological properties and the non-toxicity of the propolis sample are proven it could be used as a mild antioxidant and preservative. Due to antioxidant and preservative properties of propolis, it may not only prolong the physiological functions of some aquatic living organisms, but also contribute to the health benefit of consumers who consume aquatic animals.
Acknowledgment Nigde University Research Fund (FEB 2007/13) is gratefully acknowledged for support of this work. References Alberto, C., Alejandro, R., Esteban, M.A., Jose´, M., 2005. In vivo effects of propolis, a honeybee product, on gilthead seabream innate immune responses. Fish. Shellfish. Immun. 18 (1), 71–80. Azizoglu, A., Cengizler, I., 1996. An investigation on determination of some hematologic parameters in healthy Oreochromis niloticus (L.). Turk. J. Vet. Anim. Sci. 20, 425–431. Blaxhall, P.C., Daisley, K.W., 1973. Routine hematological methods for use with fish blood. J. Fish. Biol. 5, 771–781. Blaxhall, P.C., 1981. A comparison of methods used for the separation of fish lymphocytes. J. Fish Biol. 18, 177–181. Bankova, V.S., De Castro, S.L., Marcucci, M.C., 2000. Propolis: recent advances in chemistry and plant origin. Apidologie 31, 3–15. Banskota, A.H., Tezuka, Y., Adnyana, I.K., et al., 2001a. Hepatoprotective and antiHelicobacter pylori activities of constituents from Brazilian propolis. Phytomedicine 8, 16–23. Banskota, A.H., Tezuka, Y., Kadota, S., 2001b. Recent progress in pharmacological research of propolis. Phytother. Res. 15, 561–571. Burdock, G.A., 1998. Review of the biological properties and toxicity of bee propolis (propolis). Food Chem. Toxicol. 36 (4), 347–363. De Wilde, M.A., Houston, A.H., 1961. Hematological aspect of the therma acclamatory process in the rainbow trout, Salmo gairdneri. J. Fish Res. Biol. 24, 2267–2281. Dobrowolski, JW., Vohoraq, SB., Sharma, K., Shah, SA., Naqvi, SAH., Dandiya, PC., 1991. Antibacterial, antifungal, antiamoebic, antiinflammatory, and antipyretic studies on propolis bee products. J. Ethnopharmacol. 35, 77–82. Ivanov, DF., Tikonov, AI., Krivenchuk, PE., Liurskaia, FV., 1973. Propolis and its clinical usage. Oftolmol. Zh. 28 (2), 104–107. Jewet, M.G., Behmer, D.J., Johnson, G.H., 1991. Effects of hyperoxic rearing water on blood hemoglobin and hematocrit levels of rainbow trout. J. Aquat. Anim. Health 3, 153–160. Jingtao, Z., 1999. A refined propolis which is lowly-toxic and non-allergens. World Phytomed. 14 (1), 37. Kanbur, M., Eraslan, G., Silici, S., 2009. Antioxidant effect of propolis against exposure to propetamphos in rats. Ecotox. Environ. Saf. 72 (3), 909–915. Kleinrok, Z., Borzecki, Z., Scheller, S., Matuga, W., 1978. Biological properties and clinical application of propolis: X. Preliminary pharmacological evaluation of ethanol extract of propolis (EEP). Arzneim. Forsch./Drug Res. 28 (1), 291–292. Kolankaya, D., Selmanoglu, G., Sorkun, K., Salih, B., 2002. Protective effects of Turkish propolis on alcohol-induced serum lipid changes and liver injury in male rats. Food Chem. 78, 213–217. Lee, Y.T., Don, M.J., Hung, P.S., Shen, Y.C., Lo, Y.S., Chang, K.W., Chen, C.F., Ho, L.K., 2005. Cytotoxic of phenolic acid phenethyl esters on oral cancer cells. Cancer Lett. 223, 19–25. Lenhardt, M., 1992. Seasonal changes in some blood chemistry parameters and in relative liver and gonad weights of pike (Essox lucius) from the river Danube. J. Fish. Biol. 40, 709–718. Lopes, P.A., Pinheiro, T., Santos, M.C., Mathias, M.D., Collares-Pereira, M.J., ViegasCrespo, A.M., 2001. Response of antioxidant enzymes in freshwater fish
ARTICLE IN PRESS 1998
Z.S. Talas, M.F. Gulhan / Ecotoxicology and Environmental Safety 72 (2009) 1994–1998
populations (Leuciscus alburnoides complex) to inorganic pollutants exposure. Sci. Total Environ. 280, 153–163. McKnight, I.M.A., 1966. Hematological study on the mountain whitefish. Popium willasemi south. Fish Reb. Bd. 23, 45–64. Sakai, M., 1999. Current research status of fish immunostimulants. Aquaculture 172, 63–92. Sforcin, J.M., 2007. Propolis and the immune system: a review. J. Ethnopharmacol. 113, 1–14.
Shahidi, F., Botta, J.R., 1994. Seafoods: Chemistry, Processing Technology and Quality. Chapman & Hall, London, pp. 3–9. Strmack, M., Braunbeck, T., 2000. Isolated hepatocytes of rainbow trout (Oncorhynchus mykiss) as a tool to discriminate between differently contaminated small river system. Toxicol. Vitro 14, 361–377. Wilhelm Filho, D.M., Eble, G.J., Kassner, G., Caprario, F.X., Dafre, L.A., Ohira, M., 1992. Comparative hematology in marine fish. Comp. Biochem. Physiol. 102, 311–321.
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Effects of various propolis concentrations on biochemical and hematological parameters of rainbow trout (Oncorhynchus mykiss) Zeliha Selamoglu Talas , Mehmet Fuat Gulhan Department of Biology, Faculty of Arts and Science, Nigde University, Nigde, Turkey
a r t i c l e in f o
a b s t r a c t
Article history: Received 20 October 2008 Received in revised form 10 April 2009 Accepted 13 April 2009 Available online 9 May 2009
Biochemical and hematological parameters in blood of rainbow trout treated to various concentrations of propolis for 96 h were determined. Total leukocyte count and granulocytes values increased (po0.05) in 0.02 and 0.03 g/L propolis groups. There was a decrease in agranulocytes (po0.05) erythrocytes, hemoglobin and hematocrit values for fish exposed to 0.02 and 0.03 g/L propolis. MCV and MCH values (po0.05) were significantly increased; 0.02 and 0.03 g/L propolis caused an increase (po0.05) in the levels of glucose, blood urea nitrogen, triglyceride, total cholesterol, lactate dehydrogenase, amylase and gamma glutamyltransferase. There was a decrease in the levels of aspartate aminotransferase and alkaline phosphatase. Hematological and biochemical protective effects of 0.01 g/L propolis were investigated. Dose-dependent effects of propolis on blood of fish can be favorable, opening new perspectives of investigation on their biological properties and utilization. & 2009 Elsevier Inc. All rights reserved.
Keywords: Biochemical parameters Blood Hematological parameters Oncorhynchus mykiss Propolis
1. Introduction Propolis is a resinous material collected by bees from bud and exudates of plants, which is mixed with products of their salivary glands and wax. Its color varies from green, red to dark brown. Propolis has been used in folk medicine since ancient times. Propolis has attracted researchers’ interest in the last decades because of several biological and pharmacological properties, such as immunomodulatory, antitumor, antimicrobial, anti-inflammatory, and antioxidant, among others (Bankova et al., 2000; Kanbur et al., 2009). As produced by the bees, propolis is a strongly adhesive, resinous substance used to seal holes in their hives, smooth out the internal walls and protect the entrance against intruders. Although a common source of the resin is Populus balsamifera L. (and other Populus species), the precise composition of raw propolis varies with the source. In general, it is composed of 50% resin and vegetable balsam, 30% wax, 10% essential and aromatic oils, 5% pollen and 5% various other substances, including organic debris. Raw propolis is processed using water washing and solubilizing in 95% ethanol to remove the wax and organic debris, creating propolis tincture, ‘‘propolis balsam’’, or ethanol extract of propolis (Burdock, 1998). Besides, propolis-containing products have been intensely marketed by the pharmaceutical industry and health-food stores (Banskota et al., 2001a, b). The ethnopharmacological approach, combined with chemical and biological methods, may provide useful
Corresponding author. Fax: +90 388 2250180.
E-mail address: [email protected] (Z.S. Talas). 0147-6513/$ - see front matter & 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2009.04.011
pharmacological leads. Several researchers have reported the antitumoral property of propolis both in vivo and in vitro (Banskota et al., 2001a, b; Lee et al., 2005). Propolis has been used since ancient times as a medicine because of its biological properties as an antimicrobial, antifungal, antiprotozoan and antiviral agent (Alberto et al., 2005). Important sources of propolis include poplars, alders and birches, chestnut, ash and various Pinus and Salix species. Variations in the bees-wax content of raw propolis also affect the chemical composition. The main chemical classes found in propolis are flavonoids, phenolics and various aromatic compounds. However, propolis contains many of the B-complex vitamins, important minerals and trace elements. But its bioflavonoid content is now receiving attention. Bioflavonoids are antioxidant molecules that play very important roles in the scavenging of free radicals, which are produced in degenerative heart diseases, atherosclerosis, aging and effects of toxic substances (Kolankaya et al., 2002). Biochemical and hematological parameters of fish are determined as an index of their health status as well (Blaxhall and Daisley, 1973). Fish in particular are commonly used to estimate the influence of environmental compounds due to the sensitivity of their biochemical and hematological parameters under such conditions (Lopes et al., 2001). As a sign of stress, the use of hematological and biochemical methods provides valuable knowledge about physiological reactions occurring against changing environmental conditions, especially understanding the physiological, biochemical and hematological changes occurring at various concentrations of compounds, to predict the possible level of threat to life.
ARTICLE IN PRESS Z.S. Talas, M.F. Gulhan / Ecotoxicology and Environmental Safety 72 (2009) 1994–1998
In this study, we have tested for the first time the possible use of propolis using the rainbow trout, one of the most popular cultural fish in Turkey. To observe the effects of propolis, which is a natural product obtain from plants, rainbow trout, which is one of the most important fish in Turkey and human diet was studied. This study opens a new perspective on the investigation of propolis biological properties, mainly with respect to the hematological and biochemical parameters in blood of rainbow trout. This study was aimed at the investigation of effects and useful concentration of natural products like propolis, which has biologically benefits exists in natural environmental conditions, on living organisms that live in natural area.
1995
2.4. Hematological analysis Blood samples were collected prior to anesthetizing of fish to prevent hemolysis (McKnight, 1966). The samples were transferred to glass tubes and hemotimetric parameters were immediately determined. Red blood cell counting was done after 1:200 dilution into Hayem solution. Leukocyte counting was done in blood samples after proper dilution into Turck solution (De Wilde and Houston, 1961; Blaxhall and Daisley, 1973; Blaxhall, 1981). Hemoglobin (HGB) concentration was determined according to the cyano-methemoglobin procedure (Kit 525-A; Sigma Chemical, St. Louis, MO, USA; Blaxhall and Daisley, 1973). Nonclotted blood (20 ml aliquots) was diluted with 1 ml of Drabkin solution and left to stand for 10 min at room temperature. The absorbance was read at 540 nm and the amount of hemoglobin was calculated against a hemoglobin standard (Azizoglu and Cengizler, 1996). Hematocrit was determined as in Jewet et al., (1991) and Wilhelm Filho et al. (1992). Nonclotted blood was transferred to a microhematocrit capillary, centrifuged at 14,000g for 5 min and read against a standard chart.
2. Materials and methods
2.5. Biochemical assay
2.1. Experimental section
After treatments and propolis exposure 2 ml of blood was withdrawn from caudal vein. Blood samples were transferred to glass tubes, kept in cooled bath and promptly analyzed. The blood was centrifuged at 3000g, at 4 1C for 5 min. Alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyltransferase (GGT), lactate dehydrogenase (LDH) and amilase activities were assayed; glucose, total protein, creatinine, blood urea nitrogen (BUN), triglycerides, total cholesterol, chloride, sodium, potassium, total calcium, phosphates and ferric levels in plasma were determined. All such analyses were done (Olympus Optical Corp., Shizuoka-ken, Japan) using commercially available kits (Roche).
The rainbow trout (Oncorhynchus mykiss) were purchased from Camardı, Ecemis fish farm (Nigde, Turkey). Fish were fed for 15 days in a 8 5 1.5 m3 stock pond to be acclimatized. After the adaptation period, 8 fish were transferred to 200 L water tank filled with natural spring water. Fish used in this study had an average weight of 245.5175.22 g and length of 29.7573.81 cm. Physical and chemical properties of water are depicted in Table 1.
2.2. Preparation of propolis extractive solution 2.6. Statistical analysis Propolis extraction methods may influence its activity, since different solvents solubilize and extract different compounds. The most common extracts used in biological assays are ethanol, methanol and water. Its chemical composition is very complex; more than 300 components have already been identified, and its composition is dependent on the local flora. Moreover, propolis composition is highly variable, creating a problem the medical use and standardization. In the present work, propolis was collected from a farm at village Kocaavsar in Balikesir, Turkey. Propolis was dissolved to 30% in ethanol, protected from light and moderately shaken for 1 day at room temperature. Afterward, the extracts were filtered twice, dried and stored in sealed bottles at 4 1C until use. Three different concentrations (0.01, 0.02 and 0.03 g/L) of the dry propolis extracts were achieved in double distilled water.
2.3. Experimental design In the study, there were totally 32 rainbow trout in 0.01 g/L-propolis-exposed group, 0.02 g/L-propolis-treated group, 0.03 g/L-propolis-treated groups and control group (the average weights and lengths of rainbow trout were 240–250 g and 29.7573.81 cm, respectively). Thirty-two rainbow trout were divided into four groups, each consisting of eight animals. Each rainbow trout was weighed just before the start of the study; 0.01 g/L propolis was treated to the animals in group I for 96 h and not fed for 12 h before; 0.02 g/L propolis was treated to the animals in group II for 96 h and not fed for 12 h before; 0.03 g/L propolis was treated to the animals in group III for 96 h and not fed for 12 h before. Fish in group IV were used as a control and were killed 96 h later. Fish experiments were performed in accordance with the guidelines for fish research from the National Institute of Health and approved by the Committee of Fish Research at Nigde University, Nigde, Turkey.
Table 1 Some parameters of the water used in the experiment. Parameter (mg/L)
Before treatment
After treatment
Dissolved oxygen Chemical oxygen demand Suspended solids Calcium Sodium Chloride Total nitrogen Hardness (CaCO3) Temperature (1C) pH
7.870.2 15.170.1 36.871.2 126.071.6 22.470.8 16.071.5 6.070.2 174.373.1 11.571.0 7.870.1
7.670.1 16.270.2 40.171.8 114.171.1 19.770.8 18.071.4 6.870.3 168.272.9 1271 7.870.1
Hematological and biochemical data were analyzed with SPSS 9.0 for Windows using one-way analyses of variance (ANOVA). Differences between means were determined using Duncan’s multiple range test in which the significance level was defined as po0.05.
3. Results In order to determine the effects of propolis extracts, we used three concentrations. The effects on the fish, observed through biochemical parameters, are summarized in Table 2. Fish exposed to 0.01 g/L did not change (p40.05) the levels of glucose, total protein, creatinine, BUN, triglycerides, total cholesterol ALT, AST, LDH, ALP, amilase, and GGT as compared with the control group (Table 2). Fish exposed to 0.02 and 0.03 g/L presented significant increase (po0.05) in glucose, BUN, triglycerides, total cholesterol levels and LDH, amilase and GGT activities as compared with the control group. However there was no significant increase (p40.05) in the level of total protein and creatinine. There was significant decrease in AST and ALP as compared with the control group (Table 2). But no significant decrease in the levels of ALT was observed as compared with the control group (p40.05). It is seen (Table 3) that changes in the electrolytes in rainbow trout blood with three different concentrations of propolis administration, 0.01, 0.02 and 0.03 g/L, are no statistically significant changes (p40.05) in the levels of chloride, sodium, total calcium and phosphates as compared with the control group (Table 3). However, with 0.02 and 0.03 g/L administration there was a statistically significant decrease (po0.05) in the level of potassium as compared with the control and 0.01 g/L (Table 3). There was a statistically significant increase in the level of ferric as compared with the control and 0.01 g/L (p40.05) (Table 3). The effects of propolis on the hematological parameters are summarized in Table 4. Significant increases (po0.05) in total leukocyte (WBC), granulocyte, MCV and MCH values in fish exposed to 0.02 and 0.03 g/L propolis and significant decreases (po0.05) in agranulocyte, RBC, HGB and HCT values were observed in contrast with MCHC (Table 4).
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Table 2 Changes in the biochemical parameters in rainbow trout blood with different concentrations of propolis. Biochemical parameters
Control
0.01 g/L propolis
0.02 g/L propolis
0.03 g/L propolis
Glucose (mg dL1) Total protein (g dL1) Creatinine (mg dL1) BUN (mg dL1) Triglycerides (mg dL1) Total cholesterol (mg dL1) ALT (IU l1) AST (IU l1) LDH (IU l1) ALP (IU l1) Amilase (IU l1) GGT (IU l1)
68.570.5 2.570.1 0.170.0 4.970.2 380.2736.2 180.3711.5 30.378.4 536.5743.1 784.2740.2 54.175.9 287.5714.2 5.270.9
66.170.6 2.670.08 0.170.0 5.170.2 387.1723.1 184.4713.1 26.573.3 470.6736.5 802.1719.1 53.374.2 292.8713.2 6.070.8
74.270.8 2.770.1 0.170.0 6.770.1 458.1721.4 221.4716.5 21.673.7 332.6721.2 1070.2760.4 40.573.8 454.1724.1 8.671.2
80.371.1 2.770.1 0.270.0 7.170.3 487.7717.2 244.7715.4 28.773.0 312.5730.5 1281.7778.5 34.274.1 591.7740.6 9.270.6
Data are mean values 7sd (n ¼ 8). Denotes statistically significant groups (po0.05).
Table 3 Changes in the electrolytes in rainbow trout blood with different concentrations of propolis. Electrolytes
Control
0.01 g/L propolis
0.02 g/L propolis
0.03 g/L propolis
Chloride (mmol l1) Sodium (mmol l1) Potassium (mmol l1) Total calcium (mmol l1) Phosphates (mmol l1) Ferric (mmol l1)
117.672.6 151.672.5 3.870.6 11.870.4 20.371.5 73.374.3
123.571.9 148.671.7 2.270.5 10.970.3 19.870.6 84.276.1
122.672.2 147.671.3 1.270.3 10.170.3 21.671.8 110.279.1
119.571.5 146.571.2 1.970.3 10.270.4 18.970.9 111.7711.8
Data are mean values 7sd (n ¼ 8). Denotes statistically significant groups (po0.05).
Table 4 Changes in the hematological parameters in rainbow trout blood with different concentrations of propolis. Hematological parameters 3
Control 3
Total leukocyte—WBC (m /10 ) Granulocytes (%) Agranulocytes (%) Erytrocyte—RBC (mm3/106) Hemoglobin—HGB (g/dL) Hematocrit—HCT (%) Erythrocyte indexes MCV (m3) MCH (mg) MCHC (%)
0.01 g/L propolis
0.02 g/L propolis
7.670.1 75.470.4 24.670.3 1.570.1 8.570.2 35.671.5
7.270.2 77.570.4 22.570.4 1.470.1 8.370.1 33.971.2
8.770.1 81.170.7 18.970.7 1.270.1 7.170.2 27.670.4
238.972.3 57.071.4 23.870.7
235.472.1 57.671.7 24.470.8
240.071.9 61.771.8 25.770.6
0.03 g/L propolis 9.570.7 82.670.8 17.470.8 1.070.1 6.970.2 26.170.6 263.072.7 70.472.4 26.470.7
Data are mean values 7sd (n ¼ 8). Denotes statistically significant groups (po0.05).
In the present study, the protective effect doses of propolis were compared to control group in hematological and biochemical parameters. The data show that propolis has an important effect on biochemical and hematological parameters at 0.01 g/L levels, whereas 0.02 and 0.03 g/L concentrations appear to be unfavorable for blood tissue of rainbow trouts.
4. Discussion Fish is considered as a valuable source of protein in the human diet. The significance of long-chain polyunsaturated fatty acids has gained attention because of their prevention of human
cardiovascular diseases (Shahidi and Botta, 1994). Investigations were carried out on the rainbow trout. Due to rapid growth and easy accommodation to environment conditions, rainbow trout are the common consumed and economical important fish. In recent years, there has been a great deal of studies carried out on propolis metabolism but not on dose studies of propolis and its effects on biochemical and hematological parameters of aquatic animals. Thus, this study aimed to discuss the doses of this bee product on biochemical and hematological parameters in blood of rainbow trout. Hematological and biochemical data showed that propolis at various concentrations exerted a certain influence on some of the blood indices studied. Propolis compounds may enter aquatic
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environments from agricultural and rural fields by rain water and have long-term effects in fish. These effects appear in population or ecosystems as physiological and biochemical reactions. To understand the effects of propolis on living organisms, metric, physiological, hematological and biochemical parameters should be examined (Lenhardt, 1992; Strmack and Braunbeck, 2000). As shown in various studies, propolis has several biological and pharmacological properties, such as those of antimicrobial, antiinflammatory, antioxidant, antifungal, antiprotozoan and antiviral agents. (Bankova et al., 2000; Kanbur et al., 2009). When these features are taken into account, it is thought that propolis might harm people and animals when they are exposed to amounts higher than needed for the normal diet (Burdock, 1998; Jingtao, 1999). Due to the cytotoxic effect of high concentration of propolis it may have a role in the inhibition of erythropoiesis in hematopoietic organisms. Administration of propolis at different doses resulted in evaluation of levels of glucose, total protein, creatinine, BUN, triglyceride, total cholesterol, LDH, amilase, GGT, and ferric. The elevation in their levels in fish exposed to 0.02 and 0.03 g/L propolis may be considered as one of the markers of renal and liver dysfunctions, and changes of lipid metabolism (Dobrowolski et al., 1991); 0.01 g/L propolis caused a significant (po0.05) decrease in most of these levels, therefore appeared to have potential to maintain liver and renal function, and avoid hypercatabolism at high concentration (0.02 and 0.03 g/L) in propolis-treated animals. As propolis possesses antioxidant properties, it is also likely to behave as an antioxidant. It is known that exogen agents can change hematological parameters, such as erythrocyte number, HGB amount, hematocrit value and total leukocytes. We emphasize that effective dose of propolis to inhibit changes in hematological and biochemical parameters exists at high concentration. With 0.02 and 0.03 g/L propolis while group levels of total leukocyte count and granulocytes, MCV, MCH and MCHC increased, erytrocyte count, agranulocytes, HGB and hematocrit levels decreased. Decreases in erythrocyte, HGB and hematocrit levels at high doses (0.02 and 0.03 g/L) of propolis may be an indicator of anemia with inhibition of erythropoesis in the hemopoetic organism (Kleinrok et al., 1978). In addition, increases in MCV, MCH and MHCH values indicated that the anemia was of a macrocytic type. However, high leukocyte values depend on stimulatory effects of cytotoxic agents on the immune system (Ivanov et al., 1973; Kleinrok et al., 1978; Sforcin, 2007). The usefulness of immunostimulants has been demonstrated in modern aquaculture and fish farmers use a wide range of immunostimulants that may need to be purified (vitamins, chitin, glucans, etc.) or not (microorganisms, animal and plant extracts, subproducts of other industries, etc, Sakai, 1999; Alberto et al., 2005). According to results of our study, 0.01 g/L propolis can act as an immunostimulant. Ample stimulation of the immune system by 0.02 and 0.03 g/L doses of propolis can increase the total leukocytes value. In conclusion, it can be said that propolis is important at various concentrations for the rainbow trout and may significantly influence certain physiological functions, especially hematopoiesis. Hematological and biochemical parameters of rainbow trout treated at various doses were investigated, and the effects of 0.01 g/L propolis were outlined, evidencing their preservation role on hematological and biochemical parameters. However, there is still a need for further studies regarding the use of propolis extracts or its constituents and environmental distribution as natural antioxidant, possible food supplement and natural protective agent.
1997
5. Conclusions Most recent studies have shown that natural preventive compounds have gained popularity day by day as some of the widely used synthetic pharmaceuticals and therapeutics might have some undesirable effects. One can think that certain natural food ingredients would be better and safer than synthetic ones. Many of these compounds, such as plant phenolics, often exhibit antioxidant activities; therefore the addition of these compounds into food products may be helpful to health the of consumers and also to the stabilization of food products. Due to the presence of some of these effective compounds such as flavonoids (flavones and flavanones), phenolic acids and their esters in propolis and propolis extract, if the positive physiological properties and the non-toxicity of the propolis sample are proven it could be used as a mild antioxidant and preservative. Due to antioxidant and preservative properties of propolis, it may not only prolong the physiological functions of some aquatic living organisms, but also contribute to the health benefit of consumers who consume aquatic animals.
Acknowledgment Nigde University Research Fund (FEB 2007/13) is gratefully acknowledged for support of this work. References Alberto, C., Alejandro, R., Esteban, M.A., Jose´, M., 2005. In vivo effects of propolis, a honeybee product, on gilthead seabream innate immune responses. Fish. Shellfish. Immun. 18 (1), 71–80. Azizoglu, A., Cengizler, I., 1996. An investigation on determination of some hematologic parameters in healthy Oreochromis niloticus (L.). Turk. J. Vet. Anim. Sci. 20, 425–431. Blaxhall, P.C., Daisley, K.W., 1973. Routine hematological methods for use with fish blood. J. Fish. Biol. 5, 771–781. Blaxhall, P.C., 1981. A comparison of methods used for the separation of fish lymphocytes. J. Fish Biol. 18, 177–181. Bankova, V.S., De Castro, S.L., Marcucci, M.C., 2000. Propolis: recent advances in chemistry and plant origin. Apidologie 31, 3–15. Banskota, A.H., Tezuka, Y., Adnyana, I.K., et al., 2001a. Hepatoprotective and antiHelicobacter pylori activities of constituents from Brazilian propolis. Phytomedicine 8, 16–23. Banskota, A.H., Tezuka, Y., Kadota, S., 2001b. Recent progress in pharmacological research of propolis. Phytother. Res. 15, 561–571. Burdock, G.A., 1998. Review of the biological properties and toxicity of bee propolis (propolis). Food Chem. Toxicol. 36 (4), 347–363. De Wilde, M.A., Houston, A.H., 1961. Hematological aspect of the therma acclamatory process in the rainbow trout, Salmo gairdneri. J. Fish Res. Biol. 24, 2267–2281. Dobrowolski, JW., Vohoraq, SB., Sharma, K., Shah, SA., Naqvi, SAH., Dandiya, PC., 1991. Antibacterial, antifungal, antiamoebic, antiinflammatory, and antipyretic studies on propolis bee products. J. Ethnopharmacol. 35, 77–82. Ivanov, DF., Tikonov, AI., Krivenchuk, PE., Liurskaia, FV., 1973. Propolis and its clinical usage. Oftolmol. Zh. 28 (2), 104–107. Jewet, M.G., Behmer, D.J., Johnson, G.H., 1991. Effects of hyperoxic rearing water on blood hemoglobin and hematocrit levels of rainbow trout. J. Aquat. Anim. Health 3, 153–160. Jingtao, Z., 1999. A refined propolis which is lowly-toxic and non-allergens. World Phytomed. 14 (1), 37. Kanbur, M., Eraslan, G., Silici, S., 2009. Antioxidant effect of propolis against exposure to propetamphos in rats. Ecotox. Environ. Saf. 72 (3), 909–915. Kleinrok, Z., Borzecki, Z., Scheller, S., Matuga, W., 1978. Biological properties and clinical application of propolis: X. Preliminary pharmacological evaluation of ethanol extract of propolis (EEP). Arzneim. Forsch./Drug Res. 28 (1), 291–292. Kolankaya, D., Selmanoglu, G., Sorkun, K., Salih, B., 2002. Protective effects of Turkish propolis on alcohol-induced serum lipid changes and liver injury in male rats. Food Chem. 78, 213–217. Lee, Y.T., Don, M.J., Hung, P.S., Shen, Y.C., Lo, Y.S., Chang, K.W., Chen, C.F., Ho, L.K., 2005. Cytotoxic of phenolic acid phenethyl esters on oral cancer cells. Cancer Lett. 223, 19–25. Lenhardt, M., 1992. Seasonal changes in some blood chemistry parameters and in relative liver and gonad weights of pike (Essox lucius) from the river Danube. J. Fish. Biol. 40, 709–718. Lopes, P.A., Pinheiro, T., Santos, M.C., Mathias, M.D., Collares-Pereira, M.J., ViegasCrespo, A.M., 2001. Response of antioxidant enzymes in freshwater fish
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populations (Leuciscus alburnoides complex) to inorganic pollutants exposure. Sci. Total Environ. 280, 153–163. McKnight, I.M.A., 1966. Hematological study on the mountain whitefish. Popium willasemi south. Fish Reb. Bd. 23, 45–64. Sakai, M., 1999. Current research status of fish immunostimulants. Aquaculture 172, 63–92. Sforcin, J.M., 2007. Propolis and the immune system: a review. J. Ethnopharmacol. 113, 1–14.
Shahidi, F., Botta, J.R., 1994. Seafoods: Chemistry, Processing Technology and Quality. Chapman & Hall, London, pp. 3–9. Strmack, M., Braunbeck, T., 2000. Isolated hepatocytes of rainbow trout (Oncorhynchus mykiss) as a tool to discriminate between differently contaminated small river system. Toxicol. Vitro 14, 361–377. Wilhelm Filho, D.M., Eble, G.J., Kassner, G., Caprario, F.X., Dafre, L.A., Ohira, M., 1992. Comparative hematology in marine fish. Comp. Biochem. Physiol. 102, 311–321.