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Toxic effects and depuration after the dietary lead(II) exposure on the bioaccumulation and hematological parameters in starry flounder (Platichthys stellatus)
Accepted Manuscript Title: Toxic effects and depuration after the dietary lead(II) exposure on the bioaccumulation and hematological parameters in starry flounder (Platichthys stellatus) Author: In-Ki Hwang Kyeong-Wook Kim Jun-Hwan Kim Ju-Chan Kang PII: DOI: Reference:
S1382-6689(16)30155-7 http://dx.doi.org/doi:10.1016/j.etap.2016.06.017 ENVTOX 2552
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
26-3-2016 13-6-2016 14-6-2016
Please cite this article as: Hwang, In-Ki, Kim, Kyeong-Wook, Kim, JunHwan, Kang, Ju-Chan, Toxic effects and depuration after the dietary lead(II) exposure on the bioaccumulation and hematological parameters in starry flounder (Platichthys stellatus).Environmental Toxicology and Pharmacology http://dx.doi.org/10.1016/j.etap.2016.06.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Toxic effects and depuration after the dietary lead (II) exposure on the bioaccumulation and hematological parameters in starry flounder (Platichthys stellatus)
In-Ki Hwang, Kyeong-Wook Kim, Jun-Hwan Kim and Ju-Chan Kang
Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, Republic of Korea
Email address: [email protected] (Ju-Chan Kang) Telephone: 82 51 5944 Fax: 82 51 5938
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Highlights Lead bioaccumulation in P.stellatus was dose dependent.
The depuration of lead was not effective in most tissues.
Exposure to lead resulted in RBC count, hematocrit and hemoglobin decrease.
The recovery after the depuration period occurred in the level of glucose, total protein and ALP.
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Abstract
Platichthys stellatus (mean length 20 ± 2 cm, mean weight 160.15 ± 15 g) were exposed to the different levels of dietary lead (II) at the concentrations of 0, 30, 60, 120, 240 mg/kg for 4 weeks. Depuration was conducted for 2 weeks after exposure. The lead exposure over 60 mg Pb/kg induced the significant bioaccumulation in tissues of P.stellatus (5-30 µg/g tissue), except for brain and muscle where the exposure to 240 mg Pb/kg caused the bioaccumulation (2-4 µg/g tissue). The hematological parameters such as red blood cell (RBC) counts, hematocrit (Ht) value and hemoglobin (Hb) concentration were substantially decreased over 60 mg Pb/kg, and lasted even after the depuration period. For plasma components, calcium and magnesium levels in plasma were generally decreased over 60 mg Pb/kg, and glucose level was also mainly increased over 60 mg Pb/kg. Total protein was significantly decreased over 120 mg Pb/kg after 4 weeks exposure. Glucose and total protein showed the restoration after the depuration period in groups of fish exposed previously to over 60 and 120 mg Pb/kg, respectively. However, other parameters that changed during the exposure over 60 mg Pb/kg did not recovered. For enzymatic components in plasma, glutamic oxalate transminase (GOT), glutamic pyruvate transminase (GPT) and alkaline phosphatase (ALP) were significantly increased over 120 mg Pb/kg, and there was only restoration observed after the depuration for ALP over 120 mg Pb/kg.
Keywords: Platichthys stellatus, dietary lead(II) exposure, bioaccumulation, depuration, hematological parameters
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1. Introduction
Lead (II) is globally one of the most ubiquitous metals in the environment, which is present naturally in water (0.02 µg/L and 2µg/L in sea and river, respectively) as well as in soils (16mg/kg) and air (0.1µg/m3 and 0.3 to 2 µg/m3 in rural and urban areas, respectively) (Labrot et al., 1999). It has been used in alloys with other metals, storage battery and a protection against radiation. In the recent, the usage of lead has exceeded the total amount that had been used in the previous era (Hsu and Guo, 2002). However, this active manufacturing has brought about severe lead contamination of the aquatic ecosystem, including soils and air. Generally, it is demonstrated that the lead can not only induce the disturb effects on hematological components, but also it accumulates large amounts of lead in the internal organs of fish, exposed to dietary exposed to that metal exposure (Javed, 2012). For example, neotropical fish (Hoplias malabaricus) exposed to inorganic lead showed the abnormal shapes, such as elongation and roundness of erythrocytes (Ribeiro et al., 2006). Also, Nussey et al, (1999) reported that there were the highest metal bioaccumulations in gill (direct contact with the environment) and liver (storage and detoxification) of moggel (Labeo umbratus). Bioaccumulation of heavy metals in organs of fish is normally affected by various biotic and abiotic elements. Generally, it is dependent on the physical factors such as temperature, pH, salinity and hardness (Kim and Kang, 2015a) as well as chemical properties such as water solubility, uptake rates and bioavailability (Bandowe et al, 2014). Heavy metals can enter into the body through main three possible ways (the body surface, the gills and the internal organs digestive tract). They are easily absorbed through the gills when their concentrations are high in water (Pourang, 1994). Meanwhile, the digestive organs can be a major route in case of food contamination, potentially resulting in the biomagnifications throughout the food chain (Amundsen et al., 1997). After penetration of heavy metals into the body, a variety of tissues show significantly different degree of accumulation. In this sense, liver and kidney are not higher, but maybe they are more sensitive to accumulation than gill, muscle and swim bladder (Chi et al., 2007). This bioaccumulation phenomenon in fish can be an important indicator, because it causes directly physiological distress. A problem of heavy metals accumulation 4
in fish is important because they may be transferred to the consumers in food chain, including humans as they occupy a range of trophic levels. Hematological analysis is a basic parameter for determination of fish health and stress in case of exposure to diverse toxicants, such as metals, pesticides and chemical industrial effluents in eco-toxicology (Singh et al., 2008). Generally, hematocrit (Ht), hemoglobin (Hb) concentration and red blood cell (RBC) counts are related to a general anemic state of fish (Kochhann et al, 2013). Also, there are specific enzymes, such as serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in liver that suggest acute exposure or damages of particular tissues. In addition, plasma organic (glucose and total protein) and inorganic (calcium and magnesium) components can be used indirectly used to evaluate indirectly stress and osmotic disturbance of fish, respectively. Teleost is generally regarded as good models to evaluate the toxicity of contaminants, because their biochemical and physiological responses are similar to those of mammals. Starry flounder (Platichthys stellatus) is a euryhaline species which can adapt to a broad range of salinity concentrations, and also one of the cold-water fishes which live in the cold oceans of Korea, Japan, Alaska and California as well as Sea of Okhotsk and Bering Sea. Recently, P. stellatus is attracting attention because of its high value and flavor throughout all field and farm in Korea. However, researches on heavy metals bioaccumulation and their influence on hematological parameters of P. stellatus of heavy metals are unusual were not performed until now in P. stellatus. Therefore, the aim of this study is to analyze the bioaccumulation in each organ and biochemical properties of blood of P. stellatus, exposed to dietary lead.
2. Material and methods
2.1 Experimental fish and conditions Sixty fish (mean length 20 ± 2 cm, mean weight 160.15 ± 15 g) were obtained from a fish farm in Gijang City, Korea, and they were divided into five 250L circular tanks (15 fish per each tank). The fish were acclimatized for 1 week under constant environmental conditions (Table 1), and there was no mortality of fish during the experiment in flow-through system. The fish were fed a commercial diet (Woosung feed, Daejeon City, Korea) by one percent of body weight twice a day before the experiment. Also, they were maintained on a 12-h: 12-h 5
light/dark cycle. Lead premix was composed of 1 gram of lead (II) nitrate (Sigma chemical Co., LTD) and 99 gram of cellulose, and then thoroughly mixed by feed composition (Table 2). Manufactured fodder was pelleted to avoid the release of lead by contamination into the water during dietary lead exposure. The dietary Pb concentrations were 0, 30, 60, 120, 240 mg/kg. Whole breeding period was 6 weeks, when the fish were fed a lead-bearing diet for 4 weeks and control diet for the rest of the period.
2.2 Bioaccumulation
Four fish at each concentration were anesthetized with benzocaine (Sigma Aldrich Co., USA) and dissected after 2, 4 and 6 weeks of the exposure. The tissue samples of brain, muscle, liver, spleen, kidney, intestine and gill of P. stellatus were performed with freeze-dried to measure dry weight of the samples. The dried samples were digested by wet digestion method in 65% (v/v) HNO3, and re-dried at 120oC. The procedure was repeated until total digestion. The entirely digested samples were diluted in 2% (v/v) HNO3.The samples were filtered through a membrane filter (AdvantecMFS, Inc. 0.2µm) under pressure for analysis. For determination of total lead concentrations, the digested and extracted solutions were analyzed by ELAN 6600DRC ICP-MS instrument with argon gas (Perkin-Elmer). Total lead concentrations were determined by external calibration, ICP multi-element standard solution VI (Merck). The lead bioaccumulation in tissue samples was expressed µg/g dry wt.
2.3 Hematological parameters
Blood samples were collected through the caudal vein of the fish with 1-mL disposable heparinized syringes to prevent blood coagulation. The blood samples were kept at 4oC until the blood parameters were completely analyzed. The total red blood cell (RBC) counts, hemoglobin (Hb) concentration, and hematocrit (Ht) value were determined immediately. Total RBC counts were counted using optical microscope with hemocytometer (Improved Neubauer, Germany) after diluted by Hendrick's diluting solution. The Hb concentration was determined using Cyan-methemoglobin technique (Asan Pharm. CO., Ltd.). The Ht value was determined by the micro-hematocrit centrifugation technique (Model; 01501, Hawkslyy and sons Ltd., England).
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2.4 Plasma components
The blood samples were centrifuged to separate serum at 3000g, 4oC for 5min. The serum samples were analyzed for organic and inorganic substances, and enzyme activity using clinical kit (Asan Pharm. Co., Ltd.). For inorganic substances assay, calcium and magnesium were analyzed by the OCPC method (ocresolphthalein-complexon) and xylidyl blue –I technique, respectively. For organic substances assay, glucose and total protein were analyzed by GOD/POD technique and Biuret technique, respecively. Also, for enzyme activity assay, glutamic oxalate transaminase (GOT), glutamic pyruvate transaminase (GPT) and alkaline phosphatase (ALP) were analyzed by Kind-king method and Reitman-Frankel method, respectively.
2.5 Statistical analysis
Statistical analyses were performed using the SPSS/ PC + statistical package (SPSSInc, Chicago, IL, USA). Significant differences between groups were identified using one-way ANOVA and Duncan's test for multiple comparisons or Student's t-test for two groups (Duncan, 1955). The significance level was set at P < 0.05.
3. Results
3.1 Bioaccumulation
Lead accumulations in brain, muscle, liver, spleen, kidney, intestine and gill of S P. stellatus are shown in figure 1. The significant changes were observed at 240 mg Pb/kg after 2 weeks (control; 1.55±0.19 µg/g, 30 mg/kg; 1.50±0.14 µg/g, 60mg/kg; 1.59±0.14 µg/g, 120 mg/kg;1.63±0.14 µg/g, 240 mg/kg; 1.85±0.13 µg/g) and 4 weeks (control; 1.56±0.14 µg/g, 30 mg/kg; 1.58±0.13 µg/g, 60 mg/kg; 1.75±0.16 µg/g, 120 mg/kg; 1.81±0.15 µg/g, 240 mg/kg; 2.02±0.12 µg/g) in the brain. Relatively, there was only accumulation after 4 weeks (control; 3.22±0.53 µg/g, 30 mg/kg; 3.57± 0.41 µg/g, 60 mg/kg; 3.67±0.27 µg/g, 120 mg/kg; 3.43±0.31 µg/g, 240 mg/kg; 3.83±0.48 µg/g) in the muscle. On the other hand, the lead concentration was considerably increased over 120 mg Pb/kg after 2 weeks (control; 7
1.84±0.32 µg/g, 30 mg/kg; 2.32± 0.31 µg/g, 60 mg/kg; 4.60±0.32 µg/g, 120 mg/kg; 8.20±0.74 µg/g, 240 mg/kg; 11.08±1.04 µg/g), 30 mg Pb/kg after 4 weeks (control; 1.24±0.32 µg/g, 30 mg/kg; 3.45± 0.31 µg/g, 60 mg/kg; 6.06±0.32 µg/g, 120 mg/kg; 10.14±0.74 µg/g, 240 mg/kg; 12.88±1.04 µg/g) and 120 mg Pb/kg after the depuration period (control; 1.72±0.31 µg/g, 30 mg/kg; 2.01± 0.32 µg/g, 60 mg/kg; 2.64±0.33 µg/g, 120 mg/kg; 4.68±0.31 µg/g, 240 mg/kg; 8.56±0.53 µg/g) in the spleen. Also, there were similar changes over 60 mg Pb/kg after 2 weeks (control; 1.99±0.43 µg/g, 30 mg/kg; 3.98± 1.08 µg/g, 60 mg/kg; 9.47±1.31 µg/g, 120 mg/kg; 16.08±1.25 µg/g, 240 mg/kg; 24.86±2.17 µg/g), 30 mg Pb/kg after 4 weeks (control; 3.03±1.61 µg/g, 30 mg/kg; 8.85± 1.01 µg/g, 60 mg/kg; 12.75±1.48 µg/g, 120 mg/kg; 20.09±2.25 µg/g, 240 mg/kg; 30.28±2.25 µg/g) and 60 mg Pb/kg after the depuration period (control; 2.05±1.04 µg/g, 30 mg/kg; 4.19± 0.86 µg/g, 60 mg/kg; 8.16±1.27 µg/g, 120 mg/kg; 14.69±1.81 µg/g, 240 mg/kg; 20.93±2.42 µg/g) in the intestine. The gill showed the notable increases over 60 mg Pb/kg after 2 weeks (control; 1.73±0.27 µg/g, 30 mg/kg; 3.02± 0.55 µg/g, 60 mg/kg; 4.76±0.94 µg/g, 120 mg/kg; 7.85±1.66 µg/g, 240 mg/kg; 10.23±1.78 µg/g), 30 mg Pb/kg after 4 weeks (control; 1.78±0.50 µg/g, 30 mg/kg; 4.31± 0.35 µg/g, 60 mg/kg; 6.33±1.53 µg/g, 120 mg/kg; 9.23±2.15 µg/g, 240 mg/kg; 12.89±2.08 µg/g) and 60 mg Pb/kg after the depuration period (control; 1.09±0.20 µg/g, 30 mg/kg; 1.54± 0.26 µg/g, 60 mg/kg; 3.11±0.60 µg/g, 120 mg/kg; 4.02±0.40 µg/g, 240 mg/kg; 6.32±0.68 µg/g). The lead accumulation in the kidney was significantly increased over 60 mg Pb/kg after 2 weeks (control; 3.77±0.47 µg/g, 30 mg/kg; 6.09± 1.89 µg/g, 60 mg/kg; 8.37±1.53 µg/g, 120 mg/kg; 10.79±2.36 µg/g, 240 mg/kg; 20.59±2.49 µg/g), 30 mg Pb/kg after 4 weeks (control; 4.98±0.96 µg/g, 30 mg/kg; 6.38± 2.72 µg/g, 60 mg/kg; 11.26±3.25 µg/g, 120 mg/kg; 18.84±3.84 µg/g, 240 mg/kg; 25.50±2.47 µg/g) and 30 mg Pb/kg after the depuration period (control; 4.26±1.36 µg/g, 30 mg/kg; 5.99± 0.99 µg/g, 60 mg/kg; 7.35±1.14 µg/g, 120 mg/kg; 10.03±1.39 µg/g, 240 mg/kg; 14.99±3.36 µg/g). The liver showed a notable change over 120 mg/kg after 2 weeks (control; 1.57±0.37 µg/g, 30 mg/kg; 4.01± 0.97 µg/g, 60 mg/kg; 7.01±1.09 µg/g, 120 mg/kg; 13.05±0.75 µg/g, 240 mg/kg; 20.03±1.73 µg/g), 4 weeks (control; 1.32±0.58 µg/g, 30 mg/kg; 6.27± 0.55 µg/g, 60 mg/kg; 10.02±2.34 µg/g, 120 mg/kg; 18.37±1.95 µg/g, 240 mg/kg; 28.14±1.08 µg/g) and the depuration period (control; 0.94±0.28 µg/g, 30 mg/kg; 3.45± 1.02 µg/g, 60 mg/kg; 7.02±1.19 µg/g, 120 mg/kg; 10.04±1.27 µg/g, 240 mg/kg; 18.12±1.22 µg/g). The depuration rates at 240 mg Pb/kg (4 weeks to 6 weeks) was 85% in the brain, 79.1% in the muscle, 69.1% in the intestine, 66.5% in the spleen, 64.4% in the liver, 58.8% in the kidney and 49.1% in the gill
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3.2 Hematological parameters
Changes of total RBC counts, hematocrit values and hemoglobin concentrations in P. stellatus are shown in Table 3. The RBC counts were significantly decreased over 120 60 mg Pb/kg after 2 weeks, at 240 over 120 mg Pb/kg after 4 weeks and over 120 mg/kg after the depuration period. The notable decreases in hematocrit value were observed at 240 mg Pb/kg after 2 weeks, and over 120 mg Pb/kg after 4 weeks and at 240 mg Pb/kg after the depuration period. Hemoglobin concentrations were significantly decreased at 240 mg/kg after 2 weeks and over 120 mg Pb/kg after 4 weeks of exposure, and over 120 mg/kg as well as after the depuration period.
3.3 Plasma components
Alterations in plasma components are demonstrated in Table 4. Calcium and magnesium levels in plasma were significantly decreased over 120 mg Pb/kg and over 60 mg Pb/kg respectively after 2 weeks, over 60 mg Pb/kg after 4 weeks and at 240 mg Pb/kg after the depuration period. For blood glucose levels, there were notable increases over 60 mg/kg after 2 weeks and 4 weeks. After the depuration period, glucose level returned to the baseline level, as at 60 mg Pb/kg, at 120 mg Pb/kg and at 240 mg Pb/kg exposure. Also, blood total protein levels were considerably decreased over 120 mg/kg after 4 weeks and at 240 mg/kg after the depuration period. GOT and GPT in plasma showed significant increases at 240 mg/kg after 2 weeks, over 120 mg/kg after 4 weeks and at 240 mg/kg after the depuration period. Also, there were considerable increases of ALP level over 60 mg Pb/kg after 2 weeks and over 120 mg Pb/kg after 4 weeks of Pb exposure, and effective restoration after 2 weeks of depuration period.
4. Discussion
The study to assess the bioaccumulation patterns in fish tissues under the metal exposure is considered as a reliable and sensitive indicator of environmental metal contamination (Kim and Kang, 2015b). In this study, the heavy metal accumulation in the tissues of P. stellatus was observed in the order of this follows; intestine > kidney > liver > spleen > gill > muscle > brain after 2 weeks, and intestine > kidney > liver > spleen ≒ gill > muscle > brain after 4 weeks. 9
In this study, the significant accumulation was observed over 60 mg/kg in the intestine of P. stellatus. Generally, dietary contamination is a major cause of natural bioaccumulation in fish, because they have the high status in the food chain, and intestine has an important role in this process (Dural et al., 2007). Alves et al. (2006) reported the high accumulation in the intestine of juvenile freshwater rainbow trout (Oncorhynchus mykiss), exposed to dietary lead. Also, it was demonstrated that dietary cadmium caused the highest accumulation in the intestine of juvenile rockfish (Sebastes schlegeli) (Kim et al., 2006). It might be assumed that heavy metals in food were remained and accumulated on the wall of intestine, because of unfiltered by mucous (Glover and Hogstrand, 2002). Also, the kidney, liver and spleen indicated the significant degree of bioaccumulation over 60 mg/kg in P. stellatus (Figure 1). Łuszczek-Trojnar (2013) explained that the kidney showed the high accumulation levels in Prussian carp (Carassius gibelio), exposed to lead. Also, there was a significant bioaccumulation occurred in the liver of Atalantic salmon (Salmo salar L.) after three months exposure to dietary arsenobetaine (Amlund et al., 2006). It may be explained that these organs are commonly known as actively metabolic organs through their role in detoxification and excretion of toxicants from the body, and that is why large amounts of heavy metals released from other tissues during the detoxification process may be accumulated again in organs involved with detoxification. Furthermore, comparatively a high level of the accumulation was observed over 60 mg/kg in the gill of P. stellatus. Tao et al. (1999) reported that Carassius auratus exposed to lead showed the significant accumulation and adherence on surface of gill. Hollis et al. (1999) suggested that the gill metal burden continuously increased according to the longer metal exposure, which was due to its metal binding properties. In general, the gill has a key role for the migration of heavy metals as well as the ion exchange, pH control and excretion of nitrogenous compounds, when exposed to waterborne metals rather than dietary condition. Gill can be a major site of accumulation by dietary exposure, because the absorbed substances from intestine are entered into the side of gill epithelium (Szebedinszky et al., 2001). The lower accumulation was observed in the brain and muscle of P. stellatus compared to the other tissues. Tulasi et al. (1992) suggested that the brain was the lower accumulation than other organs such as kidney, gill, and liver of freshwater fish, Anabas testudineus exposed to the lead exposure. Jarić et al. (2011) reported that there were the lowest levels of metal accumulation occurred in muscles among analyzed organs of starlet (Acipenser ruthenus) from the Danube River in Serbia. Also, concentrations of heavy metals in muscle of 10
Leuciscus cephalus were lower than in gills (Demirak et al., 2006). For this result, Conto Cinier et al. (1999) suggested that it was because most of the metals including lead were accumulated in the active metabolic organs, such as liver and kidney, and this is the reason of lower metals concentration in muscle. However, the problem of heavy metals accumulation in this tissue is important because the muscle, as the edible part of fish, in case of contamination pose the serious risk for human as a consumer. Fish normally eliminates heavy metals from the body through the gill, mucus, bile and urine etc. after the biotransformation. The depuration rate was in the orders as follows; gill > liver > kidney ≥ spleen ≈ intestine, but it failed in complete recovery as normal status. On the other hand, brain and muscle showed the recoveries in the concentration of 240 mg/kg in P. stellatus. The higher level of lead accumulation in some tissues may need more time to eliminate more lead amounts. The results obtained in other research confirm the effects observed in the present work. For instance, the lead was excreted quickly in the muscle of Chinese sturgeon (Acipenser sinensis) (Hou et al., 2011). Also, Luszczek-Trojnar et al. (2013) announced that Prussian carp (Carassius gibelio) exposed to dietary lead showed more rapid elimination of that metal from muscle and from the intestine. The hematological parameters have been widely used to evaluate the toxic stress induced by environmental contamination (Kim and Kang, 2014). Many authors reported the effects of heavy metals on hematological parameters in various fish species such as in Hoplias malabaricus (Ribeiro et al., 2006), Clarias batrachus L. (Maheswaran et al., 2008) and Cyprinus carpio (Kandemir et al., 2010) etc. The hematological parameters of P. stellatus were decreased over 60 mg Pb/kg during the exposure period. It is thought that heavy metals increased the membrane permeability and caused the edema in red blood cells (Witeska and Kosciuk, 2003), or they suppressed the activation of hematopoietic organs (Vinodhini and Narayanan, 2009). Due to these reasons, hematological properties can be altered negatively, and anaemia can be triggered (Lohner et al., 2001). The reason to be insufficient recoveries may be due to the toxic effects on the hematopoietic organs by the accumulation. Toxicants in the aquatic environment significantly change chemical responses, due to their potential cellular and molecular toxicities, so biochemical plasma analysis is one of the most important parameters (Vutukuru, 2003). In the present study, calcium and magnesium levels of P. stellatus were considerably decreased over 60 mg/kg. MacDonald et al. (2002) explained that the lead competes with these ions in calcium-magnesium ATPase, and has higher affinity to this pump system. It is thought that the lead was not eliminated completely in from the 11
body, and then it has had a consistently bad deleterious effect on the osmoregulation. Glucose is used as a sensitive parameter for the stress in fish. The glucose levels of P. stellatus were increased over 60 mg Pb/kg during the exposure period reaching the level 123.7 mg/dL (Table 4). This result would be also compared to the results obtained in similar research presented by Kim and Kang 2015b. Cicik and Engin (2005) announced that glucose levels were increased in common carp (Cyprinus carpio), exposed to cadmium. This reaction could be explained as the result of elevated metabolic rate in the body and the gluconeogenesis by the stress (Saravanan et al., 2011). On the other hand, total protein which is an indicator of metabolism activation was significantly decreased after 4 weeks of exposure P. stellatus to over 120 mg/kg. Martinez et al. (2004) demonstrated that the neotropical fish (Prochilodus lineatus) exposed to the lead showed the decreased total protein values due to its toxicity to the metabolism. After the exposure, organic components represented the recoveries over 60 and 120 mg/kg, respectively. GOT, GPT, ALP levels of P. stellatus were increased over 120 mg/kg. The results were similar in the previous research (Kim and Kang, 2015b). Generally, these enzymes are excreted in the liver, and are present increased levels when organism is exposed to toxicants and other stress. Also, it was possibly announced that histopathological degeneration and necrosis could give rise to the alteration in cell membrane, the leakage of intracellular enzymes and the collapse of transportation system (Humtsoe et al., 2007). After the exposure, substantial recoveries were not observed over 120 mg/kg, except for that of ALP. It suggests that histological damages would last even after the depuration period. In conclusion, the active organs such as intestine, kidney, liver, spleen, and gill observed significant accumulations, and the most depuration was the brain. The hematological parameters were significantly changed for each component. Considering the above results, the exposure to the dietary lead exposure over 60 mg/kg can negatively affect the experimental fish, P. stellatus, and deleterious effects of that exposure can last until the depuration period.
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17
Brain exposure
depuration b
b
2.0 a
a
ab
ab
ab ab a a a
a a
a a
1.5
1.0
6
Muscle
0 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg 240 mg/kg
exposure
depuration b
a 4
a
a a
a
ab a a a
a
a a a a
2
30
Pb concentration (g/g)
Pb concentration (g/g)
2.5
8
0 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg 240 mg/kg
Pb concentration (g/g)
3.0
4W
6W
2W
4W
Weeks
Spleen 30
e
exposure
depuration
d
12
d
10
c
c
8 c 6
b
b b
4 2
a
a
a a
a
0 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg 240 mg/kg
Pb concentration (g/g)
16 14
0 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg 240 mg/kg
c
e
c b b
d c
b
a
6W
4W
a
6W
30
Intestine
0 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg 240 mg/kg
d exposure
depuration
d d
c 20 c
c b
b
b
10 a
b a
a a
a
a 0
2W
4W
Weeks
6W
2W
4W
6W
Weeks
Gill d depuration
d
10
a
a
b
a
exposure
12
c
c b
8
c b
6
b
b b
a
4 2
depuration
20
10
40
Kidney d
0
4W
ab a
Weeks
exposure
Weeks
18
b a
ab
2W
d
a
0 2W
b ab
10
6W
Pb concentration (g/g)
14
0 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg 240 mg/kg
c a
15
Weeks
Pb concentration (g/g)
Pb concentration (g/g)
16
depuration b
0
0 2W
exposure c
20
5
0.0
Liver b
a
0.5
18
25
0 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg 240 mg/kg
a
a
a
a
0 2W
4W
6W
Weeks
Fig. 1 Pb concentration in brain, muscle, liver, spleen, kidney, intestine and gill of starry flounder (Platichthys stellatus) exposed to the different concentrations of dietary lead for 4 weeks, followed by a depuration period of 2 weeks. Vertical bar denotes a standard error. Values with different superscript are significantly different (P < 0.05) as determined by Duncan’s multiple range test.
18
Table 1 The chemical components of seawater and experimental condition used in the experiments. Item Value Temperature (oC)
13.0±0.8
pH
8.1±0.4
Salinity (‰)
32.2±0.8
Dissolved Oxygen (mg/L)
7.1±0.3
Chemical Oxygen Demand (μg/L)
1.24±0.2
Ammonia(mg/L)
11.9±0.9
Nitrite (mg/L)
1.3±0.2
Nitrate (mg/L)
11.31±1.0
19
Table 2 Formulation of the experimental diet (% dry matter). Lead concentration (mg/kg) Ingredient (%)
Casein1 2
Fish meal
Wheat flour
3
4
Fish oil
Cellulose
1
Corn starch
3
30
60
120
240
33.0
33.0
33.0
33.0
33.0
23.0
23.0
23.0
23.0
23.0
20.0
20.0
20.0
20.0
20.0
10.0
10.0
10.0
10.0
10.0
5.0
4.7
4.4
3.8
2.6
5.0
5.0
5.0
5.0
5.0
5
2.0
2.0
2.0
2.0
2.0
6
Vitamin premix Mineral premix
0
2.0
2.0
2.0
2.0
2.0
7
0.0
0.3
0.6
1.2
2.4
Actual lead concentration
1.8
31.4
62.1
118.8
244.5
Lead premix
1. United States Biochemical (Cleveland, OH). 2. Suhyup Feed Co., Ltd., Gyeong Nam Province, Korea. 3. Young Nam Flour Mills Co., Pusan, Korea. 4. Sigma Chemical Co., St. Louis, MO. 5. Vitamin premix (mg/kg diet): ascorbic acid, 240; dl-calcium pantothenate, 400; Choinechloride, 200; inositol, 20; menadione, 2; nicotinamide, 60; pyridoxine ∙HCl, 44; riboflavin, 36; thiamine mononitrate, 120; dl-a-tocopherolacetate, 60; retinyl acetate, 20,000IU; biotin, 0.04; folic acid, 6; vitamin B12, 0.04 ; and cholecalcifero, 4000IU. 6. Mineral premix (mg/kg diet): Al, 1.2; Ca, 5000; Cl, 100; Cu, 5.1; Co, 9.9; Na, 1280; Mg, 520; P, 5000; K, 4300; Zn, 27; Fe, 40; I, 4.6; Se, 0.2; and Mn, 9.1. 7. Lead premix (mg/kg diet): 10,000mg Pb/kg diet.
20
Table 3 Changes of RBC count, hematocrit value and hemoglobin concentration in starry flounder, Platichthys stellatus exposed to the different concentrations of dietary lead for 4 weeks, followed by a depuration period of 2 weeks. Parameters
RBC count (×104mm3)
Hematocrit (%)
Hemoglobin (g/dL)
Period
Lead concentration (mg/kg)
(weeks)
0
30
60
120
240
2
263.33±10.46a
262.67±10.9ab
242.38±10.02bc
230.67±10.13c
194±15.1c
4
266.5±5.98a
256±12.54a
235±15.54ab
204.5±9.98bc
190±17.44c
6
263.33±8.77a
253.33±14.69a
255.34±10.03a
208±11.78b
198±12.97b
2
30.5±1.73a
29.25±2.36a
29±4.58a
22.67±4.73ab
19±3.46b
4
30.66±4.16a
24.67±3.79ab
25.33±3.06bc
22.67±3.31bc
19.33±2.59c
6
27.33±5.52a
30.33±5.00a
25±3.12bc
24.33±2.58bc
20.32±2.52c
2
6.09±0.23a
5.85±0.32ab
5.72±0.50ab
5.55±0.57ab
5.36±0.3b
4
5.95±0.14a
6.08±0.28a
5.60±0.32ab
5.29±0.32bc
4.83±0.23c
6
5.82±0.05a
5.87±0.27a
5.64±0.11a
5.23±0.25b
5.26±0.24b
Values are mean±S.E. Values with different superscript are significantly different (P ＜0.05) as determined by Duncan's multiple range test.
21
Table 4 Changes of serum parameters in starry flounder, Platichthys stellatus exposed to the different concentrations of dietary lead for 4 weeks, followed by the depuration period of 2 weeks. Period
Lead concentration (mg/kg)
(weeks)
0
30
60
120
240
2
8.82±0.76a
8.73±0.20a
8.23±0.32a
7.45±0.21b
5.56±0.18c
4
9.44±0.67a
8.63±0.20a
6.03±0.35b
4.44±0.21c
3.69±0.48c
6
9.58±0.40a
9.60±0.42a
9.21±0.91a
9.06±0.29a
8.39±0.17b
2
3.94±0.23a
3.86±0.28ab
3.61±0.18bc
3.49±0.21c
3.38±0.15c
4
3.82±0.21a
3.80±0.02a
3.52±0.02b
3.38±0.12c
2.89±0.21d
6
3.72±0.02a
3.76±0.06a
3.65±0.06a
3.62±0.03a
3.43±0.08b
2
96.11±3.92a
96.85±5.63a
104.38±4.70b
107.63±4.10b
110.16±4.99b
4
96.32±4.48a
95.92±4.46a
108.16±5.38b
119.92±5.69c
123.70±4.68c
6
97.05±4.44a
95.92±4.46a
99.16±5.38a
98.92±5.69a
100.70±4.68a
Total
2
2.81±0.18a
3.08±0.17a
2.80±0.10a
2.85±0.16a
2.89±0.11a
protein
4
2.96±0.21a
3.08±0.18a
3.00±0.12a
2.54±0.13b
2.05±0.11c
(g/dL)
6
2.71±0.19a
2.72±0.10a
2.80±0.14a
2.65±0.15a
2.75±0.14b
GOT
2
22.33±5.46a
25.07±2.75ab
26.25±2.76ab
28.45±3.74ab
30.92±4.66b
(karmen
4
21.70±2.03a
24.00±1.18a
25.58±2.57ab
29.20±0.81b
32.85±3.13c
unit)
6
22.48±2.64a
23.78±3.69a
22.27±1.84a
24.75±4.58a
29.57±4.87b
GPT
2
15.23±1.74a
14.09±0.52ab
16.69±2.39ab
17.28±2.42ab
21.96±1.77b
(karmen
4
14.58±1.64a
20.23±2.27ab
19.60±0.67ab
22.69±1.47b
23.91±1.61b
unit)
6
15.35±1.42a
17.72±3.44a
16.01±0.82a
16.07±0.08a
23.88±1.61b
2
4.32±0.11a
4.58±0.09ab
4.79±0.08bc
4.74±0.12bc
4.99±0.28c
4
4.14±0.15a
4.31±0.02ab
4.30±0.10ab
4.59±0.05bc
4.69±0.14c
6
4.24±0.11a
4.18±0.19a
4.26±0.18a
4.26±0.19a
4.18±0.10a
Parameters
Calcium (mg/dL)
Magnesium (mg/dL)
Glucose (mg/dL)
ALP (K-A)
Values are mean±S.E. Values with different superscript are significantly different (P ＜0.05) as determined by Duncan's multiple range test.
22
S1382-6689(16)30155-7 http://dx.doi.org/doi:10.1016/j.etap.2016.06.017 ENVTOX 2552
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
26-3-2016 13-6-2016 14-6-2016
Please cite this article as: Hwang, In-Ki, Kim, Kyeong-Wook, Kim, JunHwan, Kang, Ju-Chan, Toxic effects and depuration after the dietary lead(II) exposure on the bioaccumulation and hematological parameters in starry flounder (Platichthys stellatus).Environmental Toxicology and Pharmacology http://dx.doi.org/10.1016/j.etap.2016.06.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Toxic effects and depuration after the dietary lead (II) exposure on the bioaccumulation and hematological parameters in starry flounder (Platichthys stellatus)
In-Ki Hwang, Kyeong-Wook Kim, Jun-Hwan Kim and Ju-Chan Kang
Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, Republic of Korea
Email address: [email protected] (Ju-Chan Kang) Telephone: 82 51 5944 Fax: 82 51 5938
1
Highlights Lead bioaccumulation in P.stellatus was dose dependent.
The depuration of lead was not effective in most tissues.
Exposure to lead resulted in RBC count, hematocrit and hemoglobin decrease.
The recovery after the depuration period occurred in the level of glucose, total protein and ALP.
2
Abstract
Platichthys stellatus (mean length 20 ± 2 cm, mean weight 160.15 ± 15 g) were exposed to the different levels of dietary lead (II) at the concentrations of 0, 30, 60, 120, 240 mg/kg for 4 weeks. Depuration was conducted for 2 weeks after exposure. The lead exposure over 60 mg Pb/kg induced the significant bioaccumulation in tissues of P.stellatus (5-30 µg/g tissue), except for brain and muscle where the exposure to 240 mg Pb/kg caused the bioaccumulation (2-4 µg/g tissue). The hematological parameters such as red blood cell (RBC) counts, hematocrit (Ht) value and hemoglobin (Hb) concentration were substantially decreased over 60 mg Pb/kg, and lasted even after the depuration period. For plasma components, calcium and magnesium levels in plasma were generally decreased over 60 mg Pb/kg, and glucose level was also mainly increased over 60 mg Pb/kg. Total protein was significantly decreased over 120 mg Pb/kg after 4 weeks exposure. Glucose and total protein showed the restoration after the depuration period in groups of fish exposed previously to over 60 and 120 mg Pb/kg, respectively. However, other parameters that changed during the exposure over 60 mg Pb/kg did not recovered. For enzymatic components in plasma, glutamic oxalate transminase (GOT), glutamic pyruvate transminase (GPT) and alkaline phosphatase (ALP) were significantly increased over 120 mg Pb/kg, and there was only restoration observed after the depuration for ALP over 120 mg Pb/kg.
Keywords: Platichthys stellatus, dietary lead(II) exposure, bioaccumulation, depuration, hematological parameters
3
1. Introduction
Lead (II) is globally one of the most ubiquitous metals in the environment, which is present naturally in water (0.02 µg/L and 2µg/L in sea and river, respectively) as well as in soils (16mg/kg) and air (0.1µg/m3 and 0.3 to 2 µg/m3 in rural and urban areas, respectively) (Labrot et al., 1999). It has been used in alloys with other metals, storage battery and a protection against radiation. In the recent, the usage of lead has exceeded the total amount that had been used in the previous era (Hsu and Guo, 2002). However, this active manufacturing has brought about severe lead contamination of the aquatic ecosystem, including soils and air. Generally, it is demonstrated that the lead can not only induce the disturb effects on hematological components, but also it accumulates large amounts of lead in the internal organs of fish, exposed to dietary exposed to that metal exposure (Javed, 2012). For example, neotropical fish (Hoplias malabaricus) exposed to inorganic lead showed the abnormal shapes, such as elongation and roundness of erythrocytes (Ribeiro et al., 2006). Also, Nussey et al, (1999) reported that there were the highest metal bioaccumulations in gill (direct contact with the environment) and liver (storage and detoxification) of moggel (Labeo umbratus). Bioaccumulation of heavy metals in organs of fish is normally affected by various biotic and abiotic elements. Generally, it is dependent on the physical factors such as temperature, pH, salinity and hardness (Kim and Kang, 2015a) as well as chemical properties such as water solubility, uptake rates and bioavailability (Bandowe et al, 2014). Heavy metals can enter into the body through main three possible ways (the body surface, the gills and the internal organs digestive tract). They are easily absorbed through the gills when their concentrations are high in water (Pourang, 1994). Meanwhile, the digestive organs can be a major route in case of food contamination, potentially resulting in the biomagnifications throughout the food chain (Amundsen et al., 1997). After penetration of heavy metals into the body, a variety of tissues show significantly different degree of accumulation. In this sense, liver and kidney are not higher, but maybe they are more sensitive to accumulation than gill, muscle and swim bladder (Chi et al., 2007). This bioaccumulation phenomenon in fish can be an important indicator, because it causes directly physiological distress. A problem of heavy metals accumulation 4
in fish is important because they may be transferred to the consumers in food chain, including humans as they occupy a range of trophic levels. Hematological analysis is a basic parameter for determination of fish health and stress in case of exposure to diverse toxicants, such as metals, pesticides and chemical industrial effluents in eco-toxicology (Singh et al., 2008). Generally, hematocrit (Ht), hemoglobin (Hb) concentration and red blood cell (RBC) counts are related to a general anemic state of fish (Kochhann et al, 2013). Also, there are specific enzymes, such as serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in liver that suggest acute exposure or damages of particular tissues. In addition, plasma organic (glucose and total protein) and inorganic (calcium and magnesium) components can be used indirectly used to evaluate indirectly stress and osmotic disturbance of fish, respectively. Teleost is generally regarded as good models to evaluate the toxicity of contaminants, because their biochemical and physiological responses are similar to those of mammals. Starry flounder (Platichthys stellatus) is a euryhaline species which can adapt to a broad range of salinity concentrations, and also one of the cold-water fishes which live in the cold oceans of Korea, Japan, Alaska and California as well as Sea of Okhotsk and Bering Sea. Recently, P. stellatus is attracting attention because of its high value and flavor throughout all field and farm in Korea. However, researches on heavy metals bioaccumulation and their influence on hematological parameters of P. stellatus of heavy metals are unusual were not performed until now in P. stellatus. Therefore, the aim of this study is to analyze the bioaccumulation in each organ and biochemical properties of blood of P. stellatus, exposed to dietary lead.
2. Material and methods
2.1 Experimental fish and conditions Sixty fish (mean length 20 ± 2 cm, mean weight 160.15 ± 15 g) were obtained from a fish farm in Gijang City, Korea, and they were divided into five 250L circular tanks (15 fish per each tank). The fish were acclimatized for 1 week under constant environmental conditions (Table 1), and there was no mortality of fish during the experiment in flow-through system. The fish were fed a commercial diet (Woosung feed, Daejeon City, Korea) by one percent of body weight twice a day before the experiment. Also, they were maintained on a 12-h: 12-h 5
light/dark cycle. Lead premix was composed of 1 gram of lead (II) nitrate (Sigma chemical Co., LTD) and 99 gram of cellulose, and then thoroughly mixed by feed composition (Table 2). Manufactured fodder was pelleted to avoid the release of lead by contamination into the water during dietary lead exposure. The dietary Pb concentrations were 0, 30, 60, 120, 240 mg/kg. Whole breeding period was 6 weeks, when the fish were fed a lead-bearing diet for 4 weeks and control diet for the rest of the period.
2.2 Bioaccumulation
Four fish at each concentration were anesthetized with benzocaine (Sigma Aldrich Co., USA) and dissected after 2, 4 and 6 weeks of the exposure. The tissue samples of brain, muscle, liver, spleen, kidney, intestine and gill of P. stellatus were performed with freeze-dried to measure dry weight of the samples. The dried samples were digested by wet digestion method in 65% (v/v) HNO3, and re-dried at 120oC. The procedure was repeated until total digestion. The entirely digested samples were diluted in 2% (v/v) HNO3.The samples were filtered through a membrane filter (AdvantecMFS, Inc. 0.2µm) under pressure for analysis. For determination of total lead concentrations, the digested and extracted solutions were analyzed by ELAN 6600DRC ICP-MS instrument with argon gas (Perkin-Elmer). Total lead concentrations were determined by external calibration, ICP multi-element standard solution VI (Merck). The lead bioaccumulation in tissue samples was expressed µg/g dry wt.
2.3 Hematological parameters
Blood samples were collected through the caudal vein of the fish with 1-mL disposable heparinized syringes to prevent blood coagulation. The blood samples were kept at 4oC until the blood parameters were completely analyzed. The total red blood cell (RBC) counts, hemoglobin (Hb) concentration, and hematocrit (Ht) value were determined immediately. Total RBC counts were counted using optical microscope with hemocytometer (Improved Neubauer, Germany) after diluted by Hendrick's diluting solution. The Hb concentration was determined using Cyan-methemoglobin technique (Asan Pharm. CO., Ltd.). The Ht value was determined by the micro-hematocrit centrifugation technique (Model; 01501, Hawkslyy and sons Ltd., England).
6
2.4 Plasma components
The blood samples were centrifuged to separate serum at 3000g, 4oC for 5min. The serum samples were analyzed for organic and inorganic substances, and enzyme activity using clinical kit (Asan Pharm. Co., Ltd.). For inorganic substances assay, calcium and magnesium were analyzed by the OCPC method (ocresolphthalein-complexon) and xylidyl blue –I technique, respectively. For organic substances assay, glucose and total protein were analyzed by GOD/POD technique and Biuret technique, respecively. Also, for enzyme activity assay, glutamic oxalate transaminase (GOT), glutamic pyruvate transaminase (GPT) and alkaline phosphatase (ALP) were analyzed by Kind-king method and Reitman-Frankel method, respectively.
2.5 Statistical analysis
Statistical analyses were performed using the SPSS/ PC + statistical package (SPSSInc, Chicago, IL, USA). Significant differences between groups were identified using one-way ANOVA and Duncan's test for multiple comparisons or Student's t-test for two groups (Duncan, 1955). The significance level was set at P < 0.05.
3. Results
3.1 Bioaccumulation
Lead accumulations in brain, muscle, liver, spleen, kidney, intestine and gill of S P. stellatus are shown in figure 1. The significant changes were observed at 240 mg Pb/kg after 2 weeks (control; 1.55±0.19 µg/g, 30 mg/kg; 1.50±0.14 µg/g, 60mg/kg; 1.59±0.14 µg/g, 120 mg/kg;1.63±0.14 µg/g, 240 mg/kg; 1.85±0.13 µg/g) and 4 weeks (control; 1.56±0.14 µg/g, 30 mg/kg; 1.58±0.13 µg/g, 60 mg/kg; 1.75±0.16 µg/g, 120 mg/kg; 1.81±0.15 µg/g, 240 mg/kg; 2.02±0.12 µg/g) in the brain. Relatively, there was only accumulation after 4 weeks (control; 3.22±0.53 µg/g, 30 mg/kg; 3.57± 0.41 µg/g, 60 mg/kg; 3.67±0.27 µg/g, 120 mg/kg; 3.43±0.31 µg/g, 240 mg/kg; 3.83±0.48 µg/g) in the muscle. On the other hand, the lead concentration was considerably increased over 120 mg Pb/kg after 2 weeks (control; 7
1.84±0.32 µg/g, 30 mg/kg; 2.32± 0.31 µg/g, 60 mg/kg; 4.60±0.32 µg/g, 120 mg/kg; 8.20±0.74 µg/g, 240 mg/kg; 11.08±1.04 µg/g), 30 mg Pb/kg after 4 weeks (control; 1.24±0.32 µg/g, 30 mg/kg; 3.45± 0.31 µg/g, 60 mg/kg; 6.06±0.32 µg/g, 120 mg/kg; 10.14±0.74 µg/g, 240 mg/kg; 12.88±1.04 µg/g) and 120 mg Pb/kg after the depuration period (control; 1.72±0.31 µg/g, 30 mg/kg; 2.01± 0.32 µg/g, 60 mg/kg; 2.64±0.33 µg/g, 120 mg/kg; 4.68±0.31 µg/g, 240 mg/kg; 8.56±0.53 µg/g) in the spleen. Also, there were similar changes over 60 mg Pb/kg after 2 weeks (control; 1.99±0.43 µg/g, 30 mg/kg; 3.98± 1.08 µg/g, 60 mg/kg; 9.47±1.31 µg/g, 120 mg/kg; 16.08±1.25 µg/g, 240 mg/kg; 24.86±2.17 µg/g), 30 mg Pb/kg after 4 weeks (control; 3.03±1.61 µg/g, 30 mg/kg; 8.85± 1.01 µg/g, 60 mg/kg; 12.75±1.48 µg/g, 120 mg/kg; 20.09±2.25 µg/g, 240 mg/kg; 30.28±2.25 µg/g) and 60 mg Pb/kg after the depuration period (control; 2.05±1.04 µg/g, 30 mg/kg; 4.19± 0.86 µg/g, 60 mg/kg; 8.16±1.27 µg/g, 120 mg/kg; 14.69±1.81 µg/g, 240 mg/kg; 20.93±2.42 µg/g) in the intestine. The gill showed the notable increases over 60 mg Pb/kg after 2 weeks (control; 1.73±0.27 µg/g, 30 mg/kg; 3.02± 0.55 µg/g, 60 mg/kg; 4.76±0.94 µg/g, 120 mg/kg; 7.85±1.66 µg/g, 240 mg/kg; 10.23±1.78 µg/g), 30 mg Pb/kg after 4 weeks (control; 1.78±0.50 µg/g, 30 mg/kg; 4.31± 0.35 µg/g, 60 mg/kg; 6.33±1.53 µg/g, 120 mg/kg; 9.23±2.15 µg/g, 240 mg/kg; 12.89±2.08 µg/g) and 60 mg Pb/kg after the depuration period (control; 1.09±0.20 µg/g, 30 mg/kg; 1.54± 0.26 µg/g, 60 mg/kg; 3.11±0.60 µg/g, 120 mg/kg; 4.02±0.40 µg/g, 240 mg/kg; 6.32±0.68 µg/g). The lead accumulation in the kidney was significantly increased over 60 mg Pb/kg after 2 weeks (control; 3.77±0.47 µg/g, 30 mg/kg; 6.09± 1.89 µg/g, 60 mg/kg; 8.37±1.53 µg/g, 120 mg/kg; 10.79±2.36 µg/g, 240 mg/kg; 20.59±2.49 µg/g), 30 mg Pb/kg after 4 weeks (control; 4.98±0.96 µg/g, 30 mg/kg; 6.38± 2.72 µg/g, 60 mg/kg; 11.26±3.25 µg/g, 120 mg/kg; 18.84±3.84 µg/g, 240 mg/kg; 25.50±2.47 µg/g) and 30 mg Pb/kg after the depuration period (control; 4.26±1.36 µg/g, 30 mg/kg; 5.99± 0.99 µg/g, 60 mg/kg; 7.35±1.14 µg/g, 120 mg/kg; 10.03±1.39 µg/g, 240 mg/kg; 14.99±3.36 µg/g). The liver showed a notable change over 120 mg/kg after 2 weeks (control; 1.57±0.37 µg/g, 30 mg/kg; 4.01± 0.97 µg/g, 60 mg/kg; 7.01±1.09 µg/g, 120 mg/kg; 13.05±0.75 µg/g, 240 mg/kg; 20.03±1.73 µg/g), 4 weeks (control; 1.32±0.58 µg/g, 30 mg/kg; 6.27± 0.55 µg/g, 60 mg/kg; 10.02±2.34 µg/g, 120 mg/kg; 18.37±1.95 µg/g, 240 mg/kg; 28.14±1.08 µg/g) and the depuration period (control; 0.94±0.28 µg/g, 30 mg/kg; 3.45± 1.02 µg/g, 60 mg/kg; 7.02±1.19 µg/g, 120 mg/kg; 10.04±1.27 µg/g, 240 mg/kg; 18.12±1.22 µg/g). The depuration rates at 240 mg Pb/kg (4 weeks to 6 weeks) was 85% in the brain, 79.1% in the muscle, 69.1% in the intestine, 66.5% in the spleen, 64.4% in the liver, 58.8% in the kidney and 49.1% in the gill
8
3.2 Hematological parameters
Changes of total RBC counts, hematocrit values and hemoglobin concentrations in P. stellatus are shown in Table 3. The RBC counts were significantly decreased over 120 60 mg Pb/kg after 2 weeks, at 240 over 120 mg Pb/kg after 4 weeks and over 120 mg/kg after the depuration period. The notable decreases in hematocrit value were observed at 240 mg Pb/kg after 2 weeks, and over 120 mg Pb/kg after 4 weeks and at 240 mg Pb/kg after the depuration period. Hemoglobin concentrations were significantly decreased at 240 mg/kg after 2 weeks and over 120 mg Pb/kg after 4 weeks of exposure, and over 120 mg/kg as well as after the depuration period.
3.3 Plasma components
Alterations in plasma components are demonstrated in Table 4. Calcium and magnesium levels in plasma were significantly decreased over 120 mg Pb/kg and over 60 mg Pb/kg respectively after 2 weeks, over 60 mg Pb/kg after 4 weeks and at 240 mg Pb/kg after the depuration period. For blood glucose levels, there were notable increases over 60 mg/kg after 2 weeks and 4 weeks. After the depuration period, glucose level returned to the baseline level, as at 60 mg Pb/kg, at 120 mg Pb/kg and at 240 mg Pb/kg exposure. Also, blood total protein levels were considerably decreased over 120 mg/kg after 4 weeks and at 240 mg/kg after the depuration period. GOT and GPT in plasma showed significant increases at 240 mg/kg after 2 weeks, over 120 mg/kg after 4 weeks and at 240 mg/kg after the depuration period. Also, there were considerable increases of ALP level over 60 mg Pb/kg after 2 weeks and over 120 mg Pb/kg after 4 weeks of Pb exposure, and effective restoration after 2 weeks of depuration period.
4. Discussion
The study to assess the bioaccumulation patterns in fish tissues under the metal exposure is considered as a reliable and sensitive indicator of environmental metal contamination (Kim and Kang, 2015b). In this study, the heavy metal accumulation in the tissues of P. stellatus was observed in the order of this follows; intestine > kidney > liver > spleen > gill > muscle > brain after 2 weeks, and intestine > kidney > liver > spleen ≒ gill > muscle > brain after 4 weeks. 9
In this study, the significant accumulation was observed over 60 mg/kg in the intestine of P. stellatus. Generally, dietary contamination is a major cause of natural bioaccumulation in fish, because they have the high status in the food chain, and intestine has an important role in this process (Dural et al., 2007). Alves et al. (2006) reported the high accumulation in the intestine of juvenile freshwater rainbow trout (Oncorhynchus mykiss), exposed to dietary lead. Also, it was demonstrated that dietary cadmium caused the highest accumulation in the intestine of juvenile rockfish (Sebastes schlegeli) (Kim et al., 2006). It might be assumed that heavy metals in food were remained and accumulated on the wall of intestine, because of unfiltered by mucous (Glover and Hogstrand, 2002). Also, the kidney, liver and spleen indicated the significant degree of bioaccumulation over 60 mg/kg in P. stellatus (Figure 1). Łuszczek-Trojnar (2013) explained that the kidney showed the high accumulation levels in Prussian carp (Carassius gibelio), exposed to lead. Also, there was a significant bioaccumulation occurred in the liver of Atalantic salmon (Salmo salar L.) after three months exposure to dietary arsenobetaine (Amlund et al., 2006). It may be explained that these organs are commonly known as actively metabolic organs through their role in detoxification and excretion of toxicants from the body, and that is why large amounts of heavy metals released from other tissues during the detoxification process may be accumulated again in organs involved with detoxification. Furthermore, comparatively a high level of the accumulation was observed over 60 mg/kg in the gill of P. stellatus. Tao et al. (1999) reported that Carassius auratus exposed to lead showed the significant accumulation and adherence on surface of gill. Hollis et al. (1999) suggested that the gill metal burden continuously increased according to the longer metal exposure, which was due to its metal binding properties. In general, the gill has a key role for the migration of heavy metals as well as the ion exchange, pH control and excretion of nitrogenous compounds, when exposed to waterborne metals rather than dietary condition. Gill can be a major site of accumulation by dietary exposure, because the absorbed substances from intestine are entered into the side of gill epithelium (Szebedinszky et al., 2001). The lower accumulation was observed in the brain and muscle of P. stellatus compared to the other tissues. Tulasi et al. (1992) suggested that the brain was the lower accumulation than other organs such as kidney, gill, and liver of freshwater fish, Anabas testudineus exposed to the lead exposure. Jarić et al. (2011) reported that there were the lowest levels of metal accumulation occurred in muscles among analyzed organs of starlet (Acipenser ruthenus) from the Danube River in Serbia. Also, concentrations of heavy metals in muscle of 10
Leuciscus cephalus were lower than in gills (Demirak et al., 2006). For this result, Conto Cinier et al. (1999) suggested that it was because most of the metals including lead were accumulated in the active metabolic organs, such as liver and kidney, and this is the reason of lower metals concentration in muscle. However, the problem of heavy metals accumulation in this tissue is important because the muscle, as the edible part of fish, in case of contamination pose the serious risk for human as a consumer. Fish normally eliminates heavy metals from the body through the gill, mucus, bile and urine etc. after the biotransformation. The depuration rate was in the orders as follows; gill > liver > kidney ≥ spleen ≈ intestine, but it failed in complete recovery as normal status. On the other hand, brain and muscle showed the recoveries in the concentration of 240 mg/kg in P. stellatus. The higher level of lead accumulation in some tissues may need more time to eliminate more lead amounts. The results obtained in other research confirm the effects observed in the present work. For instance, the lead was excreted quickly in the muscle of Chinese sturgeon (Acipenser sinensis) (Hou et al., 2011). Also, Luszczek-Trojnar et al. (2013) announced that Prussian carp (Carassius gibelio) exposed to dietary lead showed more rapid elimination of that metal from muscle and from the intestine. The hematological parameters have been widely used to evaluate the toxic stress induced by environmental contamination (Kim and Kang, 2014). Many authors reported the effects of heavy metals on hematological parameters in various fish species such as in Hoplias malabaricus (Ribeiro et al., 2006), Clarias batrachus L. (Maheswaran et al., 2008) and Cyprinus carpio (Kandemir et al., 2010) etc. The hematological parameters of P. stellatus were decreased over 60 mg Pb/kg during the exposure period. It is thought that heavy metals increased the membrane permeability and caused the edema in red blood cells (Witeska and Kosciuk, 2003), or they suppressed the activation of hematopoietic organs (Vinodhini and Narayanan, 2009). Due to these reasons, hematological properties can be altered negatively, and anaemia can be triggered (Lohner et al., 2001). The reason to be insufficient recoveries may be due to the toxic effects on the hematopoietic organs by the accumulation. Toxicants in the aquatic environment significantly change chemical responses, due to their potential cellular and molecular toxicities, so biochemical plasma analysis is one of the most important parameters (Vutukuru, 2003). In the present study, calcium and magnesium levels of P. stellatus were considerably decreased over 60 mg/kg. MacDonald et al. (2002) explained that the lead competes with these ions in calcium-magnesium ATPase, and has higher affinity to this pump system. It is thought that the lead was not eliminated completely in from the 11
body, and then it has had a consistently bad deleterious effect on the osmoregulation. Glucose is used as a sensitive parameter for the stress in fish. The glucose levels of P. stellatus were increased over 60 mg Pb/kg during the exposure period reaching the level 123.7 mg/dL (Table 4). This result would be also compared to the results obtained in similar research presented by Kim and Kang 2015b. Cicik and Engin (2005) announced that glucose levels were increased in common carp (Cyprinus carpio), exposed to cadmium. This reaction could be explained as the result of elevated metabolic rate in the body and the gluconeogenesis by the stress (Saravanan et al., 2011). On the other hand, total protein which is an indicator of metabolism activation was significantly decreased after 4 weeks of exposure P. stellatus to over 120 mg/kg. Martinez et al. (2004) demonstrated that the neotropical fish (Prochilodus lineatus) exposed to the lead showed the decreased total protein values due to its toxicity to the metabolism. After the exposure, organic components represented the recoveries over 60 and 120 mg/kg, respectively. GOT, GPT, ALP levels of P. stellatus were increased over 120 mg/kg. The results were similar in the previous research (Kim and Kang, 2015b). Generally, these enzymes are excreted in the liver, and are present increased levels when organism is exposed to toxicants and other stress. Also, it was possibly announced that histopathological degeneration and necrosis could give rise to the alteration in cell membrane, the leakage of intracellular enzymes and the collapse of transportation system (Humtsoe et al., 2007). After the exposure, substantial recoveries were not observed over 120 mg/kg, except for that of ALP. It suggests that histological damages would last even after the depuration period. In conclusion, the active organs such as intestine, kidney, liver, spleen, and gill observed significant accumulations, and the most depuration was the brain. The hematological parameters were significantly changed for each component. Considering the above results, the exposure to the dietary lead exposure over 60 mg/kg can negatively affect the experimental fish, P. stellatus, and deleterious effects of that exposure can last until the depuration period.
12
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Dural, M., Göksu, M.Z.L., Özak, A.A., 2007. Investigation of heavy metal levels in economically important fish species captured from the Tuzla lagoon. Food Chemistry 102, 415-421.
Glover, C.N., Hogstrand, C., 2002. In vivo characterisation of intestinal zinc uptake in freshwater rainbow trout. Journal of Experimental Biology 205, 141-150.
Hollis, L., McGeer, J.C., McDonald, D.G., Wood, C.M., 1999. Cadmium accumulation, gill Cd binding, acclimation, and physiological effects during long term sublethal Cd exposure in rainbow trout. Aquatic Toxicology 46, 101-119.
Hou, J. L., Zhuang, P., Zhang, L. Z., Feng, L., Zhang, T., Liu, J. Y., Feng, G. P., 2011. Morphological deformities and recovery, accumulation and elimination of lead in body tissues of Chinese sturgeon, Acipenser sinensis, early life stages: a laboratory study. Journal of Applied Ichthyology 27, 514–519.
Hsu, P. C., Guo, Y. L., 2002. Antioxidant nutrients and lead toxicity. Toxicology 180, 33–44.
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Jarić, I., Višnjić-Jeftić, Z., Cvijanović, G., Gačić, Z., Jovanović, L., Skorić, S., Lenhardt, M., 2011. Determination of differential heavy metal and trace element accumulation in liver, gills, intestine and muscle of sterlet (Acipenser ruthenus) from the Danube River in Serbia by ICP-OES. Microchemical Journal 98, 77–81.
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Kim, J.H., Kang, J.C., 2014. The selenium accumulation and its effect on growth, and haematological parameters in red sea bream, Pagrus major, exposed to waterborne selenium. Ecotoxicology and Environmental Safety 104, 96-102.
Kim, J. H., Kang, J. C., 2015a. The arsenic accumulation and its effect onoxidative stress responses in juvenile rockfish, Sebastes schlegelii, exposed to waterborne arsenic (As3+). Environmental Toxicology and Pharmacology 39, 668-676.
Kim, J.H., Kang, J.C., 2015b. The lead accumulation and hematological findings in juvenile rock fish Sebastes schlegelii exposed to the dietary lead (II) concentrations. Ecotoxicology and Environmental Safety 115, 3339.
Kim, S. G., Eom, K. H., Kim, S. S., Jin, H. G., Kang, J. C., 2006. Kinetics of Cd accumulation and elimination in tissues of juvenile rockfish (Sebastes schlegeli) exposed to dietary Cd. Marine Environmental Research 62, 327–340
Kochhann, D., Brust, S.M.D.A., Domingos, F.X.Y., Val, A.L., 2013. Linking Hematological, Biochemical, Genotoxic, and Behavioral Responses to Crude Oil in the Amazon Fish Colossoma macropomum (Cuvier, 1816). Archives of Environmental Contamination and Toxicology 65, 266-275.
Labrot, F., Narbonne, J.F., Ville, P., Saint Denis, M., Ribera, D., 1999. Acute Toxicity, Toxicokinetics, and Tissue Target of Lead and Uranium in the Clam Corbicula fluminea and the Worm Eisenia fetida: Comparison with the Fish Brachydanio rerio. Archives of Environmental Contamination and Toxicology 36, 167–178.
Lohner, T.W., Reash, R.J., Willet, V.E., Rose, L.A., 2001. Assessment of tolerant Sunfish populations (Lepomis sp.) inhabiting selenium-laden coal ash effluents: 1. Hematological and population level assessment. Ecotoxicology and Environmental Safety 50, 203-216.
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MacDonald, A., Silk, L., Schwartz, M., Playle, R.C., 2002. A lead–gill binding model to predict acute lead toxicity to rainbow trout (Oncorhynchus mykiss). Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 133, 227-242.
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Martinez, C. B. R., Nagae, M. Y., Zaia, C. T. B. V., Zaia, D. A. M., 2004. Acute morphological and physiological effects of lead in the neotropical fish, Prochilodus lineatus. Brazilian Journal of Biology 64, 797-807.
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Szebedinszky, C., McGeer, J.C., McDonald, D.G., Wood, C.M., 2001. Effects of chronic Cd exposure via the diet or water on internal organ-specific distribution and subsequent gill Cd uptake kinetics in juvenile rainbow trout (Oncorhynchus mykiss). Environmental Toxicology and Chemistry 20, 597–607.
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Vinidhini, R., Narayanan, M., 2008. Bioaccumulation of heavy metals in organs of fresh water fish Cyprinus carpio (Common carp). International Journal of Environmental Science & Technology 5, 179-182.
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Vutukuru, S.S., 2003. Chromium induced alterations in some biochemical profiles of the Indian major carp, Labeo rohita (Hamilton). Bulletin of environmental contamination and toxicology 70, 118-123.
Witeska, M., Kościuk, B., 2003. The changes in common carp blood after short-term zinc exposure. Environmental Science and Pollution Research 10, 284-286.
17
Brain exposure
depuration b
b
2.0 a
a
ab
ab
ab ab a a a
a a
a a
1.5
1.0
6
Muscle
0 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg 240 mg/kg
exposure
depuration b
a 4
a
a a
a
ab a a a
a
a a a a
2
30
Pb concentration (g/g)
Pb concentration (g/g)
2.5
8
0 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg 240 mg/kg
Pb concentration (g/g)
3.0
4W
6W
2W
4W
Weeks
Spleen 30
e
exposure
depuration
d
12
d
10
c
c
8 c 6
b
b b
4 2
a
a
a a
a
0 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg 240 mg/kg
Pb concentration (g/g)
16 14
0 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg 240 mg/kg
c
e
c b b
d c
b
a
6W
4W
a
6W
30
Intestine
0 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg 240 mg/kg
d exposure
depuration
d d
c 20 c
c b
b
b
10 a
b a
a a
a
a 0
2W
4W
Weeks
6W
2W
4W
6W
Weeks
Gill d depuration
d
10
a
a
b
a
exposure
12
c
c b
8
c b
6
b
b b
a
4 2
depuration
20
10
40
Kidney d
0
4W
ab a
Weeks
exposure
Weeks
18
b a
ab
2W
d
a
0 2W
b ab
10
6W
Pb concentration (g/g)
14
0 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg 240 mg/kg
c a
15
Weeks
Pb concentration (g/g)
Pb concentration (g/g)
16
depuration b
0
0 2W
exposure c
20
5
0.0
Liver b
a
0.5
18
25
0 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg 240 mg/kg
a
a
a
a
0 2W
4W
6W
Weeks
Fig. 1 Pb concentration in brain, muscle, liver, spleen, kidney, intestine and gill of starry flounder (Platichthys stellatus) exposed to the different concentrations of dietary lead for 4 weeks, followed by a depuration period of 2 weeks. Vertical bar denotes a standard error. Values with different superscript are significantly different (P < 0.05) as determined by Duncan’s multiple range test.
18
Table 1 The chemical components of seawater and experimental condition used in the experiments. Item Value Temperature (oC)
13.0±0.8
pH
8.1±0.4
Salinity (‰)
32.2±0.8
Dissolved Oxygen (mg/L)
7.1±0.3
Chemical Oxygen Demand (μg/L)
1.24±0.2
Ammonia(mg/L)
11.9±0.9
Nitrite (mg/L)
1.3±0.2
Nitrate (mg/L)
11.31±1.0
19
Table 2 Formulation of the experimental diet (% dry matter). Lead concentration (mg/kg) Ingredient (%)
Casein1 2
Fish meal
Wheat flour
3
4
Fish oil
Cellulose
1
Corn starch
3
30
60
120
240
33.0
33.0
33.0
33.0
33.0
23.0
23.0
23.0
23.0
23.0
20.0
20.0
20.0
20.0
20.0
10.0
10.0
10.0
10.0
10.0
5.0
4.7
4.4
3.8
2.6
5.0
5.0
5.0
5.0
5.0
5
2.0
2.0
2.0
2.0
2.0
6
Vitamin premix Mineral premix
0
2.0
2.0
2.0
2.0
2.0
7
0.0
0.3
0.6
1.2
2.4
Actual lead concentration
1.8
31.4
62.1
118.8
244.5
Lead premix
1. United States Biochemical (Cleveland, OH). 2. Suhyup Feed Co., Ltd., Gyeong Nam Province, Korea. 3. Young Nam Flour Mills Co., Pusan, Korea. 4. Sigma Chemical Co., St. Louis, MO. 5. Vitamin premix (mg/kg diet): ascorbic acid, 240; dl-calcium pantothenate, 400; Choinechloride, 200; inositol, 20; menadione, 2; nicotinamide, 60; pyridoxine ∙HCl, 44; riboflavin, 36; thiamine mononitrate, 120; dl-a-tocopherolacetate, 60; retinyl acetate, 20,000IU; biotin, 0.04; folic acid, 6; vitamin B12, 0.04 ; and cholecalcifero, 4000IU. 6. Mineral premix (mg/kg diet): Al, 1.2; Ca, 5000; Cl, 100; Cu, 5.1; Co, 9.9; Na, 1280; Mg, 520; P, 5000; K, 4300; Zn, 27; Fe, 40; I, 4.6; Se, 0.2; and Mn, 9.1. 7. Lead premix (mg/kg diet): 10,000mg Pb/kg diet.
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Table 3 Changes of RBC count, hematocrit value and hemoglobin concentration in starry flounder, Platichthys stellatus exposed to the different concentrations of dietary lead for 4 weeks, followed by a depuration period of 2 weeks. Parameters
RBC count (×104mm3)
Hematocrit (%)
Hemoglobin (g/dL)
Period
Lead concentration (mg/kg)
(weeks)
0
30
60
120
240
2
263.33±10.46a
262.67±10.9ab
242.38±10.02bc
230.67±10.13c
194±15.1c
4
266.5±5.98a
256±12.54a
235±15.54ab
204.5±9.98bc
190±17.44c
6
263.33±8.77a
253.33±14.69a
255.34±10.03a
208±11.78b
198±12.97b
2
30.5±1.73a
29.25±2.36a
29±4.58a
22.67±4.73ab
19±3.46b
4
30.66±4.16a
24.67±3.79ab
25.33±3.06bc
22.67±3.31bc
19.33±2.59c
6
27.33±5.52a
30.33±5.00a
25±3.12bc
24.33±2.58bc
20.32±2.52c
2
6.09±0.23a
5.85±0.32ab
5.72±0.50ab
5.55±0.57ab
5.36±0.3b
4
5.95±0.14a
6.08±0.28a
5.60±0.32ab
5.29±0.32bc
4.83±0.23c
6
5.82±0.05a
5.87±0.27a
5.64±0.11a
5.23±0.25b
5.26±0.24b
Values are mean±S.E. Values with different superscript are significantly different (P ＜0.05) as determined by Duncan's multiple range test.
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Table 4 Changes of serum parameters in starry flounder, Platichthys stellatus exposed to the different concentrations of dietary lead for 4 weeks, followed by the depuration period of 2 weeks. Period
Lead concentration (mg/kg)
(weeks)
0
30
60
120
240
2
8.82±0.76a
8.73±0.20a
8.23±0.32a
7.45±0.21b
5.56±0.18c
4
9.44±0.67a
8.63±0.20a
6.03±0.35b
4.44±0.21c
3.69±0.48c
6
9.58±0.40a
9.60±0.42a
9.21±0.91a
9.06±0.29a
8.39±0.17b
2
3.94±0.23a
3.86±0.28ab
3.61±0.18bc
3.49±0.21c
3.38±0.15c
4
3.82±0.21a
3.80±0.02a
3.52±0.02b
3.38±0.12c
2.89±0.21d
6
3.72±0.02a
3.76±0.06a
3.65±0.06a
3.62±0.03a
3.43±0.08b
2
96.11±3.92a
96.85±5.63a
104.38±4.70b
107.63±4.10b
110.16±4.99b
4
96.32±4.48a
95.92±4.46a
108.16±5.38b
119.92±5.69c
123.70±4.68c
6
97.05±4.44a
95.92±4.46a
99.16±5.38a
98.92±5.69a
100.70±4.68a
Total
2
2.81±0.18a
3.08±0.17a
2.80±0.10a
2.85±0.16a
2.89±0.11a
protein
4
2.96±0.21a
3.08±0.18a
3.00±0.12a
2.54±0.13b
2.05±0.11c
(g/dL)
6
2.71±0.19a
2.72±0.10a
2.80±0.14a
2.65±0.15a
2.75±0.14b
GOT
2
22.33±5.46a
25.07±2.75ab
26.25±2.76ab
28.45±3.74ab
30.92±4.66b
(karmen
4
21.70±2.03a
24.00±1.18a
25.58±2.57ab
29.20±0.81b
32.85±3.13c
unit)
6
22.48±2.64a
23.78±3.69a
22.27±1.84a
24.75±4.58a
29.57±4.87b
GPT
2
15.23±1.74a
14.09±0.52ab
16.69±2.39ab
17.28±2.42ab
21.96±1.77b
(karmen
4
14.58±1.64a
20.23±2.27ab
19.60±0.67ab
22.69±1.47b
23.91±1.61b
unit)
6
15.35±1.42a
17.72±3.44a
16.01±0.82a
16.07±0.08a
23.88±1.61b
2
4.32±0.11a
4.58±0.09ab
4.79±0.08bc
4.74±0.12bc
4.99±0.28c
4
4.14±0.15a
4.31±0.02ab
4.30±0.10ab
4.59±0.05bc
4.69±0.14c
6
4.24±0.11a
4.18±0.19a
4.26±0.18a
4.26±0.19a
4.18±0.10a
Parameters
Calcium (mg/dL)
Magnesium (mg/dL)
Glucose (mg/dL)
ALP (K-A)
Values are mean±S.E. Values with different superscript are significantly different (P ＜0.05) as determined by Duncan's multiple range test.
22