Accepted Manuscript
Toxic effects on bioaccumulation and hematological parameters of juvenile
rockfish Sebastes schlegelii exposed to dietary lead (Pb) and ascorbic acid
Jun-Hwan Kim, Ju-Chan Kang
PII:
S0045-6535(17)30285-0
DOI:
10.1016/j.chemosphere.2017.02.097
Reference:
CHEM 18860
To appear in:
Chemosphere
Received Date:
27 January 2017
Revised Date:
17 February 2017
Accepted Date:
19 February 2017
Please cite this article as: Jun-Hwan Kim, Ju-Chan Kang, Toxic effects on bioaccumulation and
hematological parameters of juvenile rockfish Sebastes schlegelii exposed to dietary lead (Pb) and
ascorbic acid, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.02.097
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ACCEPTED MANUSCRIPT
Highlights

Exposure to dietary Pb induced significant bioaccumulations in specific tissues.

Hematological parameters were affected by dietary Pb exposure.

Growth performance was decreased by dietary Pb exposure.

High levels of AsA supplementation were effective to attenuate the Pb-induced toxic effects.
ACCEPTED MANUSCRIPT
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Toxic effects on bioaccumulation and hematological parameters of juvenile rockfish Sebastes
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schlegelii exposed to dietary lead (Pb) and ascorbic acid
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Jun-Hwan Kim1 and Ju-Chan Kang2*
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1West
Sea Fisheries Research Institute, National Institute of Fisheries Science, Incheon
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22383, Korea
2Department
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of Aquatic Life Medicine, Pukyong National University, Busan 48513, Korea
*Corresponding
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Author: Ju-Chan Kang, Tel: +82 51 629 5944
Fax: +82 51 629 5938, E-mail: jckang@pknu.ac.kr
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Abstract
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Juvenile rockfish, Sebastes schlegelii (mean length 11.3±1.2 cm, and mean weight 32.5±4.1 g) were exposed for
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four weeks to dietary lead (Pb2+) at 0, 120, and 240 mg/L and ascorbic acid (AsA) at 100, 200, and 400 mg/L.
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The exposure concentrations and duration of significant Pb-induced accumulations in specific tissues of S.
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schlegelii were assessed. High levels of ascorbic acid significantly attenuated accumulations following exposure
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to dietary Pb. Dietary Pb exposure caused a significant increase in blood Pb concentrations, whereas red blood
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cell (RBC) count, hematocrit, and hemoglobin were significantly decreased. Notable changes were also
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observed in plasma calcium, magnesium, glucose, cholesterol, glutamic oxaloacetic transaminase (GOT), and
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glutamic pyruvate transaminase (GPT). The growth performance of S. schlegelii was significantly decreased,
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whereas lysozyme activity was significantly increased. High doses AsA supplemention were effective in
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attenuating the changes brought about by dietary Pb exposure.
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Key words: Rockfish, Pb, Ascorbic acid, Bioaccumulation, Hematological parameters
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1. Introduction
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Exposure to heavy metals in the marine environment is a crucial environmental issue, because these metals can
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easily accumulate in humans through the consumption of marine products such as fish, shrimp, and shellfish,
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thereby creating a health risk. Among the various heavy metals, lead (Pb) is one of the most toxic. Even at low
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doses of exposure Pb can be highly toxic to aquatic organisms.
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Fish can accumulate a lot of metal due to its higher levels of the food-web, and the accumulation patterns in fish
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rely on uptake and elimination rates (Squadrone et al., 2013a). Pb exposure in the aquatic environment also
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induces accumulation of harmful substances in specific tissues of aquatic animals, and it is critical to conduct
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research on bioaccumulation in fish tissues following metal exposure, in contrasted to biotransformation and
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excretion (Cicik et al., 2004). Metal accumulation in fish tissues depends on the type of metal, exposure
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concentration and period, the temperature, salinity, and hardness of the water, as well as the species, age, and
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metabolic activity of the fish (Allen, 1995; Pelgrom et al., 1995a). Rabitto et al. (2005) reported considerable
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metal accumulation in specific tissues of fish, as evidenced by different physiological processes that depend on
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proper tissue functions. Monitoring fish tissues such as metal accumulation patterns should be a reliable and
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good indicator to assess contamination in aquatic environment (Squadrone et al., 2013b).
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Hematological parameters of fish have been shown to be sensitive and reliable indicators of the physiological
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status of aquatic animals under stress due to metal exposure, owing to the direct interface between fish blood
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and the external environment (Cazenave et al., 2005; Kim and Kang, 2014). Blood parameters have also been
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considered as pathophysiological indicators for diagnosing the structural and functional status of fish exposed to
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toxicants (Maheswaran et al., 2008). Jacob et al. (2000) suggested that Pb exposure results in damage to the
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blood system by interference in heme and hemoglobin syntheses and altering erythrocyte morphology, which
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leads to anemia and depleted hematocrit.
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The toxicity exposure causes a disturbance of the homeostasis in aquatic animals that leads to a reallocation of
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energy resources from growth to compensatory, adaptive, and pathological processes (Knops et al., 2001).
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Previous studies have found that the exposure to metals causes reductions in growth rate of aquatic animals and
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that the toxic effects on growth performance occur in a dose-dependent manner (Campbell et al., 2002;
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Clearwater et al., 2002; Shaw and Handy, 2006).
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Among various essential nutrients, ascorbic acid (AsA) is a critical nutrient for growth and development,
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metabolic function, and eliciting an immune response in aquatic animals (Kim and Kang, 2015). AsA
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supplementation may protect most animals from the metal-induced harmful effects such as reduced growth rate,
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alterations in blood hematology, and changes in plasma biochemical components, in addition to lipid
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peroxidation, free radicals generation, and neurotoxicity (Grosicki, 2004; Yousef, 2004). Many studies have
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indicated that AsA chelates Pb, thereby reducing Pb levels in tissues (Dalley et al., 1990; West et al., 1994;
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Tandon et al., 2001). AsA inhibits absorption of Pb by decreasing ferric iron to ferrous iron in the duodenum.
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Ferrous iron then competes with Pb for intestinal absorption (Patrick, 2006). In addition to the effects of AsA on
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metal accumulation, AsA supplementation also significantly reduces levels of stress-induced cortisol and other
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stress indicators in animals (O’Keefe et al., 1999; Brody et al., 2002).
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In South Korea, rockfish, Sebastes schlegelii, is a commonly cultured species in marine net cages, owing to its
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high demand, favorable flesh quality, and rapid growth. However, toxicological studies for dietary Pb exposure
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have been scarce. In addition, insufficient studies have been conducted about the effects of ascorbic acid
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supplementation on Pb toxicity. Therefore, the purpose of the present study was to evaluate Pb accumulation in
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specific tissues and the hematological parameters of experimental fish, and assess the effects of AsA on Pb
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exposure.
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2. Materials and methods
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2.1. Experimental fish and conditions
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Juvenile rockfish, S. schlegelii, were obtained from a local fish farm in Tongyeong, Korea. Fish were
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acclimatized for 2 weeks under laboratory conditions. During the acclimation period, fish were fed a Pb-free
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diet twice daily and experimental conditions were constantly maintained at all times (temperature 19.0±1.0 °C,
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pH 8.1±0.5, salinity 33.2±0.5 ‰, dissolved oxygen 7.1±0.3 mg/L, chemical oxygen demand 1.15±0.21).
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Healthy 90 fish (mean length, 11.3±1.2 cm; mean weight, 32.5±4.1 g) were selected to conduct this study. After
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exposure experiment, diets containing dietary Pb and AsA were given at a rate of 2% body weight daily (as two
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1% meals per day). At the end of each period (at 2 and 4 weeks), fish were anesthetized in buffered 3-
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aminobenzoic acid ethyl ester methanesulfonate to collect the blood and tissues of S. schlegelii. All
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experimental animals used in this study were maintained under a protocol approved by the Institutional Animal
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Care and Use Committee of the Pukyong National University.
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2.2. Feed ingredients and diets formulation
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Composition of the diets is demonstrated in Table 1. The concentrations of Pb were 0, 120, and 240 mg/kg, and
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the concentrations of AsA were 100, 200, and 400 mg/kg. In Korea, the Pb concentrations in the coastal
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sediment reached up to 92 mg kg-1 (Lim et al., 2007). Although the Pb concentrations are much higher than
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inhabited environment, this experiment can suggest the toxic effects of dietary Pb exposure. The 9 concentration
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groups were set up using the above concentrations of Pb and AsA, and the concentrations were matched using
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Pb premix (20,000 mg/kg) and AsA premix (20,000 mg/kg). After making the diets containing respective Pb
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and AsA concentrations, the actual Pb and AsA concentrations were analyzed using ICP-MS (ELAN 6600DRC)
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and HPLC (Agilent 1200 series) (Table 2).
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2.3. Pb accumulation
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Tissue samples of liver, kidney, spleen, intestine, gill, and muscle of S. schlegelii were performed with freeze-
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dried to measure dry weight of the samples. The freeze-drying samples were digested by wet digestion method
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(Arain et al., 2008). For determination of total Pb concentrations, digested and extracted solutions were analyzed
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by ICP-MS. Total Pb concentrations were determined by external calibration. The Pb bioaccumulation in tissue
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samples was expressed µg/g dry wt.
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After sampling the blood of S. schlegelii, the blood samples were diluted in 0.1M phosphate buffer. After
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preconditioning process using 65%(v/v) HNO3 on 120 °C hot plate for removing impurities except for Pb
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component, the blood Pb concentrations were analyzed using ICP-MS (ELAN 6600DRC).
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2.4. Hematological parameters
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The blood samples of S. schlegelii were collected using heparin-treated syringes. Immediately after collecting
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blood samples, the total red blood cell (RBC) count, hemoglobin (Hb), and hematocrit (Ht) were analyzed. The
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whole blood samples serially diluted in Hendrick’s solution, and the diluted samples were injected in hemo-
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cytometer for counting RBC using microscope. The Hb concentrations were analyzed using a clinical kit (Asan
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Pharm. co., Ltd.). The Ht values were analyzed using the microhematocrit centrifugation technique.
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The plasma samples were obtained by centrifuging the blood at 3000 g for 5 minutes at 4°C. The analyses of the
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plasma biochemical components (calcium, magnesium, glucose, cholesterol, total protein, glutamic oxalate
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transaminase (GOT), glutamic pyruvate transaminase (GPT), alkaline phosphatase (ALP)) were conducted using
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clinical kits (Asan Pharm. co., Ltd.).
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2.5. Growth performance
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There was no mortality during experimental periods. The growth performance of S. schlegelii was determined
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by the below methods.
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Daily length gain = (Final length – Initial length) / day
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Daily weight gain = (Final weight – Initial weight) / day
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Condition factor (%) = (weight / length3) x 100
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Hepatosomatic index (HSI) = (liver weight / body weight) x 100
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2.6. Statistical analysis
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The experiment was conducted in exposure periods for 4 weeks and performed triplicate. The limit of
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quantification (LOQ) was set at three times the limit of detection. Statistical analyses were performed using the
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SPSS/PC+ statistical package (SPSS Inc, Chicago, IL, USA). Significant differences between groups were
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identified using one-way ANOVA and Tukey's test for multiple comparisons or Student's t-test for two groups.
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The significance level was set at P < 0.05.
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3. Results
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3.1. Pb accumulation
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Pb accumulations in the kidney, liver, spleen, intestine, gill, and muscle of S. schlegelii exposed to dietary Pb
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concentrations and AsA supplementation is presented in Fig. 1. The highest levels of Pb accumulation were
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observed in the kidney. Significant accumulation in the kidney was observed following exposure to 120 mg/kg
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dietary Pb after two and four weeks, respectively. The levels of bioaccumulation in the kidney of fish exposed
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for four weeks to 120 mg/kg Pb, supplemented with 200 and 400 mg/kg AsA, respectively, were much lower
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than those in fish supplemented with 100 mg/kg AsA.
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Pb accumulation in the liver was notably increased following exposure to 120 mg/kg Pb at two and four weeks.
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At two weeks, no alterations in Pb accumulation were observed following exposure to 120 mg/kg dietary Pb,
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regardless of the levels of AsA supplementation. However, following exposure to 240 mg/kg Pb, at the
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supplementation level of 400 mg/kg AsA, Pb accumulation was much lower than it was with supplementation of
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100 and 200 mg/kg AsA. After four weeks of exposure to 120 mg/kg dietary Pb, at the supplementation level of
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400 mg/kg AsA, Pb accumulation was significantly lower than it was at the supplementation level of 100 and
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200 mg/kg AsA, respectively. An AsA dose-dependent reduction of Pb accumulation, following exposure to
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240 mg/kg dietary Pb was evident.
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Pb accumulation in the spleen was also significantly increased following exposure to 240 mg/kg Pb with
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supplementation of 200 and 400 mg/kg AsA, respectively, were substantially lower than they were with
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supplementation of 100 mg/kg AsA. At four weeks, the levels of AsA supplementation significantly reduced Pb
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accumulation, following exposure to 120 and 240 mg/kg Pb, respectively.
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Significant Pb accumulation was observed in the intestine, following exposure to 120 mg/kg Pb. At two and
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four weeks, respectively, AsA supplementation notably affected Pb accumulation. Pb accumulation in the group
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that had been exposed to 120 mg/kg Pb and supplemented with 400 mg/kg AsA much lower at two and four
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weeks, respectively, than it was in those supplemented with 100 and 200 mg/kg AsA. Pb accumulation in the
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group exposed to 240 mg/kg Pb and supplemented with 200 and 400 mg/kg AsA, was much lower than it was in
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the group supplemented with 100 mg/kg AsA.
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Significant Pb accumulation was observed in the gill tissue following exposure to dietary Pb. At two weeks, no
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significant effects of accumulation were observed, regardless of the level of AsA supplementation. After four
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weeks however, AsA supplementation at 200 and 400 mg/kg, respectively, significantly reduced Pb
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accumulation, compared to AsA supplementation at 100 mg/kg.
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No significant Pb accumulation was observed in the muscle, with the exception of the group exposed to 240
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mg/kg Pb and supplemented with 100 mg/kg AsA. After four weeks of exposure to dietary Pb and AsA
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supplementation, the profile of Pb accumulation in the tissues was kidney > liver > spleen > intestine > gill >
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muscle.
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3.2. Hematological parameters
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Blood accumulation, RBC count, hematocrit values, and hemoglobin concentration of S. schlegelii exposed to
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dietary Pb and AsA supplementation are shown in Fig. 2. Blood Pb accumulation was significantly increased
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when Pb concentration was over 120 mg/kg at both 2 and 4 weeks. In the group exposed to 240 mg/kg Pb, AsA
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supplementation at 100 mg/kg showed a significantly higher level of blood Pb accumulation at 4 weeks than
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observed for the 200 or 400 mg/kg AsA supplement groups. A significant decrease was observed in the RBC
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count of S. schlegelii exposed to over 120 mg/kg Pb at 2 and 4 weeks. The hematocrit values were notably
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reduced for S. schlegelii exposed to over 120 mg/kg Pb with 100 and 200 mg/kg AsA and in the concentration
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of 240 mg/kg Pb with 400 mg/kg AsA after 2 weeks. After 4 weeks, a significant decrease in the hematocrit
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values was observed for S. schlegelii exposed to 120 mg/kg Pb at all AsA levels compared with the controls.
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The hemoglobin concentration in S. schlegelii was significantly decreased over 120 mg/kg Pb after 2 weeks.
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After 4 weeks, a significant decrease in the hemoglobin was observed over 120 mg/kg Pb with AsA 200 mg/kg
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and in the concentration of 240 mg/kg Pb with 100 and 400 mg/kg AsA as compared with the control values.
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The alterations in plasma components of S. schlegelii with dietary Pb exposure and AsA supplementation are
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shown in Fig. 3. Of the inorganic components, calcium was reduced over 120 mg/kg Pb with 100 mg/kg AsA
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after 2 and 4 weeks, but there were no significant changes in the groups supplemented with 200 and 400 mg/kg
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AsA. Plasma magnesium was significantly decreased in the 240 mg/kg Pb treatment for all AsA
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supplementation groups as compared with the control values. Among the organic components glucose,
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cholesterol, and total protein, the glucose and cholesterol were significantly increased by the dietary Pb
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exposure at 240 mg/mL, whereas there was no alteration in total protein. The activity of the enzymes GOT and
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GPT was considerably increased by the addition of Pb; however, there was no change in ALP activity. In the
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GOT of S. schlegelii, a significant increase was observed over 120 mg/kg Pb and receiving 100 mg/kg AsA and
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upon exposure to 240 mg/kg Pb with 200 and 400 mg/kg AsA after 2 and 4 weeks as compared with the values
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observed in the control treatments. The GPT of S. schlegelii was considerably increased by the dietary Pb
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exposure over 120 mg/kg after 2 and 4 weeks.
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3.3. Growth performance
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The growth performance of S. schlegelii with dietary Pb exposure and AsA supplementation is demonstrated in
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Fig. 4. After 2 weeks, the daily length gain of S. schlegelii was notably reduced over 120 mg/kg Pb with 100 and
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200 mg/kg AsA and for the concentration of 240 mg/kg Pb with AsA 400 mg/kg. After 4 weeks, a considerable
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decrease in the daily length gain was observed upon exposure to over 120 mg/kg Pb. A decrease was observed
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in the daily weight gain, for Pb levels of 120 and 240 mg/kg exposure after 2 and 4 weeks. The condition factor
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was significantly decreased over 120 mg/kg Pb with 100 mg/kg AsA after 2 weeks, and a notable decrease was
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observed in the concentration of 240 mg/kg Pb and 200 and 400 mg/L AsA after 4 weeks. The hepatosomatic
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index of S. schlegelii showed a substantial decrease in the concentration of 240 mg/kg Pb when 100 and 200
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mg/kg AsA were used. After 4 weeks, the hepatosomatic index was considerably decreased in the concentration
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of 240 mg/kg Pb as compared with that of the control.
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4. Discussion
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Significantly higher levels of metals generally occur in aquatic animals than in the surrounding seawater,
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because of bioaccumulation. Furthermore, different patterns of metal accumulation can be observed within the
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same species, between different metals, and among the same metals in different species (Rainbow, 1997). Tissue
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specific metal accumulation is considered a critical parameter of metal exposure, and a study of the
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toxicokinetics of metal accumulation in risk assessments can offer a deeper understanding of the relationship
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between metal toxicity and exposure (McGeer et al., 2000).
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In the present study, Pb accumulation in the kidney (the highest among all tissues under investigation) of
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rockfish, S. schlegelii supplemented with AsA was substantially elevated following exposure to dietary Pb.
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Romeo et al. (2000) also demonstrated that the kidney was a major tissue for bioaccumulation in the sea bass
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Dicentrarchus labrax exposed to copper (Cu). Similar to the kidney tissue, significant accumulation was
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observed in the liver of S. schlegelii following exposure to dietary Pb. Rashed (2001) reported the highest levels
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of Cu and zinc (Zn) accumulation in the liver of Tilapia nilotica from the Nasser Lake that is contaminated by
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heavy metals. The kidney and liver represent major target organs that are suitable for histopathological
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examination to evaluate damage to tissues and cells (Oliveira Ribeiro et al., 2006). These organs are also
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comprised of tissues that are vulnerable to prolonged metal exposure, both through waterborne and dietary
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pathways (Olsvik et al., 2000).
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Exposure to dietary Pb and AsA supplementation caused significant accumulation in the spleen of S. schlegelii.
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Ciardullo et al. (2008) also observed substantial accumulation in the spleen of rainbow trout, Oncorhynchus
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mykiss exposed to mercury. Differences in metal accumulation between tissues are highly related to ecological
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needs and metabolic activities (Canli and Atli, 2003).
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Uptake routes (whether via feeding and the digestive organs or in a dissolved form through the gills) of toxic
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substances affect tissue accumulation of toxicants in aquatic animals. The uptake of toxic substances in aquatic
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animals generally occurs by two major routes; therefore, the intestines and gills should be critical tissues for
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metal accumulation (Kim and Kang, 2014). Exposure of S. schlegelii to dietary Pb reveals higher accumulation
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in the intestine than that observed in the gill tissue. Significant bioaccumulation in the intestine of S. schlegelii
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could be attributed to direct uptake from the dietary source. The gill is also a major tissue for bioaccumulation
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following metal exposure; however, uptake through dietary sources is not significant compared to that through a
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waterborne pathway (Ciardullo et al., 2008). No significant accumulation was observed in muscle tissue
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following exposure to dietary Pb, with the exception of the group subjected to 240 mg/kg Pb exposure with 100
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mg/kg AsA supplementation. Uysal et al. (2008) suggest that the muscle of aquatic animals is not a major active
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tissue for bioaccumulation of metals. Furthermore, Dural et al. (2007) reported that bioaccumulation in the
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muscle of three fish species exposed to various metals were significantly lower than that in other tissues, such as
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the gill, liver, and kidney.
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The relative bioaccumulation in the tissues of S. schlegelii exposed to dietary Pb and AsA supplementation was
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kidney > liver > spleen > intestine > gill > muscle. Dietary Pb exposure caused significant accumulation in
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specific tissues. Tissue-specific accumulation can be a sensitive indicator of metal exposure in aquatic
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toxicology (Carriquiriborde and Ronco, 2008); therefore, dietary Pb exposure in S. schlegelii could significantly
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affect experimental fish via bioaccumulation in specific tissues.
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In the present study, the levels of AsA supplementation and the various concentrations of Pb exposure notably
248
influenced the bioaccumulation in all tissues of S. schlegelii. The inhibitory effects of AsA on Pb absorption
249
have been described in various studies. In addition, AsA increases the availability of iron by decreasing ferric
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iron to ferrous iron in the duodenum. This is mechanism permits ferrous iron to compete with Pb for intestinal
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absorption (Patrick, 2006). Kumar et al. (2009) reported a significant lower concentration of Cd in the liver and
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kidney of catfish, Clarias batrachus exposed to Cd and supplemented with AsA than in the same tissues of
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catfish exposed to Cd only. These findings could be attributed to the roles of AsA to excrete metal in the bile by
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catalyzing the synthesis of glutathione, and compete for the sulfhydryl binding sites on metallothioneins.
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Dawson et al. (1999) reported that AsA supplementation in male smokers effectively lower blood Pb levels.
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AsA supplementation also significantly lower Pb levels in rats exposed to dietary Pb (Dalley et al., 1990).
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Although many studies have reported that following the Pb exposure, Pb accumulation in various tissues is
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effectively reduced by AsA supplementation, to our knowledge, no reports have focused on teleosts under
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conditions of Pb exposure. The present study demonstrates that AsA supplementation can also significantly
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reduce Pb accumulation in the tissues of teleosts.
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Hematology has been widely used to evaluate the health status of animals exposed to environmental toxicants,
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and many authors have reported hematological changes in fish exposed to various stress-inducing substances
263
(Mattsson et al., 2001; Affonso et al., 2002; Carvalho and Fernandes, 2006). In this study, the dietary exposure
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of S. schlegelii to Pb induced a significant accumulation of Pb in the blood, however, the high AsA
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supplementation was effective in attenuating the accumulation levels. Fish blood can be a notable accumulation
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section, because the absorbed metal is transported through the bloodstream to the liver to be metabolized and
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excreted by generating the metal-binding proteins such as metallothioneins. Mazon and Fernandes (1999)
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reported a considerable copper accumulation in the blood of the prochilodontidae, Prochilodus scrofa, exposed
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to excessive levels of copper. Ascorbic acid is one of the critical nutrients, and it effectively attenuates the Pb
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accumulation by reducing ferric iron to ferrous iron, which competes with Pb for absorption (Patrick, 2006).
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The hematological parameters of S. schlegelii such as RBC count, hematocrit value, and hemoglobin
272
concentration were markedly decreased by dietary Pb exposure. Many studies have reported a decrease in RBC
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count, hematocrit value, and hemoglobin concentration in fish exposed to various toxicants (Benifey and Biron,
274
2000). Heydarnejad et al. (2013) also suggested that the exposure to cadmium significantly affected the serum
275
biochemical parameters in rainbow trout, Oncorhynchus mykiss. Toxicity exposure to substances such as heavy
276
metals commonly induces the lysis of erythrocytes in aquatic animals, leading to the depiction in hemoglobin
277
and hematocrit values in addition to the deformed erythrocytes and anemia (Maheswaran et al., 2008). Toplan et
278
al. (2004) indicated that Pb, which has a high affinity with RBC, increases the osmotic and mechanical
279
susceptibility of RBC giving rise to reduced deformability and a shortened life span. High concentrations of Pb
280
in the blood also impair heme synthesis, thus inhibiting hemoglobin synthesis and anemia.
281
The inorganic calcium and magnesium components in plasma are major indicators in the assessment of metal
282
toxicity due to their functions in ion regulation and homeostasis (Kim and Kang, 2014). Our results demonstrate
283
that dietary Pb exposure caused a considerable decrease in calcium and magnesium in the plasma of S. schlegelii.
284
Suzuki et al. (2004) reported that mercury and cadmium exposure affected the calcium homeostasis of goldfish,
285
Carassius auratus. Of the organic components, the plasma glucose and cholesterol of S. schlegelii were
286
substantially increased by dietary Pb exposure; however, there was no alteration in total protein. Blood glucose
287
level is commonly increased by the elevation of carbohydrate metabolism due to toxicant exposure stress such
288
as that caused by heavy metals (Hontela et al., 1996). The elevation in blood glucose is considered as a general
289
secondary response to the stress in fish, which can be a sensitive indicator for environmental stress (Sepici-
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Dincel et al., 2009). Glucose and glycogen is utilized in aquatic animals to fulfill the energy requirement for
291
detoxification of toxic substances (dos Santos Carvalho and Fernandes, 2008). Cholesterol is a critical structural
292
component of membranes and precursor of all steroid hormones. Given that heavy metals are well known to
293
adversely affect the cell structure, the increase in cholesterol may be a sensitive indicator for metal induced
294
environmental stress (Oner et al., 2008). The cholesterol in the plasma of S. schlegelii was significantly
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increased by dietary Pb exposure. Firat et al. (2011) also reported a considerable increase in the cholesterol of
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Nile tilapia, Oreochromis niloticus, exposed to Cu and Pb, which may increase due to liver and kidney failure
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inducing the release of cholesterol into the blood. Plasma protein is synthesized in the liver, and is commonly
298
used as a critical indicator to evaluate liver impairment (Yang and Chen, 2003). However, there was no
299
observed effect of Pb on the total protein in the S. schlegelii. Alterations in the activity of enzymes such as GOT,
300
GPT, and ALP have been used in aquatic organisms to indicate the presence of tissue damage due to toxicant
301
stress from metal exposure (Lavanya et al., 2011). The levels of GOT and GPT in the plasma of S. schlegelii
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were significantly increased by the dietary Pb exposure, and the increases in plasma GOT and GPT activities
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were in agreement with the findings of other authors who concluded that the increase in GOT and GPT activity
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may be attributed to tissue damage, particularly in the liver (Zikic et al., 2001; Levesque et al., 2002; Mekkawy
305
et al., 2011). There were no changes observed in plasma ALP levels of S. schlegelii. The changes in these
306
parameters are indicative of environmental stress, and the hematological parameters are sensitive and reliable
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parameters to assess Pb toxicity.
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In this study, the dietary Pb exposure induced significant changes in the hematological parameters and plasma
309
components of S. schlegelii. The AsA supplementation was highly effective in attenuating the alterations by the
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dietary Pb exposure. The changes in the hematological parameters of RBC count, hematocrit value, and
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hemoglobin concentration, in addition to the plasma components of calcium, glucose, GOT, and GPT, in
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response to dietary Pb exposure, were effectively moderated by the AsA supplementation. Mekkawy et al. (2011)
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reported that vitamin E supplementation of Nile tilapia Oreochromis niloticus attenuated the cadmium-induced
314
toxicity in the hematological parameters. Hounkpatin et al. (2012) reported on the protective effects of vitamin
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C on hematological parameters after exposure to cadmium, mercury, and the two heavy metals combined.
316
Dietary AsA supplementation also helps decrease in the proportion of glycated insulin in circulation, which
317
alleviates insulin resistance leading to insulin-stimulated glucose uptake by peripheral tissues (Abdel-Wahab et
318
al., 2002).
319
Fish growth is influenced by various external factors such as temperature, nutrients, and toxicants. Among these
320
factors, the toxicant exposure is a critical component that inhibits growth. The growth performance of the S.
321
schlegelii was significantly decreased by the dietary Pb exposure, which may be due to the reallocation of
322
energy from the growth and development to detoxification. Hansen et al. (2002) reported growth inhibition
323
could be induced by physiological or behavioral stress during toxic substance exposure, as the stress can cause a
324
decrease in food consumption or food assimilation. Hepatosomatic index (HSI) alterations have been observed
ACCEPTED MANUSCRIPT
325
in aquatic animals in response to the damage of organs and biochemical changes brought about by exposure to
326
toxicants, and Nikam (2012) reported a significant decrease in the HSI of Channa punctatus exposed to zinc. In
327
our study, dietary Pb exposure caused a decrease in the hepatosomatic index of S. schlegelii, but this was
328
attenuated by AsA supplementation. Considering the AsA effects on the improving health status and disease
329
resistance in addition to growth and development (Kim and Kang, 2015), the high levels of AsA
330
supplementation were significantly effective in alleviating the toxicity caused by dietary Pb exposure.
331
In conclusion, exposure of S. schlegelii to dietary Pb resulted in significant Pb accumulation in specific tissues,
332
and AsA supplementation significantly reduced this accumulation. In addition, the exposure of S. schlegelii to
333
dietary Pb induced the notable Pb accumulation in the blood, decrease in hematological parameters (RBC count,
334
hematocrit value, and hemoglobin concentration), alterations in plasma components (Ca, Mg, glucose,
335
cholesterol, GOT, and GPT), and reduction in growth performance. In addition, the AsA supplementation
336
showed considerable effectiveness in attenuating the changes caused by the Pb-induced toxicity. However, even
337
high AsA supplementation has also limitation to curb Pb toxicity. The results of the present study demonstrate
338
that the exposure of S. schlegelii to dietary Pb caused substantial indications of toxicity, and that AsA
339
supplementation was able to effectively reduce the Pb-induced toxicity. From this study, we proposed that
340
dietary Pb exposure over 120 mg/kg can be highly toxic to aquatic animals, even at the 4 week exposure period.
341
In addition, accumulation patterns can suggest guideline to understand Pb toxic mechanism.
342
343
344
Acknowledgment
345
This study was supported by the project ‘The Environmental-friendly Aquaculture Technology using biofloc’
346
(R2017016) of the National Institute of Fisheries Science (NIFS), Incheon, South Korea.
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348
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50
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
Kidney
80
4 weeks
d
c
c
c
b
b
Pb accumulation (
40
b b
d
d
60
c
b
20
30
d
a aa
a
a
a
a
120
240
Control
120
240
Control
30
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
Spleen
120
Control
120
240
Intestine
25
2 weeks
4 weeks
d
c
c
c
bc
bc
b
bc
b
10
b
Pb accumulation (
cd
d
20
a
240
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
2 weeks
/g)
/g)
Pb accumulation (
a
Pb concentration (mg/kg)
20
4 weeks
a
d
d
c c
15
c c
c
bc
bc
bc
10
b
b
5
a
aa
a
aa
a
a
0
aa
0
Control
120
240
Control
120
240
Control
120
Pb concentration (mg/kg)
3.0
Control
120
240
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
Muscle
/g)
2.5
4 weeks
bc
bc
15
b
b
b
b
b
bb
a aa
abab
aa
2 weeks
4 weeks
2.0
c
Pb accumulation (
/g)
Gill
2 weeks
10
240
Pb concentration (mg/kg)
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
20
Pb accumulation (
a
0
30
5
bc
b
bbb
Pb concentration (mg/kg)
25
c
c
20
aaa
Control
30
c
c
d
10
0
40
4 weeks
/g)
2 weeks
d
Liver
2 weeks
40
/g)
Pb accumulation (
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
b
b
1.5
a
a
a
a
aa
abab
ab
a
aa
a
ab
a
a
1.0
0.5
a
0
0.0
Control
120
240
Control
Pb concentration (mg/kg)
120
240
Control
120
240
Control
120
240
Pb concentration (mg/kg)
Figure 1. Pb accumulation in rockfish, Sebastes schlegelii exposed to different concentrations of dietary lead
and ascorbic acid for four weeks. Values with different superscripts are significantly different at two and four
weeks (P < 0.05) as determined by the Tukey's multiple range test.
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AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
2 weeks
c
bc
6
b
b
c
b
b
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4
2
aa a
2 weeks
300
aa
4
8
b b
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
4 weeks
c
3
10
d
RBC count ( x 10 mm )
Pb blood accumulation (mg/L)
12
bb b
200
bc b
bc
bc bc
c
cc c
100
0
Control
120
240
Control
120
240
Control
Pb concentration (mg/kg)
a a
b
40
a
2 weeks
ab
b
a
a
bc
4 weeks
b
bc
8
bb
bc bc
c
c
30
20
Hemoglobin (g/dL)
ab
120
240
Control
120
240
Pb concentration (mg/kg)
10
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
50
Hematocrit (%)
a
a a
aaa
0
60
4 weeks
a
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
2 weeks
a a
ab
bc
ab
bb
a a
bc bc
4 weeks
b
ab
bc
c
6
bc
c c
4
2
10
0
0
Control
120
240
Control
Pb concentration (mg/kg)
120
240
Control
120
240
Control
120
240
Pb concentration (mg/kg)
Fig. 2. Pb blood accumulation and changes of RBC count, Hematocrit and Hemoglobin in rockfish, Sebastes
schlegelii combinedly exposed to the different concentration of dietary lead and ascorbic acid for 4 weeks.
Values with different superscript are significantly different in 2 and 4 weeks (P < 0.05) as determined by
Tukey's multiple range test.
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5
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
2 weeks
a a
a
ab a
b
4
4 weeks
abab
b
ab a
a
a
Magnesium (mg/dL)
Calcium (mg/dL)
30
abab
ab
b
b
20
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
a
2 weeks
a a
4 weeks
aaa
aaa
bb
a
ab
b
a
bb
b
3
2
10
1
0
0
Control
120
240
Control
120
240
Control
Pb concentration (mg/kg)
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg 2 weeks
100
Glucose (mg/dL)
bc bc
80
a a
a
b
250
4 weeks
c
bc
bc bc
bc
c
c
bc
Control
120
2 weeks
4 weeks
bbb
60
40
150
240
a aa
ababab
ab
abab
bbb
aaa
100
50
20
0
0
Control
120
240
Control
120
240
Control
Pb concentration (mg/kg)
140
100 mg/kg
200 mg/kg
400 mg/kg
2 weeks
120
4 weeks
a
a
aa
a
a
a a
a a
a
a
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
Control
2 weeks
120
4 weeks
c
240
aa
a a
a
4
a
c c
c
b
b
abab
100
GOT (KU)
a
240
bcbc
6
aa
120
Pb concentration (mg/kg)
8
Total Protein (g/dL)
240
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
200
b
a a
a
Cholesterol (mg/dL)
120
120
Pb concentration (mg/kg)
a
a a
ab
ab
80
60
40
2
20
0
0
Control
120
240
Control
120
240
Control
Pb concentration (mg/kg)
8
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg 2 weeks
4 weeks
c
bc
60
b b
aa
a
GPT (KU)
bc
bc bc
a
b
c
c c
6
240
Control
120
240
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg 2 weeks
a aa
a a
a
a
aa
a
aa
4 weeks
aa
a
a
aa
b
a
a
ALP (K-A)
80
120
Pb concentration (mg/kg)
40
20
4
2
0
0
Control
120
240
Control
Pb concentration (mg/kg)
120
240
Control
120
240
Control
120
240
Pb concentration (mg/kg)
Fig. 3. Changes of plasma parameters in rockfish, Sebastes schlegelii combinedly exposed to the different
concentration of dietary lead and ascorbic acid for 4 weeks. Values with different superscript are significantly
different in 2 and 4 weeks (P < 0.05) as determined by Tukey's multiple range test.
ACCEPTED MANUSCRIPT
600
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
2.0
ab
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
500
2 weeks
a
a
ab
1.5
4 weeks
a a
ab
b
b
b
bc
b
bc
cc
c
1.0
c c
0.5
Daily weight gain (mg/day)
Daily length gain (mm/day)
2.5
aa
b
bc
bc
c
300
c
c
c
200
0
120
240
Control
120
240
Control
Pb concentration (mg/kg)
4
a
1.2
2 weeks
a
a
b
ab
b ab
a a
ab
240
Control
120
240
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
4 weeks
ab
ab ab
bb
1.0
b
0.8
0.6
0.4
Hepatosomatic index (%)
a
ab
120
Pb concentration (mg/kg)
AsA 100 mg/kg
AsA 200 mg/kg
AsA 400 mg/kg
1.4
Condition factor (%)
b
bc
bc
100
Control
1.6
4 weeks
ab
bb
400
0.0
1.8
a a
2 weeks
ab
2 weeks
3
4 weeks
aaa
ab a a
2
a
a
a
ab
b b
ababab
b
b b
1
0.2
0.0
0
Control
120
240
Control
Pb concentration (mg/kg)
120
240
Control
120
240
Control
120
240
Pb concentration (mg/kg)
Fig. 4. Daily length, daily weight gain, condition factor, and hepatosomatic index factor of rockfish, Sebastes
schlegelii combinedly exposed to the different concentration of dietary lead and ascorbic acid for 4 weeks.
Values with different superscript are significantly different in 2 and 4 weeks (P < 0.05) as determined by
Tukey's multiple range test.
ACCEPTED MANUSCRIPT
Table 1. Formulation of the experimental diet (% dry matter)
Concentration (mg/kg)
Ingredient (%)
M0V1
M0V2
M0V3
M1V1
Casein1
33.0
33.0
33.0
33.0
33.0
33.0
33.0
33.0
33.0
Fish meal2
23.0
23.0
23.0
23.0
23.0
23.0
23.0
23.0
23.0
Wheat flour3
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
Fish oil4
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
Cellulose1
4.5
4.0
3.0
3.9
3.4
2.4
3.3
2.8
1.8
Corn starch3
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
Vitamin Premix (Vitamin C-free)5
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
Mineral Premix6
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
Lead Premix7
0.0
0.0
0.0
0.6
0.6
0.6
1.2
1.2
1.2
Ascorbic acid Premix8
0.5
1.0
2.0
0.5
1.0
2.0
0.5
1.0
2.0
1United
M1V2
M1V3
M2V1
M2V2
M2V3
States Biochemical (Cleveland, OH).
2Suhyup
Feed Co., Ltd., Gyeong Nam Province, Korea.
3Young
Nam Flour Mills Co., Pusan, Korea.
4Sigma
Chemical Co., St. Louis, MO.
5Vitamin
Premix (vitamin C-free) (mg/kg diet): dl-calcium pantothenate, 400; choline chloride 200; inositol, 20; menadione,
2; nicotinamide, 60; pyridoxine·HCl, 44; riboflavin, 36; thiamine mononitrate, 120, DL-a-tocopherol acetate, 60; biotin, 0.04;
folic acid, 6; vitamin B12, 0.04; retinyl acetate, 20000IU; cholecalciferol, 4000 IU.
6Mineral
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; Mn, 9.1.
7Lead
Premix: 20,000 mg Pb/ kg diet
8Ascorbic
acid Premix: 20,000 mg ascorbic acid/ kg diet
(M0: Pb 0 mg/kg, M1: Pb 120 mg/kg, M2: Pb 240 mg/kg; V1: AsA 100 mg/kg; V2: AsA 200 mg/kg; V3: AsA 400 mg/kg)
ACCEPTED MANUSCRIPT
Table 2. Analyzed dietary concentration (mg/kg) of each source
Diets (mg/kg)
M0V1
M0V2
M0V3
M1V1
M1V2 M1V3 M2V1 M2V2 M2V3
Pb concentration
0
0
0
120
120
Actual Pb level
2.1
1.8
2.4
121.5
117.8
AsA concentration
100
200
400
100
200
400
100
Actual AsA level
87.8
168.5
359.5
91.2
176.8
364.5
89.3
120
240
240
240
118.2 237.2 236.7 241.9
200
400
162.5 372.8