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The effects of dietary lead on growth, bioaccumulation and antioxidant capacity in sea cucumber, Apostichopus japonicus
Environmental Toxicology and Pharmacology 40 (2015) 535–540
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The effects of dietary lead on growth, bioaccumulation and antioxidant capacity in sea cucumber, Apostichopus japonicus Jing Wang a , Tongjun Ren a,∗ , Yuzhe Han a , Yang Zhao a , Mingling Liao b , Fuqiang Wang a , Zhiqiang Jiang a a b
Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea, Ministry of Agriculture, Dalian Ocean University, Dalian 116023, China State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361000, China
a r t i c l e
i n f o
Article history: Received 28 May 2015 Received in revised form 7 August 2015 Accepted 8 August 2015 Available online 12 August 2015 Keywords: Lead Sea cucumber Growth performance Bioaccumulation Antioxidant capacity
a b s t r a c t Three different diets amended with lead nitrate [Pb(NO3 )2 ] (100, 500 and 1000 mg Pb/kg dry weight) and a Pb-free control diet (1.03 mg Pb/kg dry weight) were fed to sea cucumber (Apostichopus japonicus) for 30 days. The patterns of Pb accumulation over time were determined in various tissues (body wall, intestine and respiratory tree), as well as growth performance and antioxidant enzymes activities. Pb accumulation in body wall and intestine increased with time in all dietary Pb treatments. When fed the highest Pb diet, the body wall exhibited the greatest Pb burden (16.37 mg Pb/kg tissue wet weight), while Pb content in the intestine (2.68 mg Pb/kg tissue wet weight) and the respiratory tree (1.78 mg Pb/kg tissue wet weight) were lower than Pb content in the body wall by day 30. The body weight gain (BWG), specific growth rate (SGR) and survival rate (SR) had not been affected by 30 days oral administration of Pb supplemented diet. However, the antioxidant enzymes activities [superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px)] of test groups were lower than control group in body wall and malondialdehyde (MDA) concentration in the body wall was opposite after 30 days in sea cucumbers. In summary, this work reports toxic effects in sea cucumber, A. japonicus, after dietary exposure to Pb. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Sea cucumber is a very important species in northern coastal areas in China due to its high nutritional value and desirable taste. It is also important for the coastal economy and for ecosystems. Sea cucumbers generally have slow rates of population turnover and feed on scraps found mixed in sediments (Purcell et al., 2010). However, chronic contamination of aquatic environment by heavy metals is a severe problem since these pollutants invariably persist in the environment (Velez and Montoro, 1998; Fang, 2004; Ashraf, 2005). Heavy metal contamination enters the aquatic environment from farms, urban and industrial production sites, and cause long-term eco-toxicological effects (Strmack and Braunbeck, 2000). Lead (Pb) is a cumulative, non-essential toxic element that has neither beneficial nor desirable nutritional effects on animals. However, due to many of its physical and chemical properties such as softness, malleability, ductility, poor conductivity and resistance to corrosion (Paul et al., 2014), Pb has been widely used in several industrial processes such as clothe tinge, varnish, pesticide,
∗ Corresponding author at: 52 Heishijiao Street, Dalian 116023, China. E-mail address: sea [email protected] (T. Ren). http://dx.doi.org/10.1016/j.etap.2015.08.012 1382-6689/© 2015 Elsevier B.V. All rights reserved.
explosives, batteries and painting manufacturing (Johnson, 1998) and could be responsible for death or sublethal changes in reproduction, growth and behavior of the fishes (Burden et al., 1998). Continuous exposure to Pb in the environment and occupational may cause renal, nervous, hepatic, hematological and reproductive damage in animals, including humans (Flora et al., 2006; El-Sayed and El-Neweshy, 2010; Ashry et al., 2010). Today, it is widely accepted that even small quantities of Pb are harmful to humans and animals (Ashry et al., 2010). Pb may disturb the antioxidant barrier via inhibition of the functional sulfhydryl (SH) groups present also in free radical-scavenging enzymes such as glutathione reductase (GR), glutathione peroxidase (GPx), glutathione s-transferase (GST), superoxide dismutase (SOD) catalase (CAT) and §-aminolevulinic acid dehydratase (ALAD) (Patra et al., 2001; Moniuszko-Jakoniuk et al., 2007; Olaleye et al., 2007). Weber et al. (1991) and Weber (1996) have previously reported on the effects of short-term exposure to Pb on the feeding abilities of juvenile fathead minnows (Pimephales promelas) and Pb may cause metabolic imbalances in juvenile fish. A number of studies have shown that significant gill and renal lead burden occurs after both acute exposure and chronic exposure to sublethal concentrations of waterborne lead (Davies et al., 1976; Hodson et al., 1978; Rogers et al., 2003). Moreover, some studies had showed
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that contaminants like lead can affect the phagocytic activity of macrophages in Oreochromis niloticus (Omima and Aboud, 2010) and the general immune status of Channa puncratus (Paul et al., 2014). There is no published research on the effects of dietary Pb on sea cucumbers (Apostichopus japonicus). So, the present study was conducted to determine the effects of dietary Pb on growth performance, bioaccumulation in tissues and antioxidant ability in sea cucumber to four Pb concentrations for 30 days in an attempt to explore the relationship between Pb exposure and bioaccumulation in sea cucumbers. 2. Materials and methods 2.1. Test diets The basal ingredients, Pb concentration and proximate composition of the treatment diets were shown in Table 1. Pb content is 1.03 ± 0.5 mg/kg dry weight in the basal diets. Lead nitrate [Pb(NO3 )2 ] was supplemented separately to the basal diet to obtain four Pb levels, 0, 100, 500 and 1000 mg Pb/kg dry weight. All dry ingredients were mixed thoroughly. Tap water was added to the mash at 600 g/kg and the mixture was passed through a pelletizer (Pinzheng Equipment Co. Ltd., Changzhou, China) with a 1.8-mmdiameter. Diets were then dried in a thermostatically controlled hot air oven (Jinghong Test Equipment Co. Ltd., Shanghai, China) at 40 ◦ C (Tewary and Patra, 2008) and kept in a refrigerator at −20 ◦ C until used. 2.2. Feeding trial Sea cucumber juveniles used in this experiment were obtained from the Yanhua Factory of Dalian (Dalian Yanhua Group Co. Ltd., Dalian, China) and transported to the Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea, Ministry of Agriculture of Dalian Ocean University (Dalian, China). Sea cucumber juveniles were fed with control diet for 2 weeks before the start of the experiment to acclimate the sea cucumbers to the diets and to the experimental conditions. Sea cucumbers (240 N, 10.06 ± 0.02 g) were then randomly allocated into 40-L tanks at a density of 20 sea cucumbers per tank with 24 h air supply to maintain dissolved oxygen near saturation, with three replicates in each treatment. The experiment was performed in a static aquarium system. Water temperature ranged from 16.0 to 18.0 ◦ C. Room temperature ranged from 18.0 to 20.0 ◦ C. The pH was 7.9–8.1 and the salinity was 31–32. Sea cucumbers were fed at an amount of 3% of body weight (Wang et al., 2009) once a day at 16:00. To ensure water quality, 50% of the water was replaced every day and residual diet and feces were removed using a siphon before feeding. The experiment lasted for 30 days. Sea cucumbers were sampled on days 10, 20, and 30. On each sampling date, five randomly selected sea cucumbers from each tank were dissected on ice to obtain the body wall, intestine, and respiratory tree. Tissues were stored at −80 ◦ C immediately for later determination of Pb concentrations. 2.3. Growth performance At the end of the experiment, sea cucumbers were starved for 24 h, and then every sea cucumber was counted and weighed, individually, to determine final body weight (FBW), body weight gain (BWG), specific growth rate (SGR) and survival rate (SR). Growth parameters were calculated as follows: BWG (%) = 100 × (final mean weight − initial mean weight)/initial mean weight; SGR (%/day) = 100 × (ln final mean weight − ln initial mean weight)/no.
of days; SR (%) = 100 × (final sea cucumber number)/(initial sea cucumber number). 2.4. Pb contents in tissues Pb was extracted from the tissues of the sea cucumbers by acid digestion using a method modified from Nakayama et al. (2011). Briefly, 2000 mg of respiratory tree and intestine sample were separately digested using concentrated nitric acid in a 100 ◦ C water bath until the liquid was clear. The volume of the digested samples was standardized to 25-mL using distilled water for measurements in a polarized Zeeman atomic absorption spectrophotometer (Hitachi Z-2010, Tokyo, Japan). The Pb was measured in the graphite furnace mode and the concentrations in the tissues were expressed as ug/g. 2.5. Antioxidant ability analysis Six sea cucumbers per treatment (two sea cucumbers from each tank) were selected and dissected on ice to obtain the body wall for determination of antioxidant ability on day 30. Body wall samples were thoroughly homogenized using physiological saline in ice-cold homogenizers and centrifuged immediately at 3000 rpm at 4 ◦ C for 10 min. The supernatant fluid was collected separately and stored in a refrigerator at 4 ◦ C in 1.5-mL Eppendorf tubes for subsequent analysis. The SOD activity was measured by the ability to inhibit superoxide anion generated by xanthine and the xanthine oxidase reaction system according to Wang and Chen (2005) using a SOD detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Superoxide anion radicals, produced during the reaction of xanthine with O2 catalyzed by xanthine oxidase, react with hydroxylamine producing nitric ions. After the reaction of nitric ions with naphthalene diamine, sulfanilic acid produces a colored product, which is proportional to the amount of produced superoxide anion radical. The absorbance is assayed at 550 nm (Wang et al., 2011). One unit (U) of SOD activity was defined as the amount required for inhibiting the rate of xanthine reduction by 50% in a 1-mL reaction system and specific activity was expressed as SOD unit per ml serum (Lin et al., 2011). The activity of GSH-Px in the body wall was assayed using the glutathione peroxidase kit by the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). GSH-Px catalyzes glutathione oxidation (GSH) with hydrogen peroxide and cumene hydroperoxide. In the presence of glutathione reductase and simultaneous triphosphopyridine nucleotide (NADPH), the oxidized form of glutathione (GSSG) immediately converts to its reduced form with simultaneous NADPH oxidation. The concentration of GSH-Px was assessed from the decrease in absorption at 340 nm due to the oxidation of NADPH to NADP+ (Balic et al., 2012). The malondialdehyde (MDA) concentration was determined using a test kit with a method established by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). It was measured by the thiobarbituric acid-reactive substance assay. The method is based on the measurement of the concentration of pink chromogen compound that forms when MDA reacts with thiobarbituric acid (Bayatli et al., 2013). MDA concentration was measured at 532 nm. The MDA content was expressed in units per milligram protein. 2.6. Statistical analysis Results are presented as means ± SD. One-way analysis of variance (ANOVA) was performed to determine statistical differences among groups. When overall differences were significant (P < 0.05), Duncan’s multiple range test was used to compare significant
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Table 1 Ingredients composition and proximate analysis of the experimental diets (g/kg). Ingredients
Sargassum thunbergiia Vitamin mixtureb Mineral mixturec Flourd Fish meale Soybean mealf Guar gum Microcrystalline celluloseg Pb(NO3 )2 h Total Analyzed nutrients (% in dry basis) Moisture Ash Crude protein Crude lipid
Treatments 0 mg Pb/kg
100 mg Pb/kg
500 mg Pb/kg
1000 mg Pb/kg
700 20 20 50 90 60 20 40 0 1000
700 20 20 50 90 60 20 39.9 0.1 1000
700 20 20 50 90 60 20 39.5 0.5 1000
700 20 20 50 90 60 20 39.0 1.0 1000
9.33 ± 0.12 32.27 ± 0.01 14.35 ± 0.04 7.07 ± 0.74
7.49 ± 0.10 32.83 ± 0.01 14.64 ± 0.01 4.25 ± 0.33
7.82 ± 0.05 32.40 ± 0.01 14.20 ± 0.16 5.99 ± 1.14
10.04 ± 0.03 32.12 ± 0.02 14.73 ± 0.18 5.24 ± 0.77
a
Sargassum thunbergii: the protein content is 10%. Longyuan Fishmeal Inc., Dalian, China. Vitamin mixture (per kilogram of premix): vitamin A, 1,000,000 IU; vitamin D3 , 300,000 IU; vitamin E, 4000 IU; vitamin K3 , 1000 mg; vitamin B1 , 2000 mg; vitamin B2 , 1500 mg; vitamin B6 , 1000 mg; vitamin B12 , 5 mg; nicotinic acid, 1000 mg; vitamin C, 5000 mg; Ca pantothenate, 5000 mg; folic acid, 100 mg; inositol, 10,000 mg; carrier glucose; H2 O ≤ 10%. c Mineral mixture (0.025 mg/g of premix): NaCl, 107.79; MgSO4 ·7H2 O, 380.02; NaHPO4 ·2H2 O, 241.91; KH2 PO4 , 665.20; Ca (H2 PO4 )·2H2 O, 376.70; Fe citrate, 82.38; Ca lactate, 907.10; Al(OH)3 , 0.52; ZnSO4 ·7H2 O, 9.90; CuSO4 , 0.28; MnSO4 ·7H2 O, 2.22; Ca(IO3 )2 , 0.42; CoSO4 ·H2 O, 2.77. d Flour: the protein content is 18%. New Market Co., Dalian, China. e Fish meal: the protein content is 65%. Longyuan Fishmeal Inc., Dalian, China. f Soybean meal: the protein content is 40%. g Keyuan Industrial Co. Ltd., Shanghai, China. h Lead nitrate [Pb(NO3 )2 ]. b
differences between treatments. Statistical analyses were performed using SPSS 13.0 for Windows (SPSS Inc, Chicago, IL). 3. Results 3.1. Growth performance After 30 days, the effect of dietary Pb supplementation on growth performance is found in Table 2. No significant differences (P > 0.05) were observed in SGR or BWG among all treatment groups. In addition, high survival (>98%) was observed in all dietary treatment groups. 3.2. Pb content in tissues At all dietary Pb doses, Pb accumulation is seen in the body wall (Fig. 1), intestine (Fig. 2) and respiratory tree (Fig. 3) during the course of the 30 days experiment. The body wall showed a substantial accumulation with a burden of 16.37 ug Pb/g tissue wet weight in the 1000 mg Pb/kg exposure, which was 9× greater than the respiratory tree (1.78 ug Pb/kg) on day 30. The body wall (16.37 ug Pb/g tissue wet weight), intestine (2.68 ug Pb/kg tissue wet weight), and respiratory tree (1.78 ug Pb/kg tissue wet weight) all exhibited the highest Pb burdens on day 30. Pb burden in the body wall (Fig. 1) and intestine (Fig. 2) increased with time in all dietary Pb treatments. The Pb accumulation depended upon the dietary Pb concentration and exposure periods. After 30 days of the dietary Pb exposure, the profile of tissue Pb accumulation was body wall > intestine > respiratory tree.
(P < 0.05) lower than the control group and there were no significant (P > 0.05) differences between control group and 100 mg Pb/kg treatment. The T-AOC capability was similar to the SOD activity, and the three treatments significantly (P < 0.05) lower than the control group and there were no significant differences (P > 0.05) among the three treatments. In contrast, MDA contents of the three treatments in body wall were significantly (P < 0.05) higher than the control group, but there were no significant (P > 0.05) differences among the three treatments. 4. Discussion This experiment demonstrated Pb-induced effects on growth performance, Pb contents and enzymes activities in sea cucumbers. The present study investigated that dietary exposure to Pb at these measured concentrations (100, 500, and 1000 mg Pb/kg) had few
3.3. Enzymes activities and MDA content Enzymes activities and MDA content in the body walls of sea cucumbers in each treatment after 30 days are shown in Table 3. SOD activities of the three treatments were significantly (P < 0.05) lower than the control group and there were no significant (P > 0.05) differences among the three treatments. GSH-Px activities of 500 mg Pb/kg, and 1000 mg Pb/kg treatments were significantly
Fig. 1. The Pb content in the body wall. Note: values are mean ± SD. Different letters are significantly different (P < 0.05). A/B/C denote the difference of the control group on 10, 20, 30 days. a/b/c denote the difference of the 100 mg Pb/kg group on 10, 20, 30 days. A’/B’/C’ denote the difference of the 500 mg Pb/kg group on 10, 20,30 days. a’/b’/c’ denote the difference of the 1000 mg Pb/kg group on 10, 20, 30 days.
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Table 2 Effects of dietary Pb on the growth performance of sea cucumber. Treatments
0 mg Pb/kg
IBW (g) FBW (g) BWG (%) SGR (%/day) SR (%)
10.07 17.52 73.92 1.81 93.33
± ± ± ± ±
0.01 1.23 5.71 0.23 2.98
100 mg Pb/kg 10.06 17.51 74.00 1.81 98.33
± ± ± ± ±
0.02 0.92 5.22 0.17 2.98
500 mg Pb/kg 10.06 17.44 73.25 1.82 98.33
± ± ± ± ±
0.02 0.49 5.44 0.10 2.98
1000 mg Pb/kg 10.07 17.00 72.47 1.73 100
± ± ± ± ±
0.01 0.07 0.91 0.01 0
Note: values are expressed as mean ± SD. (n = 3): IBW: initial body weight; FBW: final body weight; BWG (body weight gain) = 100 × (final mean weight − initial mean weight)/initial mean weight; SGR (specific growth rate) = 100 × (ln final mean weight − ln initial mean weight)/no. of days. Table 3 Anti-oxidative enzymes and total anti-oxidative ability in the body wall of sea cucumber. Treatments
0 mg Pb/kg
SOD GSH-Px MDA T-AOC
71.71 498.96 3.20 3.49
± ± ± ±
5.14b 4.72b 0.20a 0.36b
100 mg Pb/kg 53.14 310.27 4.03 2.48
± ± ± ±
3.47a 9.34ab 0.10b 0.23a
500 mg Pb/kg 51.99 348.40 4.04 2.26
± ± ± ±
5.04a 44.1a 0.30b 0.21a
1000 mgPb/kg 47.63 396.04 4.13 2.13
± ± ± ±
5.50a 41.60a 0.25b 0.27a
Note: values are expressed as mean ± SD. (n = 3): different superscript letters within each row indicate significant differences (P < 0.05).
Fig. 2. The Pb content in the intestine of sea cucumber. Note: values are mean ± SD. Different letters are significantly different (P < 0.05). A/B/C denote the difference of the control group on 10, 20, 30 days. a/b/c denote the difference of the 100 mg Pb/kg group on 10, 20, 30 days. A’/B’/C’ denote the difference of the 500 mg Pb/kg group on 10, 20,30 days. a’/b’/c’ denote the difference of the 1000 mg Pb/kg group on 10, 20, 30 days.
Fig. 3. The Pb content in the respiratory tree of sea cucumber. Note: values are mean ± SD. Different letters are significantly different (P < 0.05). A/B/C denote the difference of the control group on 10, 20, 30 days. a/b/c denote the difference of the 100 mg Pb/kg group on 10, 20, 30 days. A’/B’/C’ denote the difference of the 500 mg Pb/kg group on 10, 20,30 days. a’/b’/c’ denote the difference of the 1000 mg Pb/kg group on 10, 20, 30 days.
apparent adverse effects on BWG and SGR in sea cucumbers during the 30 days experiment. These results are consistent with Alves et al. (2006) who found no effects on growth and survival when juvenile rainbow trout were fed commercial trout pellets amended with various levels of Pb (7–520 ug Pb/g) for 21 days, and Alves and Wood (2006) also found chronic dietary exposure in the range of 50–500 ug Pb/g that resulted in no significant effects on the growth and survival of juvenile rainbow trout over 42 days. The study about bioaccumulation patterns in fish tissues exposed to metals can be used as effective and reliable indicators of environmental metal pollution (Kim et al., 2004). The accumulation in aquatic animals has been occurring through different mechanisms via the direct uptake from water by gills and ingestion from food by intestine (Oost et al., 2003). Between two pathways, the metal accumulation in fish was mainly from their diet (Hall et al., 1997). Dietary Pb concentrations were chosen to mimic environmentally relevant concentrations in terms of those found in benthic invertebrates at both contaminated and uncontaminated sites in the environment (0–792 ug Pb/g dw; Woodward et al., 1994; Farag et al., 1999). In our study, dietary Pb accumulation occurred in a time-dependent manner in body wall and intestine; moreover, the order of Pb concentration in three tissues was body wall > intestine > respiratory tree. The high Pb burden in the intestine in this study may be due to binding of Pb from the diet by mucus whose secretion may be stimulated by dietary metals (Glover and Hogstrand, 2002). The gill in fish is one of the most vital organs to function gaseous exchange, ionic transport, acid–base regulation, and nitrogenous waste excretion, as well as detoxification (Goss et al., 1998). The accumulation of gill in aquatic animals by the dietary metal exposure induces a switch from the bloodstream to the basolateral cell membranes (Szebedinszky et al., 2001); it is similar to the respiratory tree of sea cucumbers. It is commonly accepted that the metabolic activities of tissues in aquatic animals affect metal accumulation in tissues (Shukla et al., 2007). Alves et al. (2006) exported that the order of Pb concentration in specific tissues was intestine > carcass > kidney > liver > gills when juvenile rainbow trout were fed commercial trout pellets amended with various levels of Pb (7–520 ug Pb/g) for 21 days. The profile of tissue Pb accumulation was kidney > liver > spleen > intestine > gill > muscle after 4 weeks exposure with the different levels of dietary Pb (30–240 mg Pb/kg diet) in juvenile rock fish (Kim and Kang, 2015). However, the concentrations of heavy metals (Pb, Zn, Cu, and Cd) in the chicken tissues and feces were in the sequence: feces > liver > muscle > blood (Zhuang et al., 2009). Canli and Atli
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(2003) studied the relationships between heavy metal (Cd, Cr, Cu, Fe, Pb, and Zn) levels and the size of six Mediterranean fish species (Sparus auratus, Atherina hepsetus, Mugil cephalus, Trigla cuculus, Sardina pilchardus and Scomberesox saurus) showed that metal concentrations (as ug/g d.w.) were highest in the liver, except for Fe in the gill of S. saurus and lowest in the muscle of all the fish species. It was consistent with Martins et al. (2011), the effect of recirculating aquaculture systems on the concentrations of heavy metals in culture water and tissues of Nile tilapia O. niloticus, which reported that the accumulation of heavy metals (Al, As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) was always higher in the liver than in the muscle for 71 days (Martins et al., 2011). Moreover, Jebali et al. (2014) reported that notable differences in Cd, Pb, Zn, Mn, and Fe accumulation patterns within the digestive gland, gills and muscle of bivalve Pinna nobilis were found and this may be due to the ability of each tissue to accumulate metals. It is known that several factors have the potential to significantly affect metal concentrations such as changes in body weight during long-term exposure experiments in field that can have an influence on accumulated metals. Some studies seem to indicate that most of the seasonal variability may be due to changes in animal body weight, since total body burdens remained largely unchanged (Amiard et al., 1986). However, growth rate, age and body size can still not only significantly influence concentrations but can also affect bioaccumulation of metals in Mytilus edulis (Mubiana et al., 2006). Lead-induced disruption of prooxidant/antioxidant balance or oxidative stress in blood and other soft tissues has been postulated to be the major mechanism of Pb-associated tissue injury (El-Neweshy and El-Sayed, 2011). Previous report demonstrated that Pb can intensify liver lipid peroxidation and causes oxidative stress (Sun and Wang, 2007), inducing the generation of reactive oxygen species (ROS) (Gurer and Ercal, 2000) and inhibiting the activity of many antioxidant enzymes (Sidhu and Nehru, 2004). However, the level of antioxidant enzyme is a good indicator for the impacts of pollutants like heavy metals. One of the most important biochemical parameters of those toxicological effects is the SOD level of tissues. SOD is called the first line of the cell against ROS due to the superoxide radical being a precursor to several other highly reactive species (Fridovich, 1997). In particular, SOD works to catalyze dismutation of the superoxide anion to H2 O and H2 O2 (Shao et al., 2012). This trial showed that SOD activity and GSHPx activity of Pb dietary groups (100–1000 mg Pb/kg) were lower than the control group without Pb dietary and MDA levels were higher compared with control group in body wall of sea cucumber after 30 days. This is similar to Ates et al. (2008) who reported that SOD activity and GSH-Px activity were found to be lower and MDA levels were higher compared with control group in liver, spleen, heart and brain tissues of rainbow trout after 72-h of exposure to 2 ppm Pb2+ . Wang et al. (2011) also reported that administration of Pb decreased the activities of SOD and increased the MDA content in 30-day-old mice. Exposure to Pd has been reported to enhance oxidative stress and causes hepatotoxicity and immunotoxicity (Ashry et al., 2010), and under oxidative stress, the GSH-related enzymes merely catalyze reactions to detoxify peroxides in the water phase by reacting them with GSH (Moniuszko-Jakoniuk et al., 2007). Thus, the enhanced concentration of MDA and severe depletion in GSH activity suggests that the increased peroxidation is a consequence of depleted GSH stores and diminished GSH-related enzymes, which are otherwise capable of moderating the degree of lipid peroxidation. Pb is not able to induce the production of free radicals directly, but it indirectly does influence the processes of lipid peroxidation (Patra et al., 2001; Bandhu et al., 2006; Massó et al., 2007) In conclusion, this study has shown that dietary supplementation with Pb (0–1000 mg Pb/kg dw) in sea cucumbers appeared to be not effective on growth performance, but increases the Pb
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content in the body wall, intestine and respiratory tree, and lowering antioxidant enzymes activities. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements This work was funded by the Public Science and Technology Research Funds Project of Ocean of the State Oceanic Administration of the People’s Republic of China (Grant No. 201405003) and the Science and Technology Research Funds Project of the Dalian City Oceanic and Fishery Administration, Liaoning Province, China (Grant No. 20140101). References Alves, L.C., Glover, C.N., Wood, C.M., 2006. Dietary Pb accumulation in juvenile freshwater rainbow trout (Oncorhynchus mykiss). Arch. Environ. Contam. Toxicol. 51, 615–625. Alves, L.C., Wood, C.M., 2006. The chronic effects of dietary lead in freshwater juvenile rainbow trout (Oncorhynchus mykiss) fed elevated calcium diets. Aquat. Toxicol. 78, 217–232. Amiard, J.C., Amiard-Triquet, C., Berthet, B., Métayer, C., 1986. Contribution to the ecotoxicological study of cadmium, lead, copper and zinc in the mussel Mytilus edulis: I. Field study. Mar. Biol. 90, 425–431. Ashraf, W., 2005. Accumulation of heavy metals in kidney and heart tissues of Epinephelus microdon fish from the Arabian Gulf. Environ. Monit. Assess. 101, 311–316. Ashry, K.M., El-Sayed, Y.S., Khamiss, R.M., El-Ashmawy, I.M., 2010. Oxidative stress and immunotoxic effects of lead and their amelioration with myrrh (Commiphora molmol) emulsion. Food Chem. Toxicol. 48, 236–241. Ates, B., Orun, I., Talas, Z.S., Durmaz, G., Yilmaz, I., 2008. Effects of sodium selenite on some biochemical and hematological parameters of rainbow trout (Oncorhynchus mykiss Walbaum, 1792) exposed to Pb2+ and Cu2+ . Fish Physiol. Biochem. 34, 53–59. Balic, I.M., Milinkovic-Tur, S., Samardzija, M., Vince, S., 2012. Effect of age and environmental factors on semen quality, glutathione peroxidase activity and oxidative parameters in simmental bulls. Theriogenology 78, 423–431. Bandhu, H.K., Dani, V., Gary, M.L., Dhawan, D.K., 2006. Hepatoprotective role of zinc in lead-treated, protein-deficient rats. Drug Chem. Toxicol. 29, 11–24. Bayatli, F., Akkus, D., Kilic, E., Saraymen, R., Sonmez, M.F., 2013. The protective effects of grape seed extract on MDA, AOPP, apoptosis and eNOS expression in testicular torsion: an experimental study. World J. Urol. 31, 615–622. Burden, V.M., Sandheinrich, M.B., Caldwell, C.A., 1998. Effects of lead on the growth and §-aminolevulinic acid dehydratase activity of juvenile rainbow trout, Oncorhynchus mykiss. Environ. Pollut. 101, 285–289. Canli, M., Atli, G., 2003. The relationships between heavy metal (Cd, Cr, Cu, Fe, Pb, Zn) levels and the size of six Mediterranean fish species. Environ. Pollut. 121, 129–136. Davies, P.H., Goettl, J.P., Sinley, J.R., Smith, N.F., 1976. Acute and chronic toxicity of lead to rainbow trout Salmo gairdneri, in hard and soft water. Water Res. 10, 199–206. El-Sayed, Y.S., El-Neweshy, M.S., 2010. Impact of lead toxicity on male rat reproduction at “hormonal and histopathological levels”. Toxicol. Environ. Chem. 92, 765–774. El-Neweshy, M.S., El-Sayed, Y.S., 2011. Influence of vitamin C supplementation on lead-induced histopathological alterations in male rats. Exp. Toxicol. Pathol. 63, 221–227. Flora, S., Flora, G., Saxena, G., 2006. Chapter 4 – environmental occurrence, health effects and management of lead poisoning. Environ. Impact Health Eff., 158–228. Fang, F.C., 2004. Antimicrovial reactive oxygen and nitrogen species: concepts and controversies. Nat. Rev. Microbiol. 2, 820–832. Farag, A.M., Woodward, D.F., Brumbaugh, W., Goldstein, J.N., MacConnell, E., Hogstrand, C., Barrows, F.T., 1999. Dietary effects of metals contaminated invertebrates from the Coeuid’ Alene River, Idaho on cutthroat trout. Trans. Am. Fish Soc. 128, 578–592. Fridovich, I., 1997. Superoxide anion radical, superoxide dismutase and related matters. J. Biol. Chem. 250, 18515–18517. Glover, C.N., Hogstrand, C., 2002. In vivo characterisation of intestinal zinc uptake in freshwater rainbow trout. J. Exp. Biol. 205, 141–150. Goss, G.G., Perry, S.F., Fryer, J.N., Laurent, P., 1998. Gill morphology and acid–base regulation in freshwater fishes. Comp. Biochem. Physiol. A 119, 107–115. Gurer, H., Ercal, N., 2000. Can antioxidants be beneficial in the treatment of lead poisoning? Free Radic. Biol. Med. 29, 927–945. Hall, B.D., Bodaly, R.A., Fudge, R.J.P., Rudd, J.W.M., Rosenberg, D.M., 1997. Food as the dominant pathway of methylmercury uptake by fish. Water Air Soil Pollut. 100, 13–24.
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The effects of dietary lead on growth, bioaccumulation and antioxidant capacity in sea cucumber, Apostichopus japonicus Jing Wang a , Tongjun Ren a,∗ , Yuzhe Han a , Yang Zhao a , Mingling Liao b , Fuqiang Wang a , Zhiqiang Jiang a a b
Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea, Ministry of Agriculture, Dalian Ocean University, Dalian 116023, China State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361000, China
a r t i c l e
i n f o
Article history: Received 28 May 2015 Received in revised form 7 August 2015 Accepted 8 August 2015 Available online 12 August 2015 Keywords: Lead Sea cucumber Growth performance Bioaccumulation Antioxidant capacity
a b s t r a c t Three different diets amended with lead nitrate [Pb(NO3 )2 ] (100, 500 and 1000 mg Pb/kg dry weight) and a Pb-free control diet (1.03 mg Pb/kg dry weight) were fed to sea cucumber (Apostichopus japonicus) for 30 days. The patterns of Pb accumulation over time were determined in various tissues (body wall, intestine and respiratory tree), as well as growth performance and antioxidant enzymes activities. Pb accumulation in body wall and intestine increased with time in all dietary Pb treatments. When fed the highest Pb diet, the body wall exhibited the greatest Pb burden (16.37 mg Pb/kg tissue wet weight), while Pb content in the intestine (2.68 mg Pb/kg tissue wet weight) and the respiratory tree (1.78 mg Pb/kg tissue wet weight) were lower than Pb content in the body wall by day 30. The body weight gain (BWG), specific growth rate (SGR) and survival rate (SR) had not been affected by 30 days oral administration of Pb supplemented diet. However, the antioxidant enzymes activities [superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px)] of test groups were lower than control group in body wall and malondialdehyde (MDA) concentration in the body wall was opposite after 30 days in sea cucumbers. In summary, this work reports toxic effects in sea cucumber, A. japonicus, after dietary exposure to Pb. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Sea cucumber is a very important species in northern coastal areas in China due to its high nutritional value and desirable taste. It is also important for the coastal economy and for ecosystems. Sea cucumbers generally have slow rates of population turnover and feed on scraps found mixed in sediments (Purcell et al., 2010). However, chronic contamination of aquatic environment by heavy metals is a severe problem since these pollutants invariably persist in the environment (Velez and Montoro, 1998; Fang, 2004; Ashraf, 2005). Heavy metal contamination enters the aquatic environment from farms, urban and industrial production sites, and cause long-term eco-toxicological effects (Strmack and Braunbeck, 2000). Lead (Pb) is a cumulative, non-essential toxic element that has neither beneficial nor desirable nutritional effects on animals. However, due to many of its physical and chemical properties such as softness, malleability, ductility, poor conductivity and resistance to corrosion (Paul et al., 2014), Pb has been widely used in several industrial processes such as clothe tinge, varnish, pesticide,
∗ Corresponding author at: 52 Heishijiao Street, Dalian 116023, China. E-mail address: sea [email protected] (T. Ren). http://dx.doi.org/10.1016/j.etap.2015.08.012 1382-6689/© 2015 Elsevier B.V. All rights reserved.
explosives, batteries and painting manufacturing (Johnson, 1998) and could be responsible for death or sublethal changes in reproduction, growth and behavior of the fishes (Burden et al., 1998). Continuous exposure to Pb in the environment and occupational may cause renal, nervous, hepatic, hematological and reproductive damage in animals, including humans (Flora et al., 2006; El-Sayed and El-Neweshy, 2010; Ashry et al., 2010). Today, it is widely accepted that even small quantities of Pb are harmful to humans and animals (Ashry et al., 2010). Pb may disturb the antioxidant barrier via inhibition of the functional sulfhydryl (SH) groups present also in free radical-scavenging enzymes such as glutathione reductase (GR), glutathione peroxidase (GPx), glutathione s-transferase (GST), superoxide dismutase (SOD) catalase (CAT) and §-aminolevulinic acid dehydratase (ALAD) (Patra et al., 2001; Moniuszko-Jakoniuk et al., 2007; Olaleye et al., 2007). Weber et al. (1991) and Weber (1996) have previously reported on the effects of short-term exposure to Pb on the feeding abilities of juvenile fathead minnows (Pimephales promelas) and Pb may cause metabolic imbalances in juvenile fish. A number of studies have shown that significant gill and renal lead burden occurs after both acute exposure and chronic exposure to sublethal concentrations of waterborne lead (Davies et al., 1976; Hodson et al., 1978; Rogers et al., 2003). Moreover, some studies had showed
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that contaminants like lead can affect the phagocytic activity of macrophages in Oreochromis niloticus (Omima and Aboud, 2010) and the general immune status of Channa puncratus (Paul et al., 2014). There is no published research on the effects of dietary Pb on sea cucumbers (Apostichopus japonicus). So, the present study was conducted to determine the effects of dietary Pb on growth performance, bioaccumulation in tissues and antioxidant ability in sea cucumber to four Pb concentrations for 30 days in an attempt to explore the relationship between Pb exposure and bioaccumulation in sea cucumbers. 2. Materials and methods 2.1. Test diets The basal ingredients, Pb concentration and proximate composition of the treatment diets were shown in Table 1. Pb content is 1.03 ± 0.5 mg/kg dry weight in the basal diets. Lead nitrate [Pb(NO3 )2 ] was supplemented separately to the basal diet to obtain four Pb levels, 0, 100, 500 and 1000 mg Pb/kg dry weight. All dry ingredients were mixed thoroughly. Tap water was added to the mash at 600 g/kg and the mixture was passed through a pelletizer (Pinzheng Equipment Co. Ltd., Changzhou, China) with a 1.8-mmdiameter. Diets were then dried in a thermostatically controlled hot air oven (Jinghong Test Equipment Co. Ltd., Shanghai, China) at 40 ◦ C (Tewary and Patra, 2008) and kept in a refrigerator at −20 ◦ C until used. 2.2. Feeding trial Sea cucumber juveniles used in this experiment were obtained from the Yanhua Factory of Dalian (Dalian Yanhua Group Co. Ltd., Dalian, China) and transported to the Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea, Ministry of Agriculture of Dalian Ocean University (Dalian, China). Sea cucumber juveniles were fed with control diet for 2 weeks before the start of the experiment to acclimate the sea cucumbers to the diets and to the experimental conditions. Sea cucumbers (240 N, 10.06 ± 0.02 g) were then randomly allocated into 40-L tanks at a density of 20 sea cucumbers per tank with 24 h air supply to maintain dissolved oxygen near saturation, with three replicates in each treatment. The experiment was performed in a static aquarium system. Water temperature ranged from 16.0 to 18.0 ◦ C. Room temperature ranged from 18.0 to 20.0 ◦ C. The pH was 7.9–8.1 and the salinity was 31–32. Sea cucumbers were fed at an amount of 3% of body weight (Wang et al., 2009) once a day at 16:00. To ensure water quality, 50% of the water was replaced every day and residual diet and feces were removed using a siphon before feeding. The experiment lasted for 30 days. Sea cucumbers were sampled on days 10, 20, and 30. On each sampling date, five randomly selected sea cucumbers from each tank were dissected on ice to obtain the body wall, intestine, and respiratory tree. Tissues were stored at −80 ◦ C immediately for later determination of Pb concentrations. 2.3. Growth performance At the end of the experiment, sea cucumbers were starved for 24 h, and then every sea cucumber was counted and weighed, individually, to determine final body weight (FBW), body weight gain (BWG), specific growth rate (SGR) and survival rate (SR). Growth parameters were calculated as follows: BWG (%) = 100 × (final mean weight − initial mean weight)/initial mean weight; SGR (%/day) = 100 × (ln final mean weight − ln initial mean weight)/no.
of days; SR (%) = 100 × (final sea cucumber number)/(initial sea cucumber number). 2.4. Pb contents in tissues Pb was extracted from the tissues of the sea cucumbers by acid digestion using a method modified from Nakayama et al. (2011). Briefly, 2000 mg of respiratory tree and intestine sample were separately digested using concentrated nitric acid in a 100 ◦ C water bath until the liquid was clear. The volume of the digested samples was standardized to 25-mL using distilled water for measurements in a polarized Zeeman atomic absorption spectrophotometer (Hitachi Z-2010, Tokyo, Japan). The Pb was measured in the graphite furnace mode and the concentrations in the tissues were expressed as ug/g. 2.5. Antioxidant ability analysis Six sea cucumbers per treatment (two sea cucumbers from each tank) were selected and dissected on ice to obtain the body wall for determination of antioxidant ability on day 30. Body wall samples were thoroughly homogenized using physiological saline in ice-cold homogenizers and centrifuged immediately at 3000 rpm at 4 ◦ C for 10 min. The supernatant fluid was collected separately and stored in a refrigerator at 4 ◦ C in 1.5-mL Eppendorf tubes for subsequent analysis. The SOD activity was measured by the ability to inhibit superoxide anion generated by xanthine and the xanthine oxidase reaction system according to Wang and Chen (2005) using a SOD detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Superoxide anion radicals, produced during the reaction of xanthine with O2 catalyzed by xanthine oxidase, react with hydroxylamine producing nitric ions. After the reaction of nitric ions with naphthalene diamine, sulfanilic acid produces a colored product, which is proportional to the amount of produced superoxide anion radical. The absorbance is assayed at 550 nm (Wang et al., 2011). One unit (U) of SOD activity was defined as the amount required for inhibiting the rate of xanthine reduction by 50% in a 1-mL reaction system and specific activity was expressed as SOD unit per ml serum (Lin et al., 2011). The activity of GSH-Px in the body wall was assayed using the glutathione peroxidase kit by the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). GSH-Px catalyzes glutathione oxidation (GSH) with hydrogen peroxide and cumene hydroperoxide. In the presence of glutathione reductase and simultaneous triphosphopyridine nucleotide (NADPH), the oxidized form of glutathione (GSSG) immediately converts to its reduced form with simultaneous NADPH oxidation. The concentration of GSH-Px was assessed from the decrease in absorption at 340 nm due to the oxidation of NADPH to NADP+ (Balic et al., 2012). The malondialdehyde (MDA) concentration was determined using a test kit with a method established by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). It was measured by the thiobarbituric acid-reactive substance assay. The method is based on the measurement of the concentration of pink chromogen compound that forms when MDA reacts with thiobarbituric acid (Bayatli et al., 2013). MDA concentration was measured at 532 nm. The MDA content was expressed in units per milligram protein. 2.6. Statistical analysis Results are presented as means ± SD. One-way analysis of variance (ANOVA) was performed to determine statistical differences among groups. When overall differences were significant (P < 0.05), Duncan’s multiple range test was used to compare significant
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Table 1 Ingredients composition and proximate analysis of the experimental diets (g/kg). Ingredients
Sargassum thunbergiia Vitamin mixtureb Mineral mixturec Flourd Fish meale Soybean mealf Guar gum Microcrystalline celluloseg Pb(NO3 )2 h Total Analyzed nutrients (% in dry basis) Moisture Ash Crude protein Crude lipid
Treatments 0 mg Pb/kg
100 mg Pb/kg
500 mg Pb/kg
1000 mg Pb/kg
700 20 20 50 90 60 20 40 0 1000
700 20 20 50 90 60 20 39.9 0.1 1000
700 20 20 50 90 60 20 39.5 0.5 1000
700 20 20 50 90 60 20 39.0 1.0 1000
9.33 ± 0.12 32.27 ± 0.01 14.35 ± 0.04 7.07 ± 0.74
7.49 ± 0.10 32.83 ± 0.01 14.64 ± 0.01 4.25 ± 0.33
7.82 ± 0.05 32.40 ± 0.01 14.20 ± 0.16 5.99 ± 1.14
10.04 ± 0.03 32.12 ± 0.02 14.73 ± 0.18 5.24 ± 0.77
a
Sargassum thunbergii: the protein content is 10%. Longyuan Fishmeal Inc., Dalian, China. Vitamin mixture (per kilogram of premix): vitamin A, 1,000,000 IU; vitamin D3 , 300,000 IU; vitamin E, 4000 IU; vitamin K3 , 1000 mg; vitamin B1 , 2000 mg; vitamin B2 , 1500 mg; vitamin B6 , 1000 mg; vitamin B12 , 5 mg; nicotinic acid, 1000 mg; vitamin C, 5000 mg; Ca pantothenate, 5000 mg; folic acid, 100 mg; inositol, 10,000 mg; carrier glucose; H2 O ≤ 10%. c Mineral mixture (0.025 mg/g of premix): NaCl, 107.79; MgSO4 ·7H2 O, 380.02; NaHPO4 ·2H2 O, 241.91; KH2 PO4 , 665.20; Ca (H2 PO4 )·2H2 O, 376.70; Fe citrate, 82.38; Ca lactate, 907.10; Al(OH)3 , 0.52; ZnSO4 ·7H2 O, 9.90; CuSO4 , 0.28; MnSO4 ·7H2 O, 2.22; Ca(IO3 )2 , 0.42; CoSO4 ·H2 O, 2.77. d Flour: the protein content is 18%. New Market Co., Dalian, China. e Fish meal: the protein content is 65%. Longyuan Fishmeal Inc., Dalian, China. f Soybean meal: the protein content is 40%. g Keyuan Industrial Co. Ltd., Shanghai, China. h Lead nitrate [Pb(NO3 )2 ]. b
differences between treatments. Statistical analyses were performed using SPSS 13.0 for Windows (SPSS Inc, Chicago, IL). 3. Results 3.1. Growth performance After 30 days, the effect of dietary Pb supplementation on growth performance is found in Table 2. No significant differences (P > 0.05) were observed in SGR or BWG among all treatment groups. In addition, high survival (>98%) was observed in all dietary treatment groups. 3.2. Pb content in tissues At all dietary Pb doses, Pb accumulation is seen in the body wall (Fig. 1), intestine (Fig. 2) and respiratory tree (Fig. 3) during the course of the 30 days experiment. The body wall showed a substantial accumulation with a burden of 16.37 ug Pb/g tissue wet weight in the 1000 mg Pb/kg exposure, which was 9× greater than the respiratory tree (1.78 ug Pb/kg) on day 30. The body wall (16.37 ug Pb/g tissue wet weight), intestine (2.68 ug Pb/kg tissue wet weight), and respiratory tree (1.78 ug Pb/kg tissue wet weight) all exhibited the highest Pb burdens on day 30. Pb burden in the body wall (Fig. 1) and intestine (Fig. 2) increased with time in all dietary Pb treatments. The Pb accumulation depended upon the dietary Pb concentration and exposure periods. After 30 days of the dietary Pb exposure, the profile of tissue Pb accumulation was body wall > intestine > respiratory tree.
(P < 0.05) lower than the control group and there were no significant (P > 0.05) differences between control group and 100 mg Pb/kg treatment. The T-AOC capability was similar to the SOD activity, and the three treatments significantly (P < 0.05) lower than the control group and there were no significant differences (P > 0.05) among the three treatments. In contrast, MDA contents of the three treatments in body wall were significantly (P < 0.05) higher than the control group, but there were no significant (P > 0.05) differences among the three treatments. 4. Discussion This experiment demonstrated Pb-induced effects on growth performance, Pb contents and enzymes activities in sea cucumbers. The present study investigated that dietary exposure to Pb at these measured concentrations (100, 500, and 1000 mg Pb/kg) had few
3.3. Enzymes activities and MDA content Enzymes activities and MDA content in the body walls of sea cucumbers in each treatment after 30 days are shown in Table 3. SOD activities of the three treatments were significantly (P < 0.05) lower than the control group and there were no significant (P > 0.05) differences among the three treatments. GSH-Px activities of 500 mg Pb/kg, and 1000 mg Pb/kg treatments were significantly
Fig. 1. The Pb content in the body wall. Note: values are mean ± SD. Different letters are significantly different (P < 0.05). A/B/C denote the difference of the control group on 10, 20, 30 days. a/b/c denote the difference of the 100 mg Pb/kg group on 10, 20, 30 days. A’/B’/C’ denote the difference of the 500 mg Pb/kg group on 10, 20,30 days. a’/b’/c’ denote the difference of the 1000 mg Pb/kg group on 10, 20, 30 days.
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Table 2 Effects of dietary Pb on the growth performance of sea cucumber. Treatments
0 mg Pb/kg
IBW (g) FBW (g) BWG (%) SGR (%/day) SR (%)
10.07 17.52 73.92 1.81 93.33
± ± ± ± ±
0.01 1.23 5.71 0.23 2.98
100 mg Pb/kg 10.06 17.51 74.00 1.81 98.33
± ± ± ± ±
0.02 0.92 5.22 0.17 2.98
500 mg Pb/kg 10.06 17.44 73.25 1.82 98.33
± ± ± ± ±
0.02 0.49 5.44 0.10 2.98
1000 mg Pb/kg 10.07 17.00 72.47 1.73 100
± ± ± ± ±
0.01 0.07 0.91 0.01 0
Note: values are expressed as mean ± SD. (n = 3): IBW: initial body weight; FBW: final body weight; BWG (body weight gain) = 100 × (final mean weight − initial mean weight)/initial mean weight; SGR (specific growth rate) = 100 × (ln final mean weight − ln initial mean weight)/no. of days. Table 3 Anti-oxidative enzymes and total anti-oxidative ability in the body wall of sea cucumber. Treatments
0 mg Pb/kg
SOD GSH-Px MDA T-AOC
71.71 498.96 3.20 3.49
± ± ± ±
5.14b 4.72b 0.20a 0.36b
100 mg Pb/kg 53.14 310.27 4.03 2.48
± ± ± ±
3.47a 9.34ab 0.10b 0.23a
500 mg Pb/kg 51.99 348.40 4.04 2.26
± ± ± ±
5.04a 44.1a 0.30b 0.21a
1000 mgPb/kg 47.63 396.04 4.13 2.13
± ± ± ±
5.50a 41.60a 0.25b 0.27a
Note: values are expressed as mean ± SD. (n = 3): different superscript letters within each row indicate significant differences (P < 0.05).
Fig. 2. The Pb content in the intestine of sea cucumber. Note: values are mean ± SD. Different letters are significantly different (P < 0.05). A/B/C denote the difference of the control group on 10, 20, 30 days. a/b/c denote the difference of the 100 mg Pb/kg group on 10, 20, 30 days. A’/B’/C’ denote the difference of the 500 mg Pb/kg group on 10, 20,30 days. a’/b’/c’ denote the difference of the 1000 mg Pb/kg group on 10, 20, 30 days.
Fig. 3. The Pb content in the respiratory tree of sea cucumber. Note: values are mean ± SD. Different letters are significantly different (P < 0.05). A/B/C denote the difference of the control group on 10, 20, 30 days. a/b/c denote the difference of the 100 mg Pb/kg group on 10, 20, 30 days. A’/B’/C’ denote the difference of the 500 mg Pb/kg group on 10, 20,30 days. a’/b’/c’ denote the difference of the 1000 mg Pb/kg group on 10, 20, 30 days.
apparent adverse effects on BWG and SGR in sea cucumbers during the 30 days experiment. These results are consistent with Alves et al. (2006) who found no effects on growth and survival when juvenile rainbow trout were fed commercial trout pellets amended with various levels of Pb (7–520 ug Pb/g) for 21 days, and Alves and Wood (2006) also found chronic dietary exposure in the range of 50–500 ug Pb/g that resulted in no significant effects on the growth and survival of juvenile rainbow trout over 42 days. The study about bioaccumulation patterns in fish tissues exposed to metals can be used as effective and reliable indicators of environmental metal pollution (Kim et al., 2004). The accumulation in aquatic animals has been occurring through different mechanisms via the direct uptake from water by gills and ingestion from food by intestine (Oost et al., 2003). Between two pathways, the metal accumulation in fish was mainly from their diet (Hall et al., 1997). Dietary Pb concentrations were chosen to mimic environmentally relevant concentrations in terms of those found in benthic invertebrates at both contaminated and uncontaminated sites in the environment (0–792 ug Pb/g dw; Woodward et al., 1994; Farag et al., 1999). In our study, dietary Pb accumulation occurred in a time-dependent manner in body wall and intestine; moreover, the order of Pb concentration in three tissues was body wall > intestine > respiratory tree. The high Pb burden in the intestine in this study may be due to binding of Pb from the diet by mucus whose secretion may be stimulated by dietary metals (Glover and Hogstrand, 2002). The gill in fish is one of the most vital organs to function gaseous exchange, ionic transport, acid–base regulation, and nitrogenous waste excretion, as well as detoxification (Goss et al., 1998). The accumulation of gill in aquatic animals by the dietary metal exposure induces a switch from the bloodstream to the basolateral cell membranes (Szebedinszky et al., 2001); it is similar to the respiratory tree of sea cucumbers. It is commonly accepted that the metabolic activities of tissues in aquatic animals affect metal accumulation in tissues (Shukla et al., 2007). Alves et al. (2006) exported that the order of Pb concentration in specific tissues was intestine > carcass > kidney > liver > gills when juvenile rainbow trout were fed commercial trout pellets amended with various levels of Pb (7–520 ug Pb/g) for 21 days. The profile of tissue Pb accumulation was kidney > liver > spleen > intestine > gill > muscle after 4 weeks exposure with the different levels of dietary Pb (30–240 mg Pb/kg diet) in juvenile rock fish (Kim and Kang, 2015). However, the concentrations of heavy metals (Pb, Zn, Cu, and Cd) in the chicken tissues and feces were in the sequence: feces > liver > muscle > blood (Zhuang et al., 2009). Canli and Atli
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(2003) studied the relationships between heavy metal (Cd, Cr, Cu, Fe, Pb, and Zn) levels and the size of six Mediterranean fish species (Sparus auratus, Atherina hepsetus, Mugil cephalus, Trigla cuculus, Sardina pilchardus and Scomberesox saurus) showed that metal concentrations (as ug/g d.w.) were highest in the liver, except for Fe in the gill of S. saurus and lowest in the muscle of all the fish species. It was consistent with Martins et al. (2011), the effect of recirculating aquaculture systems on the concentrations of heavy metals in culture water and tissues of Nile tilapia O. niloticus, which reported that the accumulation of heavy metals (Al, As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) was always higher in the liver than in the muscle for 71 days (Martins et al., 2011). Moreover, Jebali et al. (2014) reported that notable differences in Cd, Pb, Zn, Mn, and Fe accumulation patterns within the digestive gland, gills and muscle of bivalve Pinna nobilis were found and this may be due to the ability of each tissue to accumulate metals. It is known that several factors have the potential to significantly affect metal concentrations such as changes in body weight during long-term exposure experiments in field that can have an influence on accumulated metals. Some studies seem to indicate that most of the seasonal variability may be due to changes in animal body weight, since total body burdens remained largely unchanged (Amiard et al., 1986). However, growth rate, age and body size can still not only significantly influence concentrations but can also affect bioaccumulation of metals in Mytilus edulis (Mubiana et al., 2006). Lead-induced disruption of prooxidant/antioxidant balance or oxidative stress in blood and other soft tissues has been postulated to be the major mechanism of Pb-associated tissue injury (El-Neweshy and El-Sayed, 2011). Previous report demonstrated that Pb can intensify liver lipid peroxidation and causes oxidative stress (Sun and Wang, 2007), inducing the generation of reactive oxygen species (ROS) (Gurer and Ercal, 2000) and inhibiting the activity of many antioxidant enzymes (Sidhu and Nehru, 2004). However, the level of antioxidant enzyme is a good indicator for the impacts of pollutants like heavy metals. One of the most important biochemical parameters of those toxicological effects is the SOD level of tissues. SOD is called the first line of the cell against ROS due to the superoxide radical being a precursor to several other highly reactive species (Fridovich, 1997). In particular, SOD works to catalyze dismutation of the superoxide anion to H2 O and H2 O2 (Shao et al., 2012). This trial showed that SOD activity and GSHPx activity of Pb dietary groups (100–1000 mg Pb/kg) were lower than the control group without Pb dietary and MDA levels were higher compared with control group in body wall of sea cucumber after 30 days. This is similar to Ates et al. (2008) who reported that SOD activity and GSH-Px activity were found to be lower and MDA levels were higher compared with control group in liver, spleen, heart and brain tissues of rainbow trout after 72-h of exposure to 2 ppm Pb2+ . Wang et al. (2011) also reported that administration of Pb decreased the activities of SOD and increased the MDA content in 30-day-old mice. Exposure to Pd has been reported to enhance oxidative stress and causes hepatotoxicity and immunotoxicity (Ashry et al., 2010), and under oxidative stress, the GSH-related enzymes merely catalyze reactions to detoxify peroxides in the water phase by reacting them with GSH (Moniuszko-Jakoniuk et al., 2007). Thus, the enhanced concentration of MDA and severe depletion in GSH activity suggests that the increased peroxidation is a consequence of depleted GSH stores and diminished GSH-related enzymes, which are otherwise capable of moderating the degree of lipid peroxidation. Pb is not able to induce the production of free radicals directly, but it indirectly does influence the processes of lipid peroxidation (Patra et al., 2001; Bandhu et al., 2006; Massó et al., 2007) In conclusion, this study has shown that dietary supplementation with Pb (0–1000 mg Pb/kg dw) in sea cucumbers appeared to be not effective on growth performance, but increases the Pb
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