Introduction

Heavy metals are widely used in agriculture, chemical, and industrial processes. Most heavy metals are biological non-essential elements [1]. Lead (Pb) is one of most ubiquitous and toxic elements in the aquatic environment, and belongs to a group of toxic metals, which have no function in the physiological processes of organisms [2, 3]. Pb can exist in water for a long time without being decomposed, so it becomes a source of pollution and present risks to human beings and other living organisms [4]. Many studies have reported that Pb is highly concentrated in the environment and even exceeds 90 mg/kg in the marine sediments [5, 6].

At low concentrations of Pb in the aquatic environment, omnivorous fish, including common carp and C. gibelio, are more likely to accumulate higher levels of Pb in tissues [7]. Pb accumulation causes hematopoietic system dysfunction [8], oxidative stress [9], immune suppression [10], neurological dysfunction [11, 12], and even death (at high concentrations). One of the most important Pb toxicity mechanisms is the hematopoietic system dysfunction, which decreased the red blood cell (RBC) count and hemoglobin concentrations [13]. In addition, many studies have reported that Pb induces oxidative stress [9, 10, 14]. Pb exposure induces the production of reactive oxygen species (ROS), damages the cell membrane and increases the level of lipid peroxidation. Recently, many studies reported effects of lead exposure in mammals; however, there is currently limited information regarding Pb exposure in fish. Although a few studies reported that some substances (including ascorbic acid and Lactobacillus plantarum) can alleviate Pb toxicity [15,16,17], none has mentioned probiotics’ ability to alleviate Pb toxicity in fish. Therefore, it is essential to formulate strategies for safeguarding aquatic organisms against Pb toxicity.

For mammals or humans, the current therapeutic strategy for heavy metal poisoning involves heavy metal excretion by chelation therapy [18, 19]. However, this therapy is limited in aquatic animals like fish. Probiotics are living microorganisms which are beneficial to the host [20]. B. subtilis is a kind of probiotic, which was widely used in aquaculture [21]. It was suggested that B. subtilis stimulate antioxidant defense mechanisms, immune-related defense mechanisms, growth performance, and disease resistance [22, 23]. Compared with Lactobacillus plantarum, B. subtilis is able to survive in low pH environments and wider temperatures [22, 24]. Currently, no study has been carried out on the protective effects of B. subtilis against Pb toxicity in C. gibelio. However, B. subtilis may have the potency for Pb toxicity attenuation due to its ability in Pb tolerance. In this study, we analyzed the effects of Pb exposure and B. subtilis administration on bioaccumulation, hematological parameters, and antioxidant stress. The results suggested that B. subtilis alleviates Pb toxicity in C. gibelio.

Materials and Methods

Bacillus subtilis and Culture

The B. subtilis strain used in this study was obtained from C. gibelio intestine. The B. subtilis strain isolated was previously reported [25].The strain was cultivated in Luria-Bertani (LB) medium and grown under aerobic conditions at 30 °C with shaking (at 180 rpm) for 18 h.

Diet Preparation

Commercial feed (crude protein 37.7%, crude lipid 7.4%, and ash 10.8%) obtained from Jinyanhong Aquarium Products Co., Hangzhou, China was used as the basal diet. The experimental diet was formulated by supplementing the basal diet with B. subtilis (at a final dose of 109 cfu/g diet) and/or Pb (120 and 240 mg/kg). The Commercial feed which was made of powder was sifted through a 120-μm mesh. B. subtilis and/or Pb at specified concentrations were mixed thoroughly in cooled conditions and then pelleted with a hand pelletizer. The concentration of B. subtilis in the feed was determined by spread plate technique (nutrient agar incubated at 30 °C for 24 h). The control diet was prepared by adding the same volume of sterile saline to the basal diet. The prepared diets were stored at 4 °C before use. The actual dietary Pb concentration is showed in Table 1 using an atomic absorption spectrometer AA-6300 (Shimadzu, Japan).

Table 1 Analyzed and actual Pb concentration (mg/kg)
Full size table

Feed and Experimental Design

Three hundred and sixty C. gibelio (62.51 ± 0.42 g) acquired from a specialized aquatic fry farm (Jilin province, China) were used in the study. Fish were acclimatized for 2 weeks under laboratory conditions. During the acclimation period, fish were fed a Pb-free diet twice daily and constantly maintaining experimental conditions at all times (dissolved oxygen: 5.91 ± 0.21 mg/L;pH: 7.4 ± 0.2; ammonia: less than 0.5 mg/L; nitrites: less than 0.05 mg/L and temperature: 23 ± 2 °C). Two hundred and seventy healthy fish were randomly distributed into six groups with 3 replications each (15 fish per replicate). Each group was kept in 80-L plastic tanks. Each group was exposed to dietary Pb and/or B. subtilis. The groups were divided as follows: CK group (control), CB group (B. subtilis, 109 cfu/g), LP group (120 mg/kg Pb), LPB group (120 mg/kg Pb plus B. subtilis, 109 cfu/g), HP group (240 mg/kg Pb), and HPB group (240 mg/kg Pb plus B. subtilis, 109 cfu/g). The fish were fed twice daily (9:00 and 15:00) for 30 days at a daily rate of 3% body weight. Lead acetate (CH3COO)2 Pb·3H2O was purchased from the Sinopharm Chemical Reagent Company (Shanghai, China). All experiments and handling of the animals were conducted according to the research protocols approved by the Institutional Animal Care and Use Committee, Jilin Agricultural University.

Tissue Sampling

Fish were fasted 24 h before collecting samples. Tissue samples were collected on days 15 and 30 of the feeding period from 6 randomly selected fish from each tank. The fish were euthanized using 300 mg/L of methane-sulfonate − 222 (MS-222). Tissue samples from the gill, liver, muscle, kidneys, gut, and spleen were subsequently collected. The blood samples of fish were collected using heparin-treated syringes. The red blood cells (RBC) were analyzed by counting using a microscope. The blood was subsequently separated by centrifugation at 4000 g for 5 min. All samples were frozen in liquid nitrogen prior to storage in a − 80 °C freezer.

Lead Accumulation Analysis

Six tissues (gill, kidney, gut, liver, spleen, and muscle) from each group were sampled on days 15 and 30 to observe Pb concentration. All tissues were dried in an oven at 65 °C before analysis. Tissues were placed in a digestion vessel and mixed with 10 mL of concentrated nitric acid (65% HNO3). The digestion reaction occurred from 60 °C to a finial temperature of 95 °C in the hot plate until the samples were completely dissolved. The so-obtained clear liquid was diluted with deionized water to 20 mL and then assayed for concentration of lead using an atomic absorption spectrometer AA-6300 (Shimadzu, Japan).

Serum Assays

The δ-aminolevulinic acid dehydratase (ALAD) was analyzed using an Elisa kit according to the method described by Dai et al. [10].The hemoglobin concentrations were analyzed using a clinical kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China).

Antioxidant Response Analysis

The liver was examined for antioxidant activity on days 15 and 30 of the feeding period. Superoxide dismutase (SOD), catalase (CAT), total antioxidant (T-AOC), and glutathione (GSH) activities were determined using commercially available Elisa kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) according to the method described by Dai et al. [10].

Statistical Analysis

All data were subjected to analysis of variance, ANOVA followed by Turkey’s test and using the data analysis software SPSS 20.0 (SPSS, Chicago, IL, USA). The results were expressed as mean ± standard deviation (S.D) and differences with P values of < 0.05 were considered to be statistically significant.

Results

Pb Accumulation

The accumulation of Pb in the kidneys, liver, spleen, gut, gill, and muscle of C. gibelio exposed to dietary Pb and/or B. subtilis is presented in Fig. 1. In the present study, lead accumulation level in the organs was kidneys > liver > spleen > gut > gill > muscle. Compared with the Pb-only treatment groups, the Pb-B. subtilis groups significantly decreased (P < 0.05) organ (except muscle) Pb levels at days 15 and 30 (Fig. 1a, b, c, d, and e). Compared with the Pb-only treatment groups, Pb accumulation in the muscle significantly decreased (P < 0.05) following Pb-B. subtilis supplementation at 120 and 240 mg/kg at 15 days and at 240 mg/kg at 30 days (Fig. 1f).

Fig. 1
figure 1

Pb accumulation (kidneys (a), liver (b), spleen (c), gut (d), gill (e), muscle (f)) of C. gibelio (n = 6) after exposure to lead and/or B. subtilis. Data are expressed as the mean ± S.D. Bar with different letters are significantly (P < 0.05) different by Tukey test on the same sampling interval

Full size image

Hematological Parameters

Blood accumulation, ALAD activity, hemoglobin concentration, and RBC count of C. gibelio exposed to dietary Pb and/or B. subtilis are shown in Fig. 2. At 15 and 30 days, compared with the Pb-only treatment groups, Pb accumulation in the blood significantly decreased (P < 0.05) in the 120- and 240 mg/kg Pb-B. subtilis groups (Fig. 2a). Compared with the Pb-only treatment group, ALAD activity in the blood significantly increased (P < 0.05) in the 120 mg/kg Pb-B. subtilis treatment group at 15 and 30 days and in the 240 mg/kg Pb-B. subtilis treatment group at 30 days (Fig. 2b). Also, hemoglobin concentration in the blood significantly increased (P < 0.05) in the 120- and 240 mg/kg Pb-B. subtilis groups at 30 days, compared with the Pb-only treated groups (Fig. 2c). There were no significant differences in RBC count between the Pb-only and Pb-B. subtilis groups at all levels (P > 0.05) (Fig. 2d). However, RBC count significantly decreased compared with the control (P < 0.05).

Fig. 2
figure 2

Hematological parameters in the blood (blood accumulation (a), ALAD activity (b), hemoglobin concentration(c), and RBC count (d)) of C. gibelio (n = 6) after exposure to lead and/or B. subtilis. Data are expressed as the mean ± S.D. Bar with different letters are significantly (P < 0.05) different by Tukey test on the same sampling interval

Full size image

Antioxidant Response

SOD, GSH, CAT, and T-AOC activities of C. gibelio exposed to dietary Pb and/or B. subtilis are presented in Fig. 3. At 15 and 30 days, SOD and GSH levels in the liver were significantly increased in the 0,120, and 240 mg/kg Pb-B. subtilis treatment groups compared with the Pb-only groups (P < 0.05) (Fig. 3a, b). At 15 days, CAT activity in the liver significantly increased (P < 0.05) in the 240 mg/kg Pb-B. subtilis treatment group, while at 30 days, it increased significantly (P < 0.05) in the 120- and 240 mg/kg Pb-B. subtilis groups compared with the Pb-only treatment groups (Fig. 3c). There were no significant differences in T-AOC activity in the liver between the Pb-only and Pb-B. subtilis groups at 15 days. However, T-AOC activity decreased significantly (P < 0.05) in the 120- and 240 mg/kg Pb-B. subtilis groups at 30 days, compared with the Pb-only treatment groups (Fig. 3d).

Fig. 3
figure 3

Antioxidant capability in the liver (SOD (a), GSH (b), CAT (c), T-AOC (d)) of C. gibelio (n = 6) after exposure to lead and/or B. subtilis. Data are expressed as the mean ± S.D. Bar with different letters are significantly (P < 0.05) different by Tukey test on the same sampling interval

Full size image

Discussion

With industrial development, Pb has become abundant in the water environment. At a low concentration of Pb in water, the concentration of Pb can be increased in the tissues of aquatic animals through accumulation [26]. Uptake routes (whether via feeding and the digestive organs or in a dissolved form through the gills) of toxic substances affect tissue accumulation of toxicants in aquatic animals [27]. Alsop et al. demonstrated that the bioaccumulation pattern of waterborne lead in different tissues is gill > gut > liver [28]. However, Ewa et al. reported that the profile of Pb accumulation in the organs was kidneys > bone > liver > scales > gills > distal intestine > muscle > proximal > skin after long-term dietary Pb exposure [7]. Similar results were also observed in the rainbow trout [13] and in rockfish [28]. In the present study, our data suggest that the profile of Pb accumulation in tissues is kidneys > liver > spleen > gut > gill > muscle after dietary supplementation with Pb and/or B. subtilis. Many studies suggest that bacteria can tolerate heavy metals, including Cd, Cu, and Pb at high concentrations [15, 17]. Indeed, in this study, we observed that B. subtilis were reasonably tolerant to the high concentrations of Pb (data not shown). At the same time, compared with the same Pb-only treated groups, Pb-plus B. subtilis groups significantly decreased in organ Pb levels at 15 and 30 days. Our results suggest that B. subtilis may absorb Pb in the gut and thus reduced the organ Pb concentrations.

Toxic elements, including Pb, Cd, Hg, and As, have high blood toxicity, and many authors have reported hematological changes in aquatic animals exposed to various toxic elements [29,30,31]. In the present study, Pb concentration in the blood of C. gibelio was significantly increased following dietary Pb exposure. However, the B. subtilis supplementation was effective in attenuating the accumulation levels. Kim et al. suggested that blood can be a notable accumulation section because the absorbed metal in the organs (including gut, liver, and kidney) are transported through the bloodstream [13]. Pb is also known to inhibit heme biosynthesis at several enzymatic levels, as well as iron utilization [32]. The results of the current study determine that the ALAD activity and hemoglobin concentration were significantly increased following B. subtilis supplementation. Many reports suggested that secondary metabolites from probiotics exert beneficial effects on the host [33,34,35]. Chen et al. suggested that secondary metabolites from Bacillus Licheniformis can stimulate antioxidant defense mechanisms, growth performance, and disease resistance in common carp [36]. In the present study, we observed that ALAD activity was increased in the blood of fish in the Pb plus B. subtilis groups. This is probably because it caused the removal of ROS by secondary metabolites in the blood. Although there were no significant differences recorded in RBC count in fish following B. subtilis plus Pb exposure, increased hemoglobin concentrations were recorded. The increased hemoglobin concentration indicates that the function of red blood cell increased.

Among the various heavy metals, many studies have reported that Pb is a critical inducer of oxidative stress [10, 37]. The antioxidant system responses play an important role in the pollution effects in aquatic animals. Endogen antioxidant enzymes, including T-AOC, SOD, GSH, and CAT, are the first line of defense against ROS [38]. Many authors have suggested that lead can reduce the activity of antioxidant enzymes in liver tissues of fish [27, 39], and so induce oxidative stress. However, a few studies reported that B. subtilis show a good effect of increasing antioxidant enzyme activity [23]. Liu et al. suggested that B. subtilis HAINUP40 is able to survive in low pH and wider temperatures, and enhance SOD, AOC, and LZM activity in tilapia (Oreochromis niloticus) [24]. Recently, Zhai et al. determined that Lactobacillus plantarum CCFM8610 supplementation prevents the autooxidation chain reaction that is associated with amelioration of the Cd-induced toxin in rat and an enhancement in antioxidant enzyme activity, including increases in the activity of SOD, GPx, and CAT [16]. In a similar study, Tian et al. reported that Lactobacillus plantarum CCFM8661 alleviates Pb toxicity in mice, by increasing the antioxidant enzyme activity, decreasing the Pb accumulation in tissues, and reducing the oxidant stress of blood [15]. Lee et al. previously suggested that B. subtilis (at a dose of 108 cfu/g) might represent a more effective probiotic in aquaculture compared with Lactobacillus plantarum [40]. In the present study, SOD and GSH levels in the liver were significantly increased following B. subtilis supplementation at 0, 120, and 240 mg/kg. However, CAT and T-AOC activity in the liver were significantly changed following B. subtilis supplementation at 120 and 240 mg/kg. Previous studies have reported that the aforementioned probiotics enhance host antioxidant ability after at least 21 days of probiotic treatment [24]. In fact, B. subtilis were already reported to have antioxidant effects via changing antioxidant enzyme activity in fish. The antioxidant ability might be one of the main mechanisms through which B. subtilis alleviates Pb toxicity.

Conclusion

The results of this study suggest that B. subtilis can reduce Pb accumulation in organs, regulate hematological parameters (RBC count, ALAD activity, and hemoglobin concentration), and change antioxidant activity (SOD, CAT, T-AOC, and GSH activity), following Pb exposure in C. gibelio. These results indicate that B. subtilis has the potential to alleviate the effects of lead toxicity in aquaculture.