Chemosphere 128 (2015) 171–177
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Lead in the soil–mulberry (Morus alba L.)–silkworm (Bombyx mori) food chain: Translocation and detoxification Lingyun Zhou a,b, Ye Zhao a,⇑, Shuifeng Wang a,c, Shasha Han a, Jing Liu a a
State Key Laboratory of Water Environment Simulation, School of Environment, Beijing Normal University, Beijing 100875, China Fujian Province Key Laboratory of Modern Analytical Science and Separation Technology, College of Chemistry and Environment, Minnan Normal University, Zhangzhou 363000, China c Analytical Testing Center, Beijing Normal University, Beijing 100875, China b
h i g h l i g h t s Biomagnification of Pb in the soil–mulberry–silkworm system was not observed. Roots sequestered most of the Pb, most of the Pb in leaves bound to the cell wall. Excretion and homeostasis protect silkworms from Pb stress. Detoxification in mulberry–silkworm regulates Pb transfer along the food chain. Pb tolerance of mulberry and silkworm indicates the bioremediation potential.
a r t i c l e
i n f o
Article history: Received 26 March 2014 Received in revised form 23 January 2015 Accepted 24 January 2015
Handling Editor: Shane Snyder Keywords: Lead Translocation Detoxification Mulberry Silkworm
a b s t r a c t The translocation of lead (Pb) in the soil–mulberry–silkworm food chain and the process of Pb detoxification in the mulberry–silkworm chain were investigated. The amount of Pb in mulberry, silkworm, feces and silk increased in a dose-responsive manner to the Pb contents in the soils. Mulberry roots sequestered most of the Pb, ranging from 230.78 to 1209.25 mg kg 1. Over 92% of the Pb in the mulberry leaves was deposited in the cell wall, and 95.29–95.57% of the Pb in the mulberry leaves was integrated with oxalic acid, pectates and protein, and it had low bioavailability. The Pb concentrations in the silkworm feces were 4.50–4.64 times higher than those in the leaves. The synthesis of metallothioneins in three tissues of the silkworms was induced to achieve Pb homeostasis under Pb stress. These results indicated the mechanism involved in Pb transfer along the food chain was controlled by the detoxification of Pb in different trophic levels. Planting mulberry and rearing silkworm could be a promising approach for the remediation of Pb-polluted soils due to the Pb tolerance of mulberry and silkworm. Ó 2015 Published by Elsevier Ltd.
1. Introduction Lead (Pb) is a non-essential and toxic trace element in the environment. The soils in some locations are contaminated by Pb due to anthropogenic activities (Stafilov et al., 2010; Jin et al., 2015). Pb pollution in soils is covert, persistent and non biodegradable. Soils polluted by Pb could be a source of contamination for water and air (Laidlaw et al., 2012). Previous studies indicated that Pb was accumulated in the ecosystem through the food chains and posed a great risk to the environment and to human health ⇑ Corresponding author at: School of Environment, Beijing Normal University, No. 19, Xinjiekouwai Street, Haidian District, Beijing 100875, China. Tel./fax: +86 10 58802875. E-mail address: [email protected] (Y. Zhao). http://dx.doi.org/10.1016/j.chemosphere.2015.01.031 0045-6535/Ó 2015 Published by Elsevier Ltd.
(Lopes et al., 2011). However, previous studies regarding Pb transfer in terrestrial ecosystems found that the Pb concentration in higher plant or higher animals was lower than that in polluted soils (Notten et al., 2005; Roodbergen et al., 2008), even though the Pb concentrations steadily declined with increasing trophic levels along a soil–plant-insect-chicken food chain at a contaminated area (Zhuang et al., 2009). It is concluded from the previous studies that metal biomagnifications are not generally applicable to all environmental ecosystems and that accumulation in the food chain varies depending on the metal concentration, biological species and trophic level (Wang, 2002). Therefore, there is an urgent need to explore both the translocation of Pb in a soil ecosystem and the remediation of Pb-polluted soils (Peralta-Videa et al., 2009; Cao et al., 2011).
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Recent research suggested that organisms might induce a whole suite of tolerance mechanisms involving metal detoxification to cope with metal stress (Shao et al., 2010). It is increasingly accepted that the chemical speciation of non-essential metals influences the internal metal detoxification of organisms and the trophic transfer of metals along a food chain (van Gestel, 2008). It has been documented that heavy metals with inorganic and water-soluble forms in plants have higher activities (Wang et al., 2008) that enable them to be easily transferred to the next trophic level. Compared to plants that are sensitive to heavy metals, tolerant plants usually accumulate more non-essential metals in the cell wall and vacuoles to prevent them from injuring the more sensitive cell metabolic sites. The non-essential metals in the cell wall often are integrated with pectates and protein, in addition to becoming water insoluble (Wu et al., 2005; Weng et al., 2012). For animals, on the one hand, the intake of non- essential metals can be decreased by the strategy of ‘‘avoidance’’ actions (Zidar et al., 2004; Lukkari and Haimi, 2005), and on the other hand, a detoxification process involving ‘‘excretion’’ and ‘‘homeostasis’’ actions can be used to cope with metals stress (Ahearn et al., 2004). Thus, a mechanistic understanding of the trophic transfer of toxic metals benefits from the detoxification of organism; however, only a few studies have examined this process. Additionally, a considerable number of studies proposed that the use of some hard woody plants can be an alternative for the removal or stabilization of metals from contaminated soils (Pulford and Watson, 2003). Those woody plants with a moderate capacity to accumulate heavy metals in the aerial parts possess characteristics of rapid growth, deep and extensive root systems and high biomass production (Unterbrunner et al., 2007; Shukla et al., 2011). Similarly, mulberry exhibits the potential for remediating metal-polluted soils. Furthermore, mulberry, which is found from the tropics to the temperate regions and from sea level to altitudes as high as 4000 m, has superior attributes of high adaptability and wide distribution (Ercisli and Orhan, 2007). Previous studies identified that mulberry exhibits tolerance to many metals (Ashfaq et al., 2009; Zhao et al., 2013). Prince et al. (2001) commented on the mulberry–silkworm food chain as being a template to assess heavy metal mobility in terrestrial ecosystems. To date, few articles have reported in detail on the transfer of Pb in the soil–mulberry–silkworm system, on the Pb detoxification mechanisms of mulberry and silkworm, and on the influence of the latter to the former. Thus, the specific aims of this study were to (i) determine the suitability of mulberry–silkworm as an alternative method to the soil remediation of Pb pollution through an investigation of the accumulation and translocation of Pb in a soil–mulberry–silkworm system and to (ii) elucidate the underlying mechanisms controlling the trophic transfer of Pb along this food chain based on the identification of the process of Pb detoxification in mulberry and silkworm involving sub-cellular distribution and the chemical forms of Pb in mulberry leaves, the excretion of Pb and metallothionein level in silkworm.
transformation of Pb added as solutions in the soil could achieve equilibrium and better contact the different components of the soil. After approximately two months, NPK fertilizer was added to the soils in the form of ammonium sulfate (0.68 g kg 1 of soil), super phosphate (0.35 g kg 1 of soil) and muriate of potash (0.09 g kg 1 of soil) and homogenized. Ten kilograms of the prepared soil was added to each pot (26 cm in upper diameter and 28 cm in height). Three pots were replicated for all the Pb treatments. The one-year-old mulberry plant stocks (var. nongsang 14), which was a common breed and planted widely in silkworm breeding areas of China, used in this study were purchased from commercial market. Two plants were planted in each pot in May and irrigated with tap water. All the pots were placed randomly on outdoor grassland, and the plants grew under natural conditions. After three months (from May, 2013 to July, 2013), the silkworm breeding experiments were performed and some plant samples were collected for other experiments and analysis. The hybrid silkworm used in this study was named Qingfeng Mingyue, whose spawns were from the silkworm spawn station in Huzhou, Zhejiang Province, China. All the larvae were reared on fresh and uncontaminated mulberry leaves until their fourth molt. At the beginning of the fifth instar, 48 healthy larvae of uniform size were isolated and divided into 4 groups that fed on the mulberry leaves richened with Pb from the treatment soils. After five days, six larvae from each treatment were selected to be individually weighed and died in liquid nitrogen, and then were stored at 80 °C until analysis. The other silkworms of each treatment were allowed to grow normally and complete their life stages. The cocoons and the adults were collected, washed with distilled water and dried in an oven at 50 °C to a constant weight. Subsequently, these samples were used for analysis. 2.2. Sub-cellular distribution and the chemical forms of Pb in the leaves To determine the Pb contents at the subcellular level of the plant leaves from each treatment, differential centrifugation was used to separate the Pb, following the methods reported by Weigel and Jäger (1980) and Wang et al. (2009). The extraction of the chemical forms of Pb in mulberry leaves was performed according to the method described by Weng et al. (2012) and Li et al. (2013). 2.3. Chlorophyll extraction and determination The Chlorophyll in mulberry leaves were extracted by ethanol, the concentration was determined by spectrophotometry. The detail procedure is in Section S1 (see supplementary material). 2.4. Extraction and determination of metallothioneins (MTs) The detailed information is supplied in Section S2 (see supplementary material).
2. Materials and methods
2.5. Pb concentration determination
2.1. Soil preparation, plant materials, and animal materials
Four chemical forms of Pb in soils were extracted according to the modified BCR procedure (Rauret et al., 1999). All of the soils, plant materials, sub-cellular distribution fractions (except the soluble fraction), the residuals from the extraction of chemical forms, and animal materials were digested with a mixture of HNO3–HF– HClO4 (3:1:1 v/v) at 160 °C for 6 h. The digested mixture was cooled to room temperature and diluted to 10 mL with super purified water. Finally, the concentrations of Pb in all of the soluble samples were directly determined by ICP-AES. The quality control
Soil for the pot experiment was collected from a cleaning field in Zhangzhou, Fujian province, China, and was air-dried and then ground with a wooden roller to pass through a 2-mm sieve. The general properties of the soil are presented in Table S1 (see supplementary material). The initial soil was incubated with 0, 200, 400, and 800 mg Pb kg 1 added in the form Pb(NO3)2. The soils were submitted to two wetting and drying cycles to ensure that the
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and quality assurance of the Pb content in these samples was based on the standard reference materials (GSS-1 (geochemical standard soil) and GBW07602 (stems and leaves of brush) obtained from the National Research Center for Certified Reference Materials of China). In addition, duplicated samples were conducted simultaneously for 15% of the samples. Blank samples were performed throughout all the experiments. The recovery percentage of standard samples ranged from 95% to 105%. 2.6. Statistics and calculations The data reported in this paper are the means ± standard deviations (n = 3) except for the Pb contents in the silkworm peel. Only plus error bar are presented in graphs. All the calculations and statistical analyses of these data were performed using Microsoft Excel 2007 and Origin 8.0 software. Significant differences were analyzed by one-way analysis of variance at a significance level of p < 0.05, followed by least significant difference multiple comparisons testing.
3.2. Accumulation and translocation of Pb in mulberry The concentrations of Pb in the different parts of mulberry increased with the increase of the available fraction of Pb in the soils (Table 1). The Pb distributions in the roots, stems and leaves of mulberry from the control group were homogeneous and were 5.58, 5.80 and 5.54 mg kg 1, respectively. Except for the control group, the order of the accumulated Pb by the different parts of mulberry was roots > stems > leaves. The Pb concentrations in the parts of mulberry from various groups are listed in Table 1. As presented in Table 2, the TF3s continuously decreased with the increase of Pb concentrations in the soils, as did TF4s, TF5s and TF6s. The TF1s of Pb were 0.38 in the control group, 1.13 in the 200 mg kg 1 group, 2.35 in the 400 mg kg 1 group and 1.60 in the 800 mg kg 1 group. For TF1, TF4 and TF6 of three groups added Pb, TF1 was the highest, followed by TF6 and TF4 from the same group, which indicated that a large amount of Pb was sequestered in mulberry roots, while the leaves accumulated the lowest amount of Pb. 3.3. Sub-cellular distribution and chemical forms of Pb in mulberry leaves
3. Results 3.1. Effect of lead on the growth of the plant and the chlorophyll content in the leaves The total Pb content and the proportions of each chemical fraction in all soil samples are presented in Fig. S1 (see supplementary). A larger liable fraction (F1 + F2, F1: exchangeable and carbonate fraction, F2: reducible fraction) and a less residual part in all the added samples were observed compared to the control sample. The plant height and chlorophyll content in mulberry leaves of the control group were 75.33 cm and 2.38 mg g 1 FW, respectively. As illustrated in Fig. 1, the plant height and the chlorophyll content in the leaves were reduced 12.82% and 6.02%, 16.55% and 10.53%, 42.03% and 36.13% for the groups at 200, 400, and 800 mg kg 1, respectively, compared to the control group. One-way ANOVA indicated that the chlorophyll values from the groups (200 mg kg 1and 400 mg kg 1) were not significantly different from the control group, and the heights between the 200 mg kg 1 group and the 400 mg kg 1 group were not significantly different. All of these results indicated that mulberry tolerated Pb, although the addition of 800 mg kg 1 Pb to the soils strongly inhibited its growth.
2.8
120 Height
chlorophll a
2.4
100
The sub-cellular distribution of Pb in mulberry leaves was dependent on the Pb concentration in the soils. Regardless of the Pb content in the soils, the sub-cellular distribution from all the groups exhibited a similar trend of FC > FA > FB. In the control group, most of the Pb content in the leaves, with approximately 80.25%, was deposited in the cell walls and 3.96% of total Pb located in the organelle. Fig. 2(a) illustrates that the proportion of Pb distributed in the cell wall of the leaves enhances remarkably, up to 92.15–92.82%, after the Pb addition treatments. Meanwhile, the proportions of Pb located in the organelle decreased and were in the range of: 1.71–2.7%. In addition, the Pb concentrations of each of the chemical forms in mulberry leaves increased with the increase of Pb in the soils. As shown in Fig. 2(b), for the control group, Pb extracted by 0.6 mol L 1 HCl was the dominant fraction (90.46%) followed by the residual (4.84%). The 1 mol L 1 NaCl extract and 2% HAc extract were lower than the detection limits of the instrument. For the 200, 400 and 800 mg kg 1 groups, the percentage of each chemical form of Pb in the leaves was changed to the order HCl extract > NaCl extract > H2O extrac > residual > ethanol extract > HAc extract, which was lower than the detection limits, as well. In addition, the addition of Pb to the soils caused the percentage of Pb extracted by NaCl to increase markedly with the increase of Pb in the soils, while the percentage of Pb in the residual reduced gradually. 3.4. Silkworms transferred and responded to Pb in the mulberry leaves
a a b
2.0
80
a b
60
Height (cm)
Chlorophll (mg g-1FW)
173
c
40
1.6
b
0
200
400
800
20
Pb treatment (mg kg-1) Fig. 1. Effects of Pb stress on the mulberry growth and the chlorophyll in the leaves (different lower case letters indicate significant differences between data derived from the same index (p < 0.05)).
The consumption of mulberry leaves by the 5th instar larva accounts for approximately 85% of the total intake. The Pb in leaves did not lead to death of the silkworms (Table S2). The mean silk weight of a silkworm derived from each group ranged from 0.218 to 0.243 g, with no significant difference. The growth rates of the body weight were 118.09%, 114.00%, 101.91%, 85.54% for the 0, 200, 400 and 800 mg kg 1 groups, respectively. The bodyweight of the silkworms reduced due to 60.26 mg kg 1 Pb in the mulberry leaves. As listed in Table 3, the accumulation abilities of Pb by larvae, feces, silk, peeling and silk moths were different. For each group, the order of Pb accumulated in these compartments was feces > peel > larvae > silk moths > silk. The Pb concentration in the silkworm feces reached 9.85, 187.96, 230.44 and 279.80 mg kg 1 for the 0, 200, 400 and 800 mg kg 1 groups,
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Table 1 Accumulation of Pb in mulberry. Pb treatment (mg kg
1
)
Available fraction in soils (mg kg
1
)
Pb content in organs of mulberry (mg kg
14.66 ± 0.03a 204.05 ± 10.54b 371.12 ± 33.37c 757.68 ± 24.28d
0 200 400 800
1
)
Roots
Stems
Leaves
5.58 ± 0.27a 230.78 ± 9.54b 873.89 ± 33.12c 1209.25 ± 36.58d
5.80 ± 0.25a 58.54 ± 8.21b 125.98 ± 10.44c 156.68 ± 3.98d
5.54 ± 0.35a 41.79 ± 3.95b 51.21 ± 3.48bd 60.26 ± 5.40cd
Available fraction in the soils is the sum of F1 and F2 (F1: exchangeable and carbonate fraction, F2: reducible fraction). The values in each column followed by different lower case letters are significantly different (p < 0.05).
Table 2 Translocation factors of Pb between the different parts of mulberry and the available fraction of the Pb in soils. Treatment (mg kg
1
)
Translocation factor (TF)
0 200 400 800
TF1 Root/AF
TF2 Stem/AF
TF3 Leaf/AF
TF4 Stem/root
TF5 Leaf/root
TF6 Leaf/stem
0.38 1.13 2.35 1.60
0.40 0.29 0.34 0.21
0.38 0.20 0.14 0.08
1.04 0.25 0.14 0.13
0.99 0.18 0.06 0.05
0.99 0.71 0.41 0.39
proportion of subcellular fraction (%)
Translocation factor: concentration of Pb in the receiving level (mg kg 1)/concentration of Pb in the source level (mg kg AF: available fraction in the soils (exchangeable and carbonate fraction and reducible fraction).
100
FA FB FC
60 3.5. Responses of metallothioneins in the different organs of the silkworm to Pb
40 20 0
0
200
400
800
Pb treatment (mg kg -1) proportion of the chemical forms (%)
).
respectively. The Pb concentrations in these parts were significantly different among the groups, which increased with the increase in the Pb concentration in the leaves, except the peel. The silk moths of all groups had a lower Pb concentration than the larva body through the spinning silk and peeling. To avoid experimental error, the peel samples from the same group were combined to only one because of its light weight.
a
80
1
100
b
95
F1 F2 F3 F4 F5 F6
90 85 80 20 15 10
4. Discussion
5 0
To understand the detoxification of Pb in the larvae body, the MTs in the mid-gut, the posterior of the silk glands, and the fat bodies of silkworms were sampled and detected. The result of the sampling is shown in Fig. 3. All of the MTs levels in the three tissues of the control group remained at a low level and indicated that MTs1 > MTs2 > MTs3. Compared to the control group, high Pb (41.79–60.26 mg kg 1) in the food significantly induced synthesis of MTs. The MTs1 level first increased and remained at a steady state, which were 0.79 nmol g 1 ww in the 200 mg kg 1 group and 0.73 nmol g 1 ww in the 400 mg kg 1 group. The level of MTs1 in the 800 mg kg 1 group was 1.19 nmol g 1 ww, higher than that in the other groups. In contrast to MTs1, MTs2 had a slight increasing in the 200 mg kg 1 group, and then it increased strongly in the 400 and 800 mg kg 1 groups and reached 1.47 and 1.51 nmol g 1 ww, respectively, as did MTs3. MTs3 in the 400 and 800 mg kg 1 groups reached 0.58 and 0.64 nmol g 1 ww, respectively.
0
200
400
800
Pb treatment (mg kg -1) Fig. 2. Proportions of sub-cellular fractions of Pb (a) and proportions of the chemical forms of Pb (b) in mulberry leaves (FA: soluble fraction, FB: organelle, FC: cell wall, F1-F6: chemical forms extracted by different extractant: F1:80% ethanol, F2: deionized water, F3: 1 mol L 1 NaCl, F4: 2% acetic acid, F5: 0.6 mol L 1 HCl, F6: residues).
4.1. Accumulation, translocation and detoxification of Pb in mulberry plant Baker (1987) indicated that metal-tolerant plants are able to function normally, even in the presence of high concentration of toxic elements by some specific physiological mechanisms. In this work, chlorophyll and biomass (plant height) of mulberry strongly decreased after the highest Pb treatment of 800 mg kg 1 in the
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L. Zhou et al. / Chemosphere 128 (2015) 171–177 Table 3 Silkworm accumulation and redistribution Pb in mulberry leaves. Treatment (mg kg
1
)
Pb content in leaves (mg kg
1
)
Pb content in different parts (mg kg
5.54 ± 0.35a 41.79 ± 3.95b 51.21 ± 3.48bd 60.26 ± 5.40cd
0 200 400 800
1
)
Larvae
Feces
Silk
Peel
Silk moth
0.63 ± 0.08a 4.08 ± 0.13b 5.74 ± 0.22c 11.16 ± 0.65d
9.85 ± 0.11a 187.96 ± 15.87b 230.44 ± 1.82c 279.80 ± 15.45d
0.56 ± 0.01a 1.63 ± 0.09b 2.04 ± 0.28b 3.28 ± 0.29c
2.76 30.84 31.22 28.10
0.6 ± 0.15a 2.95 ± 0.10b 4.39 ± 0.36c 6.23 ± 0.48d
The values in each column followed by different lower case letters are significantly different (p < 0.05)
-1
Metallothionein (nmol g ww)
2.0 MTs1 MTs2 MTs3
b
b
1.5 c
1.0
b
0.5
0.0
b a
a a
0
a
c
b a
200
400
800 -1
Pb treatment (mg kg ) Fig. 3. MTs levels of the different tissues in silkworms (MTs1: MTs in the mid-gut, MTs2: MTs in the posterior of silk gland, MTs3: MTs in the fat body. The different letters above the same series of bars indicate significant differences).
soils. However, they can survive and grow normally under higher Pb stress (200 and 400 mg kg 1 Pb in soils), which indicated that mulberry is a Pb-tolerant plant. Additionally, in our research, Pb could be accumulated in all tissues of mulberry plants cultured in soils with different Pb contents. Most of the Pb content was sequestered in the roots. Previous studies indicated that metal-tolerant plants always accumulate more toxic metals in their root and less in their shoots and leaves than non-tolerant plants. Moreover, as described above, mulberry plants isolated more Pb in the roots and stems to protect the sensitive organ (leaves) and survive, which can be an important means of detoxification of Pb in mulberry. This detoxification, which inhibits the accumulation of Pb from soils in the mulberry leaf and reduces the risk to the silkworm, must influence the transfer of Pb along the soil–mulberry–silkworm food chain.
4.2. Sub-cellular distribution, chemical forms and detoxification of Pb in leaves The organelles are the main locations for cellular metabolism. At the sub-cellular level, Pb stress resulted in most of the Pb in the mulberry leaves being stored in cells walls to keep the organelle functioning normally. Similar results were reported by other studies (Yan et al., 2012; Qiao et al., 2015). This means that the cell wall may be a key component for the detoxification of Pb. The plant cell wall contains protein, polysaccharides and pectin acid, which can provide many chelating sites to bind heavy metals and restrict the trans-membrane translocation of the heavy metal ion so that normal physiological activities are maintained in the plant cells (Wójcik et al., 2005; He et al., 2008). Toxic metals entering into the cell are transported into vacuoles and stored in them with various complex forms to not injure the organelles when the cell wall binding sites reach saturation.
The metal chemical forms can reflect the plant detoxification mechanisms. Usually, the metal fractions extracted by ethanol and purified water have the highest migration ability followed by the fractions extracted by NaCl, HAc and HCl. The residual form was considered as non-toxic. In this work, the percentages of Pb extracted by 0.6 mol L 1 NaCl increased strongly with the Pb concentration in the soil, whereas the percentages of Pb extracted by 80% ethanol clearly decreased. This result indicated that the Pb in mulberry leaves is difficult to be absorbed by the silkworm. The result of the chemical speciation of Pb in the leaves supported the hypothesis that Pb sequestration in the cell wall may be crucial for detoxification, as was inferred above. Sharma et al. (2004) demonstrated that Pb was predominantly present in the form of lead-acetate and lead–sulfur compounds in the leaf cells of Sesbania drummondii. In addition, it is often reported that Pb deposited by Pb-phosphate complexes (Kopittke et al., 2008; Zheng et al., 2012). Nevertheless, insoluble Pb-phosphate complexes (2% acetic acid extractive) were not found in this work. The fact was evidenced by a new report (Bovenkamp et al., 2013), which measured the chemical environment of lead in roots and leaves of plants from four different plant families by Pb L3-edge XANES measurements, in which no precipitation with a group (–PO4) was observed. Therefore, further studies should be conducted.
4.3. Accumulation and detoxification of Pb in silkworm No silkworms died, and adverse effects Pb in food on the silk production were not recorded during the experiment, which suggests that silkworms have a strong tolerance to Pb. On the one hand, the high migration ability fraction of Pb in the leaves was least, which was in favor of the silkworm’s toleration to Pb. On the other hand, the experimental results suggested that the silkworm has two detoxification strategies of Pb: ‘‘excrete’’ and ‘‘homeostasis’’. The metal detoxification was observed in a few studies. High Pb concentrations in the food of litter-dwelling collembolan appear to not have any harmful effects because most of the lead remains unabsorbed and is concentrated in the feces, and 30% of the uptake lead is stored in intestinal epithelium cells and excreted by periodic renovation of the intestinal epithelium (Joosse and Buker, 1979). Li et al. (2009) investigated earthworms that inhabit and survive in the pig manure with rich copper and zinc. Their results suggested the total copper and zinc concentrations in the earthworm casts were higher than those in the pig mature, and found that the increases were by factors of 1.18–3.40 and 1.29–2.46, respectively. In the present work, similar results were found. Most of the Pb passed through the silkworm alimentary canal and was enriched in feces, except for a slight uptake because of the alkaline environment in the gut of the silkworm (Jiang et al., 2012; Ponnuvel et al., 2012). This detoxification process further reduces the Pb accumulation in the silkworm larvae. Pb assimilated through the alimentary system was transferred into the other organs. Afterwards, some of the Pb was excreted through spinning and molting. Finally, the Pb contents in silk moths from each group were lower than those
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in the larvae, i.e., biomagnifications of Pb along this food chain was not observed. Of course, more investigations are required to determine whether the excrete stratagem for detoxification of Pb can be used by silkworm to detoxify other heavy metals because some previous studies found that the uptake and excretion of heavy metals by animals may be effected profoundly by the factors of the organism development phase, species, environmental temperature, exposure time and the type of metals (Spurgeon and Hopkin, 1999; Schaller et al., 2011). MTs played an important role in the tolerance mechanism of the animals against adverse environmental factors, especially heavy metal stress. MTs are a family of soluble, heat stable and low-molecular-weight proteins rich in cysteine, which exhibit a high affinity for metal ions and can detoxify non-essential metals to protect the cells and tissues (Kaegi and Schaeffer, 1988; Sauge-Merle et al., 2012). Very few studies have examined the MTs levels induced by Pb because mercury and cadmium are considered the principal inductors of MTs. Lee et al. (1983) found that there was no significant change in the urinary level of MTs in rats given up to 5 injections of saline solution of Pb. Nevertheless, the results from this study demonstrated that the MTs of the gut, the posterior of the silk gland, and the fat body were markedly synthesized by the induction of enhanced Pb stress. It is inferred that MTs are involved in the detoxification and homeostasis of Pb in the cells. This result is supported by some previous works. Pb could bind to MTs under ex vivo conditions (Waalkes et al., 1984), and tissue levels of MTs can be increased after Pb exposure (Ikebuchi et al., 1986). Similarly, on the basis of work in the laboratory, Maity et al. (2011) recorded a significant increase in the level of MTs in tissue from Lampito mauritii exposed to contaminated soil with 75, 150 and 300 mg kg 1 Pb. In addition, a strict dose–effect linear relationship was lacking in our work. MTs1 was the most sensitive to Pb stress as indicated by its obvious increase under lower concentration (200 mg kg 1 group). However, the levels of MTs2 and MTs3 were induced remarkably at higher Pb stress (400 and 800 mg kg 1 groups). As we know, the gut is the first barrier that is directly in contact with heavy metals, so MTs1 synthesis was induced under lower Pb stress. The synthesis of MTs2 and MTs3 was induced strongly when excess Pb was transported into the other organs after the Pb burden of the gut was maximum. Numerous studies have suggested that the level of MTs varies considerably depending on the species, tissue, animal age and other environmental factors (Van den Broeck et al., 2010; Fang et al., 2012). Thus, the actual mechanism of MTs under various situations is not fully understood and identified.
5. Conclusions Pb was transferred into mulberry and silkworm after exposure, although its biomagnification along the soil–mulberry–silkworm food chain was not observed. The treatment of soils with 200, 400, 800 mg kg 1 Pb resulted in the mulberry roots accumulating most of the Pb, i.e., 5.52–20.07 times higher than that in leaves. 92.15–92.82% of the Pb in the leaves was bound to the cell wall, and the low bioavailability fractions of Pb in the leaves, which were extracted by HCl, NaCl, and residual, were 95.29–95.57% of the total Pb. The Pb concentrations in the silkworm feces were 25.07–46.07 times higher than that in larva. Silk had the lowest Pb contents. The Pb contents in feces, silk, and peel and the change of the levels of MTs in various silkworm tissues indicated excretion and homeostasis are two important detoxification strategies of Pb for silkworm under high Pb stress. The process of Pb detoxification in mulberry and silkworm played an important role in the inhibition of Pb transfer along the food chain. In addition, mulberry and silkworm can be a promising approach for the remediation
of Pb-polluted soils due to the tolerances of mulberry and silkworm to Pb. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2015.01.031. References Ahearn, G., Mandal, P., Mandal, A., 2004. Mechanisms of heavy-metal sequestration and detoxification in crustaceans, a review. J. Comp. Physiol. B. 174 (6), 439– 452. Ashfaq, M., Ali, S., Hanif, M.A., 2009. Bioaccumulation of cobalt in silkworm (Bombyx mori L.) in relation to mulberry, soil and wastewater metal concentrations. Process Biochem. 44 (10), 1179–1184. Baker, A., 1987. Metal tolerance. New Phytol. 106 (s1), 93–111. Bovenkamp, G.L., Prange, A., Schumacher, W., Ham, K., Smith, A.P., Hormes, J., 2013. Lead uptake in diverse plant families, a study applying X-ray absorption near edge spectroscopy. Environ. Sci. Technol. 47 (9), 4375–4382. Cao, X., Ma, L., Liang, Y., Gao, B., Harris, W., 2011. 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Lead in the soil–mulberry (Morus alba L.)–silkworm (Bombyx mori) food chain: Translocation and detoxification Lingyun Zhou a,b, Ye Zhao a,⇑, Shuifeng Wang a,c, Shasha Han a, Jing Liu a a
State Key Laboratory of Water Environment Simulation, School of Environment, Beijing Normal University, Beijing 100875, China Fujian Province Key Laboratory of Modern Analytical Science and Separation Technology, College of Chemistry and Environment, Minnan Normal University, Zhangzhou 363000, China c Analytical Testing Center, Beijing Normal University, Beijing 100875, China b
h i g h l i g h t s Biomagnification of Pb in the soil–mulberry–silkworm system was not observed. Roots sequestered most of the Pb, most of the Pb in leaves bound to the cell wall. Excretion and homeostasis protect silkworms from Pb stress. Detoxification in mulberry–silkworm regulates Pb transfer along the food chain. Pb tolerance of mulberry and silkworm indicates the bioremediation potential.
a r t i c l e
i n f o
Article history: Received 26 March 2014 Received in revised form 23 January 2015 Accepted 24 January 2015
Handling Editor: Shane Snyder Keywords: Lead Translocation Detoxification Mulberry Silkworm
a b s t r a c t The translocation of lead (Pb) in the soil–mulberry–silkworm food chain and the process of Pb detoxification in the mulberry–silkworm chain were investigated. The amount of Pb in mulberry, silkworm, feces and silk increased in a dose-responsive manner to the Pb contents in the soils. Mulberry roots sequestered most of the Pb, ranging from 230.78 to 1209.25 mg kg 1. Over 92% of the Pb in the mulberry leaves was deposited in the cell wall, and 95.29–95.57% of the Pb in the mulberry leaves was integrated with oxalic acid, pectates and protein, and it had low bioavailability. The Pb concentrations in the silkworm feces were 4.50–4.64 times higher than those in the leaves. The synthesis of metallothioneins in three tissues of the silkworms was induced to achieve Pb homeostasis under Pb stress. These results indicated the mechanism involved in Pb transfer along the food chain was controlled by the detoxification of Pb in different trophic levels. Planting mulberry and rearing silkworm could be a promising approach for the remediation of Pb-polluted soils due to the Pb tolerance of mulberry and silkworm. Ó 2015 Published by Elsevier Ltd.
1. Introduction Lead (Pb) is a non-essential and toxic trace element in the environment. The soils in some locations are contaminated by Pb due to anthropogenic activities (Stafilov et al., 2010; Jin et al., 2015). Pb pollution in soils is covert, persistent and non biodegradable. Soils polluted by Pb could be a source of contamination for water and air (Laidlaw et al., 2012). Previous studies indicated that Pb was accumulated in the ecosystem through the food chains and posed a great risk to the environment and to human health ⇑ Corresponding author at: School of Environment, Beijing Normal University, No. 19, Xinjiekouwai Street, Haidian District, Beijing 100875, China. Tel./fax: +86 10 58802875. E-mail address: [email protected] (Y. Zhao). http://dx.doi.org/10.1016/j.chemosphere.2015.01.031 0045-6535/Ó 2015 Published by Elsevier Ltd.
(Lopes et al., 2011). However, previous studies regarding Pb transfer in terrestrial ecosystems found that the Pb concentration in higher plant or higher animals was lower than that in polluted soils (Notten et al., 2005; Roodbergen et al., 2008), even though the Pb concentrations steadily declined with increasing trophic levels along a soil–plant-insect-chicken food chain at a contaminated area (Zhuang et al., 2009). It is concluded from the previous studies that metal biomagnifications are not generally applicable to all environmental ecosystems and that accumulation in the food chain varies depending on the metal concentration, biological species and trophic level (Wang, 2002). Therefore, there is an urgent need to explore both the translocation of Pb in a soil ecosystem and the remediation of Pb-polluted soils (Peralta-Videa et al., 2009; Cao et al., 2011).
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Recent research suggested that organisms might induce a whole suite of tolerance mechanisms involving metal detoxification to cope with metal stress (Shao et al., 2010). It is increasingly accepted that the chemical speciation of non-essential metals influences the internal metal detoxification of organisms and the trophic transfer of metals along a food chain (van Gestel, 2008). It has been documented that heavy metals with inorganic and water-soluble forms in plants have higher activities (Wang et al., 2008) that enable them to be easily transferred to the next trophic level. Compared to plants that are sensitive to heavy metals, tolerant plants usually accumulate more non-essential metals in the cell wall and vacuoles to prevent them from injuring the more sensitive cell metabolic sites. The non-essential metals in the cell wall often are integrated with pectates and protein, in addition to becoming water insoluble (Wu et al., 2005; Weng et al., 2012). For animals, on the one hand, the intake of non- essential metals can be decreased by the strategy of ‘‘avoidance’’ actions (Zidar et al., 2004; Lukkari and Haimi, 2005), and on the other hand, a detoxification process involving ‘‘excretion’’ and ‘‘homeostasis’’ actions can be used to cope with metals stress (Ahearn et al., 2004). Thus, a mechanistic understanding of the trophic transfer of toxic metals benefits from the detoxification of organism; however, only a few studies have examined this process. Additionally, a considerable number of studies proposed that the use of some hard woody plants can be an alternative for the removal or stabilization of metals from contaminated soils (Pulford and Watson, 2003). Those woody plants with a moderate capacity to accumulate heavy metals in the aerial parts possess characteristics of rapid growth, deep and extensive root systems and high biomass production (Unterbrunner et al., 2007; Shukla et al., 2011). Similarly, mulberry exhibits the potential for remediating metal-polluted soils. Furthermore, mulberry, which is found from the tropics to the temperate regions and from sea level to altitudes as high as 4000 m, has superior attributes of high adaptability and wide distribution (Ercisli and Orhan, 2007). Previous studies identified that mulberry exhibits tolerance to many metals (Ashfaq et al., 2009; Zhao et al., 2013). Prince et al. (2001) commented on the mulberry–silkworm food chain as being a template to assess heavy metal mobility in terrestrial ecosystems. To date, few articles have reported in detail on the transfer of Pb in the soil–mulberry–silkworm system, on the Pb detoxification mechanisms of mulberry and silkworm, and on the influence of the latter to the former. Thus, the specific aims of this study were to (i) determine the suitability of mulberry–silkworm as an alternative method to the soil remediation of Pb pollution through an investigation of the accumulation and translocation of Pb in a soil–mulberry–silkworm system and to (ii) elucidate the underlying mechanisms controlling the trophic transfer of Pb along this food chain based on the identification of the process of Pb detoxification in mulberry and silkworm involving sub-cellular distribution and the chemical forms of Pb in mulberry leaves, the excretion of Pb and metallothionein level in silkworm.
transformation of Pb added as solutions in the soil could achieve equilibrium and better contact the different components of the soil. After approximately two months, NPK fertilizer was added to the soils in the form of ammonium sulfate (0.68 g kg 1 of soil), super phosphate (0.35 g kg 1 of soil) and muriate of potash (0.09 g kg 1 of soil) and homogenized. Ten kilograms of the prepared soil was added to each pot (26 cm in upper diameter and 28 cm in height). Three pots were replicated for all the Pb treatments. The one-year-old mulberry plant stocks (var. nongsang 14), which was a common breed and planted widely in silkworm breeding areas of China, used in this study were purchased from commercial market. Two plants were planted in each pot in May and irrigated with tap water. All the pots were placed randomly on outdoor grassland, and the plants grew under natural conditions. After three months (from May, 2013 to July, 2013), the silkworm breeding experiments were performed and some plant samples were collected for other experiments and analysis. The hybrid silkworm used in this study was named Qingfeng Mingyue, whose spawns were from the silkworm spawn station in Huzhou, Zhejiang Province, China. All the larvae were reared on fresh and uncontaminated mulberry leaves until their fourth molt. At the beginning of the fifth instar, 48 healthy larvae of uniform size were isolated and divided into 4 groups that fed on the mulberry leaves richened with Pb from the treatment soils. After five days, six larvae from each treatment were selected to be individually weighed and died in liquid nitrogen, and then were stored at 80 °C until analysis. The other silkworms of each treatment were allowed to grow normally and complete their life stages. The cocoons and the adults were collected, washed with distilled water and dried in an oven at 50 °C to a constant weight. Subsequently, these samples were used for analysis. 2.2. Sub-cellular distribution and the chemical forms of Pb in the leaves To determine the Pb contents at the subcellular level of the plant leaves from each treatment, differential centrifugation was used to separate the Pb, following the methods reported by Weigel and Jäger (1980) and Wang et al. (2009). The extraction of the chemical forms of Pb in mulberry leaves was performed according to the method described by Weng et al. (2012) and Li et al. (2013). 2.3. Chlorophyll extraction and determination The Chlorophyll in mulberry leaves were extracted by ethanol, the concentration was determined by spectrophotometry. The detail procedure is in Section S1 (see supplementary material). 2.4. Extraction and determination of metallothioneins (MTs) The detailed information is supplied in Section S2 (see supplementary material).
2. Materials and methods
2.5. Pb concentration determination
2.1. Soil preparation, plant materials, and animal materials
Four chemical forms of Pb in soils were extracted according to the modified BCR procedure (Rauret et al., 1999). All of the soils, plant materials, sub-cellular distribution fractions (except the soluble fraction), the residuals from the extraction of chemical forms, and animal materials were digested with a mixture of HNO3–HF– HClO4 (3:1:1 v/v) at 160 °C for 6 h. The digested mixture was cooled to room temperature and diluted to 10 mL with super purified water. Finally, the concentrations of Pb in all of the soluble samples were directly determined by ICP-AES. The quality control
Soil for the pot experiment was collected from a cleaning field in Zhangzhou, Fujian province, China, and was air-dried and then ground with a wooden roller to pass through a 2-mm sieve. The general properties of the soil are presented in Table S1 (see supplementary material). The initial soil was incubated with 0, 200, 400, and 800 mg Pb kg 1 added in the form Pb(NO3)2. The soils were submitted to two wetting and drying cycles to ensure that the
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and quality assurance of the Pb content in these samples was based on the standard reference materials (GSS-1 (geochemical standard soil) and GBW07602 (stems and leaves of brush) obtained from the National Research Center for Certified Reference Materials of China). In addition, duplicated samples were conducted simultaneously for 15% of the samples. Blank samples were performed throughout all the experiments. The recovery percentage of standard samples ranged from 95% to 105%. 2.6. Statistics and calculations The data reported in this paper are the means ± standard deviations (n = 3) except for the Pb contents in the silkworm peel. Only plus error bar are presented in graphs. All the calculations and statistical analyses of these data were performed using Microsoft Excel 2007 and Origin 8.0 software. Significant differences were analyzed by one-way analysis of variance at a significance level of p < 0.05, followed by least significant difference multiple comparisons testing.
3.2. Accumulation and translocation of Pb in mulberry The concentrations of Pb in the different parts of mulberry increased with the increase of the available fraction of Pb in the soils (Table 1). The Pb distributions in the roots, stems and leaves of mulberry from the control group were homogeneous and were 5.58, 5.80 and 5.54 mg kg 1, respectively. Except for the control group, the order of the accumulated Pb by the different parts of mulberry was roots > stems > leaves. The Pb concentrations in the parts of mulberry from various groups are listed in Table 1. As presented in Table 2, the TF3s continuously decreased with the increase of Pb concentrations in the soils, as did TF4s, TF5s and TF6s. The TF1s of Pb were 0.38 in the control group, 1.13 in the 200 mg kg 1 group, 2.35 in the 400 mg kg 1 group and 1.60 in the 800 mg kg 1 group. For TF1, TF4 and TF6 of three groups added Pb, TF1 was the highest, followed by TF6 and TF4 from the same group, which indicated that a large amount of Pb was sequestered in mulberry roots, while the leaves accumulated the lowest amount of Pb. 3.3. Sub-cellular distribution and chemical forms of Pb in mulberry leaves
3. Results 3.1. Effect of lead on the growth of the plant and the chlorophyll content in the leaves The total Pb content and the proportions of each chemical fraction in all soil samples are presented in Fig. S1 (see supplementary). A larger liable fraction (F1 + F2, F1: exchangeable and carbonate fraction, F2: reducible fraction) and a less residual part in all the added samples were observed compared to the control sample. The plant height and chlorophyll content in mulberry leaves of the control group were 75.33 cm and 2.38 mg g 1 FW, respectively. As illustrated in Fig. 1, the plant height and the chlorophyll content in the leaves were reduced 12.82% and 6.02%, 16.55% and 10.53%, 42.03% and 36.13% for the groups at 200, 400, and 800 mg kg 1, respectively, compared to the control group. One-way ANOVA indicated that the chlorophyll values from the groups (200 mg kg 1and 400 mg kg 1) were not significantly different from the control group, and the heights between the 200 mg kg 1 group and the 400 mg kg 1 group were not significantly different. All of these results indicated that mulberry tolerated Pb, although the addition of 800 mg kg 1 Pb to the soils strongly inhibited its growth.
2.8
120 Height
chlorophll a
2.4
100
The sub-cellular distribution of Pb in mulberry leaves was dependent on the Pb concentration in the soils. Regardless of the Pb content in the soils, the sub-cellular distribution from all the groups exhibited a similar trend of FC > FA > FB. In the control group, most of the Pb content in the leaves, with approximately 80.25%, was deposited in the cell walls and 3.96% of total Pb located in the organelle. Fig. 2(a) illustrates that the proportion of Pb distributed in the cell wall of the leaves enhances remarkably, up to 92.15–92.82%, after the Pb addition treatments. Meanwhile, the proportions of Pb located in the organelle decreased and were in the range of: 1.71–2.7%. In addition, the Pb concentrations of each of the chemical forms in mulberry leaves increased with the increase of Pb in the soils. As shown in Fig. 2(b), for the control group, Pb extracted by 0.6 mol L 1 HCl was the dominant fraction (90.46%) followed by the residual (4.84%). The 1 mol L 1 NaCl extract and 2% HAc extract were lower than the detection limits of the instrument. For the 200, 400 and 800 mg kg 1 groups, the percentage of each chemical form of Pb in the leaves was changed to the order HCl extract > NaCl extract > H2O extrac > residual > ethanol extract > HAc extract, which was lower than the detection limits, as well. In addition, the addition of Pb to the soils caused the percentage of Pb extracted by NaCl to increase markedly with the increase of Pb in the soils, while the percentage of Pb in the residual reduced gradually. 3.4. Silkworms transferred and responded to Pb in the mulberry leaves
a a b
2.0
80
a b
60
Height (cm)
Chlorophll (mg g-1FW)
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c
40
1.6
b
0
200
400
800
20
Pb treatment (mg kg-1) Fig. 1. Effects of Pb stress on the mulberry growth and the chlorophyll in the leaves (different lower case letters indicate significant differences between data derived from the same index (p < 0.05)).
The consumption of mulberry leaves by the 5th instar larva accounts for approximately 85% of the total intake. The Pb in leaves did not lead to death of the silkworms (Table S2). The mean silk weight of a silkworm derived from each group ranged from 0.218 to 0.243 g, with no significant difference. The growth rates of the body weight were 118.09%, 114.00%, 101.91%, 85.54% for the 0, 200, 400 and 800 mg kg 1 groups, respectively. The bodyweight of the silkworms reduced due to 60.26 mg kg 1 Pb in the mulberry leaves. As listed in Table 3, the accumulation abilities of Pb by larvae, feces, silk, peeling and silk moths were different. For each group, the order of Pb accumulated in these compartments was feces > peel > larvae > silk moths > silk. The Pb concentration in the silkworm feces reached 9.85, 187.96, 230.44 and 279.80 mg kg 1 for the 0, 200, 400 and 800 mg kg 1 groups,
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Table 1 Accumulation of Pb in mulberry. Pb treatment (mg kg
1
)
Available fraction in soils (mg kg
1
)
Pb content in organs of mulberry (mg kg
14.66 ± 0.03a 204.05 ± 10.54b 371.12 ± 33.37c 757.68 ± 24.28d
0 200 400 800
1
)
Roots
Stems
Leaves
5.58 ± 0.27a 230.78 ± 9.54b 873.89 ± 33.12c 1209.25 ± 36.58d
5.80 ± 0.25a 58.54 ± 8.21b 125.98 ± 10.44c 156.68 ± 3.98d
5.54 ± 0.35a 41.79 ± 3.95b 51.21 ± 3.48bd 60.26 ± 5.40cd
Available fraction in the soils is the sum of F1 and F2 (F1: exchangeable and carbonate fraction, F2: reducible fraction). The values in each column followed by different lower case letters are significantly different (p < 0.05).
Table 2 Translocation factors of Pb between the different parts of mulberry and the available fraction of the Pb in soils. Treatment (mg kg
1
)
Translocation factor (TF)
0 200 400 800
TF1 Root/AF
TF2 Stem/AF
TF3 Leaf/AF
TF4 Stem/root
TF5 Leaf/root
TF6 Leaf/stem
0.38 1.13 2.35 1.60
0.40 0.29 0.34 0.21
0.38 0.20 0.14 0.08
1.04 0.25 0.14 0.13
0.99 0.18 0.06 0.05
0.99 0.71 0.41 0.39
proportion of subcellular fraction (%)
Translocation factor: concentration of Pb in the receiving level (mg kg 1)/concentration of Pb in the source level (mg kg AF: available fraction in the soils (exchangeable and carbonate fraction and reducible fraction).
100
FA FB FC
60 3.5. Responses of metallothioneins in the different organs of the silkworm to Pb
40 20 0
0
200
400
800
Pb treatment (mg kg -1) proportion of the chemical forms (%)
).
respectively. The Pb concentrations in these parts were significantly different among the groups, which increased with the increase in the Pb concentration in the leaves, except the peel. The silk moths of all groups had a lower Pb concentration than the larva body through the spinning silk and peeling. To avoid experimental error, the peel samples from the same group were combined to only one because of its light weight.
a
80
1
100
b
95
F1 F2 F3 F4 F5 F6
90 85 80 20 15 10
4. Discussion
5 0
To understand the detoxification of Pb in the larvae body, the MTs in the mid-gut, the posterior of the silk glands, and the fat bodies of silkworms were sampled and detected. The result of the sampling is shown in Fig. 3. All of the MTs levels in the three tissues of the control group remained at a low level and indicated that MTs1 > MTs2 > MTs3. Compared to the control group, high Pb (41.79–60.26 mg kg 1) in the food significantly induced synthesis of MTs. The MTs1 level first increased and remained at a steady state, which were 0.79 nmol g 1 ww in the 200 mg kg 1 group and 0.73 nmol g 1 ww in the 400 mg kg 1 group. The level of MTs1 in the 800 mg kg 1 group was 1.19 nmol g 1 ww, higher than that in the other groups. In contrast to MTs1, MTs2 had a slight increasing in the 200 mg kg 1 group, and then it increased strongly in the 400 and 800 mg kg 1 groups and reached 1.47 and 1.51 nmol g 1 ww, respectively, as did MTs3. MTs3 in the 400 and 800 mg kg 1 groups reached 0.58 and 0.64 nmol g 1 ww, respectively.
0
200
400
800
Pb treatment (mg kg -1) Fig. 2. Proportions of sub-cellular fractions of Pb (a) and proportions of the chemical forms of Pb (b) in mulberry leaves (FA: soluble fraction, FB: organelle, FC: cell wall, F1-F6: chemical forms extracted by different extractant: F1:80% ethanol, F2: deionized water, F3: 1 mol L 1 NaCl, F4: 2% acetic acid, F5: 0.6 mol L 1 HCl, F6: residues).
4.1. Accumulation, translocation and detoxification of Pb in mulberry plant Baker (1987) indicated that metal-tolerant plants are able to function normally, even in the presence of high concentration of toxic elements by some specific physiological mechanisms. In this work, chlorophyll and biomass (plant height) of mulberry strongly decreased after the highest Pb treatment of 800 mg kg 1 in the
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L. Zhou et al. / Chemosphere 128 (2015) 171–177 Table 3 Silkworm accumulation and redistribution Pb in mulberry leaves. Treatment (mg kg
1
)
Pb content in leaves (mg kg
1
)
Pb content in different parts (mg kg
5.54 ± 0.35a 41.79 ± 3.95b 51.21 ± 3.48bd 60.26 ± 5.40cd
0 200 400 800
1
)
Larvae
Feces
Silk
Peel
Silk moth
0.63 ± 0.08a 4.08 ± 0.13b 5.74 ± 0.22c 11.16 ± 0.65d
9.85 ± 0.11a 187.96 ± 15.87b 230.44 ± 1.82c 279.80 ± 15.45d
0.56 ± 0.01a 1.63 ± 0.09b 2.04 ± 0.28b 3.28 ± 0.29c
2.76 30.84 31.22 28.10
0.6 ± 0.15a 2.95 ± 0.10b 4.39 ± 0.36c 6.23 ± 0.48d
The values in each column followed by different lower case letters are significantly different (p < 0.05)
-1
Metallothionein (nmol g ww)
2.0 MTs1 MTs2 MTs3
b
b
1.5 c
1.0
b
0.5
0.0
b a
a a
0
a
c
b a
200
400
800 -1
Pb treatment (mg kg ) Fig. 3. MTs levels of the different tissues in silkworms (MTs1: MTs in the mid-gut, MTs2: MTs in the posterior of silk gland, MTs3: MTs in the fat body. The different letters above the same series of bars indicate significant differences).
soils. However, they can survive and grow normally under higher Pb stress (200 and 400 mg kg 1 Pb in soils), which indicated that mulberry is a Pb-tolerant plant. Additionally, in our research, Pb could be accumulated in all tissues of mulberry plants cultured in soils with different Pb contents. Most of the Pb content was sequestered in the roots. Previous studies indicated that metal-tolerant plants always accumulate more toxic metals in their root and less in their shoots and leaves than non-tolerant plants. Moreover, as described above, mulberry plants isolated more Pb in the roots and stems to protect the sensitive organ (leaves) and survive, which can be an important means of detoxification of Pb in mulberry. This detoxification, which inhibits the accumulation of Pb from soils in the mulberry leaf and reduces the risk to the silkworm, must influence the transfer of Pb along the soil–mulberry–silkworm food chain.
4.2. Sub-cellular distribution, chemical forms and detoxification of Pb in leaves The organelles are the main locations for cellular metabolism. At the sub-cellular level, Pb stress resulted in most of the Pb in the mulberry leaves being stored in cells walls to keep the organelle functioning normally. Similar results were reported by other studies (Yan et al., 2012; Qiao et al., 2015). This means that the cell wall may be a key component for the detoxification of Pb. The plant cell wall contains protein, polysaccharides and pectin acid, which can provide many chelating sites to bind heavy metals and restrict the trans-membrane translocation of the heavy metal ion so that normal physiological activities are maintained in the plant cells (Wójcik et al., 2005; He et al., 2008). Toxic metals entering into the cell are transported into vacuoles and stored in them with various complex forms to not injure the organelles when the cell wall binding sites reach saturation.
The metal chemical forms can reflect the plant detoxification mechanisms. Usually, the metal fractions extracted by ethanol and purified water have the highest migration ability followed by the fractions extracted by NaCl, HAc and HCl. The residual form was considered as non-toxic. In this work, the percentages of Pb extracted by 0.6 mol L 1 NaCl increased strongly with the Pb concentration in the soil, whereas the percentages of Pb extracted by 80% ethanol clearly decreased. This result indicated that the Pb in mulberry leaves is difficult to be absorbed by the silkworm. The result of the chemical speciation of Pb in the leaves supported the hypothesis that Pb sequestration in the cell wall may be crucial for detoxification, as was inferred above. Sharma et al. (2004) demonstrated that Pb was predominantly present in the form of lead-acetate and lead–sulfur compounds in the leaf cells of Sesbania drummondii. In addition, it is often reported that Pb deposited by Pb-phosphate complexes (Kopittke et al., 2008; Zheng et al., 2012). Nevertheless, insoluble Pb-phosphate complexes (2% acetic acid extractive) were not found in this work. The fact was evidenced by a new report (Bovenkamp et al., 2013), which measured the chemical environment of lead in roots and leaves of plants from four different plant families by Pb L3-edge XANES measurements, in which no precipitation with a group (–PO4) was observed. Therefore, further studies should be conducted.
4.3. Accumulation and detoxification of Pb in silkworm No silkworms died, and adverse effects Pb in food on the silk production were not recorded during the experiment, which suggests that silkworms have a strong tolerance to Pb. On the one hand, the high migration ability fraction of Pb in the leaves was least, which was in favor of the silkworm’s toleration to Pb. On the other hand, the experimental results suggested that the silkworm has two detoxification strategies of Pb: ‘‘excrete’’ and ‘‘homeostasis’’. The metal detoxification was observed in a few studies. High Pb concentrations in the food of litter-dwelling collembolan appear to not have any harmful effects because most of the lead remains unabsorbed and is concentrated in the feces, and 30% of the uptake lead is stored in intestinal epithelium cells and excreted by periodic renovation of the intestinal epithelium (Joosse and Buker, 1979). Li et al. (2009) investigated earthworms that inhabit and survive in the pig manure with rich copper and zinc. Their results suggested the total copper and zinc concentrations in the earthworm casts were higher than those in the pig mature, and found that the increases were by factors of 1.18–3.40 and 1.29–2.46, respectively. In the present work, similar results were found. Most of the Pb passed through the silkworm alimentary canal and was enriched in feces, except for a slight uptake because of the alkaline environment in the gut of the silkworm (Jiang et al., 2012; Ponnuvel et al., 2012). This detoxification process further reduces the Pb accumulation in the silkworm larvae. Pb assimilated through the alimentary system was transferred into the other organs. Afterwards, some of the Pb was excreted through spinning and molting. Finally, the Pb contents in silk moths from each group were lower than those
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in the larvae, i.e., biomagnifications of Pb along this food chain was not observed. Of course, more investigations are required to determine whether the excrete stratagem for detoxification of Pb can be used by silkworm to detoxify other heavy metals because some previous studies found that the uptake and excretion of heavy metals by animals may be effected profoundly by the factors of the organism development phase, species, environmental temperature, exposure time and the type of metals (Spurgeon and Hopkin, 1999; Schaller et al., 2011). MTs played an important role in the tolerance mechanism of the animals against adverse environmental factors, especially heavy metal stress. MTs are a family of soluble, heat stable and low-molecular-weight proteins rich in cysteine, which exhibit a high affinity for metal ions and can detoxify non-essential metals to protect the cells and tissues (Kaegi and Schaeffer, 1988; Sauge-Merle et al., 2012). Very few studies have examined the MTs levels induced by Pb because mercury and cadmium are considered the principal inductors of MTs. Lee et al. (1983) found that there was no significant change in the urinary level of MTs in rats given up to 5 injections of saline solution of Pb. Nevertheless, the results from this study demonstrated that the MTs of the gut, the posterior of the silk gland, and the fat body were markedly synthesized by the induction of enhanced Pb stress. It is inferred that MTs are involved in the detoxification and homeostasis of Pb in the cells. This result is supported by some previous works. Pb could bind to MTs under ex vivo conditions (Waalkes et al., 1984), and tissue levels of MTs can be increased after Pb exposure (Ikebuchi et al., 1986). Similarly, on the basis of work in the laboratory, Maity et al. (2011) recorded a significant increase in the level of MTs in tissue from Lampito mauritii exposed to contaminated soil with 75, 150 and 300 mg kg 1 Pb. In addition, a strict dose–effect linear relationship was lacking in our work. MTs1 was the most sensitive to Pb stress as indicated by its obvious increase under lower concentration (200 mg kg 1 group). However, the levels of MTs2 and MTs3 were induced remarkably at higher Pb stress (400 and 800 mg kg 1 groups). As we know, the gut is the first barrier that is directly in contact with heavy metals, so MTs1 synthesis was induced under lower Pb stress. The synthesis of MTs2 and MTs3 was induced strongly when excess Pb was transported into the other organs after the Pb burden of the gut was maximum. Numerous studies have suggested that the level of MTs varies considerably depending on the species, tissue, animal age and other environmental factors (Van den Broeck et al., 2010; Fang et al., 2012). Thus, the actual mechanism of MTs under various situations is not fully understood and identified.
5. Conclusions Pb was transferred into mulberry and silkworm after exposure, although its biomagnification along the soil–mulberry–silkworm food chain was not observed. The treatment of soils with 200, 400, 800 mg kg 1 Pb resulted in the mulberry roots accumulating most of the Pb, i.e., 5.52–20.07 times higher than that in leaves. 92.15–92.82% of the Pb in the leaves was bound to the cell wall, and the low bioavailability fractions of Pb in the leaves, which were extracted by HCl, NaCl, and residual, were 95.29–95.57% of the total Pb. The Pb concentrations in the silkworm feces were 25.07–46.07 times higher than that in larva. Silk had the lowest Pb contents. The Pb contents in feces, silk, and peel and the change of the levels of MTs in various silkworm tissues indicated excretion and homeostasis are two important detoxification strategies of Pb for silkworm under high Pb stress. The process of Pb detoxification in mulberry and silkworm played an important role in the inhibition of Pb transfer along the food chain. In addition, mulberry and silkworm can be a promising approach for the remediation
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