Process Biochemistry 44 (2009) 1179–1184

Short communication

Bioaccumulation of cobalt in silkworm (Bombyx mori L.) in relationto mulberry, soil and wastewater metal concentrations

Muhammad Ashfaq a, Sajjad Ali a, Muhammad Asif Hanif b,*a Department of Agri. Entomology, University of Agriculture, Faisalabad 38040, Pakistanb Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad 38040, Pakistan

A R T I C L E I N F O

Article history:

Received 11 February 2009

Received in revised form 3 May 2009

Accepted 18 May 2009

Keywords:

Cobalt

Bioaccumulation

Toxicity

Silkworm

Mulberry

A B S T R A C T

The present study was planned to evaluate Co(II) toxicity in silkworm population. The soil was irrigated

using synthetic wastewater to determine the effects of pH and initial cobalt concentration in its

bioaccumulation in silkworm (Bombyx mori L.) food chain. The amount of cobalt in wastewater, soil,

mulberry and silkworm was determined by atomic absorption spectrophotometer (AAS) analysis. The

obtained results clearly indicate that silkworm can be used as template to indicate local cobalt pollution

as its body length, body weight and mortality rate was found to be strongly related to cobalt

concentration. Higher the cobalt amount in mulberry leaves more the toxicity to silkworm population. At

400 mg/L Co concentration and pH 4 there was maximum deposition of Co in the soil from the synthetic

effluent. However, in plants and silk worm the accumulation of Co was maximum at pH 4.5 at an initial

Co concentration of 400 mg/L in the synthetic effluent. The maximum cobalt found in wastewater, soil,

mulberry and silkworm was 400 � 0.01, 273.5 � 0.04, 42.85 � 0.01, 36.62 � 0.22 mg/kg, respectively.

� 2009 Published by Elsevier Ltd.

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Process Biochemistry

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1. Introduction

Heavy metals, absorbed through the root systems, inducechlorosis of leaf, deficiency of essential elements, and inhibitionof root penetration and growth [1]. In fact, heavy metals have asignificant toxicity for humans, animals, microorganisms and plants[2]. Excessive accumulation of heavy metals in agricultural soils,through wastewater irrigation, may not only result in soilcontamination, but also affects the food quality and safety [3].Although Co is an essential nutrient, excessive doses result in avariety of adverse responses. In higher concentrations, Co is toxic tohumans and to terrestrial and aquatic animals and plants [4]. Thedistribution of cobalt in plants is entirely species dependent. Theuptake is controlled by different mechanisms in different species.Toxic concentrations inhibit active ion transport. In higher plants,absorption of Co2+ by roots, involves active transport. Transportthrough the cortical cells is operated by both passive diffusion andactive process. In the xylem, the metal is mainly transported by thetranspirational flow. Distribution through the sieve tubes isacropetal by complexing with organic compounds [5].

The transfer of Co from soil to plants appears to be viable routeto expose lower trophic levels to biologically significant concen-trations and Co potentially accumulates in biomass and top soil [6].The behavior of Co in the environment has previously been

* Corresponding author. Tel.: +92 3338362781; fax: +92 419200764.

E-mail address: muhammadasifhanif@yahoo.com (M.A. Hanif).

1359-5113/$ – see front matter � 2009 Published by Elsevier Ltd.

doi:10.1016/j.procbio.2009.05.006

reviewed [7] and with the increased capability of analyticaltechniques and improved understanding of metal behavior in theenvironment [8], it is timely to consider further developments,particularly in light of changes to the legislative environment.Cobalt is an important part of industrial effluents and municipalwastewater. It has an ability to be accumulated in plants andanimals thus can enter into food chain. Environmental pollutionand degradation are the worst problems of the world. Bombyx mori

has long been a model organism in different studies due to its largesize and ease of culture. In this regard, the present study is plannedto evaluate the phytoremediation of Co(II) by mulberry (Morus alba

L.) plants and its subsequent effects on silkworm (as a modelorganism) which fed on the leaves of these plants.

2. Materials and methods

2.1. Production of cobalt containing mulberry and silkworm biomass

Mulberry (M. alba L.) plants were planted in cobalt impregnated soil, as outdoor

plants, under the environmental condition prevailing from September 2007 to

March 2008 at University of Agriculture, Faisalabad, Pakistan (dimension 31.42N

and 73.0 E and altitude of 184 m above sea level). The plants were irrigated using

synthetic effluents containing Co(II). The plants were planted in ditches (30 � 30

dimensions) at 5 ft row to row and plant to plant distance. Plastic sheets were used

to avoid contamination and to ensure the usage of given wastewater only. Plants

were exposed to different treatments of pH (3–5) at 100 mg/L concentration and

different concentrations of cobalt (25–400 mg/L) after 7 days of plantation by

irrigating plants using Co(II) containing wastewater. The control treatments were

maintained by irrigating with canal water. Plant leaves were collected, extensively

washed with deionized distilled water (DDW) and fed to silkworm larvae. Leaf and

soil sampling were done after a predetermined period of 15 days. Sampling of the

silkworm larvae and their excreta was done at the end of all the five larval instars.

M. Ashfaq et al. / Process Biochemistry 44 (2009) 1179–11841180

These sampled larvae, excreta, soil and leaves were dried in an oven at 70 8C to a

constant weight. Later on, these dried samples were ground into powdered form.

2.2. Chemical regents

All chemicals reagents used in this study were of analytical grade which were

purchased from Fluka chemicals. The chemicals used in this study were Co(No3)2,

HNO3, H2O2, NaOH, HCl, pH buffer solutions and cobalt atomic absorption

spectrometry standard solution (1000 mg/L).

2.3. Co(II) solutions

The stock Co(II) solution of 1000 mg/L was prepared by dissolving 4.95 g/L of

Co(NO3)2 in DDW. Co(II) solutions of required concentration were prepared by

diluting stock solution appropriately.

2.4. Digestion of sampled soil, mulberry leaves, silkworm larvae and faeces

One gram of oven dried and powdered samples of mulberry leaves, silkworm

larvae, faeces and soil were wet digested according to the described method [9].

2.5. Determination of Co

The concentrations of Co in the mulberry leaves, silkworm larvae, faeces and soil

samples were determined by flame atomic absorption spectrometry (AAS) using

PerkinElmer analyst [10].

Fig. 1. Deposition of Co(II) to soil by

Fig. 2. Relationship between Co(II) concen

2.6. Statistical analysis

All experimental treatments were triplicated. Microsoft Excel Version office

Xp was used for statistical analysis. The obtained results were discussed as

mean � S.D.

3. Results and discussion

The entrance of cobalt into terrestrial food chain is not wellunderstood yet. In this regard, the present study was planned tomonitor cobalt entrance into silkworm from mulberry plantsgrown on soil irrigated with cobalt containing wastewater.

3.1. Co(II) contents in soil

The soil, in which mulberry plants were planted, was irrigatedwith Co(II) synthetic effluents with pH ranging from 3 to 5 andwith initial Co(II) concentrations ranging from 25 to 400 mg/L. Theconcentration of cobalt in soil before irrigation using wastewaterwas 8.45 � 0.02 mg/kg. After irrigating soil using wastewater, theconcentration of Co(II) in soil increased significantly (Figs. 1 and 2).The maximum amount of Co(II) reside in soil at pH 4 and 400 mg/Linitial concentration in synthetic wastewater. It was found that the

synthetic effluent of various pH.

tration in synthetic effluent and soil.

Fig. 3. Bioaccumulation of Co(II) by Morus alba at various soil pH values.

Fig. 4. Effect of Co(II) concentration in soil on its bioaccumulation by Morus alba.

M. Ashfaq et al. / Process Biochemistry 44 (2009) 1179–1184 1181

Co(II) contents in soil were increased with increase concentration ofCo(II) effluents (Fig. 1). It reached to its maximum value of273.5 � 0.04 mg/kg at the highest concentration of 400 mg/L after75 days of irrigation. Increasing concentration effect was alsoobserved by Perez-Espinosa et al. [11]. The lowest Co(II) contentswere recorded to be 59.5 � 0.01 mg/kg at 25 mg/L dose rate after thesame time period. The Co(II) contents in soil were also increased withthe increase in pH and were maximum (206 � 0.04 mg/kg) at pH 4and minimum (19.5 � 0.01 mg/kg) at pH 3 after 75 days. The contentswere decreased after pH 4 as shown in (Fig. 2).

Fig. 5. Transportation of Co(II) to Bombyx mori from

3.2. Co(II) contents in mulberry leaves

Mulberry was found to be Co(II) non-hyper accumulator plant(Figs. 3 and 4). The maximum amount of Co(II) bioaccumulated inmulberry leaves was 42.85 � 0.01 mg/kg at 400 mg/L cobalt initialconcentration in wastewater. When the initial Co(II) concentrationwas low, the Co(II) bioaccumulation in mulberry leaves was alsoreduced. The lowest Co(II) contents in mulberry leaves were15.45 � 0.02 mg/kg at 25 mg/L Co(II) in wastewater. These resultsare reported after 75 days microplot experiment. Wastewater pH was

Morus alba leaves at various soil pH values.

Fig. 6. Effect of Co(II) concentration in soil on its transportation to Bombyx mori from Morus alba.

Fig. 7. Co(II) concentration in Bombyx mori excreta at various soil pH values.

M. Ashfaq et al. / Process Biochemistry 44 (2009) 1179–11841182

also found to be important parameter in Co(II) uptake. The maximumbioaccumulation of Co(II) in mulberry leaves occurred at a soil pH of4.5. The entrance of high concentrations of Co(II) in mulberry suggestthat it could be a source of danger for animal and human life. Theplant biomass present in highly polluted areas should be carefullydisposed off to reduce the risk of severe damage to life and ecosystem.The accumulation of Co(II) in other plants was studied by previousresearchers [5,12–15]. They observed significant effect of Co(II) onplant growth. Prince et al. [14], studied the toxicity of cadmium metalin mulberry plants.

Fig. 8. Effect of Co(II) concentration in soil

3.3. Co(II) contents in silkworm larvae

Co(II) bioaccumulation in the larval body was maximum(31.2 � 0.02 mg/kg and 28.3 � 0.03 mg/kg) at 400 mg/L Co(II) con-centration and soil pH 4.5. The lowest values were 11.7 � 0.01 mg/kgand 16.5 � 0.02 mg/kg at 25 mg/L and soil pH 3 after the completionof 5th larval instar (Figs. 5 and 6).

The life cycle of silk worm consist of five instars. The life span of1st, 2nd, 3rd, 4th and 5th silkworm instars is of 2–3, 4–5, 6–7, 6–7and 4–6 days, respectively. A detailed review of literature was

on its amount in Bombyx mori excreta.

M. Ashfaq et al. / Process Biochemistry 44 (2009) 1179–1184 1183

made to study bioaccumulation of Co(II) in insects. But accordingto the information of the authors there was not a single studycarried out in this regard. However accumulation of other heavymetals in insects is reported by many researchers [15–21].

3.4. Co(II) contents in silkworm faeces

The silkworm faeces were analyzed to evaluate the Co(II)contents (Figs. 7 and 8). The maximum Co(II) concentration infaeces was found to be 19.76 � 0.02 mg/kg at 400 mg/L of Co(II)concentration. However, most of Co(II) still reside inside the insectbody and was responsible for metal toxicity in silkworm body.Jackson et al. [22] found that non-biodegradable compounds such asheavy metals are the most dangerous due to their innate ability to

Table 1Effect of pH and Co(II) concentrations in synthetic effluent on body length (cm) of Bom

Treatment 1st instar 2nd instar

Control 0.68 � 0.01 1.30 � 0.11

Co(II) amount (mg/L)

25 0.56 � 0.11 1.12 � 0.09

50 0.52 � 0.10 0.91 � 0.08

100 0.49 � 0.09 0.72 � 0.05

200 0.45 � 0.08 0.70 � 0.04

400 0.41 � 0.02 0.65 � 0.02

pH

3 0.51 � 0.05 0.70 � 0.01

3.5 0.50 � 0.05 0.68 � 0.04

4 0.48 � 0.01 0.66 � 0.02

4.5 0.44 � 0.06 0.62 � 0.11

5 0.46 � 0.03 0.67 � 0.21

Table 2Effect of pH and Co(II) concentrations in synthetic effluent on body weight (g) Bombyx

Treatment 1st instar 2nd instar

Control 0.021 � 0.002 0.120 � 0.002

Co(II) amount (mg/L)

25 0.018 � 0.004 0.090 � 0.007

50 0.016 � 0.001 0.079 � 0.004

100 0.013 � 0.005 0.068 � 0.003

200 0.011 � 0.001 0.064 � 0.001

400 0.009 � 0.002 0.059 � 0.002

pH

3 0.012 � 0.004 0.069 � 0.002

3.5 0.011 � 0.002 0.062 � 0.004

4 0.009 � 0.001 0.057 � 0.003

4.5 0.008 � .0002 0.054 � 0.002

5 0.010 � 0.006 0.058 � 0.005

Table 3Effect of pH and Co(II) concentrations in synthetic effluent on morality rate Bombyx m

Treatment 1st instar 2nd instar

Control 09 � 0.65 07 � 0.65

Co(II) Amount (mg/L)

25 14 � 0.07 11 � 0.08

50 16 � 0.04 12 � 0.55

100 20 � 0.02 16 � 0.40

200 22 � 0.01 18 � 0.20

400 23 � 0.03 21 � 0.60

pH

3 21 � 0.40 18 � 0.40

3.5 21 � 0.62 19 � 0.50

4 23 � 0.60 21 � 0.20

4.5 25 � 0.55 22 � 0.30

5 21 � 0.40 20 � 0.35

constantly remain with the ecosystem. The same type of conditionswas drawn by Hernandez-Hernandez et al. [23] and Tyler [24]. Theresult suggested that digestive rate of silkworm larvae wassignificantly affected by presence of Co in mulberry leaves.

3.5. Body length, body weight and mortality rate of silkworm larvae

Body length and body weight of silkworm (B. mori L.) werereduced with increase in Co(II) concentration. This suggested thatincreasing Co(II) concentration in silkworm body weakened thesilkworm larvae. And subsequently the mortality rate of silkwormwas also increased. The results are presented in Tables 1–3. Thetoxicity of different heavy metals in various insects was studied bymany researchers in the past [15,25]. From the obtained results it

byx mori larvae.

3rd instar 4th instar 5th instar

1.82 � 0.10 3.98 � 0.02 5.96 � 0.02

1.60 � 0.01 3.12 � 0.01 5.10 � 0.09

1.51 � 0.02 2.51 � 0.01 4.30 � 0.08

1.39 � 0.09 2.25 � 0.08 4.10 � 0.03

1.35 � 0.05 2.20 � 0.11 4.12 � 0.01

1.26 � 0.21 2.15 � 0.05 3.98 � 0.05

1.35 � 0.01 2.38 � 0.06 4.40 � 0.02

1.37 � 0.11 2.30 � 0.04 4.15 � 0.04

1.28 � 0.07 2.21 � 0.02 4.07 � 0.02

1.25 � 0.11 2.11 � 0.01 3.90 � 0.06

1.32 � 0.12 2.18 � 0.10 4.09 � 0.17

mori larvae.

3rd instar 4th instar 5th instar

0.550 � 0.001 3.902 � 0.004 8.750 � 0.015

0.425 � 0.066 3.450 � 0.019 8.650 � 0.001

0.370 � 0.005 3.222 � 0.005 8.580 � 0.002

0.349 � 0.002 3.190 � 0.002 8.550 � 0.003

0.315 � 0.003 3.105 � 0.006 8.400 � 0.002

0.306 � 0.011 2.950 � 0.015 8.155 � 0.002

0.347 � 0.010 3.210 � 0.001 8.660 � 0.004

0.310 � 0.006 3.192 � 0.005 8.600 � 0.007

0.300 � 0.004 3.125 � 0.007 8.450 � 0.009

0.295 � 0.002 2.900 � 0.002 8.100 � 0.002

0.305 � 0.007 3.162 � 0.004 8.350 � 0.004

ori larvae.

3rd instar 4th instar 5th instar

06 � 0.55 04 � 0.60 03 � 0.65

09 � 0.50 07 � 0.55 04 � 0.55

11 � 0.45 08 � 0.65 08 � 0.65

13 � 0.50 11 � 0.45 10 � 0.50

13 � 0.60 13 � 0.50 10 � 0.45

16 � 0.60 14 � 0.45 11 � 0.35

15 � 0.45 09 � 0.45 06 � 0.45

16 � 0.50 11 � 0.35 08 � 0.35

16 � 0.35 14 � 0.65 10 � 0.45

18 � 0.50 16 � 0.55 13 � 0.55

14 � 0.45 14 � 0.25 11 � 0.70

M. Ashfaq et al. / Process Biochemistry 44 (2009) 1179–11841184

can be easily concluded that silkworm larvae can be used as auseful templet to access localized Co pollution in terrestrialecosystems by observing changes in its normal body cycle.

4. Conclusions

Bioaccumulation of Co(II) in soil, mulberry plants, silkwormlarvae and faeces was maximum at the initial Co concentration of400 mg/L. Bioaccumulation of Co(II) in the larval bodies ofsilkworm was significantly increased when they were feed uponCo polluted mulberry leaves. Although silkworm (B. mori L.) larvaeexcreted large quantity of Co(II) in its faeces, still a large amount ofCo(II) remained in its body. These findings suggested that Co(II)presence in aqueous effluents used for plant irrigation should bestrictly monitored to avoid ecological pollution.

Acknowledgment

Authors like to thank Mr. Ishaq (Laboratory Technician,Department of Agri. Entomology, University of Agriculture,Faisalabad-38040, Pakistan) for his help during the presentstudy.

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