Cu And Pb Uptake By Spinach And Carrots Biology Essay

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Several factors have been used to determine heavy metal uptake by vegetables from soil. This study investigated copper (Cu) and lead (Pb) enrichment factor (EF) and mobility factors (MF) as possible indicators of potential uptake of Cu and Pb by spinach and carrots grown on a sludge-amended luvisol. Sewage sludge was applied to the luvisol at different application rates and spinach and carrots planted. The enrichment of Cu and Pb in the luvisol after sludge application was determined, and the values regressed with values of total Cu and Pb concentrations in the spinach and carrots. Concentrations of Cu and Pb in spinach and carrots was calculated using the regression model, and the calculated values compared with the analyzed values. Lead MF values were higher than those of Cu but EF values for both Cu and Pb were < 3.0, indicating minor addition from sludge application. Whereas EF was inaccurate in predicting Cu and Pb uptake by spinach and carrots (R2 ≤ 10%), MF was more accurate (R2 ≥ 70%) in predicting Cu uptake in carrots and Pb uptake in spinach. Results indicate that whether MF or EF is used to determine possible metal uptake may depend on the vegetable and the particular cation in question.

Keywords: Bioavailability; Luvisol; sludge application; regression model, vegetables

INTRODUCTION

Soils usually contain small concentrations of heavy metals but application of sewage sludge and other forms of organic manure to soils have contributed to the elevated concentrations of heavy metals reported in some soils. The use of sludge as a soil amendment is practiced in several countries (Tlustoš et al. 2000; El-Naim and El-Housseini 2002) where it is used to improve soil physical properties (Logan et al. 1996), plant nutrient contents, and microbial activities (Chander et al., 2001). Though a valuable resource, sludge has also been documented as a major source of heavy metals including Cu and Pb in soils (Tlustoš et al. 2000). Some plants may require small concentrations of these heavy metals for their physiological processes but at high concentrations, Cu and Pb may become phytotoxic, and could bioaccumulate in the plants, presenting a health hazard to man and other animals. Health complications associated with Cu include irritation of the nose, mouth and eyes, headaches, stomach aches, dizziness, vomiting and diarrhea, weaknesses in fingers, wrists, or ankles while anemia, damage to the brain and kidneys have been associated with Pb (ATSDR 2007). Because of these health concerns, the concentrations of Cu and Pb among other metals in sewage sludge destined for agricultural use are regulated in many countries.

Several authors (Zhou et al. 2005; Rout and Das 2003; Merrington et al. 2003) have carried out studies on the uptake of heavy metals by plants grown on sludge-amended soil. Whereas some of these studies investigated the relationship between the concentration of heavy metals in these soils and the amount taken up by plants, others have focused on the relationship between the bioavailable fraction of the metal in the soils only, and the amount taken up by plants. Mingorance et al. (2007) and Yoon et al. (2006) for example used the biconcentration factor (BF) (ratio of heavy element concentration in plant to element concentration in soil) to determine heavy metal uptake by plants in soil, while Badaway & El-Motaium (2001) used heavy metal mobility factor (the ratio of an element in the mobile fractions (exchangeable + CO32-) to the inert fractions (reducible + oxidizable + residual)). Different methods have also been used to characterize the amount of heavy metal input in soils. Mingorance et al., (2007) and Mtanga and Machiwa (2007) used enrichment factor (EF) (the relative abundance of a chemical element in soil compared to the relative abundance in a local control site) to determine heavy metal contamination and uptake by plants whereas Manzoor et al. (2006) and Franco-Uría et al. (2009) used multivariate analyses.

In this paper, Cu and Pb mobility factor (MF) and enrichment factor (EF) in a sludge-amended luvisol from Botswana are used to determine the amount of Cu and Pb taken up by spinach (Spinaceae oleraceae) and carrots (Daucus carota) grown on the luvisol in an endeavor to understand which one of these two parameters could be used to estimate possible metal uptake from soils. Though sludge generated in Botswana is believed to have low concentrations of heavy metals because of a comparatively low amount of industrial effluent discharge into the sewer system, elevated concentrations of Cu and Pb have been reported in sludge generated from the waste water treatment plant (Ngole 2007). Spinach and carrots were the vegetables of choice because of their high demand and consumption in Botswana. It is anticipated that this investigation will shed more light on the estimation of heavy metal uptake by plants.

MATERIAL AND METHODS

Sampling of soil and sludge

The soil type used was a luvisol from Barolong Farms (located between latitudes 25°30'S and 25°45'S and between longitudes 25°00'E and 25°45'E) in the eastern part of Botswana. Barolong Farms area is one of the main agricultural regions in Botswana and the dominant soil type in the area is luvisol (Soil mapping and advisory services Botswana, 1990). From depths of between 0-50 cm (rooting zone of most crops), sixty samples were collected from the sampling area and homogenized to form a composite sample that was representative of the soils in the area. The properties of the luvisol used are indicated in Table 1. A three year old sludge was sampled from a sludge pile at a wastewater treatment plant in Botswana. The sludge was generated through the activated sludge method of waste water treatment, anerobically digested, air-dried, and piled at the sides of the sludge drying beds in a wastewater treatment plant. No further treatment was applied to the sludge but periodic aeration of the sludge may have occurred when the sludge pile was moved further from the drying beds to create space for younger sludge piles. Sludge samples were collected from different spots on the sludge pile and homogenized to produce a sample that was representative of the sludge pile. Properties of the homogenized sludge are indicated in Table 1.

Greenhouse experiment

The composite samples of sludge and luvisol were mixed at volume per volume percent (v/v %) ratios of 0: 100, 5: 95, 10: 90, 20:80, and: 40:60 sludge: luvisol and allowed to mature in the open. The 0:100 sludge-luvisol mixture served as the control luvisol sample. Three months after mixing the luvisol and sludge, samples were collected from each luvisol-sludge mixture and the speciation of Cu, and Pb determined as indicated in Table 2. Each luvisol-sludge mixture was individually passed through a sieve with a mesh size of 4 mm and then transferred into ten (10) different plant pots. The pots with the sludge-amended luvisol were then moved into a greenhouse where they were allowed to acclimatize for three days. Seeds of spinach and carrots were then planted directly on the sludge-amended luvisol. Five of the 10 pots were used for growing spinach and the other five for carrots. Carrots were grown for 13 weeks, and spinach for 9 weeks. At time of harvest, the edible leaves of spinach and roots of carrots were washed with tap water to remove soil particles and rinsed twice with distilled water. They were patted dry and the fresh biomass noted. The plants were then oven-dried at 70°C for 72 hours ground and used for chemical analyses (Su and Wong, 2004).

Chemical analyses

Speciation of Cu and Pb in the luvisol was determined using the five stage sequential extraction procedure described in Tessier et al. (1979). Details of the protocol used are presented in Table 2. The concentrations of Cu and Pb in the sample extracts were read using a Spectra AA Varian 220 FS flame atomic absorption spectrometer (FAAS) with deuterium correction. Copper and Pb content in spinach and carrots were determined in one gram (1 gm) of oven-dried sample that was ashed for 6 h at temperatures of between 500 and 550°C and then digested with concentrated HNO3 (Almăs and Singh, 2001). Concentrations of Cu and Pb in the digested vegetable samples were determined with the Spectra AA Varian 220 FS FAAS.

Analyses of data

The whole experiment was repeated twice with each sample analyzed in duplicate. Values reported are therefore means of data from both experiments. Copper and Pb MF in the sludge amended-luvisol were determined as directed by Badaway and El-Motaium (2001) (Equation 1).

MF = (1)

Where S1, S2, S3, S4, and S5 represent the concentration of metal in the exchangeable, carbonate, reducible, oxidizable, and residual geochemical fractions of the sludge-amended luvisol respectively.

Copper and Pb Enrichment Factor for the luvisol (EFsoil) was calculated as indicated in Equalution 2 (Mingorance et al., 2007, Mtanga and Machiwa 2007, Å muc et al., 2009).

EFsoil = (2)

Where M = concentration of metal, and Al = concentration of Aluminum

Aluminum was used as a reference lithogenic element to normalize metal concentration because its concentration in the soil was not affected by sludge application. Aluminium has also been used as the normalizing element by Mingorance et al., (2007), Mtanga and Machiwa (2007) and Å muc et al., 2009.

Copper and Pb Enrichment Factor in the vegetables (EFplant) was calculated as directed by Mingorance et al., (2007) (Equation 3)

EFplant = (3)

Where M = concentration of metal

EFsoil was correlated with EFplant to determine any relationship between metal enrichment in soil and metal enrichment in plant. Values for Cu and Pb EF and MF in the sludge-amended luvisol were each regressed with the concentrations of Cu and Pb in spinach and carrot. The regression model with the highest R2 value was used to calculate the concentration of the different metals in the vegetables. Calculated values of Cu and Pb concentrations in both vegetables were then compared with actual values to see how accurate the parameter is in determining uptake of Cu and Pb from sludge-amended soils. All analyses were done with SPSS version 17.0 for windows.

RESULTS AND DISCUSSION

Effect of sludge addition on mobility of Cu and Pb

Total Cu concentration in the sludge-amended luvisol increased with increase in SAR (Figure 1) with Cu EFsoil values being 0.58, 0.99, 1.1 and 1.1 at SAR of 5 %, 10 %, 20 % and 40 % respectively. According to Cheng et al. (2007), EF values < 3.0 indicate minor anthropogenic impact factor. With EF values of Cu at all SAR being < 3.0, it can be concluded that the enrichment of Cu by sludge addition was minor. Higher EF values of Cu in the sludge -amended luvisol at higher SAR can be explained by the difference between the concentrations of Cu in the sludge and that in the luvisol. Though total Cu concentration increased, its bioavailability reduced with increase in SAR as indicated by a decrease in Cu MF values with increase in SAR (Table 3). Ščančar et al. (2001) have also reported low availability of Cu in similar studies. The decrease in mobility may be explained by the increase in the concentration of Cu in the less available S3, S4 and S5 fractions with increase in SAR (Figure 1). Fuentes et al., (2008), have demonstrated strong affinity for OM by Cu. This affinity is further substantiated by the high stability constants that have been reported for organic matter-Cu complexes. An increase in OM content of the luvisol as a result of sludge addition (data not shown) would have increased the Cu-binding capacity of the luvisol resulting in the increase observed in the S4 fraction of Cu.

Total Pb concentration in the luvisol increased slightly with increase in SAR (Figure 1) with Pb EFsoil values of 0.7, 1.0, 1.2, and 1.0 at SARs of 5 %, 10 %, 20 % and 40 % respectively. The EF values of Pb also indicate low enrichment as a result of sludge addition. Bioavailability of Pb in the sludge-amended luvisol decreased slightly with increase in SAR as indicated by the MF values for Pb (Table 3). Increase in the concentration of Pb in the S4 fractions (Figure 1) may explain the decrease in Pb mobility observed with increase in SAR. Fractionation studies of Pb have indicated that it will preferentially sorb unto either the reducible, CO32-, or residual fraction depending on the prevailing chemical environment in the soil (Ma and Rao, 1997). Ščančar et al. (2001) have also reported binding of Pb by OM. The observations in this study are therefore concomitant with those of others.

Uptake of Cu and Pb by vegetables.

The fresh biomass of both spinach and carrots increased with increase in SAR with very little difference observed between the fresh biomass of spinach and carrots at corresponding SAR (Table 4). For both Cu and Pb, concentrations in spinach were higher than those in carrots (Figure 2). Increased uptake of heavy metals by spinach compared to carrots has also been reported in other studies (Ščančar et al. 2001). Whereas the amount of Cu taken up by spinach increased with increase in SAR, there was no significant change in the concentration of Cu in carrots with increase in SAR (Figure 2).

Copper EF values for spinach indicate a slight increase in Cu uptake due to sludge application while no clear pattern was observed for carrots (Table 4). The concentration of Pb in spinach was also higher than in carrots and decreased with increase in SAR (Figure 2). Whereas Pb EF values for carrots were higher than those of spinach, Cu EF values for spinach were higher than those of carrots (Table 4). Considering that both vegetables had similar fresh biomass (Table 4), the differences observed in the amount of Cu and Pb taken up may be explained by other factors including differences among species and enrichment of Cu and Pb in the sludge-amended luvisol. Lead concentration in spinach and carrots were lower than that of Cu (Figure 2) even though Pb content and Pb MF in the sludge-amended luvisol were higher than that of Cu. This observation may indicate that these vegetables have a tendency to take up more Cu from the soils than Pb even though the concentration of available Pb may be higher than that of Cu.

Determining Cu and Pb uptake by carrots from Cu and Pb MF and EF

Copper

The R2-values obtained from the regression between Cu MF and EF values in the sludge-amended luvisol and total Cu concentration in the spinach (Table 5) indicate that ≤ 10% of the variations observed in the amount of Cu taken up by spinach were accounted for by variations in mobility and enrichment of Cu in the sludge- amended luvisol. With such low influence, neither EF nor MF of Cu in the luvisol may be a reliable indicator of possible Cu uptake by spinach.

Copper EF values in the sludge-amended luvisol was also irrelevant in determining Cu uptake by carrots as it explained only 12 % of the variation in Cu concentration in carrots (Table 5). The inaccuracy of EF in determining Cu uptake is further enhanced by the low correlation coefficient between Cu EF in the sludge-amended luvisol and Cu EF in spinach (0.32) and carrots (-0.17). Variations in mobility of Cu in the sludge-amended soil on the other hand explain 70% of the variation in Cu concentration in carrots (Table 5). Copper MF therefore proved to be a more reliable parameter in the determination of Cu uptake by carrots as the calculated values of Cu in carrots and actual values were very similar (Figure 3).

Lead

The EF values of Pb in the sludge-amended luvisol also had low R2-values when regressed with total Pb concentration in spinach and carrots (Table 5) indicating the little influence that Pb enrichment had on the variations in the amount of Pb taken up by spinach and carrots. Pearson correlation also revealed that there was no correlation between enrichment of Pb in the sludge-amended soil and enrichment of Pb in spinach (-0.19) and carrots (0.46). Lead enrichment values may therefore not give reliable information on the potential uptake of Pb by carrots and spinach in sludge-amended soils. Mobility of Pb in the sludge-amended luvisol also had very little influence on variations observed in Pb taken up by carrots but in spinach, 74% of the variation observed in Pb uptake was accounted for by variations in the mobility of Pb in the sludge-amended luvisol (Table 5). The potential of Pb mobility in the sludge-amended luvisol as an indicator of Pb uptake by spinach is depicted by the similarity in the calculated and actual values of Pb in spinach (Figure 4).

Whereas enrichment of Cu and Pb in the sludge-amended luvisol give an idea of the degree to which sludge addition may have affected the concentration of Cu and Pb in the luvisol, it does not tell whether the element is available or not. The concentration of trace elements in plant tissue is correlated with the concentrations of the same metal in solution phase of the growth medium. The mobility factor proved to be a better indicator of metal bioavailability in the sludge-amended luvisol. Lack of correlation between Cu and Pb EF values in the sludge-amended luvisol, and Cu and Pb EF values of both spinach and carrots, coupled with the low influence of metal enrichment on metal uptake by both vegetables confirm the findings of other research which indicated that total concentration of heavy metals in soils is a poor indicator of metal bioavailability. The influence of metal mobility on the concentration of the metals in both vegetables as observed in this investigation further buttresses the opinion that the available fraction of metals in soils is a better indicator of possible uptake by plant.

CONCLUSION

Application of sludge to luvisol resulted in an increase in total Cu and Pb in the sludge-amended luvisol but using Al to normalize the concentrations of Cu and Pb in the control and sludge-amended luvisol indicate that the enrichment of both metals in the luvisol was minor. Though total concentrations of Cu and Pb were increased, their mobility was reduced, resulting in low bioavailability. Copper and Pb EF values in the sludge-amended luvisol showed no correlation with the Cu and Pb EF values of carrots and spinach, and proved not to be a reliable parameter in the determination of the amount of Cu and Pb taken up by both vegetables. Mobility factor was a more reliable indicator of Cu uptake by carrot but not by spinach. Similarly, MF proved to be more reliable than EF in indicating Pb uptake by spinach but not carrot. The implication is that MF and EF alone may not be adequate to determine metal uptake but the parameter used may depend on the element and possibly the vegetable in question. More studies however need to be carried out to validate this opinion.

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