The correlation study of several chemical extractants to assess plant copper uptake from tropical soils polluted with heavy metals

Glass-house experiments were conducted to study the correlation between Cu uptake by several plants and soil Cu extracted by several chemical extractants. Heavy-metal contaminated soils from Lampung, the southern part of Sumatra, Indonesia, with different levels of Cu were used. Eight different plants including amaranth (Amaranthus tricolor), caisim (Brassica chinensis var. Parachinensis), corn (Zea mays L.), land spinach (Ipomoea reptans Poir), lettuce (Lactuca sativa), napier grass (Penissetum purpureum), thorny amaranth (Amaranthus spinosus L.) and water spinach (Ipomoea aquatica) were employed. The uptake of Cu for amaranth is well predicted by N HCl, Buffered DTPA pH 7.30, N NH4OAc pH 7.00, and M CaCl2; for corn by N HCl and N NH4OAc pH 7.00; for land spinach by N HNO3, N HCl, Buffered DTPA, N NH4OAc pH 7.00, and M CaCl2; for napier grass by N HCl and Unbuffered DTPA. N HNO3 is good in predicting the uptake of Cu by land spinach, N HCl by amaranth, corn, land spinach, and napier grass; Buffered DTPA by amaranth and land spinach; Unbuffered DTPA by napier grass; N NH4OAc pH 7.00 by amaranth, corn and land spinach, and M CaCl2 by amaranth and land spinach. None of the tested methods is good in predicting the uptake Cu by caisim, lettuce, thorny amaranth, and water spinach


Introduction
Problems related to heavy metal contamination and pollution in the soil environment exist extensively all over the world in line with the industrialization and the development of modern lives .The contamination and pollution caused significantly different concentrations of heavy metals in soils and their effects on the living things.This situation stimulates the development of analytical techniques to assess plant available heavy metals.
To develop good soil plant available heavy metal elemental analytical technique researchers must abide to three crucial steps, i.e. chemical extractant development, greenhouse correlation study, and field calibration test [24].The development of chemical extractants is the initial step to devise particular extractants that may extract the intended forms of available heavy metals in the soil system [24,25].As previously reported [25], heavy metals in the soil system exist in two major forms: dissolved heavy metals that include all forms in the soil water comprising free cations, complexes and chelates and the structural heavy metals comprising the exchangeable forms of heavy metal bounded on the negative charges of clays and organic molecules with various binding energies and the more strongly structural 1314 (2024) 012003 IOP Publishing doi:10.1088/1755-1315/1314/1/012003 2 heavy metals in the soil secondary and primary minerals [24].The amount of the structural heavy metals is much greater than the dissolved heavy metals.
The extraction of each heavy metal form needs different chemical extractants [25].Research reported in [25] studied the soil Cu extraction of 6 different extractants and showed that N HNO3 and N HCl extracted more Cu than did buffered and unbuffered DTPA as well as the weaker extractants including M CaCl2 and N NH4OAc pH 7.00.The M CaCl2 and N NH4OAc pH 7.00 extracted only the soil exchangeable Cu from the soil particle surface negative charges, while DTPA extracted more exchangeable Cu due to enhanced release of heavy metals driven by the heavy metal chelation by DTPA molecules.The dilute acids may also extract the soil heavy metal precipitates in addition to the dissolved heavy metals.The extraction powers of these extractants follow the order of dilute acids > DTPA > dilute salts and therefore the magnitude of extracted heavy metals follows the order of dilute acids > DTPA > dilute salts [25].
Before the last field calibration, the greenhouse correlation test is more crucial.Plants are allowed to grow in soil samples with significantly different concentrations of heavy metals for a particular time span.The promising extractants must extract heavy metals and suffice significantly high correlation coefficients with those absorbed by the plants.The correlation coefficient of > 0.500 is considered relatively high.It is mentioned in [25] that high correlations existed between the Cu and Zn absorbed by land spinach and water spinach with the soil Cu extracted by N HNO3 in a preliminary study.The correlation study may reveal the chemicals extractants that may accurately predict the heavy metal absorption by particular plants.
This correlation study was to evaluate the relationship between plant uptake of Cu by 8 different plants with the tropical soil Cu extracted by several chemical extractants reported previously [25] that extract different forms of heavy metals from weakly held, moderately held to strongly held heavy metals in soils.Correlation study is hardly found in current literature more particularly in the tropical soils from Indonesia.

Soil samples and tested plants
Soil samples containing relatively wide range of Cu were taken from topsoil (0-15 cm) in 23 years old experimental plots one-time amended with a Cu-containing industrial waste from CV Metal Wares Jakarta in 1998.The industrial waste was given at 0 (Control), 15 (Low Metal) and 60 Mg ha -1 (High Metal) as reported in [20].The soil sample was sieved to 2 mm after air-drying, grinding and a thorough mixing.Gravimetric water content was determined to employ soil samples at oven-dry equivalent base (105 o C, 24 hours).Some selected physical and chemical properties of the soils are listed in Table 1.Plants used in this study were amaranth (Amaranthus tricolor), caisim (Brassica chinensis var.Parachinensis), corn (Zea mays L.), land spinach (Ipomoea reptans Poir), lettuce (Lactuca sativa), napier grass (Penissetum purpureum), thorny amaranth (Amaranthus spinosus L.), and water spinach (Ipomoea aquatica).These include 1 crop plant, 5 vegetable plants, and 2 weed plants.

Greenhouse experiment
Exactly 200 g of air-dry soil samples (24 hours 105 o C oven-dry equivalent) was put into a 300 ml pot completed with a small hole at the bottom.The pot was placed on a piece of wooden board also completed with holes to capillary moist the soil samples at the soil field water capacity through cotton wick connected to a common water reservoir beneath.Plant seeds or seedlings previously prepared were planted on the soil samples after the soil samples reaching their field water capacities.Plants were allowed to grow for 4 weeks, during which the soil field water capacity was capillary maintained by daily maintaining the water volume in the common reservoir.
To evaluate the concentrations of Cu in soil and plant, soil and plants were sampled at the end of the 4 weeks plant growth.Soil samples were air-dried, ground, thoroughly mixed, and its gravimetric water content were determined.Plant shoots were cut at the soil surface and oven dried at 60 o C for 3 x 24 hours.Before oven-drying, plant roots was rinsed with tap water to remove the remaining soil particles.After an oven drying at 60 o C for 3 x 24 hours, their dry-masses were weighed using an analytical balance.

Soil, plant, and data analysis and interpretation
Soil samples were analysed for the Cu concentration using the ICE 3000 flame atomic absorption spectrophotometry (AAS) at  = 324.7 nm after a 1-hour extraction of 10 g of oven dry equivalent soil sample with 20 ml of the related extracting solution in an end-to-end shaker.The related extracting solution is found in Section 2.2.Filtration was conducted using a whatman filter paper to obtain an AAS ready-to-analyse supernatant.This extraction was conducted with each of the chemical extractants studied in this research.Each plant sample was also analysed for Cu content using the method described in [25].
Data were converted into the same unit i.e. mg kg -1 both for soil and plant samples.The data were then plotted in computer program of Excel to obtain the linear regression equation and its correlation coefficient (R), that indicate the correlation between the uptake of Cu and the soil Cu concentration for each plant and chemical extractant tested.The R of 0.500 to 1.000 was considered good to excellent in predicting the Cu uptake by the related soil heavy metal extractant.The gradient values (b) in the regression equation y = a + bx indicate the sensitivity of the method.Higher gradient values are preferred.

Correlation between soil and plant Cu uptake
As previously stated, dilute acid HCl and also HNO3, which have the highest relative strengths, extracted the highest soil Cu compared to the other 4 chemical extractants.Plots of the soil Cu extracted by N HCl with the Cu uptake by corn, land spinach, thorny amaranth, and napier grass are depicted in Figure 1.The correlation coefficients are relatively high, greater that 0.500 i.e 0.713 for corn, 0.922 for land spinach, 0.905 for thorny amaranth, and 0.872 for napier grass.These values indicated that N HCl was excellent in predicting the uptake of Cu from soils contaminated with Cu from industrial waste a longtime amended in the soils.By considering the value of intercepts in the case of N HCl as the single extractant, i.e. 2.32 (napier grass), 0.425 (land spinach), 0.412 (corn), and 0.02 (thorny amaranth) (Figure 1), it is clear that napier grass absorbed the highest Cu from soil.By considering the gradients, which is 0.086 (land spinach), 0083 (napier grass), 0.035 (thorny amaranth) and 0.002 (corn), it is also clear that land spinach and napier grass were the more sensitive to the changes in the soil Cu concentration.These data indicate that N HCl was good in predicting the uptake of Cu from contaminated soils by napier grass.Napier grass absorbed greater soil Cu than the other 3 plants.
Comparing the 5 chemical extractants using land spinach as the tested plant, it is clear that N NH4OAc pH 7.00 and M CaCl2 predicted Cu absorption better than did dilute acids and DTPA as indicated by the value of intercepts of N NH4OAc pH 7.00 (2.29) and M CaCl2 (2.06) are greater than N HNO3 (1.91), Buffered DTPA pH 7.30 (1.78), and N HCl (0.43) (Figure 2).The gradients that show the sensitivity of the method to the changes in soil Cu for N NH4OAc (1.613) and M CaCl2 (0.907) are also greater than for N HCl (0.086), Buffered DTPA pH 7.00 (0.074), and N HNO3 (0.0293).These data show that N NH4OAc pH 7.00 and M CaCl2 are better than the other 3 chemicals in predicting the uptake of soil Cu by land spinach.

Comparison among extractants and tested plants
The linear relationship between the uptake of Cu by all plants and the soil Cu are listed in Table 2 (for extractant N HNO 3 and N HCl), Table 3 (for Buffered and Unbuffered DTPA), and Table 4 (for N NH4OAc pH 7.00 and M CaCl2).It is clearly shown that N HNO3 is good in predicting the uptake of Cu for land spinach and lettuce while N HCl for all tested plants except for thorny amaranth (Table 2).The buffered DTPA pH 7.30 is good in predicting the plant uptake of Cu by all tested plants except for lettuce, napier grass, and thorny amaranth while the unbuffered DTPA is good only for napier grass and water spinach (Table 3).Shown in Table 4 that N NH4OAc pH 7.00 is good in predicting the soil Cu absorbed by amaranth, corn, land spinach, thorny amaranth and water spinach while M CaCl2 is good in predicting the uptake of Cu from contaminated soils for amaranth, caisim, corn, and land spinach.This data indicates clearly that analysing the availability of Cu for plant management depend not only on the chemical extractant but also the tested plant.However, there are various chemical extractants available to predict the availability of Cu as summarized in Table 5.To choose the right chemical extractant, it is important to consider the value of gradient that indicate its sensitivity to the concentration of soil Cu (Tabel 6).This means that N HNO3 is good for predicting plant Cu uptake for land spinach and N HCl for land spinach (Table 2 and 6), Buffered DTPA pH 7.30 for land spinach and Unbuffered DTPA for napier grass (Table 3 and 6), N NH4 OAc pH 7.00 for land spinach, and M CaCl2 for land spinach (Table 4 and 6).Each plant also shows its own suitability for the extractants.Based on the sensitivity of the method, the uptake of Cu by amaranth was best predicted by N NH4OAc pH 7.00, caisim by M CaCl2, corn by N NH4OAc pH 7.00, land spinach by N NH4OAc pH 7.00, lettuce by N HCl, napier grass by N HCl, thorny amaranth by N NH 4 OAc pH 7.00, and water spinach by M CaCl 2 (Table 2, 3, 4, 5, and 6).Future researches can be directed to the use of various concentrations for each extractants to devise more accurate chemicals in predicting the amounts of copper and other heavy metals absorbed by each tested plant.Developing the analytical method for different soil with different characteristics will be very important to test this method in different soils with different characteristics other than their heavy metal concentrations.

Figure 1 .
Figure 1.The correlation between plant uptake and HCl-extracted Cu from heavymetal-waste amended soils.

Figure 2 .
Figure 2. The correlation between land spinach uptake of Cu and soil Cu extracted by several chemical extractants.

Table 1 .
Selected physical and chemical properties of soil samples a .
a Waste amendment was conducted in 1998 or about 23 years before soil sampling, b soil textural class was Clay Loam

Table 2 .
The correlation of plant and soil Cu extractable by 1 N HNO3 and 1 N HCl from heavy metal containing industrial waste amended soils.

Table 3 .
The correlation of plant and soil Cu extractable by buffered and unbuffered DTPA from heavy metal containing industrial waste amended soils.

Table 4 .
The correlation of plant and soil Cu extractable by N NH4OAc pH 7.00 and M CaCl2 from heavy metal containing industrial waste amended soils.

Table 5 .
The ≥ 0.500 correlation coefficients between uptake and soil Cu in heavy metal amended soils.