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Mobility, bioavailability, and toxic effects of cadmium in soil samples
Environmental Research 91 (2003) 119–126
Mobility, bioavailability, and toxic effects of cadmium in soil samples Z. Prokop,* P. Cupr, V. Zlevorova-Zlamalikova, J. Komarek, L. Dusek, and I. Holoubek Research Centre for Environmental Chemistry and Ecotoxicology, Masaryk University, Kamenice 126/3, 625 00 Brno, Czech Republic Received 14 June 2001
Abstract Total concentration is not a reliable indicator of metal mobility or bioavailability in soils. The physicochemical form determines the behavior of metals in soils and hence the toxicity toward terrestrial biota. The main objectives of this study were the application and comparison of three approaches for the evaluation of cadmium behavior in soil samples. The mobility and bioavailability of cadmium in five selected soil samples were evaluated using equilibrium speciation (Windermere humic aqueous model (WHAM)), extraction procedures (Milli-Q water, DMSO, and DTPA), and a number of bioassays (Microtox, growth inhibition test, contact toxicity test, and respiration). The mobility, represented by the water-extractable fraction, corresponded well with the amount of cadmium in the soil solution, calculated using the WHAM (r2 ¼ 0:96; Po0:001). The results of the ecotoxicological evaluation, which represent the bioavailable fraction of cadmium, correlated well with DTPA extractability and also with the concentration of free cadmium ion, which is recognized as the most bioavailable metal form. The results of the WHAM as well as the results of extraction experiments showed a strong binding of cadmium to organic matter and a weak sorption of cadmium to clay minerals. r 2003 Elsevier Science (USA). All rights reserved. Keywords: Bioavailability; Cadmium; Extractability; Mobility; Soil; Speciation
1. Introduction The behavior of metals in soils (e.g., mobility, bioavailability) cannot be reliably predicted on the basis of their total concentrations. The uptake and toxicity of many metals show marked dependence on speciation of the metals and these responses often correlate best with the activity of free metal ion (Laxen and Harrison, 1981; Knight and McGrath, 1995; Parker and Pedler, 1997). Exceptions to this generalization have been observed; however, the free metal form is considered the most bioavailable and the most active form (Janssen et al., 1997a). The bioavailability of metals in soils or sediments is often expressed in terms of concentration in a water phase. The metal distribution between a solid phase and pore water of a soil is commonly described by equilibrium partitioning (Janssen et al., 1997b). But the total dissolved metal concentration does not necessarily correspond to the amount available to biota. Ion pairs, complex ions, polymers, or microparticulates can reduce *Corresponding author. Research Centre for Environmental Chemistry and Ecotoxicology, Kamenice 126/3, 625 00 Brno, Czech Republic. Fax: +54-112-9506. E-mail address: [email protected] (Z. Prokop).
free ion species of heavy metals in solution (Green et al., 1993). The process of identifying and quantifying these different species of metals in a sample is referred to as speciation. Chemical equilibrium models, such as the Windermere humic aqueous model (WHAM) and freeion activity model (FIAM), are available to calculate metal speciation in waters, sediments, or soils (Tipping, 1994; Parker and Pedler, 1997). Many studies refer to metal speciation in terms of extractable metals related to single or sequential extraction. For example DTPA, CaCl2, or other individual extractants are frequently used for prediction of availability of metals to plants (Liang and Karamanos, 1993). Ahnstrom and Parker (2001) suggested that conventional sequential extraction procedures may be of limited utility for predicting bioavailability. The extractable fraction as well as the pore water fraction of metals does not necessarily correspond to the amount available to soil organisms. The concentrations of the bioavailable form can be related more closely to biological toxicity (Tokalioglu et al., 2000). Ecotoxicological evaluations of soils are usually associated with the measurement of aqueous or solvent extract toxicity. A bioassay in which the test organism directly interacts with untreated soil, with both the
0013-9351/03/$ - see front matter r 2003 Elsevier Science (USA). All rights reserved. PII: S 0 0 1 3 - 9 3 5 1 ( 0 2 ) 0 0 0 1 2 - 9
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aqueous and the solid phase, may provide a better assessment of bioavailability of contaminants. Microbial contact toxicity tests provide intimate contact between toxicants and test microorganisms or enzymes (Rossel et al., 1997; Shaw et al., 2000). In this study, the behavior of cadmium spiked into five selected noncontaminated soil samples was investigated following chemical and ecotoxicological procedures. The main objective was a comparison of equilibrium cadmium speciation in the studied soil samples (WHAM) with the cadmium-extractable fractions (Milli-Q water, DMSO, and DTPA) and the ecotoxicological results (Microtox test, growth inhibition test, contact toxicity test, and respiration test). A second objective was the determination of the physicochemical soil characteristics mostly influencing the behavior of cadmium in soil.
2.2. Chemical analyses
2. Materials and methods
Cadmium concentrations in the extracts were measured using atomic absorbtion spectrometr (Perkin– Elmer 306). The 228.8 nm resonance line was generated by a Cathodeon hollow cathode lamp (6 mA) or a Perkin–Elmer electrodeless discharge lamp (5 W input power), respectively. The transmitted spectral interval was 0.7 nm. Deuterium background correction was used to eliminate nonselective absorption. A 10-cm one-slot burner was used for measurements in the acetylene–air flame. The flow rates of the acetylene and air were 3.6 and 22.5 L/min, respectively. The samples were acidified by 1 M HCl to pH 2. Standard solutions of CdCl2 were prepared by dissolving 1 g of metallic cadmium of analytical grade quality in 30 mL of 6 M HCl and diluting with double-distilled water to a volume of 1 L. The solutions were standardized by chelometric titration with EDTA using xylenol orange as an indicator and contained 0.99 g Cd/L.
2.1. Sample collection and handling
2.3. Ecotoxicity tests
Soil samples were collected from five unpolluted areas in the Czech Republic (samples 1 and 3, loamy–sandy soil; sample 2, sandy–loam soil; sample 4, loam soil; sample 5, clay–loam soil). The soil samples were collected from depths of 0–20 cm, dried to constant mass at ambient temperature, ground, sieved through a 2-mm sieve, and homogenized (ISO 10 381-6, 1993). Samples were stored at 4 C until use. Background cadmium concentration in all selected samples was lower than 0.1 mg Cd/g of soil. Soil samples from each site were characterized for grain size, total organic carbon (Cox ) according to ISO 14 235 (1998), and total nitrogen (Ntot ) according to ISO 11 261 (1995). Fulvic acid (FA) and humic acid (HA) amounts were measured according to Schnitzer and Schuppli (1989). Amounts of calcium, potasium, and magnesium were measured according to Mehlich (1978). The pH was measured according to ISO 10 390, 1994. The samples were moistured, spiked with CdCl2 (0.01–5.0 mg Cd/g of dry soil), and preincubated for 72 h at 22 C prior to testing. Sample moisture was adjusted to 65% of its water holding capacity (Forster, 1995). Cadmium was extracted from soils by Milli-Q water (QW), by DMSO (10% dimethylsulfoxide, 10% methanol in deionized water) according to Dutka et al., (1993, 1994) and by DTPA (5 mM diethylenetriamine pentaacetic acid, 10 mM CaCl2, and 0.1 M triethanolamine buffered at pH 7.3) according to Bailey et al. (1995). The extraction was initiated by homogenization of the soil samples and extract reagents at a ratio of 1:1 (w:v) for water and DMSO and at a ratio of 1:2 (w:v) for DTPA. The suspension was shaken (200 rpm) for 1 h at 20 C and then filtered.
The Microtox test was conducted according to the procedures outlined in ISO 11 348 (1998). The degree of reduction of bioluminescence in the presence of a sample gives an indication of the toxicity of the sample under investigation. The growth inhibition test was performed according to ISO 10 712 (1997). Test samples were inoculated with Pseudomonas putida and incubated for 16 h. Changes in growth rate were estimated turbidimetrically. The contact toxicity test with Bacillus cereus . was performed according to Ronnpagel et al. (1995), with some modifications. This screening test is based on determination of microbial dehydrogenase activity inhibition. Test bacteria activity is proportional to the amount of reduced resazurin (oxido-reduction indicator). In the test, 2 g of each sample was transferred into a centrifuge screw-cap tube and resuspended in 2 mL of phosphate buffer. The inoculum was prepared from actively growing biomass by adjusting the cell concentration to OD601 ¼ 1:0 and added to the soil–buffer mixture. Incubation was performed for 4 h at 30 C and 70 rpm. After incubation, 2 mL of resazurin solution was added at concentration of 0.2 mg/mL of phosphate buffer. The mixture was shaken for 1 h and centrifuged (3500g, 5 min). The reduction of resazurin was measured at 601 nm. Soil respiration was measured after preincubation. Soil samples were incubated in closed jars for 48 h at 22 C. Moisture was adjusted to 60% of water holding capacity. After incubation CO2 production was measured using a gas chromatograph equipped with thermal conductivity detector. A polyethylene column 1.5 m long and 4 mm id packed with Porapac Q 80/100 mech (Supelco) was operated at laboratory temperature using hydrogen as carrier gas at head pressure 300 kPa.
Z. Prokop et al. / Environmental Research 91 (2003) 119–126
2.4. Data analysis
The fraction of cadmium in soil can be mobilized, is important with regard to the protection of groundwater quality. Water is the major carrier of metal pollutants (Bourg, 1988), so the water-soluble fraction in the water extract corresponds to the mobile fraction of the metals. Water extractability correlated well with the amount of cadmium in soil solution calculated using WHAM (Table 3). The amount of cadmium in a water extract, i.e., potential mobile fraction, increased with increasing total concentration of cadmium in the soil samples. All of the soil samples successfully held cadmium up to a concentration of 1 mg/g of dry soil. This was followed by an exponential increase in the amount of cadmium in water extracts as the total concentration in soil samples was further elevated. It is apparent that the sorption of cadmium on soil samples was limited. The sorption of cadmium reached a maximum once the sorption surfaces were saturated. The Langmuir model was fit to the water-extractability data and the sorption maximum and an affinity parameter was then calculated (Table 4). The sorption maximum correlated well with the amount of organic carbon in the soil samples (Table 3). On the other hand, no relationship was found between the solid particle distribution and the extractable fraction of cadmium from the soil samples. The weak metal binding on clay compared to binding to organic matter was previously described by Janssen et al. (1997a). There was no significant difference between water and DMSO extractability of cadmium from the soil samples. Using the paired nonparametric Wilcoxon test, there was no significant difference (a ¼ 0:05) between these two extraction procedures for Cd-spiked concentrations of 0.01, 0.1, 0.5, 1.0, and 2.5 mg/g. There was a
The effects of cadmium on the test microorganisms (activity inhibition or stimulation) were expressed as the percentage change in their activity compared to negative controls. Linear models were fit to the linear parts of the dose–response relationships. The model parameters were determined by the least-squares fitting method. EC50 values (mg Cd/g dry soil) were calculated by inverse prediction from these linear models (Cochran and Cox, 1957). The chemical speciation of cadmium in the soil samples was calculated using WHAM (Tipping, 1994). The amount of cadmium sorbed on the soil samples (mg/kg) was plotted against the cadmium concentration in the water extract (mg/L). The experimental data were fit to the Langmuir model (Samiullah, 1990) n ¼ K c M=ð1 þ K cÞ;
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ð1Þ
where n and c are cadmium concentrations sorbed on the soil and in the water extract, respectively, M is a sorption maximum of cadmium in a studied soil, and K is an affinity parameter.
3. Results and discussion The physicochemical characteristics of studied soils are shown in Table 1. The selected soils contained 2.81% to 5.47% organic carbon and 53–85% of clay (particles o2 mm). The cation exchange capacity ranged from 198 to 416 mmol eq/kg1. Acidity was similar for all soil samples. H2O pH ranged from 5.4 to 7.1. The concentrations of cadmium extracted by water, DMSO, and DTPA is shown in Table 2.
Table 1 Physical and chemical characteristics of soil samples Sample
pH
CEC (mmol eq kg1)
Ntot (%)
Composition of soil particles (%) o1 mm
H2O
KCl
1 2 3 4 5
6.9 7.1 6.9 6.5 5.4
6.6 6.8 6.5 6.4 5.1
339 367 416 219 198
0.32 0.34 0.47 0.25 0.41
Sample
Cox (%)
Humic acids (%)
Fulvic acids (%)
HA/FA (ratio)
1 2 3 4 5
2.81 3.40 4.65 5.47 3.85
0.35 0.42 0.47 0.73 0.63
0.69 0.76 0.60 0.97 0.88
0.51 0.55 0.78 0.75 0.72
1–2 mm
2–50 mm
0.05–2 mm
35.3 29.5 29.1 28.5 28.9
11.1 4.5 7.4 9.5 10.3
13.4 10.1 20.2 37.8 20.3
Ca2+ (mmol eq kg1)
K+ (mmol eq kg1)
Mg2+ (mmol eq kg1)
Na+ (mmol eq kg1)
285 302 360 142 115
5.2 13.3 9.8 7.8 4.1
25.2 26.4 20.8 24.7 27.2
40.2 55.9 43.3 24.2 40.5
0.9 1.0 0.9 1.0 1.4
Note: CEC, cation exchange capacity (ISO 13 536, 1995); Cox ; total organic carbon (ISO 14 235, 1998); Ntot ; total nitrogen (ISO 11 261, 1995).
Z. Prokop et al. / Environmental Research 91 (2003) 119–126
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Table 2 Amount of cadmium extracted from the soil samples using QW, DTPA and DMSO Extracting solvent (mg Cd/g soil)
Sample
Concentration of Cd in soil (mg g1 dry mass) 0.01
QW
1 2 3 4 5
0.07 0.05 0.05 0.07 0.07
DTPA
1 2 3 4 5
2.77 2.56 2.54 3.06 2.71
DMSO
1 2 3 4 5
0.34 0.02 0.03 0.08 0.07
0.1
0.5
0.40 0.19 0.20 0.33 0.37 28 25 25 29 26
Equation
r2
P value
N
M ¼ Cox 205 þ 4495 cfree ¼ DTPA 0:59 46 csolut ¼ QW 6:9 þ 143 Kd ¼ DTPA ð0:012Þ þ 8:7 Icont ¼ cfree 0:2 1:8 Icont ¼ DTPO 0:12 13:1 Icont ¼ Kd ð8:9Þ þ 69 PCO2 ¼ DTPA ð0:08Þ þ 100:5 PCO2 ¼ Kd ð6:6Þ þ 42
0.74 0.85 0.77 0.86 0.81 0.85 0.77 0.66 0.69
0.0388 o0.001 o0.001 o0.001 o0.001 o0.001 o0.001 o0.001 o0.001
5 30 29 30 29 29 29 25 25
Note: Icont ; inhibition of B. cereus activity in contact toxicity test; M; sorption maximum (Eq. (1)); cfree ; free concentration of cadmium in the soil samples (calculated using WHAM); DTPA, amount of cadmium extracted; Kd ; distribution coefficient (calculated using of WHAM); csolut ; Cd concentration in solution (calculated using WHAM); QW, cadmium water extractability; PCO2 ; respiration (production of CO2).
significant difference between water and DMSO extractability only at 5.0 mg/g (P ¼ 0:043). However, these findings suggest that presence of dimethylsulfoxide in the extraction solution had negligible effect on cadmium extractability. A different relationship was observed between the DTPA-extractable fraction and concentration of cadmium in soil samples, compared to the waterextractable fraction. DTPA is a chelating agent, the capacity of which is saturated as the concentration of cadmium in the soil samples increases. The percentage extractability of cadmium by DTPA decreased with increasing total concentration of cadmium in the soil
0.98 0.53 0.26 1.63 4.10
4.1 2.8 1.4 8.6 21.6
143 127 134 150 139
0.11 0.04 0.04 0.18 0.40
Table 3 Relationships between cadmium toxicity, extractability, distribution, and physicochemical characteristics of soil samples
1.0
268 265 293 287 288
0.82 0.55 0.38 1.74 4.56
4.1 3.9 1.6 8.3 20.1
2.5
5.0
56 46 4 80 201
381 342 85 350 799
485 488 485 495 498
663 615 540 638 783
59 53 17 83 198
401 350 138 359 815
Table 4 Cadmium binding to the soil samples according to Langmuir model Sample
K
M
r2
1 2 3 4 5
0.0188 0.0216 0.0911 0.0111 0.0048
5 5 5 5 5
0.99 0.99 0.98 0.99 0.99
153 212 334 730 178
Note: K; affinity factor; M; sorption maximum.
sample. Despite this, the amounts of cadmium extracted by DTPA from the soil samples corresponded well to the results of the toxicological evaluation, as well as to cadmium speciation in the soil samples. The amount extracted by DTPA correlated well with the free metal ion concentration of cadmium in the soil samples (Table 5). Cadmium speciation in the studied soil samples, calculated using WHAM, is illustrated in Table 6. The results showed a strong binding of cadmium to organic matter. Thirty to ninety percent of cadmium was bound to FA and 4–25% of cadmium was bound to HA in the soil samples, depending on the total concentration and sorption capacity of the soil samples. WHAM calculations predicted a weak sorption of cadmium to clay (o2% of total cadmium amount in the soil samples). These calculations corresponded well to the results of cadmium extractability. Thirty-six percent of total cadmium was in the solution and only 0.5–9.2% was present in free ion form. A strong correlation was observed between
0.78nn 0.41 0.53n 0.40 0.78nn 0.54n 0.64nn 0.50 0.52n 0.38 Coefficients near 1.0 indicate a strong positive correlation and coefficients near 1.0 indicate a strong negative correlation. n po0:01: nn po0:001:
0.50 0.66nn 0.35 0.77nn 0.60n 0.60n 0.27 0.68nn 0.43 0.76nn 0.72nn 0.50 0.78nn 0.26 0.39 0.30 0.72nn 0.67nn 0.76nn 0.49 0.88nn 0.43 0.51n 0.32 0.70nn 0.60n 0.87nn 0.89nn 0.66nn 0.89nn 0.45 0.58n 0.33 0.86nn 0.72nn 1.0nn 0.88nn 0.90nn 0.66nn 0.89nn 0.45 0.58n 0.32 0.86nn 0.74nn 0.99nn 0.99nn 0.90nn 0.88nn 0.63nn 0.90nn 0.46 0.57n 0.37 0.85nn 0.69nn 0.95nn 0.96nn 0.95nn 0.83nn 0.85nn 0.73nn 0.83nn 0.47 0.61n 0.40 0.87nn 0.79nn 0.97nn 0.97nn 0.98nn 0.97nn 0.86nn 0.87nn 0.67nn 0.88nn 0.49 0.60n 0.39 0.87nn 0.81nn Cd conc. (QW) Cd conc. (DMSO) Cd conc. (DTPA) Cd in solution Free Cd (WHAM) Contact tox. test Turbid. test (DTPA) Turbid. test (DMSO) Turbid. test (QW) Microtox (DTPA) Microtox (DMSO) Microtox (QW) Respiration Kd
Cd conc. (QW) N ¼ 25
Table 5 Spearman rank order correlations
Cd conc. (DMSO)
Cd conc. (DTPA)
Cd in solution
Free Cd (WHAM)
Contact tox. test
Turbid. T (DTPA)
Turbid.T (DMSO)
Turbid.T (QW)
Microtox (DTPA)
Microtox (DMSO)
Microtox (QW)
Respiration
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cadmium amount extracted by DTPA and cadmium distribution and free ion amount in the soil samples, calculated using WHAM. The concentration determined from the extraction analysis can be quite different from the amount of chemical actually available for exposure of organisms in the environment. Extract toxicity was measured using four ecotoxicity tests (Table 7). Compared to the water extracts, the DTPA extracts did not show higher toxicity, even though a higher amount of cadmium was extracted by DTPA. This can be explained by the stimulation effect of DTPA on bacterial activity and formation of Cd–DTPA complexes resulting in reduced bioavailability. The toxicity effects of solvent or alteration of sample during extraction procedures influenced the toxicological evaluations using DTPA extracts. On the other hand, a strong correlation between DTPA extractability and results of toxicity tests was observed. Water and DMSO extractability were poorly correlated with toxicological data compared to the DTPA extractability. The toxicity of the water extract evaluated using growth inhibition test corresponded well with the cadmium distribution, but when the Microtox test was used, it did not correspond with any other toxicological and speciation descriptors. Microtox medium is salt (10 g NaCl/L), therefore the fact that cadmium is forms CdCl2 complexes must be considered. The toxicity of the DMSO extract, evaluated using the Microtox test, corresponded well with the cadmium distribution. The growth inhibition test showed a weak correspondence with the other descriptors. The application of extracts for toxicological screening is problematic (Brouwer et al., 1990; Kwan and Dutka, 1992 . and 1995; Kwan, 1993; Day et al., 1995; Ronnpagel et al., 1995). Solid-phase contact toxicity tests, which allow the test organisms to come in direct contact with untreated solid samples, seem to be more appropriate procedures for toxicity and bioavailability evaluation (Brouwer et al., 1990; Kwan, 1993). The toxicity of cadmium-spiked soil samples was evaluated using the respiration test and the contact toxicity test with the bacterium B. cereus. The best correlation was observed between results of contact toxicity test and cadmium DTPA extractability. The results of the contact toxicity test also correlated well with concentration of free cadmium and with cadmium distribution in the soil samples, predicted by WHAM. The respiration of indigenous microflora also correlated well with cadmium DTPA extractability and with cadmium distribution in the soil samples (Table 3). When respiration and single-species contact toxicity tests were used to evaluate the testing organisms in the environment of untreated soil samples and the test organisms responded to the actual bioavailable fraction of cadmium in the sample.
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Table 6 Fractional distribution and speciation of cadmium in the soil samples Concentration of Cd in soil (mg g1 dry mass)
Sample
0.01
0.1
0.5
1.0
2.5
5.0
Cd in solution (%)
1 2 3 4 5
4.8 4.4 3.4 2.7 2.8
5.9 5.3 3.8 3.2 3.3
13.2 11.4 7.1 5.9 6.4
22.7 19.7 12.6 10.2 11.1
43.2 38.9 28.6 22.5 24.5
60.3 56.3 47.0 39.9 42.5
Cd bound on FA (%)
1 2 3 4 5
90.6 90.4 92.7 90.1 90.3
88.5 88.5 91.7 88.7 88.8
75.7 76.7 85.1 79.7 79.5
62.3 63.9 75.4 68.8 68.3
41.3 43.5 55.7 51.6 51.0
27.3 29.1 38.9 37.6 36.7
Cd bound on HA (%)
1 2 3 4 5
4.4 4.9 3.8 7.0 6.6
5.4 5.9 4.3 8.0 7.7
10.6 11.3 7.5 14.0 13.7
14.2 15.4 11.4 20.4 19.8
14.0 15.9 14.6 24.8 23.1
10.5 12.3 12.5 21.1 19.0
Cd bound on clay (%)
1 2 3 4 5
0.1 0.2 0.1 0.1 0.1
0.2 0.2 0.1 0.1 0.1
0.4 0.4 0.2 0.1 0.3
0.6 0.8 0.4 0.3 0.5
1.2 1.5 0.8 0.5 1.1
1.7 2.1 1.3 0.9 1.8
Free Cd in solution (mg ml1)
1 2 3 4 5
0.07 0.07 0.05 0.05 0.05
0.90 0.82 0.58 0.54 0.57
10.1 8.8 5.4 5.1 5.6
Kd (kg L1)
1 2 3 4 5
7.8 8.1 9.7 8.9 9.3
7.4 7.8 9.5 9.0 9.3
5.0 5.6 7.8 8.5 8.3
35 30 19 17 19 3.2 3.7 5.8 6.8 6.5
169 146 107 94 106
461 427 348 337 360
1.3 1.6 2.5 3.4 3.0
0.7 0.8 1.1 1.5 1.3
Table 7 EC50 values of cadmium in the soil samples evaluated using different toxicity tests (mg Cd g1 dry soil) Extracting solvent
Sample
Microtox test
GIT test
B. cereus contact testa
Respiration testb
QW
1 2 3 4 5
0.63 0.33 0.70 0.73 0.33
0.28 0.20 0.30 1.17 0.16
2.98 3.37 3.66 3.88 0.99
5.51 6.64 6.64 5.70 2.79
DTPA
1 2 3 4 5
0.66 0.52 1.63 0.80 0.74
0.53 5.78 1.72 0.36 0.44
— — — — —
— — — — —
DMSO
1 2 3 4 5
0.46 — 0.61 0.62 0.31
0.37 0.29 0.45 0.34 0.00
— — — — —
— — — — —
Note: GIT, growth inhibition test. a Tested in buffer–soil suspension. b Tested in moist soil sample.
Z. Prokop et al. / Environmental Research 91 (2003) 119–126
4. Conclusions Taken together, chemical extractability, Windermere humic aqueous model (WHAM) calculation and toxicological evaluations provided useful information about speciation and behavior of cadmium in the studied soil samples. A potential mobile fraction of cadmium in the soils, i.e., water-extractable fraction corresponded well with the amount of cadmium in the soil solution, as calculated using the WHAM. The mobility of cadmium depended on the maximum sorption capacity of the soil samples. The water extractability, soil solution concentration, and soil sorption capacity were found to be good predictors in the context of metal mobility evaluation and protection of groundwater quality. A potential bioavailable fraction of cadmium in the soils, i.e., free ion form, was in good agreement with DTPA extractability. Both the concentration of free cadmium and the DTPA-extractable fraction correlated well with cadmium toxicity. A strong correlation was observed with the contact toxicity test and the soil respiration test. The solid-phase contact toxicity procedures were very suitable for assessing the bioavailability of cadmium in the soil. The use of extract toxicity is less suitable for bioavailability assessment. Thus, DTPA was found to be a good extraction agent for chemical availability analysis, but less appropriate for toxicological assessment. The bioavailable fraction of cadmium was reduced by binding to the soil components. The results of the WHAM calculations as well as the results of cadmium extractabilty showed a strong binding of cadmium to organic matter and a weak sorption of cadmium to the clay minerals.
Acknowledgments The research was financially supported by the Grant Agency of the Czech Republic (GACR 526/98/1147) and the Ministry of Education of the Czech Republic (FRVS 0759/1998). The authors thank Dr. J. Damborsky and M. L. Vangheluwe for editing the manuscript.
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. Forstner, U. (Eds.), Chemistry and Biology of Solid Waste: Dredged Material and Mine Tailings. Springer, Berlin, pp. 3–32. Brouwer, H., Murphy, T., McArdle, L., 1990. A sediment-contact bioassay with Photobacterium phosphoreum. Environ. Toxicol. Chem. 9, 1353–1358. Cochran, W.G., Cox, G.M., 1957. Experimental Design. Wiley, New York. Day, K.E., Dutka, B.J., Kwan, K.K., Batista, N., Reynoldson, T.B., Metcalfe-Smith, J.L., 1995. Correlations between solid-phase microbial assay, whole-sediment toxicity tests with macroinvertebrates and in situ benthic community structure. J. Great Lakes Res. 21, 192–206. Dutka, B.J., Liu, D.L., Jurkovic, A., McInnis, R., 1993. A comparison of four simple water extraction–concentration procedures to be used with battery of bioassays tests approach. Environ. Toxicol Water Q. 8, 397–407. Dutka, B.J., Marsalek, J., Jurkovic, A., McInnis, R., Kwan, K.K., 1994. A seasonal ecotoxicological study of stormwater ponds. Z. Angew. Zool. 80, 364–381. Forster, J.C., 1995. Soil physical analysis. In: Alef, K., Nannipieri, P. (Eds.), Methods in Applied Soil Microbiology and Biochemistry. Academic Press, San Diego, pp. 105–119. Green, A.S., Chandler, G.T., Blood, E.R., 1993. Aqueous-, pore-water-, and sediment-phase cadmium: toxicity relationships for a meiobenthic copepod. Environ. Toxicol. Chem. 8, 1497–1506. ISO 10 381-6, 1993. Soil quality—sampling—Part 6: guidance on the collection, handling and storage of soil for the assessment of aerobic microbial processes in the laboratory. ISO 10 390, 1994. Soil quality—determination of pH. ISO 11 261, 1995. Soil quality—determination of total nitrogen— modified Kjeldahl method. ISO 13 536, 1995. Soil quality—determination of the potential cation exchange capacity and exchangeable cations using barium chloride solution buffered at pH 8.1. ISO 10 712, 1997. Water quality—Pseudomonas putida growth inhibition test—pseudomonas cell multiplication inhibition test. ISO 11 348, 1998. Water quality—determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (luminescent bacteria test), Part 3: method using freeze-dried bacteria. ISO 14 235, 1998. Soil quality—determination of organic carbon by sulfochromic oxidation. Janssen, R.P.T., Peijnenburg, W.J.G.M., Posthuma, L., Van Den Hoop, M.A.G.T., 1997b. Equilibrium partitioning of heavy metals in duch field soils, I: relationship between heavy metal partition coefficients and soil characteristics. Environ. Toxicol. Chem. 16, 2479–2488. Janssen, R.P.T., Posthuma, L., Baerselman, R., Hollander, H.A.D., Van Veen, R.P.M., Peijnenburg, W.J.G.M., 1997a. Equilibrium partitioning of heavy metals in duch field soils, II: prediction of metal accumulation in earthworms. Environ. Toxicol. Chem. 16, 2479–2488. Knight, B., McGrath, S.P., 1995. A method to buffer the concentrations of free Zn and Cd ions using a cation exchange resin in bacterial toxicity studies. Environ. Toxicol. 14, 2033–2039. Kwan, K.K., 1993. Direct toxicity assessment of solid-phase samples using the toxi-chromotest kit. Environ. Toxicol. Water Q. 8, 223–230. Kwan, K.K., Dutka, B.J., 1992. Evaluation of toxi-chromotest direct sediment toxicity testing procedure and Microtox solidphase testing procedure. Bull. Environ. Contam. Toxicol. 49, 656–662. Kwan, K.K., Dutka, B.J., 1995. Comparative assessment of two solidphase toxicity bioassays: the direct sediment toxicity testing
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procedure (DSTTP) and the Microtox solid-phase test (SPT). Bull. Environ. Contam. Toxicol. 55, 338–346. Laxen, D.P.H., Harrison, R.M., 1981. The physicochemical speciation of Cd, Pb, Cu, Fe and Mn in the final effluent of a sewage treatment works and its impact on speciation in receiving river. Water Res. 15, 1053–1065. Liang, J., Karamanos, R.E., 1993. DTPA-extractable Fe, Mn, Cu, and Zn. In: Carter, M.R. (Ed.), Soil Sampling and Methods of Analysis. CRC Press, Boca Raton, FL, pp. 87–90. Mehlich, A., 1978. New extractant for soil test evaluation of phosphorus, potassium, magnesium, calcium, sodium, manganese and zinc. Commun. Soil. Sci. Plant Anal. 9, 477–492. Parker, D.R., Pedler, J.F., 1997. Reevaluating the free-ion activity model of trace metal availability to higher plants. Plant Soil 196, 223–228. Rossel, D., Tarradellas, J., Bitton, G., Morel, J.L., 1997. Use of enzymes in soil ecotoxicology: a case for dehydrogenase and hydrolytic enzymes. In: Tarradellas, J., Bitton, G., Rossel, D. (Eds.), Soil Ecotoxicology. CRC Press, Boca Roton, FL, pp. 179–206.
. Ronnpagel, K., Lisse,, W., Ahlf, W., 1995. Microbial bioassays to assess the toxicity of solid-associated contaminants. Ecotox. Environ. Saf. 31, 99–103. Samiullah, Y., 1990. Prediction of the Environmetal Fate of Chemicals. Elsevier Science, Essex. Shaw, L.J., Beaton, Y., Glover, L.A., Kennetizi, K., Meharg, A.A., 2000. Interaction between soil, toxicant and lux-marked bacterium during solid-phase contact toxicity testing. Environ. Toxicol. Chem. 5, 1247–1252. Schnitzer, M., Schuppli, P., 1989. Method for sequential extraction of organic matter from soil fractions. Soil Sci. Soc. Am. J. 53, 1418–1424. Tipping, E., 1994. WHAM-A chemical equilibrium model and computer code for waters, sediments, and soils incorporating a discrete site/electrostatic model of ion-binding by humic substances. Comput. Geosci. 6, 973–1023. Tokalioglu, S., Kartal, S., Elci, L., 2000. Determination of heavy metals and their speciation in lake sediments by flame atomic absorption spectrometry after a four-stage sequential extraction procedure. Anal. Chim. Acta 413, 33–40.
Mobility, bioavailability, and toxic effects of cadmium in soil samples Z. Prokop,* P. Cupr, V. Zlevorova-Zlamalikova, J. Komarek, L. Dusek, and I. Holoubek Research Centre for Environmental Chemistry and Ecotoxicology, Masaryk University, Kamenice 126/3, 625 00 Brno, Czech Republic Received 14 June 2001
Abstract Total concentration is not a reliable indicator of metal mobility or bioavailability in soils. The physicochemical form determines the behavior of metals in soils and hence the toxicity toward terrestrial biota. The main objectives of this study were the application and comparison of three approaches for the evaluation of cadmium behavior in soil samples. The mobility and bioavailability of cadmium in five selected soil samples were evaluated using equilibrium speciation (Windermere humic aqueous model (WHAM)), extraction procedures (Milli-Q water, DMSO, and DTPA), and a number of bioassays (Microtox, growth inhibition test, contact toxicity test, and respiration). The mobility, represented by the water-extractable fraction, corresponded well with the amount of cadmium in the soil solution, calculated using the WHAM (r2 ¼ 0:96; Po0:001). The results of the ecotoxicological evaluation, which represent the bioavailable fraction of cadmium, correlated well with DTPA extractability and also with the concentration of free cadmium ion, which is recognized as the most bioavailable metal form. The results of the WHAM as well as the results of extraction experiments showed a strong binding of cadmium to organic matter and a weak sorption of cadmium to clay minerals. r 2003 Elsevier Science (USA). All rights reserved. Keywords: Bioavailability; Cadmium; Extractability; Mobility; Soil; Speciation
1. Introduction The behavior of metals in soils (e.g., mobility, bioavailability) cannot be reliably predicted on the basis of their total concentrations. The uptake and toxicity of many metals show marked dependence on speciation of the metals and these responses often correlate best with the activity of free metal ion (Laxen and Harrison, 1981; Knight and McGrath, 1995; Parker and Pedler, 1997). Exceptions to this generalization have been observed; however, the free metal form is considered the most bioavailable and the most active form (Janssen et al., 1997a). The bioavailability of metals in soils or sediments is often expressed in terms of concentration in a water phase. The metal distribution between a solid phase and pore water of a soil is commonly described by equilibrium partitioning (Janssen et al., 1997b). But the total dissolved metal concentration does not necessarily correspond to the amount available to biota. Ion pairs, complex ions, polymers, or microparticulates can reduce *Corresponding author. Research Centre for Environmental Chemistry and Ecotoxicology, Kamenice 126/3, 625 00 Brno, Czech Republic. Fax: +54-112-9506. E-mail address: [email protected] (Z. Prokop).
free ion species of heavy metals in solution (Green et al., 1993). The process of identifying and quantifying these different species of metals in a sample is referred to as speciation. Chemical equilibrium models, such as the Windermere humic aqueous model (WHAM) and freeion activity model (FIAM), are available to calculate metal speciation in waters, sediments, or soils (Tipping, 1994; Parker and Pedler, 1997). Many studies refer to metal speciation in terms of extractable metals related to single or sequential extraction. For example DTPA, CaCl2, or other individual extractants are frequently used for prediction of availability of metals to plants (Liang and Karamanos, 1993). Ahnstrom and Parker (2001) suggested that conventional sequential extraction procedures may be of limited utility for predicting bioavailability. The extractable fraction as well as the pore water fraction of metals does not necessarily correspond to the amount available to soil organisms. The concentrations of the bioavailable form can be related more closely to biological toxicity (Tokalioglu et al., 2000). Ecotoxicological evaluations of soils are usually associated with the measurement of aqueous or solvent extract toxicity. A bioassay in which the test organism directly interacts with untreated soil, with both the
0013-9351/03/$ - see front matter r 2003 Elsevier Science (USA). All rights reserved. PII: S 0 0 1 3 - 9 3 5 1 ( 0 2 ) 0 0 0 1 2 - 9
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aqueous and the solid phase, may provide a better assessment of bioavailability of contaminants. Microbial contact toxicity tests provide intimate contact between toxicants and test microorganisms or enzymes (Rossel et al., 1997; Shaw et al., 2000). In this study, the behavior of cadmium spiked into five selected noncontaminated soil samples was investigated following chemical and ecotoxicological procedures. The main objective was a comparison of equilibrium cadmium speciation in the studied soil samples (WHAM) with the cadmium-extractable fractions (Milli-Q water, DMSO, and DTPA) and the ecotoxicological results (Microtox test, growth inhibition test, contact toxicity test, and respiration test). A second objective was the determination of the physicochemical soil characteristics mostly influencing the behavior of cadmium in soil.
2.2. Chemical analyses
2. Materials and methods
Cadmium concentrations in the extracts were measured using atomic absorbtion spectrometr (Perkin– Elmer 306). The 228.8 nm resonance line was generated by a Cathodeon hollow cathode lamp (6 mA) or a Perkin–Elmer electrodeless discharge lamp (5 W input power), respectively. The transmitted spectral interval was 0.7 nm. Deuterium background correction was used to eliminate nonselective absorption. A 10-cm one-slot burner was used for measurements in the acetylene–air flame. The flow rates of the acetylene and air were 3.6 and 22.5 L/min, respectively. The samples were acidified by 1 M HCl to pH 2. Standard solutions of CdCl2 were prepared by dissolving 1 g of metallic cadmium of analytical grade quality in 30 mL of 6 M HCl and diluting with double-distilled water to a volume of 1 L. The solutions were standardized by chelometric titration with EDTA using xylenol orange as an indicator and contained 0.99 g Cd/L.
2.1. Sample collection and handling
2.3. Ecotoxicity tests
Soil samples were collected from five unpolluted areas in the Czech Republic (samples 1 and 3, loamy–sandy soil; sample 2, sandy–loam soil; sample 4, loam soil; sample 5, clay–loam soil). The soil samples were collected from depths of 0–20 cm, dried to constant mass at ambient temperature, ground, sieved through a 2-mm sieve, and homogenized (ISO 10 381-6, 1993). Samples were stored at 4 C until use. Background cadmium concentration in all selected samples was lower than 0.1 mg Cd/g of soil. Soil samples from each site were characterized for grain size, total organic carbon (Cox ) according to ISO 14 235 (1998), and total nitrogen (Ntot ) according to ISO 11 261 (1995). Fulvic acid (FA) and humic acid (HA) amounts were measured according to Schnitzer and Schuppli (1989). Amounts of calcium, potasium, and magnesium were measured according to Mehlich (1978). The pH was measured according to ISO 10 390, 1994. The samples were moistured, spiked with CdCl2 (0.01–5.0 mg Cd/g of dry soil), and preincubated for 72 h at 22 C prior to testing. Sample moisture was adjusted to 65% of its water holding capacity (Forster, 1995). Cadmium was extracted from soils by Milli-Q water (QW), by DMSO (10% dimethylsulfoxide, 10% methanol in deionized water) according to Dutka et al., (1993, 1994) and by DTPA (5 mM diethylenetriamine pentaacetic acid, 10 mM CaCl2, and 0.1 M triethanolamine buffered at pH 7.3) according to Bailey et al. (1995). The extraction was initiated by homogenization of the soil samples and extract reagents at a ratio of 1:1 (w:v) for water and DMSO and at a ratio of 1:2 (w:v) for DTPA. The suspension was shaken (200 rpm) for 1 h at 20 C and then filtered.
The Microtox test was conducted according to the procedures outlined in ISO 11 348 (1998). The degree of reduction of bioluminescence in the presence of a sample gives an indication of the toxicity of the sample under investigation. The growth inhibition test was performed according to ISO 10 712 (1997). Test samples were inoculated with Pseudomonas putida and incubated for 16 h. Changes in growth rate were estimated turbidimetrically. The contact toxicity test with Bacillus cereus . was performed according to Ronnpagel et al. (1995), with some modifications. This screening test is based on determination of microbial dehydrogenase activity inhibition. Test bacteria activity is proportional to the amount of reduced resazurin (oxido-reduction indicator). In the test, 2 g of each sample was transferred into a centrifuge screw-cap tube and resuspended in 2 mL of phosphate buffer. The inoculum was prepared from actively growing biomass by adjusting the cell concentration to OD601 ¼ 1:0 and added to the soil–buffer mixture. Incubation was performed for 4 h at 30 C and 70 rpm. After incubation, 2 mL of resazurin solution was added at concentration of 0.2 mg/mL of phosphate buffer. The mixture was shaken for 1 h and centrifuged (3500g, 5 min). The reduction of resazurin was measured at 601 nm. Soil respiration was measured after preincubation. Soil samples were incubated in closed jars for 48 h at 22 C. Moisture was adjusted to 60% of water holding capacity. After incubation CO2 production was measured using a gas chromatograph equipped with thermal conductivity detector. A polyethylene column 1.5 m long and 4 mm id packed with Porapac Q 80/100 mech (Supelco) was operated at laboratory temperature using hydrogen as carrier gas at head pressure 300 kPa.
Z. Prokop et al. / Environmental Research 91 (2003) 119–126
2.4. Data analysis
The fraction of cadmium in soil can be mobilized, is important with regard to the protection of groundwater quality. Water is the major carrier of metal pollutants (Bourg, 1988), so the water-soluble fraction in the water extract corresponds to the mobile fraction of the metals. Water extractability correlated well with the amount of cadmium in soil solution calculated using WHAM (Table 3). The amount of cadmium in a water extract, i.e., potential mobile fraction, increased with increasing total concentration of cadmium in the soil samples. All of the soil samples successfully held cadmium up to a concentration of 1 mg/g of dry soil. This was followed by an exponential increase in the amount of cadmium in water extracts as the total concentration in soil samples was further elevated. It is apparent that the sorption of cadmium on soil samples was limited. The sorption of cadmium reached a maximum once the sorption surfaces were saturated. The Langmuir model was fit to the water-extractability data and the sorption maximum and an affinity parameter was then calculated (Table 4). The sorption maximum correlated well with the amount of organic carbon in the soil samples (Table 3). On the other hand, no relationship was found between the solid particle distribution and the extractable fraction of cadmium from the soil samples. The weak metal binding on clay compared to binding to organic matter was previously described by Janssen et al. (1997a). There was no significant difference between water and DMSO extractability of cadmium from the soil samples. Using the paired nonparametric Wilcoxon test, there was no significant difference (a ¼ 0:05) between these two extraction procedures for Cd-spiked concentrations of 0.01, 0.1, 0.5, 1.0, and 2.5 mg/g. There was a
The effects of cadmium on the test microorganisms (activity inhibition or stimulation) were expressed as the percentage change in their activity compared to negative controls. Linear models were fit to the linear parts of the dose–response relationships. The model parameters were determined by the least-squares fitting method. EC50 values (mg Cd/g dry soil) were calculated by inverse prediction from these linear models (Cochran and Cox, 1957). The chemical speciation of cadmium in the soil samples was calculated using WHAM (Tipping, 1994). The amount of cadmium sorbed on the soil samples (mg/kg) was plotted against the cadmium concentration in the water extract (mg/L). The experimental data were fit to the Langmuir model (Samiullah, 1990) n ¼ K c M=ð1 þ K cÞ;
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ð1Þ
where n and c are cadmium concentrations sorbed on the soil and in the water extract, respectively, M is a sorption maximum of cadmium in a studied soil, and K is an affinity parameter.
3. Results and discussion The physicochemical characteristics of studied soils are shown in Table 1. The selected soils contained 2.81% to 5.47% organic carbon and 53–85% of clay (particles o2 mm). The cation exchange capacity ranged from 198 to 416 mmol eq/kg1. Acidity was similar for all soil samples. H2O pH ranged from 5.4 to 7.1. The concentrations of cadmium extracted by water, DMSO, and DTPA is shown in Table 2.
Table 1 Physical and chemical characteristics of soil samples Sample
pH
CEC (mmol eq kg1)
Ntot (%)
Composition of soil particles (%) o1 mm
H2O
KCl
1 2 3 4 5
6.9 7.1 6.9 6.5 5.4
6.6 6.8 6.5 6.4 5.1
339 367 416 219 198
0.32 0.34 0.47 0.25 0.41
Sample
Cox (%)
Humic acids (%)
Fulvic acids (%)
HA/FA (ratio)
1 2 3 4 5
2.81 3.40 4.65 5.47 3.85
0.35 0.42 0.47 0.73 0.63
0.69 0.76 0.60 0.97 0.88
0.51 0.55 0.78 0.75 0.72
1–2 mm
2–50 mm
0.05–2 mm
35.3 29.5 29.1 28.5 28.9
11.1 4.5 7.4 9.5 10.3
13.4 10.1 20.2 37.8 20.3
Ca2+ (mmol eq kg1)
K+ (mmol eq kg1)
Mg2+ (mmol eq kg1)
Na+ (mmol eq kg1)
285 302 360 142 115
5.2 13.3 9.8 7.8 4.1
25.2 26.4 20.8 24.7 27.2
40.2 55.9 43.3 24.2 40.5
0.9 1.0 0.9 1.0 1.4
Note: CEC, cation exchange capacity (ISO 13 536, 1995); Cox ; total organic carbon (ISO 14 235, 1998); Ntot ; total nitrogen (ISO 11 261, 1995).
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Table 2 Amount of cadmium extracted from the soil samples using QW, DTPA and DMSO Extracting solvent (mg Cd/g soil)
Sample
Concentration of Cd in soil (mg g1 dry mass) 0.01
QW
1 2 3 4 5
0.07 0.05 0.05 0.07 0.07
DTPA
1 2 3 4 5
2.77 2.56 2.54 3.06 2.71
DMSO
1 2 3 4 5
0.34 0.02 0.03 0.08 0.07
0.1
0.5
0.40 0.19 0.20 0.33 0.37 28 25 25 29 26
Equation
r2
P value
N
M ¼ Cox 205 þ 4495 cfree ¼ DTPA 0:59 46 csolut ¼ QW 6:9 þ 143 Kd ¼ DTPA ð0:012Þ þ 8:7 Icont ¼ cfree 0:2 1:8 Icont ¼ DTPO 0:12 13:1 Icont ¼ Kd ð8:9Þ þ 69 PCO2 ¼ DTPA ð0:08Þ þ 100:5 PCO2 ¼ Kd ð6:6Þ þ 42
0.74 0.85 0.77 0.86 0.81 0.85 0.77 0.66 0.69
0.0388 o0.001 o0.001 o0.001 o0.001 o0.001 o0.001 o0.001 o0.001
5 30 29 30 29 29 29 25 25
Note: Icont ; inhibition of B. cereus activity in contact toxicity test; M; sorption maximum (Eq. (1)); cfree ; free concentration of cadmium in the soil samples (calculated using WHAM); DTPA, amount of cadmium extracted; Kd ; distribution coefficient (calculated using of WHAM); csolut ; Cd concentration in solution (calculated using WHAM); QW, cadmium water extractability; PCO2 ; respiration (production of CO2).
significant difference between water and DMSO extractability only at 5.0 mg/g (P ¼ 0:043). However, these findings suggest that presence of dimethylsulfoxide in the extraction solution had negligible effect on cadmium extractability. A different relationship was observed between the DTPA-extractable fraction and concentration of cadmium in soil samples, compared to the waterextractable fraction. DTPA is a chelating agent, the capacity of which is saturated as the concentration of cadmium in the soil samples increases. The percentage extractability of cadmium by DTPA decreased with increasing total concentration of cadmium in the soil
0.98 0.53 0.26 1.63 4.10
4.1 2.8 1.4 8.6 21.6
143 127 134 150 139
0.11 0.04 0.04 0.18 0.40
Table 3 Relationships between cadmium toxicity, extractability, distribution, and physicochemical characteristics of soil samples
1.0
268 265 293 287 288
0.82 0.55 0.38 1.74 4.56
4.1 3.9 1.6 8.3 20.1
2.5
5.0
56 46 4 80 201
381 342 85 350 799
485 488 485 495 498
663 615 540 638 783
59 53 17 83 198
401 350 138 359 815
Table 4 Cadmium binding to the soil samples according to Langmuir model Sample
K
M
r2
1 2 3 4 5
0.0188 0.0216 0.0911 0.0111 0.0048
5 5 5 5 5
0.99 0.99 0.98 0.99 0.99
153 212 334 730 178
Note: K; affinity factor; M; sorption maximum.
sample. Despite this, the amounts of cadmium extracted by DTPA from the soil samples corresponded well to the results of the toxicological evaluation, as well as to cadmium speciation in the soil samples. The amount extracted by DTPA correlated well with the free metal ion concentration of cadmium in the soil samples (Table 5). Cadmium speciation in the studied soil samples, calculated using WHAM, is illustrated in Table 6. The results showed a strong binding of cadmium to organic matter. Thirty to ninety percent of cadmium was bound to FA and 4–25% of cadmium was bound to HA in the soil samples, depending on the total concentration and sorption capacity of the soil samples. WHAM calculations predicted a weak sorption of cadmium to clay (o2% of total cadmium amount in the soil samples). These calculations corresponded well to the results of cadmium extractability. Thirty-six percent of total cadmium was in the solution and only 0.5–9.2% was present in free ion form. A strong correlation was observed between
0.78nn 0.41 0.53n 0.40 0.78nn 0.54n 0.64nn 0.50 0.52n 0.38 Coefficients near 1.0 indicate a strong positive correlation and coefficients near 1.0 indicate a strong negative correlation. n po0:01: nn po0:001:
0.50 0.66nn 0.35 0.77nn 0.60n 0.60n 0.27 0.68nn 0.43 0.76nn 0.72nn 0.50 0.78nn 0.26 0.39 0.30 0.72nn 0.67nn 0.76nn 0.49 0.88nn 0.43 0.51n 0.32 0.70nn 0.60n 0.87nn 0.89nn 0.66nn 0.89nn 0.45 0.58n 0.33 0.86nn 0.72nn 1.0nn 0.88nn 0.90nn 0.66nn 0.89nn 0.45 0.58n 0.32 0.86nn 0.74nn 0.99nn 0.99nn 0.90nn 0.88nn 0.63nn 0.90nn 0.46 0.57n 0.37 0.85nn 0.69nn 0.95nn 0.96nn 0.95nn 0.83nn 0.85nn 0.73nn 0.83nn 0.47 0.61n 0.40 0.87nn 0.79nn 0.97nn 0.97nn 0.98nn 0.97nn 0.86nn 0.87nn 0.67nn 0.88nn 0.49 0.60n 0.39 0.87nn 0.81nn Cd conc. (QW) Cd conc. (DMSO) Cd conc. (DTPA) Cd in solution Free Cd (WHAM) Contact tox. test Turbid. test (DTPA) Turbid. test (DMSO) Turbid. test (QW) Microtox (DTPA) Microtox (DMSO) Microtox (QW) Respiration Kd
Cd conc. (QW) N ¼ 25
Table 5 Spearman rank order correlations
Cd conc. (DMSO)
Cd conc. (DTPA)
Cd in solution
Free Cd (WHAM)
Contact tox. test
Turbid. T (DTPA)
Turbid.T (DMSO)
Turbid.T (QW)
Microtox (DTPA)
Microtox (DMSO)
Microtox (QW)
Respiration
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123
cadmium amount extracted by DTPA and cadmium distribution and free ion amount in the soil samples, calculated using WHAM. The concentration determined from the extraction analysis can be quite different from the amount of chemical actually available for exposure of organisms in the environment. Extract toxicity was measured using four ecotoxicity tests (Table 7). Compared to the water extracts, the DTPA extracts did not show higher toxicity, even though a higher amount of cadmium was extracted by DTPA. This can be explained by the stimulation effect of DTPA on bacterial activity and formation of Cd–DTPA complexes resulting in reduced bioavailability. The toxicity effects of solvent or alteration of sample during extraction procedures influenced the toxicological evaluations using DTPA extracts. On the other hand, a strong correlation between DTPA extractability and results of toxicity tests was observed. Water and DMSO extractability were poorly correlated with toxicological data compared to the DTPA extractability. The toxicity of the water extract evaluated using growth inhibition test corresponded well with the cadmium distribution, but when the Microtox test was used, it did not correspond with any other toxicological and speciation descriptors. Microtox medium is salt (10 g NaCl/L), therefore the fact that cadmium is forms CdCl2 complexes must be considered. The toxicity of the DMSO extract, evaluated using the Microtox test, corresponded well with the cadmium distribution. The growth inhibition test showed a weak correspondence with the other descriptors. The application of extracts for toxicological screening is problematic (Brouwer et al., 1990; Kwan and Dutka, 1992 . and 1995; Kwan, 1993; Day et al., 1995; Ronnpagel et al., 1995). Solid-phase contact toxicity tests, which allow the test organisms to come in direct contact with untreated solid samples, seem to be more appropriate procedures for toxicity and bioavailability evaluation (Brouwer et al., 1990; Kwan, 1993). The toxicity of cadmium-spiked soil samples was evaluated using the respiration test and the contact toxicity test with the bacterium B. cereus. The best correlation was observed between results of contact toxicity test and cadmium DTPA extractability. The results of the contact toxicity test also correlated well with concentration of free cadmium and with cadmium distribution in the soil samples, predicted by WHAM. The respiration of indigenous microflora also correlated well with cadmium DTPA extractability and with cadmium distribution in the soil samples (Table 3). When respiration and single-species contact toxicity tests were used to evaluate the testing organisms in the environment of untreated soil samples and the test organisms responded to the actual bioavailable fraction of cadmium in the sample.
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Table 6 Fractional distribution and speciation of cadmium in the soil samples Concentration of Cd in soil (mg g1 dry mass)
Sample
0.01
0.1
0.5
1.0
2.5
5.0
Cd in solution (%)
1 2 3 4 5
4.8 4.4 3.4 2.7 2.8
5.9 5.3 3.8 3.2 3.3
13.2 11.4 7.1 5.9 6.4
22.7 19.7 12.6 10.2 11.1
43.2 38.9 28.6 22.5 24.5
60.3 56.3 47.0 39.9 42.5
Cd bound on FA (%)
1 2 3 4 5
90.6 90.4 92.7 90.1 90.3
88.5 88.5 91.7 88.7 88.8
75.7 76.7 85.1 79.7 79.5
62.3 63.9 75.4 68.8 68.3
41.3 43.5 55.7 51.6 51.0
27.3 29.1 38.9 37.6 36.7
Cd bound on HA (%)
1 2 3 4 5
4.4 4.9 3.8 7.0 6.6
5.4 5.9 4.3 8.0 7.7
10.6 11.3 7.5 14.0 13.7
14.2 15.4 11.4 20.4 19.8
14.0 15.9 14.6 24.8 23.1
10.5 12.3 12.5 21.1 19.0
Cd bound on clay (%)
1 2 3 4 5
0.1 0.2 0.1 0.1 0.1
0.2 0.2 0.1 0.1 0.1
0.4 0.4 0.2 0.1 0.3
0.6 0.8 0.4 0.3 0.5
1.2 1.5 0.8 0.5 1.1
1.7 2.1 1.3 0.9 1.8
Free Cd in solution (mg ml1)
1 2 3 4 5
0.07 0.07 0.05 0.05 0.05
0.90 0.82 0.58 0.54 0.57
10.1 8.8 5.4 5.1 5.6
Kd (kg L1)
1 2 3 4 5
7.8 8.1 9.7 8.9 9.3
7.4 7.8 9.5 9.0 9.3
5.0 5.6 7.8 8.5 8.3
35 30 19 17 19 3.2 3.7 5.8 6.8 6.5
169 146 107 94 106
461 427 348 337 360
1.3 1.6 2.5 3.4 3.0
0.7 0.8 1.1 1.5 1.3
Table 7 EC50 values of cadmium in the soil samples evaluated using different toxicity tests (mg Cd g1 dry soil) Extracting solvent
Sample
Microtox test
GIT test
B. cereus contact testa
Respiration testb
QW
1 2 3 4 5
0.63 0.33 0.70 0.73 0.33
0.28 0.20 0.30 1.17 0.16
2.98 3.37 3.66 3.88 0.99
5.51 6.64 6.64 5.70 2.79
DTPA
1 2 3 4 5
0.66 0.52 1.63 0.80 0.74
0.53 5.78 1.72 0.36 0.44
— — — — —
— — — — —
DMSO
1 2 3 4 5
0.46 — 0.61 0.62 0.31
0.37 0.29 0.45 0.34 0.00
— — — — —
— — — — —
Note: GIT, growth inhibition test. a Tested in buffer–soil suspension. b Tested in moist soil sample.
Z. Prokop et al. / Environmental Research 91 (2003) 119–126
4. Conclusions Taken together, chemical extractability, Windermere humic aqueous model (WHAM) calculation and toxicological evaluations provided useful information about speciation and behavior of cadmium in the studied soil samples. A potential mobile fraction of cadmium in the soils, i.e., water-extractable fraction corresponded well with the amount of cadmium in the soil solution, as calculated using the WHAM. The mobility of cadmium depended on the maximum sorption capacity of the soil samples. The water extractability, soil solution concentration, and soil sorption capacity were found to be good predictors in the context of metal mobility evaluation and protection of groundwater quality. A potential bioavailable fraction of cadmium in the soils, i.e., free ion form, was in good agreement with DTPA extractability. Both the concentration of free cadmium and the DTPA-extractable fraction correlated well with cadmium toxicity. A strong correlation was observed with the contact toxicity test and the soil respiration test. The solid-phase contact toxicity procedures were very suitable for assessing the bioavailability of cadmium in the soil. The use of extract toxicity is less suitable for bioavailability assessment. Thus, DTPA was found to be a good extraction agent for chemical availability analysis, but less appropriate for toxicological assessment. The bioavailable fraction of cadmium was reduced by binding to the soil components. The results of the WHAM calculations as well as the results of cadmium extractabilty showed a strong binding of cadmium to organic matter and a weak sorption of cadmium to the clay minerals.
Acknowledgments The research was financially supported by the Grant Agency of the Czech Republic (GACR 526/98/1147) and the Ministry of Education of the Czech Republic (FRVS 0759/1998). The authors thank Dr. J. Damborsky and M. L. Vangheluwe for editing the manuscript.
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