Phytoavailability assessment of heavy metals in soils by single extractions and accumulation by Phaseolus vulgaris

Environmental and Experimental Botany 60 (2007) 385–396

Phytoavailability assessment of heavy metals in soils by single extractions and accumulation by Phaseolus vulgaris E. Meers a,∗ , R. Samson b , F.M.G. Tack a , A. Ruttens c , M. Vandegehuchte a , J. Vangronsveld c , M.G. Verloo a a

Department of Applied Analytical and Physical Chemistry, Laboratory of Analytical Chemistry and Applied Ecochemistry, Ghent University, Coupure Links 653, 9000 Ghent, Belgium b Department of Applied Ecology and Environmental Biology, Laboratory of Plant Ecology, Ghent University, Coupure Links 653, 9000 Ghent, Belgium c Laboratory of Environmental Biology, Centre for Environmental Sciences, Hasselt University, 3590 Diepenbeek, Belgium Received 4 May 2006; received in revised form 25 November 2006; accepted 28 December 2006

Abstract In Western Europe, policy makers are currently moving towards a more integrated risk-based approach of soil contamination assessment. As part of this approach, selective single extraction procedures have been proposed to add complementary insights regarding heavy metal behaviour and phytoavailability in soils and sediments. However, there is currently a wide range of such procedures available in literature, hampering standardisation and harmonisation of phytoavailability research of heavy metals. The current study examines shoot accumulation of Cd, Cu, Ni, Pb and Zn by the test plant Phaseolus vulgaris in 21 soils, differing in soil composition and level of contamination. On these soils, 12 different commonly used extraction procedures have been compared: soil solution extraction by Rhizon soil moisture samplers, 0.01 M CaCl2 , 0.1 M NaNO3 , 1 M NH4 NO3 , 1 M NH4 NOAc, 1 M MgCl2 , 0.11 M HOAc, 0.5 M HNO3 , 0.1 M HCl, DTPA–TEA–CaCl2 , EDTA-NH4 OAc and aqua regia. The plant species used in this study has previously been proposed as a test plant in a bioassay for assessing heavy metal induced oxidative stress in contaminated soils [Van Assche, F., Clijsters, H., 1990. A biological test system for the evaluation of the phytotoxicity of metal-contaminated soils. Environ. Pollut., 66, 157–172]. Cadmium shoot accumulation correlated best with soil solution concentrations, unbuffered nitrate solutions and the dilute CaCl2 extraction procedure. The same was observed for Zn, yet for this element NH4 OAc and MgCl2 also provided significant interactions. The best prediction for Ni was observed in the cluster containing CaCl2 and NH4 NO3 . For Cd, Zn and Ni, the pseudo-total content and the aggressive chelate based and/or acidic extractants did not correlate well with shoot accumulation. Cu and Pb uptake on the other hand was found to correlate significantly (p = 0.01) with total content as well as with all aggressive extraction procedures over the range of soils used in this experiment. In general, the 0.01 M CaCl2 extraction procedure proved to be the most versatile as it provided a good indication of phytoavailability for all five metals under evaluation. © 2007 Elsevier B.V. All rights reserved. Keywords: Heavy metals; Phytoavailability; Single extractions; Environmental risk assessment; Phaseolus vulgaris

1. Introduction 1.1. Soil pollution Over the last decades, local legislations in Western Europe have come to realise that soil and water pollution with heavy metals and metalloids is more wide-spread than was initially anticipated (Ferguson and Kasamas, 1999). Before implementation of the Flemish soil protection legislation (Vlarebo, 1995),

∗

Corresponding author. Tel.: +32 9 264 59 47; fax: +32 9 264 62 32. E-mail address: [email protected] (E. Meers).

0098-8472/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2006.12.010

the number of contaminated soils in Flanders was estimated at less than 2000 sites or 0.2–0.3% of the total surface area (Geuzens and Cornelis, 1994). Current estimations of potentially polluted sites amount up to 76,200 sites (Dries et al., 2002). Total remediation costs in Flanders are estimated at D 7 billion (Ceenaeme et al., 2004). For the entire European Union, the remediation costs are estimated at D 59–109 billion (EC, 2002). Another development in environmental science and legislation over recent years, has been the insight that total soil content of metals by itself is not a good measure for assessing their bioavailability and not a very useful tool to determine potential risks from soil and sediments contamination (Rieuwerts et al., 1998). Western-European legislations are currently moving

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towards implementation of more risk-based legislative frameworks, both at the level of soil evaluation and of soil remediation. The use of single extractions for soil evaluation allows for a more nuanced assessment of heavy metal forms and their mobility in the soil. Soil remediation based on risk reduction associated with heavy metals allows the application of more economic soft-target techniques that can be used over extended surface areas. Examples of such techniques are physico-chemical stabilisation, phytoremediation techniques and innovations in in situ soil washing techniques. Adapting legal frameworks to accommodate the use of soft-target techniques can expedite riskbased soil remediation of diffuse soil contaminations. However, a prerequisite for adaptation of these frameworks is the correct understanding of bioavailability and risk assessment of heavy metals. 1.2. Bioavailable fraction, a qualitative term The ‘bioavailable fraction’ is considered as the fraction of the total contaminant in the interstitial water and soil particles that is available to the receptor organism (Vig et al., 2003). Hund-Rinke and K¨ordel (2003) describe ‘bioavailability’ as a complex term, in which both dynamic processes and speciesspecific interactions are involved. It is strongly associated to the organism under evaluation, the type of exposure and the chemical speciation of the metals. However, the term ‘bioavailable’ itself is vague and can be misleading. In the context of soil chemical fractionation, the term ‘bioavailable fraction’ deviates from the IUPAC recommended terminology, as ‘fractionation’ refers to the classification of an analyte or group of analytes accordingly to physical or chemical properties (Templeton et al., 2000). Quevauviller (1998) stated that functionally defined fractions in literature, such as ‘active’ or ‘mobile’ can also be confusing as they already reflect the interpretation of data rather than the measurements themselves. Meyer (2002) stated that although the term ‘bioavailable fraction’ is hard to define quantitatively and context-specific, the term ‘bioavailability’ itself does describe a general and useful qualitative concept. Peijnenburg et al. (1997) proposed to treat bioavalability as a dynamic process in two separate phases with (i) ‘environmental bioavailability’ defined as a physico-chemically determined fraction and (ii) ‘toxicological bioavailability’ as a measure of physiologically induced effects. Caussy et al. (2003) point out that some authors differentiate between ‘external bioavailability’ or ‘bioaccessibility’, which largely depends on the ability of metals to be dissolved and released from media or food, and ‘internal bioavailability’ which reflects the ability to be absorbed by the organism, reach target tissues and exert toxicological effects. The availability of heavy metals for plant uptake is often described as phytoavailability (Song et al., 2004). For the purposes of this study, phytoavailability refers to plant uptake and subsequent accumulation in aboveground plant parts. 1.3. Selective soil extraction procedures Water soluble metal ions can easily be mobilised, and may be considered as highly ‘bioavailable’ (Seguin et al., 2004). To

assess the readily available metal fractions under field conditions, collection and analysis of pore water has therefore become an important aspect of many environmental monitoring programs. Chapman et al. (2002) concluded that pore water testing and analyses can be effective tools provided their limitations are well understood by researchers and managers. Rhizon soil moisture samplers were originally designed soil moisture as micro-tensiometers for soil science for seepage water sampling in the unsaturated zone (Meijboom and Van Noordwijk, 1991). In previous studies, these samplers were considered to be a wellperforming and reliable extraction technique for evaluation of heavy metals in soil solutions (Meers et al., 2005a, 2006a,b). Novozamsky et al. (1993) proposed the use of 0.01 M CaCl2 as an extraction reagent to estimate bioavailability of metals and nutrients in air-dry soil samples. In Germany, exchangeable soil metals are estimated by a 1 M NH4 NO3 extraction procedure (DIN, 1997). In the Swiss legislation, a 0.1 M NaNO3 extraction procedure is used (VSBo, 1986). The use of 1 M NH4 OAc as extractant has been adopted in French legislation (French standard NF X 31-108; Afnor, 1994). Sequential extractions can be used for fractionation of heavy metals. Two of the most commonly used protocols in this regard are the BCR extraction procedure (Bureau Communautaire de R´ef´erence; Ure et al., 1993; Rauret et al., 1999) and the procedure proposed by Tessier et al. (1979). The first steps of both of these protocols were likewise evaluated in this study (0.11 M HOAc, 1 M MgCl2 ). Nitric acid-extractable metals in the soil have been suggested as a measure for geochemically active metals, present in the soil (Tipping et al., 2003). The term ‘active’ implies susceptibility to chemical interactions with the soil solids that control solution concentrations. Barreto et al. (2004), in analogy with Fiszman et al. (1984), define the 0.1 M HCl-extractable fraction as the metals operationally released by ‘moderate acid attack’. Complex extraction solutions, aimed at mimicking rhizosphere effects in the soil, have been developed to ascertain bioavailability of trace metals. A mixed ethylenediaminetetra-acetic acid (EDTA)-NH4 OAc extraction procedure was first introduced by Lakanen and Ervio (1971). Complexation by EDTA and acetic acid are thought to simulate complexing behaviour by root exudates, whereas NH4 is capable of desorbing the exchangeable soil fraction, and the pH simulates rhizosphere acidity. Lindsay and Norvell (1978) proposed a diethylenetriaminepentaacetic acid (DTPA)-based extraction solution, buffered at pH 7.3 to exclude effects involving carbonate dissolution. This protocol is also widely used, predominantly for phytoavailability studies. 1.4. Phaseolus vulgaris as test organism The test species used in this study has been selected because of its application in a bio-assay for metal induced oxidative stress assessment in soils (Van Assche and Clijsters, 1990; Vangronsveld and Clijsters, 1992; Ruttens, 2006). The species has also been used for assessment of heavy metal phytotoxicity based on growth experiments (Boisson et al., 1999; Theodoratos et al., 2002; Bleeker et al., 2003). Bennett and Buchen (1995) also described this species as one of the most frequently used test organisms for environmental assessment of air pollution effects.

Table 1 General properties of the soils under study: sample origina , most recent land use, electrical conductivity (EC), pH-H2 O, pH-KCl, carbonate content (CaCO3 ), organic matter (OM), texture, moisture content at field capacity (FC), cation exchange capacity (CEC) and texture class; intervals denote standard deviations (n = 3) Origina

Land use

EC (␮S cm−1 )

pH-H2 O

pH-KCI

CaCO3 (%)

OM (%)

Clay (%)

Silt (%)

Sand (%)

FC (ml kg−1 )

CEC (cmol kg−1 )

Texture class (USDA)

S1 S2 S3 S5 S6 S7 S9 S10 S11 S12 S17 S18 S20 S21 S22 S24 S25 S26 S27 S28 S29

DS DS DS DS DS DS DS DS DS Ctrl Ctrl Ctrl Ctrl Ctrl PA PA Ctrl PA Ctrl PA

Nature Nature Nature Nature Agriculture Nature Nature Nature Nature Agriculture Agriculture Agriculture Agriculture Clay quarry Agriculture Industrial Agriculture Nature Industrial Agriculture (Field exp.)

123 ± 9 375 ± 2 1116 ± 14 455 ± 7 348 ± 2 1963 ± 20 438 ± 9 400 ± 10 425 ± 6 216 ± 4 52 ± 1 216 ± 8 345 ± 7 2823 ± 12 119 ± 4 63 ± 4 122 ± 3 45 ± 3 97 ± 4 108 ± 4 137 ± 38

7.7 7.7 7.5 8.2 7.3 7.6 7.4 7.4 7.3 7.3 5.9 7.5 8.2 3.6 5.6 5.8 6.1 5.2 6.1 5.9 5.3

7.0 7.1 7.1 7.8 6.5 7.1 6.9 7.0 7.0 7.2 4.6 7.0 7.7 3.0 4.6 5.1 5.3 4.0 5.8 5.0 4.4

6.9 ± 1.3 7.6 ± 0.8 7.9 ± 0.8 8.3 ± 0.9 9.0 ± 1.0 3.8 ± 0.6 11.8 ± 0.7 5.9 ± 1.0 6.0 ± 0.8 7.6 ± 0.0 0.0 ± 0.2 10.6 ± 0.3 26.6 ± 0.2 0.0 ± 0.1 0.0 ± 0.1 0.0 ± 0.1 0.0 ± 0.1 0.0 ± 0.1 0.0 ± 0.4 0.0 ± 0.1 0.0 ± 0.3

5.9 ± 0.5 7.4 ± 0.5 4.3 ± 0.7 6.6 ± 0.4 9.9 ± 0.3 8.5 ± 0.6 9.7 ± 0.1 12.7 ± 0.2 10.6 ± 0.3 6.2 ± 0.4 3.5 ± 0.1 3.6 ± 0.1 12.8 ± 0.2 2.8 ± 0.1 10.0 ± 0.1 2.8 ± 0.0 4.9 ± 0.2 4.9 ± 0.1 2.4 ± 0.0 4.9 ± 0.5 7.8 ± 0.2

28 40 35 35 52 51 50 47 39 22 15 37 27 42 10 8 14 6 8 4 4

48 54 63 56 48 47 50 53 52 42 60 54 51 37 36 24 58 14 28 14 8

25 6 3 9 0 2 0 0 9 36 24 9 22 22 55 68 28 80 64 83 88

320 379 335 375 384 342 351 386 350 328 214 321 298 310 318 213 243 190 211 250 246

17.5 ± 0.9 26.2 ± 1.4 16.6 ± 1.7 23.4 ± 1.2 34.2 ± 1.6 39.2 ± 0.5 29.8 ± 1.5 27.7 ± 5.3 23.4 ± 1.3 17.8 ± 1.2 5.6 ± 0.2 22.2 ± 0.6 31.4 ± 0.7 18.7 ± 0.7 8.7 ± 0.8 3.0 ± 0.5 5.0 ± 0.4 3.7 ± 0.8 2.7 ± 0.0 3.6 ± 0.6 6.2 ± 0.7

Clay loam Silty clay loam Silty clay loam Silty clay loam Silty clay Silty clay Silty clay Silty clay Silty clay loam Loam Silt loam Silty clay loam Silt loam Clay Sandy loam Sandy loam Silt loam Loamy sand Sandy loam Loamy sand Sand

Min.

45

3.6

3.0

0.0

0.9

4

14

0

190

2.7

Max.

1963

8.2

7.8

26.6

12.8

52

60

83

386

39.2

a

E. Meers et al. / Environmental and Experimental Botany 60 (2007) 385–396

Soil

DS: dredged sediment derived soil, Ctrl: control soil, PA: soil polluted by atmospheric deposition.

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1.5. Objective This paper focuses on phytoavailability assessment of heavy metals in soils varying in their degree of contamination. In this study, heavy metals (Cd, Cu, Ni, Pb, Zn) extracted by commonly used selective extraction procedures are correlated with plant uptake by the selected test species (Phaseolus vulgaris L.) to ascertain which extraction procedure exhibits the best correlation with shoot accumulation and which can therefore be used as indicator of phytoavailability. 2. Material and methods 2.1. Sample collection For the evaluation of the plants physiological stress response to heavy metals, 21 soils were collected in Flanders, Belgium (Table 1). For comparison purposes, the same dataset of soils has also been used in previous environmental monitoring and risk assessment studies (Meers et al., 2005a, 2006a,b). Of these soils, nine were dredged sediment derived soils (S1; S2; S3; S5; S6; S7; S9; S10; S11), seven soils were from agricultural origin (S12; S17; S18; S20; S22; S26; S28), four soils were from polluted industrial locations (historical smelter activity; S24; S25; S27; S29) and one soil was collected from a commercial clay quarry used for production of building bricks (S21). Due to the current and historic state of Western-European waterways, dredged sediments generally contain elevated levels of heavy metals. At each sampling point, 10 kg of sample was collected from the top soil layer (25 cm). Subsequently, the samples were air-dried, ground and thoroughly homogenised for further experiments. 2.2. Soil characterisation Soil conductivity was measured with a WTW LF 537 electrode (Wissenschafltich-Technischen Werkst¨aten, Weilheim, Germany) after equilibration for 30 min in deionised water at a 5:1 liquid:solid ratio and subsequent filtering (white ribbon; Schleicher & Schuell, Dassel, Germany). To determine actual

soil pH, 10 g of air-dried soil was allowed to equilibrate in 50 ml of deionised water for 24 h. For potential soil pH, 50 ml of 1 M KCl was added to 10 g of air-dried soil and allowed to equilibrate for 10 min. The pH of the supernatant was then measured using a pH glass electrode (Model 520A, Orion, Boston, MA, USA), calibrated using pH 4.0 and pH 7.0 standards. Total carbonate content present in the sediment was determined by adding a known excess quantity of sulphuric acid and back titrating the excess with sodium hydroxide (Van Ranst et al., 1999). Organic matter was determined using the method described by Walkley & Black (Allison, 1965). The grain size distribution of the soil samples was determined using laser diffractometry (Coulter LS200, Miami, FL, USA) (Vandecasteele et al., 2002). The cation exchange capacity (CEC) of the sediment was determined by first saturating the soil matrix with NH4 + , then desorbing the NH4 + by K+ and measuring the quantity of the NH4 + in the leachate (Van Ranst et al., 1999). Field capacity was determined by adding an excess of water to 400 g dry weight soil. The pots were assumed to be at field capacity when formation of further droplets at the bottom of the pot after free percolation has fully ended. Soil nutritional state of P, Ca, Mg, K and Na was estimated based on NH4 OAc-EDTA extractions and soil CEC (Van Ranst et al., 1999; Meers et al., 2005b). 2.3. Single extractions Pseudo-total soil content of heavy metals was estimated by hot plate aqua regia digestion (3:1, v/v, HCl:HNO3 ) (Van Ranst et al., 1999). This digestion procedure (ISO standard 11466) is considered adequate for analyzing total-recoverable heavy metals in soils: residual elements that are not released by aqua regia digestion are mostly bound to silicate minerals and are considered unimportant for estimating the mobility and behavior of the elements (Niskavaara et al., 1997). All extraction procedures that were compared in their ability to release heavy metals from the soil are given in Table 2. All extraction protocols were performed on air-dried and ground soil. To appraise the additional effect of chloride on heavy metal mobilisation in the 0.01 M CaCl2 and the 1 M MgCl2 extraction procedures, five soils were

Table 2 Overview of single extractions used in the experiments Extraction solution

Liquid:solid ratio

Equilibration time

Reference

Rhizon soil moisture samplers 0.01 M CaCl2 0.1 M Ca(NO3 )2 1 M NH4 NO3 0.1 M NaNO3 1 M MgCl2 1 M Mg(NO3 )2

At field capacity 5:1 5:1 2.5:1 2.5:1 8:1 8:1

3 weeks incubation 2h 2h 2h 2h 1h 1h

Meers et al. (2005a) Van Ranst et al. (1999) Boisson et al. (1999) DIN (1997); Legislation Germany VSBo (1986); Legislation Switzerland Tessier et al. (1979) Adapted after Gommy et al. (1998)

0.11 MHOAc 1 M NH4 OAc (pH 7.0)

40:1 30:1

16 h Column displacement

Rauret et al. (1999), Ure et al. (1993) Van Ranst et al. (1999)

0.5 M HNO3 0.1 M HCl

5:1 25:1

30 2.5 h

Van Ranst et al. (1999) Fiszman et al. (1984)

0.005 M DTPA, 0.01 M CaCI2 , 0.1 M TEA (pH 7.3) 0.5 M NH4 OAc, 0.5 M HAc, 0.02 M EDTA (pH 4.65)

2:1 5:1

2h 30

Lindsay and Norvell (1978) Lakanen and Ervio (1971)

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389

also extracted with 0.01 M Ca(NO3 )2 and 1 M Mg(NO3 )2 (S1 , S7 , S11 , S24 , S27 ). For the soil solution extraction by Rhizon soil moisture samplers (MOM-type; Eijkelkamp Agrisearch, Giesbeek, the Netherlands), pots containing 400 g of air dry soil were brought to field capacity and incubated at constant soil moisture content for 3 weeks. Soil solution extraction was performed by inserting the inert porous tubing of the Rhizon into the soils after 3 weeks of incubation. At the other end of the soil moisture sampler, a Luer-Lock connector was attached to a needle which was subsequently inserted into a vacuum tube (vacuette) to provide the required suction to extract soil solution over the porous filter material (<0.1 ␮m diameter). Based on soil moisture content at field capacity, the Rhizon extractable concentrations were transformed from mg l−1 to mg kg−1 dry weight soil basis to allow for comparison of relative metal extractability between the various protocols. Heavy metal (Cd, Cu, Ni, Pb, Zn) analysis in the digestion and the various extractions was subsequently performed using inductively coupled plasma-optical Emission spectrometry (ICP-OES; Varian Vista MPX, Varian, Palo Alto, CA, USA). Metal levels below the analytical detection limit of the ICP-OES were analysed with graphite furnace-atomic absorption spectrometry with Zeeman correction (GF-AAS; Varian SpectrAA 800, Varian, Palo Alto, CA, USA). However, GF-AAS was not used for analysis of extractions with high ionic strength because of analytical issues. Detection limit for ICP-OES is 10 ␮g l−1 for Cd, 50 ␮g l−1 for Pb, 6 ␮g l−1 for Cu, 30 ␮g l−1 for Ni and 10 ␮g l−1 for Zn. Detection limit for GF-AAS is 0.1 ␮g l−1 for Cd, 2 ␮g l−1 for Pb, 2 ␮g l−1 for Cu and 2 ␮g l−1 for Ni (GFAAS is not used for Zn analysis).

icher & Schuell, Dassel, Germany) into a 50 ml volumetric flask and brought to volume with 1% HNO3 before element analysis using ICP-OES (Varian Vista MPX, Varian, Palo Alto, CA, USA). Seed samples were also analysed for average initial heavy metal concentrations, by the same digestion procedure.

2.4. Plant experiment

3. Results

Heavy metal phytoavailability was assessed by a biological assay using beans (P. vulgaris cv. Limburgse vroege) as a bioindicator. The experiment was performed in two separate trials, with in the first trial harvest and metal analysis of the primary leaves after 2 weeks, in analogy with Van Assche and Clijsters (1990), and with in the second trial harvest and metal analysis of the total aboveground biomass after 4 weeks. Seeds were sown in polyethylene pots of 400 ml containing the various test soils after one day vernalisation and 4 h imbibition at a rate of six seeds per pot and three pots per soil type. In the longer experiment (B), plants were thinned to three plants per pot after 7 days and subsequently to two plants per pot after 14 days. Plants were cultured in a growth chamber under controlled conditions: air temperature of 22 ◦ C, 65% relative air humidity, 12 h light per day, photosynthetic active radiation intensity of 150 ␮mol m−2 s−1 at the height. Pots were watered daily up to 40% (first week) and 60% (second week onwards) of field capacity. Harvested samples were oven dried at 60 ◦ C and subsequently weighed. Oven-dry samples were then ground using a Culatti DCFH 48 grinder and sieved with a 1 mm sieve. Plant samples were ashed for 2 h at 450 ◦ C, dissolved in 5 ml of 6 M HNO3 and placed on a hot plate (150 ◦ C, 50%). After 30 min, 5 ml of 3 M HNO3 was added and the samples were allowed to heat for another 10 min. Finally, the digested plant samples were filtered (Schle-

3.1. Soil characterisation

2.5. Quality control For quality control of total metal content determination in plant and soil samples, certified reference samples were analysed by the methods described above. Observed concentrations for the certified BCR 281 ryegrass sample were: 0.11 mg kg−1 Cd (certified 0.12 ± 0.003 mg kg−1 ), 8.97 mg kg−1 Cu (certified 9.65 ± 0.38 mg kg−1 ), 2.82 mg kg−1 Ni (certified 3.00 ± 0.17 mg kg−1 ), 2.19 mg kg−1 Pb (certified 2.38 ± 0.11 mg kg−1 ) and 29.0 mg kg−1 Zn (certified 31.15 ± 1.4 mg kg−1 ). Observed concentrations for the certified BCR 141 soil sample were: 13.2 mg kg−1 Cd (certified 14.0 ± 0.4 mg kg−1 ), 42.0 mg kg−1 Cu (certified 46.9 ± 1.8 mg kg−1 ), 90.1 mg kg−1 Ni (certified 94.0 ± 5.0 mg kg−1 ), 51.5 mg kg−1 Pb (certified 51.3 ± 2.0 mg kg−1 ) and 266.0 mg kg−1 Zn (certified 270 ± 8.0 mg kg−1 ). 2.6. Statistical analysis Soil properties, total metal content of soils/plants and single extractions were determined in triplicate. Descriptive statistics, bivariate Pearson correlations and significance analysis (p = 0.01; 0.05) were performed using SPSS 11.0 (SPSS Inc.) and Excel (Microsoft Inc.) software packages.

The soils used for this experiment varied widely in soil composition (Table 1). Soil texture varied from sandy soils, over silty soils to silty clay soils. The soil conductivity varied between 45 and 2823 mS cm−1 . Soil pH varied between 3.6 and 8.2. Carbonate content varied between 0% and 26.6% and organic matter content between 1.8% and 10.6%. Associated with the wide range in soil texture, the soil cation exchange capacity varied from 3.0 to 39.2 cmol(+) kg−1 and the moisture content at field capacity varied from 190 to 396 ml kg−1 . Total heavy metal content in the soil is presented in Table 3. In comparison, natural background levels of heavy metals in soils in Flanders are situated at around 0.8 mg kg−1 for Cd, 37 mg kg−1 for Cr, 17 mg kg−1 for Cu, 40 mg kg−1 for Pb, 9 mg kg−1 for Ni and 62 mg kg−1 for for Zn (Vlarebo, 1995). 3.2. Metal accumulation On average, seed dry weight was 0.32 ± 0.05 g and seeds contained 0.01 mg kg−1 Cd, 7.7 mg kg−1 Cu, 0.2 mg kg−1 Pb, 2.2 mg kg−1 Ni and 26.5 mg kg−1 Zn. Total aboveground biomass after 2 weeks varied between 0.09 and 0.56 g/plant, and after 4 weeks between 0.30 and 1.91 g/plant.

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Table 3 Total metal content (aqua regia) for the 21 soils under investigation; intervals denote standard deviations (n = 3) Cd (mg kg−1 )

Cr (mg kg−1 )

Cu (mg kg−1 )

Mn (mg kg−1 )

Ni (mg kg−1 )

Pb (mg kg−1 )

Zn (mg kg−1 )

S1 S2 S3 S5 S6 S7 S9 S10 S11 S12 S17 S18 S20 S21 S22 S24 S25 S26 S27 S28 S29

6.2 ± 0.4 3.7 ± 0.3 2.2 ± 0.2 3.2 ± 0.1 22.2 ± 0.5 0.92 ± 0.02 14.0 ± 0.1 41.4 ± 0.6 23.2 ± 0.4 0.25 ± 0.02 0.19 ± 0.03 0.45 ± 0.02 0.79 ± 0.05 0.22 ± 0.03 0.39 ± 0.05 13.8 ± 0.8 2.0 ± 0.4 0.26 ± 0.05 28.2 ± 3.7 1.4 ± 0.3 5.3 ± 0.5

134 ± 10 89 ± 10 60 ± 5 81 ± 3 1287 ± 31 65 ± 1 481 ± 4 779 ± 9 1914 ± 23 35 ± 0.4 12 ± 1 49 ± 2 41 ± 0.5 68 ± 1 10 ± 2 10 ± 2 10 ± 1 6.7 ± 0.8 13 ± 1 6.1 ± 0.5 3.3 ± 0.9

97 ± 7 80 ± 6 47 ± 4 71 ± 3 106 ± 2 54 ± 1 143 ± 3 99 ± 1 166 ± 1 14 ± 4 12 ± 2 22 ± 2 13 ± 2 17 ± 0.2 16 ± 0.2 231 ± 27 14 ± 2 0.017 ± 0.005 1069 ± 103 4.4 ± 0.3 32 ± 2

223 ± 16 333 ± 26 242 ± 23 324 ± 11 697 ± 26 372 ± 6 803 ± 3 349 ± 3 370 ± 6 152 ± 7 95 ± 6 413 ± 6 705 ± 22 92 ± 2 196 ± 20 268 ± 81 193 ± 18 21 ± 0 1027 ± 92 40 ± 1 43 ± 3

44 ± 3 29 ± 2 21 ± 2 27 ± 1 40 ± 1 34 ± 1 37 ± 0.4 35 ± 0.3 40 ± 0.5 18 ± 0.1 3.5 ± 0.2 22 ± 0.4 21 ± 1 33 ± 1 4.2 ± 0.8 14 ± 2 4.1 ± 0.3 1.7 ± 0.5 34 ± 14 0.9 ± 0.6 0.8 ± 0.6

116 ± 10 113 ± 7 67 ± 6 121 ± 5 655 ± 20 49 ± 1 134 ± 1 286 ± 4 334 ± 21 31 ± 1 20 ± 2 36 ± 1 39 ± 1 15 ± 0.1 34 ± 6 605 ± 46 79 ± 8 6.4 ± 0.2 1663 ± 233 55 ± 3 272 ± 19

614 ± 49 486 ± 42 301 ± 30 426 ± 23 2175 ± 59 188 ± 8 966 ± 51 2087 ± 37 2422 ± 60 60 ± 3 21 ± 1 97 ± 5 77 ± 5 62 ± 2 150 ± 13 1329 ± 33 154 ± 12 18 ± 0.4 10839 ± 2550 89 ± 2 200 ± 14

Min. Max.

0.2 41.4

3 1914

0 1069

21 1027

1 44

6 1663

18 10839

Plant shoot concentrations of heavy metals are presented in Table 4. The range of observed shoot concentrations for all elements is broad, varying 20-fold for Cd, Pb and Zn and 2–5-fold for Cr, Cu and Ni. Plants grown on sandy soil types tended to accumulate higher levels of heavy metals than did plants grown on heavier soil textures. This can be demonstrated by comparing

the sandy control soils S22, S26 and S28 with the loam control soil (S12) and the silty clay loam control soil (S18). Also, when the soil with the highest Cd content (S10, Table 3) is compared with all sandy soils, both polluted and control soils (S22–S29), a higher accumulation can be observed in plants grown on the sandy soils than on the more polluted, yet clayey S10 substrate

Table 4 Total metal content in aboveground plant parts (dry weight) after 4 weeks of growth; intervals denote standard deviations (n = 3); (–) no growth Al (mg/kg DW)

Cd (mg/kg DW)

Cr (mg/kg DW)

Cu (mg/kg DW)

S1 S2 S3 S5 S6 S7 S9 S10 S11 S12 S17 S18 S20 S21 S22 S24 S25 S26 S27 S28 S29

27 ± 5.6 21 ± 4.2 31 ± 2.5 25 ± 5.1 27 ± 4.0 22 ± 4.2 27 ± 4.0 19 ± 2.3 23 ± 6.9 28 ± 20 24 ± 3.4 18 ± 1.3 21 ± 6.2 – 20 ± 4.2 99 ± 67 26 ± 1.0 114 ± 24 – 24 ± 1.2 38 ± 1.3

0.2 ± 0.0 0.1 ± 0.0 0.2 ± 0.1 0.2 ± 0.1 0.4 ± 0.1 0.1 ± 0.0 0.2 ± 0.0 0.6 ± 0.1 0.3 ± 0.1 0.1 ± 0.0 0.1 ± 0.1 0.1 ± 0.1 0.1 ± 0.0 – 0.2 ± 0.0 3.5 ± 1.5 1.1 ± 0.4 0.6 ± 0.1 – 1.7 ± 0.2 4.0 ± 0.4

1.0 ± 0.3 0.7 ± 0.3 0.9 ± 0.3 0.7 ± 0.2 0.8 ± 0.1 0.6 ± 0.1 1.0 ± 0.1 0.9 ± 0.4 0.7 ± 0.2 0.6 ± 0.4 0.6 ± 0.1 1.0 ± 0.4 0.5 ± 0.1 – 0.7 ± 0.3 2.4 ± 1.5 0.8 ± 0.0 0.6 ± 0.1 – 0.7 ± 0.3 1.3 ± 0.5

12 ± 1 14 ± 2 16 ± 1 15 ± 2 15 ± 1 13 ± 1 22 ± 1 13 ± 0 13 ± 2 8±1 9±1 10 ± 0 9±1 – 10 ± 1 17 ± 2 10 ± 1 11 ± 1 – 13 ± 1 14 ± 2

Minimum Maximum

18 114

0.09 4.0

0.47 2.4

8.0 22

Mn (mg/kg DW)

Pb (mg/kg DW)

Ni (mg/kg DW)

Zn (mg/kg DW)

14 ± 1 34 ± 6 23 ± 3 51 ± 9 83 ± 14 61 ± 11 115 ± 6 86 ± 54 63 ± 12 29 ± 3 38 ± 7 35 ± 3 48 ± 7 – 41 ± 10 29 ± 3 55 ± 9 162 ± 42 – 54 ± 5 88 ± 12

0.8 ± 0.1 0.8 ± 0.5 0.7 ± 0.2 0.7 ± 0.4 1.0 ± 0.3 0.9 ± 0.2 1.1 ± 0.2 0.5 ± 0.4 0.3 ± 0.1 0.3 ± 0.2 0.3 ± 0.0 0.4 ± 0.2 0.4 ± 0.1 – 0.5 ± 0.3 5.4 ± 0.7 0.5 ± 0.2 0.7 ± 0.2 – 1.0 ± 0.2 1.8 ± 1.4

7.7 ± 1.6 2.4 ± 0.4 2.8 ± 0.4 3.3 ± 0.5 3.2 ± 0.4 3.1 ± 0.9 3.4 ± 0.3 2.1 ± 0.2 2.2 ± 0.5 2.1 ± 0.3 2.4 ± 0.9 4.0 ± 0.1 2.2 ± 0.0 – 1.9 ± 0.4 5.3 ± 0.9 2.5 ± 1.0 4.9 ± 0.6 – 2.6 ± 0.6 2.8 ± 0.5

65 ± 6 72 ± 4 69 ± 14 78 ± 14 136 ± 12 63 ± 10 108 ± 1 67 ± 8 88 ± 17 41 ± 32 57 ± 31 22 ± 2 32 ± 3 – 185 ± 8 382 ± 122 164 ± 30 172 ± 29 – 336 ± 22 420 ± 48

14 162

0.30 5.4

1.9 7.7

22 420

E. Meers et al. / Environmental and Experimental Botany 60 (2007) 385–396

(Table 4). A similar comparison can be made for Pb between S6 and S24, or for Zn between S6, S10 and S11 with all sandy soils. Lower metal phytoavailability in the more clayey soils is due to a higher cation exchange capacity (CEC) in these soils. Also, in general a higher soil pH was generally observed for the more clayey soils. A higher CEC results in removal from the soil solution by adsorption, while higher pH results lower overall solubility for the metals under evaluation. 3.3. Correlation between single extractions and plant accumulation Table 5 presents the observed correlations soil Cd concentrations obtained by the different single extractions and Cd concentrations in P. vulgaris. The correlations were examined based on plant concentrations on dry weight and on ash weight basis. None of the more aggressive extraction procedures, including pseudo-total soil content, presented a good measure for Cd phytoavailability under the current experimental conditions. Similar conclusions were derived based on dry weight and ash weight basis. Rhizon soil solution extractions, extractions based on unbuffered nitrate solutions and the diluted chloride solution CaCl2 provided the best measure of Cd phytoavailability in the soil. These weak extraction procedures all correlated significantly (p = 0.01) with shoot Cd accumulation. Table 6 presents the observed correlations for Cu. Unlike as was observed for Cd, the more aggressive extraction procedures and pseudo-total content exhibited good and significant (p = 0.01) correlations with plant uptake. For Ni, neither the aggressive extraction procedures, nor the very weak aimed at ascertaining soil solution levels yielded consistently significant correlations with plant uptake (Table 7). The best predictors for accumulated Ni were found in the cluster containing the unbuffered salt solutions NH4 NO3 and CaCl2 .

391

Table 6 Pearson correlations between Cu extracted from the soil following different extraction and observed concentrations in primary leaves after 2 weeks or total shoot content after 4 weeks of growth; bold indicates significant interaction Extraction

Primary leaves after 2 weeks

Total shoot content after 4 weeks

Dry weight

Dry weight

Ash −0.05

Ash

Rhizon

0.21

0.03

0.38

NaNO3 CaCl2 NH4 NO3

0.40 0.94** 0.83**

0.25 0.82** 0.70*

0.45 0.54** 0.53

0.13 0.69** 0.51

NH4 OAc MgCl2

0.45 0.92**

0.79** 0.82**

0.94** 0.48

0.86** 0.78**

HOAc

0.33

0.86**

0.92**

0.86**

HNO3 HCl

0.79**

0.65*

0.80**

0.70*

0.42 0.26

0.54* 0.63*

DTPA NH4 OAc-EDTA

0.79** 0.86**

0.64* 0.72**

0.74** 0.65**

0.56* 0.57*

Aqua regia

0.85**

0.70**

0.66**

0.60**

** Significant

at the 0.01 level, * significant at the 0.05 level.

As was also observed for Cu, pseudo-total content already provided a good measure of phytoavailability of Pb (Table 8). Since all other extractions correlated very closely with the pseudo-total content, the correlations for all tested extraction procedures exhibited very significant correlations (p = 0.01) with plant concentrations of Pb. Table 9 presents Pearson correlations between plant uptake and soil extractions of Zn. Similar to findings for Cd, correlations between chemical extractions and plant concentrations were best with the weaker extractions either aimed at ascertaining soil solution levels, or based on unbuffered salt solutions. However, unlike for Cd, chloride solutions at higher concentrations (1 M

Table 5 Pearson correlations between Cd extracted from the soil following different extraction techniques and observed concentrations in primary leaves after 2 weeks or total shoot content after 4 weeks of growth; bold indicates significant interaction

Table 7 Pearson correlations between Ni extracted from the soil following different extraction and observed concentrations in primary leaves after 2 weeks or total shoot content after 4 weeks of growth; bold indicates significant interaction

Extraction

Extraction

Primary leaves after 2 weeks

Total shoot content after 4 weeks

Dry weight

Dry weight

Primary leaves after 2 weeks

Total shoot content after 4 weeks

Dry weight

Ash

Dry weight

Ash

Rhizon

0.68**

0.74**

0.71**

0.90**

Rhizon

0.33

0.05

0.41

0.10

NaNO3 CaCl2 NH4 NO3

0.81**

0.82**

0.73**

0.91**

0.70** 0.81**

0.76** 0.82**

0.76** 0.76**

0.93** 0.92**

NaNO3 CaCl2 NH4 NO3

0.52 0.78** 0.64*

0.45 0.82** 0.73*

0.52 0.39 0.69**

0.50 0.81** 0.88**

MgCl2 NH4 OAC

0.25 0.35

0.16 0.35

0.19 0.33

0.21 0.37

MgCl2 NH4 OAc

0.22 0.32

0.15 0.14

0.70** 0.61**

0.34 0.34

Ash

Ash

HOAc

0.48

0.44

0.35

0.41

HOAc

0.10

−0.13

0.49

0.03

HNO3 HCl

0.14 0.25

0.12 0.18

0.07 0.10

0.08 0.13

HCl HNO3

0.16 0.28

−0.15 −0.04

0.41 0.44

−0.03 −0.01

DTPA NH4 OAc–EDTA

0.18 0.15

0.17 0.13

0.16 0.09

0.16 0.09

DTPA NH4 OAc-EDTA

0.13 0.38

−0.20 0.04

0.51* 0.49*

−0.05 0.05

Aqua regia

0.15

0.14

0.08

0.10

Aqua regia

0.27

−0.13

0.24

−0.21

** Significant

at the 0.01 level,

* significant

at the 0.05 level.

** Significant

at the 0.01 level,

* significant

at the 0.05 level.

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E. Meers et al. / Environmental and Experimental Botany 60 (2007) 385–396

Table 8 Pearson correlations between Pb extracted from the soil following different extraction and observed concentrations in primary leaves after 2 weeks or total shoot content after 4 weeks of growth; bold indicates significant interaction Extraction

Primary leaves after 2 weeks

Total shoot content after 4 weeks

Dry weight

Ash

Dry weight

Ash

Rhizon

0.71**

0.72**

0.71**

0.70*

NaNO3 CaCl2 NH4 NO3

MgCl2 NH4 OAc

0.97** 0.95**

0.997** 0.98**

0.92* 0.97**

0.88* 0.98**

HOAc

0.96**

0.99**

0.99**

0.998**

HNO3 HCl DTPA NH4 OAc–EDTA

0.77**

0.76**

0.88**

0.70** 0.91** 0.76**

0.72** 0.94** 0.67**

0.94** 0.89** 0.62**

0.88** 0.96** 0.86** 0.57*

Aqua regia

0.77**

0.67**

0.62**

0.58**

** Significant

at the 0.01 level, * significant at the 0.05 level.

Table 9 Pearson correlations between Zn extracted from the soil following different extraction and observed concentrations in primary leaves after 2 weeks or total shoot content after 4 weeks of growth; bold indicates significant interaction Extraction

Primary leaves after 2 weeks

Total shoot content after 4 weeks

Dry weight

Dry weight

Ash

Ash

Rhizon

0.49

0.59

0.63**

NaNO3 CaCl2 NH4 NO3

0.59 0.52 0.59

0.70* 0.64* 0.70*

0.70** 0.81** 0.73**

0.91** 0.95** 0.92**

MgCl2 NH4 OAc

0.64* 0.44

0.67* 0.52

0.81* 0.48*

0.92** 0.64**

HOAc

0.18

0.25

0.29

0.31

HNO3 HCl

0.02 0.19

0.07 0.17

0.06 0.07

0.08 0.11

DTPA NH4 OAc–EDTA

0.12 0.10

0.11 0.10

0.11 0.05

0.13 0.10

Aqua regia

0.01

0.05

−0.01

0.05

** Significant

0.88**

at the 0.01 level, * significant at the 0.05 level.

MgCl2 ) and the buffered NH4 OAc solution (1 M; pH 7.0) also exhibited significant correlations. 4. Discussion 4.1. Correlation between single extractions and plant accumulation Table 10 presents an overview of the significant correlations between the extraction procedures and heavy metal accumulation in the aboveground biomass of P. vulgaris. Distinct differences can be observed for the various metals: for Cu and

Table 10 Overview of significant interactions between extractable levels in the soil and heavy metal accumulation in the shoot of P. vulgaris Cd Rhizon NaNO3 CaCl2 NH4 NO3 MgCl2 NH4 OAc HOAc HNO3 HCl DTPA NH4 OAc-EDTA Aqua regia

X X X X

Cu

X (X) X X X X X X X X

Ni

X X (X) (X)

(X) (X)

Pb

Zn

X ND X ND X X X X X X X X

(X) X X X X (X)

X: significant interaction at two time periods under evaluation (2 weeks and 4 weeks), (X): significant interactions at only one of the two time periods; ND: not determined.

Pb, the more aggressive extraction procedures and pseudo-total content exhibited good correlations with plant content, whereas for Cd, Ni and Zn the weaker extractants resulted in higher correlations. Exchangeable metals in the soil are considered to be easily available for plant uptake (Kabata-Pendias, 1993). The challenge at hand is to identify which chemical assay is best suited to reflect this labile pool of heavy metals. In the current dataset, CaCl2 proved to be the most versatile extraction procedure exhibiting significant correlations with plant concentrations of all five heavy metals under evaluation (Table 10). Novozamsky et al. (1993) proposed the use of 0.01 M CaCl2 as extraction reagent for estimation of metals and nutrients in air dry soil samples. Degryse et al. (2003) found that the CaCl2 extraction offers a good operational prediction for soil solution levels of Cd and Zn. Pueyo et al. (2004) also found this extraction procedure as the most suitable for heavy metal assessment, in comparison with 1 M NH4 NO3 and 0.1 M NaNO3 . Sahuquillo et al. (2003) concluded that in light of harmonisation of single extraction procedures, CaCl2 was to be recommended as being more protective in terms of risk assessment because of its higher leachability capacity. Houba et al. (1996, 2000) further elaborated on the benefits of this particular protocol. An important advantage is that the ionic strength of this extraction solution is similar to that of soil solution. In addition, Ca is generally the most abundant cation present in the soil solution. Brun et al. (2001) compared correlations between extractable Cu (NH4 OAc, CaCl2 , EDTA, DTPA) with uptake by Zea mays and concluded that single extraction with 0.01 M CaCl2 yielded the best correlation with Cu concentration in aboveground parts of Zea mays. Wang et al. (2003) also observed significant interactions of CaCl2 extractable metals with plant uptake in the assessment of phytoavailable soil fractions of heavy metals. In addition, Wang et al. (2004) compared CaCl2 and DTPA-extractable heavy metals and observed significant correlation for CaCl2 with shoot content of Cu in Apium graveolens and Spinacia oleracea, yet not with Brassica compestris

E. Meers et al. / Environmental and Experimental Botany 60 (2007) 385–396

ssp. pekinensis and Brassica compestris ssp. Chinensis. In their experiment, DTPA did not exhibit significant correlation with any of the tested plant species. Brown et al. (1994) reported that weak extractants, such as 0.01 M Ca(NO3 )2 or H2 O, Zn availability to Solanum lycopersicum and Silene vulgaris better reflected than did more aggressive extractants such as DTPA. Aten and Gupta (1996) observed good correlations between shoot content of Cd, Zn and Cu in Lactuca sativa and Lolium perenne and extractable soil concentrations using dilute salt solutions with either CaCl2 or nitrate based solutions (0.1 M NaNO3 , NH4 NO3 , KNO3 or CaCl2 ; 0.05 M CaCl2 ; 0.5 M NH4 NO3 ). Similar comparison for Pb by these authors did not result in satisfactory correlations. In the current dataset, good correlations were also observed between shoot accumulation and NH4 NO3 extractable Cd, Ni and Zn and to a lesser extent also of Cu. Song et al. (2004) found that 1 M NH4 NO3 extractions exhibited the best correlations with uptake by Elsholtzia splendens or S. vulgaris in comparison to EDTA extractable Cu, total soil Cu or free Cu2+ activity. Gryschko et al. (2005) described significant correlations between the 1 M NH4 NO3 extraction procedure and uptake of Cd by Triticum aestivum, Daucus carota, S. oleracea and L. sativa, as well as between this extraction and uptake of Zn by Brassica oleracea, T. aestivum, Beta vulgaris and L. sativa. Lower, yet still significant, correlation coefficients were observed for Ni in T. aestivum and S. oleracea. Variable results were observed for Cu by these authors, while Pb accumulation correlated poorly with NH4 NO3 extractable concentrations. 4.2. Physico-chemical considerations when using unbuffered salt based extractions From the bivariate correlations, the unbuffered salt based solutions, particularly 0.01 M CaCl2 and 1 M NH4 NO3 , proved to be the most versatile extraction solutions to reflect phytoavailability of the heavy metals for P. vulgaris (Table 10). In the following section, some of the physico-chemical considerations of these extractants are discussed in further detail. 4.2.1. Influence of ionic strength of the extraction solution Dispersion of small soil colloids is favoured in soils with high pH and low ionic strength (Kretzschmar et al., 1999). The formation of colloids in soil extractions with low ionic strength is thought to hinder correct appraisal of plant availability of heavy metals in the soil because metals present in colloidal material are considered not to be taken up by plant roots directly (Gryschko et al., 2005). For this purpose, these authors supported the use of high ionic strength extraction procedures, such as 1 M NH4 NO3 , for estimating phytoavailability of trace metals. However, Houba et al. (1996, 2000) argued that in the CaCl2 extraction procedure, the divalent cation also assures good coagulation of soil solution colloids, eliminating the need for elevated concentrations of the extractant, such as is the case for extractants based on monovalent cations. The effects of colloidal material in dilute CaCl2 as opposed to concentrated NH4 NO3 extractions require further comparative investigation.

393

4.2.2. Influence of extraction pH Chemical interactions in unbuffered salt based extraction solutions, such as CaCl2 or nitrate based solutions, occur essentially at soil pH. Birke and Werner (1991) stated that generally close correlations can be observed between trace metal content in plants and extraction procedures which operate at approximately the original pH of the soils when compared to other extraction procedures. Merkel (1996) found that correlations are poor at lower soluble concentrations of trace metals. Gryschko et al. (2005) observed a slight decrease in pH of the extraction solution by 0.5–1.0 units in 1 M NH4 NO3 extractions. Pueyo et al. (2004) also observed systematically lower extraction pH in 1 M NH4 NO3 extraction in comparison with 0.1 M NaNO3 and 0.01 M CaCl2 extractions, which was attributed to the slightly acidic character of the NH4 + ion. 4.2.3. Influence of the anion in extractions The anion also plays an important role in the reaction with desorbed heavy metals. Acetate and chloride can keep mobilised metals into solution by complexation. Prevention of readsorption to the soil matrix interferes in the natural phenomena in the soil and alters cation exchange equilibria (Gommy et al., 1998). To limit this overestimation of exchangeable cations, either the use of more dilute solutions has been proposed (Arunachalam et al., 1996) or the use of the less complexing nitrate anion (Krishnamurti et al., 1995). Gommy et al. (1998) observed an 11 times higher Cd extractability with 1 M MgCl2 when compared to 1 M Mg(NO3 )2 . A similar comparison in the current dataset revealed that, when operating at this molar concentration, extractable Cd and Zn factor were 19 and 2.3 times higher for Cd and Zn, respectively, when extracted with MgCl2 compared to Mg(NO3 )2 . However, when heavy metals released by 0.01 M CaCl2 were compared with those extracted by the same molar concentration of Ca(NO3 )2 , no significant differences were observed for any of the metals under study. This implies that at more dilute ionic strength (0.01 M) the effects of chloride on additional metal mobilisation in the CaCl2 extraction procedure were negligible in the current dataset. 4.2.4. Influence of the cation in extractions Divalent cations are more competitive exchanger than monovalent ones, in the order: Ba > Ca > Mg > NH4 > K > Na (Gommy et al., 1998). In addition, previous cation exchange experiments (Meers, unpublished results) indicated that the divalent bases were able to displace larger soil fractions of metals from the oxidised dredged sediments: whereas K and Na obtained ceiling levels of extractable metals in function of increasing extractant concentrations, Ca and Mg continued to desorb metals up to far higher levels than these ceiling levels. This suggests that the divalent bases are capable of desorbing metals, which cannot be displaced by the monovalent elements. The formation of amine complexes has been described as a heavy metal mobilising mechanism in ammonium based extraction solutions (Lebourg et al., 1996; Pueyo et al., 2004). Gryschko et al. (2005) stated that dissociation of NH4 + and subsequent formation of soluble amine metal complexes can result in an overestimation of exchangeable heavy metals in 1 M

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NH4 NO3 extraction. This dissociation and subsequent metal mobilisation was observed to be significant above pH 7–7.5 for Cd and above pH 6.0 for Cu. These authors proposed 1 M KNO3 extraction as a workable alternative to avoid artefact effects induced by amine-metal complex formation. In the current dataset, the ratio NH4 NO3 /CaCl2 extractable Cd is 2.3 times higher in soils with pH above 7.0 than in soils below 7.0. Analogously, this ratio was 1.5 for Cu and 3.8 for Ni whereas no such increase was observed for Zn. 4.2.5. Analytical considerations High ionic strength solutions, such as the 1 M NH4 NO3 extractant, cause difficulties during analytical determination: when metal concentrations are below instrumental detection limits for the ICP-OES or Flame AAS, as was observed for Pb in many samples (Table 8), the high salt concentrations impair more sensitive analysis using techniques such as ICP-MS or GF-AAS. More dilute extraction solutions such as CaCl2 are not hampered by this limitation. Lebourg et al. (1996) generally found the use of salt solutions to be limiting for evaluation of Pb. The liquid to solid ratio (L:S) of the 0.01 M CaCl2 extraction procedure as proposed by Houba et al. (2000) for environmental assessment of heavy metals is 10:1. However, at this ratio the concentrations tend to fall below the detection limits of ICP-OES and Flame-AAS, particularly for Cd and Pb, requiring more sensitive analytical approaches. For routine laboratory assessment of large quantities of environmental samples, the more conservative L:S ratio of 5:1, as described by Van Ranst et al. (1999) is therefore proposed. Meers et al. (2006a) observed that for weak water-based extractions, choice of the type of filter significantly affects extractions concentrations of heavy metals. This was attributed to variation in metal adsorption to the filter surface and to variable percolation of colloidal material. However, when water filtrations were performed in the presence of competitive cations (Ca, Mg, K, Na, Fe, Mn, Al), at concentrations indicative for soil solutions, adsorption of heavy metals (Cu, Zn, Cd, Pb, Ni, Cr) was found to be negligible. This implies that, at least for adsorption, no specific filter type effect is expected for 0.01 M CaCl2 soil extractions. However, the effect of filter type selection on percolation of colloidal material requires further investigation. Houba et al. (1996, 2000) argued that CaCl2 induces good coagulation, effectively removing colloids from the soil solution. The analytical significance of heavy metals associated with residual colloidal material in weak salt based extractions such as CaCl2 is still open to debate and deserves further attention. 4.3. Implications for P. vulgaris bioassay Measurement of enzymatic activity of enzymes involved with alleviation of metal induced oxidative stress in P. vulgaris has been proposed as a sensitive instrument in for assessment of phytoavailability of heavy metals (Van Assche and Clijsters, 1990; Vangronsveld and Clijsters, 1992; Geebelen et al., 2003; Meers et al., 2005c). Phytoavailability assessment, based on accumulation in aboveground plant parts and on dry weight biomass production with this plant species has also been pro-

posed (Boisson et al., 1999; Theodoratos et al., 2002; Bleeker et al., 2003). Within the context of bioassays with this test species, CaCl2 provided the most versatile physico-chemical estimation of heavy metal phytoavailability for all metals under study. For the essential trace metal Zn, observed correlations for all significant interactions were consistently higher with total shoot content after 4 weeks in comparison to correlations with Zn in primary leaves after 2 weeks (Table 9). It was hypothesised that Zn present in the seed significantly affected shoot concentrations in the initial weeks of the bioassay. This Zn reserve in the seed is partially translocated to the shoot after germination and therefore can potentially induce a bias in the evaluation of shoot concentrations of Zn in relation to extractable Zn from the soil. Based on seed weight, plant growth, as well as seed and plant Zn concentrations, the observed seed reserve artefact effect was found to be negligible in the total shoot after 4 weeks of growth, but not in the primary leaves after merely 2 weeks of growth. For the assessment of Zn, the 4-week experiment therefore yielded more reliable information in the comparison shoot accumulation and soil extraction. This particular ‘artefact’ effect of heavy metals in the seeds was not observed for the non-essential heavy metals due to low seed content of these metals. For the essential metal Cu as well, this effect was not as pronounced as for Zn. 5. Conclusion Rhizon extractions and extractions based on unbuffered nitrate solutions provided the best measure of Cd phytoavailability in the soil. In addition CaCl2 yielded good fits. A similar observation was made for Zn, yet for this element the NH4 OAc and MgCl2 extractions also provided significant interactions. The best prediction for Ni was observed in the cluster containing CaCl2 and NH4 NO3 . The pseudo-total content and the aggressive chelate based and/or acidic extractants did not suffice to predict uptake of Cd, Zn or Ni. Cu and Pb uptake on the other hand was found to correlate significantly with pseudo-total content as well as with all aggressive extraction procedures over the range of soils used in this experiment. In general, the 0.01 M CaCl2 extraction procedure proved to be the most versatile as it provided a good indication of phytoavailability of all five metals under evaluation. In accordance with other groups (Novozamsky et al., 1993; Lebourg et al., 1996; Houba et al., 1996, 2000; Sahuquillo et al., 2003; Pueyo et al., 2004) the use of this procedure is therefore supported for evaluation of plant available heavy metals in the soil. In particular, this physico-chemical assessment of labile metals in the soil could be used in association with the P. vulgaris bioassay designed by Van Assche and Clijsters (1990) and Vangronsveld and Clijsters (1992) for assessment of heavy metal induced oxidative plant stress. In respect with technical aspects concerning instrumental detection limits, we propose an L:S ratio of 5:1 for routine assessment as opposed to 10:1 as proposed by some of the other authors supporting CaCl2 extraction for phytoavailability assessment. Further methodological conclusions derived from the current study include the observation that in short term plant growth experiments with P. vulgaris as proposed by Vangronsveld and Clijsters (1992), plant seed reserves of essen-

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