Plant and Soil 189: 21–31, 1997. 21c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

Cadmium and zinc in plants and soil solutions from contaminated soils

S.E. Lorenz1;4, R.E. Hamon1, P.E. Holm2, H.C. Domingues3, E.M. Sequeira3,T.H. Christensen2 and S.P. McGrath1

1Soil Science Department, IACR Rothamsted, Harpenden, Herts AL5 2JQ, UK, 2Institute of EnvironmentalScience and Engineering, Technical University Denmark, 2800 Lyngby, Denmark and 3Departamento Pedologia,Estacao Agronomica Nacional, 2780 Oeiras, Portugal. 4Present address: 14 Glemsford Drive, Harpenden, HertsAL5 5RB, UK�

Received 4 April 1996. Accepted in revised form 19 October 1996

Key words: bioavailability, heavy metals, metal speciation, metal uptake, radish, rhizosphere

Abstract

In an experiment using ten heavy metal-contaminated soils from six European countries, soil solution was sampledby water displacement before and after the growth of radish. Concentrations of Cd, Zn and other elements insolution (K, Ca, Mg, Mn) generally decreased during plant growth, probably because of uptake by plants andthe subsequent redistribution of ions onto soil exchange sites at lower ionic strength. Speciation analysis by aresin exchange method showed that most Cd and Zn in non-rhizosphere solutions was present as Cd2+ and Zn2+,respectively. The proportion of free ions was slightly lower in rhizosphere solutions, mainly due to an increasein dissolved organic carbon during plant growth. Solution pH increased during plant growth, although the bulksoil pH generally remained constant. Cd concentrations in leaves and tubers were more closely correlated withtheir total or free ionic concentrations in rhizosphere solutions (adjusted R2 � 0.90) than with their concentrationsin soils (adj. R2 � 0.79). Cd concentrations in non-rhizosphere solutions were only poorly correlated with Cdconcentrations in leaves and tubers. In contrast to Cd, there were no soil parameters that individually predicted Znconcentrations in leaves and tubers closely. However, multiple correlation analysis (including Zn concentrations inrhizosphere solutions and in bulk soils) closely predicted Zn concentrations in leaves and tubers (adj. R2 = 0.85 and0.70, respectively). This suggests that the great variability among soils in the solubility of Zn affected the rate ofrelease of Zn into solution, and thus Zn uptake. There was no such effect for Cd, for which solubility varied muchless. Furthermore, the plants may have partly controlled Zn uptake, as they took up relatively less at high solutionconcentrations of Zn.

Free ionic concentrations in soil solution did not predict concentrations of Cd or Zn in plants better than theirtotal concentrations in solution. This suggests that with these soils, analysis of Cd and Zn speciation is of littlepractical importance when their bioavailability is assessed.

Introduction

The importance of the soil solution as the mediumof contact between the soil reservoir of nutrient- andheavy metal-ions and the plant root has long been rec-ognized (Whitney and Cameron, 1903). Its importancein determining the ionic environment for plant rootswas first demonstrated by Olsen and Peech (1960)

� FAX No: +441582760981.E-mail: 106270.41@compuserve.com

for the uptake of Rb+ and Ca2+. Furthermore, it isthought that metal speciation and ionic activity, ratherthan the total amount of a dissolved metal, determineplant uptake (Kasawneh, 1971). Experiments involv-ing solution culture of plants have usually (e.g.Checkaiet al., 1987; Cabrera et al., 1988), but not always (Lau-rie et al., 1991), suggested that it is free ionic Cd andZn that are absorbed by plants. Nor have studies con-sidering the speciation of metals in soil solution, eitherusing computer models or experimental data, consis-

JS: PIPS No. 129304 BIO2KAP

*129304* plso6212.tex; 2/07/1997; 16:26; v.7; p.1

22

tently shown that it is only the free metal ion that istaken up, and they have usually involved only a few soiltypes (e.g. Sposito and Bingham, 1981; El-Kherbawyand Sanders, 1984).

This lack of conclusive evidence for the exclusiveuptake of free ionic Cd and Zn may be attributable tothe difficulty in obtaining a soil solution that close-ly reflects the environment of plant roots. The con-ditions in rhizosphere soil can differ considerablyfrom those in non-rhizosphere soil (Marschner andRomheld, 1983). We have shown previously (Lorenzet al., 1994a) that the displacement method of Sanders(1982) is more suitable than soil centrifugation forobtaining rhizosphere solution. It is also important tolimit any external factors that may affect soil solutionproperties, in order to obtain soil solution that is morerepresentative of the whole period of plant growth. Forexample, we have shown that in pot experiments, fer-tilizer applications can significantly affect the concen-trations (Lorenz et al., 1994b) and speciation (Hamonet al., 1995) of Cd and Zn in soil solution, and thus theiruptake by plants. If these factors are controlled, Cd andZn uptakes from a single soil are correlated with thechanges in free ion activities in rhizosphere soil solu-tion (Hamon et al., 1995). The aim of this paper is toassess whether this relationship also applies in a morecomplex situation involving a range of soils that differwidely in important characteristics such as pH, clay-and organic matter-content, and in their sources andextent of contamination with Cd and Zn.

Materials and methods

Experimental design

Ten arable or grassland soils were collected in sixEuropean countries in 1992. All soils had long-termcontamination with heavy metals. The source of con-tamination for each soil is shown in Table 1. Soil from‘A’ horizons was sampled, sieved through a 3-mm mildsteel sieve and stored moist in plastic bags in the darkat 4 �C.

All plastic and glassware was soaked in 5% HNO3

and rinsed with deionized water before use. Waterholding capacities (WHCs) were determined using twopots of each soil. Approximately equal volumes ofmoist soil, each equivalent to either 750 g or 1 kg dryweight (DW), were placed in plant pots (13 cm diame-ter) with nylon mesh in their base. Next, the soil watercontent of six replicate pots of each soil was adjusted to

60% of WHC by watering from above with deionizedwater. The pots were then enclosed in loosely-sealedblack polyethylene (PE) bags in order to reduce evap-oration, and the soils were allowed to equilibrate fortwo weeks in a growth chamber (8 h night at 15 �C, 16h day at 20 �C, light supplied at 300 �mol m�2 s�1).Soil water content was adjusted every second day dur-ing this period. Radish (Raphanus sativus cv. ‘CrystalBall’) seeds were then sown in three pots of each soil.Six days later, the radish seedlings were thinned tothree per pot, and 70 g black PE beads was placed tocover the surface of each pot, around the seedlings, inorder to reduce water loss by evaporation. Pots werewatered daily, and later twice daily to maintain an aver-age moisture content of 60% WHC. Transpiration ratesof plants in each soil were calculated by deducting theaverage volume of water lost by evaporation from twounplanted pots from the volume of water lost fromplanted pots.

Nutrient requirements were established in prelimi-nary growth tests under conditions that were identicalexcept for the use of an excess of fertilizer. Hamon etal. (1995) showed that adding fertilizer in dilute solu-tions, according to the rate of transpiration, suppliessufficient nutrients to plants to meet their requirementat any particular stage of growth. Nutrients were pro-vided from day seven after sowing by adding 780 mgKNO3, 350 mg NH4NO3 and 150 mg MgSO4 � 7H2OL�1 with the water used to replenish that which hadbeen lost through transpiration. Nutrient solutions forplants grown on two P-deficient soils (La Union andCavaqueira) also contained 66 mg KH2PO4 and 56 mgNH4H2PO4 L�1.

Sampling

Soil solutions were obtained as described below fromthree unplanted pots of each soil after two weeks ofequilibration (“non-rhizosphere”), and from three fur-ther pots of each soil immediately after radish plantshad been harvested after 21 days of growth (“rhizo-sphere”). Plants were cut just above soil level. Leavesand swollen hypocotyls (“tubers”) were separated, andtheir respective fresh weights recorded. Next, all plantparts were rinsed thoroughly with deionized water,dried at 80 �C for 24 h, and their dry weights (DW)were recorded.

Soil solutions were obtained by displacement withwater (Sanders, 1982; Lorenz et al., 1994a). Sixteenhours before sampling soil solutions, soil water con-tents were raised to WHC, and pots were kept in closed,

plso6212.tex; 2/07/1997; 16:26; v.7; p.2

23

transparent PE bags in order to limit water loss throughtranspiration. Preliminary tests had shown that at least16 h incubation at WHC was required for the equilibra-tion of added water with soil solution. During the 16h incubation, redox potential decreased slightly fromaround 270 mV to 230 mV. This is still within the rangeassociated with aerobic soil conditions (i.e. > 200 mV)and did not alter the composition of soil solution sig-nificantly (data not shown).

For solution displacement, deionized water wasadded to the soil surface at a rate of 5 mL every 5 min.After discarding the first 5 mL of leachate, 40 mL ofsolution from each pot was collected into polystyrenecentrifuge bottles. Preliminary tests on all soils, usingCl� as a tracer, had shown that no breakthrough ofunequilibrated added water occurred until after 40 mLof solution had been collected.

Analyses

Analysis of soil chemical and physical parameters(except pH) was done on a subsample of each soilafter mixing, but before potting. In order to assurethe accuracy and repeatability of the results, dupli-cate digests and measurements were done on one insix samples. Soil particle size distribution was anal-ysed using the pipette method of Day (1965). ‘Avail-able’ P was extracted with 0.5 M NaHCO3 (Olsenet al., 1954), and determined colorimetrically. Organ-ic C was determined by dichromate oxidation (Walk-ley, 1947) and total N by titration of Kjeldahl digests(Bremner, 1965). Cation exchange capacity (CEC) andconcentrations of exchangeable cations were estimatedafter the method of Schollenberger and Simon (1945),and flame-atomic absorption spectrometry analysis(Flame-AAS, Perkin Elmer 5000). Major and heavymetal concentrations were determined using oven-dried, ground soils (Fritsch agate ball mill) by Flame-AAS after digestion in a mixture of HNO3, HClO4 andHF (Pratt, 1965). The pH of air-dried soils was mea-sured on all replicate pots after each soil solution sam-pling (soil : deionized water = 1 : 2.5 w/v) (RadiometerPHM 62).

Dried plants were ground in an agate ball milland digested in a 13 : 87 (v/v) mixture of concen-trated HClO4 (Fisons AR) and concentrated HNO3

(BDH Aristar), for determination of K, Ca, Mg andZn by inductively-coupled plasma atomic emissionspectrometry (ICP-AES; ARL 34000). Cd was deter-mined by graphite-furnace atomic absorption spec-trometry (GF-AAS; Perkin Elmer 5000, deuterium

background correction, HGA 400 graphite furnace)after solvent extraction of the digests in 1.0% sodiumdiethyldithiocarbamate trihydrate in 4-methylpentan-2-one (H Mosbæk, J -C Tjell and P E Holm, unpub-lished method). Total N in plants was determinedcolorimetrically after Kjeldahl digestion, using acontinuous-flow automatic analyser (Technicon AA2).

The pH of soil solutions obtained from two of thethree replicate pots was measured in a closed-chamberflow-cell before contact with the lower pCO2 of ambi-ent air could induce pH changes in the solution as theresult of the release of dissolved CO2: these valueswill be referred to as “flow-cell pH”. Soil solutionswere then centrifuged at 2000 � g for 30 min., thesupernatant decanted, and its pH measured again: thesevalues will be referred to as “final pH”. The concentra-tion of dissolved organic carbon (DOC) in centrifugedsoil solutions was determined using a DOC analyser(Dohrmann D-80). The concentrations of K, Ca, Mg,P and Mn in centrifuged solutions were determined byICP-AES. Those of total dissolved Cd and Zn weredetermined by GF-AAS after solvent extraction, asdescribed above.

Cd and Zn species in soil solutions were measuredusing the batch-column-batch-procedure developed forsmall samples of soil solution by Holm et al. (1995),although replicate samples still had to be bulked inorder to obtain enough solution for speciation analysis.Before speciation analysis, the pH of bulked solutionswas re-adjusted to the original flow-cell pH that hadbeen measured for one of the replicates, using 0.01MHNO3. Four fractions of Cd and Zn were determined:free divalent Cd2+ and Zn2+ (M2+), labile (Ml), slow-ly labile (Msl) and stable complexes (Mst). Apart fromM2+, these fractions are operationally defined, i.e. theydo not relate to specific solution complexes. M2+, Msl,and Mst fractions were obtained by ion-exchange pro-cedures, and Cd and Zn were determined using GF-AAS, after solvent extraction as described above. Theconcentration of M2+ was obtained after equilibrating25 mL of soil solution with 50 - 400 mg Amberlite resinfor 24 h, and by comparison with data from a refer-ence experiment involving identical solution pH, ionicstrength and amounts of Amberlite, but with knownactivities of Cd2+ and Zn2+ (see Holm et al., 1995).Msl was estimated after leaching 20 mL soil solutionthrough a column packed with 2 g Chelex 100 at therate of 2 mL min�1, and deducting the concentration ofMst. The Cd and Zn that remained dissolved after equi-libration of 10 mL soil solution with 100 mg Chelex for48 h were regarded as Mst. Ml was calculated by sub-

plso6212.tex; 2/07/1997; 16:26; v.7; p.3

24

Table 1. Source and concentrations of heavy metals in soils tested (mg kg�1 soil dry matter, except Fe g kg�1 soil dry matter)

Soil Source of contamination Cd Zn Pb Ni Cu Cr Mn Fe

Woburn, UKa 19 t ha�1 a�1 sewage sludge DW from 5.85 240 149 23.5 83.0 95.0 303 26.8

1942 - 1961

Bordeaux, Francea 10 t ha�1 a�1 sewage sludge DW since 1.70 320 65.0 11.7 26.0 14.7 470 5.8

1974

Bonn, FRGa 9.8 t ha�1 sewage sludge DW 0.85 340 148 37.0 54.0 80.0 850 32.5

biannually since 1959

Braunschweig, FRGa 16 t ha�1 a�1 sewage sludge DW from 1.50 247 76.0 23.0 52.3 59.3 557 13.1

1980 - 1990

Avonmouth, UK Zn smelter 1.17 370 165 38.0 56.7 50.3 1747 51.3

Arras, France Zn smelter 16.5 1317 879 16.3 34.0 55.3 367 24.4

Sala, Sweden Zn smelter 10.8 2000 1842 11.0 49.3 34.0 1803 52.3

Cavaqueira, Portugal Geogenic 0.20 397 56.0 16.0 20.0 35.7 5225 50.6

La Union, Spain Pb/Zn mine; river flood plains 11.0 5433 7490 31.0 95.3 67.3 2507 101

San Sebastian, Spain Pb/Zn mine; river flood plains 0.53 283 201 37.3 82.3 94.0 1297 55.5

aField experiments: IACR Rothamsted (Woburn), INRA (Bordeaux), University of Bonn (Meckenheim), Forschungsanstalt fur Landwirtschaft(Braunschweig).

Table 2. Physical and chemical characteristics of soils

Soil Soil texture Clay Org. C Tot. N Extr. P CEC Exch. Ca Exch. Mg Exch. K Exch. Na

(%) (g kg�1) (g kg�1) (mg kg�1) (mmolc kg�1)

Woburn Sandy loam 9.6 13.2 1.2 83.6 129 91.4 7.6 3.4 0.5

Bordeaux Loamy sand 4.6 15.6 1.3 71.2 63.8 48.5 4.3 1.9 0.3

Bonn Loam 14.5 16.8 2.1 61.0 192 170 4.0 4.7 1.3

Braunschweig Sandy loam 7.1 9.0 0.9 85.8 118 47.9 2.7 5.1 1.0

Avonmouth Loam 15.5 24.6 2.7 104 275 228 8.0 7.0 1.1

Arras Sandy loam 14.8 8.6 1.2 72.4 108 153 2.0 5.9 0.7

Sala Loam 15.2 51.3 5.1 139 561 438 57.5 5.4 0.7

Cavaqueira Loam 17.7 13.5 1.1 5.0 92.5 39.2 10.1 2.1 0.7

La Union Clay loam 32.6 6.7 0.8 9.2 208 159 27.5 8.2 7.1

San Sebastian Silty loam 24.3 29.7 2.7 82.4 249 212 22.9 1.9 1.1

Extr. – extractable, CEC – cation exchange capacity, Exch. – exchangeable.

tracting M2+, Msl, and Mst from the total concentrationin solution.

Where appropriate, data were subjected to ANO-VA and linear regression analysis, using GENSTAT 5Release 3.0 (Payne et al., 1993). Results of regressionanalyses with GENSTAT are expressed as the propor-tion of variance accounted for (adjusted R2), whichrelates the number of coefficients in the model to thenumber of observations (Lane et al., 1987).

Results and discussion

Soils

Most soils contained moderate concentrations of Cd,Zn and other heavy metals, although three soils (Arras,Sala and La Union) were highly contaminated with Cd,Zn and Pb (Table 1). The soils also varied widely intheir other chemical and physical properties, repre-senting a cross-section of soils typical for a range ofEuropean countries (Table 2).

plso6212.tex; 2/07/1997; 16:26; v.7; p.4

25

Plant growth

Dry weight of radish plants (leaves and tubers) rangedfrom 2.24 to 3.23 g per pot in most soils, but it was low-er (p < 0.001) in pots of Cavaqueira and La Union soils(1.32 and 1.38 g per pot). Although concentrations ofN and P in plants grown in these two soils were similarto those grown in the other soils, low concentrationsof P in soil solutions, especially from Cavaqueira soil,indicated that P deficiency may have persisted despitethe fertilizer amendments. Further, radishes grown inCavaqueira soil contained less K in leaves than didplants grown in the other soils (p < 0.001), even thoughonly 68% of the K fertilizer applied had been taken upinto leaves and tubers (data not shown). The growthretardation of radishes grown in La Union soil may alsohave been caused by a combination of relatively highconcentrations of Cd, Zn (Figure 1) and Na (720 mgL�1 compared to 3 to 49 mg L�1 in other soils) in soilsolution, although concentrations in the plants did notreach toxic levels for any element (see Table 3 for Cdand Zn, and 11 g Na kg�1 leaf DW). For example, Cdconcentrations in radishes from Arras soil were high-er than in radishes from La Union soil, without beingtoxic. Similarly, much higher concentrations of Na andZn than those in radishes from La Union soil have beenreported to be non-toxic elsewhere. For example, Mar-tin and Jones (1954) found no toxicity of Na at > 50 gNa kg�1 DM (Martin and Jones, 1954), and Schallerand Diez (1991) reported that concentrations of up toalmost 500 mg Zn kg�1 DM were not toxic either.

DOC

DOC concentrations were greater in rhizosphere solu-tions than in non-rhizosphere solutions of most of thesoils (p < 0.001, Table 4), presumably because of therelease of organic root exudates, and of the result-ing increase in microbial activity in the rhizosphere(Rovira and Davey, 1974). Furthermore, solubility ofhumic materials, and thus DOC, may have been greaterin rhizosphere solutions than in non-rhizosphere solu-tions because of their higher pH (Table 4) (McBride,1989) and lower ionic strength (Table 5) (Gobran andClegg, 1992). DOC tended to remain similar, or lower,in solutions from Cavaqueira and La Union soils, inwhich plant growth was poor, and changes in solutionpH and the concentrations of dissolved metals wererelatively small.

Table 3. Concentrations of Cd and Zn in radish leaves andtubers. Results are means of three replicates

Soil Cd (mg kg�1) Zn (mg kg�1)

Leaves Tubers Leaves Tubers

Woburn 2.7 0.9 90 70

Bordeaux 1.6 0.5 194 95

Bonn 0.7 0.3 53 38

Braunschweig 1.4 0.5 213 104

Avonmouth 0.4 0.2 41 32

Arras 30 4.8 340 117

Sala 5.9 1.0 351 125

Cavaqueira 0.7 0.3 57 50

La Union 14 2.6 446 107

San Sebastian 0.4 0.3 84 64

S.E.D. soil 1.83 0.17 24.1 11.1

pH

The flow-cell pH and final pH in soil solutions werehigher in samples from rhizosphere than from non-rhizosphere soils (p < 0.001) (Table 4). In contrast, pHin bulk soils was generally unaffected by plant growth,or the effect was much smaller than in soil solutions(Table 4). The increase in solution pH was probably theresult of the plants taking up N predominantly in theNO3-N form, and concurrent excretion of OH� ionsin order to maintain electrical neutrality within theirroots (Hedley et al., 1982). In the present experiment,flow-cell pH in solutions was always lower than bulksoil pH (p < 0.001) (Table 4), probably because bulksoil pH was measured in a more dilute salt matrix.Nevertheless, pH in non-rhizosphere and rhizospheresoils closely correlated with the flow-cell pH in theirrespective soil solutions (adj. R2= 0.903 and 0.873, fornon-rhizosphere and rhizosphere soils, respectively;p< 0.001; n = 10).

In the experiment described here, and in simi-lar experiments (Hamon et al., 1995; Lorenz et al.,1994a), final pH was consistently higher than flow-cell pH, particularly for solutions obtained from rhi-zosphere soils. This is because respiratory activity byplant roots and micro-organisms results in the accumu-lation in soil solution of HCO�

3 , which generates CO2

when exposed to ambient pCO2 (Appelo and Postma,1993). Hence, only the solution pH measured beforedegassing (i.e. the flow-cell pH) represents the condi-tion of the soil solution in situ.

plso6212.tex; 2/07/1997; 16:26; v.7; p.5

26

Table 4. Dissolved organic carbon (DOC) concentrations, pH measured before contact withair (flow-cell pH) and after equilibration in ambient air (final pH) in non-rhizosphere (NR)and rhizosphere (R) soil solutions, and pH in bulk soils. Data are means of three replicates

Soil DOC (mg L�1) Flow-cell pH Final pH Bulk soil pH

NR R NR R NR R NR R

Woburn 23.3 37.0 5.1 6.3 5.2 7.4 6.8 6.6

Bordeaux 78.3 86.7 5.0 6.3 5.2 7.0 6.3 6.4

Bonn 28.3 39.7 6.4 7.3 7.2 7.9 7.6 7.7

Braunschweig 38.7 40.0 4.7 6.3 4.8 7.1 6.1 6.2

Avonmouth 17.7 20.3 5.6 6.5 6.2 7.4 7.1 7.0

Arras 12.7 21.0 6.7 7.0 7.2 7.8 7.8 7.8

Sala 123 136 5.7 6.4 6.3 7.1 6.7 6.8

Cavaqueira 6.8 6.5 4.9 6.6 5.1 7.0 6.4 6.7

La Union 24.3 22.3 7.3 7.6 7.4 7.7 7.7 7.9

San Sebastian 13.3 23.3 4.4 5.7 4.6 6.5 5.8 5.7

Means 36.7 43.2 5.6 6.6 5.9 7.3 6.8 6.9

S.E.D. 2.03 0.03 0.04 0.02

sampling type

S.E.D. soil � 6.41 0.11 0.14 0.05

sampling type

Table 5. Potassium, calcium, magnesium, phosphorus and manganese concentrations in non-rhizosphere(NR) and rhizosphere (R) soil solutions. Data are means of three replicates

Soil Potassium Calcium Magnesium Phosphorus Manganese

Soil (mg L�1) (mg L�1) (mg L�1) (mg L�1) (�g L�1)

NR R NR R NR R NR R NR R

Woburn 33 1.8 328 74 33 7.5 4.8 1.9 58 15

Bordeaux 90 6.7 667 277 92 42 5.4 2.8 248 67

Bonn 33 9.0 388 356 29 27 0.7 0.3 4.5 2.5

Braunschweig 95 7.9 355 113 26 8.3 15 5.7 368 46

Avonmouth 43 4.2 392 262 55 39 1.8 0.7 24 7.6

Arras 63 18 487 381 17 12 0.8 0.3 bd bd

Sala 42 6.9 556 367 115 81 6.9 3.5 74 26

Cavaqueira 4.2 2.0 117 62 37 21 0.6 0.1 232 66

La Union 41 30 838 746 222 235 0.4 0.4 bd 11

San Sebastian 20 0.6 135 34 25 6.8 1.5 0.4 91 28

Means 46 8.8 426 267 65 48 3.8 1.5 110 27

S.E.D. 0.84 16.1 2.9 0.13 9.58

sampling type

S.E.D. soil� 2.66 50.8 9.1 0.41 30.3

sample type

bd = below limit of detection.

plso6212.tex; 2/07/1997; 16:26; v.7; p.6

27

Figure 1. Speciation of (a) Cd and (b) Zn in soil solutions from non-rhizosphere (1st bar) and from rhizosphere soils (2nd bar). Results of totalconcentrations are means from three replicate pots. Metal speciation was done using solution obtained by bulking replicate samples.

Elements in soil solution

Concentrations of K, Ca, Mg, P, Mn, Cd and Zn weregenerally lower (p < 0.001) in rhizosphere than in non-rhizosphere solutions, with exceptions in some soils(Table 5 and Figure 1). The differences were general-ly greatest for K and Zn, except in Cavaqueira and LaUnions soils, in which metal concentrations in solutiondecreased less during plant growth. This suggests thatnutrient uptake by plants, and the consequent changesin the rhizosphere, were important factors in determin-ing element concentrations in soil solution. Concen-trations of Cd in non-rhizosphere solutions correlated(p < 0.01) with Cd concentrations in bulk soils, butZn concentrations in soils and solutions did not cor-relate (data not shown). This may have been due todifferences in the relative solubility between the twoelements and among soils. Calculation of the ratio (ordistribution constant Kd) of total Zn in soil (mg kg�1)to Zn in non-rhizosphere solutions (mg L�1) showedgreat variability among the soils (Kd 69 to 36222 Lkg�1) (calculated from Figure1b, Table 1). In compar-ison, solubility of Cd varied less among the soils (Kd

156 to 2069 L kg�1) (calculated from Figure 1a, Table1).

Speciation of Cd and Zn in soil solutions

The majority of the Cd and Zn in soil solutionsfrom both, non-rhizosphere and rhizosphere soils waspresent in the ionic Cd2+ and Zn2+ forms (Figure 1).Complexes of Cd and Zn were labile (Ml) (Figure1).Slowly labile (Msl) and stable (Mst) complexes werealways close to, or below, the detection limits (0.1�g Cd L�1 and 1 �g Zn L�1). Since translocation ofCd and Zn in soils occurs mainly in stable complexes(Christensen, 1989), these results confirm the commonobservation that leaching is minimal (e.g. McGrath andLane, 1989). Our findings on Cd and Zn speciation insoil solutions also correspond broadly to those calculat-ed by other workers, using the programme GEOCHEM(e.g. Mahler et al., 1980).

These findings contrast with an earlier study, inwhich we found that the amount of complexed Cdand Zn in solution can exceed the free concentrationsof these metals due to an increase in DOC and low

plso6212.tex; 2/07/1997; 16:26; v.7; p.7

28

concentrations af competing cations in solution, suchas Ca and Mg (Hamon et al., 1995). Concentrationsof competing cations remained relatively high in thepresent study, and therefore prevented significant com-plexation of Cd and Zn. However, increasing concen-trations of DOC during plant growth were correlatedwith a decrease in the proportions of Cd2+ and Zn2+

in soil solution (adj. R2 = 0.469 and 0.355 for Cd andZn, respectively; p< 0.05; n = 10) (data not shown).

Assessment of Cd and Zn availability

Cadmium concentrations in radish leaves and tuberswere best predicted by total or free Cd concentrationsin rhizosphere solutions, but were also well-predictedby Cd concentrations in bulk soils (soil Cd) (Table6). The closeness-of-fit of all relationships was similarfor leaves and tubers. The correlation between Cd inplants and in bulk soils was not improved when soil pHwas included as an additional parameter in a multipleregression analysis. This suggests that soil pH was notthe main determinant of Cd solubility and availabilityin the soils tested. The relationship between Cd inplants and in soils or soil solutions was influenced bythe leverage caused by the highly contaminated Arrasand La Union soils (Table 1), although correlationsremained significant when data from these two soilswere excluded (p < 0.01; n = 8; results not shown). Cdconcentrations in non-rhizosphere solutions correlatedpoorly at best with Cd concentrations in leaves andtubers (Table 6).

In contrast to Cd, Zn concentrations in leaves andtubers were not closely predicted by individual soil orsolution parameters. Zn concentrations in leaves cor-related with those in bulk soils (soil Zn), but not withtotal or free Zn concentrations in rhizosphere or non-rhizosphere solutions (Table 6). In contrast, there wasa weak but significant correlation between Zn in tubersand total and free Zn in rhizosphere solutions; butnot with soil Zn (Table 6). We therefore investigatedwhether Zn concentrations in leaves and tubers werealso influenced by (1) differences in the rate of releaseof Zn into solution from soil colloids; (2) control ofuptake by the plants; and/or (3) interacting ions in soilsolutions.

Differences among soils in the rate of release ofZn into solution may have affected Zn uptake, becauseof the great variability in the solubility of Zn in thesesoils, as discussed above. This was suggested by thefact that Zn concentrations in leaves and tubers werebest predicted by a multiple linear regression equa-

tion including Zn in rhizosphere solutions and in bulksoils (adj. R2 = 0.851 and 0.704 for leaves and tubers,respectively; p < 0.01; n = 10). Therefore, Zn concen-trations in the plants growing in soils with high soilZn concentrations were higher than predicted from Znconcentrations in solution alone. Similarly, Tiller et al.(1972) suggested that quantity/intensity relationships(i.e. rates of desorption) determined the availabilty ofZn in soils. More recently, in a study of Ni uptakefrom three soils, Dunemann et al. (1991) concludedthat the total amount in the soil, and the rate of releaseinto solution should be considered along with solutionconcentrations when assessing the potential uptake ofNi by plants.

In addition, the amount of Zn taken up may alsohave been controlled partly by the plants themselves.This possibility was investigated by calculating theratios (R) of mean concentrations of Cd or Zn in leavesand tubers versus rhizosphere soil solution concentra-tions. This calculation was made without data fromCavaqueira soil, which had very large RCd and RZn

values due to very low Cd and Zn concentrations insoil solution. With respect to the remaining 9 soils,there was no relationship between RCd and total Cd inrhizosphere soil solution (Figure 2a). In contrast, RZn

values were higher at low Zn concentrations in soilsolution (Figure 2b). The results suggest that uptakeof Zn, an element that is essential for plant growth,may be more closely controlled than that of the non-essential Cd.

Finally, we investigated how interacting ions insolutions affected Zn uptake by the plants, using mul-tiple regression analysis. Ion interactions may occurbetween the two metals, with other trace metals (e.g.Mn, Fe) or with major elements (Ca, K, Mg) in solution(Chaudhry and Loneragan, 1972a, 1972b; Hardimanand Jacoby, 1984). Interactions are often competitive,so they decrease uptake of Cd and Zn. However, therewere no apparent effects of interactions among ions(Ca, K, Mg, Mn, Cd) in solution on Zn uptake in thepresent study (data not shown).

Total Cd concentrations in rhizosphere solutionspredicted plant Cd concentrations slightly better thandid Cd2+ concentrations alone, and total concentra-tions of Zn in solution predicted Zn in plants as poorlyas did Zn2+ concentrations (Table 6). This finding con-flicts with those from other studies, for plants grownin solution culture (Cabrera et al., 1988; Checkai etal., 1987), which have suggested that only M2+ ions(and not solution complexes) are absorbed by plants.Some work on heavy metal uptake from soil has also

plso6212.tex; 2/07/1997; 16:26; v.7; p.8

29

Table 6. Linear relationships (adj. R2) for Cd and Zn between concentrations in radish and soil Cd and Zn or total orfree Cd and Zn in non-rhizosphere (NR) and rhizosphere (R) soil solutions. Correlations are based on means of threereplicates from 10 soils

Soil Solution Cd Solution Cd (R) Soil Solution Zn Solution Zn

Cd (NR) Zn (NR) (R)

Total Free Total Free Total Free Total Free

Leaf 0.79*** 0.47* 0.24 0.98*** 0.90*** 0.61** 0.00 0.00 0.25 0.25

Tuber 0.80*** 0.51** 0.28 0.98*** 0.92*** 0.15 0.07 0.07 0.53** 0.55**

*p < 0.05, **p< 0.01, ***p< 0.001.

Figure 2. Relationship between R (the ratio of Cd or Zn concentrations in plants over Cd or Zn in rhizosphere soil solutions) and theirconcentrations in rhizosphere soil solutions, for (a) Cd and (b) Zn. Data for Cavaqueira soil are excluded (for explanation see text). Data aremeans of three replicates.

indicated that the closest relationship exists betweenplant metal concentrations and those of M2+ (Binghamet al., 1984), but our findings are supported by otherwork involving either estimation of ion speciation fromcomputer calculations (Sposito and Bingham, 1981) ordirect experimental measurement (El-Kherbawy andSanders, 1984). The failure of many experiments withsoil to demonstrate the importance of solution Cd2+

or Zn2+ concentrations for their respective uptake intoplants suggests that complexed Cd and Zn may also beabsorbed by plants. However, this failure could alsohave been due to other factors. In some publications,uncontrolled changes in metal speciation may haveoccurred before analysis because of the increase in pHthat occurs when solutions are exposed to ambient air.In our experiment, we attempted to anticipate thesepossible changes by re-adjusting the solution pH tothat measured in the flow-cell before speciation anal-ysis. Finally, it is also possible that Cd2+and Zn2+ insoil solution are replenished from Cd and Zn complex-es in soil solution, and also partly from exchangeably-adsorbed Cd and Zn, as soon as they are taken upby plants, so that a near constant ratio of total and free

metals in solution is maintained. In this case, the wholepool of Cd and Zn in soil solution should be consid-ered as being bio-available, although it is still possiblethat only the free ion is taken up. This suggests thatspeciation analysis of Cd and Zn may be of little prac-tical importance in assessing Cd and Zn availability toplants.

In conclusion, the experiment demonstrated thatstudies of the potential bio-availability of heavy met-als should consider the effect of the rhizosphere onsoil solution. This is because soil solution propertieschange during rhizosphere development,and the extentof this change varies greatly among soils. Accordingly,concentrations of Cd in plants were best predicted byCd concentrations in rhizosphere soil solutions. How-ever, Zn uptake also seemed to be affected by factorsother than Zn concentrations in soil solution, includ-ing the large differences in the solubility of Zn amongsoils, and, possibly, the ability of the plants partly tocontrol Zn uptake. There has been little previous pub-lished work on the mechanisms involved in Zn and Cdabsorption, and more research is needed to investigatethese.

plso6212.tex; 2/07/1997; 16:26; v.7; p.9

30

Acknowledgements

This work was partly funded by the Commission of theEuropean Communities Science and Technology forEnvironmental Protection programme. IACR is grantaided by the UK Biotechnology and Biology ScienceResearch Council. We thank A Crosland and J Futtrupfor the analysis of soil solutions and plants. We alsothank all those who provided us with soil samples,in particular Dr X Ansorena (San Sebastian), Dr JCegarra (La Union), Dr C Leyval (Arras), Dr M Mench(Bordeaux), Dr E Rietz (Braunschweig), Prof Dr DrW Werner (Bonn) and Dr E Witter (Sala).

References

Appelo C A J and Postma D 1993 Geochemistry, Groundwater andPollution. A A Balkema, Rotterdam. 536 p.

Bingham F T, Sposito G and Strong J E 1984 The effect of chlorideon the availability of cadmium. J. Environ. Qual. 13, 71–74.

Bremner J M 1965 Total Nitrogen. In Methods of Soil Analysis. Part2: Chemical and Microbiological Properties. Eds. C A Black,D D Evans, Y L White, L E Susminger and F E Clark. pp1149–1178. American Society of Agronomy, Madison, WI.

Cabrera D, Young S D and Rowell D L 1988 The toxicity of cadmiumto barley plants as affected by complex formation with humicacid. Plant Soil 105, 195–204.

Chaudhry F M and Loneragan J F 1972a Zinc absorption by wheatseedlings: I. Inhibition by macronutrient ions in short-termexperiments and its relevance to long-term zinc nutrition. SoilSci. Soc. Am. Proc. 36, 323–327.

Chaudhry F M and Loneragan J F 1972b Zinc absorption by wheatseedlings: II. Inhibition by hydrogen ions and by micronutrientcations. Soil Sci. Soc. Am. Proc. 36, 327–331.

Checkai R T, Corey R B and Helmke P A 1987 Effects of ionic andcomplexed metal concentrations on plant uptake of cadmiumand micronutrient metals from solutions. Plant Soil 99, 335–345.

Christensen T H 1989 Cadmium soil sorption at low concentrations.VII: Effect of stable solid waste leachate complexes. Water AirSoil Pollut. 44, 43–56.

Day P R 1965 Particle fractionation and particle-size analysis. InMethods of Soil Analysis. Part 1: Physical and mineralogicalproperties, including statistics of measurement and sampling.Eds. C A Black, D D Evans, L E White, F E Clark and R C Din-auer. pp 545–567. American Society of Agronomy, Madison,WI.

Dunemann L, Wiren N von, Schulz R and Marschner H 1991 Spe-ciation analysis of nickel in soil solutions and availability to oatplants. Plant Soil 133, 263–269.

El-Kherbawy M I and Sanders J R 1984 Effect of pH and phosphatestatus of a silty clay loam on manganese, zinc and copper con-centrations in soil fractions and in clover. J. Sci. Food Agric. 35,733–739.

Gobran G R and Clegg S 1992 Relationship between total dissolvedorganic carbon and SO2�

4 in soil and waters. Sci. Total Environ.117/118, 449–461.

Hardiman R T and Jacoby B 1984 Absorption and translocationof Cd in bush beans (Phaseolus vulgaris). Physiol. Plant. 61,670–674.

Hamon R E, Lorenz S E, Holm P E, Christensen T H and McGrathS P 1995 Changes in trace metal species and other componentsof the rhizosphere during growth of radish. Plant Cell Environ.18, 749–756.

Hedley M J, Nye P H and White R E 1982 Plant-induced changes inthe rhizosphere of rape (Brassica napus var. Emerald) seedlings.II. Origin of the pH change. New Phytol. 91, 31–44.

Holm P E, Christensen T H, Tjell J C and McGrath S P 1995 Specia-tion of Cd and Zn with application to soil solutions. J. Environ.Qual. 24, 183–190.

Kasawneh F E 1971 Solution ion activity and plant growth. Soil Sci.Soc. Am. Proc. 35, 426–436.

Lane P, Galway N and Alvey N 1987 Genstat 5 - An Introduction.Clarendon Press, Oxford. 163 p.

Laurie S H, Tancock N P, McGrath S P and Sanders J R 1991Influence of complexation on the uptake by plants of iron, man-ganese, copper and zinc. III. Effect of DTPA in a multi-metaland computer simulation study. J. Exp. Bot. 42, 514–519.

Lorenz S E, Hamon R E and McGrath S P 1994a Differences betweensoil solutions obtained from rhizosphere and non-rhizospheresoils by water displacement and soil centrifugation. Eur. J. SoilSci. 45, 431–438.

Lorenz S E, Hamon R E, McGrath S P, Holm P E and Christensen TH 1994b Applications of fertilizer cations affect cadmium andzinc concentrations in soil solutions and uptake by plants. Eur.J. Soil Sci. 45, 159–165.

Mahler R J, Bingham F T, Sposito G and Page A L 1980 Cadmium-enriched sewage sludge application to acid and calcareous soils:relation between treatment, cadmium in saturation extracts, andcadmium uptake. J. Environ. Qual. 9, 359–364.

Martin J P and Jones W W 1954 Greenhouse plant response to vinylacetate-maleic acid copolymer in natural soils and in preparedsoils containing high percentages of sodium and potassium. SoilSci. 78, 317–324.

Marschner H and Romheld V 1983 In vivo measurement of root-induced pH changes at the soil-root interface: effect of plantspecies and nitrogen source. Z. Pflanzenphysiol. 111, 241–251.

McBride M B 1989 Reactions controlling heavy metal solubility insoils. Adv. Soil Sci. 10, 1–56.

McGrath S P and Lane P W 1989 An explanation for the apparentlosses of metals in a long-term field experiment with sewagesludge. Environ. Pollut. 60, 235–256.

Olsen R A and Peech M 1960 The significance of the suspensioneffect in the uptake of cations by plants from soil-water systems.Soil Sci. Soc. Am. Proc. 24, 257–260.

Olsen S R, Cole C V and Adams S N 1954 Estimation of availablephosphorus in soils by extraction with sodium bicarbonate. USCirc. 939.

Payne R W, Lane P W, Todd A D, Digby P G N, Thompson R,Harding S A, Tunnicliffe-Wilson G, Leech P K, Welham S J,Morgan G W and White R P 1993 GENSTAT 5 Release 3Reference Manual. Clarendon Press, Oxford. 796 p.

Pratt P F 1965 Digestion with hydrofluoric and perchloric acids fortotal potassium and sodium. In Methods of Soil Analysis: Part I:Chemical and Microbiological Properties. Eds. C A Black, D DEvans, J L White, L E Ensminger, F E Clark and R C Dinauer.pp 1019–1021. American Society of Agronomy, Madison, WI.

Rovira A D and Davey C B 1974 Biology of the rhizosphere. InThe Plant Root and its Environment. Eds. J L Harley and R SRussel. pp 153–204. Proceedings, Virginia Polytechnic Instituteand University. University Press, Virginia.

plso6212.tex; 2/07/1997; 16:26; v.7; p.10

31

Sanders J R 1982 The effects of pH upon the copper and cupric ionconcentrations in soil solution. J. Soil Sci. 33, 679–689.

Schaller A and Diez T 1991 Pflanzenspezifische Aspekte derSchwermetallaufnahme und Vergleich mit den Richt- undGrenzwerten fur Lebens- und Futtermittel. In Auswirkun-gen von Siedlungsabfallen auf Boden, Bodenorganismen undPflanzen. Eds. D Sauerbeck and S Lubben. pp 92–125.Forschungszentrum Julich GmbH, Berichte Okol. Forsch. 6.

Schollenberger C J and Simon R H 1945 Determination of exchangecapacity and exchangeable bases in soil - ammonium acetatemethod. Soil Sci. 59, 13–24.

Sposito G and Bingham F T 1981 Computer modeling of trace metalspeciation in soil solutions: correlation with trace metal uptakeby higher plants. J. Plant Nutr. 3, 35–39.

Tiller K G , Honeysett J L and De Vries P C 1972 Soil zinc and itsuptake by plants. II. Soil chemistry in relation to prediction ofavailability. Aust. J. Soil Res. 10, 165–182.

Walkley A 1947 A critical examination of a rapid method for deter-mining organic carbon in soils - effect of variations in digestionconditions and of organic soil constituents. Soil Sci. 63, 251–264.

Whitney M and Cameron F K 1903 The chemistry of the soil as relat-ed to crop production. US Department of Agriculture, BureauSoils -Bull. 22.

Section editor: L V Kochian

plso6212.tex; 2/07/1997; 16:26; v.7; p.11