Heavy Metals in the Environment

A Biotest for Evaluating Copper Bioavailability to Plants in a Contaminated Soil

V. Chaignon and P. Hinsinger* ABSTRACT
This work aimed at defining the optimal conditions for a novel ecotoxicological test designed for evaluating the bioavailability and phytotoxicity of metals to plants. This biotest, which provided easy access to roots, shoots, and rhizosphere soil, was applied to a vineyard calcareous soil that had been contaminated by the application of Cu fungicides. A preliminary hydroponic experiment comparing various levels of solution Cu concentration enabled us to determine the no observable adverse effects concentration (NOAEC), which was in the range 5 to 20 M total Cu (0.010.06 M free Cu ion) for rape (Brassica napus L. cv. Goeland). For the biotest, rape was grown in hydroponic conditions for 21 d in pots designed so that plants developed a planar mat of roots at the surface of a polyamide mesh. By then, the plants were transferred for 4 or 8 d onto a 1- or 3-mm-thick soil layer that was separated from the root mat by the mesh and connected to a reservoir of nutrient solution or deionized water via a filter paper wick. An 8-d period was the best option as it enabled plant growth to be significant. The use of 1-mm soil thickness was recommended if the biotest aimed at investigating root-induced changes in the rhizosphere. Although it may cause some artifacts, compared with deionized water, nutrient solution provided better standardized conditions for comparing widely differing soil samples. The studied soil did not induce any Cu phytotoxicity in spite of its fairly large total Cu content.

he contamination of agricultural land can arise from various origins such as the use of fertilizers and pesticides, the application of sewage sludge, the disposal of industrial wastes or the deposition of atmospheric contaminants (Alloway, 1995). In all cases, the potential risks for terrestrial and aquatic ecosystems need to be estimated. Although this problem is of increasing concern, it is surprising that, compared with aquatic ecosystems, risk evaluation of terrestrial ecosystems still relies on only a few standardized ecotoxicological tests. Short-term plant toxicity tests have originally been developed from simple measurements used in plant physiology and weed science (Cushman and Meyer, 1990). The two short-term phytotoxicity tests that involve terrestrial plants and are currently used are (i) the seed germination test and (ii) the root elongation test (Kapustka, 1997; International Organization for Standardization, 1999). The seed germination test is relatively insensitive to many toxic substances: many of these are not readily taken up into the plants because at this stage, the seedlings derive their nutrients mostly from seed reserves rather than from the soil. This test thus provides a poor indication of soil contamination,
` ENSA.M-INRA, UMR Rhizosphere & Symbiose, Place Viala, F-34060 Montpellier Cedex 1, France. Received 12 Apr. 2001. *Corresponding author (philippe.hinsinger@ensam.inra.fr). Published in J. Environ. Qual. 32:824833 (2003).

while the root elongation assay provides a more sensitive measure of phytotoxic effects (Kapustka, 1997). However, the root elongation test is conducted in quartz sand with soil eluates (water extracts) and is therefore quite far from realistic exposure conditions. In comparison with these two standardized tests, the early seedling growth test (American Society for Testing and Materials, 1994) and related growth tests based on pot experiments (International Organization for Standardization, 1999) are closer to real-world conditions of exposure (Kapustka, 1997). In addition to their duration and cost, the major drawbacks of these tests are that the exposure conditions, relevant endpoints, interspecies extrapolation, and lab-to-field extrapolation are not clearly defined (American Society for Testing and Materials, 1994; Kapustka, 1997; International Organization for Standardization, 1999). Standardized plant tests (as early seedling growth tests) that are published in the literature dealing with phytotoxicity assessment authorize modifying the test procedure to make the test more relevant (American Society for Testing and Materials, 1994). However, in all cases, only aerial parts of plants are considered while root contamination and bioaccumulation or bioavailability and speciation of the metal in the soil are disregarded. The fraction of soil metal that can be taken up by plants (i.e., the bioavailable fraction) (Singh and Rakipov, 1988) is most often estimated with chemical extractions that are routinely used in soil testing (Lebourg et al., 1996). Many chemical extractants (CaCl2 and other dilute salts, DTPA [diethylene triaminepentaacetic acid] and EDTA [ethylenediaminetetraacetic acid]) have been used to identify the portion of metals in the soil that is the most readily available (Beckett, 1989; Lebourg et al., 1996). Such chemical extractions can hardly account for the various, complex processes that are involved in the acquisition of metals by plants (Marschner, 1995; McLaughlin et al., 1998; Hinsinger, 2001), and are thus not satisfactory for estimating soil metal bioavailability to plants. Biotests conducted with plants provide an alternative approach to estimate metal bioavailability in contaminated soils. However, the biotests that have been used by various authors for this purpose are based on pot experiments, for which conditions vary considerably. Being poorly standardized, they cannot easily be compared with one another. The present work is focused on the case of a vineyard soil that has been contaminated by the long-term field application of copper salts (Bordeaux mixture) as fungiAbbreviations: DM, dry matter; DTPA, diethylene triaminepentaacetic acid; EDTA, ethylenediaminetetraacetic acid; NOAEC, no observable adverse effects concentration.

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cides against powdery mildew. For most plant species, the critical toxicity level of copper in the leaves is 20 to 30 mg kg 1 dry matter (Reuter and Robinson, 1997). This criterion is, however, not a sufficient indication of the copper toxicity and bioavailability for the plant (Mitchell et al., 1978; Lexmond, 1980; Brun et al., 2001). Indeed, a large copper supply can inhibit root growth before shoot growth (Marschner, 1995) and Cu can accumulate in the roots without any significant increase in the Cu content of the aerial parts (Mitchell et al., 1978; Brun et al., 2001). Therefore, a proper biotest should provide easy access not only to the shoots, but also to the roots to evaluate Cu contamination of plants (Brun et al., 2001). But when plants are grown in soils, especially the plants with fine roots (Brun et al., 2001), root collection is easily biased and tedious. Indeed, at harvest, small amounts of soil can adhere to roots, resulting in overestimates of root Cu content (Brun et al., 2001), and the finest roots are often discarded from the analysis because of their difficult and almost impossible recovery. The aim of this work was to define the optimal conditions (time duration, amount of soil, and nutrients supplied) for a novel biotest that provides an easy access to roots, shoots, and rhizosphere soil to evaluate the bioavailability and phytotoxicity of Cu to oilseed rape plantlets. A preliminary biotest was performed in nutrient solution over a range of Cu concentrations to evaluate the sensitivity of rape roots and shoots to Cu toxicity. MATERIALS AND METHODS General Principles of the Proposed Biotest
The cropping device that was used for the present biotest was adapted from that of Guivarch et al. (1999), which evolved from those designed by Kuchenbuch and Jungk (1982) and Niebes et al. (1993). The major advantage of this technique is that roots are physically separated from the soil, which enables an easy recovery of the shoots, roots, and soil. Another advantage of this device is that the thickness of the soil layer used and, hence, the distance from the roots in the soil compartment can be as little as a few millimeters. It can thereby be considered as rhizosphere soil and provide enough rhizosphere material to evaluate root-induced changes in metal speciation. In that respect, the proposed biotest is just a simplified version of the root mat technique that has been extensively used in rhizosphere research over the last decades (Jaillard et al., 2003). Despite the physical separation between roots and soil, intense chemical interactions can occur (e.g., reviews by Hinsinger, 1998; Jaillard et al., 2003) and plants can thereby successfully take up nutrients (e.g., Kuchenbuch and Jungk, 1982; Niebes et al., 1993; Morel and Hinsinger, 1999) and pollutants (Guivarch et al., 1999; Kruyts et al., 2000) from the soil, at fairly large fluxes. This root mat approach has also been successfully used to study the toxicity of Al in the rhizosphere of maize (Zea mays L.) (Calba et al., 1996). The proposed biotest is a two-step procedure: in the first step, plants are grown for three weeks in hydroponic conditions (preculture) and in the second step, they are transferred onto the soil for another few days of growth (soil experiment). In a preliminary experiment, preculture plants were grown for another two weeks in hydroponic conditions with various levels of Cu (hydroponic experiment).

Hydroponic Preculture
The plant containers were prepared as follows: a polyamide grid (900- m pore diameter) was pasted at the bottom side of a PVC cylinder (25-mm i.d.). Another PVC cylinder (32-mm i.d.) was closed by a fine polyamide mesh (30- m pore diameter, Fyltis/Nytel 03-30/18; Sefar Filtration, Ruschlikon, Swit zerland) at the bottom side. The small cylinder was inserted into the large one. A space of 3 mm was left between the grid and the mesh so that roots could develop as a planar mat (roughly 3-mm-thick disk with a 32-mm diameter) in between. Seeds of oilseed rape (Brassica napus L. cv. Goeland) were surface-sterilized with 6% H2O2 for 10 min and rinsed with deionized water. Ten seeds were sown in each container onto the surface of the grid. Plants were grown in hydroponic conditions for 21 d to obtain a large planar mat of roots that fully covered the polyamide mesh. Ten containers were placed on top of a 6-L bucket, which contained an aerated nutrient solution (Fig. 1). A daily supply of nutrient solution was necessary to constantly wet the mesh during the 21-d preculture period. During the first week, plants were supplied with 600 M CaCl2 and 2 M H3BO3. The buckets were then filled for two weeks with a complete nutrient solution of following composition: 2 mM KNO3, 2 mM Ca(NO3)2, 1 mM MgSO4, 0.5 mM KH2PO4, 0.1 mM FeNaEDTA, 10 M H3BO3, 2 M MnCl2, 1 M ZnSO4, 0.05 M NaMoO4, and 0.2 M CuCl2. The nutrient solution initially was pH 5.5 and was renewed weekly to avoid large pH changes and nutrient depletion. The experiment was conducted in a growth chamber with the following daynight conditions: 16 h, 25 C, relative humidity 75%, and a light intensity of about 500 mol photons m 2 s 1 (in the range 400700 nm) during the day, and 8 h, 20 C, and relative humidity of 100% at night. After 21 d, the containers (taken from all the buckets) were ranked according to the size of the plants and those containing the smallest and tallest plants were discarded. Groups of five replicates were then selected randomly among the leftover containers that contained plants with homogeneous sizes. At this stage (i.e., at 0 d), a group of five replicates was harvested to determine the biomass and Cu concentration of shoots and roots. The

Fig. 1. Cropping device used (i) during the first step of the biotest (hydroponic preculture period) to obtain a dense mat of roots and (ii) during the hydroponic experiment.

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analyses of these containers provided references (starting point or control) for either the hydroponic or soil experiment. The other containers with 3-wk-old rape plants were then used for either the hydroponic or soil experiment.

Hydroponic ExperimentBiotest for Assessing Copper Phytotoxicity

For the purpose of this experiment, containers with 21d-old plants were grown in the same design and conditions as in the preculture period for another two weeks during which plants were exposed to seven different levels of Cu concentration (supplied as CuCl2). Five containers (replicates) were placed on top of a 6-L bucket, which contained the same nutrient solution as above (renewed every third day) with one of the following Cu concentrations: 0.2, 5, 20, 80, 100, 120, and 200 M. Free Cu ion concentrations were estimated with the SOILCHEM computer code (Sposito and Coves, 1988). Because of the supply of Fe as EDTA and the strong affinity of EDTA for Cu, a major proportion of solution Cu was present as Cu-EDTA and free Cu ion represented only 0.2, 0.25, 0.3, 1.3, 5, 16, and 22%, respectively, of the total solution Cu applied. The pH of the nutrient solutions ranged between 4.5 and 5.5.

Soil ExperimentBiotest for Assessing Soil Copper Bioavailability

This experiment corresponds to the second step of the biotest during which 21-d-old plants (preculture) were transferred onto a contaminated soil for estimating the bioavailability and phytotoxicity of soil Cu. One day before being placed in contact with the plants for either 4 or 8 d, soil containers were prepared as follows: the lower part consisted of a 3-mmthick PVC plate (7 by 5 cm) while the upper part was either a 1.1- or 3-mm-thick PVC plate that had the same dimensions and a central hole with a 32-mm i.d. An ashless filter paper (#40; Whatman, Maidstone, UK) was inserted between the two plates and connected to a solution reservoir to function as a wick (Fig. 2). Before being poured in the soil containers, the soil had been incubated for 6 d in the growth chamber by

adding 44 mL of solution to 200 g air-dried soil in a polyethylene bag. Two different bags were prepared: one with deionized water, the other one with a nutrient solution that had the same composition as the above-mentioned complete solution minus CuCl2 and FeNaEDTA. The FeNaEDTA was omitted, as EDTA might have affected the speciation of Cu in the soil. Copper was not supplied so that soil Cu was the only source of Cu for the plants at this stage of the biotest. The assembled soil container was then filled with either 2.4 or 4.4 g of incubated, wet soil (treatments called 1 or 3 mm, respectively) and left for incubating for another day in the growth chamber. They were covered with an aluminium foil to prevent their dehydration and the development of algae. When filling the soil containers, some extra soil was added so that the soil layer was actually thicker than the hollow PVC plate, to ensure a good contact with the plant container at the last stage of the biotest. This means that the actual thickness of the soil layers was close to 1.5 to 2 and 3.5 to 4 mm. Nevertheless, these two treatments will thereafter be called 1 mm and 3 mm, respectively, which are the dimensions of the corresponding soil containers. The studied soil was a calcareous soil (calcaric cambisol, pHwater 8.1, and 87 g CaCO3 kg 1 soil) that had been sampled in February 1999 in a vineyard from a small agricultural watershed at Roujan (southern France, 60 km west of Montpellier, 43 30 N, 3 19 E). It was sampled from the topsoil (02 cm), air-dried, and sieved to 2 mm before being analyzed by a routine soil testing laboratory (INRA Laboratoire dAnalyse des Sols Arras, France), according to French standardized procedures (Association Francaise de Normalisation, 1994). Its sand and clay contents (determined by sieving and the pipette method; Procedure NF X 31-107; Association Francaise de Normalisation, 1994) amounted to 419 and 204 g kg 1, respectively. Its cation exchange capacity (determined by the Metson method [ammonium acetate]; Procedure NF X 31130; Association Francaise de Normalisation, 1994) was 11 cmolc kg 1. The total copper content (determined by HF HClO4 extraction; Procedure NF X 31-151; Association Francaise de Normalisation, 1994) amounted to 398 mg kg 1. The soil contained 175 mg kg 1 of EDTA-extractable Cu (determined according to Procedure NF X 31-120; Association Francaise de Normalisation, 1994). The design of this experiment consisted of two time durations (4 and 8 d) two soil thicknesses (1 and 3 mm) two nutrient treatments (nutrient solution and deionized water). For each of these eight treatments, five replicates were prepared by placing five containers with preculture plants onto five soil containers. Five additional soil containers were prepared for each treatment, covered with aluminium foil, and incubated in the same conditions as for the soil experiment (same procedure as above but without plants) to serve as control soils, to evaluate the root-induced changes occurring in the rhizosphere soil. In this experiment, as in the hydroponic experiment, the five replicates were not fully independent, as they were sharing the same reservoir of nutrient solution. As this was large compared with their requirements, negligible interactions between the replicates were expected and the replicates were assumed to be independent when conducting the statistical tests.

Plant and Soil Analyses

The roots and shoots were harvested at the end of the hydroponic preculture period (0 d), and after 14 d of hydroponic culture (in the hydroponic experiment) or after 4 and 8 d of soilplant contact (in the soil experiment). For each treatment, the five replicates were collected and analyzed

Fig. 2. Cropping device used during the second step of the biotest, that is, soil experiment with plants being grown on top of a thin layer of soil.

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separately. They were weighed fresh; shoots were oven-dried at 105 C and roots were frozen at 20 C. Copper bound to the outer root cell walls (apoplastic Cu) was determined as follows: a subsample of 0.8 g of fresh roots (after thawing) was shaken end over end with 40 mL of 0.001 M HCl during 3 min; then 360 L of 1 M HCl were added to yield a final concentration of 0.01 M HCl. After shaking for another 5 min, the suspensions were filtered with an ashless filter paper (Whatman 40) and Cu was assayed by flame atomic absorption spectrometry (FAAS) (SpectrAA-600; Varian, Palo Alto, CA). Iwasaki et al. (1990) and preliminary experiments designed to measure K efflux from roots with a microelectrode showed that such experimental conditions (acid concentrations and duration of extraction) did not damage the plasma membrane of either fresh or frozen or thawn roots (which would have resulted in overestimating apoplastic Cu). Similar results were obtained for both fresh and frozenthawn roots, which indicated that roots could be frozen before apoplastic Cu determination. The remaining portion of roots was ovendried at 105 C. The plant roots and shoots were digested separately in a 1:1 mixture of hot, concentrated HNO3 and HClO4 (Association of Official Analytical Chemists, 1975). Copper in the digest was assayed by FAAS. The soils were collected at harvest and oven-dried at 105 C. Two independent chemical extractions were performed with two subsamples of soils collected from either cropped soil containers or uncropped, control soil containers (without plants). Copper was extracted with 0.05 M CaCl2 to estimate the exchangeable fraction of soil Cu (McLaren and Crawford, 1973). The amount of soil used in the present work was modified, but the soil to solution ratio was the same: 1 g of dry soil was shaken for 2 h with 5 mL of 0.05 M CaCl2. The suspensions were then centrifuged at 15 000 g for 3 min. The other extraction was done with DTPA. This method had been initially designed by Lindsay and Norvell (1978) for assessing the plant-available fraction of soil Cu in calcareous soils that were low or deficient in Cu. However, it has since been used also for Cu-contaminated soils (e.g., Beckett, 1989; Brun et al., 2001). Briefly, the extractant solution was composed of 0.005 M DTPA, 0.01 M CaCl2, and 0.1 M triethanolamine (TEA) at pH 7.3. The CaCl2 and TEA were added in the extractant solution to prevent CaCO3 excessive dissolution and metal release (OConnor, 1988). In comparison with the initial procedure, the amount of soil used here was much reduced to adapt to the small amount of soil collected from each soil container, but the soil to solution ratio was the same: 500 mg of dry soil was shaken for 2 h with 1 mL of extractant solution. The suspensions were then centrifuged at 15 000 g for 3 min. Copper in all soil extracts was assayed by FAAS.

of three relative to that at 0.2 M and was equal to that of preculture plants, indicating that no growth had occurred. When considering biomass as a simple endpoint, the no observable adverse effect concentration (NOAEC), was thus lying somewhere between 20 and 80 M. In comparison, for maize the NOAEC was lying between 0.16 and 1.6 M Cu concentration as deduced from shoot and root growth measurements by Lexmond and van der Vorm (1981). This was found in their experiment that most closely compared with the present experiment in terms of solution composition (only NO3N supplied and pH at 5.66.1). However, a major difference was that they supplied Fe as FeSO4 instead of FeNaEDTA and hence most of solution Cu was occurring as free Cu ion (9095%). The NOAEC found for rape in the present work was ranging between 0.06 and 1.1 M free Cu ion concentration, which thus compares fairly well with that deduced from Lexmond and van der Vorm (1981) for maize. Figures 3a and 3b also show that the EC50, that is, the exposure concentration at which 50% of the maximum biomass is achieved, was about 80 M for shoots and 50 M for roots. This further shows a larger sensitivity of roots relative to shoots. An even more sensitive indicator than root growth might have been used, in light of the approach that has been developed for assessing Al toxicity, such as the relative elongation of selected roots (e.g., Kinraide and Parker, 1987; Parker, 1995; Calba et al., 1996). This indicator is more sensitive because decreased root elongation, the primary effect of Al rhizotoxicity, is often accompanied by some root thickening that somewhat compensates the ultimate effect on root growth as assessed via the whole biomass. This indicator recently proved applicable to evaluate the response of plant roots to Cu toxicity (Parker et al., 1998) and an EC50 of about 0.3 M of free Cu ion was found for wheat (Triticum aestivum L.), to compare with an EC50 of about 0.25 M for root biomass in the present work (equivalent to 50 M total Cu). Comparison of the two approaches for an identical plant species grown in identical hydroponic conditions would be needed to evaluate which is the best suited (most sensitive) method for measuring Cu rhizotoxicity. Concentration of Copper in Plant Parts An alternative to using root or shoot biomass as an endpoint for assessing Cu phytotoxicity is the evaluation of concentration of Cu in plant parts. Shoot Cu concentration constantly increased from 6 to a maximum of 1280 mg kg 1 dry matter (DM) with increasing Cu concentration in the nutrient solution (Fig. 3c). However, shoot Cu concentrations significantly increased only between 20 and 80 M and between 120 and 200 M (Fig. 3c). Compared with reported phytotoxic levels of Cu in shoots of young rape plants (about 16 mg kg 1 DM; Reuter and Robinson, 1997), only the two smallest Cu concentrations in the nutrient solution (0.2 and 5 M) gave lower or equal values (6 and 16 mg kg 1 DM, respectively), while a concentration of 49 mg kg 1 DM was found at 20 M. Shoot Cu concentration then rapidly exceeded this value at larger solution concentra-

RESULTS AND DISCUSSION Hydroponic ExperimentBiotest for Assessing Copper Phytotoxicity

Plant Growth Response The biomass of both shoots and roots showed a maximum at 5 M and then decreased when Cu concentration further increased (Fig. 3a,b). This decrease was significant, however, only for concentrations above 20 M. For roots and shoots, no significant biomass difference was observed between 0.2, 5, and 20 M. Similarly, no significant biomass differences were observed for concentrations between 80 and 200 M (Fig. 3a,b). For shoots, at 200 M the biomass had decreased by a factor

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Fig. 3. Dry biomass of shoots (a ) and roots (b ) expressed as g per container (per 10 plants), and Cu concentrations (mg kg 1 dry matter) of shoots (c ) and roots (d ) as a function of Cu concentration in the nutrient solution ( M ) in the hydroponic experiment. Open symbols stand for values of preculture plants. Corresponding free Cu ion concentrations ( M ) as calculated from the SOILCHEM computer code (Sposito and Coves, 1988) are also indicated. Mean values with different letters are significantly different ( p 0.05) as measured by the RyanEinot GabrielWelschRange test.

tions (Fig. 3c). This would suggest that the NOAEC would lie between 5 and 20 M rather than 20 and 80 M, as deduced from the biomass measurements. The concentration of Cu in the roots might be considered an even more sensitive endpoint, as several authors have pointed out that Cu can accumulate in the roots to a large extent while Cu concentration in plant shoots remains unaltered (Mitchell et al., 1978; Lexmond and van der Vorm, 1981; Marschner, 1995; Brun et al., 2001). Indeed, much larger Cu concentrations have been found in the roots, ranging between 24 and 24 700 mg kg 1 DM, than in the shoots (Fig. 3c,d). Although constantly increasing with increasing Cu concentration in the nutrient solution, they were not significantly different for concentrations ranging from 0.2 to 80 M. Root Cu concentration peaked at 100 M, reaching 24 700 mg kg 1 DM (Fig. 3d). Because of much larger variabilities in the measured values for roots, a significant increase in root Cu concentration was found only at a solution Cu concentration of 100 M. It thus appears for this plant species that this indicator is not best suited for an

early detection of Cu phytotoxicity. It is nonetheless interesting to point out that when Cu concentration in the shoots increased from 6 mg kg 1 DM (marginal adequate range) at 0.2 M, to 16 mg kg 1 DM (critical range) at 5 M, and to 49 mg kg 1 DM (phytotoxic range) at 20 M, Cu concentrations in the roots increased in the meantime from 24 to 65 and 533 mg kg 1 DM, respectively. When plant growth started to be significantly altered (at 80 M), Cu concentration had reached about 400 mg kg 1 DM in the shoots and more than 5000 mg kg 1 DM in the roots, which is manyfold higher than phytotoxic levels (Fig. 3c). The present experiment thus clearly confirms that Cu preferentially accumulates in the roots and more so when solution Cu concentration increases, in agreement with former results obtained for maize (Mocquot et al., 1996). Shoot to root Cu concentration ratios decreased from 0.25 at 0.2 M to 0.07 at 80 M, which compares fairly well with the observations for maize by Lexmond and van der Vorm (1981). However, a striking difference between the two species was the much larger increase in Cu

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concentration in the shoots of rape exposed to slightly phytotoxic concentrations, relative to maize. In addition, the ratio of shoot to root bioaccumulated Cu in rape (3.4, 2.5, 1.3, and 1.1 for 0.2, 5, 20, and 80 M Cu concentrations, respectively) was about 5- to 10-fold larger than that deduced from the results obtained for maize by Lexmond and van der Vorm (1981). Rape was thus more prone than maize to translocate a substantial proportion of Cu into its shoots. This propensity to translocate metals to shoots has been observed in many species of the Brassica genus and offers some prospects for using these species for phytoextraction purposes as shown, for instance, by Ebbs et al. (1997).

Soil ExperimentBiotest for Assessing Copper Bioavailability

Plant Growth and Copper Concentration in Plant Parts The biomass of both shoots and roots was greater than that of rape harvested at the end of the preculture (Table 1). This increase was, however, significant only for plants supplied with nutrient solution for 8 d. Plant growth did not seem to be affected by soil layer thickness. The poor growth of plants relative to preculture plants (Table 1) might be interpreted at first sight as a response to Cu phytotoxicity. Plant growth was notably reduced when deionized water was supplied (Table 1). However, it is likely that the growth was restricted as a consequence of a shortage of nutrients supplied by the small amount of soil when deionized water was used. Indeed, supplying nutrient solution alleviated those nutrient deficiencies and significantly improved plant growth (after 8 d at least, see Table 1). After contact with the soil, Cu concentrations in rape plants varied from 6.6 to 8.5 mg kg 1 DM for the shoots and from 38 to 127 mg kg 1 DM for the roots, depending on the treatment (Fig. 4a). Copper concentrations in roots and shoots were not significantly correlated (n 40, R2 0.10). Copper concentration in the shoots significantly increased relative to preculture plants only after 8 d of growth while a systematic increase was found for root Cu concentration. No significant difference in shoot Cu concentration was observed between the various treatments. The Cu concentrations observed in the shoots of rape were all far below the critical value of 16 mg kg 1 DM provided by Reuter and Robinson (1997), which suggested that this soil, in spite of its large total and EDTA-extractable Cu content, was not
Table 1. Dry biomass of shoots and roots.
0d Preculture plants Shoot biomass Root biomass 0.6 0.06 0.1a 0.01a 1.1 0.16 Nutrient solution 1 mm 0.1ab 0.02ab 3 mm 1.1 0.19 0.1ab 0.03b 4d

inducing any Cu phytotoxicity in the conditions of the present test. Root Cu concentration was always larger with deionized water than with nutrient solution. Lorenz et al. (1994) had shown that using a nutrient solution for pot experiments may alter the composition of soil solution and shift reaction equilibria, resulting in artificially increased concentrations of free metal ions such as Zn and Cd. Using nutrient solution in pot experiments might thereby lead to overestimated plant contamination. In contrast, lower concentrations of Cu were observed in the present experiment when nutrient solution was supplied, possibly because of a dilution effect related to increased biomass on nutrient supply. Therefore, we recommend use of a nutrient solution for promoting adequate plant growth, considering the small amount of soil used in our biotest (and thus restricted reservoir of nutrients). A major interest of doing so is that it should enable easier comparison of a range of soils as these would probably differ not only by their level of metal contamination but also by their content of essential nutrients. This is a critical point that is poorly addressed in the description of the conditions of ecotoxicological tests such as the early seedling growth test (American Society for Testing and Materials, 1994) or its equivalents (International Organization for Standardization, 1999). Although it may cause some artifacts, using a nutrient solution definitely provides better standardized conditions that favor the comparison of widely differing soil samples. Root Cu concentrations did not exceed 127 mg kg 1 DM when deionized water was supplied and half of this (about 60 mg kg 1 DM) when nutrient solution was supplied (Fig. 4a). These values remain in the order of Cu concentrations observed in the roots of hydroponically grown plants supplied with nonphytotoxic Cu concentrations (24 mg kg 1 DM at 0.2 M Cu, 65 mg kg 1 DM at 5 M Cu). This is an additional indication that this soil did not induce any Cu toxicity in rape plants. These results confirm that when adequate shoot Cu concentrations (unchanged relative to uncontaminated soils) are found, fairly large Cu concentrations can occur in roots of plants grown in Cu-contaminated calcareous soils, although much larger concentrations have been found in roots in earlier reports (Mitchell et al., 1978; Brun et al., 2001). There is thus some prospect for using the concentration of Cu in roots as an early indicator of plant contamination for soil-grown plants, as suggested by Mitchell et al. (1978). However, for this purpose, references on the critical levels of root Cu that

8d Deionized water 1 mm 0.7 0.11 3 mm Nutrient solution 1 mm 3 mm 1.4 0.32 0.2b 0.04c 0.9 0.12 Deionized water 1 mm 0.2a 0.06ab 1.0 0.12 3 mm 0.1ab 0.03ab 0.05) as

g per container (per 10 plants) 0.1a 0.8 0.1a 1.4 0.4b 0.02a 0.08 0.04a 0.35 0.07c

Values are means standard deviations of five replicates. Mean values with different letters within a line are significantly different ( p measured by the RyanEinotGabrielWelschRange test.

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Fig. 4. Concentrations (a ) and amounts (b ) of Cu in plant shoots and roots expressed as mg kg 1 dry matter and g per container (per 10 plants), respectively. NS and H2O stand for nutrient solution and deionized water treatments, respectively. Percentages of total root Cu bound to root cell walls are indicated in (a ). Mean values with different letters are significantly different ( p 0.05) as measured by the RyanEinotGabrielWelschRange test.

are indicative of a toxicity need to be established. In addition, the present experiment showed that a substantial proportion (2678%) of total root copper was bound to the cell wall fraction (see apoplastic Cu in Fig. 4a), which is in line with previous results (Iwasaki et al., 1990; Parker et al., 1998). Lower proportions of apoplastic Cu

were found in plants supplied with nutrient solution, possibly because of increased competition with cations (such as Ca or Mg) supplied by the solution, as previously shown for wheat by Parker et al. (1998). As stressed by these authors, there is a need to better understand the processes involved in the rhizotoxicity of Cu

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Table 2. Copper uptake by rape and Cu extracted with CaCl2 0.05 M and diethylene triaminepentaacetic acid (DTPA).
4d Nutrient solution 1 mm Plant Cu CaCl2Cu Control Rhizosphere DTPA-Cu Control Rhizosphere 3.7 3.5 5.5 182 200 0.8ab 1.7ab 0.5c* 3 mm 2.8 2.5 5.9 0.7ab 2.9 Deionized water 1 mm 1.1ab 2.2 3 mm Nutrient solution 1 mm 3 mm 5.7 1.5 2.6 2.2cd 4.7 8d Deionized water 1 mm 1.9bc 0.2ab 0.3a* 3.2 1.8 2.1 3 mm 0.4ab 0.1ab 0.5a (NS) 34bc 12ab (NS)

mg Cu kg 1 soil 0.8a 7.6 1.9d 0.5ab 1.7 0.3cd (NS) 3.8 14c 175 11cd (NS) 177 0.1a 0.8b**

1.4ab 4.0 0.6c** 6.8 13ab 191 11d** 197

2.9b 2.1 0.7d (NS) 6.1 11c 188 16cd (NS) 195

0.1a 2.0 0.2a** 2.4 18a 12a* 189 189

10bc 162 31cd (NS) 215

14bc 150 5ab (NS) 173

13c 176 6bc (NS) 175

* Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. Values are means standard deviations of five replicates. Mean values with different letters within a line are significantly different (p 0.05) as measured by the RyanEinotGabrielWelschRange test. For each treatment, control and rhizosphere mean values are compared by a Student test. Not significant.

and its relationship with the partitioning of root Cu between the apoplasm and symplasm compartments, similar to research completed for Al rhizotoxicity. This is a prerequisite for selecting which of total, symplasmic, or apoplasmic Cu in roots is the best indicator of the Cu toxicity in soil-grown plants. The absence of any Cu phytotoxicity in the present soil, in spite of its large total Cu content, might partly be due to its calcareous nature, as Cu is known to be more bioavailable in acidic than neutral or calcareous soils (McLaren and Crawford, 1973; Mitchell et al., 1978; Alloway, 1995). Alternatively, it could be due to the procedure used in the present biotest, which is based on a rather short duration of exposure to the contaminated soil (48 d). This might have resulted in a dilution effect in the 3-wk-old plants used in this biotest. No periods of contact longer than 4 to 8 d were considered here, because a major advantage of the present biotest relative to conventional pot experiments was to provide a short-term, rapid, and thus reasonably inexpensive test. There is thus a need to investigate other soils with different properties, including noncalcareous soils and soils that are known to be responsible for phytotoxicity. Uptake and Bioavailability of Copper Little or no significant differences in amounts of bioaccumulated Cu were found between the various treatments (Fig. 4b). The uptake of Cu, however, increased when nutrient solution was supplied compared with deionized water. As Cu concentration tended to decrease in the meantime (Fig. 4a), the enhanced uptake of Cu was due to the increased growth of plants supplied with nutrient solution relative to those supplied with deionized water. For similar reasons, Cu uptake also increased with increasing soilplant contact period. Although twice as much soil (and hence Cu) was supplied in the 3-mm treatment relative to the 1-mm treatment, no significant increase in Cu uptake was observed. Copper uptake by rape per unit of soil mass supplied, which is an estimate of soil Cu bioavailability (Table 2), was deduced from the previous figures (amounts of bioaccumulated Cu, Fig. 4b) by subtracting the initial amount of Cu in the preculture plants and expressing the final result relative to the mass of dry soil supplied (which

differed between the two soil thickness treatments). Bioavailable Cu amounted to 0.5 to 2% of total soil Cu. The effect of time duration was clearly visible (Table 2). Again, soil thickness (i.e., the amount of soil supplied) had little significant effect on Cu bioavailability (Table 2). This indicates that a major proportion of the amount of Cu taken up by rape plants was derived from the first millimeter of the rhizosphere, which is not surprising owing to the poor mobility of Cu in soils. In that respect, working with 1 mm would be justified, especially if the biotest also aims at investigating the rootinduced changes that occur in the rhizosphere, as was the case in the present work. Rhizosphere effects typically occur as gradients as a function of the distance from the root surface (Hinsinger, 1998). They might therefore be increasingly diluted and eventually become hardly detectable with increasing distance from the roots (i.e., investigated soil thickness). Extractability of Copper in the Soil and in the Rhizosphere The amount of CaCl2extractable Cu in the control soil was generally lower when the soilplant contact period was 8 d and nutrient solution was supplied (Table 2). The amount of CaCl2extractable Cu in the rhizosphere was significantly lower when the soilplant contact period was 8 instead of 4 d, while soil thickness had little effect in general. The amount of CaCl2extractable Cu systematically increased in the rhizosphere, relative to the control (uncropped soil). A significant accumulation occurred only when nutrient solution was supplied. This increase in exchangeable Cu (CaCl2extractable Cu) in the rhizosphere relative to the control, uncropped soil occurred in spite of plant Cu uptake. This suggests that the solid-phase speciation of Cu was altered as a consequence of root activity, resulting in an increase of the exchangeable Cu fraction at the expense of another, unknown fraction of soil Cu. In most cases the DTPAextractable Cu did not significantly change in the rhizosphere compared with the control, uncropped soil (Table 2). Whenever it changed, it increased in the rhizosphere, suggesting again that the extractability of soil Cu was altered in the rhizosphere in a way that could not be simply explained by Cu uptake. After 8 d of

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growth, the increase in CaCl2extractable Cu in the rhizosphere was larger for 1 than 3 mm, although significantly only when nutrient solution was supplied. This confirms that a larger rhizosphere effect is observed when a lower soil thickness is used. Another advantage of working with the smaller soil thickness is that it requires lower amounts of contaminated soil, which may be of practical importance when heavily toxic or radioactive metals or trace elements are to be investigated for the purpose of ecotoxicological testing.

CONCLUSIONS
The present work aimed at (i) defining the responses of oilseed rape to Cu phytotoxicity as assessed in hydroponic conditions and (ii) optimizing the operational conditions of a novel ecotoxicological test designed for evaluating soil bioavailability and phytotoxicity of metals such as Cu. The NOAEC ranged between 5 and 20 M total Cu (0.01 and 0.06 M free Cu ion). The soil used for investigating the operational conditions of the soil-based biotest did not induce any significant phytotoxicity, in spite of its large total Cu content. It can nonetheless be concluded that the best conditions for conducting this biotest are the following: (i) the duration should be 8 rather than 4 d to be able to use plant growth as an indicator of phytotoxicity, (ii) the thickness of the soil layer (container) should be 1 mm (roughly 2 g dry soil per container) rather than 3 mm, as most of the bioavailable Cu is derived from this portion of the rhizosphere and (iii) nutrient solution should be supplied instead of deionized water, for a better comparison of soils with different nutrient status. Compared with glasshouse pot experiments that are conventionally used in terrestrial ecotoxicology, this biotest has several definitive advantages: (i) easy recovery of all the roots, (ii) cost effectiveness, as it lasts for only 8 d and does not require manual watering, (iii) small size that is adapted for conducting a large number of treatments and replicates over a small surface area, that is, in growth chambers that can provide standardized and reproducible climatic conditions. The proposed biotest, however, suffers of several disadvantages, especially related to the particular arrangement of soil and roots: (i) the roots are not growing within the soil and may therefore be less exposed to toxicities, (ii) some plant species are poorly adapted as they hardly form a dense, planar mat of roots (e.g., species with a strong, thick tap root), (iii) the small ratio of soil to plant requires the addition of a nutrient solution with associated, potential artifacts; therefore, the soil is almost water-saturated, being connected to a large reservoir of nutrient solution that may affect the cation exchange, complexation, and redox equilibria. This biotest would now need to be further evaluated for a range of soils varying in properties and levels of metal contamination. It should also be applied to conventional pot experiments that are currently being used in terrestrial ecotoxicology and, ultimately, to in situ measurements. In that respect, it is interesting to point out that a slightly different biotest derived from the same original method of

Niebes et al. (1993) has been recently validated against a conventional pot experiment for evaluating the bioavailability of 137Cs in a range of soils (Kruyts et al., 2000; Thiry et al., 2000). This biotest has also been used for studying chemical changes occurring in the rhizosphere that ultimately affect the bioavailability of trace elements to plants (Guivarch et al., 1999; Hinsinger, 2001). It is a unique tool for assessing responses of roots to metal toxicities in soil-grown plants, as it provides an easy access to the roots and hence to sensitive endpoints (e.g., root elongation, as shown for Al by Calba et al., 1996) or biomarkers (biochemical responses) of metal toxicities that would express in the roots. The proposed biotest thus can be seen as a research tool that also accommodates the requirements of standardized ecotoxicological testing procedures.
ACKNOWLEDGMENTS The authors thank Dr. P. Andrieux for providing access to the experimental vineyard watershed, G. Souche and M. Clairotte for their help in sampling the soil on this site, and Dr. C. Plassard for designing and conducting the K efflux measurements. The help provided by Professor P. Schwab and anonymous referees for improving the manuscript is gratefully acknowledged. This work was made possible by the financial support of the French Ministry of Environment, within the framework of its PNETOX program.

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