Capability of Selected Crop Plants for Shoot Mercury Accumulation From Polluted Soils: Phytoremediation Perspectives

By Rodriguez, Luis Rincon, Jesusa; Asencio, Isaac; Rodriguez- Castellanos, Laura

High-biomass crops can be considered as an alternative to hyperaccumutator plants to phytoremediate soils contaminated by heavy metals. In order to assess their practical capability for the absorption and accumulation of Hg in shoots, barley, white lupine, lentil, and chickpea were tested in pot experiments using several growth substrates. In the first experimental series, plants were grown in a mixture of vermiculite and perlite spiked with 8.35 [mu]g g^sup -1^ d.w. of soluble Hg. The mercury concentration of the plants’ aerial tissues ranged from 1.51 to 5.13 [mu]g g^sup -1^ d.w. with lentil and lupine showing the highest values. In a second experiment carried out using a Hg-polluted soil (32.16 [mu]g g^sup – 1^ d.w.) collected from a historical mining area (Almaden, Spain), the crop plants tested only reached shoot Hg concentration up to 1.13 [mu]g g^sup -1^ d.w. In the third experimental series, the Almaden soil was spiked with 1 [mu]g g^sup -1^ d.w. of soluble Hg; as a result, mercury concentrations in the plant shoots increased approximately 6 times for lupine, 5 times for chickpea, and 3.5 times for barley and lentil, with respect to those obtained with the original soil without Hg added. This marked difference was attributed to the low availability of Hg in the original Almaden soil and its subsequent increase in the Hg-spiked soil. The low mercury accumulation yields obtained for all plants do not make a successful decontamination of the Almaden soils possible by phytoremediation using crop plants. However, since the crops tested can effectively decrease the plant-available Hg level in this soil, their use could, to some extent, reduce the environmental risk of Hg pollution in the area. KEY WORDS: mercury availability, barley, lupine, chickpea, lentil, phytoextraction

INTRODUCTION

The contamination of soils by heavy metals has become one of the most important environmental problems in developed and developing countries. Mining operations, metal smelting, electroplating, gas exhausts, energy and fuel production, downwash from power lines, intensive agriculture, and sludge dumping are some of the numerous human activities that contaminate soils with large quantities of toxic metals (Kumar et al., 1995). Conventional “ex situ” cleanup technologies are extremely expensive and have a negative effect on desirable soil properties, making them unsuitable to restore such sites (Kayser et al., 2000). In an effort to develop alternative cleanup technologies, numerous studies over the last decade have focused on using plants to uptake heavy metals from soils and their subsequent accumulation in the plant tissue (Pivetz, 2001; Terry and Banuelos, 2000). This technique, known as phytoextraction, offers several advantages including low cost and minimal environmental disturbance-this method does not adversely alter the soil matrix. Two approaches are generally proposed for the phytoextraction of heavy metals: the use of natural hyperaccumulator plants that have exceptional metal-accumulation capacities and the utilization of high-biomass crops, e.g., maize, peas, oats, barley, wheat, Indian mustard, and cabbage, in conjunction with the appropriate chemical treatment to enhance the solubility of metals in soils (Chen et al., 2004). In general, the availability of heavy metals to plant roots is considered a key factor that may limit successful soil remediation (Kabata Pendias and Pendias, 2001; Kayser etal., 2000). Many authors have demonstrated this close relationship for the absorption of a range of metals (e.g., Pb, Cd, Zn, Cu, Cr, and Ni) by different plants, including some legumes and graminaceous species (Chen et al., 2004; Evangelou et al., 2004; Hammer and Keller, 2002; Kos et al., 2003; Walker et al., 2004; Wang et al., 2004; Zhao et al., 2004).

Mercury is outstanding among the global environmental pollutants that are of continuing concern. Numerous industrial activities, including the mining of gold, silver, and mercury itself, have caused mercury contamination of both terrestrial and aquatic ecosystems. Thus, it has been suggested that the amount of mercury mobilized and released into the biosphere has increased continuously since the beginning of the industrial age (Ebinghaus et al., 1999). Most of the mercury in the atmosphere is elemental mercury vapor, which circulates in the atmosphere for up to a year and, hence, can be widely dispersed and transported thousands of miles from likely sources of emission. Wet deposition is the primary mechanism for transporting mercury from the atmosphere to surface waters and land. As a result of both the direct release of mercury from industrial activities and from the deposition of atmospheric Hg, numerous cases of mercury pollution in soils have been reported throughout the world (US EPA, 1997). On the other hand, mercury undergoes a series of complex chemical and physical transformations as it cycles among the atmosphere, land, and water. During this cycle, humans, plants, and animals are routinely exposed to and accumulate mercury, potentially resulting in a variety of ecological and human health impacts.

The uptake of mercury by plants has been studied by a number of researchers. The background levels of Hg in plants grown in noncontaminated soils do not exceed 0.1 [mu]g g^sup -1^ (Kabata- Pendias and Pendias, 2001). However, the shoot Hg concentration values that are reported for plants grown in Hg-polluted soils are usually higher. Lindberg et al. (1979) grew alfalfa in Almaden soils and found 2.3 and 1.4 [mu]g Hg g^sup -1^ in leaves and stems, respectively. Huckabee etal. (1983) reported mercury-loading values in the range of 0.37-3.10 [mu] g^sup -1^ for five wild plant species collected in the vicinity of the mercury mine at Almaden. Greger et al. (2005) found mercury levels in the range of 0.16-1.40 [mu]g g^sup -1^ for five herbaceous plants grown in a nutrient solution containing 200 [mu]g Hg L^sup -1^. Several authors have shown that certain plants, e.g., garlic vine (Pseudocallyma alliacium), avocado (Persea americana), haole-koa (Leucaena glauca), and reed (Phragmites communis), can take up mercury through their roots and translocate it to their leaves before releasing it to the air via the stomata (Kozuchowsky and Johnson, 1978; Siegel et al., 1974). However, other studies suggest that the mercury absorbed by plants is accumulated almost exclusively in root tissue and that its translocation and release to the air is insignificant or nonexistent (Greger et al., 2005; Linberg et al., 1979; Suszcynsky and Shan, 1995). In spite of these investigations, the use of plants for the decontamination of Hg-polluted soils has only been recently reported by Moreno et al. (2004,2005a, 2005b).

Mercury uptake from soils by common crops of high biomass has not been reported adequately. So, the main purpose of this article is to assess the potential of several crop plants-barley, lupine, lentil, and chickpea-for the shoot accumulation of mercury from contaminated substrates with a view to their possible use for soil phytoremediation. Likewise, we also wanted to investigate the influence of mercury availability on shoot accumulation. To this aim, three pot experiments were conducted using three different substrates for plant growth. In the first experimental series, a vermiculite-perlite mixture spiked with soluble Hg was used in order to evaluate the maximum accumulation capability of the crop plants. second, the phytoextraction capability of the plants was also checked in natural conditions using contaminated soil from a historical mercury-mining area (Almaden, Spain). Finally, the influence of mercury availability on shoot accumulation was assessed by spiking the Almaden soil with soluble Hg.

MATERIALS AND METHODS

Substrates Preparation

Three substrates were used in the phytoextraction tests. In the first series of experiments, plants of the four crops were grown in a mixture of vermiculite and perlite (2:1 v/v). Plastic pots were filled with 1.5 L of this mixture and spiked with aqueous Hg 35 d after planting. The Hg spiking was made by uniformly adding 400 mL of an aqueous solution of HgCh to the pots. It used a dose of 2 mg of Hg per pot, corresponding to a theoretical Hg concentration of approximately 9 mg per kg d.w. of substrate (density of substrate, 148 g/L). No leaching was observed during the spiking process. The mixture of vermiculite and perlite will be called “VP substrate” in this article.

Another two series of phytoextraction experiments were conducted using contaminated soil collected from a site close to a historical mercury-mining center (Almaden, Ciudad Real) located in the central part of Spain, 300 km southwest of Madrid (Figure 1). The continuing Almaden mining and refining operations (more than 2000 years) together with natural emissions from the ore deposits have contributed to the high mercury loading of the soil in this area (Ferrara et al., 1998; Huckabee et al., 1983). Soil was collected from the 0-20-cm depth, air dried, crushed, and sieved to <5 mm prior to use in the pot experiments. The soil was a loam with 9.1 % clay, 50.1 % silt, and 40.8% sand, with a pH (in water) of 7.65,2.26% organic matter (OM), cation exchange capacity (CEC) of 27.1 cmolc kg"1, and 0.19% CaCOa. In both second and third experimental series of this work, plants of the four crops were grown in a 2:1 (v/v) mixture of Almaden soil and perlite. Perlite was used to improve drainage of the pots to some extent. Since the low density (c.a. 125 g/L) and nature of perlite, pH of the soil was not altered by the mixture and other physicochemical properties, e.g., total Hg, CEC, OM, and CaCOa, were only slightly decreased by the effect of the dilution (data not shown). In the third experimental series, 35 after planting, the pots were spiked with aqueous HgCb using a dose of 2 mg of Hg per pot. This treatment involved the addition of approximately 1 mg Hg per kg d.w. of substrate (density of the substrate, 1.3 kg/L). The Hg-spiking method was the same as that of the VP substrate. In order to evaluate the Hg concentration (total and CaCh-extractable) of the substrates after the spiking, nonplanted pots were used in both the first (spiked VP substrate) and the third (spiked Almaden soil) experimental series (one pot per experiment).

Phytoextraction Experiments

Four common crop plants widely used in Spain were employed in pot experiments: barley (Hordeum vulgare), white lupine (Lupinus albus), lentil (Lens esculenta), and chickpea (Cicer aretinum). In all the experiments, seeds of each species were soaked in a saturated CaSO4 solution (Shenker et al., 2001) and then wrapped in watered-filter paper for 3-4 d. After this period, the seedlings were transferred to the pots and grown under greenhouse conditions with natural light, day/night temperature of 28/10 [degrees]C, and irrigated with tap water. After growth for 150 d, shoots of the plants were cut at the substrate surface. The plant samples were rinsed with distilled water and air dried. After drying, all the plant samples were ground by a ball mill (Retsch MM200, Haan, Germany) and sealed in plastic bags for subsequent Hg analysis. On the same date, all the pots were sampled taking the upper 10 cm of the substrate. Substrate samples were air dried and milled (Retsch MM200 ball mill) for subsequent analysis. Nonplanted pots were only sampled S d. after spiking. To evaluate the dry weights, subsamples of both plant and substrate were oven dried at 70[degrees]C until a constant weight was obtained.

In the VP substrate series, plant nutrients were supplied during the growth period by means of several additions of a commercial fertilizer (Actigil from Rhone-Poulenc, Madrid, Spain; 8% N, 8% P^sub 2^O^sub 5^, 6% K^sub 2^O). On the other hand, no fertilizer was used during the growth period in both experiments carried out using the Almaden soil.

Four replicates per plant species were used in all the experimental series. Statistical analysis of the results was carried out by performing an ANOVA test using Microsoft Excel 2002. Statistical significance was defined as p

Substrate Analysis

The physicochemical parameters of the Almaden soil were determined using the standard analytical methods of the Spanish Ministry of Agriculture, Fishing and Foods (MAPA, 1994).

The total mercury loading of the substrates was directly determined on the solid samples using a LUMEX RA-915+ mercury analyzer equipped with a two-chamber pyrolysis block (RP-91 C). The samples were heated to 800[degrees]C in a first heating chamber, leading to the volatilization of the Hg and organic compounds of the sample. All products were transported from the first chamber to a second by an air flow. The second chamber was continuously heated at about 800 [degrees]C. There, smoke and interference compounds were burst, producing mostly carbon dioxide and water. Hg in the gas flow was determined by flameless atomic absorption spectrometry. A Zeeman corrector of the spectrometer eliminated the rest of the background absorption. With this technique, the limit of detection for Hg in soil samples was 0.5 ng g^sup -1^. All the samples were analyzed in triplicate. The analytical method was assessed by using the 2711 Standard Reference Material (Montana Soil, from LGC Promochem) with an agreement of c.a. 95% between the certified value and the concentration we obtained (n = 3).

The CaCl^sub 2^-extractable mercury was determined following a method reported by Novozamsky et al. (1993). Briefly, 5 g of dry soil were added to 50 ml of 0.01 M CaCl^sub 2^ solution in triplicate, shaken for 3 h, and then centrifuged for 20 min at 4000 rpm. The resultant supernatant was analyzed by means of hydride- generation atomic absorption spectroscopy using a Varian 220FS AAS (Madrid, Spain). The instrument was zeroed with 1% HNO^sub 3^ blanks. The Hg concentration values measured by the spectrometer were the mean of three readings with less than 5% of variation. Calibration solutions were prepared from a certified stock solution of 1000 mg Hg/L (Panreac, Barcelona, Spain).

Plant Analysis

The total Hg content of plant samples was determined using the aforementioned LUMEX RA-915+ mercury analyzer. The limit of detection for Hg in plant samples was 2 ng g^sup -1^. The analytical method was assessed by using the CTA-VTL-2 Reference Material (Virginia Tobacco leaves, from LGC Promochem) with an agreement of c.a. 92% between the Hg concentration we obtained (n = 3) and the certified one.

RESULTS

Vermiculite-Perlite Substrate with Spiked Mercury

All the plant species grown in the Hg-spiked VP substrate were able to take mercury up and translocate it to the aerial part (Figure 2). Shoot Hg concentrations ranged from 1.51 to 5.13 [mu]g g^sup -1^ being the order found for the four species lupine [asymptotically =] lentil > chickpea > barley. A similar trend can be seen in Table 1 for the concentration of total mercury in the pots after harvesting. The concentration of CaCl^sub 2^-extractable Hg in the growth substrates at the end of the experiment was very low compared to that of the nonplanted pot after 5 d from the Hg spiking (Table 1). Nevertheless, taking into account the experimental procedure used in the pot experiment, this marked decrease during the experiment must be attributed to some extent to the Hg leaching caused by irrigation. On the other hand, the higher values of the standard deviation obtained for the CaCl^sub 2^- extractactable Hg concentration values do not permit us to determine differences between plant species.

Almaden Soil and Hg-Spiked Soil

Shoots of the plants grown in the contaminated soil from the Almaden area showed Hg concentrations in the range of 0.16-1.13 [mu]g g^sup -1^ (Figure 3). Mercury shoot concentration differed between the four plant species tested; lentil and barley showed a significantly (p < 0.05) higher shoot concentration than lupine and chickpea. Due to the low mercury uptake by the plants and the size of the pots, the total Hg loading of the growth substrates at the end of the experiment did not change significantly with respect to the initial conditions (Table 2). Likewise, no significant differences existed between plant species. With regard to CaCl^sub 2^-extractable Hg concentration of the substrates, the growth of plants decreased the extractable concentration for all plant species; however, there were no significant differences between them (Table 2). CaCl^sub 2^-extractable Hg concentrations of the Almaden soil samples were much lower than those of the spiked VP substrate, both before and after the experiment (Tables 1 and 2).

The Hg concentration of the plant shoots for the experiment conducted with the spiked Almaden soil is shown in Figure 3, together with those of the soil without added Hg. It must be noted that the previous experiment with the Almaden soil can be considered to be control for this experimental series. Therefore, the addition of ca. 1 [mu] g^sup -1^ of soluble Hg led to a significant enhancement in the shoot mercury concentration of the four plant species used. These results indicate more than a 6-fold increase in the mercury loading for lupine, more than a 5-fold increase for chickpea, and an increase by a factor of more than 3.5 for barley and lentil with respect to the natural soil experiment. Shoot mercury loadings of the four species tested followed the same trend as that found for the experiment with natural soil (lentil [asymptotically =] barley > lupine [asymptotically =] chickpea). Again, plant growth did not lead to a significant decrease in the initial Hg loading of the substrates (Table 2). CaCl^sub 2^- extractable Hg concentration of the substrate from the nonplanted pot after 5 d of the spiking was decreased by the plants (with the exception of chickpea). Moreover, it can be observed that plants with the higher Hg shoot concentrations (lentil and barley) showed the lower values for the extractable Hg of the substrates after harvesting (Figure 2 and Table 2). Finally, it must be noted that the CaCl^sub 2^-extractable Hg concentration of the spiked substrate from the nonplanted pot after 5 d was only moderately higher than that of the natural Almaden soil without added Hg. This fact will be discussed later.

DISCUSSION

According to the values reported in the Introduction, it can be noted that the shoot Hg concentrations of the plants tested in this work are in the same order of magnitude as those reported previously and greatly exceed background levels of Hg in plants (Kabata- Pendias and Pendias, 2001). It has been reported by several authors (Greger et al., 2005; Lindberg et al., 1979; Moreno et al. 2005a) that mercury absorbed by plants is mainly accumulated in the roots and only a small amount of the absorbed Hg is translocated from roots to the aerial part. In this investigation, since we focused on the phytoremediation perspectives, Hg loading of the roots has not been analyzed. Nevertheless, we obtained later results that are consistent with this finding (data yet to be published).

The aim of using an artificial substrate (vermiculite/perlite) spiked with mercury was to initially determine the maximum capability of Hg accumulation by plants, avoiding the problems derived from the availability of the metal. Thus, the shoot Hg concentrations obtained with the spiked VP substrate (Figure 2) were significantly higher (p < 0.05) than those obtained for Almaden natural soil (Figure 3). Lupine and chickpea were the highest of the three experimental series. The differences in the Hg accumulation of the different plant species in the spiked VP substrate experiment could be related to the root-specific features of the plants. Cocking et al. (1995) showed that the Hg root loading of several grasses increased with the increasing surface area of the roots. Also, Heeraman et al. (2001) found a strong relationship between Hg uptake and root-length density of Zorro fescue (Vulpia myuros L.). Analogous results were reported by Moreno et al. (2005a) for mercury uptake by Phaseolus vulgaris (bush bean), Brassica juncea (indian mustard), and Vicia villosa (hairy vetch). In our experiment with the spiked VP substrate, we found that the order for Hg shoot concentration (Figure 2) was similar to the total Hg concentration remaining in the pot substrates after harvesting (Table 1). Therefore, taking into account that leaching of mercury was caused, to some extent, by the irrigation water that occurs in our experiment, this relationship suggests that the differences in Hg accumulation could be related to the different capacity of each plant to retain mercury in its root system. Nevertheless, other factors derived from the genetic traits of the crops tested, e.g., differences in the root-to-shoot translocation mechanism, cannot be discarded (Wang et al., 2002). As was noted in the Introduction, the availability of metals in soil may be the key factor controlling the uptake of heavy metals by plants. The results obtained for the experiments using the Almaden soil agree with this fact. Shoot Hg concentrations of the plants grown on the Almaden soil without added Hg were much lower than those of the spiked VP substrate and spiked Almaden soil (Figures 2 and 3). Similar findings were reported by Moreno et al. (2004) for the phytoremediation of mine tailings treated with soluble Hg.

Spiking the Almaden soil involved an improvement in Hg availability with respect to the original soil; subsequently, absorption and translocation by the plants was significantly increased for all the crops tested. The measured CaCl^sub 2^- extractable Hg concentrations of the substrates support this conclusion. The Hg availability (measured by CaCb extraction) in the original Almaden soil was very low (ca. 0.05% of the total Hg) and was increased in the spiked soil (Table 2). It has been reported that mercury generally is relatively immobile in natural soils, remaining bound to organic matter to a large extent (Di Giulio and Ryan, 1987; Wallschlager et al., 1996). In general, only trace concentrations of Hg are found in soil solution, mostly as uncharged complexes (Schuster, 1991). In a previous article, we showed that the Hg of Almaden soils is mainly associated to humic and fulvic acids (Rodriguez et al., 2003). It is interesting to note that, contrary to what we expected, the extractable Hg concentration of the spiked soil in the nonplanted pot after 5 d was only moderately higher than that of the untreated soil (Table 2). Two phenomena can be suggested to explain this fact. Efroymson et al. (2004) have recently reviewed the differences between plant metal availability in soils when the element is aged in the field and when it is freshly added to soil in a salt solution. Using data from a large number of published works, they concluded that the common wisdom that salt amendments are generally more bioavailable than elements in field-contaminated soils is not supported by the experimental data available. Taking this into account, it can be suggested that the soluble Hg added to the Almaden soil is partially redistributed between the different geochemical fractions of the soil in the absence of plants. Likewise, the increase of shoot Hg in the plants by spiking Hg (Figure 3) means that, if the plants are present in the soil when the soluble Hg is added, some fraction of the mercury will be absorbed by the roots before its redistribution and immobilization in the plant-soil system. On the other hand, Moreno et al. (2005b) reported on the significant role of the plants in the volatilization of mercury from polluted soils by means of Hg- resistant bacteria living at the rhizosphere or inside the roots. Moreover, they observed a low rate of Hg volatilization in experiments without plants, proposing that Hg emission were due to biological transformations and photoreduction processes occurring in the substrate. This bacterial activity could, to some extent, take place in the Almaden soil, leading to a partial Hg volatilization and a subsequent decrease in the available Hg pool of the soil. Additional experimental work must be carried out to assess and quantify the contribution of these two processes to the decrease of available Hg in the Almaden soil. Anyway, in this work we have found that the concentration of available Hg in the substrate is decreased to some extent by the plant growth in all the experimental series. Therefore, our results seem to indicate that the presence of the crops can effectively reduce the plant-available Hg level of the Almaden soil.

From the phytoremediation perspective, it is necessary to estimate the Hg phytoextraction yields of the crops tested. Assuming a normal biomass production of the crops for the Almaden area, i.e., 2285, 6225, 2435, and 2400 kg (d.w.) per ha for lupine, barley, lentil, and chickpea, respectively (MAPA, 2003), and using the Hg shoot concentrations obtained in the experiment with the natural Almaden soil (the second experimental series), we calculated Hg phytoextraction yields of 4.7 g ha^sup -1^ for barley, 2.8 g ha^sup – 1^ for lentil, and 0.4 g ha^sup -1^ for chickpea and lupine. Such amounts of Hg extracted yearly (all the plant species tested are annual crops) are negligible in comparison to the magnitude of Hg contamination in the Almaden soil (more than 100 kg ha^sup -1^ of total Hg in the 0-25-cm layer). It is clear that, with this yield, the number of years required to completely extract Hg from the Almaden soil is very high, making phytoextraction unsuitable for soil-remediation purposes. However, it must be taken into account that the mercury removed by plants corresponds to the available Hg, the most toxic fraction of the soil; therefore, it can be considered to be an important first step toward the final goal, which is a reduction in the environmental risk caused by mercury pollution. As the low yield obtained is mainly due to the low availability of the mercury in the Almaden soil, the rate of Hg recovery could be improved by using some chemical-solubilization agents. This hypothesis has been successfully demonstrated by Moreno et al. (2004, 2005a, 2005b). These authors have reported the use of thiosulfate-containing solutions to induce the solubilization of mercury from mine tailings, effectively increasing Hg root uptake and shoot translocation in Brassicajuncea. With this method, a maximum Hg extraction yield of ca. 25 g ha^sup -1^ was achieved for B. juncea grown in a Hg-contaminated mine tailing treated with ammonium thiosulphate. Thus, the application of chelating agents to soils could, to a great extent, reduce the time required for soil remediation by phytoextraction. Nevertheless, the use of these chemicals amendments must be carefully considered due to the potential contamination of groundwater via leaching of the mobilized metals.

CONCLUSIONS

Four species of crop plants-barley, white lupine, chickpea, and lentil-commonly used in Spain were investigated for their ability to accumulate mercury in the aerial part of plants from contaminated substrates. The four crops were capable of mercury absorption and its accumulation in the aboveground plant tissue. In the first experiment, plants were grown in a vermiculite-perlite substrate spiked with soluble mercury. Shoot Hg concentrations up to 5.13 [mu]g g^sup -1^ were observed. However, when the same plant species were cultivated using a contaminated soil from historical mining area (Almaden, Spain), the Hg concentration of the shoot tissues was significantly lower. The decrease in mercury accumulation was attributed to the low availability of mercury in the Almaden soil. This hypothesis was confirmed by spiking soluble mercury in the same Almaden soil. With this treatment, the shoot Hg concentration of the plants was enhanced by a factor of more than 6 for lupine, more than 5 for chickpea, and more than 3.5 for barley and lentil. Analysis of CaCl^sub 2^-extractable Hg concentration of the substrates before and after plant growth confirmed that the plants were able, to some extent, to decrease the available mercury concentration of the soils. The low Hg accumulation yield in the shoot tissues would limit the use of these species for phytoextraction of contaminated soils from the Almaden area. However, since the mercury removed by the plants came from the available fraction of the soil-and, thus the most toxic-the use of these crops could be helpful to reduce the environmental risk caused by the presence of Hg in the soils of the Almaden area.

ACKNOWLEDGEMENTS

Special thanks are due to Dr. Pablo Higueras for his assistance in the use of the LUMEX RA-915+ mercury analyzer. The authors also acknowledge M. J. Lopez, A. Fernandez-Layos, and A. Garcia for their help with the pot experiments.

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Luis Rodriguez, Jesusa Rincon, Isaac Asencio, and Laura Rodriguez- Castellanos

Department of Chemical Engineering, Faculty of Environmental Sciences,

University of Castilla-La Mancha, Toledo, Spain

Address correspondence to Luis Rodriguez, Departamento de Ingenieria Quimica, Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, Avda. Carlos III, s/n, 45071, Toledo, Spain. E-mail: Luis.RRomero@uclm.es

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