Chemosphere 84 (2011) 18

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Review

Soil plate bioassay: An effective method to determine ecotoxicological risks

R. Boluda , L. Roca-Prez, L. Marimn
Facultat de Farmcia, Departament de Biologia Vegetal, Universitat de Valncia, Av. Vicent Andrs i Estells s/n., 46100 Burjassot, Valncia, Spain

a r t i c l e

i n f o

a b s t r a c t
Heavy metals have become one of the most serious anthropogenic stressors for plants and other living organisms. Having efcient and feasible bioassays available to assess the ecotoxicological risks deriving from soil pollution is necessary. This work determines pollution by Cd, Co, Cr, Cu, Ni, Pb, V and Zn in two soils used for growing rice from the Albufera Natural Park in Valencia (Spain). Both were submitted to a different degree of anthropic activity, and their ecotoxicological risk was assessed by four ecotoxicity tests to compare their effectiveness: Microtox test, Zucconi test, pot bioassay (PB) and soil plate bioassay (SPB). The sensitivity of three plant species (barley, cress and lettuce) was also assessed. The results reveal a different degree of effectiveness and level of inhibition in the target organisms growth depending on the test applied, to such an extent that the one-way analysis of variance showed signicant differences only for the plate bioassay results, with considerable inhibition of root and shoot elongation in seedlings. Of the three plant species selected, lettuce was the most sensitive species to toxic effects, followed by cress and barley. Finally, the results also indicate that the SPB is an efcient, simple and economic alternative to other ecotoxicological assays to assess toxicity risks deriving from soil pollution. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 26 November 2010 Received in revised form 2 February 2011 Accepted 5 February 2011 Available online 9 March 2011 Keywords: Heavy metals Bioassays Rice soils Barley Cress Lettuce

Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Soil samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Microtox test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Phytotoxicity assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Soil pollution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Ecotoxicological assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Microtox test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Zucconi test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3. Pot bioassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.4. Soil plate bioassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 2 3 3 4 4 4 4 4 5 5 5 7 7 7

3.

4.

1. Introduction Generally, the effect of increasing soil pollution is a phenomenon that is noted in soils in Spain and the rest of Europe. Because of increasing anthropogenic emissions of heavy metals from agriculture (fertilizers and pesticides), metallurgy (mining and
Corresponding author. Tel.: +34 963544286.
E-mail address: boluda@uv.es (R. Boluda). 0045-6535/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.02.013

foundry works), energy production and fuel burning, microelectronic production and waste disposal, heavy metals have become one of the most serious anthropogenic stressors for plants and other living organisms (Seregin and Ivanov, 2001). Current Spanish legislation on polluted soil denes such soil as that whose characteristics have been negatively altered by the presence of dangerous human-derived chemical components whose concentration is such that it is an unacceptable risk for human health or the environment and has been expressly declared as such by legal ruling (Boluda

R. Boluda et al. / Chemosphere 84 (2011) 18

et al., 2009). In Spain, Royal Decree 9/2005 (BOE, 2005: Ofcial Gazette of the Spanish State) has established generic reference levels in polluted soil matters, except for heavy metals (Martnez and Prez, 2007). Spanish Autonomous Communities will promote measures to obtain generic reference values to declare soils to be polluted by heavy metals; yet these reference values still do not exist in the Valencian Community. Therefore it is necessary to prepare protocols to assess the ecotoxicological risks caused by soil pollution. On the other hand, soil pollutants, like heavy metals, may be associated with soil constituents in several ways: within the crystalline networks of primary minerals and of the secondary minerals that derive from edaphic alteration; adsorbed in the change complex, occluded in iron oxides and hydroxides, aluminum and manganese; sequestered or bound to organic matter; co-precipitates, and in soil solution (Gimeno-Garca et al., 1995; Rodrguez et al., 2009). Heavy metals can move from some compartments to others under the inuence of external factors like chemical weathering or the effect of plant roots metabolism. Therefore, the presence of heavy metals in soil lowers the number of living organisms living in it, alters its diversity and makes the system more fragile. Moreover, the cleaning and ltering functions in which soil participates may be affected (Frstner, 1995), and crops may absorb and accumulate these pollutants with the consequent toxicological risks for consumer health since they enter the trophic chain (Kabata-Pendias, 1995). Besides, soil uses may become limited as soils may pose unhealthy problems for users (Pierzynski et al., 2000; Martnez and Prez, 2007). Thus if the priority is to declare a soil to be polluted and to assess the level of risk to protect ecosystems, then emergency toxicity tests and seed growth in land plants are resorted to. Tests aim to provide a realistic prediction of the behavior of substances in the environment. It is certainly reasonable to investigate whether further ecotoxicity bioassays provide important information whereby a possible hazard from an environmental sample can be recognized (Boluda et al., 2002, 2009). An ecotoxicological approach using biological tests on target organisms at different trophic levels has been recommended for the sophisticated assessment of environmental hazards to complement chemical analyses (Plaza et al., 2005). Ecotoxicity tests are usually employed to detect the acute or chronic effects of substances on representative organisms such as luminescent marine bacteria (Boluda et al., 2002, 2009; Alvarenga et al., 2008; Vidic et al., 2009). Plants are also possible target organisms as they strongly depend on soil composition; alterations in their growth and various physiological and metabolic processes may reect the presence of toxic substances (Gyuricza et al., 2010). Cress is the most popular plant used in phytotoxicity studies (Zucconi et al., 1981, 1985; Wong et al., 2001; Hoekstra et al., 2002; Alburquerque et al., 2006; Walter et al., 2006; Oleszczuk, 2010; Zahir and Farrukh, 2010). Furthermore, researchers have used other plant species besides cress; for instance, barley (Vassilev et al., 2004; Katerji et al., 2006; Tariq et al., 2007; Widodo et al., 2009) and lettuce (Stevens et al., 2003; Inaba and Takenaka, Al-Maskri et al., 2010). With all this mind, the objectives of this work were to: (i) determine the pollution status by Cd, Co, Cr, Cu, Ni, Pb, V and Zn in wetland soils strongly affected by human activities; (ii) estimate the level of risk by using four ecotoxicity assays: Microtox test, Zucconi test, pot bioassay and soil plate bioassay; and compare their effectiveness; and (iii) assess the sensitivity of the three plant species: barley, cress and lettuce.

Spanish wetlands protected in Europe. Of its 21 000 ha, 14 500 ha are used for growing rice, and this practice has permitted its maintenance as a wetland. The ANP was declared as such in 1986, was also declared a Special Protection Zone for Birds (SPZB) in virtue of Guideline 79/409/CEE, and has been proposed as a Place of Community Interest (PCI) in accordance with Guideline 92/43/CEE (CMA, 2002). Serious aggressions occur in response to the industrialization of neighboring areas, demographic expansion in nearby villages, tourist urbanization in coastal areas, and the construction of a dense road network occupying 40 ha. Waste and sewage deriving from human activities are dumped into the irrigation channels and the lagoon. Excessive use of fertilizers and pesticides are other damaging factors (Boluda et al., 1993; Gimeno-Garca et al., 1995, 1996; Boluda et al., 2002; Gamn et al., 2003). The soil samples studied in this work (MAf and MSu) were taken from the rice elds in the ANP in accordance with the level of effects of human activities. The UTM coordinates of the plots are: MAf 7274367 and MSu 7334345. Soils were classied as gleyiccalcareous Fluvisol according to Gamn et al. (2003). The MAf soil was affected by urban and industrial wastewater spills, while the MSu sample was taken from the area where human activities had less impact and was used as a control. By following the UNE (1996) procedure, a reference soil was used, corresponding to a sample of commercial washed sand (MA), to conrm the reproducibility of the assays. Samples were dried, ground, homogenized and stored according to UNE regulations (1997). The following were determined: pH, electrical conductivity (EC), clay content, soil organic matter (SOM), calcium carbonate and cationic exchange capacity (CEC). Soil pH was measured in a 1:5 v/v (soil/distilled water) extract, then shaken for 5 min and measured after 2 h (UNE, 1999). SOM was analyzed by the WalkleyBlack method, while clay content was determined by sedimentation as previously reported by Day (1965). EC was measured in the 1:5 (w/v) extract. Carbonates content and CEC were determined according to the methods described in MAPA (1994). Table 1 shows the soils physic-chemical characteristics. On the one hand, we can see that MAf and MSu are soils with similar characteristics: both have a basic pH (7.5 and 8.0, respectively), a ne texture, are highly calcareous and base saturated; this is logical as they are the same soil type. On the other hand they differ in terms of EC, SOM and CEC; these parameters were higher for the MAf sample; which, as indicated by Boluda et al. (2002), could relate to irrigating MAf with urban wastewater spills. The MA sample corresponds to an inert soil. Cd, Co, Cr, Cu, Ni, Pb, V and Zn were determined according to the EPA method (1996). A representative soil sample of up to 0.5 g was digested with 9 mL nitric acid (68% w/v), 1 mL hydrogen peroxide (30% w/v), 3 mL hydrouoric acid (48% w/v), 2 mL hydrochloric acid (37% w/v) and 5 mL deionized water. Once the vessel was capped, it was placed in the microwave oven system (Mars, CEM

Table 1 Physical and chemical characteristics of soil samples. Parameters pH EC (dS m1) Clay (%) SOM (%) CaCO3 (%) CEC cmolc kg1 V (%) MAf 7.5 0.1 1.36 0.11 21 1 9.51 0.12 25 1 35 3 100 MSu 8.0 0.1 0.50 0.08 35 2 2.29 0.09 37 2 26 2 100 MA 9.1 0.2 0.05 0.01 00 nd nd nd nd

2. Materials and methods 2.1. Soil samples For this study, soil samples from the Albufera Natural Park (ANP) (Valencia, Spain) were selected. The ANP is one of the few

EC, electrical conductivity. SOM, soil organic matter. CEC, cationic exchange capacity. V, base saturation percentage. nd, not detected.

R. Boluda et al. / Chemosphere 84 (2011) 18

Corp, Matthews, NC), and the following program was run: 20 min at 200 C. Once cooled to room temperature, 30 mL of boric acid (4%) were added and heated in the microwave oven with the following program: 5 min at 170 C. The last step was performed to prevent hydrouoric acid from forming tetrauoroboric acid since hydrouoric acid attacks the glassware used in the equipment. Elements were performed and measured by ICPOES (Perkin Elmer Model Optima 5300 DV spectrometer, Norwalk, CT, USA) (Roca-Prez et al., 2010). 2.2. Microtox test This is an acute toxicity test that uses a standard suspension of marine luminescent bacteria Vibrio scheri (formerly known as Photobacterium phosphoreum) as an indicator organism (Boluda et al., 2002). The expression of inhibited luminescence in relation to the control for each assayed sample concentration was expressed as Gamma (C) following USA regulations (ASTM, 1995). Gamma is dened as the relationship between the post-exposure light lost and the post-exposure surplus light. By denition, EC50 is the concentration at which Gamma is equal to one (Microbics Corporation, 1992). The test employed estimates the toxicity of the polluted soil by means of a bioassay in water or lixiviated soil extracts. Soil lixiviates were obtained according to the procedure proposed by modied Spanish regulations based on the EP Protocol of EPA (BOE, 1989). To 50 g of fresh soil, 16 times its weight in deionized water was added, which were shaken for 24 h. Then pH was adjusted to 5.0 0.2 with acetic acid, 0.5 N (up to a maximum of 200 mL), and temperature was maintained between 20 and 30 C. Finally, the volume was completed with deionized water to reach 1 L. The obtained solution was centrifuged at 3500 rpm for 5 min and ltered using Millipore cellulose esters, 0.45 lm pore size and 47 mm in diameter, with the help of the Millipore All Glass ltering equipment and a vacuum pump. To avoid the lter from silting, a 47 mm-diameter glass ber prelter was used. Filtrates were refrigerated at between 2 C and 7 C for a maximum period of 48 h prior to the analysis. All the ecotoxicity analyses were performed using an Optocomp I luminometer (MGM Instruments, Hamden, CT, USA). To maintain the bacterium reconstituted at 5 C until the analysis commenced, and to incubate the samples with the bacterium at 15 C, a Peltier 60-well incubator block was used (Berotza, S.L., Ezkirotz, Navarre, Spain), equipped with two independent refrigeration units. The lyophilized bioluminescent bacterium, V. scheri, and the reconstitution solution were supplied by Azur Environmental (Carlsbard, CA, USA). All the solutions were prepared with reagents of analytical grade and with deionized water. A 2% NaCl solution was employed to prepare the serial samples dilutions. Acute toxicity was assessed by the basic test and the 100% test. The amended basic test was performed with a control (2% NaCl solution) and six 1:2 serial sample dilutions (79%, 40%, 20%, 10%, 5% and 2.5% v/v of the original concentration). The 100% test was done with duplicates, and with a control and four 1:2 serial dilutions (99%, 50%, 25% and 12% v/v). Immediately before the analysis, all the samples were salted at 2% with solid NaCl, and their pH were recorded. The diluted samples and controls were left at 15 C. All the tests were done with recently reconstituted bacteria and maintained at 5 C until the assay began. The luminescent bacteria reagent was hydrated with 1 mL reconstitution solution. In the amended basic test, aliquots of the reconstituted bacteria were taken (10 lL) and placed into tubes with 200 lL of the 2% NaCl solution previously left at 15 C. The light emitted by the bacterial suspensions was measured for the purpose of obtaining initial light readings. Then the control aliquots (800 lL) and the sample dilutions were added to their corresponding tubes with the bacteria. Light readings were performed 15 min after adding the sample to

the bacterial suspension. The control, or blank, was used to estimate variance with the time with which the bacteria emitted light, in the absence of toxins, and to correct bacteria-sample suspensions readings. In the 100% test, the reconstituted bacteria were directly added to the sample dilutions, and light readings were performed after 15 min. All the assays were done at the samples original pH for the purpose of determining its total toxic effect. Bacteria status and the set up of the measurement procedures were veried by reference toxins (ZnSO4 7H2O and phenol) in accordance with the AFNOR T 90-320 regulation specications (AFNOR, 1991). The ZnSO4 7H2O and phenol solutions were prepared according to the procedures detailed in the Microtox manual, and to calculate the results, the Microtox MTX7 data acquisition software was employed (Microbics Corporation, 1992). EC50 was corrected at dry weight (grams of dry soil per liter) from the humidity content in the samples. All the samples were assayed at the 99% v/v concentration. 2.3. Phytotoxicity assays To conduct this study, three phytotoxicity methods were followed, along with seeds of cress (Lepidium sativum L.) (Zucconi et al., 1981; Alburquerque et al., 2006), barley (Hordeum vulgare L.) (UNE, 1996) and lettuce (Lactuca sativa L.). They were all obtained from Verdecora (Paterna, Valencia). There are many advantages to using crop species in phytotoxicity assays: they are readily available, require no special treatment prior to sowing, and usually have consistent and reliably high germination rates (Pallet et al., 2007; White et al., 2008). The rst phytotoxcicity assay done was an amended Zucconi test (ZT) (Alburquerque et al., 2006) as the original method was used for compost. ZT was adapted for its use in soils. It is based on a germinated seeds count and on the measurement of the length of the Lepidium sativum L. radicle that had grown in the soil extracts. To obtain these extracts, 4 g of dried sample were weighed and were left at up to 60% humidity. Then they were left for 30 min and 54 mL of distilled water were added. After mechanical shaking for 30 min, the soil paste was ltered with 0.45 lm Whatman papers. The atomic absorption spectrophotometry technique was used (Thermo Solar S2-2006) to determine the concentration of the heavy metals (Cd, Co, Cr, Cu, Ni, Pb, V and Zn) in the obtained extracts. The EC for each extract was measured (Conductimeter Basic 30 Crison). Ten Petri plates were used for each soil sample, each containing a lter paper, 8 cress seeds and 1 mL of extract. To check seed viability, 10 additional Petri plates were used and 1 mL of distilled water was added on the lter paper. To maintain humidity, all the Petri plates were closed with paralm and they were incubated under dark conditions for 48 h at 28 C. Afterward, the number of germinated seeds was counted and radicle length was measured. The results are expressed as the percentage of relative seed germination (RSG), relative radicle growth (RRG) and the germination index (GI), in accordance with Hoekstra et al. (2002) (Walter et al., 2006):

RSG %

number of seeds germinated in extract 100 number of seeds germinated in distilled water mean radicle length in extract 100 mean radicle length in distilled water

RRG %

GI %

RSG RRG 100

The second phytotoxicity assay was the pot bioassay (PB), based on UNE regulations (1996), to assess the pollution of one soil. This method compares the growth rate of barley roots in the study soils

R. Boluda et al. / Chemosphere 84 (2011) 18 Table 2 The growth chamber conditions maintained. Parameters Min T (C) 23 Max T (C) 27 Min AH (%) 40 Max AH (%) 78 Humidity (%) 7072 Day length (h) 12

under standard conditions. Pots with an approximate diameter of 8 cm and a height of 12 cm were prepared. The bottom of these pots was perforated and covered with lter paper. They were then lled with 500 g of soil up to a height of 10 cm. Soil water holding capacity was maintained at 70% during the assay. To obtain this gure, the amount of water required was determined in accordance with ISO 11274. Thus, 105 mL of water were added to the pots and this amount remained throughout the assay. Previously, seeds were germinated in Petri plates on top of lter paper moistened with distilled water for 48 h at 23 C. Then when radicles emerged, but providing their length was under 2 mm, six seeds were sown into each pot with radicles placed downward at approximately 10 mm below the soil surface. Table 2 presents the growth chamber conditions maintained over the 5-d growth period. After this time, each pot was placed on its side in a 5-cm cuvette with water to carefully remove the soil from the pots, and each plant was washed. The root length and the aerial part of them all were measured. The third phytotoxicity assay was the soil plate bioassay (SPB). The Microtox test, ZT and PB are the acute toxicity assays normally employed in the scientic literature. The SPB test is our proposal as an alternative to the aforementioned tests. SPB is based on the study of seed germination and seedling growth under controlled conditions over a specic time period and is performed directly on the soil sample placed inside a Petri plate (Boluda et al., 2009). Barley, cress and lettuce seeds were tested. Two Petri plates were prepared for each soil sample and for all three plant species. Then 20 g of soil were added in each one, to which water was added to 70% of their soil water holding capacity. In parallel, and with a view to checking seed viability, two additional plates were used and 1 mL of distilled water was added to them on top of Whatman 0.45 lm lter paper. After sowing 20 seeds per Petri plate, plates were closed with paralm and incubated for 5 d under dark conditions at 27 C. After this growth period, soil was removed from plates and each seedling was washed. The root length and aerial part of them all were measured. For both PB and SPB, the results are expressed as a tolerance index, I/Io = Ln/L0, where Ln is the root or shoot length for the problem sample (MAf) and for the reference soil (MA), and L0 is the root or shoot length for the control soil (MSu). All the experiments were done per triplicate. 2.4. Statistical analysis For ZT, PB and SPB, the means, standard deviations and coefcients of variation were calculated. Statistical analysis was performed by one-way analysis of variance (ANOVA) using SPSS 17.0. For PB and SPB, ANOVA was applied separately to data of the roots and shoots. Means were compared by a Tukeys or a CDunnets test depending on variance homogeneity using signicance level of 0.05. When the level of signicance was <0.05, a CDunnets test was done, but a Tukeys test was performed if it was >0.05. The EC50 values from the Microtox test were obtained by a linear regression analysis on bioluminescence inhibition at the various toxicant concentrations. 3. Results and discussion 3.1. Soil pollution Table 3 displays the total concentration (mg kg1) of Cd, Co, Cr, Cu, Ni, Pb, V and Zn in the soils under study where a large degree of variability is seen among the three samples. Apart from Co and V, the concentration of the remaining metals in the MAf sample is very high, especially if compared with the two control soils (MSu and MA). Besides, these concentrations are much higher than those

Min AH, minimum ambient humidity; Max AH, maximum ambient humidity.

Table 3 Cd, Co, Cr, Cu, Ni, Pb, V and Zn concentrations (mg kg1) in soil samples, and the reference value (RV) from Roca-Prez et al. (2010). Element Cd Co Cr Cu Ni Pb V Zn nd, not detected. MAf 3.01 0.16 11 3 675 44 538 9 136 13 381 54 45 1 823 39 MSu 0.62 0.17 81 46 0 20 2 20 1 10 2 43 2 55 5 MA nd nd nd nd nd nd nd nd RV 0.97 35.5 217 46 50 137 120 246

obtained by other authors (Boluda et al., 1993; Gimeno-Garca et al., 1995; Andreu and Boluda, 1995) for rice eld soils from the ANP. This is due to the vast contribution of urban and industrial wastewaters that spill into the irrigation channel of this plot, as described by Boluda et al. (2002). As there is no legislation on heavy metals pollution in the Valencian Community, our levels were compared with the reference values (RV) proposed by Roca-Prez et al. (2010). As Table 3 shows, the MAf sample presented a very high level of pollution for Cd, Cr, Cu, Ni, Pb and Zn. In this sample, all the metals were above the RV by as much as three times, except Cu which exceeded the RV by as much as ten times. According to Jones and Jarvis (1981), the combined presence of Cu, Ni and Zn enhances its toxic effects and, its validity is restricted only for pH values higher than 6.5 (Andreu and Boluda, 1995). It is well-known that heavy metals adsorption is directly controlled by pH. Nonetheless, there are some exceptions, like Cr whose availability is favoured by an alkaline pH (Rodrguez et al., 2009). What this indicates is that MAf is a soil that is highly polluted by Cd, Cr, Cu, Ni, Pb and Zn and can, therefore, suggesting possible ecotoxicological risks. 3.2. Ecotoxicological assays 3.2.1. Microtox test The bacterial verication test performed with phenol provided a mean EC50-150 value of 28.0 mg L1, with a coefcient of variation (COV) of 11.1% (n = 10). The mean EC50-150 value obtained for zinc sulfate heptahydrate (expressed as ZnSO4 7H2O) was 9.2 mg L1 with a COV of 14.8% (n = 12). According to French norms (AFNOR, 1991), under normal conditions and with reagents in a good state, the test with phenol results in a EC50-150 value of between 13 and 40 mg L1, while that done with ZnSO4 7H2O results in a EC50-150 value of between 2.2 and 11.0 mg L1 (expressed as ZnSO4 7H2O), and between 0.5 and 2.5 mg L1 (expressed as Zn2+). With these obtained results, it was concluded that the bacteria lot fullled the validation specications according to AFNOR (1991). To set up the toxicity bioassay, preliminary variability studies were conducted with real lixiviated soil samples, resulting in a COV of 16.0% (n = 6) for EC50-150 . The acute toxicity results obtained with lixiviates are shown in Table 4. The values of 1.1 UT50-150 (units of toxicity) correspond to samples MSu and MA, while a value of 4.1 UT50-150 was obtained for sample MAf. The similarity of the UT between MSu and an inert

R. Boluda et al. / Chemosphere 84 (2011) 18 Table 4 Results obtained by Microtox test. Soil samples MAf MSu MA UT50-150 (units of toxicity) 4.1 1.1 1.1 EC50-150 (g L1) 8.6 1.9 29.1 7.1 34.2 3.1

the results. The concentrations obtained were as follows (mg L1): Cd (<0.05), Cu (0.42), Cr (<0.05), Ni (0.08), Pb (<0.05) and Zn (0.01). EC was also low (0.95 dS m1 25 C) as it was a highly diluted extract: 1:13.5 (w/v). This fact conrms the phytotoxicity results obtained with ZT, and indicates once more that this test is not sensitive to this pollution type in the soils studied in this work. 3.2.3. Pot bioassay Fig. 2 depicts the results obtained after performing the pots bioassay using barley in accordance with UNE (1996) norms. As expected, barley seedlings development in the reference soil (MA) presented a tolerance index that is suitable and similar to the values obtained for the corresponding MSu sample soil. However, this tolerance index value in the plants corresponding to the polluted sample had a higher value for both root and shoot elongation. This scenario indicates that barley growth was not inhibited in the same way as in the two bioassays used formerly. Indeed, the ANOVA showed no signicant differences. This lack of induction of the inhibition in plant growth of the polluted sample suggests the poor sensitivity of this test when faced with a simultaneous pollution by Cd, Cr, Cu, Pb, Ni and Zn at the levels of our assessed soil. 3.2.4. Soil plate bioassay Both ZT and PB are the phytotoxicity assays normally used in the scientic literature. As previously mentioned, the SPB test aims to be a new proposal and an alternative to these assays. The results obtained to assess the possible inhibition of plant growth in the three study plant species (barley, cress, lettuce) are offered in Fig. 3. In general, roots grew less than the aerial part, except for barley in samples MAf and MA. Contrary to the results obtained in the former bioassays, the control (MSu) and the reference (MA) soils showed a different behavior pattern, and signicant differences were found for all three plant species. Therefore, other than cress root elongation, the plants in the other two cases grew better in our control soil given its improved properties (Table 1). This indicates the need to use a soil whose characteristics are similar to the soil being assessed when a comparison standard is required. When comparing the plant growth of these two soils, inhibition was 5% for barley and 20% for lettuce for root elongation; and 35% for barley, 8% for cress and 30% for lettuce for shoot elongation (Fig. 3ac, respectively). The only exception was the case of cress roots which displayed a positive induction (30%) in the reference soil (MA). In terms of the polluted soil (MAf), growth was clearly inhibited for all three plant species, and the fact that lettuce seeds did not germinate is worth stressing (Fig. 3c). Therefore, when comparing plant growth with the control soil (MSu), inhibition was 55% for barley, 57% for cress and 100% for lettuce for root elongation; and 62% for barley, 43% for cress and 100% for lettuce in the case of shoot elongation (Fig. 3ac, respectively). A further aspect to point out is the fact that the seedlings roots in all cases were shorter than shoots (data not shown); according to the data reported by most authors, root growth is more sensitive to the impact of heavy metals than shoot growth (Seregin and Ivanov, 2001). In addition, the statistical analysis (ANOVA) revealed clearly signicant differences for plant growth between MSu and MAf. It is also worth mentioning that in this test, the most sensitive plant species to toxic soil effects of this soil was lettuce. All these results evidence that the Microtox test and the phytotoxicity ZT and PB assays are not sensitive enough to the adverse characteristics present in the polluted soil assessed in this work, unlike the SPB test which proved much more sensitive. This result may be due to several factors inuencing the soilplant system relationships and the ecotoxicity assay conditions. Under our experimental conditions, the most important factors to consider are: edaphic properties, bioavailability of the pollutants present

soil (MA) conrms that the control sample was a good choice. In accordance with the toxicity criterion followed (Vasseur et al., 1986), the lixiviates corresponding to samples MSu and MA were not toxic, while the lixiviate of sample MAf was slightly toxic. If samples were ordered from greater to lesser toxicity in accordance with the EC50-150 value obtained and expressed as g of dry soil per liter, then the following is obtained: MAf (8.6 1.9 g L1) > MSu (29.1 7.1 g L1) = MA (34.2 3.1 g L1). According to this test, and in order to consider that a soil is toxic, the EC50-150 value must be under 3 g L1, a value that is almost three times lower than that obtained for sample MAf. Therefore by considering the concentrations of Cd, Cr, Cu, Ni, Pb, and Zn present in sample MAf, it can be undoubtedly stated that this test is not sensitive enough to assess this type of pollution in soils with similar characteristics (pH, texture, SOM and calcium carbonate). 3.2.2. Zucconi test The RSG and RRG values obtained as a percentage were: in sample MAf (109 0 and 135 6), in sample MSu (106 1 and 131 2) and in sample MA (97 4 and 119 3), respectively. In all three cases, we can see how relative seed germination and relative radicle growth present similar values that are around 100%. What these values indicate is the good viability of the seeds, a good choice of reference soil and control soil and, therefore, assay validity. Likewise, the similarity of these parameters between samples MSu and MAf is closer than between MA and Maf; this is logical because MSu and MAf correspond to the same soil type. The GIs were calculated with these data, and their values are provided in Fig. 1. Surprisingly, and as seen, the GIs of the three samples were high, which suggests that radicle growth was not inhibited in cress since, according to Zucconi et al. (1985), values over 80% indicate that the material presents no phytotoxicity problems. For the purpose of assessing the possible differences for this index, an ANOVA was done and, as expected, this statistical analysis showed no signicant differences among the results obtained. Consequently, the metals concentration was determined, as was the EC in the ZT extract used with the MAf sample which, in theory, should present a greater phytotoxic risk, with a view to conrming the reliability of

Fig. 1. Germination index (%). MAf, contaminated soil; MSu, control soil; MA, reference soil. Values followed by the same letter do not differ signicantly at the 5% level. Vertical bars are standard deviation.

R. Boluda et al. / Chemosphere 84 (2011) 18

Fig. 2. Tolerance index (I/Io = Ln/L0) for root elongation and for the aerial part of barley seedlings grown in soils by means of PB, where Ln is root length or shoot length for the problem sample (MAf) and for the reference soil (MA), and L0 is the root or shoot length for the control soil (MSu). Values followed by the same letter do not differ signicantly at the 5% level. Vertical bars are standard deviation.

in soil, and the relationships established among the solid phase, the liquid phase of the edaphic environment, and the plants root metabolism in which they take place, as well as the sensitivity of the organism used in the bioassay. Thus, our soil is strongly calcareous and base saturated, it has a ne texture, a high organic matter content and cationic exchange capacity, a basic reaction and it presents high salinity; this suggests that the bioavailability of pollutants like heavy metals will be low, which was demonstrated when their concentration in the aqueous ZT-extract was analyzed. Along these lines, several authors have shown the low bioavailability of heavy metals under our soil conditions, which may be strongly xed to clay minerals, organic matter or to co-precipitates that take the form of carbonates, suldes, and of oxides and hydroxides of iron and aluminum (Gimeno-Garca et al., 1995; Kabata-Pendias, 1995; Rodrguez et al., 2009). So it is very hard for them to enter the soil solution, which must have occurred during the process followed to obtain the soil-lixiviate in the Microtox test and the ZT-extract, thus avoiding inhibition of bacteria development or cress germination. However, it is also necessary to point out that ZT was designed to assess phytotoxicity and the degree of compost maturation (Zucconi et al., 1981, 1985; Wong et al., 2001; Alburquerque et al., 2006), as well as the phytotoxic effects of different organic residues (Hoekstra et al., 2002; Walter et al., 2006). Regarding PB and SPB, another striking aspect to consider is that seedlings developed in direct contact with the soils liquid and solid phases; thus the soilplant interaction in these experiments must be considered when interpreting the results. Metal availability depends on several soil and plant factors (Alegria et al., 1991). On the one hand, and as mentioned earlier, soil properties and characteristics affect the reactions, transformations and mobility of heavy metals (Gimeno-Garca et al., 1995; Kabata-Pendias, 1995; Rodrguez et al., 2009). On the other hand, the rhizosphere receives appreciable amounts of organic material from roots, including exudates, mucilage, sloughed-off cells and their lysates (Alloway, 1990; Marschner, 1995). Researchers have found that these low-molecular-weight organic acids in the rhizosphere are capable of forming complexes with metal ions in solution, and can thus enhance the mobilization of metals and their uptake by plants (Inaba and Takenaka, 2005; Koo et al., 2010). So although sample MAf has physico-chemical characteristics that permit the

retention and precipitation of most heavy metals in the soils solid phase, root activity must have favored the mechanisms that enable the solubilization and absorption of these elements, thus bringing about toxic effects. Therefore, the plant growth inhibition observed in SPB (Fig. 3) must have been the result of the phytotoxic effect triggered by these mechanisms. Nevertheless, this inhibition was not seen in PB (Fig. 2); in this case, apart from the aforementioned edaphic factors, which are capable of diminishing the metals bioavailability, another very important factor to contemplate is the selectivity of roots and their capacity to explore the soil environment. In other words, the containers used in PB were larger, and had a much larger volume of soil available for roots (larger by some 25 times) than in SPB. The uptake of metals from soils is greater in plants grown in pots with soil in greenhouses than from the same soil in the eld. This is probably due to differences in microclimate and soil moisture, and to the roots of container-grown plants growing solely in polluted soil, whereas those of eld-grown plants may reach down to less polluted soil (Alloway, 1990; Marschner, 1995). Finally, it is necessary to consider another factor to account for the high degree of variability noted in plant growth. Indeed, the fact that lettuce seeds did not germinate may be due to the combined effect of the metals concentration with soil salinity. From the results obtained, the following classication from lesser to greater sensitivity may be made in relation to the heavy metals and the salinity of sample MAf: barley > cress ) lettuce. When assessing cationic metal toxicity in soils, metals are often added to soil as chloride, nitrate or sulfate salts. Phytotoxicity could be the effect of increased salinity associated with counter-ions applied with metal salts (Stevens et al., 2003). It is well-known that seed germination, seedling growth and physiological activities of some cultivated plants are strongly affected by soil type and salinity stress. As pointed out by Zahir and Farrukh (2010), salinity causes osmotic stress or specic ion effects, which delay, reduce or completely inhibit seed germination. Thus, the variability obtained in the growth of the roots and the shoots among barley, cress and lettuce could also be due to differences in the sensitivity or tolerance of the three plant species under study to the phytotoxicity of heavy metals combined with the salinity of sample MAf. Lots of works have recently reported that barley tolerates high Cd, Cu, Ni and Zn concentrations present in the solution and accu-

R. Boluda et al. / Chemosphere 84 (2011) 18

salt-tolerant at the germination and seedling growth stages (Zahir and Farrukh, 2010). Salinity levels of more than 2.0 and 2.6 dS m1 reduce lettuce fresh yield and plant growth, respectively (De Pascale and Barbieri, 1995; Al-Maskri et al., 2010). All these aspects, together with the analysis of the procedures of the four ecotoxicological assays assessed in this work, conrm that SPB is a sensitive, simple and economic method and is an alternative to other toxicity assays that assess soil pollution status by considering both the solid and the liquid phases of the edaphic environment. 4. Conclusions If the goal of phytotoxicity testing is the protection of wildlife habitats and biodiversity, as well as plants of agronomic interest, the range of testable species must be expanded, just as this study has done with barley, cress and lettuce, and a reference base for a comparative risk analysis must be established. The bioassays used in this work allowed obtain relevant results. The Microtox test, ZT and PB were found to be less sensitive methods than SPB to assess the toxicity of a clearly calcareous soil polluted with Cd, Cr, Cu, Ni, Pb and Zn. The variations in roots and shoots length during the germination stages of barley, cress and lettuce seeds is a good parameter to assess the effect of inhibited plant growth deriving from a phytotoxicity effect in a polluted soil. It has been possible to deduce that this toxic effect is not merely related with Cd, Cr, Cu, Ni, Pb and Zn concentrations, but could also be due to a combined effect owing to the salinity of the soil under study. The results enabled an order of increasing sensivity decreasing tolerance for the three plant species: barley > cress > lettuce. Furthermore, SPB offers important advantages if compared to the other three as it is simple, sensitive, rapid and economic. Future studies must be conducted with other soil types and polluting agents to conrm the results obtained in this work. Acknowledgment This research work has been supported by the Spanish government MICINN, Project CGL2006-09776. References
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Fig. 3. Tolerance index (I/Io = Ln/L0) for root elongation and the length of the aerial part of the barley (a), cress (b) and lettuce (c) seedlings grown by means of the SPB test in soils, where Ln is the root length or shoot length for the problem sample (MAf) and for the reference soil (MA), and L0 is the root or shoot length for the control soil (MSu). Values followed by the same letter do not differ signicantly at the 5% level. Vertical bars are standard deviation.

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