ment 378 (2007) 53–57www.elsevier.com/locate/scitotenv

Science of the Total Environ

Ecotoxicological tests assessment of soils polluted bychromium (VI) or pentachlorophenol

Esther Martí ⁎, Jordi Sierra, Mónica Sánchez,Robert Cruañas, Maria Antonia Garau

Laboratori d'Edafologia, Facultat de Farmàcia, Universitat de Barcelona, Av Joan XXIII s/n. Barcelona 08028, Spain

Available online 26 March 2007

Abstract

Carbon mineralisation and plant germination and growth (Lactuca sativa seeds) tests have been performed in two soils of differentproperties, experimentally spiked with pentachlorophenol (PCP) or Cr (VI), in concentrations between 0.001 and 1000 mg kg−1. Theevaluation has been done considering the following parameters of carbon transformation test: soil cumulative basal respiration after14 days incubation, substrate induced respiration after 12 h of glucose addition, and, in the plant germination and growth test: number ofgerminated seeds, root elongation and total biomass produced. The most sensitive assay found in our work has been carbonmineralisation test, from which the lowest toxic concentrations were obtained (especially substrate induced respiration test). In the plantgermination and growth test, the measurement of root elongation has shown the best sensitiveness, followed by plant biomass and seedgermination numbers. Regarding the contaminants, the highest toxicity, considering the minimum concentration with toxic effect, hasbeen found in PCP (0.01 mg kg−1) in C mineralisation test in the granitic soil. For Cr, the minimum concentration with toxic effect hasbeen 0.1 mg kg−1, also in the C mineralisation test and the granitic soil. The granitic soil has shown more vulnerability to the pollutantsassayed in the respiration test, whereas the calcareous soil has shown more vulnerability in the plant germination and growth test.© 2007 Elsevier B.V. All rights reserved.

Keywords: Soil pollution; Pentachlorophenol; Cr (VI); Germination test; Carbon mineralisation

1. Introduction

The availability of experimental data from ecotox-icological assays allows the development of ecologicalor human health risk studies to derivate tolerableenvironmental pollutants’ concentrations. These exper-imental data have to be adjusted by the consideration ofsome uncertainty factors, to obtain, ultimately, target orreference values, for ecosystem protection or soilpollution assessment purposes (Predicted No Effect

⁎ Corresponding author. Tel.: +34 93 4024494; fax: +34 934024495.

E-mail address: marti@ub.edu (E. Martí).

0048-9697/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.scitotenv.2007.01.012

Concentration, Reference Doses, etc.; ECB, 2003),among others.

In some countries legislation, as Spain, the regulationof contaminated soils (Ministerio de la Presidencia,2005) establishes, for ecosystem protection purposes,the need to perform OECD or equivalent ecotoxicolo-gical assays in order to identify a polluted soil and to setreference values. The tests must be done on soilorganisms (plants, invertebrates, microorganisms),aquatic organisms (fishes, daphnia, unicellular algae)or terrestrial vertebrates (birds and mammalian species).

The aim of this work is the analysis of theinformation obtained from the use of some ecotoxico-logical assays performed on different soil organisms, to

54 E. Martí et al. / Science of the Total Environment 378 (2007) 53–57

know the sensitiveness and magnitude of the adverseeffects’ concentrations for two pollutants: chromium(VI) and pentachlorophenol, in two soil types usuallyfound in the Mediterranean area, with differentproperties.

2. Materials and methods

The soil samples come from the superficial layer (Ahorizon) of two air dried and sieved (2 mm) soils. Soil Mcorresponds to an Arenosol haplic (FAO-UNESCO,1998), of granitic origin and sandy texture. Its pH valueis 6.3 and it has 1.2% of oxidizable carbon. The othersample, soil T, is a Regosol calcaric (FAO-UNESCO,1998), of calcareous origin (23% CaCO3) and clayey-loamy texture, with a pH value of 8 and a 1.7% ofoxidizable carbon. Soil M has the characteristicsrequired by the OECD method to test carbon miner-alisation process (OECD, 2000).

For the tests, the pollutants, Cr (VI) and pentachlo-rophenol (PCP), have been added to the two soils at0.001, 0.01, 0.1, 1, 10, 100 and 1000 mg kg−1,including in the assay non-spiked soil samples ascontrols. Chromium has been added as an aqueoussolution of K2CrO4, and PCP by spiking fine siliceoussand due to the low water solubility of PCP. The sandwas previously treated with a methanol solution of PCPfollowed by solvent evaporation. The proportionalamount of clean sand was added to the control toachieve similar conditions.

In the Basal Respirometry assays (BR) 50 g of soilsample was used, adjusting the humidity to the 60% ofthe respective soil water holding capacity. Thesesamples were incubated in manometric respirometers,which allow the determination of the sample oxygenconsumption (Oxitop®, WTW). They were kept at30 °C in the darkness, in a termostated incubator, during

Table 1Mean variation (%), in relation to controls, of the cumulative oxygen consu(SIR) tests for the polluted soil samples

Conc. mg kg−1 % BR

Cr (VI) PCP

T M T M

0.001 −6.3 −5.4 +12.6 −10.01 −6.3 +5.0 +0.7 −0.1 −7.1 +0.8 −3.4 −11 −1.9 −7.0 −5.4 −110 −26.7 a −21.8 a −17.8 −1100 −54.8 a −32.7 a −23.6 a −41000 −59.3 a −79.6 a −80.0 a −5

Positive values indicate increases, negative values indicate inhibitions.a Significant differences compared to controls ( pb0.05).

14 days. The oxygen consumption was periodicallyregistered. Basal Respiration (BR) was determined bythe cumulative oxygen consumption at the end of theincubation period. Once the incubation was completed,Substrate Induced Respiration (SIR) was determined,according to OECD 217, carbon transformation testmethod (OECD, 2000). It was done by the addition of anaqueous solution equivalent to 4000 mg glucose per kgof soil to the incubated samples and the determination ofthe oxygen consumed during the 12 h following glucoseaddition. All the experiments have been done intriplicate.

The plants’ germination and growth tests have beenperformed with Lactuca sativa seeds, in about 20 mLplastic pots, with four replicates of 5 seeds, in 15 g ofpolluted soil in each application rate. The pollutantconcentrations and humidity conditions are those usedin the respirometric experiment, and the water lossesduring the assay were periodically restored. The plantgermination and growth assay lasted between 17 and19 days, and was performed according to OECD 208guideline (OECD, 2003). Once concluded, the follow-ing parameters were registered: number of germinatedseeds (NG), root length (RL), and 60 °C dry biomass(BM).

In order to know the statistical significance of thedifferences between treated samples and controls anANOVA test was done. Based on that, and for eachtest, a minimum adverse effect concentration of thepollutant has been established in the conditions of theexperiments.

3. Results and discussion

Respirometric results are shown in Table 1, aspercentages of variation of BR and SIR, observed in thepolluted samples related to controls. Plant germination

mption in the basal respiration (BR) and substrate induced respiration

% SIR

Cr (VI) PCP

T M T M

0.6 +2.3 0.0 0.0 −0.27.7 −3.4 −3.7 +3.3 −15.2 a

2.5 a +5.5 −14.2 a −2.8 −15.0 a

4.9 a −2.4 −13.6 a −13.3 a −19.9 a

3.7 a −38.0 a −31.5 a −19.2 a −29.0 a

0.9 a −72.2 a −37.7 a −56.7 a −25.8 a

0.2 a −86.9 a −99.9 a −94.4 a −84.1 a

Table 2Mean variation (%), in relation to controls, of the number of germinated seeds (NG), root length (RL) and biomass (BM) in the plant germination andgrowth test for the polluted soil samples

Conc. mg kg−1 % NG % RL % BM

Cr (VI) PCP Cr (VI) PCP Cr (VI) PCP

T M T M T M T M T M T M

0.001 −5 −10 −10 −15 −6 −21 −15 −15 −39 −32 +95 a +100.01 −10 −15 −15 −15 −23 −24 +3 +13 −24 −31 +43 a +380.1 −5 −5 −10 −15 −9 −13 −31 −9 −7 −22 +6 +151 −15 −15 −15 −14 −4 −21 −23 +2 −25 −18 +12 +53 a

10 0 −10 −15 0 −71 a −11 −45 a −60 a −65 a −17 +10 −18100 −5 −5 −15 −15 −92 a −15 −70 a −93 a −52 a −38 a −42 a −43 a

1000 −100 a −70 a −90 a −100 a −100 a −100 a −100 a −100 a −100 a −56 a −95 a −100 a

Positive values indicate increases, negative values indicate inhibitions.a Significant differences compared to controls ( pb0.05).

55E. Martí et al. / Science of the Total Environment 378 (2007) 53–57

and growth test results can be found in Table 2, as control-referred percentages of seed germination numbers, rootelongation and biomass. Table 3 shows the lower adverseeffect concentrations found in all the tests assayed.

As it can be observed, in the case of Cr pollution, asignificant inhibition effect can be observed at 10 mgkg−1 in soil T, in all the respirometry and plant testparameters, except for NG (1000 mg kg−1). For soil Mthe results vary, with toxicity being higher in therespirometric tests (inhibitions at 0.1 mg kg−1 in SIRand 10 mg kg−1 in BR tests), and lower in the otherassays (between 10 and 1000 mg kg−1). The resultsshow that soil M is more sensitive for Cr, and SIR is themost sensitive test in this case.

For PCP the results are more variable. In soil T, thereis a significant inhibition of SIR starting on 1 mg kg−1,of BR starting on 10 mg kg−1, of RL starting on 10 mgkg−1, of BM starting on 100 mg kg−1, and of NG on1000 mg kg−1. For soil M the inhibitions occur startingon 0.01 and 0.1 mg kg −1, respectively in SIR and BRtests, on 10 mg kg−1 in RL, on 100 mg kg−1 in BM andon 1000 mg kg −1 in NG. Moreover, a significantincrease of BM in PCP can be seen. So, also for this

Table 3Lower observed adverse effect concentrations (LOAEL values, in mgkg−1) in the tests performed

Carbonmineralisation

Plant germination-growth

BR SIR NG RL BM

T M T M T M T M T M

Cr (VI) 10 10 10 0.1 1000 1000 10 1000 10 100PCP 10 0.1 1 0.01 1000 1000 10 10 100 100

pollutant the soil M is more sensitive than soil T, andSIR test is more suitable than others.

Generally speaking, the carbon mineralisation assaysare more sensitive than plant germination and growthtest, being in this sense better SIR than BR, consideringthe inhibitory concentration. However, the monitoringof respiration activity allows seeing possible responsechanges during the incubation period (Montserrat et al.,2006).

From all the registered parameters in the plantgermination and growth test, the less interesting one,due to its low sensitiveness is NG, in all cases. Thisagrees with other authors results (Gong et al., 2001).The best, regarding its sensitiveness, has been RL,followed by BM. Also, from all of them only the BMmeasure has been able to quantitatively detect aconcentration level with stimulatory effect in one ofthe pollutants, effect known as hormesis (Chapman,2002), which was, besides, clearly observable “de visu”.We must consider that the observed effects depend, notonly on the interaction between the pollutant and thesoil, but also on the vegetal species used. Some factors,as seed properties, root morphology, physiology andothers, may modify the plant response to pollution.

So, the tests based on the effects on soil microorgan-isms clearly seem to be more sensitive for the detectionof ecotoxicity in the soil than those based on the plants’germination and growing response (Römbke andWeeks, 2003). The soil microbiota is immediatelyaffected when a pollutant reaches the soil. In thegermination-growth test there is a delay in the effectcorresponding to the time of emergence. This delayallows the soil physicochemical and biological process-es to attenuate the initial toxicity in a certain degree. Thepossible attenuation of the toxic effects of the pollutants

56 E. Martí et al. / Science of the Total Environment 378 (2007) 53–57

in the soils depend on the pollutants’ properties and thesoils’ characteristics.

For respirometric tests soil M shows more sensitive-ness for the two pollutants assayed than soil T. This isdue to the low biological activity of soil M that suggestsa sensitive microbiota (cumulative O2 consumption in14 days: 75 mg 100 g−1, compared to 120 in soil T).

Moreover, the lower toxicity of Cr on T compared toM for respirometric tests, may also be due to a short termpartial blocking effect of the soluble chromate anion, bymeans of Ca bridges on the negative charges of clayparticles, which are much more abundant in thecalcareous soil T. The low initial blocking ability ofsoil M justifies its high vulnerability in front of Cr.Besides that, in the plant germination and growth testthe toxicity on soil T is higher than on soil M. Themedium term attenuation mechanisms are important inthe decrease of toxicity in this case. In soil M, thepresence of soluble organic matter and low pH favourthe chemical reduction of Cr (VI) (Kozuh et al., 2000),leading to the formation of Cr III insoluble compoundsin the soil medium (oxides and hydroxides). This is inagreement with the decrease of soluble Cr (VI) foundalong the incubation, which is higher in soil M than insoil T (95% and 53%, respectively).

PCP is more toxic than Cr in both soils, with toxicitybeing lower in soil T. For PCP, given its low watersolubility and its lipophilic character, the adsorptiononto the soil solid phase particles could take place,mainly on organic matter, but also on clays (Pu andCutright, 2006; He et al., 2006), so avoiding themanifestation of its toxic properties. This pollutant inthe two soils is ionised, due to its low dissociationconstant (pKa 4.7). In its anionic form it may adsorbonto the soil matrix by means of cation bridges, as Ca(Shimizu et al., 1992). Ca and clays are abundant in soilT but not in soil M. So, it may become more toxic forsoil M (sandy) than for soil T (clay–loam and calciumcarbonates presence).

4. Conclusions

From the results’ analysis it can be concluded that theassays performed on soil microorganisms show higherlevel of sensitiveness than those of plant germinationand growth tests made on L. sativa seeds. This may beobserved as lower minimum significant adverse effectconcentrations. From this point of view, the toxicityobtained by means of basal respiration is lower than theobtained through the substrate induced respiration test.

From the assays based on germination and vegetaldevelopment, the determination of root length seems to

be the most sensitive parameter. However, it is the onlyone that allows to observe the occurrence of hormesis inbiomass determination.

From the assayed pollutants, PCP presents highertoxicity than Cr (VI) in the studied soils. The minimumtoxic concentrations found are 0.01 mg kg−1 for PCP,and 0.1 for Cr (VI), both for SIR test and granitic soil.The granitic sandy soil seems to be more vulnerable topollution than the clayey calcareous one for biologicalactivity measures, due to the microbiological propertiesof this soil. The calcareous clayey soil seems to be morevulnerable for the plant germination and growth testdone.

These different vulnerability characteristics lead tothe need that diverse ecotoxicological tests should beperformed in different soils to comprehensively assessecotoxicological risk of soil pollution, which is the usualtool to establish reference values for soil contamination.It seems especially convenient to test more than one typeof soil (standards), with diverse biological and physi-cochemical properties, as the results may be differentdepending not only on the pollutant and the targetorganism assayed but also on the soil properties.

Acknowledgement

This work is supported by the Spanish Ministerio deCiencia y Tecnología, REN 2003-09513-C02-01.

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