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Environmental Pollution 159 (2011) 722e728

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Environmental Pollution

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Lumbricus terrestris L. activity increases the availability of metalsand their accumulation in maize and barley

E. Ruiz a, J. Alonso-Azcárate b, L. Rodríguez a,*

aDepartment of Chemical Engineering, School of Civil Engineering, University of Castilla-La Mancha, Avenida Camilo José Cela, s/n, 13071 Ciudad Real, SpainbDepartment of Physical Chemistry, Faculty of Environmental Sciences, University of Castilla-La Mancha, Avenida Carlos III, s/n, 45071 Toledo, Spain

Lumbricus terrestris L. activity increases the bioavailability of metals in

soils and total, shoot and root metal concentrations for maize and barley.

a r t i c l e i n f o

Article history:Received 17 July 2010Received in revised form13 November 2010Accepted 24 November 2010

Keywords:Soil pollutionLumbricus terrestrisEarthwormsSequential extractionMetal availability

* Corresponding author.E-mail address: Luis.Rromero@uclm.es (L. Rodrígu

0269-7491/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.envpol.2010.11.032

a b s t r a c t

The effect of the earthworm Lumbricus terrestris L. on metal availability in two mining soils was assessedby means of chemical extraction methods and a pot experiment using crop plants. Results from singleand sequential extractions showed that L. terrestris had a slight effect on metal fractionation in thestudied soils: only metals bound to the soil organic matter were significantly increased in some cases.However, we found that L. terrestris significantly increased root, shoot and total Pb and Zn concentrationsin maize and barley for the soil with the highest concentrations of total and available metals. Specifically,shoot Pb concentration was increased by a factor of 7.5 and 3.9 for maize and barley, respectively, whileshoot Zn concentration was increased by a factor of 3.7 and 1.7 for maize and barley, respectively.Our results demonstrated that earthworm activity increases the bioavailability of metals in soils.

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1. Introduction

The impact of earthworms on soil properties and plant growthhas been widely studied during the last two decades (Edwards andBater, 1992; Zhang and Schrader, 1993; Scheu, 2003; Amador andGorres, 2005). Earthworm soil activities such as burrowing, castingand mixing of litter and soil (bioturbation) have important conse-quences for soil processes and properties. Earthworms improvesoil structure due to the increase of macroporosity with effects onaeration, water dynamics, soil organic matter dynamics and provi-sion of paths for root exploration (Amador and Gorres, 2005; Wenet al., 2006; Suthar et al., 2008). They can alter the ecology of thesoil by suppressing plant pathogens and/or promoting growth ofmicroflora and fauna that may be beneficial to crops (Clappertonet al., 2001). Moreover, earthworms are capable of increasing thebioavailability of several soil nutrients such as C, N and P (DevliegherandVerstraete,1996; Cheng andWong, 2002; Chaoui et al., 2003), aswell as metal availability (Devliegher and Verstraete, 1996; Chengand Wong, 2002; Ma et al., 2002; Wen et al., 2004; Udovic andLestan, 2007; Sizmur and Hodson, 2009).

Furthermore, earthworms are relatively efficient accumulatorsof certain essential and non-essential metals, such as Cd, Cu, Zn and

ez).

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Pb (Morgan et al., 1989; Morgan and Morgan, 1999; Lanno et al.,2004; Yu et al., 2005). Chemical analysis of earthworms exposedto or collected in soils containing toxic substances such as metalscan also give an indication of the bioavailable amount of a selectedtoxicant (Maleri et al., 2008).

Availability of metals in soils is considered an importantparameter for the effectiveness of the uptake and accumulation ofmetals by plants (Zhang and Shan, 2000). Chelating agents, such asEDTA and its structural analogues, e.g. DTPA, EGTA and NTA, havebeen used to enhance metal solubility in soils and hence increasemetal accumulation in plants (Meers et al., 2005a). However, side-effects related to the addition of chelates, e.g. metal leaching, maycause additional health, safety and environmental concerns (Kosand Lestan, 2003). Some researchers have assessed the feasibilityof using earthworms to modify the metal availability in pollutedsoils and enhance plant uptake (Cheng andWong, 2002;Wen et al.,2006; Ruiz et al., 2009). However, further research is necessaryto study the differences due to different earthworm species andto assess the mechanisms for metal mobilization (Sizmur andHodson, 2009).

The main objective of the present study is to assess the abilityof the earthworm species Lumbricus terrestris L. to change metalavailability in polluted soils from an old PbeZn mining area bycombining chemical extraction methods, i.e. single extraction with0.01 M CaCl2 and water and BCR sequential extraction, and a plantbioassay using crop plants.

Table 1Land use, selected physicochemical properties (<2 mm fraction) and total metalconcentration (<63 mm fraction) of the two soils used in this study.

Parameter Soil

S1 S2

Land use Arable land Mine tailingpH-H2O 6.30 7.51EC (ms cm�1) 150 83.9Clay (%) 16.2 12.1Silt (%) 51.1 21.4Sand (%) 32.7 66.5CECa (meq 100 g�1) 16.7 8.56TOCb (%) 0.94 1.46Water-Holding Capacity (%) 38.5 26.1Total Cu (mg kg�1) 34.9 38.7Total Pb (mg kg�1) 133 821Total Zn (mg kg�1) 299 674

a Cation exchange capacity.b Total organic carbon content.

E. Ruiz et al. / Environmental Pollution 159 (2011) 722e728 723

2. Materials and methods

2.1. Soil sample collection and preparation

Two different soils, taken from arable land (S1) and mine tailings (S2) in thesurrounding area of an abandoned PbeZn mine in Spain (San Quintín mine), wereused in this study. Soils were sampled from the top 0e25 cm soil layer, air-dried atambient temperature for 72 h, disaggregated and, finally, sieved to <2 mm and<63 mm. The <2 mm fraction was used for plant pot experiments and for themeasurement of the soil’s physico-chemical properties. The <63 mm fraction wasused to analyze the total metal content, as well as BCR sequential extraction, waterand 0.01 M CaCl2 extractions.

Soil pH and electrical conductivity (EC) were measured in a 1:5 soil/watermixture; organic carbon was determined using a total organic carbon analyzer(Shimadzu TOC-VCSH, Columbia, USA); particle-size distribution (clay, silt and sandcontent) was determined using laser diffractometry (Beckman Coulter LS, Fullerton,USA); cation exchange capacity (CEC) was measured by the ammonium acetatesaturation method (MAPA, 1994). To determine the total concentration of Cu, Pb andZn in the soils (<63 mm fraction), 0.5 g of sample was digested with a mixture ofacids (9 mL of concentrated HNO3þ 3 mL of concentrated HCl) in a microwave unit(CEMMARS 5, Matthews, USA), according to the EPA 3051A method (USEPA, 1996a).

2.2. Pot experiments

Plastic pots were filled with 1 kg of the air-dried soils sieved to <2 mm. Sixdifferent experimental series were carried out simultaneously for each soil.The experimental series were: control soil, without plants and earthworms (C); soilwith earthworms and without plants (E); soil with maize, without earthworms (M);soil with barley, without earhworms (B); soil withmaize plus earthworms (ME); andsoil with barley plus earthworms (BE). Each series consisted of three replicates(using three separate pots); earthworms from each pot were treated separately.

Barley (Hordeum vulgare) and maize (Zea mays) seeds were pregerminated ina germination chamber (ASL IBERCEX, Madrid, Spain) for 3 days at 20e24 �C, 60%relative humidity and 46.8 mmol/m2/s light intensity. Next, a total of 20 barley and 10maize seeds were sown per pot and later reduced to 10 and 5 seeds per pot,respectively. The soil was adjusted to approximately 60% of its water-holdingcapacity, and water loss was compensated daily by the addition of deionized water.After three days the E, ME and BE series were inoculated with 12 L. terrestrisworms.The earthworms were obtained from a local commercial supplier (Armeria20,Toledo, Spain), who imported them from a commercial vermiculture supplier(Vivastic, Elsenheim, France). All individuals were adult worms with clitellum anda mean fresh weight of 436� 51 mg.

The pot experiment was carried out with continuous light (24 h day�1) to keepthe earthworms in the soil. Since the optimal temperature for L. terrestris develop-ment is between 15e20 �C (Lowe and Butt, 2005), the pot experiments wereconducted at a constant temperature of 17 �C.

Plants were harvested 4 weeks after planting. Plant shoots and roots werethoroughly washed with de-ionized water, weighed, stored at �80 �C, freeze-dried,finely ground to fine powder by a ball mill (Retsch MM200, Haan, Germany) andsealed in plastic bags for subsequent heavy metal analysis. The dry plants weredigested in the above mentioned microwave unit using a mixture of HNO3/HCl/H2O2, according to the 3052 EPA method (USEPA, 1996b). The total metal accumu-lation rate (TMAR) in maize and barley was calculated by the following equation:

TMAR ¼�½M�rootDWroot

�þ �½M�shootDWshoot�

ðDWroot þ DWshootÞwhere [M]shoot and [M]root represent the total metal concentration in plant shootsand roots (mg kg�1), respectively, while DWroot and DWshoot represent the weight ofplant roots and shoots (kg), respectively.

At the end of experiment, the earthworms were washed and placed in Petridishes for 48 h to remove ingesta residues in the guts (Cheng and Wong, 2002;Udovic and Lestan, 2007); earthworm casts were removed each day. Later, theearthworms were weighed, stored at �80 �C and freeze-dried. The dried earth-worms were digested in the microwave unit using 10 mL of HNO3 (EPA 3051Amethod). As was pointed out above, all the earthworms from each pot were treatedas separate pools. Simultaneously, three specimens of L. terrestris, not used in anexperiment, were washed, depurated, dried and digested as described above.These specimens were used as a control (control earthworms, CE) to establish theaccumulation of metals by earthworms in the pot experiment.

The concentrations of Cu, Pb and Zn in plant, soil and earthworm extracts wereanalyzed by ICP-OES using a Thermo ICAP 6500 spectrometer (Thermo Electron,Cambridge, UK).

2.3. Single and sequential extraction procedures

Single extractionwith water and 0.01 M CaCl2 and the BCR sequential extractionmethod were applied to the air-dried soil samples (<63 mm fraction) collected afterharvesting the plants.

The BCR sequential extraction scheme was applied in this work to assess thechanges in the chemical partitioning of Cu, Pb and Zn in the soil after the treatments(Sahuquillo et al., 1999). It basically consisted of three extraction steps, namely:(i) Step 1, extraction with acetic acid 0.11 M (to obtain the F1 fraction); (ii) Step 2,extraction with hydroxylamine hydrochloride 0.5 M at pH 1.5 (F2 fraction); and (iii)Step 3, reaction with 8.8 M H2O2 followed by an extraction with 1.0 M ammoniumacetate at pH 2 (F3 fraction). Residue from the third step was digested by the above-mentioned 3051A EPA method (to obtain the F4 fraction). The F1 fraction is usuallycalled ‘exchangeable’ and accounts for water-extractable, exchangeable andcarbonate-boundmetals; the F2 fraction is called ‘reducible’ and it mainly consists ofmetals bound to FeeMn oxides; the F3 fraction is called ‘oxidizable’ and it accountsfor metals bound to organic matter and sulphides; finally, the F4 fraction is called‘residual’ and consists of metals bound to mineral lattices (Zemberyová et al., 2006).

The quality of the analytical data for the BCR sequential extractionprocedurewasassessed by carrying out analyses of the BCR-701 certified reference material(LGC Promochem, Barcelona, Spain). At least one reference material and a blanksample (taken through the complete procedure but in the absence of a sample) wereincluded in each batch of samples. Measured values of the reference material weretypically �15% of certified values; an accuracy that can be considered as acceptable(Marguí et al., 2004; Cappuyns et al., 2007).

Two additional single extraction methods were used. Water-extractable metalswere determined by shaking 2 g of soil with 20 mL of milli-Q water for 2 h and thencentrifuging at 3000g for 10 min. The 0.01 M CaCl2-extractable metals were deter-mined according to the method reported by Novozamsky et al. (1993): 2 g of soil in20 mL of 0.01 M CaCl2, shaking for 2 h.

All the extractions were carried out in triplicate and reagent blanks wereincluded in every batch. Finally, water- and CaCl2-extractable Cu, Pb and Znconcentrations were measured by ICP-MS in a Thermo XSeries II spectrometer.The concentration of metals extracted in the BCR sequential extractionwas analyzedby ICP-OES using the above-mentioned Thermo spectrometer.

2.4. Statistical analysis

All statistical analyses were carried out with the program Statistica 6.0 (StatsoftInc., Tulsa, USA). A Shapiro-WilkW-test for normality was performed to determine ifthe data came from a population with normal distribution. Then, one-way ANOVAwas used to assess the effect of earthworm presence on metal fractionation instudied soil and to compare earthworm weights before and after the experiments.ANOVA followed by Tukey’s HSD tests were performed to determine significantdifferences between the Cu, Pb and Zn accumulation in the earthworms belonging todifferent experimental series. The same statistical method was used to comparemetal uptake of barley and maize in the S1 and S2 soils with and withoutearthworms.

3. Results and discussion

3.1. Soil characterization

Table 1 shows themain physico-chemical properties, total metalconcentrations and mineralogical analysis for the S1 and S2 soilsamples.

The S1 soil was almost neutral while the S2 soil had a slightlyalkaline pH value. Total organic carbon (TOC) content of the S1

0.00

1.00

2.00

3.00

4.00

5.00

E ME BE

**

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E ME BE

**

A

B

Initial

After experiment )g(thgie

whserf

mrowhtrae

naeM

)g(thgie

whserf

mrowhtrae

naeM

Fig. 1. Changes in theweight of Lumbricus terrestris earthworms before and after the potexperiment (4 weeks at 17 �C): A) S1 soil; B) S2 soil. E: pots with only earthworms; ME:pots with earthworms and maize; BE: pots with earthworms and barley. Mean values(n¼ 3) and standard deviation (error bars). * denotes statistically significant differencesin earthworm fresh biomass before and after the pot experiment (p< 0.05, ANOVA).

0

100

200

300

400

500

600

Cu Pb Zn

CE

E

ME

BE

CE

E

ME

BE

a bab b

bbb

a

a

ab

bb

0

100

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300

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Cu Pb Zn

a a

a

b

ab

b b b

ab

b b b

A

B

gkg

m(s

mrowhtrae

ninoitartnecnoclate

M-1

)gk

gm(

smro

whtraeni

noitartnecnoclateM

-1)

Fig. 2. Metal accumulation in Lumbricus terrestris tissues after 4 weeks of exposure:A) S1 soil; B) S2 soil. CE: Control earthworms (worms as received); E: pots with onlyearthworms; ME: pots with earthworms and maize; BE: pots with earthworms andbarley. Mean values (n¼ 3) and standard deviation (error bars). Bars sharing the sameletter are not significantly different (p< 0.05, Tukey’s HSD test).

E. Ruiz et al. / Environmental Pollution 159 (2011) 722e728724

sample can be considered a normal value for an arable land soil.However, TOC content of the mine tailing sample S2 was signifi-cantly higher; it may be attributed to the presence of extensivevegetation cover in the sampled area (an old pond). The S1 soil wascharacterized by high clay and silt content compared with S2.The different content of finer size soil fractions was reflected in thedifferent CEC values found in these soils with S1 showing highervalues than S2. This may be also attributed to the presence ofsmectite in S1 (data not shown), as this clay mineral has a very highCEC value. Total metal concentrations were higher in S2 comparedwith S1 for all the tested metals. S2 was classified as polluted soilfor Pb and Zn according to the threshold values (300 mg kg�1 forboth metals) established by European Directive 86/278/EEC for theaddition of sludge as an amendment to agricultural soils (Council ofthe European Communities, 1986).

3.2. Earthworm biomass and metal accumulation

The earthworm species L. terrestris was chosen for the studybecause this species dominates earthworm biomass in varioustemperate ecosystems and strongly affects organic matter trans-formation and soil development (Kizilkaya, 2004).

In general, thewormswere alive at the end of the pot experimentsand their casts and the holes burrowed were clearly visible in all thepots. Nevertheless, some worms died during the experiments. Sixand two worms, respectively, were found dead in two of the threereplicates of the BE series for the S1 soil. For the S2 soil, earthwormsonly showedmortality in one replicate of the BE experimental series(with two dead worms). Although the heavy metal content of the S2soil was higher than S1, the latter showed higher mortality. Sinceorganic matter content of the S2 soil was higher than that of the S1soil, the difference in the earthworm mortality may be attributed tothe inability of the soil organicmatter to sustain theworms (Shipitaloet al., 1988; Kennette et al., 2002).

The fresh weight of the earthworms decreased during the potexperiments (Fig. 1A and B). Moreover, that decrease was statisti-cally significant (p< 0.05) for the majority of the experimentalseries (ME and BE series of the soil S1 and E and BE series of the soilS2). These results may be explained taking into account that theearthworms did not receive food during the experiments. The dietof earthworms mainly consists of organic material in various stagesof decay (Lee, 1985). Moreover, the earthworm body mass decreasein soils with low organic matter content has been reported forL. terrestris (Marhan and Scheu, 2005) and other earthworm species(Cheng and Wong, 2002; Ruiz et al., 2009).

Average metal concentrations accumulated in the earthwormsare presented in Fig. 2A and B for S1 and S2, respectively. The E, MEand BE experimental series were compared between them andwithrespect to the control earthworms (CE). Compared with Pb, L. ter-restris from the E, ME and BE experiments seemed to regulate theaccumulation of Zn and Cu to some extent. Thus, earthworm metalconcentrations only increased 25e48% for Cu and 23e71% for Zncompared to control earthworms (CE). However, earthworm Pbconcentrations were increased significantly (p< 0.05) for all theexperimental series (5-fold for the S1 soil and up to 18-fold for the S2soil, Fig. 2). No significant differences were found between the E, MEand BE experimental series. These results agree with those previ-ously reported by us for metal accumulation in Eisenia fetida earth-worms (Ruiz et al., 2009). Earthworms havewell-developed, specifictrafficking and storage pathways and redistribution capacity toregulate metals, especially essential trace metals such as Cu andZn, in their bodies that may lead to a balance between uptake andexcretion (Morgan and Morgan, 1999; Lukkari et al., 2005). On thecontrary, lead, which is not an essential metal, accumulates in theearthworm body reaching higher concentrations as soil metal

Table 2Water- and CaCl2-extractable fractions (mg kg�1� standard deviation and, in brackets, percentage of the total metal, n¼ 3) of both control soil (C) and soil with Lumbricusterrestris earthworms (E) experimental series. Results after 4 weeks of incubation/exposure at 17 �C.

Soil fractions Soils and experimental series

S1 S2

C E C E

Water-extractable fractionCu 0.20� 0.02a (0.62� 0.02) 0.19� 0.04a (0.56� 0.04) 0.15� 0.04a (0.44� 0.06) 0.15� 0.02a (0.44� 0.03)Pb 0.14� 0.02a (0.11� 0.02) 0.15� 0.05a (0.13� 0.02) 0.41� 0.15a (0.05� 0.01) 0.22� 0.05a (0.027� 0.003)Zn 0.87� 0.19a (0.24� 0.04) 1.00� 0.36a (0.62� 0.02) 1.03� 0.28a (0.15� 0.02) 2.54� 1.27a (0.37� 0.09)

CaCl2-extractable fractionCu 0.09� 0.01a (0.27� 0.03) 0.07� 0.01a (0.20� 0.01) 0.07� 0.01a (0.20� 0.04) 0.08� 0.01a (0.22� 0.03)Pb n.d.* n.d. n.d. n.d.Zn 1.49� 0.14a (0.40� 0.02) 0.94� 0.15b (0.27� 0.04) 1.57� 0.06a (0.23� 0.01) 5.87� 0.97b (0.85� 0.09)

* n.d.: Not detectable.** Different letters (a,b) indicate significant differences between C and E experimental series (p< 0.05).

E. Ruiz et al. / Environmental Pollution 159 (2011) 722e728 725

concentration increases. The latter agreewith the results reported byKennette et al. (2002) for L. terrestris earthworms incubated in urbanpolluted soils.

3.3. Effect of earthworm activity on metal fractionation

To assess the changes produced by earthworm activities inheavy metal fractionation, samples from control soil (C) andsoil with L. terrestris (E) experimental series were subject to single(water- and CaCl2-extractable fractions) and BCR sequentialextractions. Tables 2 and 3 show the metal concentration in all thefractions obtained by single and sequential extraction, respectively.

Extractions with water, CaCl2 and acetic acid have been widelyused for assessing mobility and bioavailability of metals in soils(Houba et al., 1996). Water extraction provides an estimate of thesoluble fraction of metals in the soil; this pool of metals is consid-ered to be themost accessible for plant uptake (Meers et al., 2005b).The ionic strength of the 0.01 M CaCl2 extraction is comparable tothat of the average soil solution (Houba et al., 2000) and can mimicmetal uptake by plants or by earthworms (Novozamsky et al., 1993;Houba et al., 1996; Peijnenburg and Jager, 2003). Taking intoaccount the sum of both water-extractable and 0.01 M CaCl2

Table 3BCR sequential extraction fractions (concentration in mg kg�1� standard deviation and, iLumbricus terrestris earthworms (E) experimental series. Results after 4 weeks of incuba

BCR fractions Soils and experimental series

S1

C E

Exchangeable (F1)Cu n.d.* n.d.Pb 1.20� 0.01a (1.06� 0.01) 1.45� 0.83a (1.35� 0Zn 7.98� 1.05a (2.17� 0.29) 9.08� 0.82a (2.46� 0

Reducible (F2)Cu 2.66� 0.23a (7.69� 0.66) 2.63� 0.20a (7.38� 0Pb 70.4� 1.7a (62.1� 1.5) 69.1� 4.6a (64.1� 4Zn 16.8� 0.4a (4.55� 0.11) 15.5� 0.7a (4.20� 0

Oxidizable (F3)Cu 2.83� 0.58a (8.17� 1.66) 2.63� 0.27a (7.38� 0Pb 13.1� 5.5a (11.69� 4.9) 11.0� 2.1a (10.2� 1Zn 26.6� 1.1a (7.2� 0.3) 25.5� 1.8a (6.90� 0

Residual (F4)Cu 29.1� 0.2a (84.1� 0.6) 30.2� 0.9a (84.7� 2Pb 28.6� 4.1a (25.2� 3.6) 26.2� 0.6a (24.3� 0Zn 317� 4a (86.1� 1.1) 319� 6a (86.4� 1

* n.d.: Not detectable.** Different letters (a,b) indicate significant differences between C and E experimental se

fractions, our results showed that the pool of easily available metalsaccounts for less than 1% of all the metals in the two studiedsoils (with the exception of Zn after earthworm inoculation in the S2soil). Addition of L. terrestris did not significantly change the water-extractable fraction (Table 2). We found a statistically significantincrease in the Zn extracted with 0.01 M CaCl2 in the S2 soil(p< 0.01) in the presence of earthworms (Table 2).

The acetic acid extractant used in the first step (F1 fraction) ofthe BCR method is one of the organic acids secreted by plant roots.It extracts the more mobile metal fraction representing the metalthat is bound to exchangeable positions, water-extractable andcarbonate bound, so this fraction may be considered similar to thatavailable to the plants (Li et al., 1998; Marguí et al., 2007). Thisfractionwas the highest for the zinc present initially in the S2 minesoil (36.63%, Table 3). Zinc showed low mobility in the S1 soil(2.17% in the F1 fraction) while Pb was poorly extracted in thisfraction for both soils (Table 3). Copper was not detected in the F1fraction of either soil. The addition of earthworms did not lead toany significant change in the studied soils.

Lead was mainly associated with the Fe and Mn oxides in bothsoils (62.11e71.59%, Table 3) while Cu appeared mainly in theresidual fraction (68.03e84.13%). The later fraction was the highest

n brackets, percentage of the total metal, n¼ 3) of both control soil (C) and soil withtion/exposure at 17 �C.

S2

C E

n.d. n.d..77) 11.3� 2.7a (1.70� 0.40) 8.4� 0.6a (1.33� 0.09).23) 206� 18a (36.6� 3.2) 217� 17a (40.0� 3.2)

.67) 3.68� 0.22a (11.5� 0.7) 3.14� 0.37a (8.44� 1.00)

.2) 476� 33a (71.6� 5.0) 443� 18.5a (70.2� 2.9)

.18) 143� 7a (25.4� 1.2) 137� 4.7a (25.3� 0.9)

.77) 6.51� 0.17a (20.4� 0.5) 13.1� 1.0b (35.2� 2.7)

.9) 30.2� 3.8a (4.53� 0.56) 33.8� 2.7a (5.34� 0.43)

.48) 59.6� 0.7a (10.6� 0.1) 64.7� 3.6b (11.9� 0.7)

.6) 21.7� 1.6a (68.0� 5.0) 21.5� 1.8a (57.8� 4.8)

.6) 148� 8a (22.2� 1.2) 148� 18a (23.4� 2.9)

.7) 154� 6a (27.4� 1.0) 150� 14a (27.6� 2.7)

ries (p< 0.05).

0.00

0.50

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*

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4.50Plants

Plants+earthworm

Maize shoots Barley shoots Maize roots Barley roots

Maize shoots Barley shoots Maize roots Barley roots

A

B

)g(thgie

wyrd

tnalpnae

M)g(

thgiew

yrdtnalp

naeM

*

Fig. 3. Effects of earthworm activity on shoot and root biomass of maize and barleyafter 4 weeks of growth: A) S1 soil; B) S2 soil. Mean values (n¼ 3) and standarddeviation (error bars). * denotes statistically significant differences between experi-mental series with and without earthworms (p< 0.05, ANOVA).

E. Ruiz et al. / Environmental Pollution 159 (2011) 722e728726

for Zn in the S1 soil (Table 3). Results from the BCRextraction appliedto the control soils (C series) showed that, in general, availability ofmetals was higher for the mine soil S2 (Table 3). No statisticallysignificant differences were found betweenmetals extracted in bothresidual (F4) and reducible (F2) fractions in the control soil (C series)compared with the soil in the presence of L. terrestris (E series).Similar results were reported by Udovic and Lestan (2007) who didnot find significant changes in the major Pb bearing fractions afterthe exposure of earthworms to polluted soil.

The statistically significant changes in metal distribution wereonly found in the F3 fraction (metals bound to organic matter andsulphides) of the S2 soil. We found significant increases in both Cu(p< 0.0001) and Zn (p< 0.05) concentration associated with the F3fraction in the presence of earthworms (E series) as compared to thecontrol soil. Nevertheless, taking into account the relative accuracyof the BCRmethod, those increases can be considered asmoderatelylow. Pb in F3 fractionwas slightly, but not significantly, increased forthe S2 soil. Cheng and Wong (2002) also reported significantincreases in the Zn bound to Fe andMn oxides and bound to organicmatter fractions after the use of the earthworm Pheretima sp. It iswell documented that earthworm activity enhances the fragmen-tation and humification of coarse particulate organic matter intofiner fractions and influences the distribution of organic matter insoil (Martin, 1991; Wen et al., 2006).

In summary, the use of chemical extractions showed that theL. terrestris earthworm had a very slight effect onmetal fractionationin the studied soils; only metals bound to the soil organic matterwere significantly increased in some cases. Sizmur and Hodson(2009), in a review about the impact of earthworms on metal avail-ability in soils, strongly recommended the combination of chemicalmethods and plant bioassays in order to strengthen conclusions andaid explanations of the mechanics involved in the soil processes.

3.4. Effect of earthworm activity on maize and barley growth

Fig. 3 shows both root and shoot mean weight, on a dry basis,reached by maize and barley grown without (M and B series) andwith earthworms (ME and BE series) for both S1 (Fig. 3A) and S2(Fig. 3B) soils.

Earthworm presence did not significantly change the shootweight for barley and maize grown in the two studied soils. Barleyroot biomass was not significantly changed by earthworms. But formaize we found a statistically significant decrease in root biomassfor both soils. Results reported by the different researchers aboutthe effect of earthworms on plant growth are not uniform (Scheu,2003); some studies showed that the presence of earthwormsincreases plant biomass (Edwards and Bater, 1992), others showedno effect (Eisenhauer et al., 2008), and others showed a decrease inplant growth (Derouard et al., 1997). Our results do not seem toclarify this issue.

3.5. Effect of earthworm activity on plant metal accumulation

The total metal accumulation rate (TMAR) and shoot and rootmetal concentrations for barley and maize grown on S1 and S2 areshown in Table 4.

The order of plant metal uptake was Zn>>Pb> Cu whichagrees with the order of total metal concentrations in the twostudied soils (Table 1). Moreover, Zn was found in the most avail-able fractions (water and exchangeable fraction and F1 fraction ofthe BCR sequential extraction) in higher extent than Pb and Cu.Some metals, such as Zn and Cd, are often primarily present assoluble or exchangeable forms in the soils which can easily be takenup by plant roots (Wang et al., 2004). The low Cu plant concen-tration may be attributed, on the one hand, to the low Cu soil

concentration and, on the other hand, to the antagonistic interac-tion between Zn and Cu that lead to a low Cu uptake when Zn soilconcentrations are high, as in our case (Kabata-Pendias andPendias, 2000; Bose and Bhattacharyya, 2008; Bose et al., 2008).

Results from Table 4 showed that L. terrestris activity led tostatistically significant increases of root, shoot and total (TMAR) Pband Zn concentrations in both barley andmaize for the S2 soil (withthe only exception of Pb root concentration in maize). Cu shootconcentration was also significantly increased for maize in the S2soil. Specifically, Pb shoot concentrationwas increased by a factor of7.5 and 3.9 for maize and barley, respectively, while Zn shootconcentrationwas increased by a factor of 3.7 and 1.7 for maize andbarley, respectively. For the S1 soil, the enhancement in plant metalconcentration was only statistically significant for Cu and Zn rootconcentrations in maize (p< 0.05), for Pb and Zn root concentra-tions in barley (p< 0.05), and for TMAR of Zn in barley and maize(p< 0.05). The effect of earthworms was greater in the S2 soilprobably due to the higher organic matter content and the higherconcentration of total and available metals.

The increase of metal accumulation by plants in the S2 soil maybe related to the observed increase of metals associatedwith the soilorganic matter (F3 fraction of the BCR sequential extraction). It has

Table 4Effect of earthworm activity onmetal concentration (mg kg�1� standard deviation, n¼ 3) in shoot, root and total accumulation in bothmaize and barley growing on S1 and S2soils. Experimental series shown are soil with only maize (M), soil with maize and Lumbricus terrestris earthworms (ME), soil with only barley (B), soil with barley andLumbricus terrestris earthworms (BE). Results after 4 weeks of growth at 17 �C.

Soils and experimental series

S1 S2

M ME B BE M ME B BE

CuShoot 6.89� 2.15a 9.45� 4.25a 18.4� 4.2a 15.2� 3.0a 9.11� 0.95a 16.9� 4.2b 14.5� 5.2a 28.2� 11.5aRoot 7.16� 1.94a 17.7� 4.7b 16.8� 2.0a 19.0� 8.0a 20.3� 6.0a 21.5� 7.5a 125� 61a 136� 52aTMAR 7.02� 2.04a 12.5� 4.2a 18.1� 3.2a 16.4� 3.3a 14.1� 2.8a 18.7� 5.6a 46.5� 18.9a 52.0� 16.4a

PbShoot 13.8� 5.0a 13.2� 3.7a 17.0� 3.6a 21.9� 4.5a 13.9� 3.4a 105� 32b 13.4� 2.9a 52.2� 14.6bRoot 15.3� 5.4a 30.6� 16.8a 27.9� 14.7a 51.7� 6.5b 58.9� 7.1a 74.4� 27.0a 38.0� 10.5a 154� 90bTMAR 14.5� 5.2a 19.6� 6.6a 20.4� 7.2a 31.1� 2.3a 34.0� 2.5a 94.0� 10.0b 20.0� 2.8a 73.8� 23.9b

ZnShoot 77.1� 3.3a 86.6� 11.8a 76.9� 3.8a 86.2� 7.3a 193� 20a 373� 26b 92.7� 9.1a 143� 25bRoot 60.3� 4.2a 137� 14b 144� 17a 198� 8.7b 287� 16a 387� 38b 238� 69a 504� 171bTMAR 68.8� 0.2a 105� 12b 95.4� 10.3a 121� 7b 235� 4a 381� 3b 134� 21a 224� 19b

Different letters (a,b) indicate significant differences between experimental series with and without earthworms (p< 0.05).

E. Ruiz et al. / Environmental Pollution 159 (2011) 722e728 727

been reported that earthworms play a humifying role in the soil assome humic acids, not present in the starting soil, were detected inthe resulting earthworm-worked soil (Sizmur and Hodson, 2009).It is generally accepted that organic-associated metals are easilyavailable to plants because the organic components are easilydecomposed by soil microorganisms with the consequent release ofthe metals into solution (Cheng and Wong, 2002). Metals could bechelated by these organic materials making their transport fromthe root to the shoot easier (Dandan et al., 2007). Wen et al.(2006) reported that DOC could significantly enhance the bio-accumulation of rare earth elements in wheat roots and shoots.

Therefore, in spite of the small changes in metal fractionationassessed by chemical extraction methods, the results from plantbioassays showed that the L. terrestris earthworm can changethe metal bioavailability in soils to some extent, thus significantlyimproving their absorption by plants. It is well-known that nochemical extraction methods can be used as a measure of ‘bioavail-able metals’; they are operationally defined (Sizmur and Hodson,2009). Our results agree with this idea; they suggest that a staticassessment of chemical metal availability is not indicative of thedynamics of metal uptake by plants. Moreover, we have applied thechemical methods to the bulk soil; as we have previously reported, ifwe had analyzed the earthworm casts, the changes in metal frac-tionation would have been greater (Alonso-Azcárate et al., in press).

It has been previously suggested that some earthworm species,i.e. E. fetida, Pheretima sp., could potentially be used for improvingthe phytoextraction of metals from polluted soils (Cheng andWong, 2002; Wen et al., 2004; Yu et al., 2005; Ruiz et al., 2009).Multiplying the increases found for both shoot biomass and shootmetal concentration, we found that extraction yields of Pb in themine soil S2 were increased by a factor of 4.4 and 7.6 for barley andmaize, respectively. Of course, we are aware that the improvementsobtained in this study cannot be extrapolated to field conditions.First, in order to obtain measurable changes, we have used anunrealistic density of earthworms, and secondly, we would have tobe able to confine the worms in the contaminated area and todevelop their life cycle there.

4. Conclusions

The results presented here show that L. terrestris is able to changesoilmetal availability to someextent. Thiswas later assessedbymeansof chemical methods, i.e. single extractions with water and CaCl2 andBCR sequential extraction, and a plant bioassay. The changes observed

in metal fractionation in the two studied soils were rather moderate:only metals bound to the soil organic matter were significantlyincreased in some cases. However, the use of plants showed the abilityof L. terrestris earthworm to significantly enhance plant metal uptakeand shoot concentrations. This highlights the need for both bioassaysand chemical extraction methods for assessing the bioavailability ofmetals in contaminated soils. From a practical standpoint, it has beensuggested that L. terrestris earthworms could be used to improve themetal extraction yields in the phytoremediation of polluted soils,preserving or even enhancing soil quality.

Acknowledgments

This research has received financial support from the SpanishMinistry of Education and Science (Project REN2003-04788/TECNO), the Spanish Ministry of the Environment (Projects 1.2-089/2005/3-B 097/20062-1.2 and A140/2007/1-01.2) and theRegional Government of Castilla-La Mancha (Project PREG-05-13).We thank them all for their support.

References

Alonso-Azcárate, J., Ruiz, E., Rodríguez, L. Changes in heavy metal speciationinduced by earthworm feeding activity. Fresenius Environmental Bulletin, inpress.

Amador, J.A., Gorres, J.H., 2005. Role of the anecic earthworm Lumbricus terrestrisL. in the distribution of plant residue nitrogen in a corn (Zea mays)esoil system.Applied Soil Ecology 30, 203e214.

Bose, S., Bhattacharyya, A.K., 2008. Heavy metal accumulation in wheat plant grownin soil amended with industrial sludge. Chemosphere 70, 1264e1272.

Bose, S., Chandrayan, S., Rai, V., Bhattacharyya, A.K., Ramanathan, A.L., 2008.Translocation of metals in pea plants grown on various amendment of elec-troplating industrial sludge. Bioresource Technology 99, 4467e4475.

Cappuyns, V., Swennen, R., Niclaes, M., 2007. Application of the BCR sequentialextraction scheme to dredged pond sediments contaminated by PbeZn mining:a combined geochemical and mineralogical approach. Journal of GeochemicalExploration 93, 78e90.

Chaoui, H.I., Zibilske, L.M., Ohno, T., 2003. Effects of earthworm casts and composton soil microbial activity and plant nutrient availability. Soil Biology andBiochemistry 35, 295e302.

Cheng, J., Wong, M.H., 2002. Effects of earthworms on Zn fractionation in soils.Biology and Fertility of Soils 36, 72e78.

Clapperton, M.J., Lee, N.O., Binet, F., Conner, R.L., 2001. Earthworms indirectly reduceeffects of take-all (Gaeumannomyces graminis var. tritici) on soft white springwheat (Triticum aestivum cv. Fielder). Soil Biology & Biochemistry 33,1531e1538.

Council of the European Communities,1986. Directive (86/278/EEC) on the protectionof the environment, and in particular of the soil, when sewage sludge is used inagriculture. Official Journal of European Communities L181, 6e12 [04/07/86].

Dandan, W., Huixin, L., Feng, H., Xia, W., 2007. Role of earthworm-straw interactionson phytoremediation of Cu contaminated soil by ryegrass. Acta Ecologica Sinica27, 4.

E. Ruiz et al. / Environmental Pollution 159 (2011) 722e728728

Derouard, L., Tondoh, J., Vilcosqui, L., Lavelle, P., 1997. Effects of earthworm intro-duction on soil processes and plant growth. Soil Biology and Biochemistry 29,541e545.

Devliegher, W., Verstraete, W., 1996. L umbricus terrestris in a soil core experiment:effects of nutrient-enrichment processes (nep) and gut-associated processes(gap) on the availability of plant nutrients and heavy metals. Soil Biology andBiochemistry 28, 489e496.

Edwards, C.A., Bater, J.E., 1992. The use of earthworms in environmental manage-ment. Soil Biology and Biochemistry 24, 1683e1689.

Eisenhauer, N., Marhan, S., Scheu, S., 2008. Assessment of anecic behavior inselected earthworm species: effects on wheat seed burial, seedling establish-ment, wheat growth and litter incorporation. Applied Soil Ecology 38, 79e82.

Houba, V.J.G., Lexmond, Th.M., Novozamsky, I., Van der Lee, J.J., 1996. State of the artand future developments in soil analysis for bioavailability assessment. Scienceof the Total Environment 178, 21e28.

Houba, V.J.G., Temminghoff, E.J.M., Gaikhorst, G.A., Van Vark, W., 2000. Soil analysisprocedures using 0.01 M calcium chloride as extraction reagent. Communica-tions in Soil Science and Plant Analysis 31, 1299e1396.

Kabata-Pendias, A., Pendias, H., 2000. Trace elements in Soils and Plants. CRC Press,Boca Raton.

Kennette, D., Hendershot, W., Tomlin, A., Sauvé, S., 2002. Uptake of trace metals bythe earthworm Lumbricus terrestris L. in urban contaminated soils. Applied SoilEcology 19, 191e198.

Kizilkaya, R., 2004. Cu and Zn accumulation in earthworm Lumbricus terrestris L.in sewage sludge amended soil and fractions of Cu and Zn in casts andsurrounding soil. Ecological Engineering 22, 141e151.

Kos, B., Lestan, D., 2003. Induced phytoextraction/soil washing of lead usingbiodegradable chelate and permeable barriers. Environmental Science andTechnology 37, 624e629.

Lanno,R.,Wells, J., Conder, J., Bradham,K., Basta, N., 2004. Thebioavailability of chemicalsin soil for earthworms. Ecotoxicology and Environmental Safety 57, 39e47.

Lee, K.E., 1985. Earthworms. Their Ecology and Relationships with Soils and Landuse. Academic Press, New York, Sydney, London.

Li, F., Shan, X., Zhang, T., Zhang, S., 1998. Evaluation of plant availability of rare earthelements in soils by chemical fractionation and multiple regression analysis.Environmental Pollution 102, 269e277.

Lowe, C.N., Butt, K.R., 2005. Culture techniques for soil dwelling earthworms:a review. Pedobiologia 49, 401e413.

Lukkari, T., Aatsinki, M., Vaisanen, A., Haimi, J., 2005. Toxicity of copper and zincassessed with three different earthworm tests. Applied Soil Ecology 30, 133e146.

Ma, Y., Dickinson, N.M., Wong, M.H., 2002. Toxicity of Pb/Zn mine tailings to theearthworm Pheretima and the effects of burrowing on metal availability. Biologyand Fertility of Soil 36, 79e86.

Maleri, R.A., Reinecke, A.J., Reinecke, S.A., 2008. Metal uptake of two ecophysio-logically different earthworms (Eisenia fetida and Aporrectodea caliginosa)exposed to ultramafic soils. Applied Soil Ecology 38, 42e50.

MAPA, 1994. Métodos oficiales de análisis de suelos y aguas. Ministerio deAgricultura, Pesca y Alimentación (España), Madrid.

Marguí, E., Salvadó, V., Queralt, I., Hidalgo, M., 2004. Comparison of three-stagesequential extraction and toxicity characteristic leaching tests to evaluate metalmobility in mining wastes. Analytical Chimica Acta 524, 151e159.

Marguí, E., Queralt, I., Carvalho, M.L., Hidalgo, M., 2007. Assessment of metalavailability to vegetation (Betula pendula) in PbeZn ore concentrate residueswith different features. Environmental Pollution 145, 179e184.

Marhan, S., Scheu, S., 2005. Effects of sand and litter availability on organic matterdecomposition in soil and in casts of Lumbricus terrestris L. Geoderma 128,155e166.

Martin, A., 1991. Short- and long-term effects of the endogeic earthworm Millsoniaanomala (Omodeo) (Megascolecidæ, Oligochæta) of tropical savannas, on soilorganic matter. Biology and Fertility of Soils 11, 234e238.

Meers, E., Ruttens, A., Hopgood, M., Samson, D., Tack, F.M.G., 2005a. Comparison ofEDTA and EDDS as potential soil amendments for enhanced phytoextraction ofheavy metals. Chemosphere 58, 1011e1022.

Meers, E., Lamsal, S., Vervaeke, P., Hopgood, M., Lust, N., Tack, F.M.G., 2005b.Availability of heavy metals for uptake by Salix viminalis on a moderatelycontaminated dredged sediment disposal site. Environmental Pollution 137,354e364.

Morgan, J.E., Morgan, A.J., 1999. The accumulation of metals (Cd, Cu, Pb, Zn and Ca)by two ecologically contrasting earthworm species (Lumbricus rubellus andAporrectodea caliginosa): implications for ecotoxicological testing. Applied SoilEcology 13, 9e20.

Morgan, J.E., Norey, C.G., Morgan, A.J., Kay, J., 1989. A comparison of the cadmium-binding proteins isolated from the posterior alimentary canal of the earth-worms Dendrodrilus rubidus and Lumbricus rubellus. Comparative Biochemistryand Physiology 92, 15e21.

Novozamsky, J., Lexmond, T.M., Houba, V.J.G., 1993. A single extraction procedure ofsoil for evaluation of uptake of some heavy metals in plants. InternationalJournal of Environmental Analytical Chemistry 51, 47e58.

Peijnenburg, W.J.G.M., Jager, T., 2003. Monitoring approaches to assess bio-accesibility of metals: Matriz issues. Ecotoxicology and Environmental Safety56, 63e77.

Ruiz, E., Rodríguez, L., Alonso-Azcárate, J., 2009. Effects of earthworms on metaluptake of heavy metals from polluted mine soils by different crop plants.Chemosphere 75, 1035e1041.

Sahuquillo, A., López-Sánchez, J.F., Rubio, R., Rauret, G., Thomas, R.P., Davidson, C.M.,Ure, A.M., 1999. Use of a certified reference material for extractable trace metalsto assess sources of uncertainty in the BCR three-stage sequential extractionprocedure. Analytical Chimica Acta 382, 317e327.

Scheu, S., 2003. Effects of earthworms on plant growth: patterns and perspectives.Pedobiologia 47, 846e856.

Shipitalo, M.J., Protz, R., Tomlin, A.D., 1988. Effect of diet on the feeding and castingactivity of Lumbricus terrestris and L. rubellus in laboratory culture. Soil Biology& Biochemistry 20, 233e237.

Sizmur, T., Hodson, M.E., 2009. Do earthworm impact metal mobility andavailability in soil? e a review. Environmental Pollution 157, 1981e1989.

Suthar, S., Singh, S., Dhawan, S., 2008. Earthworms as bioindicator of metals (Zn, Fe,Mn, Cu, Pb and Cd) in soils: is metal bioaccumulation affected by theirecological category? Ecological Engineering 32, 99e107.

Udovic, M., Lestan, D., 2007. The effect of earthworms on the fractionation andbioavailability of heavy metals before and after soil remediation. EnvironmentalPollution 148, 663e668.

USEPA, 1996a. EPA Method 3051A: microwave assisted acid digestion of sediments,sludges, soils, and oils. In: Test Methods for Evaluating Solid Waste. WashingtonDC, USA.

USEPA, 1996b. EPA Method 3052: microwave assisted acid digestion of silicious andorganically based matrices. In: Test Methods for Evaluating Solid Waste.Washington DC, USA.

Wang, X., Shan, X., Zhang, S., Wen, B., 2004. A model for the evaluation of thephytoavailability of trace elements to vegetables under the field conditions.Chemosphere 55, 811e822.

Wen, B., Hu, X.Y., Liu, Y., Wang, W.S., Feng, M.H., Shan, X.Q., 2004. The role ofearthworms (Eisenia fetida) in influencing bioavailability of heavy metals insoils. Biology and Fertility of Soils 40, 181e187.

Wen, B., Liu, Y., Hu, X.-Y., Shan, X.-Q., 2006. Effect of earthworms (Eisenia fetida) onthe fractionation and bioavailability of rare earth elements in nine Chinese soils.Chemosphere 63, 1179e1186.

Yu, X., Cheng, J., Wong, M., 2005. Earthwormemycorrhiza interaction on Cd uptakeand growth of ryegrass. Soil Biology and Biochemistry 37, 195e201.

Zemberyová, M., Barteková, J., Hagarová, I., 2006. The utilization of modified BCRthree-step sequential extraction procedure for the fractionation of Cd, Cr, Cu, Ni,Pb and Zn in soil reference materials of different origins. Talanta 70, 973e978.

Zhang, H., Schrader, S., 1993. Earthworm effects on selected physical and chemicalproperties of soil aggregates. Biology and Fertility of Soils 15, 229e234.

Zhang, S., Shan, X.-Q., 2000. Is the bioavailability index applicable for trace elementsin different types of soil? Chemical Speciation and Bioavailability 12, 117.