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Journal of Hazardous Materials 233– 234 (2012) 244– 253

Contents lists available at SciVerse ScienceDirect

Journal of Hazardous Materials

j our na l ho me p age: www.elsev ier .com/ locate / jhazmat

ffects of heavy metal concentrations (Cd, Zn and Pb) in agricultural soils nearifferent emission sources on quality, accumulation and food safety in soybeanGlycine max (L.) Merrill]

aría Julieta Salazar, Judith Hebelen Rodriguez ∗, Gastón Leonardo Nieto, María Luisa Pignataultidisciplinary Institute of Plant Biology, Pollution and Bioindicator Section, Faculty of Physical and Natural Sciences, National University of Córdoba, Av. Vélez Sársfield 1611,

5016CGA Córdoba, Argentina

i g h l i g h t s

Soybean grown near metal sources presents a toxicological hazard from heavy metals for Chinese consumers.Rhizosphere soil is the most suitable compartment for toxicological studies.Soil guidelines should be modified considering the exchangeable metals.

r t i c l e i n f o

rticle history:eceived 17 February 2012eceived in revised form 4 June 2012ccepted 9 July 2012vailable online 20 July 2012

eywords:lycine maxeavy metals

a b s t r a c t

Argentina is one of the major producers of soybean in the world, this generates a high global demand forthis crop leading to find it everywhere, even close to human activities involving pollutant emissions.

This study evaluated heavy metal content, the transfer of metals and its relation to crop quality, andthe toxicological risk of seed consumption, through soil and soybean sampling. The results show thatconcentrations of Pb and Cd in soils and soybeans at several sites were above the maximum permissi-ble levels. The heavy metal bioaccumulation depending on the rhizosphere soil compartment showedsignificant and high regression coefficients. In addition, the similar behavior of Cd and Zn accumulationby plants reinforces the theory of other studies indicating that these metals are incorporated into the

ood safetymissionsrgentina

plant for a common system of transport. On the other hand, the seed quality parameters did not show aclear pattern of response to metal bioacumulation. Taken together, our results show that soybeans grownnearby to anthropic emission sources might represent a toxicological hazard for human consumption ina potential Chinese consumer. Hence, further studies should be carried out taking into account the poten-tial negative health effects from the consumption of soybeans (direct or indirect through consumptionof meat from cattle) in these conditions.

. Introduction

Deteriorating environmental conditions resulting from urban-ndustrial activities (metallurgical industries, chemical plants,

aste incineration) and agricultural practices (application of phos-hate fertilizers, pesticides and sewage sludge) are the mainnthropogenic sources of heavy metals in agricultural soils [1,2]nd have led to a growing public concern over food safety due

o the potential accumulation of heavy metals in agricultural soils3,4].

∗ Corresponding author. Tel.: +54 351 4344983x6; fax: +54 351 4334139.E-mail addresses: jrodriguez@com.uncor.edu, judheberodri@gmail.com

J.H. Rodriguez).

304-3894/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jhazmat.2012.07.026

© 2012 Elsevier B.V. All rights reserved.

Food consumption has been identified as the major source ofmetal exposure in humans [5]. In addition, studies on the transferof heavy metals from soil into crops [6,7] have shown that soybeanmay accumulate more potentially toxic elements than other crops.Soybean (Glycine max) is, in fact, one of the most widespread cropsworldwide and represents the most important protein crop; withArgentina being one of the major producers of genetically modi-fied organism (GMO) variety (Roundup Ready) of G. max, its mostimportant crop in terms of production levels [8]. The majority of itstotal production (deriving oil and by-products such as protein-richmeal and raw materials for animal feed) is exported to China and theEuropean Union [9,10]. In addition, the increasing global demand

for animal feed has put pressure on arable land [11], leading to anagricultural production system with little crop rotation that maycause a lack of nutrients in the soil hence, a common practice is theapplication of fertilizers. The high demand of soybean has resulted

dous Materials 233– 234 (2012) 244– 253 245

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M.J. Salazar et al. / Journal of Hazar

n this crop being cultivated near routes with high vehicular traffics well as in areas close to human activities such as industrial pro-uction [12]. Many of the industrial activities, for example, cementroduction and waste incineration; have been cited as importantources of heavy metals in agricultural soils in Argentina [13].

The metal uptake by plants is subject to antagonistic, addi-ive, and synergetic effects [14], with contaminated soils having

reduced quality in terms of the physical and chemical propertieshat determine the metal retention capacity [15]. The soil–plantransfer of metals is a very complex process governed by severalactors, both natural and anthropogenic, such as sorptive capacityf soil, redox conditions, organic matter and pH. These control therocesses of mobility and availability of metals in soils, which ineneral have a geochemical, climatic or biological origin [16–19].n addition, the mobility of metals plays an important role in thehizosphere compartment, which corresponds to the whole inter-ace between roots and soil where interactions between the soil

icroorganisms, roots and soil constituents takes place [20]. Theelease of protons, exudates and metabolites into the rhizospherey the roots and the microorganisms can modify the pH, dependingn the available source of nitrogen nutrients or iron deficiency [21],hus modifying the fixation and mobility of heavy metals in soils18].

Currently there are insufficient toxicological studies related toood safety of soybeans grown in proximity to industries [7,14,22].herefore, the objectives of this study were: (i) to determine theeavy metal content in soils and soybean crops near pollutant emis-ion sources; (ii) to evaluate the transfer of metals between soil andlant and its relation to crop quality; and (iii) to determine the tox-

cological risk of the consumption of the seeds that grow in thetudy area.

. Materials and methods

.1. Study area

The study region included nine areas in Córdoba province,entral Argentina (Fig. 1). The climate is mild, with an annualean temperature of about 15 ◦C and an average annual rainfall of

00–900 mm. The soybean crop areas were subjected to zero tillage,n which the soil was cropped under direct drilling with a mechan-cal drill using a 4 cm depth of seeding. The fertilizers applied ineneral were simple superphosphates (P, S and Ca) in proportionsanging from 50 to 100 kg ha−1.

The areas were chosen taking into account the presence of soy-ean crops and different anthropogenic activities. The samplingreas and their characteristics such dominant vegetation, anthro-ogenic activities, soil type, location, population, and pollutioneferences [12,13,23–26] are described in Table 1.

.2. Sampling procedure

Sampling was carried out during the soybean harvest seasont the maturity (R8) stage as defined by Fehr and Caviness [27].oybeans plants and topsoil samples were collected at the elevenampling sites (April–May), with each site consisting of a 25 m2

ith 9 subsampling points, systematically arranged with a 2.5 map between them. Three plants were collected at each point, mak-ng a total of 27 plants at every sampling site. Plants were collectedsing a stainless steel shovel to extract the whole root and the soilround it, which was treated here as rhizosphere soil. In addition,

ulk topsoil subsamples were collected at the nine points using atainless steel spatula at a depth of 0–10 cm, with stones and for-ign objects being removed by hand. Soil and plants were kept inlastic bags until being processed in the laboratory. Ta

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246 M.J. Salazar et al. / Journal of Hazardous Materials 233– 234 (2012) 244– 253

Fig. 1. Location of sampling sites in the study area, Córdoba, Argentina. Sampling sites: Malagueno and Yocsina (M and Y); General Paz and Pozo del Tigre (GP and PT);F 2 diff4

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erreyra (F); Los Molinos and Despenaderos (LM and D); Bouwer (BH and BL), with0 mg kg−1; Río Tercero (RT); and General Cabrera (GC).

Plant samples were separated into roots, shoots, seeds and pods,nd then washed and sonicated with ultrapure water with the pur-ose of removing soil remains attached to the organs. Subsequently,he samples were oven-dried at 60 ◦C to dry weight (DW), ground,omogeneously mixed, and each composite sample from each sitend plant (N = 27) was stored in the dark until analytical proceduresere carried out.

Rhizosphere soil samples were isolated from roots by shak-ng them in a plastic box. Then, bulk and rhizosphere soilubsamples were sieved to <2 mm (polyethylene sieve), homo-eneously mixed, and stored in the dark until used for heavyetal determination [28]. Each composite sample was made byixing and homogenizing 150 g of each of the topsoil sieved

ubsamples.

.3. Physico-chemical analyses

.3.1. Electrical conductivity, pH and organic matter percentagen topsoils

Soil pH and electrical conductivity (EC) were measured in 1:5oil:water suspension in triplicates [29]. In order to calculate the

ry weight, samples were oven-dried for four hours at 105 ◦C toonstant weight [30]. The percentage of organic matter (OM%) wasetermined according to Peltola and Åström [31] by the combustionf the samples at 500 ◦C for four hours.

erent Pb concentration in soil, High (BH) with c. 300 mg kg−1 and Low (BL) with c.

2.3.2. Exchangeable heavy metals in topsoilsFor the purpose of analyzing exchangeable metal fractions

in topsoils, a 0.5 M hydrochloric acid extraction was performed[28,32,33]. In this method, 0.5 M HCl is used to release relevant(labile) fractions of elements [32]. Topsoils were sieved with a63 �m stainless steel mesh [33] and each sample was extractedusing 7 g DW of topsoil with 25 mL 0.5 M HCl and shook it at roomtemperature for 30 min. After 24 h, the solution was filtered andanalyzed using a Perkin-Elmer flame Atomic Absorption 3110 spec-trophotometer (Norwalk, CT, USA) to measure CdExch, PbExch, andZnExch.

2.3.3. Pseudototal metal concentrations in topsoilsWith the aim of analyzing the pseudototal metals in topsoils,

5 g DW of topsoil were reduced to ashes at 450 ◦C for 4 h, and thendigested with hydrochloric and nitric acid extraction (HCl:HNO3,3:1, v/v) and shook it at room temperature for 30 min. After 24 h,the solution was filtered and analyzed using a Perkin-Elmer flameAtomic Absorption 3110 spectrophotometer (Norwalk, CT, USA) tomeasure CdPst, PbPst and ZnPst.

2.3.4. Metal concentrations in plantsThe concentrations of Cd, Pb and Zn were analyzed in seeds,

pods, shoots and roots (3 g DW) using 20% HCl, and HNO3. The sam-ples were reduced to ashes at 450 ◦C for 4 h and then digested using

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M.J. Salazar et al. / Journal of Hazar

.5 mL 20% HCl and 0.5 mL HNO3, according to Rodriguez et al. [4].he heavy metal concentrations in plant samples were measuredsing a Perkin-Elmer AA3110 spectrophotometer.

.4. Soybean quality analyses

.4.1. Standard germination testSeed germination was measured by placing 20 seeds from each

ample in sterile sand at 25 ◦C for 14 days, and counting dailyrom the fourth day. The germination percentage (proportion ofeeds which produced seedlings classified as normal) was calcu-ated according to ISTA [34].

.4.2. Tetrazolium testTwenty seeds from each sample were stained with a 0.075%

olution of tetrazolium chloride according to de Barros Franc a Netot al. [35], and vigor, germination potential and damage level wereetermined.

.4.3. Morphological parametersThe morphological parameters analyzed were biomass of stems,

ods, seeds and roots expressed as dry weight, weight of 1000eeds, number of seeds per plant, number of pods per plant, andumber of seeds per pod.

.5. Quality control

As a quality control, blanks and samples of the standard ref-rence material “CTA-OTL-1” (oriental tobacco leaves, Institute ofuclear Chemistry and Technology) for plants, and reference cer-

ificated material (CRM GBW07405 Soil-NRCCRM, China) for soils,ere prepared in the same way described for plants and soils

espectively, and were run after ten determinations to calibrate thenstrument and monitor the potential sample contamination dur-ng analysis. These results were found to be between 90% and 92%f the certified value for CTA-OTL-1 and between 87% and 91% forRM GBW07405 Soil-NRCCRM, with the data indicating a low errorf typically less than 15%. The coefficient of variation of replicatenalyses was calculated for different determinations, and variationsere found to be less than 10%.

.6. Data analyses

.6.1. Statistical analysesThe Shapiro–Wilks test for normality was applied, with

on-normal distributed elements being log-transformed beforearametric statistics were performed. An analysis of varianceANOVA) was used to compare metal concentrations, and pH andM percentages among rhizosphere and bulk topsoils at sampling

ites. ANOVA was also used to compare metal concentrations inoybean in organs and sites. Whenever the ANOVA indicated sig-ificant effects (p < 0.05), a pairwise comparison of means wasndertaken using the Tukey test. The pearson correlation matrixas used to identify the relationships between soil variables in the

hizosphere and between the same variables in the bulk topsoil,n order to explain differences between these compartments. This

ethod was also used to evaluate possible relationships amongetal exchangeable concentrations in rhizospheric soil, morpho-

ogical variables and heavy metal content in soybean organs. Simpleegressions were performed between the soil metal concentrationsnd the presence of metal in soybean organs.

.6.2. Bioconcentration factorIn this study, a modified bioconcentration factor (BCF) was cal-

ulated using the ratio of the element concentration in seeds and

aterials 233– 234 (2012) 244– 253 247

the “exchangeable” or potentially available portions of metals inrhizospheric soils [36].

2.6.3. Risk assessmentThe risk to human health resulting from consumption of soy-

bean grown in the different sample soil sites was calculated byemploying the estimated dietary intake (EDI �g kg−1 day−1 Bw),and target hazard quotients (THQ) as described by Zheng et al.[22] and US EPA [37]. For the present study, Chinese, European andArgentine inhabitants were considered to be potential consumers,taking into account that Argentina exports soybean and its productsmainly to China and the countries of the EU.

The EDI exposure is expressed as the mass of a substance perunit body weight per unit time, averaged over a long period of time(a lifetime), and is calculated as follows:

EDI = C × Con × EF × EDBw × AT

where C is the median concentration of a heavy metal in soybean(�g kg−1); Con is the ingestion rate of soybean (g person−1 day−1);EF is the exposure frequency (365 days year−1); ED is the exposureduration (70 years for adults); Bw is the average body weight (65 kgfor Chinese adults and 70 kg for European or Argentine adults),and AT expresses the average exposure time for non-carcinogeniceffects (ED × 365 days year−1). Keinan-Boker et al. [38] reportedthat the average daily intake of traditional soy products for a Chi-nese individual was 100 g person−1 day−1, while the daily intake inWestern inhabitants was less than 1 g person−1 day−1 [38,39].

THQ gives the potential non-cancer risk for individual heavymetals and can be calculated as follows:

THQ = EDIRfD

where RfD is the reference oral dose, and represents an estimationof the daily exposure to which the human population is likely tobe subjected to without any appreciable risk of deleterious effectsduring a lifetime. The RfD values used were 1 and 300 �g kg−1 day−1

for Cd and Zn, respectively [40]. However, as the US EPA has notyet established RfD values for Pb, the one used in this paper was4 �g kg−1 day−1 [41].

In order to assess the overall potential for non-carcinogeniceffects from more than one heavy metal, a hazard index (HI) hasbeen formulated based on the Guidelines for Health Risk Assess-ment of Chemical Mixtures of EPA [37] as follows:

HI =∑

THQ = EDIRfD1

+ EDIRfD2

+ · · · + EDIRfDi

If either TQH or HI exceeds unity, a high risk of non-carcinogeniceffects is implied.

3. Results and discussion

3.1. Analysis of pH, EC, OM and heavy metals in soils

Exchangeable and pseudototal Cd, Pb and Zn concentrations,along with the pH and OM percentages in rhizospheric and bulksoil are shown in Fig. 2 (see Supplementary material, Tables S1 andS2).

According to soil quality guidelines (Table 2) the maximumpseudototal Pb concentration was within the limits for agriculturalland use [42,43] in all the study areas in the rhizospheric compart-ment except for BL and BH. Bouwer also presented Pb concentration

above the limits established in the Canadian and Ontario soil qual-ity guidelines [44,45]. The following descending order representsthe Pb concentration in soils: BH, BL > M > GP, F, PT, RT, GC, Y > LMand D.

248 M.J. Salazar et al. / Journal of Hazardous Materials 233– 234 (2012) 244– 253

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ig. 2. Concentrations of exchangeable and pseudototal Pb (A), Zn (B) and Cd (C), anampling sites: Malagueno (M); Yocsina (Y); General Paz (GP); Pozo del Tigre (PT);b concentration in soil, High (BH) with c. 300 mg kg−1 and Low (BL) with c. 40 mg k

Cd and Zn concentrations were within the limits for agricul-ural land use in all the studied areas, but the values found wereot homogeneous among sites. Considering Zn, the concentrationrder in sites was M, Y, GC, RT > GP, F, PT and B (H and L) > D andM. Differences in Cd soil concentration were not very pronouncedmong sites, except for the exchangeable Cd concentration in bulkopsoil; where the order was M > F > GP, PT, Y > RT, BH, BL, D, GC > LM.

hese general trends in Cd, Pb and Zn concentration are consistentith those found in previous studies, which evaluated soil trace

lement pollution in most of the sampling sites taken into accountn the present work [13,25].

able 2aximum allowable total concentrations of Cd, Pb and Zn in agricultural soils

mg kg−1 DW).

Soils guidelines Metal

Cd Pb Zn

Argentinean soilsa 3 375 600Canadian soilsb 1.4 70 200Canadian soilsc 3 375 600Ontario soilsd 1.6 15 220

a Argentinean legislation (Law 24051).b CCME (Canadian Council of Ministers of the Environment) (1991) Soil Qualityuidelines.c CCME (2007) Soil Quality Guidelines for Environmental Health.d Ontario Ministry of Environment (1996) Guidelines for the utilization of

iosolids and other wastes on agricultural land.

and percentage of organic material (OM%) (D) in rhizospheric (Rz) and bulk topsoil.yra (F); Los Molinos (LM); Despenaderos (D); Bouwer (BH and BL), with 2 differentío Tercero (RT); and General Cabrera (GC).

It is important to note that from the ten cases with significantdifferences in exchangeable metal concentrations between bulkand rhizospheric soil, eight showed higher values in the rhizo-sphere. Therefore, this general trend could indicate that soybeanwas acting like a labile metal pump in soils, via the convectionor mass flow and diffusion process as reported by Lasat [46] andby Crowley et al. [47]. In addition, plants have evolved specializedmechanisms to increase the concentration of metal ions in soil solu-tion, such as rhizosphere acidification due to H+ extrusion fromroots and rhizosphere exudates of organic compounds [47,48].

Regarding pH significantly more acidic values were found inbulk topsoils from D and Y than in rhizospheric soil samples. Incontrast, LM, GP, PT, M and RT showed more acidic values in rhizo-spheric soils than in bulk topsoils. These results, therefore, indicatethat the rhizosphere compartment is in general more acidic thanbulk topsoils, which could be due to root exudates [49].

Pearson’s correlation coefficients between soil parameters andmetal concentrations are shown in Table 3. The pH was significantlycorrelated with exchangeable Cd and Zn concentration, both in bulkand rhizospheric soil, but it did not correlate with Pb as previouslyreported by McBride et al. [50]. The OM percentage was signifi-cantly correlated with exchangeable Cd, Pb and Zn concentrationsin the rhizosphere. These results suggest that OM and pH are impor-

tant factors in the differences between bulk and rhizospheric soilexchangeable metal concentration, and they may be related to thebioavailability of these metals for plants. Similar trends were foundfor Cd, Pb and Zn by Kashem and Singh [51] and McBride et al.

M.J. Salazar et al. / Journal of Hazardous Materials 233– 234 (2012) 244– 253 249

Table 3Pearson correlation coefficients between soil parameters pH, OM (%), EC (�s cm−1) and metal concentrations (�g g−1 DW) in rhizosphere and bulk topsoil.

PbExch PbPst ZnExch ZnPst CdExch CdPst pH OM EC

RhizospherepH 0.07 −0.09 0.5* −0.23 0.74*** −0.07 1OM 0.68*** 0.57** 0.72*** 0.34 0.74*** 0.26 0.45* 1EC 0.51* 0.29 0.29 −0.15 0.41 −0.16 0.37 0.71*** 1

BulkpH 0.21 0 0.48* 0.34 0.84*** 0.7*** 1OM 0.34 0.56* 0.52* 0.58* 0.59* 0.71*** 0.45* 1EC 0.29 0.22 0.54* 0.45* 0.87*** 0.66*** 0.8*** 0.66*** 1

Abbreviations. Exch, exchangeable soil fraction; Pst, pseudototal soil fraction; OM %, organic matter percentage; EC, electric conductivity.*

[tatEwdc

3

otw

snnDooetiIsbme03h(daeZtAtpbw

sufts

p < 0.05.** p < 0.01.

*** p < 0.001.

50], and for other metals by Jansen et al. [52]. Electric conduc-ivity correlated significantly and positively with exchangeable Pbnd OM in rhizosphere soil, and with exchangeable and pseudoto-al Zn and Cd, pH and OM in bulk soil. These results indicate thatC is more related to metals in bulk soil than in rhizospheric soil,hich could be indicating that root exudates maintain regular con-itions in rhizosphere buffering EC changes, despite heavy metaloncentration.

.2. Heavy metal concentrations in roots, stems, seeds and pods

Table 4 shows the concentrations of Cd, Pb and Zn in the organsf the soybean plants at the maturity stage in different sites and alsohe ANOVA results for each organ among sites and among organsithin a site.

In general, the concentration of Pb in roots, stems and pods wasignificantly higher in BH, however the Pb content in seeds showedo significant differences among areas. However it is important toote that the values found in all samples exceeded 0.2 �g g−1 PbW, the level given by the EU directive relating to maximum levelsf certain contaminants in foodstuffs [53]. The comparison amongrgans within each site generally indicated that pods had the high-st Pb values. However, this result can not be easily compared, ashe vast majority of the studies on the distribution of heavy metalsn soybean have evaluated roots, shoots and seeds but not pods.n contrast, the Pb values for seeds and stems, at all the samplingites, were higher than those reported by Lavado et al. [6] for soy-ean in Argentina. Lavado et al. [6] studied the distribution of heavyetals in soybean plants, considering fertilizer sources and differ-

nt agricultural management, and they found, in the worst case,.85 �g g−1 Pb DW for seeds, 0.69 �g g−1 Pb DW for stems and.51 �g g−1 Pb DW for roots. In addition, Lavado et al. [7] studiedeavy metal concentration in soybean seeds considering a big cityBuenos Aires, Argentina) and high traffic roads as sources, and theyetected only 0.4 �g g−1 Pb DW. With regard to Zn, the comparisonmong sites within each organ generally showed significant differ-nces in roots, stems and seeds, however, it should be noted thatn concentrations in seeds did not exceed the maximum permit-ed levels and were within the normal values quoted for soybean inrgentina [7]. Despite Zn was within the thresholds, it is important

o note that it was accumulated in seeds more than in the otherarts of the plant, with significant differences (Table 4); this shoulde considered in future studies and policy development for caseshen Zn concentration in soils is higher than in the present study.

Regarding the comparison among organs within each site, allites showed higher Cd values in seeds and pods, and lower Cd val-

es in roots. Taking into account that the maximum levels allowedor Cd in soybean (according the EU directive and the Codex Alimen-arius Commission) is 0.2 �g g−1 DW [53,54], the samples of theites BH and BL were above the permitted values, whereas GP and Y

grains were close to the maximum. In this context, it is remarkablethat the concentration of Cd in those soils did not exceed the max-imum levels allowed [42–45], despite this, Cd found its way intosoybeans seeds. These results highlight the necessity to know indeep the factors involved in Cd bioavailability. Similar results werefound by the authors Arao et al. [55] which found, even for low Cdconcentration in substrate, more than 0.2 �g g−1 DW in soybeanseeds, which grew in greenhouse and under hydroponic conditions.In addition, the authors Cataldo et al. [56] investigated the chemicalbehavior of Cd in soybean plants growing in hydroponic culturesand found that 8% of Cd in the substrate is transported to seeds.Moreover, Shute and Macfie [57] conducted experiments in green-house exposing soybean plants to different concentration of Cd, Zn,and both together, the mean Cd content found for seeds in controlsoils was 0.65 �g g−1 DW. Therefore, it was important to confirmthose results in our study under field conditions “in situ”. Never-theless, more studies to monitor concentrations of this toxic metalin food crops are still required.

3.3. Soybean quality analyses

The seed germination rate demonstrated the lowest values forsites D, PT and Y, while the tetrazolium test results revealed thelowest values of seed vigor in BH, PT and GP. In addition, PT, Dand GP showed mechanical damage and serious restrictions of seedgermination.

Considering that the quality parameters measured were corre-lated with neither the content of metals in soils nor the contentof metals in seeds, it is important to note that high metal contentin seeds does not necessarily reflect deterioration in the quality ofseeds. Therefore the absence of seed quality adverse effects doesnot ensure food safety.

3.4. Bioconcentration factor

The bio-concentration factor (BCF) is an important parameterin heavy metal uptake studies. In the present study, the biocon-centration values in seeds found for Zn (4.95 ± 1.84) were higherto those reported by Bermudez et al. [58] in wheat crops grown inagricultural lands, and than those reported by Lavado [7] in soybeancultures in Argentina. Pb and Cd also showed higher BCF in grains(Pb 0.13 ± 0.11; Cd 0.57 ± 0.20) than in Lavado [7] study, reveal-ing that these metal inputs ocurred mainly through anthropogenicactivities.

Regressions performed between metal concentrations in soilwith those in soybean organs and with BCF in different organs are

presented in Table 5. The regression coefficient between metal con-centration in soils and BCF was higher than the same coefficientbetween metal concentration in soils and that for plants (exceptfor Pb in roots). For example, Cd, Pb and Zn concentrations in seeds

250 M.J. Salazar et al. / Journal of Hazardous Materials 233– 234 (2012) 244– 253

Table 4Mean values and standard deviation (±SD) of the concentrations (in �g g−1 DW) of Cd, Pb and Zn in roots, stems, seeds and pods of Glycine max at different sites in theprovince of Córdoba.

Heavy metal Site Mean ± SD ANOVAa

Root Stem Seed Pods

Cd BH 0.04 ± 0.01BCc 0.21 ± 0.02BCb 0.2 ± 0.02Ab 0.40 ± 0.01Aa ***

BL 0.06 ± 0.03BCb 0.25 ± 0.01ABa 0.2 ± 3.90 × 10−04Aa 0.32 ± 0.06Aa *

D 0.071 ± 0.01BCc 0.32 ± 9.20 × 10−04Aa 0.1 ± 4.30 × 10−03Ec 0.12 ± 0.00Bb ***

F 0.07 ± 0.01BCb 0.17 ± 0.01BCDa 0.12 ± 0.02DEab 0.11 ± 0.03BCab *

GC 0.04 ± 0.01BC 0.11 ± 2.30 × 10−03 0.12 ± 0.01DE 0.09 ± 0.00BC nsGP 0.10 ± 0.01Bc 0.25 ± 0.02ABa 0.17 ± 3.40 × 10−03ABb 0.12 ± 0.00BCc ***

LM 0.01 ± 0.01 Cc 0.07 ± 0.01Eb 0.09 ± 2.90 × 10−03Eab 0.10 ± 0.00BCa ***

M 0.10 ± 0.00B 0.13 ± 0.02CDE 0.13 ± 0.01BCDE 0.10 ± 0.00BC nsPT 0.07 ± 0.01BCb 0.13 ± 0.02CDEab 0.13 ± 2.60 × 10−03CDEa 0.10 ± 0.01BCab *

RT 0.05 ± 0.02BCb 0.21 ± 0.02BCa 0.14 ± 4.00 × 10−03BCDa 0.01 ± 0.01Cb **

Y 0.30 ± 0.00A 0.17 ± 0.06BCD 0.17 ± 0.02ABC 0.10 ± 0.01BC nsANOVAb *** *** *** ***

Pb BH 7.19 ± 1.62Aa 5.20 ± 1.15Aab 1.52 ± 0.11b 5.48 ± 0.01Aab *

BL 0.37 ± 0.20Bb 1.90 ± 0.21Ba 1.71 ± 0.04a 1.99 ± 0.63Ca **

D 0.64 ± 0.59Bb 0.93 ± 0.56Bb 2.11 ± 0.29b 4.17 ± 0.21Aa **

F 1.12 ± 0.12B 1.47 ± 0.36B 1.63 ± 0.39 2.78 ± 0.58BC nsGC 1.26 ± 0.47Bb 1.46 ± 0.42Bb 2.23 ± 0.23ab 3.50 ± 0.40BCa *

GP 1.27 ± 0.51B 2.32 ± 1.08B 1.91 ± 0.32 3.67 ± 0.22B nsLM 1.39 ± 0.08Bc 1.18 ± 0.20Bc 2.24 ± 0.16b 3.00 ± 0.12BCa ***

M 1.04 ± 0.11Bb 1.98 ± 0.52Bab 2.53 ± 0.24a 3.01 ± 0.15BCa *

PT 2.56 ± 0.78B 2.12 ± 0.60B 1.76 ± 0.68 2.95 ± 0.62BC nsRT 1.49 ± 0.67Bb 2.70 ± 0.20Bab 2.55 ± 0.29ab 3.35 ± 0.25BCa *

Y 2.71 ± 0.11Bab 1.79 ± 0.15Bab 1.54 ± 0.74b 3.17 ± 0.04BCa *

ANOVAb *** *** ns ***

Zn BH 6.29 ± 0.51CDb 4.25 ± 0.12ABCb 30.00 ± 0.89a 5.09 ± 0.19Cb ***

BL 4.26 ± 0.69Dc 2.35 ± 0.10 Cd 32.14 ± 0.33a 7.23 ± 1.70BCb ***

D 10.59 ± 0.03Ab 4.89 ± 0.22ABd 29.52 ± 0.75a 8.08 ± 0.45ABCc ***

F 7.11 ± 0.81BCDb 5.74 ± 1.13Ab 34.39 ± 2.83a 11.72 ± 2.33Ab ***

GC 9.27 ± 0.17ABb 5.15 ± 0.30ABd 36.49 ± 0.39a 6.94 ± 0.36BCc ***

GP 7.38 ± 0.94BCb 4.15 ± 0.11ABCc 36.90 ± 0.87a 7.74 ± 0.37BCb ***

LM 5.61 ± 0.08CDb 5.62 ± 0.65Ab 32.73 ± 0.86a 7.44 ± 0.51BCb ***

M 6.74 ± 0.38BCDb 4.58 ± 0.27ABc 31.31 ± 0.44a 6.69 ± 0.01BCb ***

PT 10.79 ± 0.05Ab 5.70 ± 0.01Ac 38.36 ± 0.25a 9.99 ± 0.49ABb ***

RT 6.88 ± 0.23BCDbc 4.23 ± 0.20ABCc 32.71 ± 1.47a 7.65 ± 0.65BCb ***

Y 8.41 ± 1.98ABCb 3.64 ± 1.02BCb 31.53 ± 0.88a 5.43 ± 0.22Cb ***

ANOVAa *** *** ns **

Abbreviations: Sites: BH, Bouwer High; BL, Bouwer Low; GP, General Paz; F, Ferreyra; PT, Pozo del Tigre; D, Despenaderos; M, Malagueno; RT, Río Tercero; GC, General Cabrera;Y, Yocsina; LM, Los Molinos. ns, not significant.

a Values in each row (ANOVA) and column (ANOVA in capital letters) followed by the same letter do not differ significantly at p < 0.05.*

wocrarafiBbh

afpimeTicpt

p < 0.05.** p < 0.01.

*** p < 0.001.

ere not explained by metal concentration in soil whereas the BCFf Cd, Pb and Zn in seeds were. It should be noted that the asso-iations between BCF and metals in soils were negative, while theelationships between the metal concentrations in plants and met-ls in soils, when present, were positive. Similar results were alsoeported for Cd, Pb, Zn and other metals in algae and animals inquatic environments [59]. In our work, despite regression coef-cients being better for BCF, it is important to consider both theCF and metal concentration in plants, as a negative relationshipetween BCF and exposure concentrations could be misleading forazard identification.

Table 5 also shows a comparison among regression results usings regressor variables the metal concentrations in different soilractions and different soil compartments. The purpose of this com-arison was to assess the importance of the sampling procedure

n this type of study. The first column employs the exchangeableetal concentration in the rhizosphere as regressor variable to

xplain plant tissue concentration or BCF of the same heavy metal.he second column employs the exchangeable metal concentration

n bulk soil; the third column employs pseudototal metal con-entration in rhizospheric soil; and the fourth column employsseudototal metal concentration in bulk soil. Results for Pb showedhat regression predicting models could be obtained using any soil

compartment and fraction. On the other hand, both Zn and Cdconcentrations and BCF in soybean plants presented almost nonesignificant regression coefficient in the second, third, or fourthcolumns. Zn and Cd concentrations and the BCF in soybean plantsare only explained by exchangeable Zn and Cd concentration inrhizosphere compartment. This demonstrates that studies aboutheavy metal content in soils and its transference to crops, as wellas legislation and policies development in this area, should con-template not only pseudototal metal concentration in soils but alsoexchangeable fraction and rhizospheric influences of each crop.

These results also indicated a similar pattern for Zn and Cd, thusreinforcing the theory that they are acquired by the plant in thesame way [60,61]. Nevertheless, the Pb uptake by plants might alsobe due to an unknown mechanism [50,62].

3.5. Risk assessment

The THQ values for each metal, corresponding to an individualChinese, European, or Argentine adult are shown in Fig. 3. THQs

of less than one were found in the European or Argentine indi-viduals, whereas for a Chinese individual consumer this value wassignificant (higher than one) for Pb in soybeans from M and RTsites. Finally, the HI calculations for aggregate non-cancer risks

M.J.

Salazar et

al. /

Journal of

Hazardous

Materials

233– 234 (2012) 244– 253251

Table 5Simple regressions for metal concentration and BCF in soybean organs and metal concentration in soil.

Y X Exchangeable metal in rhizosphere Exchangeable metal inbulk soil

Pseudototal metal inrhizosphere

Pseudototal metal inbulk soil

R2 Model R2 Model R2 Model R2 Model

Pb Root 0.763*** Lineal Y = 1.195 ± 0.216*** + 0.02 ± 0.002***·X 0.751*** Lineal 0.760*** Lineal 0.761*** LinealBCF root 0.490*** Power Y = 0.640 ± 0.265*·X−0.699±0.152*** 0.491*** Power 0.456*** Power 0.49*** PowerShoot 0.692*** Lineal Y = 1.663 ± 0.155*** + 0.120 ± 0.002***·X 0.688*** Lineal 0.691*** Lineal 0.701*** LinealBCF shoot 0.828*** Power Y = 0.870 ± 0.162***·X−0.694±0.069*** 0.773*** Power 0.807*** Power 0.807*** PowerSeed ns ns ns nsBCF seed 0.962*** Power Y = 2.249 ± 0.283***·X−1.065±0.046*** 0.939*** Power 0.907*** Power 0.962*** PowerPods 0.472*** Lineal Y = 3.100 ± 0.154*** + 0.008 ± 0.002***·X 0.461*** Lineal 0.486*** Lineal 0.446*** LinealBCF pods 0.941*** Power Y = 2.742 ± 0.378***·X−0.930±0.051*** 0.912*** Power 0.870*** Power 0.908*** Power

Zn Root ns 0.319** Power ns nsBCF root 0.44*** Power Y = 5.967 ± 2.459*·X−0.868±0.208*** ns ns nsShoot ns ns ns nsBCF shoot 0.722*** Power Y = 5.680 ± 1.701**·X−1.126±0.151*** ns ns nsSeed ns ns ns nsBCF seed 0.946*** Power Y = 31.919 ± 3.232***·X−0.980±0.051*** 0.403** Lineal 0.325** Power 0.327** PowerPods ns ns ns nsBCF pods 0.745*** Power Y = 9.464 ± 2.687**·X−1.129±0.143*** ns 0.337** Power 0.379** Power

Cd Root 0.330** Lineal Y = 0.416 ± 0.123**·X 0.436*** Power ns nsBCF root ns ns ns nsShoot ns ns ns nsBCF shoot 0.423** Power Y = 0.154 ± 0.0.57*·X−1.058±0.261** ns ns 0.392** PowerSeed ns ns ns nsBCF seed 0.546*** Power Y = 0.199 ± 0.0.40***·X−0.726±0.142*** 0.307** Lineal ns 0.525*** LinealPods ns ns ns nsBCF pods 0.387** Lineal Y = 2.609 ± 0.356*** − 4.527 ± 1200**·X ns ns 0.388** Lineal

Abbreviations. BCF, bioconcentration factor; ns, not significant. In model column, “Y” is plant concentration of corresponding metal in the row and “X” is exchangeable metal concentration in rhizosphere.* p < 0.05.

** p < 0.01.*** p < 0.001.

252 M.J. Salazar et al. / Journal of Hazardous Materials 233– 234 (2012) 244– 253

THQA THQ

0 007

0,008

0,009

0,01

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ne

0 8

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0,002

0,003

0,004

0,005

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LM B H B L GP F PT D M RT GC Y

Sites

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Pb Zn Cd Pb Zn Cd

HIB

0,012

0,014

0,016

tin

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ig. 3. Target hazard quotients (THQ) (A) and hazard index (HI) (B) of Cd, Pb and Znines) inhabitants. Sampling sites: Malagueno (M); Yocsina (Y); General Paz (GP); Po

ith 2 different Pb concentration in soil, High (BH) with c. 300 mg kg−1 and Low (BL

hrough soybean consumption showed only values greater thanne at almost all sites for Chinese consumers (Fig. 3). Similarly, theuthors Bermudez et al. [58] reported values greater than unity forI in Argentine consumers of wheat in a study conducted in somef the same sites of this study. Therefore, it is important to highlighthe health risk related to heavy metal incorporation into crops dueo soil contribution.

. Conclusions

The metal concentrations (Cd, Pb and Zn) in soybean cropsrown close to different emission sources in Córdoba, Argentina,evealed a toxicological risk for a potential Chinese consumer. Tak-ng into account that the maximum levels in agricultural soils werexceeded in some areas by Pb, it is important to perform furthertudies related to the maximum permitted levels for heavy metalsn agricultural soils with different physical and chemical condi-ions. For our particular samples, the seed quality parameters wereot affected by a higher metal content, reflecting no relationshipxisting between physiological or morphological quality with toxicetal accumulation.Significant differences were found in the physical (pH, OM, EC)

nd chemical parameters (metal concentrations) between the rhi-osphere and bulk topsoils, indicating that rhizosphere soil is anmportant soil compartment with respect to food safety, due to theioaccumulation of toxic metals. Considering these findings, it isecessary to perform an evaluation and possible modification ofgricultural soil guidelines, which should in fact only consider thexchangeable fraction of metals and the physical characteristics ofifferent soil types, rather than the pseudototal fraction.

Finally, considering the bioaccumulation and magnification ofoxic heavy metals such as Pb and Cd, the potential risk of con-uming meat from cattle whose main sources of food are productserived from soybeans should be taken into account in futureesearch.

cknowledgments

This work was supported by Consejo Nacional de Investiga-iones Científicas y Técnicas (CONICET) through scholarships to theuthors M.J. Salazar and J.H. Rodriguez. The author M.J. Salazar is

Ph.D. student in Biological Sciences at the National University ofórdoba. Special thanks to L. Steffan for field surveys, to Bouwermelter neighbors (M.R. Pavani and S. Herrera) and to land ownernd mayor of Bouwer (J. Lupi), and to Dr. P. Hobson (native speaker)or language revision.

[

[

[

gh soybean consumption for Argentine, European (dashed lines) and Chinese (fulll Tigre (PT); Ferreyra (F); Los Molinos (LM); Despenaderos (D); Bouwer (BH and BL),

c. 40 mg kg−1; Río Tercero (RT); and General Cabrera (GC).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2012.07.026.

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