Eur Food Res Technol (2005) 221:361–366DOI 10.1007/s00217-005-1179-3

O R I G I N A L P A P E R

Biljana �krbic · Svetlana Cupic

Toxic and essential elements in soft wheat grain cultivated in Serbia

Received: 9 December 2004 / Revised: 15 February 2005 / Published online: 31 March 2005� Springer-Verlag 2005

Abstract A survey was carried out with the aim of as-sessing the level of some toxic (Cd, Pb, Hg and As) andessential (Cu, Zn, Fe and Mn) elements in wheat grown inSerbia. A total of 433 samples of soft winter wheat grainfrom all the Serbian wheat-growing regions were pooledinto 52 representative samples for 2002 harvest. After wetdigestion, Cd, As, and Pb were determined by graphitefurnace atomic absorption spectrometry (GFAAS),whereas Cu, Zn, Fe and Mn were determined by flameatomic absorption spectrometry (FAAS). Vapour gener-ation accessory was used for Hg determination. The meanand median contents of all samples were (on a dry weightbasis) 29 and 24 mg/kg for Cd, 137 and 87 mg/kg for Pb, 7and <0.1 mg/kg for Hg, 55 and 59 mg/kg for As, 4.1 and4.0 mg/kg for Cu, 24 and 24 mg/kg for Zn, 53 and 53 mg/kg for Fe, 34 and 33 mg/kg for Mn. The calculated meanand median levels of contamination were compared withthe recommended or regulated maximum levels accordingto the European Commission and the national legislation.The average intake of eight selected elements fromwheat-based products was estimated for the Serbianpopulation.

Keywords Soft winter wheat · Trace elements · Averageintake · Serbia

Introduction

In the last few decades cereal products, and among themwheat-based products, have received considerable atten-

tion in view of their potential role of vehicles of toxicmicroelements like Cd, Pb, Hg, and As into the humandiet.

Cd, Pb, Hg, As, Cu, Zn, Fe, and Mn are the mostcommon microelement contaminants in the environment.Cd, Pb, Hg, and As are nonessential elements in meta-bolic processes and may be toxic or lethal to organismswhen absorbed in certain amounts, while As is beingconsidered as carcinogenic one. Cu, Zn, Fe, and Mn areessential elements in metabolic processes, but they maybe toxic to organisms when they are higher in the envi-ronment, especially in organisms [1, 2].

The existing data on trace elements of wheat refer tocrops cultivated in pilot fields nearby Novi Sad, Vojvo-dina [3]. This study was on a limited, local scale and todate no survey exists in Serbia concerning the levels oftoxic and essential trace elements in soft winter wheatgrain representative of the national production. So, thereis a lack of information on the actual levels of trace ele-ments in Serbian wheat. This is worth noting, as wheat-based products play an essential role in the Serbian dietand constitute one of its most important characteristicfeatures. The average daily consumption figures forbread, wheat flour, pasta and bakery products were about270, 42, 8, and 5 g/person, respectively, in 2002. Most ofthe wheat used for bakery and pasta production is of na-tional origin.

With regard to Cd and Pb information on the averagecontent in wheat crops is particularly useful becauseFAO/WHO Codex Alimentarius Commission, the Euro-pean Commission (EC) and the Serbian national legisla-tion are evaluating proposed maximum limits for theseelements in various foodstuffs, including cereals. For Asand Hg there are neither maximum levels proposed by theEC nor any kind of recommendation. The Serbian legis-lation [4] sets up maximum levels for Cd, Pb, Hg and Asas 0.1, 0.4, 0.05 and 1 mg/kg dry matter, respectively. Inaddition to Cd, Pb, Hg, and As, Cu, Zn, Fe and Mn wereselected for this study, as the consumption of wheatproducts is also an important source of the dietary intakeof these essential trace elements and can also influence

B. �krbic ())Faculty of Technology,University of Novi Sad,Bulevar cara Lazara 1, 21000 Novi Sad, Serbiae-mail: biljana@tehnol.ns.ac.yuTel.: +381-21-450288Fax: +381-21-450413

S. CupicVinca Institute of Nuclear Sciences,Belgrade, Serbia

trace element reference values in the tissues of theworld’s population [5]. Data presented here are the resultof a national-wide survey aimed at investigating the levelsof the eight selected elements in grain samples repre-sentatively collected as specified bellow from all theSerbian-wheat growing regions.

Materials and methods

Sampling

Grain samples of soft winter wheat were collected in the frame ofthe yearly monitoring of wheat quality performed by the NationalCenter for Cereal Technology-Novi Sad, Serbia.

For the purpose of this study, the Serbian territory was subdi-vided into its 14 administrative regions; regions were in turn sub-divided into provinces. Within each province having a significantwheat production, samples of grains belonging to the varieties mostcultivated were collected at harvest 2002 in different representativefields located in the whole provincial territory. Individual samplescoming from the same province–each weighing 1 kg–were pooledtogether to obtain the test samples. In this way 52 compositesamples of soft winter wheat (coming from 433 individual samples)were prepared: Table 1 reports the number of individual samplesand pools.

The relative number of samples was proportional to its per-centage in the national production and accounted for mostly all ofthe Serbian production of soft winter wheat.

Apparatus

Samples were analyzed depending on the type of elements and theirconcentration by Perkin-Elmer atomic absorption spectrometer(AAS) model 5000 equipped with HGA 400 heated graphite at-omizer and vapour generation accessory. Software HG Graphics II(Perkin - Elmer, Norwalk, CT, USA) was employed for spectrumacquisition and data processing.

Sample preparation and determination of elements by AAS

Aliquots from the pooled grain samples were manually cleaned andmilled in a laboratory mill (Buhler MLU 202, Switzerland) to ob-tain a fine wholemeal flour. Flours were stored in clean polyeth-ylene flasks at 5 �C pending analysis.

Wet procedure was selected for sample destruction. The anal-yses were performed in two parallel determinations using testportions of approximately 1 g (fresh weight) dissolved in 10 ml ofconcentrated nitric acid and heated under reflux. After dissolution,10 ml of concentrated perchloric acid was added and heated untilformation of nitrous fumes stopped. The digestion temperature didnot exceed 85 �C to prevent the loss of As and Hg (the range oftemperature was from 70 to 85 �C). The solution was placed in a50 ml volumetric flask and made up to volume with deionised

water Milli-QTM type (18 MW). The same procedure (withoutwheat sample) was applied for the blank analysis.

Cu, Zn, Fe and Mn concentrations were determined throughdirect aspiration of the aqueous solution into air–acetylene flame ofAAS at 324.7, 213.9, 248.3 and 279.5 nm, respectively. Mi-croelements like Pb, As and Cd were determined by flamelessatomic absorption spectrometer (FAAS) with heated graphite at-omizer at 283.3, 193.7 and 228.8 nm, respectively, while vapourgeneration accessory was used for Hg determination at 253.7 nm.Background correction was performed using a deuterium lamp. Theoperating conditions were based on those suggested by the manu-facturer [6] and were presented elsewhere [7]. Standards used in thecalibration procedures were prepared in the same acids as samples.

Sample aliquots were taken for the determination of the mois-ture contents. Approximately 1 g of the sample was kept at 80 �Cuntil constant weight and moisture content were determined on thebasis of the mass losses. The concentrations of elements in wheatsamples were calculated on a dry mass basis.

Reagents

All the chemicals used in the sample treatments were of ultrapuregrade. The glassware was cleaned prior to use by soaking overnightin 10% v/v HNO3 (Suprapur, Merck) and rinsed with Milli-Q water.Standard stock solutions of analyzed elements were purchased fromMerck (Germany).

Analytical quality control

All the elements were analyzed according to the SANCO guide [8].The validation of applied analytical method for residues was per-formed on fortified samples, five at the limit of quantification(LOQ) level, and five at 10 times LOQ. Two blank samples wereincluded in the set, and their values did not exceed 30% LOQ. Theachieved recovery for all examined microelements were in therange 70–110%, with a relative standard deviation less than 20%.This validation also included testing of linearity.

The limits of detection (LOD) and quantification (LOQ) cal-culated on the basis of three and 10 times the standard deviation ofthe mean of blank determinations from 15 measurements were,respectively, 0.3 and 1.0 mg/kg for Cd; 5 and 16 mg/kg for Pb; 0.1and 0.3 mg/kg for Hg; 20 and 50 mg/kg for As; 0.9 and 3.0 mg/kgfor Cu; 1.0 and 3.0 mg/kg for Zn; 1.1 and 3.5 mg/kg for Fe; 0.01and 0.03 mg/kg for Mn.

Wheat sample from a province of region 3V was selected for therepeatibility validation of the analytical procedure.

Repeatability of measurement was checked by the analysis ofCd, Pb, Hg, As, Cu, Zn, Fe and Mn in six parallel samples after wetdigestion applying the recommended analytical procedure. Re-peatabilities as relative standard deviation values were as follows4.9% for Cd, 5.3% for Pb, 11.0% for Hg, 5.1% for As, 6.2% for Cu,3.2% for Zn, 4.3% for Fe, and 2.9% for Mn.

Table 1 Sign and number ofcomposite and individual sam-ples for each administrativeregion

Region Region sign na Region Region sign na

Northern Banat 1V 4 (33) Macva 8S 3 (30)Middle Banat 2V 4 (31) Kolubara 9S 2 (26)Southern Banat 3V 5 (34) Branicevo 10S 3 (29)Northern Backa 4V 5 (35) �umadija 11S 2 (25)Southern Backa 5V 6 (39) Pomoravlje 12S 3 (28)Western Backa 6V 5 (36) Belgrade 13S 2 (25)Srem 7V 6 (40) Bijeljina 14S 2 (22)a Number of composite samples (individual samples in parenthesis)

362

Results

The element concentrations in soft winter wheat aresummarized in Table 2, where samples are grouped byadministrative region. All figures are calculated on a dryweight basis; the average moisture contents of wheatturned out to be 11.5%.

The mean total content in soft wheat grain cultivated inSerbia were (€standard deviation): 28.8€18.3 mg/kg forCd, 137€117 mg/kg for Pb, 7.02€15.1 mg/kg for Hg,55.4€28.9 mg/kg for As, 4.12€0.79 mg/kg for Cu,23.8€2.3 mg/kg for Zn, 53.5€3.7 mg/kg for Fe,33.9€3.9 mg/kg for Mn. The contents of Cd, Zn, Fe andMn were distributed normally; the frequence distributionsof the Pb, Hg and Cu were positively, and of As wasnegatively skewed. The medians were: 24.3 mg/kg for Cd,86.8 mg/kg for Pb, <0.1 mg/kg for Hg, 58.7 mg/kg for As,3.98 mg/kg for Cu, 24.1 mg/kg for Zn, 52.6 mg/kg for Feand 32.8 mg/kg for Mn.

The mean total and median of low-level contaminationof food regarding the number of samples below LOD [9]were calculated as follows: in the case of As, it was as-sumed that their content were equal to LOD/2 as thenumber of samples with the determined content belowLOD was less than 60%, while in the case of Hg, it wasassumed to be equal LOD as the number of results belowLOD was in the range 60–80%.

Grain concentrations of elements concerned, especiallyHg and Cd, varied widely between sites. The maximumlevels of Hg, Cd, Pb, As, Cu, Mn, Zn and Fe were up to548, 14, 11, 9, 2, 1.5, 1.4 and 1.3 higher than their min-imum levels, respectively, while only for administrativeregion 14S the maximum level of Hg was slightly higherthan the allowed one by national regulation [4]. The meanand median Cd, Pb, Hg and As contents found in softwheat in the present survey are below maximum limits setby Serbian legislation.

The most frequently occurring pattern for wheat is Fe> Mn > Zn > Cu > Pb > As > Cd > Hg.

The mean element contents in soft wheat grain are infair agreement with those reported by various recentsurveys conducted in different countries (Table 3). Cd,Hg, Cu, Zn and Mn levels are very near to those reportedin the literature and summarized in Table 3, whereas thePb, As and Fe levels found in this study definitely fall inthe high concentration range.

Discussions

A maximum level of 0.2 mg/kg for Cd in wheat grain and0.1–0.2 mg/kg for Pb in cereals have been proposed andare presently under evaluation by the EC [23], while for Hgand As, there are no maximum levels proposed or rec-ommendation. Also the FAO/WHO Codex AlimentariusCommission is evaluating similar maximum levels for Cdand Pb in the same staple foods (0.2 and 0.1 mg/kg, re-spectively) [24, 25]. The median Cd content found in softwinter wheat in the present study is well below the valueproposed by EC and FAO/WHO. The median content ofPb is lower than the proposed one by EC and FAO/WHO,while the mean value is in the range proposed by EC andslightly higher than the one proposed by FAO/WHO.

On the basis of the available data (2002) from theCentral Institute of Statistics, a mean monthly consump-tion of about 9750 g of soft wheat-based products (in-cluding bread and bakery products) can be inferred forSerbia. From these data, using the mean total metalcontent in Serbian soft wheat found in this study, a roughestimation of the average intake of the eight selectedmetals from wheat-based products for the Serbian popu-lation can be drawn. Calculations were made taking intoaccount the moisture content of the different wheat-basedproducts.

In this estimation, we have ignored the amount ofimported crop and assumed that all the soft wheat em-ployed in Serbian production shows the mean levelsfound in this study.

Table 2 Mean contents of Cd, Pb, Hg, As, Cu, Zn, Fe and Mn in representative wheat grain samples from fourteen administrative growingregions of Serbia harvested in 2002 (mean € standard deviation)

Regiona nb mg/kg mg/kg

Cd Pb Hg As Cu Zn Fe Mn

1V 4 (33) 24.1€2.6 123€60.2 <0.1 61.6€5.5 4.69€1.53 21.9€0.9 60.7€5.2 35.5€2.82V 4 (31) 21.9€3.5 34.3€10.5 <0.1 <20 6.11€1.95 21.0€0.7 58.2€5.0 31.5€1.63V 5 (34) 39.5€9.9 66.7€23.4 4.50€0.27 81.2€7.2 2.99€1.03 21.4€1.1 47.0€4.2 31.8€1.34V 5 (35) 24.6€14.3 44.3€25.7 20.2€1.9 44.5€3.2 3.23€0.71 20.2€0.7 49.2€5.9 31.5€1.75V 6 (39) 7.70€2.2 65.6€13.2 <0.1 41.7€4.1 4.58€0.92 23.5€3.5 50.3€14.1 32.0€2.06V 5 (36) 26.4€5.8 173€47.1 <0.1 73.4€9.1 3.93€1.59 22.8€4.6 53.5€6.6 40.1€4.47V 6 (40) 50.0€15.0 62.4€15.3 <0.1 52.2€3.5 3.76€1.12 24.2€4.7 52.4€5.8 33.6€0.88S 3 (30) 14.2€1.2 54.6€7.8 <0.1 81.2€10.7 4.46€1.34 29.3€6.3 55.7€4.8 30.6€2.39S 2 (26) 37.8€11.7 107€41.9 <0.1 55.8€4.3 3.64€0.55 25.5€0.5 51.8€4.9 41.0€3.7

10S 3 (29) 44.8€6.7 351€78.4 4.00€0.51 90.6€9.5 3.96€0.32 25.0€1.2 51.3€5.2 31.1€2.611S 2 (25) 72.1€8.7 135€67.2 <0.1 84.7€7.5 3.39€1.41 24.2€3.6 52.4€5.7 37.3€3.212S 3 (28) 15.1€6.2 42.7€9.8 13.9€0.8 79.0€7.1 4.82€0.80 25.2€1.0 52.9€3.2 27.1€2.213S 2 (25) 5.12€1.1 379€61.5 <0.1 <20 3.99€0.08 24.0€0.5 56.1€3.7 34.8€5.314S 2 (22) 20.2€5.6 279€40.9 54.8€3.0 <20 4.11€0.42 24.6€1.7 57.0€2.3 36.5€7.5a Region sign is explained in Table 1b Number of composite samples (individual samples in parenthesis)

363

The weekly intake of Cd calculated in this way is52 mg/person, i.e. 13% of the provisional tolerable weeklyintake (PTWI) for Cd of 7 mg/kg bw or 420 mg for anadult (60 kg person) established by the Joint FAO/WHOExpert Committee on Food Additive (JECFA). ThisPTWI was reconfirmed by JECFA in 1993, pending fur-ther research, with the statement that, “there is only arelatively small safety margin between exposure in thenormal diet and exposure that produces deleterious ef-fects” [26].

A contribution of 13% of the wheat-based products tothe intake of Cd may be regarded as very high, but it isworth noting that this calculation probably overestimatesthe actual Cd content in the milling products used forbakery and pasta production. Chaudri et al. [12] reporteda reduction of 31% for Cd content in white flour withrespect to wholemeal flour as a consequence of millingprocedures, whereas the content in bran was doubled.Bruggemann and Kumpulainen [27] found that Cd con-tent in flour decreased by 2–25%, the highest decreasebeing recorded in flours with the lowest ash content.Therefore, it is reasonable to suppose that even in Serbianwhite flour mostly used for bread making, Cd contents arelower than those present in the grains from which theyoriginate. Therefore, taking into consideration the re-ported reduction of Cd content (2–31%), the weekly in-take of Cd could be between 9 and 12% of PTWI. Asimilar range of intakes by wheat-based products wasfound in Italy [14].

With regard to Pb, for which JECFA established aPTWI of 25 mg/kg bw, or 1.5 mg for an adult [26], a dailyintake of 36 mg/person can be calculated from our data,

i.e. 46% of the typical exposure (77 mg/day) estimated forthe European countries [28].

Anyway, as Pb is mostly located in the outer layers ofthe kernel, its content in white flour may be greatly re-duced by milling. Reductions of 30–50%, depending ofthe ash content of flour, have been reported in comparisonto whole grains, even if in practice contamination duringmilling and bread-making procedures may always occuras Pb is a ubiquitous element [27]. In that way, the intakeof Pb with wheat-based products would be approximately10% of PTWI. In comparison to the study of Conti et al.[14], where intake of Pb by these products was about 1%,the calculated high value could be explained by the pre-dominant usage of leaded gasoline in Serbia and subse-quent atmospheric deposition of Pb.

The weekly intake of 13 mg Hg per person, for whichJECFA established a provisional tolerable weekly intakeof 5 mg/kg bw [29], can be calculated from our data, i.e.4.3% of PTWI. This calculation overestimated the actualHg content in the final bakery products, i.e. the hightemperatures in the oven cause the volatilization of Hg.

With regard to As, for which JECFA established aprovisional tolerable daily intake (PTDI) of 2 mg/kg bw[26], a daily intake of 14 mg/person can be calculatedfrom our data, i.e. about 12% PTDI. Cubadda et al. [15]showed a 36% average decrease in As concentration inwhite flour after milling with respect to wholemeal flour.Therefore, taking into consideration the reported reduc-tion of As content, the daily intake of As could be 8% ofPTDI.

In the case of Hg and As, it should be pointed out thatthe estimations were overestimated due to the way of

Table 3 Mean and ranges of metal concentrations in wheat grain reported by various studiesa

Cd(mg/kg)

Pb(mg/kg)

Hg(mg/kg)

As(mg/kg)

Cu(mg/kg)

Zn(mg/kg)

Fe(mg/kg)

Mn(mg/kg)

Country Reference

Soft wheat (Triticum aestivum)4.06b

(4.23–4.40)25.1b

(23.4–26.9)31.3b

(27.2–33.7)40.6b

(27.1–46.0)US,Canada

[10]

5.12(2.79–7.53)

28.6(16.1–55.0)

41.4(28.6–74.7)

27.9(11.0–62.8)

UK [11]

44 (4–310) UK [12]58 (15–146) France [13]40 (13–94) 16

(6–47)5.14(3.15–7.64)

33 (22–51) Italy [14]

14 12 10 4.65 29.5 34.5 38.7 Italy [15]14 (n.d–20) 135

(n.d.–304)n.d 19

(n.d.–154)4.6(4.2–5.1)

21.8(16.8–30.6)

47.6(43.3–55.8)

45.9(41.7–47.4)

Vojvodina,northernprovinceof Serbia

[3]

28.8(5.1–72)

137(34.3–379)

7.0(<0.1–54.8)

55.4(<20–90.6)

4.1(3.0–6.1)

23.8(20.2–29.3)

53.5(47.0–60.7)

33.9(27.1–41.0)

Serbia This study

Wheatc (Triticum sp.)43(<0.2–207)

37(<0.8–716)

4.4(2.2–8.7)

27(9.3–67)

32.5(17–60)

38(13–67)

USA [16, 17]

70b

(20–350)160b

(30–650)5b

(<0.1–29)45b

(5–285)TheNetherlands

[18]

55 26 Finland [19]51 34 3.8 32 Germany [20]70 170 6.41 40.2 Bulgaria [21]58 <200 5.19 24.4 Slovenia [22]

n.d.-Not detecteda On a dry weight basis unless otherwise specifiedb On a fresh weight basisc Not specified

364

calculating the mean total concentrations of these ele-ments.

The daily intake of Cu, Zn, Fe and Mn from the con-sumption of wheat-based products averages 1.1, 6.2, 13.9and 8.8 mg/person, respectively.

However, in this case the intakes calculated from themean metal levels in grains are certainly overestimated, asmilling greatly decreases Cu and Zn contents in processedproducts. With regard to white flour, reductions as high as50–70% for Cu and 73–84% for Zn have been reported inthe literature, with smaller losses in the less refinedproducts [27]. Similar losses are reported also for Fe, Mnand other metals. Therefore, milling leads to a deterio-ration of the nutritional value of flour with respect to theircontent in essential trace elements.

In the light of the foregoing remarks, a more realisticestimate of Cu and Zn would be 0.3–0.5 mg/day and 1.0–1.7 mg/day, respectively, while Fe and Mn intake fromwheat-based products may lead to half the values reportedabove (i.e. obtaining about 7.0 mg/day for Fe and 4.4 mg/day for Mn).

However, it may be observed that the recommendeddietary allowances (RDA) of Cu and Zn for adults ac-cording US Food and Nutrition Board of the NationalAcademy of Science/National Research Council, aremuch higher and are in the range of 1.5–3 mg Cu/day and8–15 mg Zn/day. The above calculated figures for Cu andZn represent a maximum of 17 and 11% of the recom-mended dietary allowances for Cu and Zn, respectively,for the adult population and coincide with the ones ob-tained in Italy [14].

The recommended nutrient intake value for Fe is59 mg/day [30]. From our data a daily intake represents12% of the recommended exposure.

The Food and Nutrition Board of the US NationalResearch Council proposed an estimated safe and ade-quate daily dietary intake of Mn to be 2–5 mg/day [31]while the WHO [32] recommended 2–9 mg/day for anadult. Our calculated value of dietary daily intake for Mnis very high representing 88% and 49% of the above-mentioned upper limits, respectively.

Therefore, the results of this nationwide survey on Cd,Pb, Hg, As, Cu, Zn, Fe and Mn levels in the Serbianwheat allowed us to clarify some issues relating to theactual contents of these elements in such an importantstaple food. The mean and median Cd, Pb, Hg, and Ascontents lay below the maximum limits according to theSerbian legislation and also below the maximum levelspresently under evaluation by some international bodies.Nevertheless, the contribution of wheat-based products tothe total intake of these elements is significant and turnedout to be particularly high in the case of Cd and Pb. Asregarding the essential elements (Cu, Zn, Fe and Mn), thelevels determined confirm the important role of wheat-based products in meeting the dietary requirements for thegeneral population. However, it is recognized that moreinformation is needed on the degree of reduction in thecontents of essential elements with respect to wholegrains as a consequence of industrial processing.

Acknowledgements This work was supported by the ScientificFund of Serbia as a part of the Project No. 1775

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