Food Chemistry 122 (2010) 1338–1343

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Food Chemistry

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Analytical Methods

Copper contents in grapes and wines from a Mediterranean organic vineyard

Maria Rosaria Provenzano a,*, Hamid El Bilali a, Vito Simeone b, Nuray Baser b, Donato Mondelli a,Gianluigi Cesari b

a Dipartimento di Biologia e Chimica Agro-Forestale ed Ambientale, University of Bari, Via G. Amendola 165/a, 70126 Bari, Italyb Mediterranean Agronomic Institute of Bari (IAM-B), Via Ceglie 9, 70010 Valenzano (Bari), Italy

a r t i c l e i n f o

Article history:Received 6 November 2008Received in revised form 14 October 2009Accepted 24 March 2010

Keywords:CopperOrganic viticultureSoilBerriesWine

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.03.103

* Corresponding author. Tel.: +39 080 5442929; faxE-mail address: provenza@agr.uniba.it (M.R. Prove

a b s t r a c t

This work was conducted in an organic vineyard in Bari (South-Eastern Italy) to evaluate copper concen-trations in grapes and wines of four local and international varieties (Chardonnay, Primitivo, Uva di Troiaand Negroamaro) and to relate these contents to total and available copper concentrations in soil.Approximately 7.4 kg/ha of copper were used in the trial year. Soil copper availability was higher inthe 0–20 cm (10.3%) than in the 20–40 cm layer (4.7%). No copper phytotoxicity was observed on leaves.Copper residues on berries and in wines resulted below the maximum residue levels (MRLs). Concentra-tions in berries increased with applications number and varied according to the variety. Copper contentin the white wine (Chardonnay) was higher with respect to red ones (Primitivo, Uva di Troia and Negro-amaro). Results provided evidence that in the natural conditions typical of a Mediterranean environment,characterised by calcareous soils and a dry climate, the use of copper formulations in vineyards accordingto European legislation guidelines should not raise any concern with regards to human health.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

The Italian organic wine sector has dramatically increased. Inthe last years, the organic vineyard area has doubled, reachingmore than 40,000 ha, while organic wineries account for more than9000 farms (Rossetto, 2007). In Apulia region (South-Eastern Italy)organic agriculture has been rapidly growing mainly due to policysupport. Apulia represents 9% of the Italian organic land. Organicland area represents 6.9% of the regional agricultural area. Vine-yards cover 3% (2888 ha) of the regional organic agriculture landarea (Pugliese, 2006).

Copper (Cu) is one of the most important biopesticides used inorganic farms. It is effective against a high number of crops pestsand it is utilised as a fungicide, a bactericide and also as a herbi-cide. Copper formulations are effective against grapevine (Vitisvinifera L.) downy mildew and they have a secondary effect ongrapevine powdery mildew and on a wide range of other grapevineinsect pests and diseases (Boubals, 2001; Ferreira, Strecht, Ribeiro,Soeiro, & Cotrim, 1998; Morando, Morando, Bevione, & Lembo,1997). Copper use for pest management has a very long history.Bordeaux Mixture (i.e. Bouillie Bordelaise), a simple mixture of cop-per sulphate and lime, was discovered by Alexis Millardet in Francein the 1880s and it was the world’s first commercially successfulfungicide (Ayres, 2004).

ll rights reserved.

: +39 080 5442850.nzano).

Copper is an essential micronutrient for all living organisms,including humans, who require it for normal metabolic processesalong with amino and fatty acids as well as vitamins. However,as the body cannot synthesise copper, the human diet must supplyregular amounts. Copper deficiency leads to an increased risk ofdeveloping coronary heart disease whereas high levels of coppercan be harmful. The symptoms of acute copper poisoning includenausea, vomiting and abdominal and muscle pain (Britton, 1996;Olivares & Uauy, 1996).

Since copper deficiency and copper excess produce adverse ef-fects, monitoring copper level in soil and food is of crucial impor-tance for human health.

Determination of total copper contents is an important step inestimating its hazards and negative impacts on terrestrial ecosys-tems. However, from the eco-toxicological point of view, it isimportant as well to determine copper bioavailability, which rep-resents the fraction that can be utilised by microorganisms andplants (Lepp, 2003).

Regarding crops, some authors reported that no correlation wasobserved between total and/or extractable (available) copper con-centrations and its contents in plants or plants organs (Hinsinger,2001; Marschner, 1995; McLaughlin, Smolders, & Merckx, 1998).However, Romic, Romic, and Ondrašek (2004) found that DTPA(diethylenetriamine pentaacetic acid)-extractable copper was lar-gely explained by total copper contents in Croatian vineyards.Brun, Le Corff, and Maillet (2003) stated that plants aerial partswould not be a good indicator of vines Cu uptake from French soils,

M.R. Provenzano et al. / Food Chemistry 122 (2010) 1338–1343 1339

as they provide no insight into the real amount of Cu transferredfrom soils to plants.

On the other hand, the accurate determination of copper inwine is of crucial importance for wine quality. Elevated copperconcentrations can cause oxidative spoilage leading to pinking ofred wine, browning of white wine as well as haze formation. Thegenerally recommended ‘‘safe” copper concentration (total copper)is below 0.3–0.5 lg ml�1 (Clark & Scollary, 2000).

The objective of the present research is to evaluate total coppercontents in grapes and wines of four local and international varie-ties namely Chardonnay, Negroamaro, Primitivo and Uva di Troiagrown in a Mediterranean organic vineyard and to relate thesecontents to total and available copper concentrations in soil.

2. Experimental

2.1. Field trial

This work was conducted in an experimental spur-pruned or-ganic vineyard established in 2002 at the Mediterranean Agro-nomic Institute of Bari (IAM-B) (approximate coordinates: 41�020

as latitude and 16�530 as longitude), Apulia region, South-EasternItaly. In the vineyard four renowned counter espalier-trained winevarieties are grown: Primitivo, Negroamaro, Uva di Troia and Char-donnay. The vineyard was divided into eight plots each covering aland area of about 150 m2 and containing 15 plants for each of thefour varieties. Each statistical treatment (control and copper-trea-ted) was repeated on four plots. Copper treatments, carried outusing a formulation containing 20% of copper as oxychloride(Ossiclor�), were initiated as the rule of the three tens was fulfilled(10 �C; 10 mm of rainfall; 10 cm-long shoots) and carried on onlywhen climatic conditions resulted favourable to fungus develop-ment (high humidity and rainfall). Approximately 1000 L of spray-ing mixture (copper-based formulation + water) were used in eachapplication. Temperature (max, min and mean), humidity (max,min and mean) and rainfall were constantly recorded during thewhole period of the trial at the agro-meteorological station of theIAM-B.

2.2. Soil and bunches sampling

During April 2006, before starting treatments during the newvine growing season, soil samples were collected at 0–20 cm and20–40 cm depths. Three sub-samples were collected from differentplaces and were bulked to obtain a representative sample for thesite.

Four different bunches of each variety were sampled in July 19,August 20, September 19, and 26. The same number of bunches ofeach variety were also collected from the vineyard part that hasnot been treated so that copper determined in these samples canbe considered as natural.

Copper predicted environmental concentrations in soil (PECs)are evaluated assuming that no copper degradation occurs be-tween its application and sampling dates, that the whole quantityof copper sprayed remains in the top 10 cm horizon and that thesampled soil has a bulk density of 1.25 g/cm3. It is also assumedthat even if vines intercept a part of copper during treatments, thatfraction ends up in soil after rain-induced washing-off and/or afterleaves fall. Of course, it is supposed that the copper-based pesticidedistribution was homogeneous in the whole field. In addition, it isalso assumed that the amount of copper absorbed by plants andstored in the different organs (leaves, bunches, shoots, etc.) thatare removed by some cultural operations (hard and green pruning,bunches thinning, harvest, etc.) is so small that it can be neglected.

2.3. Soil physical and chemical analyses

Prior to analysis, soil samples were air-dried at green housetemperature (approximately 25–30 �C), lightly ground, mixedthoroughly in order to homogenise them, and sieved through a2 mm stainless steel sieve. Soils chemical and physical analyseswere carried out in accordance with internationally recommendedand accepted analysis procedures and/or Italian official methods(MIPAAF, 1999). Particles sizes distribution was determined usingthe pipette method (Gee & Bauder, 1986), after dispersing soilsamples with sodium hexametaphosphate and sodium carbonatesolutions. Textural classes were determined in accordance withUSDA (United States Department of Agriculture) classification. SoilpH was measured by a pH meter Crison Basic 20� in distilledwater pHH2O using a suspension 1:2.5 soil to water phase ratio.Electrical conductivity (EC) was determined by a conductimeter(XS instruments cond. 510�) on an aqueous extract (soil filtrate)of a mixture soil to water 1:2 ratio. Organic carbon was deter-mined by Walkley and Black method modified by Jackson(1958). Total carbonate was determined using a volumetric meth-od based on its dissolution with hydrochloric acid (HCl) followedby measurement in Dietrich–Fruehling calcimeter. Available Cuwas determined by DTPA (0.05 M DTPA + 0.01 M CaCl2 + 0.1 M tri-ethanolamine (TEA) solution buffered at pH 7.3 with HCl) with 2 hof continuous shaking (Lindsay & Norvell, 1978). Extracts were fil-tered with Whatman 42 before their analysis by ICP-OES – induc-tively coupled plasma-optical emission spectrometry (ThermoElectron, ICAP 3000�) at 324.75 nm wavelength. All chemicalsused were of analysis grade and purchased from Sigma Aldrich,Milan, Italy.

2.4. Total copper analysis

For soil analysis the following procedure was carried out: 0.5 gof fine soil were put in 50 ml Teflon vessels, with automated pres-sure and temperature regulation, then 3.0 ml of 70% HNO3, 1.0 ml37% HCl and 1.0 ml of 30% H2O2 were poured in the vessels. Soilswere digested in a microwave oven (CEM�, MarsX Press) as follow:3 min at 140 �C; 3 min at 165 �C, and 20 min at 190 �C. Soil extractswere then filtered by Whatman 42 paper filter.

Berries were analysed as follow: 1 g of the homogenised samplewas poured in a 250 ml Erlenmeyer beaker, acidified with concen-trated HNO3 and added with 20 ml of H2O2. The total volume wasreduced to 3 ml on a heating plate and 9 ml of concentrated HCland 3 ml of concentrated HNO3 were added. A condenser with awater-based cooling system was applied to the flask and heatedfor 2 h. The volume was adjusted to 50 ml with distilled water.Only 10 ml of the solution were submitted to analysis. Five deter-minations were made for each grape variety and each sample.

Bunches were submitted to micro-vinification at the Santi Dim-itri winery in Galatina (Lecce, Italy). Micro-vinification was carriedout according to a standard winemaking protocol set for organicgrape cultivars and designed to limit, as much as possible, techno-logical interventions and remedial actions during all winemakingstages: crushing, de-stemming, pressing, fermentation, decanting,filtration, clarification, etc. The aim is not to obtain excellent wines,but ‘‘natural” wines that are as much as possible the expression ofgrape variety and the terroir (mainly soil and climate) interaction.White wine (Chardonnay) making included: soft vacuum pressing,static cold settling and fermentation at 13 �C. Red wines (Primitivo,Negroamaro, and Uva di Troia) making comprised: entire berriesmaceration for 6 days, fermentation at 28 �C, soft vacuum pressingand short ageing. Since the objective was that of producing a ‘‘nat-ural” wine no selected yeast strains were added. All the instru-ments used during the pressing, fermentation, decanting,filtration, clarification, fermentation or short ageing do not contain

1340 M.R. Provenzano et al. / Food Chemistry 122 (2010) 1338–1343

copper. Moreover, the same instruments and utensils were used toprocess and handle all the wine samples. Therefore, we can excludethat during the micro-vinification process any copper contamina-tion occurred. Wine samples were analysed just before bottlingin March/May 2007. The sample pretreatment procedure adoptedwas that suggested by Brainina et al. (2004) for not aged wineswith a 3:10 dilution in 5% HNO3.

All total copper determinations were performed using an opti-cal emission spectrometer (Thermo Electron, ICAP 3000) at324.75 nm wavelength.

2.5. Statistical analyses

Data were analysed, when necessary, by one-way analysis ofvariance (ANOVA) and Student’s T-test. Duncan test was used formultiple range separation of means. Significant differences weredetermined at a significance level equal to or lower than 5%(p 6 5%). An SPSS (Statistical Package for the Social Sciences) soft-ware version (SPSS 12.0 for Windows) was used to carry out all sta-tistical tests.

3. Results and discussion

3.1. Soil physical and chemical properties

Results of chemical and physical analyses are shown in Table 1.As data show, the soil is alkaline, with a balanced texture and isquite rich in organic matter (OM). Clay content increases withdepth. EC is higher in the shallow sampling layer. OM contentsare slightly higher in the subsoil. Total carbonate (TC) contentsare quite high in the 0–20 cm layer probably due to the fact thatApulian vine growers are used to crashing rocks, especially calcar-eous ones, in their vineyards.

Table 1Soil chemical and physical properties. Averages were calculated considering all the samplesexpressed as means ± standard errors.

Depth (cm) Clay (g/kg) Texture class (USDA) pHH2 O (1:2.5)

0–20 233 Loam 7.99 ± 0.0120–40 314 Silt loam 7.93 ± 0.01

0

20

40

60

80

100

120

Feb

. I

Feb

. II

Feb

. III

Mar

ch I

Mar

ch II

Mar

ch II

I

Apr

il I

Apr

il II

Apr

il III

May

I

May

II

May

III

Rainfall (mm) Tmean

Fig. 1. Climatic conditions in IAM-B vineyard, 2006. Climatic

3.2. Copper quantities used in the vineyard

In the year of the trial, climatic conditions produced two impor-tant infectious cycles when important rainfall events occurred dur-ing the growing season (Fig. 1).

Disease levels were medium on leaves and medium–low onbunches. As for leaves, Negroamaro resulted the most susceptiblecultivar and Chardonnay the most resistant. Regarding bunches,the least susceptible varieties were Chardonnay and Negroamaro.No infection was observed on shoots. Apart from shoots, a signifi-cant statistical difference (ANOVA and Student’s T-test) was alwaysobserved between the control and the copper-treated statisticaltreatment in terms of disease incidence.

Copper treatments performed during the trial are reported inTable 2. Nine treatments were carried out with rates that rangedfrom 350 ml/hl, at the beginning of the season, to 450 ml/hl inJune, which is the month with maximum disease pressure andhighest plant organs susceptibility. Intervals between treatmentsranged from 5 to 12 days.

The organic farm was established in 2002 and copper was usedfor the first time in 2004. The vineyard has been managed accord-ing to organic agriculture rules since its establishment. Copperquantities used were: 5 kg/ha in 2004 and 13 kg/ha in 2005. Thetotal amount of copper used in the year of the trial, 2006, was7.4 kg/ha corresponding to a PEC of 5.9 mg/kg. Moreover, med-ium-term PEC (2004–2006) should be equal to 20.3 mg/kg.

The European Commission regulation No. 473/2002, amendingthe European Council regulation No. 2092/91 on organic agricul-ture, limited copper use to 6 kg/ha/year from 2006 and furtherreductions are not excluded in the new Council regulation No.834/2007. This limit is calculated considering the whole organicfarm land area and not each hectare of that farm or each field. Thismeans, in practical terms and for organic certification purposes,that even when the total quantity of copper used on a specific crop(grapevine in the present case) is higher than 6 kg/ha/year, organic

collected at the same depth. pHH2 O, EC, organic matter and total carbonate values were

Organic matter (g/kg) EC (dS/m) Total carbonate (g/kg)

20.7 ± 0.7 0.30 ± 0.02 228 ± 2226.1 ± 1.6 0.19 ± 0.01 31 ± 10

June

I

June

II

June

III

July

I

July

II

July

III

Aug

ust I

Aug

ust I

I

Aug

ust I

II

Sep

t. I

Sep

t. II

Sep

t. III

Oct

. I

(°C) Hmean (%)

parameters are reported as decades’ (10 days) averages.

Table 2Copper-based treatments in IAM-B vineyard.

Treatments No. Dates Dose (ml/hl) Phenological stage Interval between treatments (days)

1 April 20 350 Separate leaves –2 May 02 12

3 May 12 400 Bunches differentiation 104 May 22 Separate bunches 105 May 29 Bloom 7

6 June 03 450 End bloom 67 June 09 Berries setting 68 June 14 59 August 19 Veraison 66

M.R. Provenzano et al. / Food Chemistry 122 (2010) 1338–1343 1341

farmers can stay in line with the organic regulation reducing theuse of copper on another grape cultivar or even on a different crop(e.g., olive).

3.3. Total copper in soil

Copper concentrations were statistically similar in the 0–20 and20–40 cm layers and resulted 93.3 ± 4.6 mg/kg and 111.0 ± 5.5 mg/kg, respectively. In other works, higher concentrations in the 0–20 cm layer with respect to 20–40 cm layer were found (Pietrzak& McPhail, 2004). Our result may be explained considering thattillage, to which vineyards are subjected in Apulia region, resultin mixing up the different soil profile layers leading to a kind ofhomogenisation of the heavy metal concentrations along the soilprofile.

Concentrations found in this work were lower than those foundin Northern Italy which is characterised by different climatic con-ditions. In a survey carried out in Italy it resulted that average Cucontents of vineyard soils were 297 mg/kg in the wet Northernmountainous regions, where fungicidal treatments against downymildew are frequent, 200 mg/kg in the wet plains, and only75 mg/kg in the dry areas of the South, where fungicidal treat-ments, especially those for controlling downy mildew, are muchless frequent (Deluisa et al., 1996).

In addition, our values are comparable to those found by Rusjan,Strlic, Pucko, and Korošec-Koruza (2007) in the Sub-Mediterraneanvineyards in Slovenia (71–160 mg/kg) and Brun, Maillet, Richarte,Hermann, and Remy (1998) in French Mediterranean vineyards(31–251 mg/kg) and much lower than those found in some re-nowned winegrowing regions (Pietrzak & McPhail, 2004) whereconcentrations up to 1500 mg/kg were reached.

3.4. Soil available copper

Available copper was 9.6 mg/kg in 0–20 cm layer and 5.25 mg/kg in 20–40 cm layer thus exceeding the threshold of 0.1 mg/kgCu-DTPA (Sims & Johnson, 1991) considered as an indicator of cop-per deficiency. Copper availability (available copper/total copperratio) was also significantly influenced by depth (10.3% in 0–20 cm and only 4.7% in 20–40 cm). In French Mediterranean vine-

Table 3Copper content (mg/kg) on berries of the four wine grapes varieties. Sampling on 26 SepteValues are expressed as means ± standard errors of the means (n = 3). Concentrations of coptreatment (control or treated) followed by different letters are significantly different acco

Sampling dates July 19 August 20

Cultivars Control Treated Control Treated

Negroamaro 2.26 ± 0.02 c 3.31 ± 0.02 d 2.02 ± 0.03 c 5.81 ± 0Primitivo 2.58 ± 0.08 b 4.71 ± 0.05 c 2.35 ± 0.01 b 11.26 ±Chardonnay 4.95 ± 0.08 a 5.77 ± 0.08 b 5.07 ± 0.04 a 11.61 ±Uva di Troia 1.65 ± 0.01 d 6.49 ± 0.07 a 1.85 ± 0.03 d 9.32 ± 0

yards, DTPA-extractable Cu ranged from 5% to 52% of total copper(Flores-Veles, Ducaroir, Jaunet, & Robert, 1996). In general, copperavailability was quite high despite the soil pHH2O was almost 8.0.

3.5. Copper contents in berries

Copper contents in berries are reported in Table 3. All the un-treated bunches showed lower copper contents which did notchange significantly during the whole growing season. Differentcopper contents were observed among the different varieties likelydue to variety-specific genetic factors controlling the plant abilityto uptake copper. The highest total copper concentrations were re-corded on Chardonnay (5 mg kg�1) while the lowest were deter-mined on Uva di Troia (1.7 mg kg�1). Anyway, content in allgrapes cultivars was below the maximum allowed residue level(MRL) of copper in fresh grapes (20 mg kg�1) (Italian Health Minis-try Decree, 2004).

Copper contents increased with increasing copper applicationsnumber but were always below the MRL. Residue values found inJuly were slightly lower than those recorded in August. No copperdecrease was observed from August to September in spite of somerain events. On September 19, one month after the last treatmentperformed on August 19 – that is to say 10 days longer than copperpre-harvest interval (20 days) – copper contents in bunches re-mained, more or less, the same therefore the pre-harvest intervalcan result sometimes insufficient to reduce copper contents belowthe MRL.

3.6. Relationship between soil copper and its concentrations in berries

In Fig. 2 total copper, copper availability and the correspondingtotal copper concentrations in untreated berries are shown. Sam-pling carried out on September 19 was selected because it wasthe last one in which bunches of all the four cultivars weresampled.

Total copper concentrations in grapes represented a half (char-donnay cultivar) to a fifth (Uva di Troia cultivar) of available copperdetermined in 0–20 cm soil layer. In all cultivars, copper contentsdid not change significantly during the whole growing season.

mber was carried out only on cv. Uva di Troia which is the only late-ripening variety.per in berries of different cultivars sampled the same day and from the same statisticalrding to Duncan test at p 6 5%.

September 19 September 26

Control Treated Control Treated

.04 d 2.19 ± 0.05 c 6.43 ± 0.03 c – –0.09 b 2.51 ± 0.02 b 11.91 ± 0.21 a – –0.04 a 5.04 ± 0.03 a 14.02 ± 0.12 a – –.10 c 1.72 ± 0.01 c 9.97 ± 0.06 b 1.64 ± 0.02 10.39 ± 0.12

2.52.24.710.35.259.6

111.0

93.3

0

20

40

60

80

100

120

0-20

cm

20-4

0 cm

0-20

cm

20-4

0 cm

Neg

roam

aro

Prim

itivo

Cha

rdon

nay

Uva

di

Troi

a

Soil total copper (mg/kg) Soil copper availability(%) Total copper in grapes (September 19)

Fig. 2. Total copper and copper availability in IAM-B organic vineyard soil and corresponding total copper concentrations in berries grown on the same soil. Bars representstandard errors of the means.

1342 M.R. Provenzano et al. / Food Chemistry 122 (2010) 1338–1343

In our study no copper phytotoxicity symptoms were observedon plants organs especially leaves and shoots, which are consid-ered the most sensitive organs. In general, vine is tolerant to highcopper doses since roots are deep and it possesses quite a widerange of potential cellular mechanisms that may be involved inheavy metals detoxification and tolerance to heavy metals-inducedstresses (Hall, 2002; Hoof et al., 2001; Leopold, Gunther, Schmidt,& Neumann, 1999). In addition, it must be considered that in cal-careous soils, as those of Apulia, Cu should be precipitated ashydroxides or carbonates (Delas, 1963).

3.7. Copper concentrations in wines

Total copper concentrations in wines resulted: 462.3 ± 2.9 lg/Lin Chardonnay; 267.0 ± 4.0 lg/L in Uva di Troia; 255.0 ± 8.6 lg/L inPrimitivo; and 116.7 ± 0.6 lg/L in Negroamaro. All values were be-low the MRL (1 mg/L) for both white and red wines (EEC regulationNo. 1410 of 7/8/2003, Annex IV). A significantly higher (Duncantest, p 6 5%) copper concentration was found in Chardonnay whitewine. The lowest copper concentration was measured in Negroa-maro red wine. The marked difference in copper content betweenwhite and red wines might be due to the fact that, unlike whitewines, red wines undergo higher must aeration during the wine-making process. Therefore, their higher oxygen content may in-crease copper oxidation and copper salts precipitation. Thiscauses, usually, a higher copper content in white wines as reportedby Ostapczuk, Eschnauer, and Scollary (1997).

Copper concentrations in the wines were approximately 6 (Uvadi Troia) to 19 (Negroamaro) times lower than those determined inthe grape berries therefore it can be assumed that copper absorbedby the vine is likely sequestered in the berries pulp and skinswhich are discarded after the bunches crushing.

4. Conclusions

Total copper concentrations found in this work were within val-ues typical of Mediterranean soils whereas available copper ex-ceeded in both sampling depths the threshold of 0.1 mg/kgconsidered as an indicator for copper deficiency. No copper phyto-toxicity symptoms were observed on vines organs especially leavesand shoots. Different copper contents were determined in berriesamong the four cultivars likely due to variety-specific genetic fac-tors controlling the plant ability to uptake copper. However, all val-ues were below the allowed MRL in grapes and represented a fifth

to a half of available copper in the shallow layer (0–20 cm). Copperconcentrations in the wines were lower than those found in thegrape berries. Residues in the wines were always lower than theMRL.

Results obtained in this paper point toward the general conclu-sion that in the natural conditions typical of a Mediterranean envi-ronment characterised by calcareous soils and a dry climate, theuse of copper formulations in vineyards according to the Europeanregulation on copper use in organic farms should not raise any con-cern with regards to human health.

Acknowledgement

Funding for this work was provided by the University of Bariand the Mediterranean Agronomic Institute of Bari (IAM-B) underthe Community initiative INTERREG III A Italy–Albania 2000–2006: ‘‘integrated project for the diffusion and technical assistancein the implementation of methodologies for the production of or-ganic products”, Acronym: PAB.

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