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SUPPLEMENTARY MATERIAL
Chemometric analysis of minerals and trace elements in Sicilian wines from two
different grape cultivars
Angela Giorgia Potortί, Vincenzo Lo Turco*, Marcello Saitta, Giuseppe Daniel Bua, Alessia
Tropea, Giacomo Dugo, Giuseppa Di Bella.
Dipartimento di Scienze Biomediche, Odontoiatriche e delle Immagini Morfologiche e Funzionali
(BIOMORF), Università di Messina, Viale Annunziata – Polo Universitario 98168 Messina, Italy.
*Address correspondence to Vincenzo Lo Turco; Dipartimento di Scienze Biomediche,
Odontoiatriche e delle Immagini Morfologiche e Funzionali (BIOMORF), Università di Messina,
Viale Annunziata – Polo Universitario 98168 Messina, Italy;
phone: +39 090 3503 997;
e-mail: [email protected].
Abstract
Chemometric analysis are used for food authenticity evaluation, correlating botanical and
geographical origins with food chemical composition.
This research was carried out in order to proved that it is possible linked red wines to Nero d'Avola
and Syrah cultivars of Vitis vinifera according to their mineral content, while the values of the
physical and chemical parameters do not affect relevantly this discrimination.
The levels of mineral elements were determined by ICP-OES and ICP-MS.
Samples from cv Nero d’Avola had the highest content of Zn, Cr, Ni, As and Cd, whereas the
highest mineral concentration in cv Syrah samples was represented by K, Mg, Cu, and Sb. The
research highlights that it is possible linked red wines to Nero d'Avola and Syrah cultivars of Vitis
vinifera according to their mineral contents, adding knowledge to the determination studies of the
wine botanical origin.
Keywords: Red wines; Minerals; Trace elements; Chemometric analysis; Botanical discrimination.
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Experimental
Reagents and materials
High purity water with resistivity of 10 MΩ cm (J.T. Baker, Milan, Italy), was used throughout.
Concentrated (65%, v/v) nitric acid trace metal analysis grade (J.T. Baker, Milan, Italy) was used
together with concentrated (30%, v/v) hydrogen peroxide (J.T. Baker, Milan, Italy) for samples
digestion. The first one was also employed for cleaning glassware.
Single element standards (1000 mgL−1
in 2% nitric acid) were purchased from Fluka (Milan, Italy)
and from Merck (Darmstadt, Germany) and were mixed to prepare a multi-element standard
solution that has been subsequently diluted for calibration analysis.
To correct instrumental drift and variations due to the matrix, standard solutions of 45
Sc, 103
Rh and
209Bi (1000 mgL
−1 in 2% nitric acid) were purchased from Fluka (Milan, Italy) and were used as
on-line internal standards (at level of 1.5 mgL-1
).
To verify the digestion of sample and to correct the volumetric changes, standard solution of Re at
1000 mgL-1
in 2% nitric acid was acquired by Fluka (Milan, Italy) and was used as preparation
standard (at level of 0.5 mgL-1
).
A solution containing 1 gL-1
of 7Li,
59Co,
80Y and
205Tl in 2% HNO3 was obtained from Agilent
(Santa Clara, CA) and was used to tune the ICP-MS instrument.
A diagnostic standard solution containing 1000 mgL-1
of Ba, Mg and Zn in 5% HNO3 (JYICP-
DIAG) was obtained from Horiba Jobin Yvon (Longjumeau, France) and used for the periodic
check of the ICP-OES instrument.
Argon (N 5.0) of 99.9990% purity and helium (N 5.5) of 99.9995% purity were supplied by Rivoira
gases (Milan, Italy).
All reagents used for enological parameters determination, provided by Sigma Aldrich, were
analytical grade.
Wine samples
The analyses were carried out on 39 sicilian red wines, obtained from grapes of 100 % cv Nero
d’Avola and 34 from 100% cv Syrah, and cultivated in the same geographic areas in province of
Syracuse. All samples were obtained directly from producers and were from 2015 vintage. The wine
glass bottles (750 mL) were stored in the dark at 2°C and opened before analysis.
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Analytical procedure for minerals and trace elements determination
From the freshly opened bottles, about 1 mL of each wine was transferred and accurately weighed
into acid-prewashed PTFE vessels; it was added with internal Re standard and then digested with 6
mL of HNO3 (69%, v/v) and 2 mL of H2O2 (30%, v/v) in a microwave digestion system Ethos 1
(Milestone, Bergamo, Italy) equipped with sensors for temperature and pressure control.
Instrumental parameters and settings were: 10 min for 1000W up to 200°C, 10 min for 1000W at
200°C. Allowed to cool, each sample was made up to volume of 10 mL with HNO3 (2%, v/v). Each
sample was digested in triplicate.
The K, Ca, Mg, Na, Zn, Fe, Mn and Cu determination was carried out by Horiba Jobin Yvon
ULTIMA 2 (HORIBA Scientific, Longjumeau, France) ICP-OES spectrometer, equipped with a
glass concentric pneumatic nebulizer (i.d. 0.3mm) coupling with a quartz cyclonic type spray
chamber (50mL).
For Cr, Pb, Ni, Co, Se, As, Cd and Sb measurements an Agilent 7500cx (Agilent Technologies,
Santa Clara, CA) ICP-MS spectrometer, equipped with a MicroMist glass concentric pneumatic
nebulizer coupling with a cooled Scott double pass type spray chamber made of quartz, was used.
To minimize polyatomic interferences resulting from plasma and matrix, an octopole collision
system with 4 mLmin-1
helium as collision gas and kinetic energy discrimination mode was used
(collision mode) for almost all the elements.
The instrument operating parameters for ICP-OES and ICP-MS analyses are presented in Table S1.
According to literature, ethanol may affects the quantification of the different elements in wine
samples since its content could influence the transport properties towards atomization devices of the
instrument as well as the viscosity and the density of the samples (Aceto et al. 2002). Thus, the
possible interference of alcohol on elemental measurements was corrected by addiction of 1.3%
ethanol to all standard solutions used for calibration since wine samples were diluted 10 times
before analysis (Rodriguez et al. 2011).
The evaluation of the linearity was based on the 6 standard solutions injections. Each solution was
injected three times (n=3). The instrumental detection limits (LODs) and quantification (LOQs)
were experimentally calculated as 3.3σ/S and 10σ/S, respectively, where σ is the standard deviation
of the response of six blanks and S is the slope of the calibration curve (EURACHEM 2000).
A lab-made wine containing 5 gL-1
tartaric acid and 13% ethanol in water was used as blank
solution. It was digested as describe above and it was run with each batch of wines. Moreover, for
recovery studies, 18 spiked lab-made wine solutions were prepared: 9 were used for ICP-OES
analysis (3 at level of 50 mgL-1
, 3 at level of 100 mgL-1
and 3 at level of 300 mgL-1
) and 9 were
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used for ICP-MS analysis (3 at level of 10 µgL-1
, 3 at level of 20 µgL-1
and 3 at level of 50 µgL-1
).
Each solution was analyzed in triplicate. For repeatability estimation and intermediate precision,
each spiked level was prepared and analyzed in 12 replicates in the same batch and in 24 replicates
in different days.
Enological parameters determination
The main enological parameters, among which alcohol content, pH, total acidity, volatile acidity,
malic acid, SO2 and total SO2 contents, were determined following the procedures specified in detail
in EC Regulation 2676/90 (1990).
Finally, according to Ribéreau Gayon and Stonestreet (1965), anthocyanins quantification was
carried out in wine samples.
Validation of ICP-MS and ICP-OES analysis
Method linearity, sensitivity, accuracy, precision and repeatability are reported in Table S2. Results
showed that the adopted procedures were suitable for the research. Indeed, good linearity was
observed in each investigate concentration range with R2 ≥ 0.99943. Instrumental LOD values
ranged from 0.003 to 0.750 mgL-1
for ICP-OES analysis and from 0.009 to 0.030 µgL-1
for ICP-
MS analysis, while instrumental LOQ values ranged from 0.010 to 2.50 mgL-1
and from 0.029 to
0.100 µgL-1
, respectively. Thus the analytical limit of quantification for elements analyzed by ICP-
OES were between 0.1 to 25 mgL-1
(Cu and K, respectively), while for elements analyzed by ICP-
MS varied from 0.290 (value determined for Sb) to 1.000 µgL-1
(value determined for Se and As).
The recovery for all elements was always within the interval of 75.2-126.6%. The repeatability
RSD% was lower or equal to 5.1%, while for intermediate precision it was lower or equal to 9.5%.
Statistical analysis
The SPSS 13.0 statistical software package for Windows (SPSS Inc., Chicago, IL, USA) was used
for all statistical calculations.
Statistical methods were conducted on starting multivariate matrix where variables were the
concentrations of 24 detected parameters (8 were the enological parameters and 16 were the
concentrations of minerals and trace elements) and the cases were the 73 analyzed wine samples.
Data below LOQ were replaced with the LOD/2 values and all concentrations were loge-
transformed to reduce the effect of outliers on skewing the data distribution and to bring the
concentrations of element within the same range (Škrbić et al. 2010).
The data were subdivided in two groups, according to the cultivar of Vitis vinifera: the first one (39
samples) consisting of wines from Nero d’Avola grapes, and the second one (34 samples)
represented by wines from Syrah grapes.
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Initially, the non-parametric Mann–Whitney U test was applied to study the significances of
differences. Successively, the data set was normalized and Principal Component Analysis (PCA)
was performed to differentiate samples belonging to the two red wine types based on the enological
parameters values and the concentrations of minerals and trace elements. In addition to PCA, Linear
Discriminant Analysis (LDA) in the stepwise mode was carried out to discriminate among wine
cultivars in according to F-value.
References
Aceto M, Abollino O, Bruzzoniti MC, Mentasti E, Sarzanini C and Malandrino M. 2002.
Determination of metals in wine with atomic spectroscopy (flame-AAS, GF-AAS and ICP-AES): a
review. Food Addit Contam 19:126-133.
D.M. 29 December 1986. Decree of the Minister of Agriculture and Forests, published on
Official Gazette, No. 13, January 17th 1987.
EC Regulation 2676/90 (17 Sept. 1990). Determining Community methods for the analysis of
wines. Off J Eur Communities 1990, No. 272 (Oct 3), 1-192.
EURACHEM 2000, Guide. (2nd
Ed.). Editors: S L R Ellison (LGC, UK), M Rosslein (EMPA,
Switzerland), A Williams (UK).
European Directive EC/1881/2006/. Commission Regulation, No. 1881, December 19th
2006.
OIV (Organisation Internationale de la Vigne et du Vin). 2011. Compendium of international
methods of wine and must analysis.
Ribéreau GP and Stonestreet E. 1965. Le dosage des anthocyanes dans le vin rouge. Bull Soc
Chim Fr 9:2649-2652.
Rodriguez SM, Otero M, Alves AA, Coimbra J, Coimbra MA, Pereira E and Duarte AC. 2011.
Elemental analysis for categorization of wines and authentication of their certified brand of origin. J
Food Comp Anal 24:548-562.
Škrbić B, Szyrwińska K, Đurišić-Mladenović N, Nowicki P and Lulek J. 2010. Principal
component analysis of indicator PCB profiles in breast milk from Poland. Environ Int 36:862-872.
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Table S1 – Instrument operating parameters for ICP-OES and ICP-MS analyses.
ICP-OES analysis
Rf power 1000W
Auxiliary/nebulizer/plasma Argon flow rate 0.2/1/12 L·min-1
Nebulization pressure 2.98 bar
Nebulizer pump 20 rpm
Sample introduction flow rate 1 mL·min-1
Acquisition mode maxima
Integration time 2 sec for K, Ca, Mg and Na; 4 sec for Zn, Fe, Mn and Ca
Monitored isotopes and wavelengths (nm) K, 766.490; Ca, 393.366; Mg, 279.553; Na, 588.995; Zn, 213.856; Fe, 259.940; Mn, 257.110; Cu, 324.754
ICP-MS analysis
RF power 1500W
Plasma/auxiliary/carrier gas flow rate 15/0.9/1.1 Lmin-1
Helium collision gas flow rate 4 mLmin-1
Spray chamber temperature 2 °C
Sample depth 9 mm
Sample introduction flow rate 1 mLmin-1
Nebulizer pump 0.1rps
Extract lens 1 1.5 V
Octopole collision system setting He mode for Cr, Ni, Co, Se, As and Cd; No-gas mode for Pb and Sb
Monitored isotopes 52Cr, 59Co, 60Ni, 75As, 80Se, 114Cd, 121Sb and 208Pb
On-line internal standards 45Sc for Cr, Co, Ni, As and Se; 103Rh for Cd and Sb; 209Bi for Pb
Integration times 0.8 s/point and for Se; 0.5 s/point for As; 0.2 s/point for Cr, Co and Ni; 0.1 s/point for Cd, Sb and Pb
Point for mass 3 (3 replicates acquisitions)
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Table S2 – Validation parameters for ICP-OES and ICP-MS analyses.
ICP-OES analysis
Element R2 LODi (mg/L) LOQi (mg/L) LOQa (mg/L) Accuracy (% ± RSD%, n=9) Repeatability (RSD%, n=12) Intermediate precision (RSD%, n=24)
Level I
(50 mgL-1)
Level II
(100 mgL-1)
Level III
(300 mgL-1)
Level I
(50 mgL-1)
Level II
(100 mgL-1)
Level III
(300 mgL-1)
Level I
(50 mgL-1)
Level II
(100 mgL-1)
Level III
(300 mgL-1)
K 0.99993 0.750 2.500 25.000 76.6 ± 2.6 79.4 ± 2.2 99.8 ± 2.8 3.3 3.5 4.0 6.6 7.0 7.6
Ca 0.99943 0.625 2.083 20.833 106.7 ± 3.1 110.8 ± 2.2 126.6 ± 2.5 3.4 2.7 2.5 5.6 4.2 4.0
Mg 0.99973 0.540 1.800 18.000 98.3 ± 4.2 102.4 ± 2.3 119.4 ± 2.4 3.6 2.7 2.6 4.5 3.0 2.7
Na 0.99969 0.300 1.000 10.000 100.8 ± 2.8 104.4 ± 2.2 120.0 ± 3.2 3.6 3.0 2.8 3.9 4.3 6.0
Zn 0.99991 0.035 0.117 1.167 80.5 ± 2.7 85.0 ± 2.1 105.2 ± 2.3 4.5 4.7 5.1 8.8 7.6 7.4
Fe 0.99947 0.029 0.097 0.967 75.2 ± 3.2 78.4 ± 2.5 96.4 ± 1.2 3.8 3.3 3.1 8.2 8.6 6.5
Mn 0.99993 0.018 0.060 0.600 98.2 ± 3.4 101.4 ± 2.8 123.5 ± 1.2 2.8 2.3 2.1 5.8 6.1 6.9
Cu 0.99961 0.003 0.010 0.100 86.4 ± 2.7 90.0 ± 3.0 105.6 ± 1.2 2.6 2.8 3.4 8.1 9.5 6.2
ICP-MS analysis
Element R2 LODi (mg/L) LOQi (mg/L) LOQa (mg/L) Accuracy (% ± RSD%, n=9) Repeatability (RSD%, n=12) Intermediate precision (RSD%, n=24)
Level I
(10 µgL-1)
Level II
(20 µgL-1)
Level III
(50 µgL-1)
Level I
(10 µgL-1)
Level II
(20 µgL-1)
Level III
(50 µgL-1)
Level I
(10 µgL-1)
Level II
(20 µgL-1)
Level III
(50 µgL-1)
Cr 0.99986 0.014 0.047 0.467 86.7 ± 5.6 90.5 ± 4.1 107.8 ± 4.1 4.6 3.9 3.8 8.0 8.3 9.8
Pb 0.99995 0.010 0.033 0.333 80.5 ± 3.2 84.0 ± 4.8 102.1 ± 2.1 2.9 3.1 3.8 10.3 8.7 8.3
Ni 0.99967 0.020 0.067 0.667 83.1 ± 2.2 86.7 ± 3.8 105.4 ± 2.1 3.5 3.7 3.9 7.5 7.8 9.2
Co 0.99995 0.020 0.067 0.667 88.7 ± 4.6 92.2 ± 3.3 110.1 ± 3.1 3.0 2.5 2.4 8.4 7.6 7.3
Se 0.99943 0.030 0.100 1.000 90.2 ± 4.1 93.6 ± 2.2 111.6 ± 3.1 0.9 1.1 1.8 8.2 7.0 6.6
As 0.99996 0.030 0.100 1.000 86.2 ± 2.8 89.8 ± 3.5 106.9 ± 5.1 3.1 2.9 2.7 4.0 4.2 5.6
Cd 0.99993 0.009 0.030 0.300 86.3 ± 1.8 90.0 ± 3.4 106.8 ± 2.1 4.2 3.6 3.4 5.6 6.0 7.7
Sb 0.99973 0.009 0.020 0.290 82.1 ± 7.6 85.6 ± 3.9 103.5 ± 2.1 1.6 1.1 1.0 7.9 7.1 6.8
R2, determination coefficient; LODi, instrumental detection limit; LOQi, instrumental quantification limit; LOQa, analytical quantification limit.
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Table S3 – Contents and significant differences of the mineral and trace elements composition of wines from cv Nero d’Avola and cv Syrah grapes.
K (mg·L-1) Ca (mg·L-1) Mg (mg·L-1) Na (mg·L-1) Zn (mg·L-1) Fe (mg·L-1) Mn (mg·L-1) Cu (mg·L-1)
Nero d’Avola (n=39)
Min 506.613 68.412 100.434 10.298 3.801 1.006 0.461 0.121
Max 1519.418 216.643 151.667 204.659 11.793 9.446 1.866 1.525
Mean 887.522 124.126 122.234 41.833 6.607 4.661 1.307 0.289
S. D. 258.326 30.412 14.812 44.663 1.982 1.978 0.402 0.264
Syrah (n=34)
Min 1025.955 75.174 125.789 10.787 1.234 2.424 0.659 0.139
Max 2220.167 162.891 223.748 100.806 6.571 9.576 1.776 1.820
Mean 1450.543 110.113 158.865 37.187 3.571 4.706 1.204 0.744
S. D. 266.901 22.795 23.791 24.255 1.771 2.024 0.309 0.500
Mann Whitney U 1228.500 487.000 1207.000 702.000 196.000 625.000 534.500 1157.000
Wilcoxon W 1823.500 1082.000 1802.000 1297.000 791.000 1220.000 1129.500 1752.000
Asymp. Sign. 0.000 0.052 0.000 0.666 0.000 0.674 0.155 0.000
Cr (µg·L-1) Pb (µg·L-1) Ni (µg·L-1) Co (µg·L-1) Se (µg·L-1) As (µg·L-1) Cd (µg·L-1) Sb (µg·L-1)
Nero d’Avola (n=39)
Min 10.127 11.565 17.212 0.743 1.295 1.513 n.d. n.d.
Max 106.434 97.891 202.937 7.617 10.548 13.913 0.871 n.d.
Mean 35.099 30.855 66.801 4.193 3.841 4.018 0.515 _
S. D. 25.816 19.137 51.357 1.824 2.072 2.636 0.144 _
Syrah (n=34)
Min 10.450 10.283 10.004 1.070 1.333 0.961 n.d. n.d.
Max 29.675 59.952 52.853 6.505 6.116 3.268 0.780 0.936
Mean 18.042 30.053 28.271 3.447 3.437 1.764 0.348 0.572
S. D. 5.190 15.784 9.898 1.183 0.975 0.588 0.224 0.214
Mann Whitney U 334.000 678.000 256.000 502.000 655.000 151.000 459.500 916.500
Wilcoxon W 929.000 1273.000 851.000 1097.000 1250.000 746.000 1054.500 1511.500
Asymp. Sign. 0.000 0.868 0.000 0.075 0.930 0.000 0.019 0.000
Asymp. Sign. bold values indicate element concentrations significantly different at 95%.
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Table S4 – Legal limits of Zn, Cu, Pb, As and Cd, and comparison with results obtained in this study.
Element Legal limits (mg/L) Concentrations in wine (mg/L) Samples exceeded legal limits (%)
Nero d'Avola Syrah Nero d'Avola Syrah
Mean value Max value Mean value Max value
Pb 0.2a 0.03 0.10 0.03 0.06 0 0
0.15b 0 0
Cu 1c,d 0.3 1.5 0.7 1.8 5 24
Zn 5c,d 6.6 11.8 3.6 6.6 74 24
As 0.2d 0.004 0.014 0.002 0.003 0 0
Cd 0.1d 0.0005 0.0009 0.0003 0.0008 0 0
a Regulation EC/1181/2006; b OIV, 2006; c Ministerial Decree of 29 December 1986; d OIV, 2011.
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Table S5 – Values and significant differences of the enological parameters of wines from cv Nero d’Avola and cv Syrah grapes.
Alcoholic grade
(% vol)
pH
Total acidity
(g·L-1)
Volatile acidity
(g·L-1)
Malic acid
(g·L-1)
SO2
(mg·L-1)
Total SO2
(mg·L-1)
Anthocyanins
(mg·L-1)
Nero d’Avola (n=39)
Min 12.0 3.1 5.0 0.34 0.01 9.90 41.58 125.00
Max 14.4 3.7 6.8 0.91 1.49 38.38 131.30 1.076.00
Mean 13.0 3.5 5.6 0.52 0.29 26.13 85.36 222.20
S. D. 0.5 0.2 0.4 0.16 0.29 8.53 21.37 144.35
Syrah (n=34)
Min 11.6 3.1 4.2 0.35 0.02 9.80 41.16 120.00
Max 14.2 3.8 6.7 0.90 0.80 51.00 150.00 672.00
Mean 12.9 3.6 5.3 0.55 0.24 23.84 85.19 232.24
S. D. 0.67 0.1 0.5 0.14 0.22 10.48 24.46 104.09
Mann Whitney U 518.000 725.500 388.500 799.000 582.500 544.000 615.000 703.500
Wilcoxon W 1113.000 1320.500 983.500 1394.000 1177.500 1139.000 1210.000 1298.500
Asymp. Sign. 0.108 0.489 0.002 0.132 0.373 0.188 0.595 0.654
Asymp. Sign. bold values indicate element concentrations significantly different at 95%.
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Table S6– The Recommended Dietary Allowance (RDA) of non toxic elements and RDA shares from consumption of Nero d’Avola and Syrah wines.
Non Toxic Elements RDA (mg×day-1)a Mean concentrations in wine (mg/L) % of RDA estimated by mean value
Nero d'Avola Syrah Nero d'Avola Syrah
Zn 10 6.607 3.571 14.5 7.9
Fe 14 4.661 4.706 7.3 7.4
Se 0.055 0.004 0.003 1.5 1.4
Cu 1 0.289 0.744 6.4 16.4
Cr 0.040 0.035 0.018 19.3 9.9
Mn 2 1.307 1.204 14.4 13.2
Ca 800 124.126 110.113 3.4 3.0
K 2000 887.522 124.126 9.8 1.4
Mg 375 122.234 158.865 7.2 9.3
Na 1500b 41.833 37.187 0.6 0.5
a Commission Directive 2008/100/EC; bAI (Adeguate intake) EFSA 2005.
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Table S7 – Protection Limits (TDI, TWI, PTWI and BMDL01) of potentially toxic elements and Protection Limits shares from consumption of Nero d’Avola and Syrah wines.
Potentially Toxic Elements Mean concentrations in wine (mg/L) % of Protection limit estimated by mean
value Reference
Nero d'Avola Syrah Nero d'Avola Syrah
Pb PTWI (mg×kgb.w.-1×week-1) 0.025 0.031 0.030 3.2 3.1 EFSA, 2010
BMDL01(µg×kgb.w.-1×day-1) 1.5 7.5 7.3
As PTWI (mg×kgb.w.-1×week-1) 0.015 0.004 0.002 0.7 0.3 EFSA, 2009
BMDL01(µg×kgb.w.-1×day-1) 0.3 4.9 2.2
BMDL01(µg×kgb.w.-1×day-1) 8 0.2 0.1
Cd TWI (µg×kgb.w.-1×week-1) 2.5 0.515 0.348 0.5 0.4 EFSA, 2012
Ni TDI (µg×kgb.w.-1×day-1) 22 66.801 28.271 1.1 0.5 WHO, 2005
Sb TDI (µg×kgb.w.-1×day-1) 6 n.d. 0.572 n.d. 0.035 WHO, 2003
n.d., not determinable.