87Sr/86Sr isotopic ratios in vineyard soils and varietal wines from Douro Valley

The assessment of wine authenticity is of utmost importance in the current context of a growing market globalization. The strontium isotopic ratio 87Sr/86Sr is a well-established tool for dating and tracing the origin of rocks and minerals with special interest for wine traceability. A study was developed to examine the variation of 87Sr/86Sr in wines from Douro Valley taking into account the effects of vineyard location and grape variety. The 87Sr/86Sr of soils and wines from six vineyards were determined by using an ICP-MS based analytical procedure. A total of twenty-two monovarietal wines, obtained at micro vinification scale, from relevant white and red grapevine varieties for Douro region, were analysed. The range of 87Sr/86Sr values observed in soils and wines was of 0.708–0.725 and 0.711–0.717, respectively. The present study updates the scarce knowledge available on strontium isotopic ratios in soils and wines from Douro Valley, and its results will enlarge global databank on wine composition and support comparison with other world regions.


Introduction
Wine traceability and the assessment of its authenticity are of utmost importance in the current context of a growing market globalization [1]. The main issues concern adulterations, geographic origin, grape varieties, and vintage year. Stable isotope analyses, recognized by the International Organisation of Vine and Wine (OIV) for detecting adulterations, are limited in terms of interpreting the data and relating them to the wines provenance. The development of analytical methodologies which can positively identify the geographic origin of a given product is one of the most challenging issues for scientific community.
The strontium isotopic ratio 87 Sr/ 86 Sr is a wellestablished tool for dating and tracing the origin of rocks and minerals with special interest for wine traceability. Literature on the progress made since the first application of 87 Sr/ 86 Sr isotopic ratio for wine traceability purposes in the 1990's is available, with a significant increase number of studies developed in the last years, and involving wine producing regions worldwide [2][3][4][5][6][7][8][9][10][11][12][13]. Within [14]. The reliability of 87 Sr/ 86 Sr for wine fingerprinting is evidenced by the studies. Nevertheless, the heterogeneity of some wine regions and PDO in terms of soils and geological materials is well known, making it difficult to match wines with their substrata data. Furthermore, in some cases, soils from different wine regions and countries have been originated from similar geological formations, which can be a limitation in terms of interpreting the data and relating them to the wines provenance. Thus, aiming to use 87 Sr/ 86 Sr for traceability and authentication of wine, it is crucial to develop further studies, on other lithological situations and other world regions (where similar geological formations can occur), to confirm the feasibility of 87 Sr/ 86 Sr fingerprinting and enlarge data.
Sr has been claimed to be absorbed by the vine in the same isotopic proportions in which they occur, under available forms (labile Sr); biological processes involved in vine metabolism do not significantly fractionate Sr isotopes [15,16]. Nevertheless, studies on the hypothetical influence of rootstock and grapevine variety are limited. More, a better understanding of the impacts of anthropogenic factors and technologic processes on this marker is required for its validation. Tailored studies on its robustness to traditional and new technological processes, namely wood aging and nanofiltration for dealcoholisation, were recently published [17,18] and other studies are underway.
Portugal is the eleventh largest producer of wine and the nine largest exporter, with over six hundred millions of liters produced in 2017 [19]. Nevertheless, there is limited information on 87 Sr/ 86 Sr of Portuguese wines for their classification according to geographical origin. Located in the northeast of Portugal, the Douro Wine Region was created in 1756 as the first wine appellation in the world, being famous by Porto fortified wines and more recently for high quality Douro dry wines. The region is characterized by terraces that surround the Douro River and its tributaries, in some cases with walls made of schist supporting vineyards, and by the diversity of native grapevine varieties. Most of the vineyards are installed in soils developed on schists. There is little information on strontium isotopic ratio of soils and wines from Douro [3,5,20]. On the basis of these considerations, a study was developed to examine the variation of 87 Sr/ 86 Sr in wines from Douro region taking into account the effect of vineyard location and grapevine variety.

Vineyards and soils sampling
Six vineyards, selected based on their geographical location (Douro Region) and on the availability of certain grapevine varieties, were considered: Quinta da Boavista (QB, 41 • (Fig. 1).
For comparison purposes between 87 Sr/ 86 Sr values of wines and soils of provenance, soil samples were collected (vineyards QB, QCG, QC and QS) considering two points in the row of vines (for each grape variety), from the depth layer of 50-80 cm. The vineyards located in the Douro valley, are characterized by very hard soil structure as they are mainly originated from schist, a slate-like metamorphic rock, thus imposing the use of a heavy machinery backhoe and drill to access the required soil depth. Soil samples were dried, ground and then forced to pass through a 50 µm sieve.

Grape varieties and wines
Twenty-two V. vinifera varieties were selected from national and international white and red grapevine varieties, and based on their importance to the Portuguese wine sector, more particularly to the Douro region (Table 1). Grape samples from each grapevine variety were harvested in the production year 2012, and were vinified at the National Institute for Agricultural and Veterinary Research (INIAV) in Dois Portos, Portugal.
White wines were produced by applying the conventional technology, being exclusively obtained by the fermentation with grape must extraction preceding alcoholic fermentation. Red wines resulted from the alcoholic fermentation of musts in the presence of the solid parts of the berry (skins and seeds), by application of the conventional red winemaking. More detailed information was described by Pereira et al. [21].

Strontium isotopic analysis
Prior to 87 Sr/ 86 Sr determination by quadrupole inductively coupled plasma mass spectrometry (Q-ICP-MS), the following three main analytical steps in sequence were performed as described by Martins et al. [14]: sample digestion by high pressure microwave digestion (HPMW) (1), chromatographic separation of 87 Sr and 87 Rb (2) and determination of Sr and Rb content by Q-ICP-MS (3).
(1) For elimination of organic substances in order to prevent any interference during chromatographic separation, soils and wines were treated by high pressure microwave digestion (HPMW). A subsample of 0.20 g of dried soil was acid digested using a microwave system as described by Martins et al. [14]. Wines were acid digested following a digestion program previously optimized by Catarino et al. [22].
(2) Separation of Sr from Rb is an essential step for correct determination due to isobaric overlapping of 87 Rb and 87 Sr. An HIPEX Duran column was filled up by using Dowex 50W-X8/400 (Sigma-Aldrich) mesh resin and EDTA as eluent. Separation consists of four phases which are resin activation/pre-treatment; resin conditioning; sample preparation/dilution and elution. Separation was done in duplicates.
(3) Sr and Rb total content and 87 Sr/ 86 Sr isotopic ratio were measured by the ICP-MS equipment previously described. After first two steps, Sr containing fraction was used for Q-ICP-MS analysis. Determination of Sr and Rb total contents in Sr-containing fractions previously to the isotopic measurement is important in order to keep Rb concentration below 1% of the Sr content in Sr-fraction. The SRM 987 (SrCO 3 ) from National Institute of Standards and Technology (NIST) was used as an isotopic reference material for external correction of mass bias phenomenon. Analytical steps for Sr and Rb and isotopic determination are described in Kaya et al. [18].
After separation of Rb and Sr by ion-exchange chromatography, analytical determinations of Rb and Sr concentration and 87 Sr/ 86 Sr were carried out by ICP-MS Table 1. List of 22 grapevine varieties used for wine production followed by the corresponding official code*, abbreviation, berry color, and vineyard of origin. using a Perkin-Elmer SCIEX 9000 ICP-MS equipment. The detailed analytical protocol can be found in the study of Martins et al. [14]. In addition, total strontium concentration was analysed in wines by ICP-MS semiquantitative method as described by Catarino et al. [23].

Statistical analysis
The statistical treatment of soil and wines 87 Sr/ 86 Sr ratios was performed by one-way analysis of variance and comparison of means (Fisher LSD, significance level of 0.05) using Statistica version 7 software (StatSoft Inc., Tulsa, USA).

87 Sr/ 86 Sr of the wines from Douro Valley
The values of 87 Sr/ 86 Sr found in the experimental monovarietal wines from the six vineyards from Douro region are shown in Fig. 2. The 87 Sr/ 86 Sr range of variation, from 0.713 (MF, QCav) to 0.717 (FP, QCG), is very narrow reflecting the geographical proximity of the wines. These values are in line with those reported by Almeida and Vasconcelos [3] for two fortified wines from Douro, of 0.716 and 0.721, using the same analytical strategy applied in this study (ICP-MS and external calibration for mass bias correction).
In comparison to Dão region, mainly granitic, these values are lower than those reported by the same authors,  0005) a,b (0.0008) b (0.0008) a (0.0006) a,b (0.0003) a (0.001) a,b Results are based on average values (QB: n = 4 wines; QCG: n = 2 wines; QCav: n = 2 wines; QC: n = 3 wines; QS: n = 10 wines; QV: n = 1 wine). Means followed by the same letter are not significantly different at 0.05* level of significance. Relative standard deviations are presented in parenthesis.

87 Sr/ 86 Sr of vineyard soils from Douro Valley
The values of 87 Sr/ 86 Sr found in soils from the four vineyards (selected for evaluation of soil-wine correlation) ranged from 0.715 (QS) to 0.7183 (QCG and QC), without significant differences between vineyards.

Comparison of the 87 Sr/ 86 Sr obtained for vineyards soils and wines from Douro Valley
No significant difference was observed between vineyards soils and wines in what concerns strontium isotopic ratios. The 87 Sr/ 86 Sr mean value corresponding to the soils (n = 19) was of 0.7157 (0.0007), while for wines (n = 22) the value was of 0.7142 (0.0007). These results are in line with the trend observed in several studies, of lower 87 Sr/ 86 Sr values in wines than in the soils of provenance [5,6,12]. In fact, recent research indicates that 87 Sr/ 86 Sr of wines is mainly determined by the bioavailable fraction of the soils on which the vineyards are settled instead of by the bulk soil [7]. For each vineyard, 87 Sr/ 86 Sr values of soils and wines were compared (Table 3). With exception of Quinta do Cidrô no significant differences were observed between soils and wines.

Conclusions
The present study represents a development background for building an authentic wine reference database (e.g. official or wine organisation, PDO consortium) to evaluate the provenance of wine labelled as Douro, or to be integrated in a global wine database (e.g. EU wine databank) of great usefulness for industry. Nevertheless, despite the potential of 87 Sr/ 86 Sr for determining the provenance of wines, it seems it can be difficult to differentiate them both at the country and regional level only through 87 Sr/ 86 Sr, indicating that it should be used together with other discriminating parameters. Finally, the results of this study indicate that it is crucial to know the soil geochemistry background so a reliable wine origin relationship can be established.