Assessment of Physicochemical, Macro- and Microelements, Heavy Metals, and Related Human Health Risk from Organically, Conventionally, and Homemade Romanian Wines

Abstract

From the consumers’ perspective, organic and homemade products have become more attractive than conventional ones. However, scientific data regarding the characteristics, properties, and composition of these products are scarce. This study assessed the elemental composition of organic, conventional, and homemade Romanian wines. The physicochemical composition, SO₂ (free and total) and total concentration of macroelements, microelements, and heavy metals from nine wine regions containing 165 samples of white (38 organic/70 conventional/57 homemade), 67 red (22/31/14) and 7 rosé (2/2/3) wines were analyzed by inductively coupled plasma mass spectrometry. Dietary intake and target hazard quotient were also evaluated. The phytochemical and elemental compositions of the wine samples varied across regions and wine types. The highest levels of Ca, K, Fe and Al were detected in conventional wines, while homemade wines recorded high concentrations of Na, Mg, V, Ba and Rb. In the case of the rosé wine samples, the levels of trace elements and heavy metals were below the admissible limit. The estimated daily intake of a glass of wine provided less than 0.5% of the tolerable daily intake of the analyzed elements. No health concerns were identified. All wine samples can be safely consumed, regardless of the culture system used for production, and homemade wines are not of a lower quality than organic or conventional wines.

1. Introduction

The environment and agriculture have always been closely linked, which is why every agricultural activity has a more or less negative impact on the environment [1]. This negative impact has increased since the intensification of agricultural production [2]. Organic farming systems through their direct approach can be seen as an opportunity to resolve these issues and minimize the negative impact of agriculture on the environment [3]. Research on organic farming helps to enlarge knowledge on how the agricultural system adapts to local factors [1]. The extension of knowledge, in this case, is achieved by evaluating the effect of the production system on the ecosystem, soil, plants, food quality, and economic performance under different conditions [1]. Sustainable management in vineyards is an approach of primary importance for grapes’ quality and for the greater preservation of the ecosystem [4,5]. The main role of organic farming is to produce cleaner food, which is more suitable for the human metabolism, while it does not harm the environment [6]. In the last few decades, the consumption and production of organic products have increased exponentially all over the world (e.g., in 1999, 11 million hectares (ha.) of land was intended for organic production, while in 2017 that area was increased six times and amounted to 69.845.243 ha) [6]. In Romania, organic agricultural land registered a slight decrease by 2016, (e.g., 289,252 ha in 2014, 245,924 ha in 2015, 226,309 ha in 2016, and 258,471 ha in 2017) [7].

Together with olives and coffee, grapes are among the most important perennial crops, representing approximately 3.2 million ha of agricultural land worldwide [1]. According to the Food and Agriculture Organization Corporate Statistical Database (FAOSTAT), more than 403,000 hectares of organic grapes are grown, constituting 5.7% of the world’s grape-growing area (7.1 million hectares in 2016). In Europe, over 340,000 hectares (8.7% of the harvested grape area) are organic [6]. Organic viticulture is gaining more importance, but in most of the non-European countries, organic viticulture is still in the initial stages. Worldwide, 2.3% of all vineyards are managed according to organic standards [5]. Regarding the area cultivated with vines in the organic farming system in Romania, 2169 ha of vines was recorded in 2017, which represents 1.2 percent of the total area cultivated with vines, and 1652 out of the 2169 ha is completely transformed into organic agriculture [7].

As compared to conventional systems, organically farmed soil has a significantly higher content of soil organic matter [8,9,10], less soil erosion, larger topsoil depth [11], increased biological activity [12], lower bulk density [1], and higher soil quality [13].

Wine is an alcoholic beverage made from fermented grape juice [14]. In many countries, alcoholic beverages, especially wine, account for more than 12% of beverage consumption, so knowledge of metal concentration in alcoholic drinks such as wine is very important [15,16]. The regular monitoring of certain elements in wine has gained special attention due to the toxic effects of these elements on the human body in cases of excessive intake [17]. In recent years, numerous scientific studies have been conducted to assess the concentrations of trace elements [14,18,19,20,21,22] and heavy metals [23,24,25,26,27], as well as the stable isotope ratio, as indicators of the origin of products in the food industry [28,29,30,31]. These elements play a significant role in the efficient alcoholic fermentation of must, and directly influence, through the processes of precipitation and clouding, its organoleptic characteristics (taste, aroma, color, etc.) [14,32].

The primary source of metals is the soil on which vines are grown. Additionally, metals might also be present due to contamination, such as environmental pollution, the use of fertilizers, pesticides and fungicides, as well as enological contamination at different stages of the winemaking process [16]. The scientific literature has indicated that the level of Na, Ca, and K can be easily modified by the use of additives during the winemaking process (e.g., tartaric acid) [14,33]. Bentonites used for wine clarification can contaminate wine with Al and Na, and contribute to an increase in the concentration of rare-earth metals. Contamination with Pb, Cd, Cr, Al, Fe, Ni, and V might occur as a result of the material of the wine-making equipment [14]. Some elements, such as Ni, Cr, Li, Cd, Co, Ti, Fe, and Mn, may migrate into the drink from the glass bottles during the production of glass as color additives [34,35].

Since 2014, wine consumption in Romania has decreased by 4.5%. In 2019, Romania ranked 13th place in terms of wine consumption, with 3.725 thousand hectoliters, being surpassed by South Africa, which occupied 12th place with 4.525 thousand hectoliters, followed by the Netherlands with 3.453 thousand hectoliters [36]. The United States ranked first place with 33.032 thousand hectoliters, while France, Italy, and Germany ranked second, third, and fourth, respectively. Similarly, in Romania, the consumption of wine per capita has decreased from 2014, by 2% per year, reaching 2.9 L of pure alcohol in 2019 [37]. France ranked first, with a consumption of 7.74 L of pure alcohol per capita, followed by Portugal, Moldova, and Slovenia [37].

To obtain wines with a high degree of food safety and low concentrations of heavy metals, but also to ensure adequate quality, it is necessary to control the beverage industry from the origin to the final product. This problem has been solved by the introducing of Maximum Acceptable Limit (M.A.L.) concentrations of hazardous substances by the International Organization of Vine and Wine (O.I.V.) [38]. This organization is made up of 47 member states (including Romania and the three biggest international wine producers, France, Italy, and Spain).

The main objective of the study was to compare the levels of macroelements (¹⁹K, ²³Na, ²⁴Mg, ⁴³Ca), microelements (trace elements (⁷Li, ²⁷Al, ⁵⁶Fe, ⁶⁴Cu, ⁶⁵Zn, ⁸⁸Sr), ultra-trace elements (⁹Be, ⁵¹V, ⁵²Cr, ⁵⁵Mn, ⁵⁹Co, ⁶⁰Ni, ⁷⁰Ga, ⁷⁹Se, ⁸⁵Rb, ²⁰⁴Tl, ¹⁰⁸Ag, ²⁰⁹Bi, ¹¹⁵In, ¹³³Cs, ¹³⁷Ba)) and heavy metals (⁷⁵As, ¹¹¹Cd, ²⁰¹Hg, ²⁰⁸Pb, ²³⁸U) in wines obtained in organic, conventional, and homemade systems, since they play an important role in the Romanian wine market. The results of this study may provide useful baseline data on the concentrations of and temporal variations in the macroelement, microelement, and heavy metal contents of wines.

2. Materials and Methods

2.1. Samples

A total of 239 wine samples (62 organic wine, 103 conventional, and 74 homemade wine samples) from 9 wine-producing regions (Dealu Bujorului 47, Târnave 36, Murfatlar 33, Cotnari 33, Sarica Niculițel 33, Panciu 17, Huși 11, Drăgășani 12, Halmeu 17) were selected. There were 165 white samples (38 organic, 70 conventional, and 57 homemade), 67 red samples (22 organic, 31 conventional, and 14 homemade), and 7 rosé (2 organic, 2 conventional, and 3 homemade). The selected organic and conventional wine samples were mostly of brands of wine that are widely available on the Romanian market and can be easily found in wine bars and supermarkets. Homemade wine samples were purchased directly from amateur wine producers. Wines obtained from hybrid vines are distilled to obtain alcohol for wine vinegar production unless they can be commercialized for consumption, but it must be specified that it is a wine obtained from a hybrid variety. The selected wine samples came from unusual origins, and were obtained from native, international, and hybrid vines—American vines (Noah, Delaware, Othello, Isabelle) and European hybrids (Terras, Seibel, and Couderc). The wine samples were collected directly from shops, wine exhibitions or the internet, or purchased directly from producers. After opening the original bottles, they were transferred to plastic containers and stored at 3–4 °C until analysis.

Details about the origins and types of the wine samples are given in Supplementary Table S1. The wine trade names, wine variety code, and manufacturer’s names are not given in this paper.

2.2. Reagents and Solutions

The reagents used in the analysis were analytical-grade nitric acid (65% HNO₃—suprapure for trace analysis Merk, Darmstadt, Germany) and hydrogen peroxide (H₂O₂ ≥ 30% for trace analysis Sigma-Aldrich, Steinheim, Germany). The analysis was performed following an appropriate dilution, using an external standard calibration method [27,28]. For calibration of the inductively coupled plasma mass spectrometry (ICP-MS), high-purity ICP Multi-Element Standard Solution XXI CertiPUR (Merk, Darmstadt, Germany) and individual standard solutions of Cr and Hg were used. Moreover, Sc, Rh, Tb, and Ge in suprapure 1% HNO₃ (Merk, Darmstadt, Germany) and deionization water (Milli-Q Integral Ultrapure Water-Type 1 (maximum resistivity of 18.2 MΩ × cm⁻¹)) were used as internal standards for sample preparation. The internal standard was added at 50 µg/L to all samples, blanks, and standards. The control samples and working standards were prepared daily from the intermediate standards stored in polyethylene bottles, which were prepared from the stock solution. The polyethylene bottles and glassware were cleaned by soaking in 10% v/v HNO₃ for 24 h, followed by rinsing with ultrapure water. The accuracy of the analyses was verified by replicate analyses of known concentrations of CRMs (certified references materials). The obtained values ranged between 0.1% and 6.2%, depending on the element, and the global recovery for each element ranged between 98% and 10%.

To control the quality of the analyses, samples were analyzed in triplicates considering a coefficient variation below 5%. The limit of detection (LoD) and limit of quantification (LoQ) were determined from calibration curves, and were calculated as LoD = 3 × SD/s and LoQ = 10 × SD/s, where SD is the estimation of the standard deviation of the regression lines and s is the slope of the calibration curve. Instrumental conditions for the determination of each element in ICP-MS are given in Supplementary Table S2. The calibration standards were prepared from the ICP Multi-Element Standard Solution XXI CertiPUR at five different concentrations (2.5, 5, 10, 25, and 50 µL). The accuracy and precision of the analytical procedure were investigated by spiking a known amount of analyte metal into a test portion of the sample and analyzing the test portion along with the original sample [39]. The precision of the method was expressed as the percent relative standard deviation (RSD%) of the triplicate analyses. The recovery assays for a wine sample of 5 µL concentration, for three replicates of this level of concentration (n = 3), gave an average recovery R% between 91.52% and 115.78%.

2.3. Physicochemical Characterization of the Organic, Conventional, and Homemade Wine Samples

The physical and chemical analyses of wine samples obtained in the organic, conventional, and homemade system were performed in the Physico-Chemical and Biochemical Laboratory of the Research and Development Station for Viticulture and Vinification Bujoru (S.C.D.V.V. Bujoru), Galați, Romania, using the analytical methods described in the Compendium of International Methods of Analysis of Wines and Musts [40] and the Romanian STAS methods. The methods were optimized as described previously [41]. During the physicochemical characterization, the following parameters were determined: ALC = alcohol strength (vol.%) (ebulliometer method—STAS 6182/6-70 and OIV-MA-AS312-01A); TA = titratable acidity (g tartaric acid/L) (titrimetric method—STAS 6182-1:2008 and OIV-MA-AS313-01); VA = volatile acidity (g acetic acid/L) (STAS 6182-2:2008 and OIV-MA-AS313-02); RS = reducing sugars (g/L) (STAS 6182-18:2009); DM = total dry matter (g/L) (densitometric method—STAS 6182/9-80 and OIV-MA-AS2-03B). The total phenolic concentration (TPH (mg gallic acid/L)) was determined using the Folin-Ciolateu method (OIV-MA-AS2-10) and the total concentration of anthocyanins (ANT (mg malvidin-3-glucoside/L)) using the method of pH variation. Both methods have been presented and optimized in a previous work [42]. pH was determined using a WTW inoLab pH meter 7110.

Wine color intensity (CI (420 + 520 + 620 nm) (AU)) is the amount of color, and it varies greatly in different types of wine. Wine hue (Hue (AU)) indicates the development of color towards orange. Young wines have a value between 0.5 and 0.7, which increases throughout aging, reaching an upper limit of around 1.2–1.3. The chromatic structure is the contribution (in %) of each of the three components to the total color (DO 420 = optical density at 420 nm using 1 cm quartz tub (AU); DO 520 = optical density at 520 nm using 1 cm quartz tub (AU); DO 620 = optical density at 520 nm using 1 cm quartz tub (AU)). The wine color intensity, hue, and chromatic structure were determined with a UV-VIS spectrometer (SpectronicHelios Gamma UV-Vis, ThermoSisher Scientific, Waltham, MA, USA). The methodology used to determine these parameters has been optimized and disclosed previously [42,43].

Free SO₂ and total SO₂ were determined spectrophotometrically (MIURA ONE I.S.E. S.R.L., Rome, Italy) based on methods described in the Compendium of International Methods of Analysis of Wines and Musts (free SO₂—OIV-MA-AS323-04B; total OIV-MA-AS323-04B) and the Romanian STAS methods (free SO₂—SR 6182-13: 2009; total SO₂—SR 6182-13: 2009). The other parameters determined by spectrophotometric methods were ACA = acetic acid (g/L); AMA = amino acid (g/L); TAA = tartaric acid (g/L); LLA = L (+) lactic acid (g/L); DLA = D (-) lactic acid (g/L); LMA = L (-) malic acid (g/L); DGA = D (-) gluconic acid (g/L); PA = pyruvic acid (mg/L); G = glycerol (g/L); ACDE = acetaldehyde (mg/L). The operating parameters of the MIURA ONE I.S.E. S.R.L. are described in Supplementary Table S3.

2.4. Sample Preparation and Digestion for Determination of Elements Using ICP-MS

For the determination of elements, 0.5 mL wine samples, 7 mL HNO₃ 65%, and 1 mL H₂O₂ were put into PTFE digestion cells. Digestion was performed in a microwave system, Milestone START D Microwave Digestion System, using the program described in Supplementary Table S4. After 15–35 min, the digestion cells were cleaned by microwaving 5 mL of 65% HNO₃ for 15 min at 600 W. After mineralization, the samples were filtered through a 0.45 mm filter and brought to a volume of 25 mL. The preparation and microwave digestion methods were optimized as described previously [27,28].

2.5. General ICP-MS Instrumental Parameters of Analysis

Analytical measurements of the macroelements (¹⁹K, ²³Na, ²⁴Mg, ⁴³Ca), microelements (trace elements (⁷Li, ²⁷Al, ⁵⁶Fe, ⁶⁴Cu, ⁶⁵Zn, ⁸⁸Sr), ultra-trace elements (⁹Be, ⁵¹V, ⁵²Cr, ⁵⁵Mn, ⁵⁹Co, ⁶⁰Ni, ⁷⁰Ga, ⁷⁹Se, ⁸⁵Rb, ²⁰⁴Tl, ¹⁰⁸Ag, ²⁰⁹Bi, ¹¹⁵In, ¹³³Cs, ¹³⁷Ba)) and heavy metals (⁷⁵As, ¹¹¹Cd, ²⁰¹Hg, ²⁰⁸Pb, ²³⁸U) were performed using an inductively coupled plasma mass spectrometer (iCAP Q ICP-MS Thermo Fisher Scientific, Waltham, MA, USA) as described earlier [27,28,41]. The ICP-MS was equipped with an ASX-520 autosampler, a micro-concentric nebulizer, nickel cones, and a peristaltic sampled delivery pump, and was used in quantitative analysis mode. Each sample was analyzed in duplicate, and each analysis consisted of seven replicates. A nebulizer fitted to a cyclonic spray chamber was used for introducing the sample solution into the ICP-MS plasma. The argon (Ar 5.0) and helium (He 6.0) used were of 99.99% purity (Messer, Austria). The ICP-MS method was optimized daily to give a maximum sensitivity for M⁺ ions and the double ionization and oxides monitored by means of the ratio between Ba²⁺/Ba⁺ and Ce²⁺/CeO⁺, respectively, these always being less than 2%. The operating parameters of the ICP-MS are given in Supplementary Table S5.

2.6. Estimated Dietary Intake and Target Hazard Quotient

In this study, an adult per capita consumption rate of 250 mL wine per day and an average weight of 70 Kg per adult were adopted. Estimated diary intake (EDI) was measured in µg/kg body weight (b.w.) as follows:

EDI = (FIR × C)/Bwa

(1)

where FIR—average daily consumption of alcohol (mL/kg), C—the average concentration of the heavy metals in the samples (µg/mL), and Bwa—average body weight (Kg) [44].

The U.S. Environmental Protection Agency (EPA) has developed target hazard quotients (THQ) to estimate the potential health risks associated with long-term exposure to chemical pollutants [45]. THQ was calculated as per the US EPA Region III Risk-Based Concentration Table [46], using the following equation:

THQ = 10⁻³ × (Efr × EDtot × Fir × C)/(RfDo × Bwa × ATn)

(2)

where Efr—exposure frequency (365 days/year), EDtot—exposure duration (70 years), FIR—average daily consumption of alcohol (mL/kg), C—the average concentration of the metals in samples (mg/L), RfDo—oral reference dose (mg/kg/day), Bwa—average body weight (kg) and ATn—average exposure to non-carcinogens in the year (365 days/year × 70 years).

2.7. Statistical Analysis

Mean values and standard deviations were calculated using Excel 2019 (Microsoft, New York, NY, USA) and Addinsoft version 15.5.03.3707 (Microsoft, New York, NY, USA), and data were interpreted with the analysis of variance (ANOVA). The average separation was performed with the Duncan test at p ≤ 0.005 using SPSS Version 24 (SPSS Inc., Chicago, IL, USA).

3. Results and Discussions

3.1. Physicochemical Analyses of Organic (org.), Conventional (conv.), and Homemade (home.) Wine Samples

The results of the physicochemical analyses of organic, conventional, and homemade wine samples are presented in

Table 1. Physicochemical composition of wine samples by wine type.

Wine Samples/ Parameters	White Wine Samples			Red Wine Samples			Rosé Wine Samples			ANOVA		Management
	Org	Conv	Home	Org	Conv	Home	Org	Conv	Home	F-Value	Sig.	Org	Conv	Home
ALC	12.73 d	13.01 c	11.35 e	13.51 b	13.62 b	12.46 d	14.48 a	13.03 c	10.33 f	9.257	**	**	**	***
TA	5.03 i	5.12 h	5.64 f	6.07 b	5.83 e	5.89 d	5.51 g	6.06 c	6.19 a	9.662	**	ns	ns	ns
VA	0.47 e	0.47 e	0.51 c	0.48 d	0.55 b	0.58 a	0.34 f	0.55 b	0.58 a	6.177	*	ns	ns	*
RS	4.34 f	5.17 d	17.36 a	4.74 e	2.80 g	14.77 b	<2.00 h	<2.00 h	13.58 c	61.734	***	**	**	**
DM	20.15 h	20.46 g	19.64 f	20.94 d	21.24 b	19.83 e	22.00 a	21.00 c	18.33 i	4.839	**	ns	ns	ns
pH	3.40 f	3.36 g	3.45 d	3.49 b	3.48 c	3.45 d	3.29 h	3.44 e	3.52 a	3.490	ns	ns	ns	ns
TPH	322.64 g	310.44 h	308.09 i	1302.87 c	1394.28 a	1359.50 b	533.97 d	432.55 e	411.59 f	397.603	***	*	*	**
ANT	0	0	0	93.31 c	98.90 a	91.22 b	36.97 d	33.23 f	34.22 e	260.877	***	ns	ns	ns
ACA	0.28 g	0.26 h	0.32 e	0.35 d	0.38 b	0.38 a	0.23 i	0.36 c	0.29 f	11.530	**	ns	ns	*
AMA	0.46 g	0.41 h	0.47 f	1.01 a	1.01 a	0.86 b	0.84 c	0.55 e	0.58 d	17.039	**	ns	ns	ns
TAA	2.32 g	2.12 i	2.21 h	3.88 d	3.03 e	2.80 f	4.23 b	4.26 a	4.22 c	4.026	ns	*	*	ns
LLA	0.27 i	0.29 h	0.30 g	1.17 a	1.14 b	1.16 c	0.48 f	0.51 e	0.56 d	91.900	***	**	**	***
DLA	0.17 c	0.14 e	0.15 d	0.22 b	0.22 b	0.23 a	0.14 e	0.14 e	0.15 d	2.440	ns	ns	ns	ns
LMA	0	0	1.09 c	0	0	1.85 b	0	0	2.62 a	55.905	***	*	*	***
DGA	0.24 b	0.22 c	0.21 d	0.24 b	0.25 a	0.19 e	0.21 d	0.12 g	0.16 f	15.142	**	ns	ns	*
PA	64.75 i	67.41 g	66.89 h	87.96 b	96.65 a	81.08 e	87.82 c	83.56 d	79.10 e	18.371	**	ns	ns	ns
G	6.05 e	5.99 g	5.70 h	6.06 d	5.60 i	6.00 f	6.40 a	6.22 b	6.14 c	7.751	ns	ns	ns	ns
ACDE	42.21 g	41.74 h	51.46 f	62.80 d	60.61 e	73.58 a	40.79 i	64.10 c	72.09 b	82.906	***	**	**	***
OD (420 nm)	0	0	0	3.33 a	3.09 b	2.63 c	1.36 f	1.54 d	1.48 e	168.582	***	*	*	*
OD (520 nm)	0	0	0	4.90 a	4.77 b	3.86 c	2.14 f	2.18 e	2.19 d	422.218	***	ns	ns	*
OD (620 nm)	0	0	0	0.62 d	0.68 c	0.60 e	0.98 a	0.73 b	0.55 e	175.266	***	ns	ns	*
CI	0	0	0	8.85 a	8.54 b	7.09 c	4.49 d	4.15 f	4.22 f	550.976	***	ns	ns	**
Hue	0	0	0	0.68 b	0.66 d	0.68 b	0.64 e	0.70 a	0.67 c	88.282	***	ns	ns	ns

ALC = alcohol strength (vol.%); TA = titratable acidity (g tartaric acid/L); VA = volatile acidity (g acetic acid/L); RS = reducing sugars (g/L); DM = total dry matter (g/L); TPH = total phenolics (mg gallic acid/L); ANT = anthocyanins (mg malvidin-3-glucoside/L); ACA = acetic acid (g/L); AMA = amino acid (g/L); TAA = tartaric acid (g/L); LLA = l(+) lactic acid (g/L); DLA = D(-) lactic acid (g/L); LMA = l(-) malic acid (g/L); DGA = d(-) gluconic acid (g/L); PA = pyruvic acid (mg/L); G = glycerol (g/L); ACDE = acetaldehyde (mg/L); DO 420 = optical density at 420 nm using 1 cm quartz tub (AU); DO 520 = optical density at 520 nm using 1 cm quartz tub (AU); DO 620 = optical density at 520 nm using 1 cm quartz tub (AU); CI = color intensity (420 + 520 + 620 nm) (AU); ANOVA = analysis of variance; F = Fisher’s function values; Sig. = significant level of confidence determined with Duncan’s multiple range test; Management = the influence of the vine culture systems on the studied parameter; ns = insignificant; p > 0.05; p ≤ 0.05 (*); p ≤ 0.01 (**), and p ≤ 0.001 (***).Values are mean ± SD, n = 3; in each column, mean values with different letters are significantly different at p < 0.05.

, while average values for organic, conventional, and homemade systems are displayed in Supplementary Table S6. All values were within the ranges recommended by the Romanian legislation [47].

Alcohol content (ALC) was higher in the conventional samples than in organic samples of both white and red wines, though the difference was only significant in the case of white wines. In all three wine varieties, homemade wines had the lowest ALC (Table 1). Titratable acidity and pH are of great importance for grape juice and wine stability, and both parameters are commonly used as indicators of quality. Volatile acidity refers to volatile fatty acids, present in a free state or in the form of salts, and represents about one-tenth of the total acidity. In healthy wines, volatile acidity consists of 55% acetic acid, 45% propionic acid, and 5% other acids [41]. Volatile acidity is formed during alcoholic fermentation, and red wines always have a higher volatile acidity due to the process of maceration and fermentation of the must. The lack of acidity in wines, mainly caused by lactic and propionic bacteria, facilitates the appearance of a flat taste accompanied by weak storage persistence. In this study, both titratable and volatile acidity were highest in homemade wine of all three wine varieties, and were significantly higher than in organic samples, while pH was comparable in all samples. The elevated concentration of titratable acidity may be ascribed to the presence of l(-)-malic acid (only detected in homemade samples), as concentrations of tartaric acid did not differ significantly between varieties. The concentration of l(-)-malic acid was 2.62 g/L in rosé, 1.85 g/L in red, and 1.09 g/L in white wines, and this trend can be observed in the levels of titratable acidity as well. l(-)-malic acid was not detected in organic and conventional wine samples due to its complete conversion into lactic acid during malolactic fermentation. As expected, volatile activity levels are also reflected in the concentrations of acetic acid, homemade white and red wines having higher values than organic and conventional wines. In addition, wines are also classified according to their sugar content: dry (maximum of 4 g/L sugar), medium-dry (between 4 g/L and 12 g/L sugar), semi-sweet (between 12 g/L and 45 g/L sugar), and sweet (minimum of 45 g/L sugar) [48]. Based on the sugar content, homemade wines of all three varieties can be considered semi-sweet, with values ranging between 13.58 g/L (rosé) and 17.36 g/L (white). The organic and conventional wines were medium-dry with a residual sugar content of less than 5.17 g/L. Total dry matter includes all non-volatile substances present in a dissolved state or as colloidal suspensions. The conventional white and red wines had the highest total dry matter levels, at 20.46 g/L and 21.24 g/L, respectively. The homemade wines presented the lowest values (19.64 g/L (white), 19.83 g/L (red), 18.33 g/L (rosé)). In the case of total phenolics, red wines contain more phenols than white or rosé wines. If the culture system is considered, organic white and rosé wine samples had significantly higher values as compared to other systems within the same variety. In red wines, both total phenols and anthocyanins were highest in the conventional samples. During alcoholic fermentation, the amino acids present in grapes are consumed by yeasts and might yield higher levels of alcohols, esters, aldehydes, and other volatile compounds, influencing the final aroma of the wine [49,50]. Of the total nitrogen in wines, amino acids represent up to 40% after the completion of alcoholic fermentation [51]. The amino acid concentration ranged between 0.86 g/L and 1.01 g/L in red wines, 0.55 g/L and 0.84 g/L in rosé wines, and 0.41 g/L and 0.47 g/L in white wines. Of the lactic acid enantiomers, l(+)-lactic acid was more abundant in all wine varieties and among culture systems than d(-)-lactic acid, and was the highest in red wines, followed by rosé and white wines. Concentrations of d(-)-lactic acid were similar across all samples. Gluconic acid is a product of enzymatic glucose oxidation mediated by glucose oxidase, and is an indicator of grape infection with fungi (e.g., the concentration of gluconic acid >3 g/L in botrytized must) [52]. The values obtained in the present analysis ranged between 0.16 g/L and 0.25 g/L, indicating no infection of the grape with fungi.

Pyruvic acid is generally present in wine as a secondary product of alcoholic fermentation, and imparts a slightly sour taste. The conventional red wine samples (96.65 mg/L) and organic rosé samples (87.82 mg/L) presented high pyruvic acid concentrations. Glycerol is an extractor of taste and a carrier of aromatic substances. After water and alcohol, glycerol is most abundant in wine (5–15 g/L), depending on the health of the harvest and the type of wine [53]. Slightly higher values were obtained in organic samples as compared with conventional and homemade samples for all three wine varieties. Conversely, the acetaldehyde level was highest in the homemade samples—remarkably higher than in samples processed via the organic or conventional systems. Acetaldehyde usually has a negative impact on the quality of the wine. Wines obtained from moldy grapes or must with high SO₂ content are rich in acetaldehyde, though acetaldehyde can also form during the wine storage period due to the oxidation of alcohol [53]. The normal range of acetaldehyde in wine is 25–40 mg/L, but in the case of sweet wines, it can exceed 200 mg/L. The highest values were recorded for homemade samples, which were significantly higher than in other samples. As we expected, the highest color intensity (420 nm + 520 nm + 620 nm) and hue were recorded for red wines, particularly in organic and conventional samples.

Regarding the influence of the wine culture system on the physicochemical parameters, organic and conventional dominated over handmade. The average values indicate that homemade wine samples had higher residual sugar and acetaldehyde levels, but lower alcohol content, dry matter, total phenols, and color intensity as compared to organic and conventional samples (Supplementary Table S6). The Fisher function values indicate significant differences between the organic, conventional, and homemade wine samples in terms of all physicochemical parameters, except for pH, tartaric acid, d(-)-lactic acid, and glycerol. Red wines had the highest total phenol, anthocyanin, l(+)-lactic acid, acetaldehyde, and color intensity, with F values ranging between 55.9a nd 550.9. For homemade wine samples, high F values were obtained for residual sugar (F = 61.7) and l(-)-malic acid (F = 55.9) (Table 1). These differences obtained between quality parameters from organic and conventional wines compared to homemade wine were also identified in research conducted by Parpinello et al., 2015, when evaluating the differences between biodynamic and organic management [54].

3.2. Sulfur Dioxide (SO₂) Concentrations in Organic (org.), Conventional (conv.), and Homemade (homew.) Wine Samples

The general use of sulfur dioxide (SO₂) appears to date back to the end of the 18th century, and over the decades, it has become a common practice in the winemaking process [55]. Yeasts produce small amounts of SO₂ during the process of alcoholic fermentation, generally less than 10 mg/L, but in some cases, this can exceed 30 mg/L. Thus, the complete absence of SO₂ in wine samples is very rare. The presence of SO₂ in wine has a direct effect on the shelf life of wine. Under normal conditions, the practice has shown that wines must contain a minimum of 25 mg/L free SO₂, except sweet wines, in which the concentration of free SO₂ must be much higher than 40–60 mg/L. SO₂ content decreases regularly during storage, due to its progressive oxidation and losses in the atmosphere. The storage of wines in big vessels such as tanks reduces the monthly loss of SO₂ 2–3 times compared to small vessels (barrels) [56]. Besides its role as a general antiseptic, SO₂ has a beneficial effect associated with its antioxidant activity. During the fermentation process, the use of SO₂ prevents oxidation processes, which become more dangerous the higher the proportion of moldy and rotten grapes is, and the slower the operations of processing the grapes and extracting the must are [57]. SO₂ is an excellent preservative that prevents the microbial and oxidation spoilage of wine; additionally, it is cost-effective and easy to apply [58]. Among the inconveniences, it must be mentioned that SO₂ delays or even prevents malolactic fermentation, and sometimes imprints a certain hardness on the wine, which is why the must should not have excessive sulfites [57]. Sulfites in wine are found in free form, or in the form of hydroxy sulfonate complexes with carbonyl groups of wine constituents, such as phenolic compounds, acids, and sugar. By complexation with these compounds, sulfites lose both their antioxidative and their antimicrobial properties.

Additional problems with sulfites arise from the instability of their complexes under acidic conditions. In the gastrointestinal system, they rapidly decompose and release sulfites that are readily available for absorption [58]. A high concentration of SO₂ in wine affects the quality of wines and the health of consumers, so the SO₂ content of wines is regulated in all countries producing and importing wines. According to the O.I.V., the legal limit for SO₂ is 150–200 mg/L for dry wines, while in exceptional cases it can reach up to 400 mg/L for some sweet wines [59]. The legal limits for SO₂ recommended by the EU [60] and O.I.V. [40] are detailed in Supplementary Table S7.

The SO₂ concentrations (free and total) were in line with Romanian legislation [47]. When organic, conventional, and homemade wine samples were tested individually according to variety, a significant difference was identified in SO₂ free and SO₂ total levels (

Table 2. The ANOVA, which presents the separation of mean SO₂ (free and total) concentration from organic, conventional, and homemade wine samples.

Wine Samples/ Parameters	White Wine Samples			Red Wine Samples			Rosé Wine Samples			ANOVA		Management
	Org	Conv	Home	Org	Conv	Home	Org	Conv	Home	F-Value	Sig.	Org	Conv	Home
SO2 free (mg/L)	7.40	9.68	6.38	19.44	22.23	12.85	21.15	28.63	14.43	36.685	***	***	***	***
SO2 total (mg/L)	75.96	132.48	75.70	87.81	126.02	88.51	100.93	129.66	130.84	60.026	***	***	***	***
Wine samples/ Parameters	Dealu Bujorului Vineyard			Târnave Vineyard			Murfatlar Vineyard			ANOVA		Management
	Org	Conv	Home	Org	Conv	Home	Org	Conv	Home	F-value	Sig.	Org	Conv	Home
SO2 free (mg/L)	18.26	20.01	13.37	8.39	10.86	11.78	7.24	9.96	12.08	25.164	***	***	***	***
SO2 total (mg/L)	94.19	136.49	106.92	99.88	133.63	82.96	93.58	123.78	104.73	47.478	***	***	***	***
Wine samples/ Parameters	Cotnari Vineyard			Sarica Niculițel Vineyard			Panciuee-way Vineyard			ANOVA		Management
	Org	Conv	Home	Org	Conv	Home	Org	Conv	Home	F-value	Sig.	Org	Conv	Home
SO2 free (mg/L)	10.16	6.86	4.05	16.54	16.98	12.61	13.50	23.19	20.19	16.027	***	***	***	***
SO2 total (mg/L)	123.96	125.22	95.34	70.04	134.80	79.24	88.96	120.36	91.88	21.744	***	***	***	***
Wine samples/ Parameters	Huși Vineyard			Drăgășani Vineyard			Halmeu Vineyard			ANOVA		Management
	Org	Conv	Home	Org	Conv	Home	Org	Conv	Home	F-value	Sig.	Org	Conv	Home
SO2 free (mg/L)	14.44	14.19	17.52	11.09	17.18	8.97	5.48	8.99	4.26	11.001	***	***	***	***
SO2 total (mg/L)	60.74	127.33	51.21	70.51	131.87	69.01	87.32	133.53	51.96	62.945	***	***	***	***

Management (M) = the influence of the vine culture system on the studied parameter. F = Fisher’s test. p ≤ 0.001 (***).

). Homemade wines showed significant free SO₂ reductions for all three wine varieties, as compared to organic and conventional samples. The free SO₂ concentration was highest in conventional samples. The total SO₂ was highest in conventional samples of white and red wines, and similarly high in conventional and homemade samples of rosé wines. In the case of organic wines, a maximum of 100 mg/L total SO₂ is allowed for red wines and 120 mg/L SO₂ for white wines, while dry wines for diabetics should not exceed 20 mg/L free SO₂ [56]. In this study, the organic white wine samples from the Dealu Bujorului and Huși vineyards were within the limit (94.19 mg/L and 60.74 mg/L, respectively), while samples from Cotnari slightly exceeded the threshold (123.96 mg/L). All organic red wine samples had a total SO₂ concentration below the limit. For all vineyards, the conventional samples contained the highest total SO₂ concentrations as compared with other culture systems, regardless of wine variety.

Oral exposure to sulfites has been reported to induce a range of adverse clinical effects in sensitive individuals; therefore, acceptable daily intake (ADI) for sulfites has been set at 0.70 mg/kg b.w. [61]. This means that ADI could be exceeded by an intake of approximately 700 mL of the white wine produced conventionally at the Dealu Bujorului vineyard (the highest sulfite content among all investigated samples).

Concerning the management of wine production, organic and conventional dominated over homemade, as indicated by the 61% (average value) increase in free SO₂ (18.09 mg/L free SO₂ in combined organic and conventional wines, versus 11.22 mg/L in homemade wine), and the 11% (average value) increase in total SO₂ (108.81 mg/L total SO₂ versus 98.35 mg/L) (Supplementary Table S8). The differences obtained between free and total SO₂ from organic and conventional wines compared to homemade wine are in contradiction with the study conducted by Čepo et al., 2018, wherein no difference was observed between free and total SO₂ concentrations in conventional and organic wine samples from Croatia [61].

3.3. Level of Elemental Concentrations in Organic, Conventional, and Homemade Wine Samples

Wine has a much more complex chemical composition than other alcoholic beverages, containing water, alcohol, esters, acids, and aldehydes, which determine the undeniable nutritional and physiological value of wine [62]. Chemical elements such as Na, Mg, Ca, K, P, S, Cu, Co, I, Zn and Fe are natural constituents of must and play a key role during several stages of the wine-making process, along with coenzymes and enzymes. The compositions of the wine samples analyzed during this study are presented in

Table 3. Concentration (mean ± standard deviation) of elements in analyzed white, red, and rosé wines prepared under organic, conventional, and homemade culture system.

Wine Type	White Wine Samples			Red Wine Samples			Rosé Wine Samples
Element	Org	Conv	Home	Org	Conv	Home	Org	Conv	Home
23Na (mg/L)	42.81 ± 7.25	44.22 ± 7.38	44.78 ± 11.69	37.33 ± 5.42	38.42 ± 7.87	36.14 ± 12.14	25.29 ± 5.96	25.69 ± 6.59	48.87 ± 5.80
24Mg (mg/L)	90.38 ± 18.10	114.78 ± 20.36	107.34 ± 18.93	100.71 ± 38.71	101.61 ± 35.02	105.07 ± 37.62	100.64 ± 1.29	98.63 ± 1.03	132.21 ± 9.98
43Ca (mg/L)	65.20 ± 9.53	65.78 ± 13.98	60.95 ± 14.15	42.51 ± 18.52	46.17 ± 18.99	47.71 ± 13.88	24.32 ± 1.38	32.47 ± 0.59	22.19 ± 1.09
19K (mg/L)	284.02 ± 55.25	274.10 ± 65.05	303.07 ± 57.64	818.82 ± 195.12	801.22 ± 124.02	747.22 ± 168.52	696.12 ± 50.54	748.19 ± 46.12	644.94 ± 0.39
7Li (µg/L)	8.71 ± 1.86	9.48 ± 2.76	10.30 ± 2.74	19.88 ± 13.93	17.51 ± 11.11	13.48 ± 4.44	16.79 ± 6.40	17.17 ± 1.49	20.22 ± 3.41
64Cu (mg/L)	0.55 ± 0.16	0.60 ± 0.14	0.43 ± 0.12	0.29 ± 0.11	0.44 ± 0.24	0.48 ± 0.31	0.17 ± 0.06	0.14 ± 0.02	0.19 ± 0.03
55Mn (mg/L)	0.38 ± 0.14	0.54 ± 0.23	0.48 ± 0.20	0.51 ± 0.25	0.53 ± 0.22	0.72 ± 0.40	0.74 ± 0.17	0.96 ± 0.03	0.65 ± 0.19
56Fe (mg/L)	1.03 ± 0.50	1.55 ± 0.37	0.92 ± 0.40	1.03 ± 0.53	1.79 ± 1.02	0.76 ± 0.43	0.66 ± 0.27	1.40 ± 0.47	1.15 ± 0.04
27Al (µg/L)	220.95 ± 75.94	354.08 ± 136.92	282.78 ± 139.99	417.89 ± 203.47	452.31 ± 275.53	283.44 ± 167.22	309.16 ± 22.50	234.88 ± 85.77	191.37 ± 66.56
52Cr (µg/L)	454.93 ± 200.28	430.03 ± 206.84	512.20 ± 219.58	400.19 ± 184.95	310.23 ± 47.01	318.12 ± 55.95	160.60 ± 42.16	203.63 ± 53.58	205.36 ± 2.76
59Co (µg/L)	5.42 ± 1.36	5.88 ± 2.09	7.21 ± 2.11	5.63 ± 4.73	5.57 ± 6.21	5.46 ± 6.50	7.60 ± 0.25	10.75 ± 2.48	9.62 ± 0.62
60Ni (µg/L)	72.65 ± 23.55	71.52 ± 17.84	60.87 ± 13.72	52.14 ± 31.42	50.14 ± 38.42	32.75 ± 24.98	33.93 ± 0.69	34.29 ± 1.80	31.86 ± 0.61
65Zn (µg/L)	2534.74 ± 898.93	3128.98 ± 798.44	2490.90 ± 868.07	1829.72 ± 372.31	2137.620.01	2373.02 ± 682.20	3289.42 ± 46.82	3231.60 ± 351.19	2641.73 ± 800.39
51V (µg/L)	282.68 ± 141.17	310.48 ± 194.40	306.62 ± 194.20	186.93 ± 126.38	161.69 ± 90.06	181.21 ± 88.58	162.99 ± 9.73	147.56 ± 0.97	156.56 ± 1.96
108Ag (µg/L)	3.91 ± 1.54	5.52 ± 2.33	4.87 ± 0.56	2.95 ± 0.87	2.52 ± 0.37	2.18 ± 0.02	LOQ	LOQ	LOQ
9Be (µg/L)	0.94 ± 0.22	0.88 ± 0.11	0.64 ± 0.04	2.99 ± 1.18	3.36 ± 2.70	3.10 ± 2.25	LOQ	LOQ	LOQ
209Bi (µg/L)	3.00 ± 1.82	3.60 ± 0.79	4.11 ± 097	0.69 ± 0.02	0.45 ± 0.06	0.13 ± 0.02	16.12 ± 2.45	20.90 ± 1.56	0.66 ± 0.22
133Cs (µg/L)	7.24 ± 1.92	7.71 ± 2.84	8.28 ± 2.21	5.07 ± 2.68	6.40 ± 1.34	3.17 ± 0.46	LOQ	LOQ	LOQ
137Ba (µg/L)	181.17 ± 55.61	169.48 ± 45.29	181.00 ± 42.65	371.63 ± 65.88	377.22 ± 37.16	342.28 ± 29.75	160.16 ± 14.62	250.93 ± 14.12	311.90 ± 57.74
70Ga (µg/L)	2.82 ± 0.48	2.86 ± 0.20	2.98 ± 1.07	1.26 ± 0.56	1.24 ± 0.32	0.93 ± 0.39	1.11 ± 0.13	1.40 ± 0.50	1.52 ± 0.24
88Sr (µg/L)	538.69 ± 267.08	518.81 ± 218.71	535.40 ± 253.97	486.11 ± 231.95	515.20 ± 209.86	512.10 ± 187.55	598.87 ± 12.11	742.78 ± 11.02	686.66 ± 26.95
85Rb (µg/L)	509.26 ± 224.12	530.28 ± 227.23	524.20 ± 229.48	739.07 ± 162.77	798.73 ± 141.73	802.13 ± 127.28	1377.27 ± 56.80	1314.40 ± 79.28	1356.57 ± 58.71
79Se (µg/L)	7.34 ± 5.16	11.30 ± 9.15	10.94 ± 9.44	4.68 ± 2.39	6.16 ± 2.19	3.22 ± 2.37	1.17 ± 0.17	1.39 ± 0.04	1.33 ± 0.10
204Tl (µg/L)	0.68 ± 0.14	1.02 ± 0.35	0.59 ± 0.08	0.94 ± 0.47	0.75 ± 0.12	0.72 ± 0.12	LOQ	LOQ	LOQ
111Cd (µg/L)	0.16 ± 0.10	0.32 ± 0.17	0.22 ± 0.16	0.36 ± 0.59	0.90 ± 0.43	0.71 ± 0.05	0.23 ± 0.09	0.38 ± 0.06	0.29 ± 0.05
208Pb (µg/L)	23.50 ± 10.19	34.05 ± 15.68	28.27 ± 19.46	47.10 ± 25.69	53.70 ± 29.17	45.61 ± 27.12	16.01 ± 2.51	19.75 ± 4.90	19.16 ± 4.30

Average value ± standard deviation. M.P.L. (maximum permissible limit) for Na 60 mg/L; M.P.L. for Cu 1 mg/L; M.P.L. for Zn 5 mg/L; M.P.L. for As 0.2 mg/L; M.P.L. for Cd 0.01 mg/L; M.P.L. for Pb 0.15 mg/L. LOQ = limit of quantitation (lower than the limit of quantification); LOQ for As 0.7776 µg/L; LOQ for Be 0.0030 µg/L; LOQ for Bi 0.0223 µg/L; LOQ for Cs 0.0040 µg/L; LOQ for Ga 0.035 µg/L; LOQ for In 0.010 µg/L; LOQ for Tl 0.006 µg/L; LOQ for Cd 0.067 µg/L; LOQ for Pb 0.0053 µg/L; LOQ for Hg 0.137 µg/L; LOQ for U 0.084 µg/L. Note: ⁷⁵As, ¹¹⁵In, ²⁰¹Hg and ²³⁸U were also analyzed but not detected in any sample.

, while average values per culture system are disclosed in Supplementary Table S9.

The Na content of wines gained great interest among oenologists when it was reported that people suffering from hypertension should follow diets low in Na. The sources of Na in wine can be natural or industrial [63]. Proximity to the sea constitutes a major and natural source of Na in wine [64,65], while artificial sources include Na-based compounds (e.g., bentonites) used as fining and clarifying agents, sodium bisulfite used for keeping the barrels and tanks in good condition, sodium carbonate for reducing the natural acidity of wines, and sodium metabisulfite for sterilizing and preserving wines, whereas the sodium salt of ascorbic acid is a sterilizing and preserving agent, which can also be used for the inhibition of secondary fermentation and mold growth [63]. In this study, Na levels were comparable in both white and red wine samples when processed by any culture system, and ranged between 42.81 and 44.78 mg/L in white samples and 36.14 and 38.42 mg/L in red samples (Table 3). In rosé samples, homemade wines contained more Na (48.87 mg/L) as compared to organic (25.29 mg/L) or conventional samples (25.69 mg/L). Regarding the distribution of Na concentration according to the vineyard area, values are heterogeneous between geographical areas (Supplementary Tables S10–S12). Homemade wine produced in the Murfatlar vineyard has a Na level (62.05 mg/L) above the admissible limit set by O.I.V (60 mg/L). This might be explained by the fact the Murfatlar vineyard is situated in the vicinity of the Black Sea. Other Romanian studies reported similar Na concentrations for white wine samples from Dealu Bujorului and Murfatlar as in our study [28]. The Na level of 10.50 mg/L was found in conventional wines from Drăgășani [66], which is lower than our values obtained for conventional samples (44.04 mg/L, Supplementary Table S12). Other Romanian studies reported similar Na concentrations for white wine samples from Dealu Bujorului and Murfatlar as in our study [28], while the Na level of 10.50 mg/L was found for conventional wines from Drăgășani [66], which is lower than our values obtained for conventional samples (44.04 mg/L, Supplementary Table S12). Homemade wines from different regions of Transylvania contained Na between 6.88 and 22.5 mg/L [67]. The Na levels of our conventional samples were slightly higher than those reported from Serbia (mean Na level of 8.48 mg/L for red wines and 17.6 mg/L for white wines) [68] and Poland (5.33 µg/L–3.823 mg/L) [17], and were similar to samples from Ethiopia (24.0–24.4 mg/L) [39] or Argentina (36–138 mg/L (median values)) [18]. Portuguese wines have shown significantly higher concentrations of sodium (92.20 mg/L) compared to our study, concentrations that far exceed the maximum admissible limit set by O.I.V (60 mg/L) [69].

The amount of Mg in wine is influenced by the grape variety, the time and temperature of the maceration process, the pressing rate, the pH, the use of carbonates for deacidification, as well as the use of ion exchange resins [63]. The Mg levels ranged between 90.38 and 114.78 mg/L in white wines, 100.71 and 105.07 mg/L in red wines, and 98.63 and 132.21 mg/L in rosé wines (Table 3). Homemade rosé wines had remarkably higher concentrations of Mg as compared to homemade white or red wines. High Mg levels were detected in conventional samples from Cotnari (153.12 mg/L), and homemade wine samples from Dealu Bujorului (127.93 mg/L) and Cotnari (132.85 mg/L). Overall, between vineyards, samples from Panciu contained lower Mg levels, ranging between 72.75 and 79.13 mg/L, as compared to Cotnari (91.36–153.12 mg/L) and Drăgășani (115.14–124.91 mg/L) (Supplementary Tables S10–S12). The results obtained for Mg are in line with previously analyzed samples from Dealu Bujorului and Murfatlar [28], or Transylvanian homemade wines [67], while our samples from Drăgășani had higher Mg levels than the concentrations reported by Geana et al. (46.40 mg/L compared to 124.91 mg/L measured in this study) [66]. White and red wine samples from other Romanian vineyards also had similar Mg levels as our samples [30,70]. Studies performed in several parts of the world, such as Croatia (74.3–91.8 mg/L in white and 83.1–101.7 mg/L in red wines) [71], Serbia (66.6 mg/L in white and 94.9 mg/L in red wines) [68], Poland (42.7–161.0 mg/L), United States (123–156 mg/L in red wines) [72] and Brazil (82.2 mg/L in red wines) [73], have reported concentrations in line with our findings.

Ca is a natural component of must and wine. Well-drained viticultural soils enriched with Ca retain heat better, quicken the grown of vines and ripening of grapes, and produce grapes with higher concentrations of Mg and K compared to those produced in neutral or slightly acidic soils [63]. Ca is necessary for the normal course of alcoholic fermentation, though its high concentration influences the performance of yeast, resulting in fermentation suppression [74]. This problem can be solved by maintaining a high ratio of Mg/Ca. the Ca level decreases during the fermentation process [75] and continues during the storage and stabilization stages [76]. Ca levels were comparable between organic, conventional, and homemade samples within wine types, except for rosé wines, where conventional samples contained more Ca (32.47 mg/L) than organic (24.32 mg/L) or homemade (22.19 mg/L) ones. Nevertheless, white wine samples had the highest Ca concentrations (ranging between 60.95 and 65.78 mg/L), slightly higher than in red wines (42.51–47.71 mg/L) (Table 3). In terms of vineyards, Ca concentrations were highest in Cotnari samples regardless of the processing system. These data are in line with the Ca levels reported for other Romanian white and red samples [28,30,31,66,70], and are comparable with samples from South Africa [77], Italy [78], Brazil [73], United States [72], Portugal [69] and Ethiopia [39]. Somewhat lower Ca levels were found in Greek white (6.3 mg/L) and red (6.1 mg/L) wines [20]. Slightly higher Ca values were reported for red (83.1 mg/L) and white (84.6 mg/L) wines from Serbia [68], red (80.8 mg/L) and white (77.9–83.9 mg/L) wines from Croatia [71], and wines (31–286 mg/L) from Argentina [18].

K constitutes about 75% of the total cation content of the wines, and is required for yeast growth and fermentation [63]. K is treated as the main positive ion in wine, and its concentration in must and wine is directly influenced by the soil, climate, grape variety, the date on which the grapes were harvested, fermentation temperature, storage conditions, alcohol content, pH, ion exchange resins, and fining agents [79]. The K concentration of wines is increased by pressing, by adding potassium metabisulfite or carbonate to the crushed grapes during vinification, or by adding potassium caseinate to finish the wine [63]. In our study, the K levels were between 274.10 and 303.07 mg/L in white wines, 747.22 and 818.82 mg/L in red wines, and 644.94 and 748.19 mg/L in rosé wines. This trend has already been confirmed by our previous studies conducted on red and white samples from Dealu Bujorului and Murfatlar [28,41]. High K levels were detected in samples from Panciu and Dealu Bujorului, while the lowest values were obtained from Cotnari, Halmeu, and Târnave (Supplementary Tables S10–S12). Wine samples from other Romanian vineyards contained higher (1647–1714 mg/L [31]) or similar (142.8–482.6 mg/L [30] or 145.2–301.8 mg/L [70]) concentrations. The results obtained are also comparable to those obtained by Dalipi et al. [78] (757.00 mg/L in conventional wines from Italy), Ðurđić et al. [68] (289.0 mg/L—average value for conventional wines from Serbia), and Skendi et al. [20] (123.20 mg/L in conventional red wines from Greece). Research conducted by Leder et al. [71] (1284.0 mg/L in conventional wines from Croatia), Płotka-Wasylka et al. [17] (3250.0 mg/L in conventional wines from Poland) and Fabani et al. [18] (1537.0 mg/L in conventional wines from Argentina) showed significantly higher K levels than those presented in this study.

In must and wine, Zn can be derived from the soil, fungicides, insecticides, Bordeaux mixture, or the winemaking equipment [63]. According to the research by Karadjova et al. (2002), less than 15% of the Zn present in wine is complexed with polyphenols, while more than 60% is present as an active labile ion; if its concentration exceeds the recommended limit of 5 mg/L, established by O.I.V., Zn can create cloudiness [80]. In our study, the mean Zn concentrations were comparable between wine types and culture systems, and were relatively high in samples from Cotnari and Târnava and low in the Murfatlar region. Conventional wine produced in the Târnava vineyard resulted in a value (4493.00 µg/L, mean value) close to the maximum admissible limit set by O.I.V. (5000 µg/L). A possible explanation might be the excessive use of fungicides and insecticides in conventional vineyards. Previously analyzed Romanian samples found slightly lower mean Zn concentrations in the Valea Călugărească, Murfatlar, Moldova [29], Drăgășani, Recaș [66], Vrancea and Terasele Dunarii [31] vineyards, as well as in homemade Transylvanian wines [67]. In contrast, white wines from northern Romania (Baia Sprie and Baia Mare) presented Zn levels (5.36–8.87 mg/L) that exceeded the admissible limit. These elevated concentrations can be ascribed to the proximity of the Baia Mare Mining and Smelting Complex [27]. As compared to these concentrations, lower values were reported in wine samples from Argentina [18], Serbia [68], Croatia [71], Slovakia [81], Greece [20], Portugal [69], and Italy [78], and these were in similar ranges to wine samples from Ethiopia [39] and South Africa [77].

The concentration of Cu in must and wine is normally low (approximately 0.1–0.3 mg/L), and plays a catalytic role in yeast fermentation. Concentrations higher than 0.5 mg/L might lead to haze formation; values higher than 1 mg/L change the organoleptic properties, and concentrations exceeding 9 mg/L are toxic to the fermentation process [63]. The casse of wine appears mainly in bottled white wines when small levels of dissolved Cu and SO₂ are present; in the absence of oxygen and ferric ions, it induces a bluish-white glistering hue, and may add a bitter taste to the wine. Exposure to high temperatures and light accelerates the casse formation. In this work, Cu was detected at 0.43–0.55 mg/L in white wines, 0.29–0.48 mg/L in red wines, and 0.14–0.17 mg/L in rosé wines. Organic and conventional samples from Târnave vineyard and conventional samples from Sarica Niculițel and Drăgășani vineyards had the highest Cu concentrations, though the values were below the limit set by O.I.V. (1 mg/L). These concentrations were in similar ranges as in other Romanian wines produced in different vineyards [28,29,30,31,66], except for wines produced in polluted areas [27], and in the findings of most of the studies reviewed from the literature [20,69,71,72,73,78,82,83,84]. Conversely, research conducted by Durguti et al. [85] (5.668 mg/L in Kosovo) and Al Nasir et al. [86] (2601.00 µg/L in homemade wines from Jordan) showed significantly higher concentrations of Cu compared to the present study—concentrations that far exceed the maximum admissible limit set by O.I.V (1 mg/L).

The Mn and Fe concentrations in wine depend on the composition of the viticultural soil, air pollution, the use of herbicides, the vinification process, and the use of wine fining agents [87]. During wine fermentation, the mitochondria from the grapes, which are composed of the iron–porphyrin–protein complex, are degraded, and are considered a natural source of Fe [88]. Mn and Fe are responsible for the browning of white wines, especially at high concentrations [63]; Mn directly aids in the formation of acetaldehyde, while Fe catalyzes the reaction of acetaldehyde with the polyphenolic compounds to form polymers that precipitate [89]. Mn concentrations tended to be higher in organic and conventional rosé samples than in organic and conventional white and red samples (Table 3). The lowest values (0.17–0.19 mg/L) were obtained in Murfatlar samples, while wine samples from Sarica Niculițel (0.64–0.73 mg/L) and Drăgășani (0.56–0.95 mg/L) had the highest values. Overall, the Fe concentrations were similar among wine types and culture systems, though differences among vineyards were detected. High values were identified for samples from Dealu Bujorului (0.97–2.18 mg/L) and Huși (0.63–2.87 mg/L), while samples from Panciu had the lowest values, ranging 0.61–0.85 mg/L. Moreover, conventional wine samples from almost all vineyards had slightly higher mean Fe concentrations as compared to organic and homemade samples, except for Drăgășani (highest in organic). These values are in line with other studies conducted on Romanian samples [28,29,30,31,66].

The concentrations of Li, Rb, and Sr do not seem to be influenced by the vinification process—their presence is related to the soil composition and the ability of the Vitis vinifera to absorb these elements [63]. Li is an important trace element with multiple potential health benefits; it can be absorbed into the wine during prolonged storage in glass bottles [90]. Rb has a biochemical characteristic similar to K, but it has no known biological function. Sr is present in almost all biological systems due to its ability to exchange rapidly with K [91]. Sr is neither a nutrient nor an essential element for the vine, and is continuously accumulated throughout the plant’s growth by being absorbed from the water-soluble fraction of the soil [92]. The importance of determining Li, Rb, and Sr in wines has increased because they can be employed for the geographical classification of wines [93]. Regarding the distribution of these elements in our samples, Li and Sr presented comparable concentrations in white, red, and rosé wine samples (8.71–20.22 µg/L and 486.11–686.66 µg/L, respectively), except for the Sr concentration in conventional rosé wines, which was slightly higher (742.78 µg/L) (Table 3). In terms of Rb concentrations, white wines had the lowest values (509.26–530.28 µg/L), slightly lower than red wines (739.07–802.13 µg/L) and remarkably lower than rosé wines (1314.40–1377.27 µg/L). Considerable differences were observed in terms of the vineyard distribution. Organic, conventional, and homemade wines produced in Halmeu had a notably lower Li but higher Sr levels, and wines from Dealu Bujorului had higher Rb concentrations when compared to samples from other vineyards.

Al has a direct effect on the stability and also the toxicity of wine; high concentrations can damage wine properties and create an unwanted and unpleasant metallic taste [63]. Ag has been used as an antimicrobial agent for centuries as it has the property of reducing biological bioburden and preventing infection. Its use has been diminished with the introduction of antibiotics, but it has remained one of the most popular agents for wound infections, especially for burned patients [94]. Low amounts of Ag were assimilated from food, water, and drinks, with no substantial biomagnification [95]. Within each wine type, the mean concentrations of Al were highest in conventional samples (white and red wines) and organic samples (homemade wines). Regarding the distribution between vineyards, high values were detected for wine sampled from Panciu, Murfatlar, and Cotnari, while samples from Târnava and Sarica Niculițel had the lowest mean concentrations (Supplementary Tables S10–S12). The concentration of Ag ranged 2.18–2.95 µg/L in red wines, which is slightly lower than the concentrations in white wines (3.91–5.52 µg/L). Ag was not detected in any rosé samples.

The use of equipment and machinery made of stainless steel has led to an increased concentration of metals such as Cr, Co, Ni, and V [96]. The Cr concentration in the wine is increased during periods of aging due to glass bottle leaching, reaching concentrations of mg/L levels [97]. In the case of Ni and Co, they can come from the contact of glass wine bottles if they are made from either nickel oxide or blue glass, which can contain up to 0.02–0.05 wt. % Co [63]. Cr, Ni, and V concentrations tended to be highest in white wine samples, followed by red and rosé samples. Conversely, rosé samples had slightly higher Co concentrations, especially conventional and homemade wines. When assessed according to the vineyards, Cr concentrations were high in Târnava and Murfatlar samples, and Co was high in Dealu Bujorului and Huși samples. Surprisingly, Ni concentrations in samples from Halmeu Wine Center were much higher (317.34 µg/L (organic), 328.83 µg/L (conventional), 306.52 µg/L (homemade)) than in wine samples from any other vineyard (<68.06 µg/L (organic), <73.97 µg/L (conventional), 55.58 µg/L (homemade)) (Supplementary Tables S10–S12). A similar trend was observed for the V concentrations, conventional and homemade samples from Târnava vineyard having remarkably higher V concentrations (720.82 µg/L and 750.29 µg/L, respectively) than other samples (<397.14 µg/L in conventional and 371.54 µg/L in homemade samples).

Cd can be found in grapes in traces, though there are cases when must and wine can be contaminated with Cd, such as when the vineyards are positioned near agrochemical factories, air pollution, using winery processing equipment made of Cd-containing alloys, the metal joints of winery processing equipment, and painted surfaces [53]. Pb poisoning from homemade wine has been previously reported [98], and the use of inappropriate materials for the preparation and storage of wines would become the main cause of contamination with Pb. All analyzed samples and wine types had Cd and Pb levels in similar ranges, well below the admissible limit.

White wine samples had slightly lower mean Be and Ba concentrations and slightly higher Bi and Cs concentrations when compared with red wine samples. No Be or Cs were detected in any rosé samples. Regarding the culture system, the Ba concentrations in rosé samples presented some remarkable differences. Homemade rosé wines had 0.66 µg/L Bi, while organic rosé wines had 16.12 µg/L and conventional wines 20.90 µg/L. This latter value for conventional wines is much higher than for any other white or red wine samples (Table 3). Elevated Be concentrations were detected in wines from Huși vineyard, Bi in conventional and homemade wines from Târnava, Cs in wines from Târnava and Murfatlar, and Ba in wines of Panciu and Huși vineyards. There are limited published data on the levels of Ga exposure to the environment. In our analyses, Ga and Se concentrations were in similar ranges between wine types and culture systems, and only conventional and homemade white wines tended to have higher mean concentrations of Ga and Se.

When culture systems were considered without stratification by wine types (as presented in Supplementary Table S9), most chemical elements were present at similar concentrations in organic, conventional, and homemade wines. Organic and conventional wines dominated over homemade wines in the case of Ni, Bi, Al, and Tl, while homemade wines tended to have higher Na, Mg, Fe, and Ba concentrations.

3.4. Estimated Dietary Intake (EDI) and Estimated Target Hazard Quotient (THQ) of Organic, Conventional, and Homemade Wine

Metals have dual roles, related to the dietary requirement of essential elements and the toxicity associated with overloading with essential and toxic metals, and these are the main reasons for the determination of metal concentrations in foods [99]. It is necessary to quantify trace elements in beverages and foods regularly consumed by the general population in order to identify the real risk to public health and the tolerable levels [100]. The concentrations of toxic and less toxic elements in wine, as well as the concentrations of micro- and macroelements, are of great interest to researchers because these elements influence wine quality, dietetic characteristics, and hygienic and toxicological factors [101]. To assess the potential hazards resulting from the long-term daily consumption of wine containing these elements, we used the concept of an EDI of elements [44]. In Romania, there are no related data on the daily consumption of wine; therefore, we assumed that an adult drinker consumes a daily volume of 250 mL of wine (approximately a glass of wine). For calculation of the EDI, we selected an average weight of 70 kg. Similarly, there is very little information at the national level on the dietary intake of metals and the long-term effects of the ingestion of multiple metal ions, which can be expressed as a function of the quantified level of concern in the form of THQ. THQ values below 1 indicate no adverse effects related to a certain element, while values above 1 mean potential adverse effects [45]. Nevertheless, THQ is not a measured risk, but rather indicates a level of concern [102].

The values of EDI and THQ are presented in

Table 4. Estimated dietary intake (mg/kg body weight per day) and target hazard quotients based on a 250 mL wine per day per ca consumption.

Element	White Wine Samples			Red Wine Samples			Rosé Wine Samples			Average
	Org	Conv	Home	Org	Conv	Home	Org	Conv	Home	Org	Conv	Home
Estimated dietary intake
23Na	0.15	0.16	0.16	0.13	0.14	0.13	0.09	0.09	0.17	0.13	0.13	0.15
24Mg	0.32	0.41	0.38	0.36	0.36	0.38	0.36	0.35	0.47	0.35	0.38	0.41
43Ca	0.23	0.23	0.22	0.15	0.16	0.17	0.09	0.12	0.08	0.16	0.17	0.16
19K	1.01	0.98	1.08	2.92	2.86	2.67	2.49	2.67	2.30	2.14	2.17	2.02
7Li	0.03	0.03	0.04	0.07	0.06	0.05	0.06	0.06	0.07	0.05	0.05	0.05
56Fe	N/A	N/A	N/A	N/A	0.01	N/A	N/A	0.01	N/A	N/A	0.01	N/A
65Zn	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01
Target hazard quotient
7Li	0.02	0.02	0.02	0.03	0.03	0.02	0.03	0.03	0.03	0.03	0.03	0.02
64Cu	0.05	0.05	0.04	0.03	0.04	0.04	0.01	0.01	0.02	0.03	0.03	0.03
55Mn	0.05	0.08	0.07	0.07	0.08	0.10	0.11	0.14	0.09	0.08	0.10	0.09
56Fe	0.01	0.01	0.005	0.01	0.01	0.004	0.003	0.01	0.01	0.004	0.01	0.004
27Al	0.001	0.001	0.001	0.001	0.002	0.001	0.001	0.001	0.001	0.001	0.001	0.001
59Co	0.06	0.07	0.08	0.06	0.06	0.06	0.09	0.12	0.11	0.07	0.08	0.08
60Ni	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01
65Zn	0.03	0.04	0.03	0.02	0.02	0.03	0.04	0.04	0.03	0.03	0.03	0.03
51V	0.19	0.21	0.21	0.13	0.11	0.12	0.11	0.10	0.11	0.15	0.14	0.15
108Ag	0.003	0.004	0.003	0.002	0.002	0.002	N/A	N/A	N/A	0.002	0.002	0.002
137Ba	0.003	0.003	0.003	0.006	0.007	0.006	0.003	0.004	0.005	0.004	0.005	0.005
88Sr	0.003	0.003	0.003	0.003	0.003	0.003	0.004	0.004	0.004	0.003	0.003	0.003
111Cd	0.001	0.001	0.001	0.001	0.003	0.002	0.001	0.001	0.001	0.001	0.002	0.001

Note: Estimated dietary uptake could not be calculated for ⁶⁴Cu, ⁵⁵Mn, ²⁷Al, ⁵²Cr, ⁵⁹Co, ⁶⁰Ni, ⁵¹V, ¹⁰⁸Ag, ⁷⁵As, ⁹Be, ²⁰⁹Bi, ¹³³Cs, ¹³⁷Ba, ⁷⁰Ga, ¹⁵In, ⁸⁸Sr, ⁸⁵Rb, ⁷⁹Se, ²⁰⁴Tl, ¹¹¹Cd, ²⁰⁸Pb, ²⁰¹Hg, or ²³⁸U. Target hazard quotients could not be calculated for ²³Na, ²⁴Mg, ⁴³Ca, ¹⁹K, ⁵²Cr, ⁷⁵As, ⁹Be, ²⁰⁹Bi, ¹³³Cs, ⁷⁰Ga, ¹¹⁵In, ⁸⁵Rb, ⁷⁹Se, ²⁰⁴Tl, ²⁰⁸Pb, ²⁰¹Hg, or ²³⁸U.

. The intake values for Na were similar within white and red wine samples, while homemade rosé samples had higher values (0.17 mg/kg b.w. per day) as compared to organic and conventional rosé wine samples (0.09 mg/kg b.w. per day). Higher intakes of Na are likely to occur in wine consumers who regularly consume wine obtained in the Târnava and Murfatlar vineyards (Supplementary Table S13). A possible explanation for the Murfatlar samples might be related to the high Na level identified, which exceeds the admissible limit of O.I.V. The estimated Mg intake was also highest in the homemade rosé samples, while samples of other wine types provided similar values. The Ca intake seemed to be consistently lower in all rosé samples (0.08–0.12 mg/kg b.w. per day) than in red (0.15–0.17 mg/kg b.w. per day) or white samples (0.22–0.23 mg/kg b.w. per day). In contrast, K and Li intake values tended to be lower for white samples as compared to red and rosé samples. The recommended dietary allowance (RDA) values are 400–420 mg (male) and 310–320 mg (female) per day for Mg, 1000 mg per day for Ca, and 3500 mg per day for K [99]. Based on the average values, we can conclude that homemade samples had slightly increased EDI Na and Mg, but slightly decreased EDI K versus organic and conventional samples (Table 4). The EDI values obtained for Ca (0.16 mg/kg body weight/day—average value) and Mg (0.38 mg/kg body weight/day—average value) are close to those presented for wine samples from the Slovak market [81].

It is not possible to calculate the THQ values for Na, Mg, Ca, K, Rb, Pb [78], and Cr [103], for which an oral reference dose is not given by the EPA. For all other elements, THQ values were below 1 and did not raise any health concerns (Table 4).

4. Conclusions

The results indicate that wines produced in Dealu Bujorului, Târnava, Murfatlar, Cotnari, Sarica Niculițel, Panciu, Huși, Drăgășani, and Halmeu vineyards differ in their physicochemical parameters and elemental composition. If we assume that the ecoclimatic conditions are the same in these areas, then the results obtained in this research reflect differences in the winemaking process adopted by each area and by the amateur winemaker.

Our research shows that organic, conventional, and homemade wines all contained low levels of trace elements and heavy metals, thus they can be safely consumed.

Based on the results obtained, several conclusions can be drawn. As expected, the most abundant elements in the organic, conventional, and homemade wine samples were K, Mg, Ca, and Na. The highest Na and Mg concentrations were recorded in homemade wines, while for Ca and K, the highest were recorded in conventional wines. Organically produced wine contained significantly lower levels of Mg, while for Ca and Na, the concentration was comparable between the conventional and homemade wines. The major conclusions of this work are that the concentrations of Cr, Co, V, Ba, and Rb in amateur vinification (homemade wine) are higher than in professional winemaking (conventional and organic wine). Possible explanations for these concentrations would be the non-controlled use of enological additives in amateur vinification (the interventions are done without the presence of an oenologist), or the use of low-quality galvanization materials. In the case of Cu, the concentration in conventional and homemade wine is higher than in organic wine samples. An explanation for this concentration may again be the fact that amateur winemaking is often not monitored by oenologists, and also, the use of fertilizers, insecticides, and enological additives become more uncontrollable, without the advice of appropriate scientists.

As for trace metals, Cr, Co, Ni, V, Ag, Be, Bi, Cs, Ba, Ga, Sr, and Tl were the most concentrated elements, probably due to their use in phytosanitary treatments, pollution, or assimilation through plant roots, while heavy metals As, Cd, Pb, Hg, and U have a very low concentration, and in the case of As, Cd, Pb, Hg, and U, they are below the detection limit. The analyzed homemade wine samples, especially red ones, are distinguished by red and blue hues, while white wines fall in the moderate light category, from which we concluded that they have been stored properly. The most important conclusion that we can draw from this research is that although big winemakers are those who have the expertise and the professional knowledge about quality wines, even amateur winemaking can produce wine that is not only safe from the point of view of heavy metals, but also contains comparable or even higher polyphenol compound levels than in commercial wines, as reported in the scientific literature. This is mainly related to the increased awareness of the general public about safety issues, and the individual efforts of amateur winemakers. The estimated daily intake of a glass of wine containing the average and maximum concentrations of Na, Mg, Ca, K, Fe, and Zn provides less than 0.5% of the tolerable daily intake. The concentrations of the analyzed wine samples highlight the safety characteristics of the wines obtained in Romania; these heavy metals identified in the analyzed wine samples do not endanger the health of wine consumers. The main purpose of this research is to compare the levels of macroelements (¹⁹K, ²³Na, ²⁴Mg, ⁴³Ca), microelements (trace elements (⁷Li^{, 27}Al, ⁵⁶Fe, ⁶⁴Cu, ⁶⁵Zn, ⁸⁸Sr), ultra-trace elements (⁹Be, ⁵¹V, ⁵²Cr, ⁵⁵Mn, ⁵⁹Co, ⁶⁰Ni, ⁷⁰Ga, ⁷⁹Se, ⁸⁵Rb, ²⁰⁴Tl, ¹⁰⁸Ag, ²⁰⁹Bi, ¹¹⁵In, ¹³³Cs, ¹³⁷Ba)) and heavy metals (⁷⁵As, ¹¹¹Cd, ²⁰¹Hg, ²⁰⁸Pb, ²³⁸U) in wines obtained in organic, conventional, and homemade systems, and not to analyze the methanol concentration. The concentration of methanol in the wine samples has not been determined; it is currently being developed. Very important to note is that the conclusions presented refer strictly to the concentrations of trace and heavy metals. Regarding the acetaldehyde concentration, some wine samples showed traces of oxidation, but this oxidation does not make their consumption dangerous in the case of homemade wine.