**2. Case study: Spatial distribution of copper concentrations in vineyard soils of Croatia: Wine–growing subregion of Plesivica**

Elements inherited from the bedrock are partitioned within the soil through specific process‐ es. Besides the parent material characteristics, geomorphology and landscape features con‐ tribute greatly to the variability of elements distribution. The topography is especially important, since it affects water infiltration and drainage on the one hand and soil erosion on the other. This variability is additionally enhanced in cultivated soils, especially in moun‐ tain regions and on sloping terrains where erosion processes are more expressed [34].

Steep southern slopes of the mountains in north-western Croatia have been used for centu‐ ries to grow vine and produce wine (Figure 1). Wine quality is almost always associated with the location, which means with the specific natural viticultural environment where soil is one of the major factors: firstly in terms of soil physical properties, and secondly in terms of soil inorganic chemistry [35, 36].

Although the systems of vineyard planting and maintenance in north-western Croatia have been changing with time and modern plantations prevail nowadays, parcels under tradi‐ tional cultivation and old, almost forgotten, cultivars can still be found. Therefore, the aim of this study was to explore the distribution and retention pattern of copper concentrations in vineyard soil and, and to study the copper speciation and its distribution within five opera‐ tionally defined fractions to assess its bioavailability and possible downward movement.

**Figure 1.** Grapevine plantation in the study region (Site Lokosin dol, Plesivica wine-producing subregion, Croatia, pho‐ to M. Romic)

#### **2.1. Study area**

Research was carried out on different wine-producing subregions in Croatia, in the wider Zagreb region: the wine-growing subregion of Plesivica (approximate coordinates: latitude, 45° 42' and longitude, 15° 37'). The area of Žumberak, which is a mountainous territory lo‐ cated to the west of Zagreb, is bordered to the north and west by Slovenia, and to the south by Kupa River (Figure 3). Soils are developed on Pannonian sediments. This sediments con‐ sist of limy marls, sands, sandstones, conglomerates and breccias [37-39]. Week consistency of those sediments, solid Triassic dolomite as bedrock and periodic streams led to fast ero‐ sion and filling in valleys and formation of amphitheater-shaped valleys. Namely, those landscape shapes were formed by the distinct climatic oscillations and pulsation between glacial and stadial periods, along with constant tectonic activity and elevation of terrain [39]. The landscape reflects the features of the Dinaric, so numerous formations characteristic of the karst are found on highly dissected limestone terrains. As the Zumberacka Mt. piedmont spreads perpendicularly to the mountains, some slopes are firmly interlinked by ridges, forming well protected, amphitheatre shaped, vineyard areas in the wine-growing subre‐ gion of Plesivica.

Anthropogenic vineyard soils, classified as Aric Anthrosols [40], have been developed on Tertiary sediments and Pleistocene loams. Owing to exceptional geomorphological and agro-ecological conditions, these locations have been occupied almost exclusively by vine‐ yards for many decades.

The climate of the wider area is humid and the average annual rainfall is 836 mm. The mean annual temperature is 10.3 °C, ranging from –0.6 °C (January) to 20.4 °C (July).

To investigate the spatial variability of surface soils, 67 soil samples were taken at the nodes of a square grid at intervals of 1 km (Figure 3). The samples were defined as composite sam‐ ples made up of 10 increments collected from the soil upper 10 cm in a cross pattern, with a 5 m distance between increments (Eijkelkamp soil sampling kit used). Site descriptions were registered at the time of sampling to record the sample location in relation to vineyard char‐ acteristics and major environmental features.

Three soil profiles were then located according to the landscape feature: profile 1 (Aric An‐ throsols) was dug in the vineyard plot down to the parent material (80 cm depth) at the 258 m asl, profile 2 (Aric Anthrosols) was located in vineyard plot at 231 m asl, and profile 3 (Colluvic soil) was located in the meadow at the foot of the hill 200 m asl, with the altitude difference of 28 % between P1 and P2, and 13 % between P-2 and P-3. Table 1 reports the selected physical and analytical features of soil profiles, and Figure 2 shows a sequence of horizons.


**Table 1.** Selected physical and chemical features of soil profiles

#### **2.2. Chemical analysis**

**2.1. Study area**

804 Environmental Risk Assessment of Soil Contamination

to M. Romic)

gion of Plesivica.

yards for many decades.

Research was carried out on different wine-producing subregions in Croatia, in the wider Zagreb region: the wine-growing subregion of Plesivica (approximate coordinates: latitude, 45° 42' and longitude, 15° 37'). The area of Žumberak, which is a mountainous territory lo‐ cated to the west of Zagreb, is bordered to the north and west by Slovenia, and to the south by Kupa River (Figure 3). Soils are developed on Pannonian sediments. This sediments con‐ sist of limy marls, sands, sandstones, conglomerates and breccias [37-39]. Week consistency of those sediments, solid Triassic dolomite as bedrock and periodic streams led to fast ero‐ sion and filling in valleys and formation of amphitheater-shaped valleys. Namely, those landscape shapes were formed by the distinct climatic oscillations and pulsation between glacial and stadial periods, along with constant tectonic activity and elevation of terrain [39]. The landscape reflects the features of the Dinaric, so numerous formations characteristic of the karst are found on highly dissected limestone terrains. As the Zumberacka Mt. piedmont spreads perpendicularly to the mountains, some slopes are firmly interlinked by ridges, forming well protected, amphitheatre shaped, vineyard areas in the wine-growing subre‐

**Figure 1.** Grapevine plantation in the study region (Site Lokosin dol, Plesivica wine-producing subregion, Croatia, pho‐

Anthropogenic vineyard soils, classified as Aric Anthrosols [40], have been developed on Tertiary sediments and Pleistocene loams. Owing to exceptional geomorphological and agro-ecological conditions, these locations have been occupied almost exclusively by vine‐ Soil samples were air-dried, sieved at 2-mm, and subjected to the following analyses: pH in a 1:5 soil/water ratio (MettlerToledo MPC 227 pH- meter), soil organic carbon (SOC) by sul‐ fochromic oxidation [41], calcium carbonate (CaCO3) by the volumetric calcimeter method after HCl attack, and effective cation exchange capacity (CEC) using BaCl2 solution. Particle

**Figure 2.** Soil profiles description (Site Lokosin dol, Plesivica wine-producing subregion, Croatia, photo M. Romic)

size distribution was determined by the pipette method after disaggregation in sodium py‐ rophosphate (HRN ISO 11277:2004). Soil samples were also digested in aqua regia [42] with the microwave technique on a MARSXpress system (CEM).

Copper concentrations in soil digests were determined by inductivey coupled plasma opti‐ cal emission spectroscopy (ICP-OES) on a Vista MPX AX (Varian). All concentrations were calculated on the basis of dry weight of samples (105 °C, 24 h). Quality control procedure consisted of reagent blanks, duplicate samples and several referenced soil and sediment samples with similar matrix from the inter-laboratory calibration program [43]. Maximum allowable relative standard deviation between replicates was set to 10 %.

Two other methods were used for evaluating soil available copper: DTPA extraction [44] and calcium chloride extraction [45].

DTPA extraction: 10 g of soil were extracted with 20 ml DTPA 0.005 M + TEA 0.1 M + CaCl2 0.01 M for 2 h at 20 °C under stirring (Heidolph PROMAX 2200 used), prior to being filtered.

Calcium chloride extraction: 0.5 g of soil were extracted with 50 ml of CaCl2 0.01 M for 2 h at 20 °C under stirring, prior to being filtered.

#### **2.3. Metal fractionation**

The selective sequential dissolution procedure was employed to divide metals into five sol‐ id-phase fractions [46, 47]. Chemical reagents and the experimental conditions applied are summarized in Table 2. In this procedure, 0.5 g of each sample was weighted into 50 ml polyethylene centrifuge tube and the extractions were carried out directly in the tubes, ex‐ cept in the last step where Teflon tubes for microwave digestion were used. At each extrac‐ tion step, after shaking and equilibration, solid-liquid separation was achieved by centrifugation at 3500 rpm (2000 x g) for 10 min (centrifuge Sigma 3-15 used). The superna‐ tant was filtered through S&S 583 filter paper and placed to an acid-washed polyethylene tube. The solid residue was washed three times successively with 5 ml of ethanol and the liquid was discarded leaving the residue soil for the next step. The supernatant obtained at each step was analysed for metals using inductively coupled plasma optical emission spec‐ troscopy (ICP-OES) on a Vista MPX AX (Varian). Single element standards were prepared for each extraction in the same solution as the extracting agent to minimise matrix effects. Blanks were used for background correction and other sources of error.


**Table 2.** The selective sequential dissolution procedure; chemical reagents and the experimental conditions.

#### **2.4. Metal statistical analysis and data management**

size distribution was determined by the pipette method after disaggregation in sodium py‐ rophosphate (HRN ISO 11277:2004). Soil samples were also digested in aqua regia [42] with

**Figure 2.** Soil profiles description (Site Lokosin dol, Plesivica wine-producing subregion, Croatia, photo M. Romic)

Copper concentrations in soil digests were determined by inductivey coupled plasma opti‐ cal emission spectroscopy (ICP-OES) on a Vista MPX AX (Varian). All concentrations were calculated on the basis of dry weight of samples (105 °C, 24 h). Quality control procedure consisted of reagent blanks, duplicate samples and several referenced soil and sediment samples with similar matrix from the inter-laboratory calibration program [43]. Maximum

Two other methods were used for evaluating soil available copper: DTPA extraction [44]

DTPA extraction: 10 g of soil were extracted with 20 ml DTPA 0.005 M + TEA 0.1 M + CaCl2 0.01 M for 2 h at 20 °C under stirring (Heidolph PROMAX 2200 used), prior to being filtered. Calcium chloride extraction: 0.5 g of soil were extracted with 50 ml of CaCl2 0.01 M for 2 h at

The selective sequential dissolution procedure was employed to divide metals into five sol‐ id-phase fractions [46, 47]. Chemical reagents and the experimental conditions applied are

the microwave technique on a MARSXpress system (CEM).

and calcium chloride extraction [45].

806 Environmental Risk Assessment of Soil Contamination

**2.3. Metal fractionation**

20 °C under stirring, prior to being filtered.

allowable relative standard deviation between replicates was set to 10 %.

Linear regression: Relations between extractible copper in soils and soil properties were ana‐ lyzed by simple and multiple linear regression [48]. General conditions for model applica‐ tion were verified after parameter determination. Thus, residuals should follow normal distribution, the assumption of homoscedasticity of variable variances should be proven, and they should be independent of one another. Distribution normality was tested using the Shapiro-Wilk test, while the other hypotheses were checked visually after the graphs were drawn. Details given by [49]. All soil data were incorporated into the GIS database.
