**3. Copper concentrations in surface vineyard soil**

Total copper concentrations in vineyard soils ranged from 30 to 700 mg kg-1, while total cop‐ per in 88 % samples exceeded the maximum tolerant concentration under the Croatian regu‐ lation of 100 mg kg-1 [50] (Figure 3).

**Figure 3.** Study area, surface soil sampling scheme and interpolated map of total surface soil copper concentrations [51]

The amount of copper built up in the surface vineyard soil was estimated on a *per hectare* basis taking into account the total copper concentration and the weight of 10 cm thick sur‐ face layer of soil, assuming a bulk density of 1.5 (Table 3). Approximate plantation age (with within-decade precision) was estimated for most plots, and a detailed history for some plots: time of the first and possibly second deep ploughing, time of vineyard restoration or supple‐ mentary planting, common growing practice in the past, and thereby also approximate fre‐ quency of copper fungicide applications. More than 64 % vineyards are more than 40 years old and it is assumed that the same percent of all plots received similar annual amounts of copper, ranging from 2 to 5 kg ha-1. This certainly allows only an estimation of the overall copper input into soil throughout the vineyard history. The upper layer of plots planted with about a century old vines exhibited high copper contents.


Copper Accumulation in Vineyard Soils: Distribution, Fractionation and Bioavailability Assessment http://dx.doi.org/10.5772/57266 


1Location: All the data available from the GIS database

**Figure 3.** Study area, surface soil sampling scheme and interpolated map of total surface soil copper concentrations

The amount of copper built up in the surface vineyard soil was estimated on a *per hectare* basis taking into account the total copper concentration and the weight of 10 cm thick sur‐ face layer of soil, assuming a bulk density of 1.5 (Table 3). Approximate plantation age (with within-decade precision) was estimated for most plots, and a detailed history for some plots: time of the first and possibly second deep ploughing, time of vineyard restoration or supple‐ mentary planting, common growing practice in the past, and thereby also approximate fre‐ quency of copper fungicide applications. More than 64 % vineyards are more than 40 years old and it is assumed that the same percent of all plots received similar annual amounts of copper, ranging from 2 to 5 kg ha-1. This certainly allows only an estimation of the overall copper input into soil throughout the vineyard history. The upper layer of plots planted

**Location1 CuTOT(mg kg-1) CuDTPA (mg kg-1) CuCaCl2 (mg kg-1) CuTOT/ha (kg ha-1)2** 363 136 1.15 491 154 48 0.63 208 380 142 1.17 514 389 121 0.74 525 181 56 0.73 244 166 54 0.55 224 655 368 1.45 884 586 296 1.96 790 369 135 0.94 498 565 296 1.35 763 633 340 2.08 855 641 228 1.68 866

with about a century old vines exhibited high copper contents.

[51]

Environmental Risk Assessment of Soil Contamination

2Cu/ha (kg ha-1) Calculated from the data on total Cu (mg kg-1) in the top 10 cm assuming a bulk density of 1.5

**Table 3.** Total Cu, extractable Cu and total amount of Cu per ha in the upper layer (10 cm) of vineyard soils (n=67) (Romic et al., 2004)

Determination of the total metal content in soils is an important step in assessing the haz‐ ards to the vital roles of soils in the ecosystem, but also in comparing them with quality standards referring to the effects of contamination and system sustainability. However, the mobility and availability of soil copper are governed by the processes of dynamic equilibri‐ um, and not only by its total concentration [52]. This study shows that the mobile (CaCl2 extractable) fraction of copper in vineyard soils amounted only to 0.2 – 3.1 % of its total content. As Cu accumulation in the studied soils is restricted to surface layer the risk of Cu phytotoxicity for grapevine is small, since grapevine develops most of its roots at a depth >30 cm, depending on the soil type and the profile depth. Material suspended by erosion, however, carries away also a part of the applied copper, and redistribution of this material depends on a number of factors (relief, size and shape of the drainage basin, etc.). Halamic et al. [53] applied factor analysis in geochemical investigations of stream sediments in drain‐ age basins in the Mt. Medvednica region, which also includes part of the studied area, with‐ out determining correlation of copper concentrations with any lithological unit, so they assumed anthropogenic influence, mostly grapevine production. Ribolzi [54] carried out the research in the Mediterranean drainage basins of wine-growing regions in France with the aim to characterize copper forms in suspended material and recorded an average total con‐ centration as high as 245 mg Cu kg-1, but Brun et al. [55] reported the maximum of 250 mg kg-1 of copper in vineyard soils of the Mediterranean part of France.

#### **3.1. Correlation between total, extractable Cu and soil properties**

In the total copper concentration extracted with aqua regia, DTPA-extractable copper amounted to 12-81 % and CaCl2- extractable to 0.2-3.1 %. Both DTPA- and CaCl2-extractable copper were largely explained by the total copper concentrations, as confirmed by their high correlation coefficient (R = 0.899 and R = 0.896). They were also highly correlated to each other (R = 0.763) (Table 4).


ns - not significant

\* Correlation significant at p < 0.05

\*\* Correlation significant at p < 0.01

\*\*\* Correlation significant at p < 0.001

**Table 4.** Correlation matrix, upper triangle

Highly significant positive correlation was also determined between total copper and organ‐ ic matter in soil, and a weaker correlation, but still significant, between total copper and cati‐ on exchange capacity and carbonate content (Table 4). In the case of extractable copper, a significant correlation was determined between CuDTPA and organic carbon content and pH, but not between cation exchange capacity and carbonate content. Significant correlation was recorded between all the analyzed soil properties and CuCaCl2.

#### **3.2. Linear regression of total and extractable copper and selected soil properties**

Determination of the total metal content in soils is an important step in assessing the haz‐ ards to the vital roles of soils in the ecosystem, but also in comparing them with quality standards referring to the effects of contamination and system sustainability. However, the mobility and availability of soil copper are governed by the processes of dynamic equilibri‐ um, and not only by its total concentration [52]. This study shows that the mobile (CaCl2 extractable) fraction of copper in vineyard soils amounted only to 0.2 – 3.1 % of its total content. As Cu accumulation in the studied soils is restricted to surface layer the risk of Cu phytotoxicity for grapevine is small, since grapevine develops most of its roots at a depth >30 cm, depending on the soil type and the profile depth. Material suspended by erosion, however, carries away also a part of the applied copper, and redistribution of this material depends on a number of factors (relief, size and shape of the drainage basin, etc.). Halamic et al. [53] applied factor analysis in geochemical investigations of stream sediments in drain‐ age basins in the Mt. Medvednica region, which also includes part of the studied area, with‐ out determining correlation of copper concentrations with any lithological unit, so they assumed anthropogenic influence, mostly grapevine production. Ribolzi [54] carried out the research in the Mediterranean drainage basins of wine-growing regions in France with the aim to characterize copper forms in suspended material and recorded an average total con‐ centration as high as 245 mg Cu kg-1, but Brun et al. [55] reported the maximum of 250 mg

In the total copper concentration extracted with aqua regia, DTPA-extractable copper amounted to 12-81 % and CaCl2- extractable to 0.2-3.1 %. Both DTPA- and CaCl2-extractable copper were largely explained by the total copper concentrations, as confirmed by their high correlation coefficient (R = 0.899 and R = 0.896). They were also highly correlated to each

**Cutotal** 1 0.899\*\*\* 0.896\*\*\* 0.388\*\* 0.601\*\*\* 0.277\* 0.314\*\* **CuDTPA** 1 0.763\*\*\* 0.321\*\* 0.395\*\*\* 0.084ns 0.236ns **CuCaCl2** 1 0.392\*\*\* 0.734\*\*\* 0.257\* 0.341\*\* **pHH2O** 1 0.121ns 0.315\*\* 0.587\*\*\* **Org. C** 1 0.339\*\* 0.173ns **CEC** 1 0.130ns **CaCO3** 1

**Cutotal CuDTPA CuCaCl2 pHH2O Org. C CEC CaCO3**

kg-1 of copper in vineyard soils of the Mediterranean part of France.

**3.1. Correlation between total, extractable Cu and soil properties**

other (R = 0.763) (Table 4).

810 Environmental Risk Assessment of Soil Contamination

ns - not significant

 Correlation significant at p < 0.05 \*\* Correlation significant at p < 0.01 \*\*\* Correlation significant at p < 0.001

**Table 4.** Correlation matrix, upper triangle

\*

To establish the relation between copper fractions after particular extractions (aqua regia, DTPA and CaCl2) and soil properties that may affect their behavior in soil and availability to plants, the multiple linear regression analysis was done. The model included those variables for which correlation probability p<0.05 was determined [48]. For the regression model of aqua regia extracted copper, this condition was met by the following properties: CuDTPA, CuCaCl2 and cation exchange capacity (CEC).

For total copper, the regression model explains 92 % of total variance (Table 5). The largest contribution to the variance in regression was that of CuDTPA.


Regression equation: CuTOT = -63.42 + 0.956\* CuDTPA + 170.9\*CuCaCl2 + 3.761\*CEC R2 = 0.92

**Table 5.** Linear regression of total copper (CuTOT) as a function of DTPA-extractable (CuDTPA), CaCl2-extractable (CuCaCl2) and cation exchange capacity (CEC).

Concentration of total copper in vineyard topsoil went up with an increase in the cation ex‐ change capacity. Square root transformation (SQRT) was applied tothe regression model of DTPA-extractable copper, whereby the model conditions were satisfied, and the trans‐ formed variables SQRT(CuDTPA) were predominantly dependent on Cutot. As this relation was already determined, it was omitted from the model. Two other properties met the con‐ dition of correlation probability p<0.05: organic matter content (Org-C) and CaCl 2-extracta‐ ble copper (CuCaCl2), and they were included into the model. For CuDTPA, the regression model explains 85 % of total variance (Table 6).


R2 = 0.853

**Table 6.** Linear regression of DTPA-extractable (CuDTPA), as a function of CaCl2-extractable (CuCaCl2) and soil organic matter (Org. C)

Two parameters were included into the regression model of CaCl2-extractable copper: or‐ ganic matter content (Org-C) and pH, and the model explains 62 % of total variance (Table 7). Concentrations of CaCl2- extractable copper mainly depend on pH, which relation was also confirmed by this investigation. However, since these are predominantly alkaline soils, this relation is not as strong as in the case of soils with a more varying pH [55].


R2 = 0.621

**Table 7.** Linear regression of CaCl2-extractable copper as a function of soil organic matter (Org. C) and pH

Over 65 % of vineyard plots under study were more than 40 years old, and some have been continuously cultivated for more than 100 years. It is assumed that the same percent of par‐ cels received a similar annual amount of copper, ranging from 2 to 5 kg ha-1. This, naturally, does not allow an exact estimate of the overall copper input into soil throughout the vine‐ yard history. Vineyard age parameter was not therefore included in the multiple linear re‐ gression model. There is, however, strong statistical evidence that an increase in vineyard age is related to the increase of expected total copper content (Figure 4). According to histor‐ ical documents, the vine downy mildew infection started spreading in the vineyards of northwestern Croatian in 1882, and the Bordeaux mixture application became indispensable during the wine boom period at the end of the 19th century. Recognizing the benefits, winegrowers often did not observe the recommended concentrations and application times, and a large number of treatments, as many as 8 to 14, were often applied at positions exposed to disease attacks. However, numerous other factors, such as scattering during applications, washing off the leaves by rain, input of treated plant residues into soil, tillage and erosion, make it difficult to establish the relation between vineyard age and accumulated copper. In France, for example, the Bordeaux mixture has been used since 1855, and it was found that after several decades of its continuous application the soil total copper reached a concentra‐ tion of as much as 1.0 g kg-1 [56]. Research done by Deluisa et al. [11] on 43 plots in a humid region of northern Italy revealed an average copper accumulation in topsoil of 297 mg kg-1. Moolenaar and Beltrami [57] have calculated that organic protection of grapevine, which im‐ plies exclusive use of the Bordeaux mixture, can result in an increase of soil copper concen‐ tration up to 600 mg kg-1 after 100 years.

**Figure 4.** Vine age as a function of total soil copper

**Variable explained: SQRT (CuDTPA)**

812 Environmental Risk Assessment of Soil Contamination

CuCaCl2 Org-C Total

R2 = 0.853

matter (Org. C)

**Variable explained: CuCaCl2**

Org-C pH Total

R2 = 0.621

**Source of variation Degree of freedom Sum of squares F Pr > F**

**Table 6.** Linear regression of DTPA-extractable (CuDTPA), as a function of CaCl2-extractable (CuCaCl2) and soil organic

this relation is not as strong as in the case of soils with a more varying pH [55].

1 1 64

**Table 7.** Linear regression of CaCl2-extractable copper as a function of soil organic matter (Org. C) and pH

Two parameters were included into the regression model of CaCl2-extractable copper: or‐ ganic matter content (Org-C) and pH, and the model explains 62 % of total variance (Table 7). Concentrations of CaCl2- extractable copper mainly depend on pH, which relation was also confirmed by this investigation. However, since these are predominantly alkaline soils,

**Source of variation Degree of freedom Sum of squares F Pr > F**

Over 65 % of vineyard plots under study were more than 40 years old, and some have been continuously cultivated for more than 100 years. It is assumed that the same percent of par‐ cels received a similar annual amount of copper, ranging from 2 to 5 kg ha-1. This, naturally, does not allow an exact estimate of the overall copper input into soil throughout the vine‐ yard history. Vineyard age parameter was not therefore included in the multiple linear re‐ gression model. There is, however, strong statistical evidence that an increase in vineyard age is related to the increase of expected total copper content (Figure 4). According to histor‐ ical documents, the vine downy mildew infection started spreading in the vineyards of northwestern Croatian in 1882, and the Bordeaux mixture application became indispensable during the wine boom period at the end of the 19th century. Recognizing the benefits, winegrowers often did not observe the recommended concentrations and application times, and a large number of treatments, as many as 8 to 14, were often applied at positions exposed to disease attacks. However, numerous other factors, such as scattering during applications, washing off the leaves by rain, input of treated plant residues into soil, tillage and erosion,

685.7 44.54 1224

8.30 1.62 17.34 240.0 15.59

83.37 16.23 0.0000 0.0002

0.0000 0.0002

1 1 63

Regression equation: SQRT(CuDTPA) = 5.534 + 9.400\*CuCaCl2 – 1.326\* Org-C

Regression equation: CuCaCl2 = -2.673 + 0.379\* Org-C + 0.337\* pH

Diethylenetriaminepentaacetic acid (DTPA) is a potent synthetic chelating agent, and the method of extraction with DTPA was developed for the purpose of determining zinc, iron, manganese or copper deficiency in neutral and carbonate soils [58]. Haq and Miller [59] re‐ ported negative results of the DTPA test, which they explained by their failure to determine sufficiently significant relations between concentrations of metals (copper and manganese) extracted from soil and those found in the tested plants. Mention should be also made of the research done by O'Connor [60], who gave a number of comments on the DTPA test, based also on non-significant correlation between DTPA-extractable metals in soil and their con‐ centrations in plants. Regardless of the above considerations, DTPA is the most widely used agent for extraction of "available" cadmium, copper, nickel and zinc, and thereby also the most standardized one [44, 61, 62]. Starting from the fact that the data on total copper con‐ tent reveals very little about its bio-availability, such strong correlation between copper ex‐ tracted with aqua regia and DTPA actually indicates that neither the latter extraction method is suitable for assessing copper availability to plants.

Merry et al. [63] in vineyard soils recorded 25-35 times higher contents of copper, lead and arsenic, originating from plant protection agents, than their common values in uncontami‐ nated soils; they also determined a strong correlation between total and DTPA-extractable copper (0.93 < r < 0.96). Also Brun et al. [55] found that the regression model CuDTPA ex‐ plained 90 % of total variance in vineyard soils, its largest part referring to total copper con‐ tent.

When cation exchange capacity was included into the model, it was found that the DTPAextractable copper decreased with increasing cation exchange capacity.

Soil extraction 0.01 M CaCl2 is the method that was increasingly used in the last decade for soil testing to determine soil fertility and the behavior of nutrients and contaminants in the soil. The capabilities of instrumental chemical analysis have improved to such an extent, even in the last few years, that it become possible to determine very low concentrations of nutrients and pollutants in soil extracts [45]. The advantage of this method for determining metal concentrations in soil is that the concentration of electrolytes stays practically constant and metal concentrations reflect the difference in binding strength or solubility between soils. The extractant is an unbuffered solution and therefore the measured metals reflect their availability at the pH of the soil.

The best criterion of the efficiency of the method for determining the soil bioavailable frac‐ tion is the high correlation between the Cu content observed in plants grown in situ, at least for neutral to acid soils [55].

#### **3.3. Vertical distribution of total copper in soil profiles**

High copper concentrations were found in vineyard soils down to 20 cm depth (to 800 mg kg-1 in profile 1, and to 500 mg kg-1 in profile 2) (Figure 5). Profile 1 is situated at a higher altitude, erosion is more pronounced, and the anthropogenic horizon is less thick. Marl ap‐ pears already at 45 cm depth, so that the root zone extends into horizon C as well, thus opening the transport routes of water, dissolved substances and solid particles deeper into the profile. In profile 2, the anthropogenic horizon is much thicker, while total copper con‐ centrations are lower down to 30 cm depth. Erosion material was deposited at the base of the slope, so that as much as 100 mg kg-1 of copper was found in the topsoil of colluvial soil, to which no copper agents for plant protection had ever been directly applied. Accumula‐ tion of copper in colluvial soil (profile 3) was recorded down to 30 cm depth, that is, over the entire depth of the humus-accumulative horizon. Uniform copper concentrations of <25 mg kg-1 were found at greater depths.

Land use for agriculture causes great changes in the natural properties of soil. Translocation of soil by tillage may be the key reason for redistribution of soil particles within the profile and over the entire site, while erosion due to tillage is especially present in hilly landscapes [64]. Tillage and homogenization of several natural horizons alter both morphological and physicochemical characteristics of soil.

#### **3.4. Sequential extraction**

Results of the five-step sequential extraction are presented in Figure 6, which shows the dis‐ tribution of exchangeable (Exch), carbonate-associated (Carb), Fe-Mn oxides-bound (Fe-Mn

Copper Accumulation in Vineyard Soils: Distribution, Fractionation and Bioavailability Assessment http://dx.doi.org/10.5772/57266 815

**Figure 5.** Verical distribution of total copper within the soil profiles

nated soils; they also determined a strong correlation between total and DTPA-extractable copper (0.93 < r < 0.96). Also Brun et al. [55] found that the regression model CuDTPA ex‐ plained 90 % of total variance in vineyard soils, its largest part referring to total copper con‐

When cation exchange capacity was included into the model, it was found that the DTPA-

Soil extraction 0.01 M CaCl2 is the method that was increasingly used in the last decade for soil testing to determine soil fertility and the behavior of nutrients and contaminants in the soil. The capabilities of instrumental chemical analysis have improved to such an extent, even in the last few years, that it become possible to determine very low concentrations of nutrients and pollutants in soil extracts [45]. The advantage of this method for determining metal concentrations in soil is that the concentration of electrolytes stays practically constant and metal concentrations reflect the difference in binding strength or solubility between soils. The extractant is an unbuffered solution and therefore the measured metals reflect

The best criterion of the efficiency of the method for determining the soil bioavailable frac‐ tion is the high correlation between the Cu content observed in plants grown in situ, at least

High copper concentrations were found in vineyard soils down to 20 cm depth (to 800 mg kg-1 in profile 1, and to 500 mg kg-1 in profile 2) (Figure 5). Profile 1 is situated at a higher altitude, erosion is more pronounced, and the anthropogenic horizon is less thick. Marl ap‐ pears already at 45 cm depth, so that the root zone extends into horizon C as well, thus opening the transport routes of water, dissolved substances and solid particles deeper into the profile. In profile 2, the anthropogenic horizon is much thicker, while total copper con‐ centrations are lower down to 30 cm depth. Erosion material was deposited at the base of the slope, so that as much as 100 mg kg-1 of copper was found in the topsoil of colluvial soil, to which no copper agents for plant protection had ever been directly applied. Accumula‐ tion of copper in colluvial soil (profile 3) was recorded down to 30 cm depth, that is, over the entire depth of the humus-accumulative horizon. Uniform copper concentrations of <25

Land use for agriculture causes great changes in the natural properties of soil. Translocation of soil by tillage may be the key reason for redistribution of soil particles within the profile and over the entire site, while erosion due to tillage is especially present in hilly landscapes [64]. Tillage and homogenization of several natural horizons alter both morphological and

Results of the five-step sequential extraction are presented in Figure 6, which shows the dis‐ tribution of exchangeable (Exch), carbonate-associated (Carb), Fe-Mn oxides-bound (Fe-Mn

extractable copper decreased with increasing cation exchange capacity.

their availability at the pH of the soil.

814 Environmental Risk Assessment of Soil Contamination

mg kg-1 were found at greater depths.

physicochemical characteristics of soil.

**3.4. Sequential extraction**

**3.3. Vertical distribution of total copper in soil profiles**

for neutral to acid soils [55].

tent.

ox), organic-bound (Org) and residual (Resid) copper found in the soil samples. These five metal fractions were separated in soil samples from 10-cm profile layers of Aric Anthrosols and master horizons of Calcaric Regosols. The data were used to calculate the relative error (RE %), which for most metals amounted to ±10 %.

In the surface 10 cm of profile 1, 877 mg kg-1 of copper was determined by extraction in *aqua regia*. The sum of copper fractions separated by selective sequential extraction is slightly lower and amounts to 816 kg-1 (RE = 7 %). As much as 47 % of total copper was bound in the organic fraction, 20 % in the residual fraction, and 18 % and 16 % in the reductive and carbo‐ nate fractions, respectively. Down to 30 cm depth, the share of copper in exchangeable frac‐ tion was <1 %, whereas it was not detected in deeper layers (Figure 6). However, copper distribution per fractions changes with the profile depth. As the total concentration decreas‐ es to background values, the share of copper in residual fractions increases, since complex‐ ing ability decreases with the reduced amount of organic matter.

Considering the high content of the organic fraction (54 % in the 0–10 cm layer and 27 % at 60–80 cm depth), a very probable mechanism is translocation through complexation with soil organic matter. However, copper is certainly not translocated in the same way with sur‐ face eroded material as vertically through the profile depth. Eroded material deposited at the base of hillsides under vineyards is richer in silt, and copper is mostly strongly bound in the residual fraction. In the surface 10 cm of soil of the profile 1, 20 % of total copper content is bound in the residual fraction. In the profile 2, copper content in the residual fraction was increased to 21 %, while it amounted to 50.3 % in colluvial soil (profile 3).

**Figure 6.** The distribution of exchangeable (Exch), carbonate-associated (Carb), Fe-Mn oxides-bound (Fe-Mn ox), or‐ ganic-bound (Org) and residual (Resid) copper in soils

Copper distribution per fractions in profile 2 resembled that in the preceding profile. High copper concentrations decrease with depth, the profile being deeper as well. Copper content of <1 % was found in the exchangeable fraction, and that of 3 % at 60–80 cm depth. It is obvi‐ ous that copper translocation occurs within the profile, but this is also the zone where most of the vine roots develop.

Although no copper fungicides had ever been directly applied to the area on which profile 3 was dug, as much as 100 mg Cu kg-1 was determined in topsoil by extraction in aqua regia. Background concentrations were reached at 30 cm depth, and copper was predominantly bound in the residual fraction (47 %-63 %). There was a significant share of the organic frac‐ tion, but it decreased with depth from 33 % to 15 %. The content of exchangeable copper was higher than in vineyard soils – 2 % to 5 %.

Research has shown that soil type is the main factor of accumulation and distribution of both natural and anthropogenic concentrations of heavy metals. In natural profiles, the in‐ digenous element distribution is generated by long-term pedogenesis [65]. In the case of cul‐ tivated soils, their characteristics in time and space change in dependence on the ecological environment and land use and management, whereby also their production capacity and environmental impact get changed [66, 67]. When growing woody crops, and thus also grapevine, soil is homogenized to a greater depth, first by deep ploughing to xx cm depth before the setting up of the plantation, and then by regular tillage. This somewhat disrupts the morphogenetic soil properties, changes the sequence of genetic horizons, and often deepens the active part of the profile. Such changes are naturally reflected in the distribution of elements, changes in their mobility and bioavailability. Selective sequential extraction was used in this research to determine the way and strength of binding and retention of heavy metals in soil, which under certain conditions enables the estimation of potential mobility and bioavailability.

The spatial variability of trace metals in agricultural surface soils of the wider Zagreb area has shown that the application of agrochemicals has caused high accumulation of copper and zinc [51, 68]. This especially applies to vineyard soils, but also to orchard and vegetable garden soils. The presence of a buffer material, such as carbonate, can be particularly impor‐ tant in the retention of heavy metals. The trace metal retention capacity of silty soils with high carbonate content can be as high as, or higher than, the retention capacity of certain clayey soils [69].

In soils and sediments that were receiving high concentrations of copper (along with other metals) for at least 6 years, Hickey and Kittrick [70] established that about 28 % copper was bound in the organic fraction. It is also in the soils treated with waste sludge or stable man‐ ure rich in copper that most of this metal is bound in the organic fraction [71, 72]. Affinity of humic and fulvo acids to copper sorption was, among others, reported by Senesi et al. [73] while McLaren et al. [74], pointed to the importance of soluble copper chelates in soil solu‐ tion. In soils of lighter texture, poorer in organic matter, the added copper is initially re‐ tained in the exchangeable fraction, whereafter it is translocated into the carbonate fraction [75]. Incubation in the laboratory experiment revealed that translocation of copper to more stabile fractions was much slower in texturally light, but acid, vineyard soil, also poor in or‐ ganic matter [76]. However, Flores-Velez et al. [10] report that in the case of sandy acid vine‐ yard soils the selective sequential extraction procedure was not selective enough to specify the form of copper. The same authors report that copper in anthropogenic vineyard soil, originating from Cu-fungicides, was concentrated in the coarse organic fraction (plant resi‐ dues) and in the mineral colloid fraction. In soils or sediments deficient in organic matter, a larger part of copper was bound to Fe and Mn oxides that it was found in this research. Thus, Szarek–Gwiazda and Mazurkiewicz–Boron [77] found that 40.2-54.1 % of total copper in fluvial sediment was bound to Mn oxides and amorphous Fe hydroxides, and only about 10 % to the organic fraction. These authors maintain that Fe(III) and Mn(IV)-oxides can oc‐ cur either as coats on detritus particles, as cement between them or as pure concretions.

Copper distribution per fractions in profile 2 resembled that in the preceding profile. High copper concentrations decrease with depth, the profile being deeper as well. Copper content of <1 % was found in the exchangeable fraction, and that of 3 % at 60–80 cm depth. It is obvi‐ ous that copper translocation occurs within the profile, but this is also the zone where most

**Figure 6.** The distribution of exchangeable (Exch), carbonate-associated (Carb), Fe-Mn oxides-bound (Fe-Mn ox), or‐

Although no copper fungicides had ever been directly applied to the area on which profile 3 was dug, as much as 100 mg Cu kg-1 was determined in topsoil by extraction in aqua regia. Background concentrations were reached at 30 cm depth, and copper was predominantly bound in the residual fraction (47 %-63 %). There was a significant share of the organic frac‐ tion, but it decreased with depth from 33 % to 15 %. The content of exchangeable copper

of the vine roots develop.

was higher than in vineyard soils – 2 % to 5 %.

ganic-bound (Org) and residual (Resid) copper in soils

816 Environmental Risk Assessment of Soil Contamination

Their ability to adsorb and control heavy metal distribution between the solution and the matrix has been thoroughly explained in scientific literature (e.g., [78]).

Romic et al. [49] did not establish, either by individual correlations or by factorial analysis in the vineyard soils of NW Croatia, the importance of the contents of clay, Fe and Mn oxides or cation exchange capacity for copper sorption in soil, though some authors stress the im‐ portance of these fractions [74]. In their investigations, the relation between the content of metals and soil properties was assessed on the basis of their total contents. In this research, correlations were determined between copper fractions and the selected soil properties: sig‐ nificant correlation was recorded between the contents of organic C and ORG and RESID fractions, or its total content, whereas no significant correlation was found between copper in the said fractions and cation exchange capacity, or total carbonates. This corroborates the reports that the distribution of copper of anthropogenic origin among fractions depends pre‐ vailingly on soil organic matter.

Finally, the use of copper containing fungicides is allowed in the organic agriculture by the European Union regulation, and the official guidelines for soil copper content are usually derived from the total soil copper content. However, these guidelines should be modified according to the soil properties, such as pH and organic matter content, which will affect the Cu solubility, and consequently its bioavailability. Furthermore, soil copper thresholds should be confirmed with toxicological data obtained for biota (e.g. plants, microorganisms, invertebrates). Above mentioned implies that site-specific guidelines should developed for the risk assessment of soil copper toxicity.
