Relationships between extractable copper, soil properties and copper uptake by wild plants in vineyard soils

ENVIRONMENTAL POLLUTION

Environmental Pollution 102 (1998) 151±161

Relationships between extractable copper, soil properties and copper uptake by wild plants in vineyard soils L.A. Brun a, J. Maillet a,*, J. Richarte a, P. Herrmann b, J.C. Remy b a

ENSAM/INRA, Department of Plant Biology, Ecology and Pathology, 2 Place Viala, 34060 Montpellier Cedex 1, France b ENSAM/INRA, Department of Soil Science, 2 Place Viala, 34060 Montpellier Cedex 1, France Received 20 October 1997; accepted 6 June 1998

Abstract The repeated use of Cu fungicides to control vine downy mildew has been responsible for the heavy increase of total Cu content in vineyard soils. In a French Mediterranean region (DeÂpartement de l'HeÂrault), the total Cu content observed in the upper layer of vineyard soils ranged from 31 to 250 mg kgÿ1, versus 14 to 29 mg kgÿ1 in woodland plots. Cu distribution with soil depth showed that Cu input from Cu treatment was essentially concentrated in the upper layers, except for two soil pro®les which presented Cu enrichments in deep horizons. EDTA, DTPA and ammonium acetate-extractable Cu were highly correlated with soil total Cu, and were weakly correlated with Cu content in wild plants grown in the plots. These observations suggested that these extraction methods are of limited interest for predicting the availability of Cu to plants in Cu-contaminated soils. In comparison, CaCl2 extractable Cu was not correlated to total Cu and depended mainly on soil pH. When the soil pH increased the quantities of extractable Cu decreased. For soil with neutral to acid pH, CaCl2 extractable Cu was correlated with the contents observed in wild plants. It thus appeared as the best way of predicting plant-available Cu in vineyard soils. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Copper; Vineyard; Soil pollution; Extractable copper; Wild plants

1. Introduction The repeated use of copper fungicides to control cultivated plant diseases has led to long-term accumulation of Cu in the surface of some agricultural soils throughout the world. In Europe, systematic spraying of Bordeaux mixture (Ca(OH)2 + CuSO4) since the end of the 19th century to control vine downy mildew has been responsible for a heavy pollution of vineyard soils. Arable land usually presents quantities of Cu varying between 5 and 30 mg kgÿ1, whereas the soils of numerous wine-growing areas can contain as much as 200 to 500 mg kgÿ1 of Cu (Drouineau and Mazoyer, 1962; Delas, 1963; Geo€rion, 1975; Deluisa et al., 1996; Flores Velez, 1996). In the Mediterranean regions of the south of France, wine-growing is the main agricultural activity and vineyards are covering an extensive portion of arable land. However the Cu content of these vineyard soils is largely unknown. A substantial proportion * Corresponding author. Tel.: +33-4-99-61-25-16; fax: +33-4-6754-59-77; e-mail: [email protected] 0269-7491/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S0269 -7 491(98)00120 -1

of copper sprayed on the vines eventually reaches the soil where it often remains in the top soil as a consequence of the strong ®xation of copper in many types of soils (Merry et al., 1983; Deluisa et al., 1996; Flores Velez et al., 1996). The bioavailability of Cu accumulated in vineyards can be de®ned as its ability to be transferred from the soil to the plant roots. It depends on the soils properties, temperature, water content and aeration, and also on plant species (Juste et al., 1995). Soil pH heavily in¯uences Cu bioavailability, which is higher as the soil pH decreases (Delas, 1984; Gupta and Aten, 1993). In a ®eld experiment on an acid, Cu-rich, sandy soil, Lexmond (1980) showed that the Cu content in corn leaves decreased dramatically once the soil pH had been increased by adding CaCO3. In an acidic, sandy soil treated with Cu, Citrus-root Cu content also dropped with increasing soil pH (Alva et al., 1993). The bioavailability of Cu has also been reported to decrease when the cation exchange capacity or when the level of soil organic matter increases (Delas, 1984; Gupta and Aten, 1993).

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The total amount of Cu in the soil gives little information on its bioavailability. Di€erent chemical extractants can be used to estimate the bioavailability of Cu in soils: water, bu€ered or unbu€ered salt solutions (ammonium acetate, CaCl2, Ca(NO3)2, NH4NO3, etc.), chelating agents (EDTA, DTPA), diluted acids or a mix of these reagents (Juste, 1988; Gupta and Aten, 1993; Lebourg et al., 1996). The Cu accumulated in the soil can be responsible for phyto-toxicity above a threshold which depends on both plant species and soil properties. The toxicity of Cu is essentially observed in acid soils, and is the most likely to occur at low soil pH and for soils exhibiting a small cation exchange capacity (Drouineau and Mazoyer, 1962; Gupta and Aten, 1993). The toxic threshold content for a pH below 6 is generally accepted to be 25 mg kgÿ1 of Cu extracted with ammonium acetate 1M in sandy soils and above 100 mg kgÿ1 in clayey soils (Delas, 1984). In calcareous soils and for Cu contents as high as those reported in vineyard soils, no Cu toxicity has been observed, Cu being precipitated as hydroxides or carbonates (Delas, 1963). The main objectives of this study are: (1) to obtain data on the amounts of Cu built-up in the vineyard soils of the South of France (DeÂpartement de l'HeÂrault) where few studies have been carried out, (2) to quantify the Cu taken up by the plants which naturally grow in this soil, and (3) to establish relationships between the results of chemical extractions of Cu and soil properties. These results will then be discussed in order to evaluate how e€ective the various chemical extractants are in estimating the bioavailability of Cu in vineyard soils. 2. Material and methods 2.1. Study area Twenty-®ve vineyard plots (or former vineyard plots) and four woodland plots (control areas having never received Bordeaux mixture applications) were used for this study (Table 1). The plots are located in a winegrowing area north of BeÂziers (HeÂrault, southern France) in the foothills of the eastern side of the Montagne Noire. Vines are grown (1) in calcareous soils developed on middle lacustrine Miocene sediments (Plots 1±9), (2) in regolithic acidic soils developed on Ordovician and Visean sandstoneÐslate complex (Plots 10±22), or (3) in degraded acidic ferrimorphic soils developed on old Villafranchian, stony, alluvial deposits (Plots 23±29) (Bon®ls, 1984, 1993). 2.2. Soil samples For each plot, 10 soil samples (15 cm deep by 8 cm wide) were randomly collected, then mixed together, air-

dried and ground to pass through a 2 mm sieve. The resulting sample was analyzed. During each analysis residual humidity was measured by weight loss of an aliquote of soil after oven drying at 105 C for 12 h. The results are given per unit of dried soil. All the subsequent soil analysis were realized according to the French standard procedures (Afnor, 1994). The particule size distribution was determinated by sedimentation. Soil pH was measured in deionized water using a 1:2.5 soil/solution ratio or in 1M KCl solution using a 1:2.5 soil/solution ratio. Organic matter (OM) was determined by sulfochromic oxidation, cation exchange capacity (CEC) by ammonium acetate method and total CaCO3 by HCl attack. Selected soil properties are presented in Table 2. Soils samples were collected every 15 cm along a soil pro®le to a depth of 60 cm for 10 vineyard plots representative of the study area. These samples were treated as described above. 2.3. Plant samples Plants which have grown on the soil in situ can be considered as the best indicators of the bioavailability of a soil element, compared with plants obtained under greenhouse conditions which may not be representative of ®eld conditions (Barona and Romero, 1997). Therefore nine species of plants commonly found on all the plots were sampled: Dactylis glomerata L., Poa annua L., Andryala integrifolia L., Hypochoeris radicata L., Senecio vulgaris L., Sanguisorba minor Scop. s.l., Rumex acetosella L., Allium polyanthum Schultes & Schultes f. and Rubia peregrina L. The plants were collected in situ in March±April 1996 and 1997, that is before the vines were chemically weeded and Cu fungicides were applied to control vine downy mildew. For each species, 10 plants were randomly collected from the plot. When the root system or the below-ground organs were also collected they were quickly and carefully washed to remove adhering soil particles. The plants were then oven dried at 70 C for 48 h before being analyzed. 2.4. Chemical analysis The soil samples were digested with aqua regia to measure total Cu content (French standard NF X 31151; Afnor, 1994).: in brief, 0.5 g of soil was digested with 7.5 ml concentrated HCl+2.5 ml concentrated HNO3 for 12 h at room temperature and 2 h at 150 C, ®ltered and diluted to 100 ml with distilled water. Four other methods were used for evaluating soil available Cu: 1. EDTA extraction (French standard NF X 31-120; Afnor, 1994): 7.5 g of soil were extracted with 50

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153

Table 1 Description of the 29 plots studied Plot no.

Topography

Plant cover and agricultural practices in January 1996

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Terraces Terraces Terraces Terraces Terraces Terraces Terraces Terraces Terraces Slight slope Slight slope Plain ®eld Slight slope Steep slope Steep slope Steep slope Steep slope Steep slope Steep slope Steep slope Slight slope Slight slope Slight slope Plateau Plateau Slight slope Slight slope Plateau Plateau

Vine (goblet, chemical weeding) Vine (trained, chemical+mechanical weed control within the row) Vine (goblet, chemical weeding) Vine (goblet, chemical weeding) Vine (trained, chemical+mechanical weed control within the row) Vine (goblet, chemical weeding) Vine abandoned for approximately 10 years Vine abandoned for 3 years Pine forest (control) Vine (goblet, mechanical weed control) Vine (goblet, mechanical weed control) Vine abandoned for 3 years Vine (goblet, mechanical weed control) Vine (goblet, mechanical weed control) Oak forest (control) Vine abandoned for approximately 10 years Vine abandoned for 14 years Vine (goblet, chemical weed control) Vine (goblet, chemical weed control) Vine (goblet, chemical weed control) Vine abandoned for 8 years Oak forest (control) Vine abandoned Vine pulled up Vine (goblet, chemical weed control) Vine pulled up 4 years before Vine (trained, mechanical weed control, organic farming since 1988) Vine (trained, mechanical weed control, organic farming since 1988) Pine forest (control)

ml of Na2-EDTA 0.01 M+CH3COONH4 1 M for 2 h at 20 C under stirring, prior to being ®ltered, 2. DTPA extraction (French standard NF X 31-121; Afnor, 1994): 10 g of soil were extracted with 20 ml of DTPA 0.005 M+TEA 0.1 M+CaCl2 0.01 M for 2 h at 20 C under stirring, prior to being ®ltered, 3. ammonium acetate extraction (French standard NF X 31-121; Afnor, 1994): 10 g of soil were extracted with 200 ml of CH3COONH4 1 M for 1 h at 20 C under stirring, prior to being ®ltered, 4. calcium chloride extraction (Lebourg et al., 1996): 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 ®ltered. Plant samples were wet digested according to Mench et al. (1992): 1 g of plant material was digested with 5 ml concentrated HNO3+10 ml H2O2 (30% v/v) for 12 h at room temperature, 50 min at 60 C, 35 min at 90 C, 35 min at 150 C and 150 min at 250 C. The ®nal volume was diluted to 100 ml with distilled water. Cu concentrations in soil extracts and plant digests were determined by ¯ame atomic absorption spectrometry (AAS) or inductively-coupled plasma atomic emission spectrometry (ICP) depending on concentrations.

Number of years since the last vine has been planted 45 years 36 years ? 52 years 16 years 106 years ? 100 years ? ? ? 100 years 4 years ? 30±35 years 50 years 50 years 93 years 80 years ? ? ? 40 years 9 years 18 years

2.5. Statistical analysis The relationship between extractable Cu and soil properties was analysed by simple or multiple linear regression (SAS, 1985). After estimation of the model parameters, the general conditions for model application had to be checked. The residuals were required to (1) be normally distributed, (2) con®rm the homoscedasticity hypothesis, and (3) be mutually independant (Tomassone, 1987). Residual normality was checked by the Shapiro±Wilk test (Shapiro and Wilk, 1965), the other hypotheses were checked visually by using the residuals/predicted values graph. 3. Results 3.1. Total contents in the upper layer of vineyard soil Comparing the upper layer of a vineyard soil with the upper layer of a woodland soil sampled nearby showed a systematic distinct increase in soil Cu content (Table 3). In vineyard soils, Cu contents ranged from 31 to 251 mg kgÿ1, versus 14 to 29 mg kgÿ1 in woodland soils (Plots 9, 15, 22 and 29).

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L.A. Brun et al./Environmental Pollution 102 (1998) 151±161

Table 2 Selected soil properties of the top 15 cm of soil of the 29 plots studied Plot no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Particle size (g kgÿ1)

Stones >2 mm (g kgÿ1)

30 170 30 10 110 130 90 160 100 660 610 590 460 700 650 630 660 450 460 640 410 460 360 430 550 550 470 550 640

pH pH H2O KCl

Clay <2 mm

Silt 2±50 mm

Sand 0.05±2 mm

266 173 190 187 224 249 169 240 279 182 216 87 169 215 152 175 309 327 358 262 341 211 303 265 50 102 345 143 57

588 292 514 548 485 435 441 367 582 402 389 236 380 405 443 405 424 475 494 441 395 423 232 157 85 219 140 141 136

146 535 296 265 291 316 390 393 139 416 395 677 451 380 405 420 267 198 148 297 264 366 465 578 865 679 515 716 807

The amount of Cu contained in the top 15 cm of the soil on a per ha basis was estimated assuming a bulk density of 1.5 and taking the percentage of gravel and stones into account. For a given total Cu content in sieved soil, the estimate of total Cu content per hectare in the upper layer would thus be higher for less stony soils. This was the case for calcareous soils which exhibited 300±400 kg haÿ1 (Table 3). The upper soil layer of plots planted with extremely old vines (about a century old, i.e. Plots 6, 8, 13 and 20) exhibited high Cu contents ranging from 127 to 177 mg kgÿ1, that is from 80 to 300 kg haÿ1. The plots which had been planted more recently, after clearing (Plots 14 and 17), showed little increase in Cu content compared with control soils (7 and 9 mg kgÿ1 for Plots 14 and 17, respectively). With the exception of these cases where the plot history was known, it was dicult to establish a link between vine age and Cu content. Cu treatments could have been applied for more than a century but it was not possible to ascertain the history of the plot over such a long period. The soil Cu content was thus observed without knowing the periods of application of Cu. For example, the plot which exhibited the highest

8.5 8.3 8.4 8.6 8.4 8.3 8.3 8.3 8.2 4.8 4.9 5.5 7.1 7.3 5.6 5.2 5.2 4.7 4.9 4.5 4.6 6.1 4.8 6.6 5.1 5.0 6.9 6.5 7.1

7.6 7.6 7.7 7.7 7.5 7.4 7.4 7.5 7.1 3.6 3.6 3.9 6.3 5.6 4.1 3.7 3.8 3.5 3.5 3.4 3.4 4.3 3.4 5.0 3.8 4.0 5.8 5.6 6.5

Organic matter (g kgÿ1)

CEC (cmolc kgÿ1)

Total CaCO3 (g kgÿ1)

17.5 24.9 17.5 14.6 14.1 16.7 32.9 24.3 28.4 15.8 15.7 16.7 18.4 12.7 22.9 18.7 10.0 14.8 15.1 23.2 9.8 44.7 12.7 13.1 16.9 13.4 6.0 15.5 36.3

12.9 11.0 9.9 9.6 11.8 12.6 12.4 14.7 16.6 8.9 10.9 6.0 10.0 9.2 10.1 9.1 11.0 10.9 14.4 13.3 12.1 11.7 12.4 10.2 3.7 4.2 12.2 5.9 6.7

319 322 278 252 299 321 276 258 95 <1 <1 <1 5 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1

Cu content (Plot 2) was planted with a fairly young vine which might, however, have replaced an old vine. 3.2. Cu accumulation in deep horizons The vertical distribution of Cu in calcareous soil (Plots 2 and 8) showed that Cu remained mostly concentrated in the topsoil (Fig. 1). For four Cu-rich, neutral to acidic soils (Plots 11, 13, 16 and 20), the Cu content of the 15± 30 cm soil horizon ranged between 60 and 80 mg kgÿ1 which was about twice that normally found in this type of soil. Plot 20 had a high content (56 mg kgÿ1) in the 45± 60 cm horizon. Two acid soils (Plots 24 and 26) showed distinct Cu enrichments at depth, with a peak in the 30± 45 cm horizon. In Plot 26, the Cu content at 45±60 cm was still large, close to that of the upper layer. Plots 17 and 23 showed little Cu enrichment in the topsoil and almost no Cu enrichment at depth. 3.3. Total and extractable Cu EDTA-extractable Cu amounted to 8±65% of total Cu, while DTPA-extractable Cu ranged from 5 to 52%

L.A. Brun et al./Environmental Pollution 102 (1998) 151±161

of total Cu. Ammonium acetate-extractable Cu amounted to 2±17% of total Cu whereas CaCl2-extractable Cu ranged from 0.1 to 7.2% of total Cu (Table 3). Both EDTA- and DTPA-extractable Cu were largely explained by the total Cu contents, as attested by their high correlation coecient (R=0.94 and R=0.93, respectively). They were also highly correlated to each other (R=0.99) (Table 4). Ammonium acetate-extractable Cu seemed less strongly correlated to total Cu content (R=0.66). Plot 26 exhibited a markedly higher value of CuNH4OAc than the other plots and did not conform to the same relationship between CuNH4OAc and total Cu (Fig. 2). This soil is an acid, sandy loam soil with a low CEC (CEC=4.2 cmolc kgÿ1) and a high level of total Cu (CuTotal=168 mg kgÿ1). In comparison, the calcium chloride-extractable Cu was neither correlated to total content nor to the values of EDTAor DTPA-extractable Cu (Table 4). 3.4. Extractable Cu and soil properties Extractable Cu was regressed against total Cu and soil variables that were assumed to a€ect the bioavailability of Cu to plants, i.e. pH, CEC and the level of OM (Delas, 1984; Gupta and Aten, 1993), in a multiple linear regression model. These variables were included

155

in the models if the probability associated to the variable was <0.05 (GLM procedure; SAS, 1985). Whatever the form of extractable Cu considered, the level of OM did not meet this requirement and was thus discarded from the regression. For EDTA- and DTPA-extractable Cu, the regression models accounted for 93 and 90% of the total variance, respectively (Tables 5 and 6). However, total Cu itself accounted for a major proportion of the variance in the regression. For a given total Cu content, the amounts of EDTA- and DTPA-extractable Cu decreased with increasing CEC. In addition, for EDTA-extractable Cu, soil pH was introduced in the linear regression model whereas it was not for DTPA-

Table 4 Linear correlation coecients between total Cu and the di€erent forms of extractable Cu for the 29 plots studied

CuTotal CuEDTA CuDTPA CuNH4OAc

CuEDTA

CuDTPA

CuNH4OAc

CuCaCl2.

0.94***

0.93*** 0.99***

0.66*** 0.77*** 0.81***

0.16ns 0.12ns 0.18ns 0.51**

ns, non signi®cant; *p<0.05; **p<0.001; ***p<0.0001.

Table 3 Total Cu, extractable Cu and total amount of Cu per ha in the tilled horizon (0±15 cm) of the 29 plots studied Plot no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

CuTotal (mg kgÿ1)

CuEDTA (mg kgÿ1)

CuDTPA (mg kgÿ1)

CuNH4OAc (mg kgÿ1)

CuCaCl2 (mg kgÿ1)

Cu/ha (kg haÿ1)

134 251 118 128 78 165 210 173 20 100 103 126 177 33 26 102 38 77 47 127 72 29 60 92 93 168 31 81 14

43.6 95.1 49.1 44.1 22.4 63.2 74.6 56.0 4.6 24.5 23.9 33.5 68.8 5.7 4.2 26.5 3.1 18.5 5.5 36.2 14.5 2.3 18.0 38.8 44.2 79.2 16.2 52.5 5.5

34.1 82.1 35.9 33.8 17.9 52.1 57.1 51.1 4.5 20.6 20.5 27.8 44.5 3.0 3.6 23.3 2.4 13.0 4.1 30.8 11.6 1.5 17.9 33.7 36.6 71.4 7.4 41.8 2.7

4.5 14.4 6.4 5.6 3.4 9.0 6.4 6.9 1.7 4.2 5.1 5.9 9.1 0.6 1.6 5.6 1.8 2.9 0.9 7.0 2.5 1.2 3.3 4.4 6.4 29.7 2.5 9.3 1.2

0.17 0.56 0.23 0.16 0.10 0.22 0.43 0.39 0.10 2.86 4.25 4.46 0.35 0.10 0.30 3.76 0.40 3.98 0.58 6.40 5.21 0.10 3.54 0.14 4.36 9.24 0.10 0.44 0.10

293 469 257 285 157 322 429 327 41 76 90 116 215 22 21 85 29 95 57 103 96 36 86 117 95 170 37 82 11

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L.A. Brun et al./Environmental Pollution 102 (1998) 151±161

Fig. 1. Vertical distribution of total Cu along soil pro®les of 10 vineyard plots.

L.A. Brun et al./Environmental Pollution 102 (1998) 151±161

157

Table 7 Linear regression of ammonium acetate-extractable Cu (its Log value, Ln CuNH4OAc) as a function of total Cu and cation exchange capacity (CEC) for the 29 plots studied Variable explained: Ln(CuNH4OAc)a Source of variation CuTotal CEC Total

Fig. 2. Ammonium acetate-extractable Cu as a function of total Cu for the topsoils of the 29 plots studied.

extractable Cu; when the other variables remained constant EDTA-extractable Cu increased with increasing pH (Table 5). For the regression models of ammonium acetateextractable Cu, the conditions of residual normality were not satis®ed. As variations in ammonium acetateextractable Cu increased with increasing total Cu (Fig. 2), a change of variable was carried out by considering the variable Ln(CuNH4OAc.) (Tomassonne, Table 5 Linear regression of EDTA-extractable Cu as a function of total Cu, cation exchange capacity (CEC) and soil pH for the 29 plots studied Variable explained: CuEDTA Source of variation CuTotal CEC pH Total

Degree of freedom

Sum of squaresa

F observed*

Pr > F*

1 1 1 28

12413.2 751.3 338.4 18578.1

222.86 13.49 6.08

0.0001 0.0011 0.0209

Degree of freedom

Sum of squaresb

F observed*

Pr > F*

1 1 28

13.69 1.62 20.63

73.62 8.69

0.0001 0.0067

CuTotalÿ0.0798 Regression equation: Ln(CuNH4OAc)=0.0116 CEC+1.08 (R2=0.77). a As the application conditions of the model were not satis®ed, a change of variable was carried out. b The sum of squares is that calculated when the variable is added last to the model.

1987). Application conditions of the model were then satis®ed (Table 7). The transformed variable Ln(CuNH4OAc) was again largely dependent on total Cu. The CEC was incorporated in the model: for a given total Cu content, ammonium acetate-extractable Cu increased with decreasing CEC (Table 7). The amount of CaCl2-extractable Cu depended mainly on soil pH. It decreased with increasing soil pH. Total Cu was also incorporated in the regression. It had, however, less in¯uence than soil pH, the sum of squares associated with total Cu variable being roughly three times less than that associated with soil pH (Table 8). This method of extraction discriminated a group of soils with extractable Cu contents above 2 mg kgÿ1. This group consists of all the acid soils (pH <6.4) that had been enriched in Cu. It was also observed that when the pH was >6.4, the CaCl2-extractable Cu was <0.6 mg kgÿ1 whether the soil had been enriched in Cu or not (Fig. 3). 3.5. Cu assimilated in situ by plants

Regression equation: CuEDTA=0.373 CuTotalÿ1.81 CEC+2.56 pHÿ0.957 (R2=0.93). a The sum of squares is that calculated when the variable is added last to the model.

Cu contents in plants sampled in situ were determined on 10 plots (Table 9). All the species studied (except

Table 6 Linear regression of DTPA-extractable Cu as a function of total Cu and cation exchange capacity (CEC) for the 29 plots studied

Table 8 Linear regression of calcium chloride-extractable Cu as a function of total Cu and soil pH for the 29 plots studied

Variable explained: CuDTPA

Variable explained: CuCaCl2

Source of variation CuTotal CEC Total

Degree of freedom

Sum of squaresa

F observed*

Pr > F*

1 1 28

11101.7 374.5 13049.4

213.71 7.21

0.0001 0.0125

Regression equation: CuDTPA=0.329 CuTotalÿ1.21 CEC+7.24 (R2=0.90). a The sum of squares is that calculated when the variable is added last to the model.

Source of variation CuTotal pH Total

Degree of freedom

Sum of squaresa

F observed*

Pr > F*

1 1 28

29.19 106.3 166.0

13.74 50.04

0.0010 0.0001

Regression equation: CuCaCl2=0.0179 CuTotalÿ1.36 pH+8.88 (R2=0.67). a The sum of squares is that calculated when the variable is added last to the model.

158

L.A. Brun et al./Environmental Pollution 102 (1998) 151±161

Table 9 Total Cu (mg kgÿ1) contained in the di€erent organs of nine species of wild plants harvested on 10 of the studied plots Species

Plant stage

Organs

Plot no. 2

Dactylis glomerata Poa annua Andryala integrifolia

End of tillering Flowering Rosette of leaves

Hypochoeris radicata

Rosette of leaves

Senecio vulgaris

Flowering

Sanguisorba minor

Rosette of leaves

Rumex acetosella

Bolting

Allium polyanthum

Leafy stage

Rubia peregrina

Adult aerial organs

Aerial part Aerial part Tap root Aerial part Tap root Aerial part Roots Aerial part Tap root Aerial part Rhizomes Aerial part Bulb Aerial part Stolons Aerial part

Fig. 3. Calcium chloride-extractable Cu as a function of total Cu for the topsoils of the 29 plots studied.

Sanguisorba minor) had the bulk of their root system in the top 15 cm of the soil. They could therefore be considered as good indicators of the bioavailability of Cu in the topsoil. In contrast, S. minor had a tap root growing down to a depth of one meter. This species then did not only absorb minerals from the upper layer and its Cu contents could therefore not be considered as representative of the bioavailability of Cu in the topsoil. Total contents observed at the below-ground organ level should be considered with caution. Despite careful washing of these organs, small amounts of soil might have remained attached to the roots and led to overestimating copper content in the plant tissue. In non-calcareous soils (Plots 11±26), the Cu contents observed in the aerial parts of the plants were always larger for asteraceae (Andryala integrifolia, Hypochoeris radicata and Senecio vulgaris) than for poaceae (Dacty-

8

14.9 14.7 9.81 11.3

37.4 22.6

12.3 3.80 17.5 4.53

11

13

11.4 12.2 19.1 14.4 17.5

10.9 18.0 17.5 10.2 11.2 28.3 19.8

5.50 10.2

16

17

7.46

6.67

11.1 16.2 16.5 14.1

17.0 32.7 9.16 7.80

4.56 9.44 9.91 12.8

20

37.4 20.0 16.2 21.6

23

24

12.2

3.54

11.2 12.4 10.8 14.2

26

9.66 14.7 12.6 26.6 7.40 8.48

13.1 9.01

5.34 9.78 71.0 16.0

lis glomerata and Poa annua). The largest Cu contents registered in the aerial parts were found in A. integrifolia (26.6 mg kgÿ1) growing on Plot 26 and Rumex acetosella (32.7 mg kgÿ1) on Plot 16 (Table 9). S. vulgaris, Allium polyanthum and Rubia peregrina were collected from both calcareous soils (Plots 2 and 8) and neutral to acidic soils (Plots 13, 16 and 20). In calcareous soil, Cu contents in the aerial parts were often smaller than those observed in other soils, with comparable amount of total Cu, except for R. peregrina (22.6 mg kgÿ1) in Plot 2 (Table 9). No heavy accumulation of Cu was found in roots, values being generally lower than 20 mg kgÿ1, except for S. vulgaris in Plot 13 (28.3 mg kgÿ1) and A. integrifolia in Plot 20 (37.4 mg kgÿ1). High Cu contents were found in the stolons of R. peregrina in Plot 20 (71 mg kgÿ1) and Plot 2 (37.4 mg kgÿ1; Table 9). 3.6. Extractable Cu and Cu assimilated by the plants The correlations between the di€erent forms of extractable Cu and the Cu assimilated by plants were calculated for A. integrifolia, H. radicata, R. peregrina and D. glomerata (Table 10). Except for the aerial parts of R. peregrina, the Cu extracted with 0.01M calcium chloride solution was often correlated with the contents of Cu found in the plants grown in situ. These correlations were large when only considering the Cu contents of the aerial parts of plants grown in acid to neutral soils (A. integrifolia, H. radicata and D. glomerata; Table 10). The correlations obtained for EDTA-, DTPA- and ammonium acetate-extractable Cu were generally weak (except for the aerial parts of A. integrifolia), as were those obtained for total Cu.

L.A. Brun et al./Environmental Pollution 102 (1998) 151±161

159

Table 10 Linear correlation coecients between the values obtained for soil Cu using the di€erent extraction methods and the amounts of Cu assimilated by the plants in situ

Cu A. integrifolia (Aerial part) Cu A. integrifolia (Tap root) Cu H.radicata (Aerial part) Cu H.radicata (Tap root) Cu R. peregrina (Aerial part) Cu R. peregrina (Stolons) Cu D. glomerata (Aerial part)

Number of observations

CuTotal

CuEDTA

CuDTPA

CuNH4OAc

CuCaCl2.

8 8 7 7 4 4 4

0.82** 0.50ns 0.10 ns 0.22 ns 0.61 ns 0.06 ns ÿ0.34 ns

0.76* 0.32 ns ÿ0.20 ns ÿ0.10 ns 0.63 ns 0.05 ns ÿ0.43 ns

0.82** 0.29 ns ÿ0.14 ns ÿ0.05 ns 0.57 ns 0.02 ns ÿ0.36 ns

0.85** 0.15 ns 0.18 ns 0.31 ns 0.83 ns 0.19 ns ÿ0.18 ns

0.84** 0.45 ns 0.92** 0.86** 0.07 ns 0.60 ns 0.76 ns

ns, non signi®cant; *p<0.05; **p<0.001.

The Cu contents observed in the aerial parts of A. integrifolia were highly correlated with the di€erent forms of extractable Cu and with total Cu. 4. Discussion 4.1. Accumulation of Cu in vineyard soils In the Mediterranean area studied, soils did not contain more than 250 mg kgÿ1 of total Cu, which is less than the values reported for northern, wine-making regions of France, with values reaching 400±500 mg kgÿ1 in vineyards of Alsace, Champagne or Burgundy (Drouineau and Mazoyer, 1962; Flores Velez, 1996) and as much as 800 mg kgÿ1 in the Bordeaux area (Delas, 1963). Such di€erences can be attributed to the dry climatic conditions occuring in Mediterranean areas which are less favorable to the development of vine downy mildew and are responsible for lesser applications of Bordeaux mixture (Geo€rion, 1975). In Italy, a study carried out on 43 plots demonstrated that the Cu contents of vineyard soils were on average 297 mg kgÿ1 in the wet mountainous regions of the north where mildew fungicidal treatments are frequent, 200 mg kgÿ1 on the wet plains and only 75 mg kgÿ1 in the dry areas of the south where fungicidal treatment is much less frequent (Deluisa et al., 1996). The French standard NF U 44-041 (Afnor, 1985) states a maximal, acceptable soil Cu content at 100 mg kgÿ1. Above this value, soil cannot be used for application of sewage sludge. In the present study 14 soils, i.e. more than half of the 25 vineyard soils studied, had a total Cu content above this standard. If we consider according to French standard NF U 44-041 that the soil pH after sewage sludge application should exceed 6, only 4 plots (5, 14, 24 and 27) would be suitable for sewage sludge application. Three major processes contribute to the redistribution of anthropic Cu in the soil: soil erosion and leaching (Yingming and Corey, 1993), and deep ploughing

before replanting vines which can extend the contamination of topsoil to a depth of 40 cm (Flores Velez, 1996). Grape harvesting only removes a tiny fraction of the Cu applied by spraying while leaf fall restores the rest (Flores Velez, 1996). Plot 8 is a vine dating back to the last century. In this calcareous soil, no deep ploughing had occurred since Cu was applied and therefore no Cu enrichment was found in the soil below 15 cm depth. It seems that in such conditions Cu remained strongly ®xed in the topsoil and no leaching occured. In this plot, total Cu in the topsoil amounted to approximately 300 kg haÿ1, which suggests that over the last century Cu application was about 3 kg haÿ1 yearÿ1 on average. Surprisingly though, a single application of Bordeaux mixture introduces 3 to 5 kg haÿ1 of Cu and between three and 10 applications of Bordeaux mixture were applied annually until the 1970s. This would correspond to applications of 1500 to 3000 kg haÿ1 since the end of the last century (Geo€rion, 1975). The bulk of Cu applied to vineyard plots was therefore not recovered in the soil and must have been removed from the plot by soil erosion. Such a phenomenon of erosion driven removal of Cu (and other heavy metals) has been frequently reported after application of polluted sludge (McGrath and Lane, 1989; Juste and Mench, 1992; Yingming and Corey, 1993). Two plots (24 and 26) exhibited higher Cu concentrations in the subsoil than in the topsoil. As the vines in these plots had been pulled out and replanted several times during the last century, the enrichment was likely to be due to burial of enriched topsoil deep in the soil pro®le. On sludge-amended soils, Miner et al. (1997) also concluded that leaching of Cu was limited and that the Cu accumulation in deep horizons was the result of sludge injection depth. 4.2. Cu bioavailability As total Cu in the soil provides little information on copper bioavailability, the high correlations between

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total Cu and EDTA- and DTPA-extractable Cu suggested that these extraction methods have little interest for predicting the availability of Cu to plants. The same conclusion was made by Merry et al. (1983) in orchard soils with Cu concentrations similar to those observed in vineyard soils in the present study. They observed strong correlations between total Cu and EDTA-extractable Cu (0.95
present study. In Cu contaminated soil, Gupta and Aten (1993) also concluded that unbu€ered salt solutions were providing the best results for estimating the transfer of soil Cu into plants. Acknowledgements The authors thank the French Environment Agency (Ademe) for funding this research project (convention 9675020), Mrs I. Feix and Mr T. Sterckeman for comments on the manuscript, and Mr P. Hinsinger for reviewing this manuscript. References Afnor, 1985. Norme franc,aise NF U 44-041. MatieÁres fertilisantes. Boues des ouvrages de traitement des eaux useÂes urbaines. DeÂterminations et speÂci®cations. Association franc,aise de normalisation, Paris. Afnor, 1994. Qualite des sols. MeÂthodes d'analysesÐRecueil de normes franc,aises. Association franc,aise de normalisation, Paris. Alva, A.K., Graham, J.H., Tucker, D.P.H., 1993. Role of calcium in amelioration of copper phytotoxicity for citrus. Soil Science 155, 211±218. Aoyama, M., Nagumo, T., 1996. Factors a€ecting microbial biomass and dehydrogenase activity in apple orchard soils with heavy metal accumulation. Soil Sci. Plant Nutr. 42, 821±831. Barona, A., Romero, F., 1997. Relationships among metals in the solid phase of soil and in wild plants. Water, Air and Soil Pollution 95, 59±74. Bon®ls, P., 1984. Les sols acides du versant meÂridional de la montagne noire. ReÂgion de Saint-Chinian, FaugeÁres et CabrieÁres. Le ProgreÁs Agricole et Viticole 101, 102±108. Bon®ls, P., 1993. Carte peÂdologique de France aÁ 1/100000: LodeÁve. INRA (Institut National de la Recherche Agronomique), Paris. Delas, J., 1963. La toxicite du cuivre accumule dans les sols. Agrochimica 7, 258±288. Delas, J., 1984. Les toxiciteÂs metalliques dans les sols acides. Le ProgreÁs Agricole et Viticole 101, 96±102. Deluisa, A., Giandon, P., Aichner, M., Bortolami, P., Bruna, L., Lupetti, A., Nardelli, F., Stringari, G., 1996. Copper pollution in Italian vineyard soils. Commun. Soil Sci Plant Anal. 27, 1537±1548. Drouineau, G., Mazoyer, R., 1962. Contribution aÁ l'eÂtude de la toxicite du cuivre dans les sols. Ann. Agron. 13, 31±53. Flores Velez, L.M., 1996. Essai de speÂciation des meÂtaux dans les sols: cas du Cu dans les vignobles. Ph.D. thesis, Universite Paris XII, Val-de-Marne. Flores Velez, L.M., Ducaroir, J., Jaunet, A.M., Robert, M., 1996. Study of the distribution of copper in an acid sandy vineyard soil by three di€erent methods. European Journal of Soil Science 47, 523±532. Geo€rion, R., 1975. L'alteÂration des terres aÁ vigne par une longue reÂpeÂtition des traitements aÁ base de cuivre et de soufre. PhytomaÐ DeÂfense des cultures 267, 14±16. Gupta, S.K., Aten, C., 1993. Comparison and evaluation of extraction media and their suitability in a simple model to predict the biological relevance of heavy metal concentrations in contaminated soils. Intern. J. Environ. Anal. Chem. 51, 25±46. Haq, A.U., Bates, T.E., Soon, Y.K., 1980. Comparison of extractants for plant-available zinc, cadmium, nickel, and copper in contaminated soils. Soil Sci. Soc. Am. J. 44, 772±777. Juste, C., 1988. Appreciation de la mobilite et de la biodisponbilite des eÂleÂments en trace du sol. Science du Sol 26, 103±112.

L.A. Brun et al./Environmental Pollution 102 (1998) 151±161 Juste, C., Mench, M., 1992. Long-term application of sewage sludge and its e€ects on metal uptake by crops. In: Adriano, D.C. (Ed.), Biogeochemistry of trace metals. Lewis, Boca Raton, USA, pp. 159±193. Juste, C., Chassin, P., Gomez, A., LineÁres, M., Mocquot, B., 1995. Les micro-polluants meÂtalliques dans les boues reÂsiduaires des stations d'eÂpuration urbaines. Ademe, Angers, France. Lebourg, A., Sterckeman, T., Ciesielski, H., Proix, N., 1996. InteÂreÃt de di€eÂrents reÂactifs d'extraction chimique pour l'eÂvaluation de la biodisponibilite des meÂtaux en trace du sol. Agronomie 16, 201±215. Lexmond, Th.M., 1980. The e€ect of soil pH on copper toxicity to forage maize grown under ®eld conditions. Neth. J. agric. Sci. 28, 164±183. Lindsay, W.L., Norvell, W.A., 1978. Development of a DTPA soil test for zinc, iron, manganese, and copper. Soil Sci. Soc. Am. J. 42, 421±428. McGrath, S.P., Lane, P.W., 1989. An explanation for the apparent losses of metals in a long term ®eld experiment with sewage sludge. Environ. Pollut. 60, 235±256. Mench, M., Juste, C., Solda, P., 1992. E€ets de l'utilisation de boues urbaines en essai de longue dureÂe: accumulation par les veÂgeÂtaux supeÂrieurs. Bull. Soc. Bot. Fr. 139, 141±156.

161

Merry, R.H., Tiller, K.G., Alston, A.M., 1983. Accumulation of copper, lead and arsenic in some Australian orchard soils. Aust. J. Soil Res. 21, 549±561. Miner, G.S., Gutierrez, R., King, L.D., 1997. Soil factors a€ecting plant concentrations of cadmium, copper, and zinc on sludgeamended soils. J. Envir. Qual. 26, 989±994. O'Connor, G.A., 1988. Use and misuse of the DTPA soil test. J. Envir. Qual. 17, 715±718. SAS, 1985. SAS Procedures Guide for Personal Computers, Version 6 Edition. SAS Institute Inc., Cary NC. Shapiro, S.S., Wilk, M.B., 1965. An analysis of variance test for normality. Biometrika 52, 591±611. Singh, S.P., Rakipov, N.G., 1988. E€ects of Cd, Cu and Ni on barley and their removal from the soil. Intern. J. Environmental Studies 31, 291±295. Tomassone, R., 1987. Comment interpreÂter les reÂsultats d'une reÂgression lineÂaire? Institut Technique des CeÂreÂales et des Fourrages, Paris. Yingming, L., Corey, R.B., 1993. Redistribution of sludge-borne cadmium, copper, and zinc in a cultivated plot. J. Envir. Qual. 22, 1±8.