Published online 24 October 2007
Published in J Environ Qual 36:1760-1764 (2007)
DOI: 10.2134/jeq2006.0476
© 2007 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
TECHNICAL REPORTS
Ecological Risk Assessment
Tolerance (PICT) of the Bacterial Communities to Copper in Vineyards Soils from Spain
M. Díaz-Raviñaa,*,
R. Calvo de Antab and
E. Bååthc
a Instituto de Investigaciones Agrobiológicas de Galicia (CSIC), Avda Vigo s/n, Apartado 122, E-15780 Santiago de Compostela, Spain
b Universidad de Santiago de Compostela, Departamento de Edafología y Química Agrícola, Facultad de Biología, 15782 Santiago de Compostela, Spain
c Dep. of Microbial Ecology, Lund Univ., Ecology Building, SE- 223 62 Lund, Sweden
* Corresponding author (mdiazr{at}iiag.cesga.es).
Received for publication November 2, 2006.
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ABSTRACT
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To detect effects of Cu pollution, the Cu tolerance of soil bacterial communities extracted from several vineyards located in NW Spain was measured. Bacterial community tolerance was estimated by means of the thymidine (TdR) and leucine (Leu) incorporation techniques using either IC50 values (the log of the metal concentration that reduced incorporation to 50%) or the percentage of activity at one specific Cu concentration (10–6 mol L–1). The tolerance measurements by the TdR incorporation technique were similar to those obtained by the Leu incorporation method, indicating that the two methods were equivalent in terms of suitability for detecting the toxicity of Cu to soil bacterial communities. The two tolerance indices considered (IC50 values and percentage of activity) were closely correlated (r = 0.975, P < 0.001), showing that both were equally good in measuring Cu tolerance of the bacterial community. An increased bacterial community tolerance to Cu, indicating a pollution effect, was observed in vineyard soils with more than 100 mg Cu kg–1 soil. Thus, the long-term use of Cu in vineyards has a toxic effect on the soil bacterial community, resulting in an increased tolerance. An effect of increased levels of Cu could not be detected when measuring bacterial community activity, pointing to the increased sensitivity to detect toxicity in field studies using tolerance measurements.
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INTRODUCTION
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HEAVY metals are a serious threat to soil quality due to their toxicity and persistence after entering the soil. Risk assessment associated with heavy metal–polluted soils should therefore be evaluated to preserve the environment. The use of analytical chemistry techniques can indicate exposure, but can be questioned because of the difficulties in determining the "bioavailability" of metals in soils. Bioassays using single species will overcome this problem, but might not always be relevant for the particular environment and might not include the most sensitive species. Thus, there is a need to use bioassays that reflect the actions of many species in the microbial community of a soil and that allow an integrative evaluation of ecotoxicological effects of contaminated soils (Boivin et al., 2002).
A number of microbiologically mediated processes (e.g., C and N transformations) and groups of microorganisms (denitrification, nitrification, and N-fixing bacteria) have been suggested as bioindicators when screening for heavy metal toxicity in terrestrial ecosystems (Bååth, 1989; Brookes, 1995; Wuertz and Mergeay, 1997). However, although these methods can detect toxicity, the drawback is that they are sensitive to other changes in the environment. Such confounding effects make it difficult to draw firm conclusions on effects of heavy metals in field studies. One way of overcoming the problem with confounding environmental factors is to measure PICT (pollution-induced community tolerance) (Blanck, 2002). This involves measuring the tolerance of a community to a toxicant, where an increased tolerance indicates that the compound has exerted a toxic effect on the community. This also circumvents the necessity of measuring available metal concentrations, since increased tolerance per se is an indication of earlier toxicity. The ratio of metal tolerant to sensitive bacteria determined by plate counts (Jansen et al., 1994) was an initial attempt to use the PICT concept for metal pollution in soil and more recently the bacterial community tolerance, measured by either plate counts (Olson and Thornton, 1982; Huysman et al., 1994; Kelly et al., 1999), the thymidine and leucine incorporation techniques (Bååth, 1992b; Pennanen, 2001; Shi et al., 2002; Almås et al., 2004), or Biolog plates (Rutgers et al., 1998; Van Beelen et al., 2004), have been successfully employed as monitoring tools to detect heavy metal effects.
In Mediterranean countries, the prolonged use of copper-based fungicides to combat plant diseases can result in copper accumulation in vineyards soils far in excess of the trace amounts that are required for healthy plant growth, causing harmful effects on soil organisms. The present work is the first attempt to determine Cu tolerance measurements in vineyards located in Galicia (NW Spain) and thus to assess the risk of soil contamination affecting soil microorganisms. This was made by measuring PICT of the bacterial community to Cu using the thymidine and leucine incorporation techniques in several vineyard soils with different soil Cu contents.
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Material and Methods
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Soils
The soil samples used were collected from the 0- to 20-cm top layer of 21 vineyards of different ages located in Galicia (NW Spain) that have been treated with copper salts to combat fungal disorders (samples 1–21). Additionally, an agricultural control soil non-affected by anthropogenic impact of copper was sampled in the same area to estimate the background soil properties and copper concentration (sample 22). The soils samples were sieved (2 mm) and kept in cold storage (4°C) for no longer than 3 mo. Organic matter content (loss on ignition) and pH (soil/water ratio of 1:2.5) were determined in triplicate for each sample as described by Guitián-Ojea and Carballas (1976). The total copper concentration in all dried samples was determined by flame atomic absorption spectrometry after HNO3/HF/HCl digestion in a microwave (ZEM, MDS-810) (Hossner, 1996). Bacterial activity and tolerance measurements were measured at two occasions using soil samples stored for 2 and 3 mo. No major differences between the two occasions were found and thus the mean values of two measurements are reported.
Bacterial Activity Measurements
Bacterial activity was simultaneously determined by the incorporation of thymidine (TdR) and leucine (Leu) into bacteria, following the procedure previously described (Bååth, 1992a, 1994). A 10 g portion of soil was homogenized with 200 mL distilled water, centrifuged at 750 g for 10 min and the supernatant was collected. The extracted bacterial suspension (2 mL) was incubated with 100 nmol L–1 [3H]-thymidine (925 Gbq mmol–1, Amersham, UK) and 395 nmol L–1 L-[U14–C] Leucine (11.9 Gbq mmol–1, Amersham, UK) at 20°C for 2 h and then the reaction was stopped by adding 1 mL of 5% formalin. Filtration, washing of filters, and scintillation counting were performed as described by Bååth (1994).
Tolerance Measurements
Bacterial community tolerance to Cu was determined using both thymidine and leucine incorporation rates as previously described (Bååth, 1992b; Díaz-Raviña and Bååth, 1996b). Bacteria were first extracted from soil by homogenization-centrifugation as described above. The bacterial solutions (1.8 mL) thus obtained were amended with different amounts of Cu solutions added as CuSO4 (0.2 mL) in a range of concentrations (10–8 to 10–4 mol L–1, final concentration) where the highest concentration gave almost total inhibition of incorporation rates. These suspensions were incubated with thymidine and leucine and the incorporated radioactivity was measured. The data were expressed as a percentage of the treatment with no metal added (distilled water control). The logarithm of the concentration (mol L–1) resulting in 50% inhibition (IC50) of the thymidine and leucine incorporation was then determined by plotting the percentage of inhibition against the log metal concentration and calculating the IC50 values from the slope of the decreasing linear part. Community tolerance was also evaluated in a more simple way as percent of activity in the presence of 10–6 mol L–1 Cu (as CuSO4) to that in the presence of no added Cu. The latter data were also used to calculate EC20 values (effective concentration of soil Cu giving a 20% change in tolerance) using a logistic model with the soil Cu concentrations logarithmically transformed.
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Results and Discussion
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Physical and chemical properties varied considerably between the samples (Table 1
). The pH(H2O) of the vineyard soils ranged from 4.9 to 7.1 and the organic matter from 58.1 to 144.8 g kg–1. The values of the control soil (pH of 5.4 and organic matter of 61.4 g kg–1) were within the range for vineyard soils. The total copper concentration in the control soil was 5 mg kg–1, which is within the range reported for different natural soil types (Calvo de Anta, 1997; Kabata-Pendias and Pendias, 2001). Vineyard soils showed values ranging from 35 to 550 mg kg–1; in half of the soil samples copper concentration exceeded 100 mg kg–1. Some of these values are higher than those earlier reported from vineyards more than 100 yr old, located in the same area (17–301 mg total Cu kg–1) (Arias et al., 2004). Since maximum permitted levels in the EU range from 50 to 150 mg kg–1, with lower levels permitted in acid soils, most of the soils studied here can be considered Cu polluted (Table 1). A positive significant correlation between copper concentration and organic matter was observed (r = 0.593, P < 0.005, n = 22). Arias et al. (2004) also found a similar relationship between these variables in the soils mentioned above, which is consistent with the recognized affinity of copper for organic matter (Senesi et al., 1989; Ponizovsky et al., 1999).
Bacterial activity varied considerably between vineyard samples but were not correlated to the total copper content (Fig. 1
, only TdR incorporation values are shown). Thymidine (TdR) and Leu (Leu) incorporation into cold-acid insoluble material (total macromolecules) of bacteria extracted from unpolluted control was 8.8 x 10–14 mol TdR h–1 mL–1 and 1.37 x 10–13 mol Leu h–1 mL–1, respectively. Although bacterial growth rates determined with the thymidine and leucine incorporation techniques have been found to be sensitive to heavy metal pollution in short-term experiments (Rajapaksha et al., 2004), this will not necessarily be the case for long-term experiments in the laboratory (Díaz-Raviña and Bååth, 1996c; Rajapaksha et al., 2004) or under field conditions (Bååth et al., 2005). In the former case this will be due to the selection of tolerant bacteria growing on fresh substrate from dead bacteria in the polluted soils and in the latter case confounding effects from other variables affecting bacterial growth rates might also make it difficult to register inhibitory effects.
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Fig. 1. Thymidine (TdR) incorporation in vineyard soils with different total Cu content (mean of the two measurement occasions ± SE).
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There was a good correlation between the IC50 obtained at the two measurement occasions using either thymidine or leucine incorporation techniques (r = 0.933, P < 0.001, n = 22 and r = 0.909, P < 0.001, n = 22 for TdR and Leu, respectively). Thus, IC50 values calculated by means of the TdR incorporation technique (first measurement –6.52 mol L–1 for the unpolluted soil and –6.11 ± 0.25 mol L–1 for the vineyard soils; second measurement –6.46 mol L–1 for the unpolluted soil and –6.13 ± 0.22 mol L–1 for the vineyard soils) were also similar to those obtained by the Leu incorporation method (first measurement –6.42 mol L–1 for the unpolluted control and –6.13 ± 0.29 mol L–1 for the vineyard soils; second measurement –6.54 mol L–1 for the unpolluted soil and –6.10 ± 0.22 mol L–1 means of vineyard soils). According to simple regression analysis, IC50 calculated by the TdR and Leu incorporation techniques were closely related (r = 0.977, P < 0.001, n = 22, Fig. 2
). Thus, both techniques had equal potential for estimating metal tolerance of the bacterial community. This is consistent with studies on the effect of diverse toxic substances on bacterial assemblages from water (Riemann and Lindgaard-Jörgensen, 1990; Tubbing, 1993). Likewise, similar IC50 values were also reported for the two methods in a previous study performed with soil bacterial communities from an agricultural soil contaminated artificially in the laboratory (Díaz-Raviña and Bååth, 1996b).
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Fig. 2. Bacterial community tolerance (PICT) to Cu measured by the thymidine (TdR) and leucine (Leu) incorporation techniques in vineyard soils with different total Cu content (r = 0.977, n = 22).
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Tolerance measurements on the basis of percentage of control activity in samples with 10–6 mol L–1 Cu were also similar independently if Leu or TdR incorporation methods were used (data not shown, r = 0.975, P < 0.001, n = 22). This resulted in a strong correlation between the estimated community tolerance, using percentage of activity and the IC50 values (r = 0.975, P < 0.001, n = 22 for TdR and r = 0.988, P < 0.001, n = 22 for Leu) (Fig. 3
, only TdR data are shown). This indicated that the two methods were equivalent in terms of suitability for measuring tolerance. In the routine short-term toxicity test used to calculated IC50 values, six concentrations of Cu were used. The use of only one metal concentration, as in the present study (10–6 mol L–1 Cu), to compare with a control sample was thus less laborious and time consuming and reduced the cost of the analyses considerably. However, considering the biological interpretation of the two measurements as well as the errors associated with the determinations, IC50 values are probably preferable. Nevertheless, when processing a large amount of samples the use of tolerance measurements on the basis of only one specific metal concentration can be very useful as a first approximation for monitoring heavy metal pollution effects.
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Fig. 3. Relationship between two indices of bacterial community tolerance (PICT) to Cu, IC50 or percentage of control values when 10–6 mol L–1 Cu was added (100% inhibition), measured using the thymidine incorporation technique (r = 0.975, n = 22).
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Higher Cu tolerance of the bacterial community was found in soil samples with higher Cu pollution levels, while the unpolluted soil showed similar Cu tolerance levels to vineyard soils with low Cu concentrations (Fig. 4
). This relationship, however, has to be interpreted with some caution since IC50 values could differ between soils due to, for example, different soil pH, organic matter contents, or bacterial community composition. Such problems have also been encountered in aquatic systems (Blanck et al., 2003). This might also be the reason for bacteria from two soils (soil 5 and 6) having higher tolerance to Cu than other soils with similar pollution levels. On the other hand, IC50 values obtained here for bacterial communities in the unpolluted soil were similar to those obtained for bacterial communities from an agricultural sandy loam soil from southern Sweden (4.5% organic matter, pH 7.8, IC50 for Cu –6.48 mol L–1) (Díaz-Raviña et al., 1994), indicating a similar sensitivity of bacterial communities to Cu despite of differences in soil characteristics.
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Fig. 4. Bacterial community tolerance (PICT) to Cu measured using the thymidine incorporation technique, as determined by IC50 (A) or percentage of control values when 10–6 mol L–1 Cu was added (100% inhibition) (B), in vineyard soils with different total Cu content (mean of the two measurement occasions ± SE).
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In our study, two main soil groups could be distinguished on the basis of the ability of bacteria to tolerate Cu; one group was formed by the unpolluted soil and nine soils with total Cu concentration lower than 100 mg kg–1, and the second group was formed by 12 vineyard soils: 10 soils with total Cu concentration higher than 100 mg kg–1 and samples 5 and 6 that exhibited a higher tolerance than expected from total Cu concentration (55–60 mg Cu kg–1) (Fig. 4). An EC20 value (effective concentration of soil Cu giving a 20% change in tolerance) of 92 mg Cu kg–1 soil could also be calculated, using a logistic model and the data in Fig. 4B but excluding these two soils (r2 = 0.459). Within each group, despite a wide range of Cu pollution (5–100 mg kg–1 and 100–550 mg kg–1 for the first and second group, respectively), no clear differences in Cu tolerance were observed. This can be explained on the basis of the Cu availability of the different soils studied since total Cu content does not reflect the effective Cu concentration present in soil solution that exerts an influence on soil bacterial communities. Saeki et al. (2002) reported that soil solutions with high Cu and Zn concentrations were not necessarily found in the soils containing the largest amounts of total metals. This is also consistent with the studies of Almås et al. (2004), who found that the correlation of IC50 values for Cd and Zn, determined by the thymidine incorporation technique, and the metal concentration were much poorer for the total metal pool (aqua regia) than for the labile pools (pore water, effective concentration), indicating that total metal concentrations are inaccurate for predicting biological effects. It may be pointed out that this relationship can improve considerably when samples from the same soil type are considered; thus, in previous studies performed under laboratory and field conditions, a linear or exponential increase of tolerance measurements with the total soil metal pollution content was observed (Díaz-Raviña et al., 1994; Díaz-Raviña and Bååth, 1996a; Bååth et al., 2005).
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Conclusions
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Our results showed that increased bacterial community tolerance to Cu, indicating a pollution effect, was observed in vineyards exposed to Cu pollution. Although the use of a full range of metal concentrations to determine IC50 values was preferable, the close correlation to the result with a more simple method calculating percentage activity using only one level of metal addition would make it possible to easily process a great number of soils in a short time. Further experiments including both tolerance assays of soil bacterial communities to Cu by means of the TdR and Leu incorporation techniques and chemical analyses of Cu distribution in soils (speciation or bioavailability) by sequential extraction techniques should be conducted in a wide range of vineyard soils to confirm these preliminary data and determine the usefulness of these measurements as monitoring tools in risk assessment studies.
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ACKNOWLEDGMENTS
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This study was supported by grants from Ministerio Español de Educación y Ciencia to M. Díaz-Raviña and from the Swedish Research Council to E. Bååth.
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NOTES
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All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher.
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ABSTRACT
INTRODUCTION
