Soil moisture pre-treatment effects on enzyme activities as indicators of heavy metal-contaminated and reclaimed soils

Soil Biology & Biochemistry 36 (2004) 1559–1568 www.elsevier.com/locate/soilbio

Soil moisture pre-treatment effects on enzyme activities as indicators of heavy metal-contaminated and reclaimed soils M. Bele´n Hinojosaa,*, Jose´ A. Carreiraa, Roberto Garcı´a-Ruı´za, Richard P. Dickb a

Departamento de Biologı´a Animal, Vegetal y Ecologı´a, Facultad de Ciencias Experimentales, Universidad de Jae´n, Campus Las Lagunillas s/n, 23071 Jae´n, Spain b Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331-7306, USA Received in revised form 23 April 2004

Abstract Heavy metal contamination can inhibit soil functions but it is often difficult to determine the degree of pollution or when soil reclamation is complete. Enzyme assays offer potential as indicators of biological functioning of soils. However, antecedent water content of soil samples may affect the outcome of biological measurements. In Mediterranean regions, for much of the year ‘field moist’ surface soil can have water content similar to that of air-dry samples. The objectives of this study were to: (1) determine the sensitivity of a range of enzyme assays to detect the degree of pollution from a heavy metal mine spill; (2) evaluate rewetting field-dry soil as a pre-treatment for enzyme assays; and (3) test multivariate analysis for improving discrimination between polluted, reclaimed and non-polluted soils. The Aznalco´llar mining effluent spill provided a unique opportunity to address these objectives. This accident released toxic, heavy metal-contaminated (As, Bi, Cd, Cu, Pb, Tl, Zn.) and acid tailings into the Guadiamar watershed (SW Spain) in 1998, severely affecting the riparian zone along more than 4000 ha. Contaminated soils were collected from the highly polluted upper watershed and less polluted lower watershed along with reclaimed soil at both sites. Enzyme activities (phosphatases, arylsulfatase, b-glucosidase, urease and dehydrogenase) were assessed on both field-moist samples and soils rewetted to 80% of water-holding capacity and then incubated at 21 8C for 7 d prior to the assay. The reclaimed soils had higher activities than polluted soils but, typically, 1.5–3 times lower levels of activity than the non-polluted soil. Regardless of the moisture pre-treatment, all enzymes showed significant effects due to pollution, with urease and b-glucosidase showing the greatest discrimination between degrees of contamination. In general, rewetting field-dried soils increased activities on non-polluted and reclaimed soils which improved discrimination with polluted soils. Another method to increase the potential of soil enzyme activities to detect soil contamination could be to combine them in multivariate analysis, which provides a more holistic representation of the biochemical and microbial functionality of a soil. q 2004 Elsevier Ltd. All rights reserved. Keywords: Enzyme activities; Xeric moisture regime; Mediterranean region; Heavy metals; Soil pollution; Rewetting pre-treatment

1. Introduction Soil enzyme activities can be sensitive and early indicators of both natural and anthropogenic disturbances (Nannipieri, 1994; Dick, 1997; Giller et al., 1998). As Brookes (1995) pointed out, applications of European Community standards for heavy metal concentrations result in significant negative impacts on microbial biomass and activity, indicating the greater sensitivity of the soil to these impacts in comparison with plants or animals. * Corresponding author. Tel.: C34-953-212551; fax: C34-953-212141 E-mail address: [email protected] (M.B. Hinojosa). 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.07.003

Important questions are what constitutes a significant environmental impact on soils and when is reclamation complete. Indicators are needed, not only as surrogates for reflecting the functionality of soils, but also to guide reclamation. The soil microbial component and soil enzyme activities are attractive as indicators for monitoring disturbance or pollution of soils because of their central and crucial role in the functions of the soil ecosystem. Most studies on heavy metals have been carried out with soils incubated in the laboratory. In studies where a wide range of trace elements have been tested individually, Hg, Ag, Cr, and Cd have generally caused the greatest inhibition in enzyme assays (Frankenberger and Tabatabai, 1981,

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1991a,b; Eivazi and Tabatabai, 1990; Deng and Tabatabai, 1995). However, as reported in a recent review by Speir and Ross (2002), there is relatively little data from long-term studies (with no reports at all from Mediterranean regions) on enzyme responses to heavy metal contamination caused by chronic industrial pollution or acute spills under field conditions. On the other hand, different results for biological assays might be expected, depending on soil moisture status. This is reflected in the work of Brynhildsen et al. (1988) who found that stress conditions, such as dry soils, caused metal uptake by microorganisms in their energy driven transport system to be less toxic. Sampling, pre-treatment and storage conditions must be considered when soil enzyme assays are conducted. It is generally recommended that soils should be assayed immediately after sampling in the field (Anderson, 1987; ¨ hlinger, 1995; Dick et al., 1996) and not allowed to freeze, O dry out or become waterlogged during storage (ISO 10381-6, 1993). These general recommendations were developed on soils with udic moisture regimes in temperate regions (Anderson, ¨ hlinger, 1995). The udic moisture regime is common 1987; O to soils under climates that have: a relatively low seasonal variability in rainfall (Soil Survey Staff, 1999); enough rain in summer so that the amount of stored moisture plus rainfall is approximately equal to, or exceeds, the amount of evapotranspiration; and adequate winter rains to recharge the soils. Conversely, soils of Mediterranean ecosystems have a xeric moisture regime and typically experience rapid rewetting and drying cycles that have low predictability and high variability depending on the intensity of rainfall events (Soil Survey Staff, 1999). Thus, to develop protocols depending of enzyme activities as soil quality indicators of xeric Mediterranean soils, there is need to determine whether soil moisture levels affect the sensitivity of the assays for detecting heavy metal-pollution in soils. This also has implications for adoption of soil quality indicators by commercial analytical laboratories that, for convenience, prefer to use air-dried soils. Previous research has shown that enzyme assays can effectively discriminate between different types of tillage and organic matter management using air dried soils (Bandick and Dick, 1999; Ndiaye et al., 2000). However, there is no information on rewetting field-dry soils as a pre-treatment to standardize moisture levels prior to enzyme assays in heavy metal-contaminated soils. The objectives of this study were to: (1) determine the sensitivity of a range of enzyme assays to detect the degree of contamination of polluted and reclaimed soils, 4 years after an industrial heavy metal mine spill; (2) evaluate rewetting field-dry soil as a pre-treatment for enzyme assays on soil collected in a xeric moisture regime; and (3) test multivariate analysis for improving discrimination between polluted, reclaimed and non-polluted soils. The study was carried out at sites affected by the Aznalco´llar mining effluent spill that released toxic, acid

and heavy metal-contaminated tailings into the Guadiamar river basin (SW Spain). The Aznalco´llar ore was processed by grinding and treating the particles with SO2 followed by separation of Cu, Pb and Zn by adjusting solution pH to selectively float and remove each metal (Simo´n et al., 1999). The residues from this process were stored in a very large dammed pond. In 1998, the dam broke and approximately 36!105 m3 of polluted water and 9!105 m3 of toxic tailings spilled into the Guadiamar river basin. The tailings spread about 50 km down-river, affecting 4634 ha of alluvial soils. A general overview of the accident can be found in Grimalt et al. (1999), Gallart et al. (1999), Alastuey et al. (1999) and Lo´pez-Pamo et al. (1999). This watershed provided a unique opportunity to study a long-term, realworld pollution site in a Mediterranean environment and to determine the potential of enzyme assays to detect and assess the impact of heavy metal contamination in soils.

2. Material and methods 2.1. Study sites The Aznalco´llar mine spill in the Guadiamar river watershed is shown in Fig. 1. Two sections of the basin were selected, upper watershed (sandy loam) and lower watershed (loam). In the upper watershed (nearest the original holding pond), five sampling sites were selected, two in an adjacent

Fig. 1. Location of the study plots.

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non-polluted area (NP), two in the polluted but subsequently reclaimed area (PR), and another polluted plot (P) that was left by the Regional Government with the pyrite mud on the surface for research purposes. In the lower watershed, five plots also were selected, two plots of polluted soil and two reclaimed plots, but in this case, only one non-polluted plot was possible. The PR soil remediation involved intensive clean-up by removal of the pyrite slurry deposited on top of the soil using bulldozers and by soil treatments involving the addition of calcium carbonate and iron oxy-hydroxides to prevent metal solubilization. All sites had comparable riparian ecosystems except that the PR treatment in the upper watershed had minimal vegetation at the time of sampling. This location has a Mediterranean climate, with a 3-month hot, dry summer and mild winter. Mean annual temperature is 18.6 8C; the months of July and August are the warmest with a mean of 27.3 8C, although maxima of 40–42 8C can be reached. January has the lowest temperature (10.7 8C) and freezing is absent (Direccio´n General del Instituto Nacional de Meteorologı´a, 2002). 2.2. Soil sampling and experimental design Soils were sampled at the end of the summer (September 2002). Each soil sample was composited by pooling four subsamples randomly collected in each plot, using a 7 cm diameter and 0–5 cm depth core sampler. Soil samples of polluted plots were collected just underneath the pyrite mud layer, which was removed prior to sampling. Field moisture content of soil samples was 4.0% (G2.8). Soil samples were sieved to pass a 2 mm screen. One-half was air-dried for subsequent physico-chemical analysis and the remaining soil was retained at field moisture level and stored at 4 8C. Enzyme assays were carried out within 7 d of sampling. The experimental Randomized Complete Block design (RCB) 2!3 factorial was as follows: (i) two pre-treatments (field-moist or re-wetted soil), and (ii) three levels of contamination [non-polluted (NP), polluted (P), and reclaimed (PR)] where the locations (soil types) were treated as blocks (2 replications). For the rewetting treatment, water content was adjusted to 80% of water-holding capacity and the soil incubated at 21 8C for 7 d. Incubations were in plastic containers with a perforated plastic cover to restrict evaporation and yet permit gas exchange. Moisture levels were maintained gravimetrically every 2 days using deionized water. 2.3. Laboratory analyses Chemical and physical measures were performed as follows: pH in a 1:1 soil/CaCl2 solution (McLean, 1982); organic matter by measuring weight lost on ignition (550 8C for 2 h) (Nelson and Sommers, 1982); extractable heavy metals with DTPA (1:2 soil:extractant ratio) according to Lindsay and Norvell (1978); particle distribution by the pipette method (Gee and Bauder, 1986); and water-holding capacity (WHC) at 33 kPa in a Richard’s membrane-plate extractor (Klute, 1986).

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Enzyme assays were selected to represent a range of processes involved in decomposition and nutrient cycling or to reflect microbial activity. Acid phosphatase (EC 3.1.3.2, orthophosphoric-monoester phosphohydrolase, acid optimum), alkaline phosphatase (EC 3.1.3.1, orthophosphoric-monoester phosphohydrolase, alkaline optimum), arylsulfatase (EC 3.1.6.1, arylsulfate sulfohydrolase) and b-glucosidase (EC 3.2.1.21, b-D-glucoside glucohydrolase) activities were determined as described by Tabatabai (1982) where the activity is reported as mg r-nitrophenol (pNP) gK1 hK1. Urease activity (EC 3.5.1.5, urea amidohydrolase) was determined according to Gianfreda et al. (1994) where the product, NHþ 4 , was measured colorimetrically using Indophenol Blue Method (Keeney and Nelson, 1982). Controls were performed in all cases by adding the substrate after the reaction was stopped and before filtration of the soil suspension. Dehydrogenase was determined as described by Casida et al. (1964) with the following modification: 1 g soil mixed with 0.01 g of CaCO3 was incubated with 1 ml 3% 2,3,5 tri-phenyl tetrazolium chloride (TTC) and 3 ml water at 37 8C in darkness. After 6 h 10 ml methanol was added, and the suspension homogenized, filtered and washed with methanol until the reddish colour caused by the reduced TTC (triphenyl formazan) had disappeared from soil. The optical density at 485 nm was compared to those of triphenyl formazan standards. Results of enzyme activities are reported on oven-dry soil weight, determined by drying the soils for 24 h at 105 8C. All the measurements (except textural analysis, and WHC) were carried out in triplicate. 2.4. Statistical analyses Multivariate analysis of variance (MANOVA) was carried out to test for the effects of pre-treatment, degree of pollution and soil type, on total enzyme activities. The statistical treatment analysis was done as an RCB, 2!3 factorial design to test the effects of pre-treatment, pollution and their interactions on each enzyme assayed. Sun ray plots (Dilly and Blume, 1998) were constructed to show graphically the mean enzyme activities of restored and polluted soils scaled against the reference point (i.e. the non-polluted soil). The star shape and area for each treatment allow a comparison of visual and numeric presentations of multivariate data. The resulting areas of these sun ray plots were compared using Student’s t-tests. Principal Component Analysis (PCA) was carried out on the six enzyme activities for both field-moist and rewetted soil to determine whether the pre-treatments affected the degree of separation between polluted and non-polluted soil (on a multivariate basis) and to determine which enzymes were most important in differentiating between polluted and reclaimed soils. Statistical analyses were performed using STATISTICA’99 edition (StatSoft, 1999).

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Table 1 General characteristics of 0–5 cm surface soils (meanGSE) at the sampling sites Soil property

Upper watershed

pH % OM* Cda Pba Cua Zna % Sand % Clay WHCb

Lower watershed

Non-polluted

Reclaimed

Polluted

Mean

Non-polluted

Reclaimed

Polluted

Mean

7.1G!0.01 8.8G0.1 0.2G!0.01 7.7G2.5 4.4G1.4 22.8G11.4 81.1G5.6 9.6G3.4 0.2G!0.01

2.6G!0.01 8.4G0.2 1.4G0.2 2.0G0.6 35.5G1.4 111.3G8.5 70.8G1.5 11.6G1.6 0.2G!0.01

2.5 7.1 2.2 15.3 31.0 113.4 60.6 8.0 0.2

4.4G0.6 8.3G0.2 1.1G0.2 6.9G1.7 22.1G3.9 76.3G12.9 72.9G4.2 10.1G1.4 0.2G!0.01

7.6 8.5 0.1 3.1 8.7 29.9 42.6 26.2 0.3

7.3G0.1 8.7G0.1 0.4G0.0 4.9G1.2 7.9G0.5 51.7G21.6 46.2G4.9 21.9G2.2 0.3G!0.01

3.3G0.3 8.2G0.1 0.6G0.2 1.8G0.4 17.1G1.9 41.7G11.5 47.8G0.7 15.3G4.7 0.3G!0.01

5.7G0.5 8.5G0.1 0.4G0.1 3.3G0.6 11.7G1.4 43.5G9.5 46.1G1.8 20.1G2.7 0.3G!0.01

a

mg kgK1. cm3 cmK3. * Organic matter. b

between non-polluted and polluted soils (both with and without restoration). In the lower watershed, reclaimed soil generally showed similar heavy metal concentrations to nonpolluted soil.

3. Results 3.1. Soil characteristics Physico-chemical characteristics of the soils are shown in Table 1. Soil texture changed from a sandy loam to a loam between the upper and lower watersheds. The pH and organic matter content did not show differences between two study areas of the river basin. Extractable heavy metal contents generally were higher in the upper watershed (nearest the mine pond) than in the lower watershed, for both polluted and reclaimed soils. Within each study area, pH and heavy metal concentrations indicated significant differences in the degree of pollution; pH was always lowest and heavy metal concentration highest in polluted soils. Reclaimed soils in the upper watershed also had low pH, whereas reclaimed soils in the lower watershed had a similar pH to non-polluted soils. In the upper watershed, Pb concentration was not significantly different between non-polluted and reclaimed soils, whereas, extractable Cd, Cu and Zn were significantly different

3.2. Univariate tests Factorial analysis (Table 2) confirmed that the rewetting pre-treatment and degree of pollution had highly significant effects on soil enzyme activities, and that generally there were significant interactions between these factors. Furthermore, there was a block effect suggesting there was a spatial or soil type influence on soil enzyme activities. All enzyme activities, except b-glucosidase, were significantly greater for pre-treated rewetted and incubated soils. Such increases ranged from 72 and 67% for alkaline phosphatase and urease activities, respectively; to 34 and 24% for acid phosphatase and arylsulfatase. Thus, the rewetting pre-treatment did not change treatment ranking among the different levels of pollution, but allowed for better discrimination between polluted and non-polluted soils.

Table 2 Effects of pre-treatment (field-moist soil or rewetted soil), degree of pollution and interaction on soil enzyme activities (meanGSE) by a two-way ANOVA (*, p%0.05; **, p%0.01; NS, not significant) Factor

Treatment

Acida phosphatase

Alkalinea phosphatase

Arylsulfatasea

b-Glucosidasea

Ureaseb

Dehydrogenasec

Pre-treatment soil moisture (PM) Degree of pollution (DP)

Field-moist

** 73.7G7.8 98.5G12.9 ** 139.7G12.9 64.6G6.7 52.9G4.5 * NS

** 104.0G15.1 178.5G36.2 ** 259.8G37.9 98.9G15.8 58.4G8.3 ** **

NS 19.6G3.8 24.4G6.5 ** 51.8G4.7 13.8G3.1 1.58G0.5 ** **

NS 92.9G13.3 96.6G15.9 ** 161.5G12.3 95.5G14.1 25.8G4.6 ** NS

* 29.8G7.3 49.8G13.9 ** 76.4G14.3 53.0G0.5 2.9G12.1 ** NS

* 32.8G4.7 41.7G7.9 ** 71.4G5.2 53.0G0.5 2.9G12.1 NS NS

Block (location) Interaction a b c

Rewetted NP PR P PM!DP

mg pNP g dw hK1. K1 mg NHþ dw hK1. 4 g mg TPF gK1dw hK1. K1

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Enzyme activities decreased significantly with increasing degree of pollution: non-polluted soil showed the highest enzyme activity values, polluted the lowest, and restored soil intermediate values. The greatest differences between the three levels of pollution were for urease, b-glucosidase and dehydrogenase activities (Table 2). In many cases there were significant interactions among factors (Table 2). Because the interaction between main

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effects can distort, conceal or exaggerate their differences, further analysis is required. Therefore, enzyme activities were analysed independently using one-way ANOVA, which showed (Fig. 2) that activities, for both field moist and rewetted soils, successfully discriminated the pollution treatments. However, rewetting generally increased enzyme activities of non-polluted and reclaimed soils which improved discrimination from polluted treatments.

Fig. 2. Effects of pre-treatment (field-moist or rewetted soil) on enzyme activities of soil affected by different degrees of pollution (non-polluted, NP; polluted and restored, PR; polluted, P).

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Fig. 3. Sun ray plots for enzyme activities in restored and polluted soils scaled against the reference situation (non-polluted soil, 100%). Plots are shown for both pre-treatment: field-moist soil (A) and rewetted soil (B). acP, acid phosphatase; alkP, alkaline phosphatase; aryl, arylsulfatase; gluc, b-glucosidase; dehy, dehydrogenase; urea, urease.

Polluted soils had much smaller increases or even decreases in enzyme activities with rewetting. For example, in the sandy loam soil, b-glucosidase decreased from 26.1 (G0.7) to 11.5 (G2.6) mg pNP gK1 hK1 in the polluted soil because of the rewetting pre-treatment. This also occurred for arylsulfatase activity in restored soils, where field-moist soil was 29.1 (G4.8) and rewetted soil was 20.3 (G1.3) mg pNP gK1 hK1. 3.3. Multivariate analysis A method of increasing the potential of soil enzyme activities to detect soil contamination could be to combine them in multivariate analysis, which provides a more holistic representation of the biochemical and microbial functionality of a soil. MANOVA, followed by the Wilk’s lambda test, showed that the two main effects (degree of pollution and pre-treatment) had significant impacts on enzyme activities on a multivariate basis. Sun ray plots have been proposed by Dilly and Blume (1998) and Nannipieri et al. (2002) as a way to simultaneously display all the measured parameters and relative differences among treatments. Two sun ray plots for enzyme activities are shown in Fig. 3 for the restored and the polluted sites either field-moist soils or rewetted soil samples. For each enzyme activity, the corresponding values in the non-polluted sites were taken as a reference point (100%), and the values in the restored and the polluted sites were scaled against it. For field-moist soils (Fig. 3A), the probability value for differences in the area on the sun ray plot between restored and polluted soils was pZ0.016 and for rewetted soils (Fig. 3B) the level of significance increased to pZ0.005. Thus, rewetting pre-treatment increased the probability of finding significant differences. On the other hand, there were no significant (Student t-test, pO0.05) differences between areas on the sun ray plots for fieldmoist soils vs. rewetted soils, neither for restored sites nor for the polluted sites.

Table 3 shows the results of Principal Component Analysis (PCA), which was performed separately for field-moist and rewetted soils to determine which enzymes were most useful (on a multivariate basis) for accounting the variation among pollution treatments. Using field-dry soil samples, factor loadings of enzyme activities on the PCA showed the following ordination: arylsulfataseOb-glucosidaseOalkaline phosphataseOacid phosphataseOureaseO dehydrogenase. For rewetted soil samples the ordination of enzyme activities was: alkaline phosphataseOb-glucosidaseO arylsulfataseOacid phosphataseOdehydrogenaseOurease. Although absolute values of the loading factors for each enzyme activity were different for both soil pre-treatments (field-dry and rewetted), the resulting overall ordination was qualitatively similar: arylsulfatase, b-glucosidase and alkaline phosphatase were the most important variables for PC1. It is interesting that the percentage of variance explained by the PC1 was higher for the rewetted soil than for the field-moist soils confirming our previous observations that rewetting provides a better measure of the degree of pollution.

4. Discussion Results showed that soil enzyme activities were affected by the pollution from the Aznalco´llar mine spill. Table 3 Principal components analysis for enzyme activities in both field-moist soil and rewetted soil. Loading factors and total variance (%) in the first principal component (PC1) are showed

Acid phosphatase Alkaline phosphatase Arylsulfatase b-Glucosidase Urease Dehydrogenase Total variance (%)

Field-moist soil, PC1

Rewetted soil, PC1

0.902 0.923 0.979 0.974 0.764 0.718 77.9

0.943 0.977 0.944 0.946 0.841 0.940 87.0

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Furthermore, reclaimed soils still showed significantly lower enzyme activities, indicating that these soils have not been fully restored and that the microbial community is still affected by the spill. Besides the direct effect of the mine spill on soil properties, initially plants were completely absent from the polluted soils. Thus, it is possible there could have been some treatment effects due to lack of vegetation on the polluted soils due to reduced C inputs. However, this likely had a small effect on the microbial community compared to the heavy metal contamination. This is because when we sampled 4 yrs after the spill, most of the research sites were re-vegetated, even soils with a thin layer of pyrite mud on top (the only exception being reclaimed plots from the upper watershed). Furthermore, organic C levels were very similar across all treatments (Table 1) making it less likely that lack of C was inhibiting the microbial community. Another line of evidence for a direct effect of metals, rather than lack of plant C inputs, was in a previous laboratory experiment (unpublished data, M.B. Hinojosa, 2003). In this case, non-polluted soils from upper and lower watersheds were mixed with a 4% pyrite mud (rate of amendment accepted by environmental regulatory agency at which a soil is considered recovered) and incubated 92 days. Enzymes activities (phosphatases, arylsulfatase, b-glucosidase, urease and dehydrogenase) were significantly reduced more than 40% on average by the addition of pyrite mud over unpolluted soil (data not shown). This suggests a major contribution of heavy metal and/or lower pH on the direct inhibition of enzyme activities. The results shown in Table 1 indicate there was a significant effect on pH in the polluted soils. The field soil pH would not directly affect the enzyme assay as the assays are run using buffers at their optimal pH. However, the long term effect of low pH would probably cause shifts in microbial community composition and size (Killam, 1994) and this, in turn, would affect enzyme dynamics at the time of sampling. There were fairly high correlations of soil pH with the whole set of enzyme activities for field-moist soils (rZ0.64) and rewetted soils (rZ0.69). Part of the enzyme response may have been due to a smaller and different community that developed as a response to the low pH of polluted soils. In combination with pH, heavy metals pollution is important in suppressing enzyme activity. Simple correlations across all the data showed that alkaline phosphatase, arylsulfatase and b-glucosidase activities had significant negative correlation coefficients (rZK0.64 to K0.75) with extractable Cd, Cu and Zn for field moist soils. Similar results were found for rewetted soils except for Zn where these same enzymes had lower r values ranging from K0.41 to K0.49. Lead had very low and non-significant correlations with all the enzyme assays, which indicated that there were no significant pollution treatment effects on extractable Pb concentration. The results for Cd, Cu and Zn correspond to other studies where enzyme activities were

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shown to reflect the extent of heavy metal contamination (see reviews by Dick, 1997; Giller et al., 1998; Speir and Ross, 2002). Enzyme reactions are inhibited by metals which may complex with the substrate, combine with the protein-active groups of the enzymes, or react with the enzyme-substrate complex. Sulfhydral groups of enzymes serve as catalytic sites or as groups involved in maintaining the correct conformation of the protein. Metals can react with sulfhydral groups causing inactivation or inhibition of enzyme activity (Shaw and Raval, 1961). The mode of action of metals varies with enzymes and little is known about the exact mechanisms by which metals interact with the multitude of enzymes that exist in soils. Enzymes vary in their degree of inhibition by trace elements. Previous research has shown that arylsulfatase activity is sensitive to heavy metals whereas acid phosphatase, urease, and invertase were less affected by them (Al-Khafaji and Tabatabai, 1979; Bardgett et al., 1994; Yeates et al., 1994). These results run somewhat contrary to what we found with urease, b-glucosidase and dehydrogenase showing the greatest difference between polluted and non-polluted soil. In fact, all the enzymes we assayed were significantly affected by pollution treatments. On the other hand, when the data were analysed on a multivariate basis using PCA, arylsulfatase ranked high in both field moist and rewetted soil. Although dehydrogenase was a good indicator of polluted soil in our study, it should be used with caution because of several confounding factors. In theory it should only operate as an enzyme (or group of enzymes), involved in electron transport during oxidative metabolism in viable cells. However, there are extracellular phenol oxidases and alternative electron acceptors (such as nitrate or humic acids) in soils, that can cause an over estimation of dehydrogenase activity (Bremner and Tabatabai, 1973; Dick, 1997; Rossel et al., 1997). Furthermore, Cu reduces the measured dehydrogenase activity by interfering with the assay procedure (Chander and Brookes, 1991). Our polluted soil did have elevated Cu levels which likely did affect the assay. Indeed, unlike the other enzymes, dehydrogenase activity showed virtually no correlation with concentration for any of the metals (data not shown). The nature and degree of inhibition by heavy metals on soil enzymes are strongly related to soil type (Speir et al., 1992). Greater inhibition of arylsulfatase and phosphatase has been shown on soil with low surface area, CEC and organic matter content. All of these may reduce the potential of the soil to inactivate metals via complexation or sorption reactions and increases the availability of metals and their impact on enzyme processes (Speir et al., 1995). It is difficult to separate the effect of soil type in our study because one soil was closer to the source and presumably had greater potential for contamination, as evidenced by higher levels of extractable metals, than did the soil in the upper part of the watershed. On the other hand, the lower

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watershed had a higher clay content which would likely have greater potential to complex or adsorb metals. Before soil enzyme activities can be used as soil quality indices, soil sample pre-treatment procedures and units of measurement must be standardized (Dick, 1994). The antecedent soil moisture is a potentially confounding factor when biological assessments of soil quality are used. Our results showed that in general, rewetting field-dry soils increased enzyme activities except on the lighter texture, metal-contaminated soil which had further inhibition of some enzymes. This is consistent with other studies on microbial activity and mineralization rates in dried and rewetted soil (Bloem et al., 1992; Bandick and Dick, 1999; Fierer and Schimel, 2002; Mamilov and Dilly, 2002). The increases in the enzyme activities in rewetted soils may be attributed to the biological response to a dryrewetting cycle from mineralization of: (i) non-biomass soil organic matter rendered accessible to microbial attack (Utumo and Dexter, 1982; Appel, 1998; Denef et al., 2001); and (ii) microbial C released from lysed cells (Bottner, 1985; Kieft et al., 1987). These two influences on microbial activity have been supported by other studies (van Gestel et al., 1993; Scheu and Parkinson, 1994; Fierer and Schimel, 2003). However, increase of enzyme activity not always takes place in rewetted contaminated soils where there may be no change in enzyme activity or sometimes even a decrease. This may be due to an increase in the solubility of heavy metals and their inhibitory effect in rewetted soils (Janssen et al., 1997; Bordas and Bourg, 2001). In our study, we found a somewhat different response to rewetting for each enzyme. Alkaline phosphatase and urease activities showed the highest increase after soil rewetting whereas b-glucosidase did not show any significant change. Other researchers have shown the rewetting response is enzyme specific and that it varies among different studies. For example, Harrison (1983) and Speir and Cowling (1991) reported significant correlation of phosphatase activity with changes in soil moisture over time under field conditions. However, Rastin et al. (1988), Kra¨mer and Green (2000), and Wick et al. (2002) showed that seasonal variation in soil moisture had a limited influence on phosphatase activities. Rastin et al. (1988) and Wick et al. (2002) concluded that alkaline phosphatase and b-glucosidase activity were not controlled by climatic conditions over the course of one dry and two rainy seasons. This variability of results on the effect of soil moisture among researchers could be explained by the differences in regional ecosystem characteristics (soil texture, moisture regime, temperature regime, etc.). Nonetheless, this variability makes it difficult to provide universal standards or thresholds for interpreting enzyme assays as soil quality indicators. Enzyme activities may be most useful for the monitoring of trends over time. In our study, the assays of acid phosphatase, dehydrogenase and b-glucosidase consistently showed the same relative

differences in activity among the polluted treatments for fielddry and rewetted samples. This is important in selecting soil quality indicators because, although these enzyme activities increased with rewetting, in all cases this increase was similar for each treatment and the use of either field-dry soils or rewetted soils does not change the conclusions about the impacts of heavy-metal pollution. Bandick and Dick (1999), in a study of soil quality relative to fertilizer amendments and crop rotations, also found that air-dried and field-moist samples provide the same ranking of activities for a number of enzymes. They concluded that b-glucosidase was most consistent in showing treatment effects but, as with our results, showed only small differences in activity between moist and air-dried soils. Indicators of soil quality and health, to be useful and practical, must meet certain criteria. These include: sensitivity to perturbation or contamination; a relationship to soil function; reproducibility and low temporal and spatial variability; and have simple sampling and analytical methods (Doran and Parkin, 1996; Doran and Zeiss, 2000; Nortcliff, 2002). Enzyme assays have potential to meet all these criteria and our results show that all the enzymes tested were appropriate for detecting heavy metal pollution in soils. Whilst arylsulfatase showed the greatest separation between polluted and non-polluted soils, b-glucosidase offers potential to be a more universal indicator because both control and polluted soils were unaffected by rewetting. However, it is clear there is no universal enzyme assay that can be recommended for all soils under all conditions and it is best if the polluted soil can be compared to an adjacent non-polluted soil of the same soil type. The water content of the soil prior to sampling is an important consideration when using enzyme activity as an indicator of soil pollution, furthermore, the soil moisture response is enzyme-specific. We recommend a rewettingincubation pre-treatment period when enzyme assays are used as soil quality indicators of heavy metal pollution for soils in xeric moisture regimes. However, this type of research should be expanded to other soils and ecosystems to further verify the appropriate soil moisture pre-treatment protocol. Furthermore, rewetting the lighter textured soil caused a greater inhibition of some enzymes due to metal contamination, reinforcing the need for this pre-treatment and provides evidence for the role of soil moisture in affecting bioavailability of metals. The latter merits further investigations from a microbial ecology perspective.

Acknowledgements This work was supported by Consejerı´a de Medio Ambiente of Junta de Andalucı´a, as a project of the Green Corridor Research Plan (PICOVER). Authors are grateful to J. Sandeno, M. Fernandes and J.M. Rodrı´guez Maroto for their help in laboratory analyses.

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