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

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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 Aznalcóllar 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, β-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 °C 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 β-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.

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.

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, Frankenberger and Tabatabai, 1991a, Frankenberger and Tabatabai, 1991b, 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, Öhlinger, 1995, Dick et al., 1996) and not allowed to freeze, 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, 1987, Öhlinger, 1995). The udic moisture regime is common 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 Aznalcóllar mining effluent spill that released toxic, acid and heavy metal-contaminated tailings into the Guadiamar river basin (SW Spain). The Aznalcóllar 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 (Simón 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, López-Pamo et al., 1999. This watershed provided a unique opportunity to study a long-term, real-world 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.

Section snippets

Study sites

The Aznalcóllar 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 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

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

Discussion

Results showed that soil enzyme activities were affected by the pollution from the Aznalcóllar mine spill. 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

Acknowledgements

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

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