Leaching and aging decrease nickel toxicity to soil microbial processes in soils freshly spiked with nickel chloride

Nickel is a trace metal that exhibits pronounced long-term immobilization reactions in soil. It is unknown if the slowly decreasing solubility of Ni in soil on aging correlates with decreased toxicity to soil biota. Three uncontaminated soils (pH 4.5-7.6) were contaminated with NiCl2 and experimentally leached or incubated outdoors with free drainage for up to 15 months. Nickel toxicity was measured for three microbial processes (potential nitrification rate, glucose-induced respiration, and C mineralization of maize residue). Results for leached and aged samples were compared with results for these soils tested immediately after spiking. Experimental leaching increased Ni ED50s (Ni dose to inhibit process by 50%) with a median factor of 2.0, whereas Ni ED50s in soils aged 15 months were a factor 1 to 23 (median, 4.6) larger compared to freshly spiked soils. Changes in soil Ni toxicity on aging generally were largest in the soil with the highest pH, consistent with the largest relative decreases of soil solution Ni concentration or predicted Ni2+ activity. Soil solution Ni concentrations explained part, but not all, of the reduction in Ni toxicity. The predicted soil solution Ni2+ activity also did not fully explain the reduced toxicity, which was ascribed to the variable concentrations of ions competing with Ni2+ at biological membranes (e.g., H+, Mg2+, or Ca2+) among treatments. It is concluded that testing Ni toxicity to soil microbial processes immediately after spiking soils in the laboratory overestimates Ni toxicity compared to aged soils. Soil solution composition in freshly spiked soils clearly is different from that in leached or aged soils; therefore, soil spiked with metal salts should be leached before toxicity tests begin.     

Comparison of toxicity of zinc for soil microbial processes between laboratory-contamined and polluted field soils

Soil microbial processes are readily disturbed by added zinc (Zn) in laboratory ecotoxicity tests. This study compares Zn toxicity between freshly spiked soils and soils that have been contaminated with Zn in the field. Soils were sampled in three transects (< 80 m) toward galvanized electricity transmission towers (pylons). The soil total Zn concentrations gradually increased in each transect from background values (25-82 mg Zn/kg) to elevated Zn concentrations near the pylon (226-595 mg Zn/kg). Soil samples taken at the furthest distance from the Zn source were spiked with ZnCl2 to a range of total Zn concentrations similar to those in the transect. Nitrification, respiration, and N-mineralization rates were significantly reduced by added Zn in laboratory-spiked soils and were 9 to 95% (mean 32%) of the control values at largest doses depending on soil type and the microbial process. In contrast, these processes were either unaffected by soil Zn (p > 0.05) or increased significantly with soil Zn concentrations in the transect soils. These increases could not be explained by soil pH or % soil organic carbon. Leaching soils after spiking significantly lowered the toxic effects of Zn on nitrification or on substrate-induced respiration. The soil solution Zn concentrations of field soils were always smaller than in spiked soils at equivalent total Zn. Highest soil solution Zn concentrations were always lower than the soil-solution EC50s of spiked soils. It is concluded that there is a large discrepancy in microbial responses to elevated Zn between spiked soils (unleached) and field-contaminated soils and there is a need to explain this discrepancy in terms of Zn availability, adaptation processes, and additional soil factors controlling the microbial processes.     

Soil properties affecting toxicity of zinc to soil microbial properties in laboratory-spiked and field-contaminated soils

The effects of soil properties and zinc (Zn) availability on the toxicity of Zn to soil microbial processes are poorly understood. Three soil microbial processes--potential nitrification rate (PNR), substrate (glucose)-induced respiration (SIR), and a maize residue respiration (MRR)--were measured in 15 European topsoils (pH 3.0-7.5; total Zn 7-191 mg/kg) that were freshly spiked with ZnCl2. The Zn toxicity thresholds of 20 to 50% effective concentrations (EC20s and EC50s) based on total concentrations of Zn in soil varied between 5- and 26-fold among soils, depending on the assay. The Zn toxicity thresholds based on Zn concentrations in soil solution varied at least 10-fold more than corresponding total metal thresholds. Soil pH had no significant effect on soil total Zn toxicity thresholds, whereas significant positive correlations were found between these thresholds and background Zn for the PNR and SIR test (r = 0.74 and 0.71, respectively; log-log correlations). No such trend was found for the MRR test. Soil solution-based thresholds showed highly significant negative correlations with soil pH for all assays that might be explained by competition of H+ for binding sites, as demonstrated for aquatic species. The microbial assays were also applied to soils collected under galvanized pylons (three sites) where concentrations of total Zn were up to 2,100 to 3,700 mg Zn/kg. Correlations between concentrations of total Zn and microbial responses were insignificant at all sites even though spiking reference samples to equivalent concentrations reduced microbial activities up to more than 10-fold. Differences in response between spiked and field soils are partly but not completely attributed to the large differences in concentrations of Zn in soil solution. We conclude that soil pH has no significant effect on Zn toxicity to soil microbial processes in laboratory-spiked soils, and we suggest that community tolerance takes place at both background and elevated Zn concentrations in soil.     

Influence of aging on copper bioavailability in soils

Because of long-term chemical processes, metal bioavailability in field soils decreases with time. Metal toxicity may, therefore, be overestimated if toxicity data with freshly spiked soils are used to derive soil quality criteria, a current practice. In the present study, effects of the long-term processes, called aging, on copper partitioning and ecotoxicity are investigated. Twenty-five field soils contaminated by copper runoff from bronze statues and 25 uncontaminated control soils sampled at 5-m distance from these statues were collected in Flanders (Belgium). The soils were selected so that parameters affecting copper bioavailability (pH, cation-exchange capacity, organic matter content, etc.) varied considerably. To assess the effect of aging on copper toxicity, control soils were spiked at total copper concentrations comparable to those of historically contaminated soils. Pore-water copper concentrations and 0.01 M CaCl2-extracted copper concentrations were significantly higher in freshly spiked soils compared to contaminated field soils. However, this could be a pH effect, because pH decreased after spiking. Acute toxicity to Enchytraeus albidus (14 d) as well as chronic toxicity to Folsomia candida (28-d reproduction) and Trifolium pratense (14-d growth) indicated a dose-response relationship between copper toxicity and pore-water copper concentration or the CaCl2-extracted copper fraction.     

Soil properties affecting the toxicity of CuC12 and NiC12 for soil microbial processes in freshly spiked soils

It generally is unknown to what extent the toxicity of Cu and Ni for soil microbial processes varies among different soils. A comparative study was made using three different microbial assays (nitrification potential, glucose-induced respiration, and C-mineralization of a plant residue) in 19 (for Cu) or 16 (for Ni) soils with contrasting soil properties. Each soil was spiked with CuCl2 or NiCl2 at seven different concentrations, and the bioassays were started after a 7-d equilibration period. The Cu and Ni toxicity thresholds varied 19- to 90-fold among soils. The differences in both Cu and Ni toxicity among soils were neither explained by soil solution metal concentrations nor by free ion activities calculated from soil solution composition. Copper toxicity thresholds (total concentrations) increased with increasing organic matter content or cation exchange capacity (CEC) and, surprisingly, decreased with increasing pH depending on the assay. Nickel toxicity thresholds consistently increased with increasing CEC, background Ni, and clay content for all three assays. Thresholds expressed as soil solution free ion activities all significantly decreased with increasing soil solution pH (r2 = 0.57-0.93), consistent with a decreased H+:M2+ competition at the biological membrane. That competition largely counteracts the H+:M2+ competition for sorption, effectively explaining the insignificant or weak effect of pH on total Ni or Cu toxicity thresholds. It is concluded that free metal-ion activity alone does not explain variation in metal toxicity among soils.     

Comparing soil chemistries of leached and nonleached copper-amended soils

Leaching metal-spiked samples has been proposed as a means to reduce the artifacts of the spiking procedure (e.g., salt effect, increased metal solubility) that can artificially increase metal bioaccessibility and toxicity in laboratory ecotoxicity tests. The effects on soil chemistry from leaching Cu-spiked samples were investigated by comparing chemistries of freshly spiked samples to samples that underwent the spike/leach procedure. Chemical parameters investigated included electrical conductivity (EC), pH, ethylenediaminetetraacetic acid- and CaCl(2) -extractable Cu, soil-solution Cu, Cu(2+) activity (estimated using Visual MINTEQ), and other solution parameters (dissolved organic carbon [DOC], Ca, Mg, Al). In leached samples, the electrical conductivity values of the spiked samples did not vary significantly from those of the control samples (p?>?0.05), confirming that the leaching procedure had sufficiently minimized the salt effect. In the range of soil Cu concentrations where Cu ecotoxicity is expected, the pH in freshly spiked samples was as much as 0.52 units lower than the pH from leached samples at the same total-soil Cu concentration. The CaCl(2) -extractable fraction was up to 2.3-fold smaller in leached samples and inversely related to the pH of the spiked soil. Despite little to no difference in soil-solution Cu, up to 100-fold less Cu(2+) activity was observed in leached samples. Reduced Cu(2+) activity was related to less Al(3+) competition for DOC. Leaching resulted in solution chemistries that were more consistent with those of the control samples and reduced the artifacts of traditional soil-spiking procedures. Environ. Toxicol. Chem. 2012; 31: 2253-2260. ? 2012 SETAC.     

An in situ respirometric technique to measure pollution-induced microbial community tolerance in soils contaminated with 2,4, 6-trinitrotoluene

Long-term exposure to 2,4,6-trinitrotoluene (TNT) can induce changes in the structure and activities of soil microbial communities. Such changes may be associated with an elevated microbial tolerance. An in situ respirometry technique based on the analysis of the substrate-induced respiration response to freshly added TNT was used to examine soil microbial tolerance to TNT at the community level. The specific growth rate derived by fitting an exponential equation to respiration data was taken as the measurement endpoint. Microbial tolerance was evaluated using a tolerance index defined as the ratio of the specific growth rate at a spiking dose of 2000 microg TNT/g soil to that of the control with no spiked TNT. Three soils with long-term exposure histories (TNT level in soil: 1.5, 32, and 620 microg TNT/g, respectively) exhibited significantly higher microbial community tolerance to TNT than two uncontaminated control soils. A soil containing 29,000 microg TNT/g exhibited the highest tolerance. Findings from this study support the hypothesis that pollution-induced community tolerance can be used as a means of identifying those compounds that have exerted selective pressure on the community.     

Influences of soil properties and leaching on nickel toxicity to barley root elongation

The influence of soil properties on Ni toxicity to barley root elongation was investigated using 17 Chinese soils treated with soluble Ni salts, with and without leaching. The effective concentration of added Ni causing 50% inhibition (EC50) in barley root elongation ranged from 48 to 2519 mg/kg in unleached soils and 46 to >2381 mg/kg in leached soils. Leaching decreased Ni phytotoxicity significantly for approximately 90% of soils, and the effect was most pronounced for soils with pH ≥8.2. Soil pH was the most important factor controlling Ni toxicity in soils, explaining approximately 68% of the variance in unleached and leached EC50 values. Regression models between toxicity thresholds and soil pH, soil organic carbon content, or effective cation exchange capacity were developed. The models showed good agreement with those developed previously for European soils (r²=0.87). These quantitative relationships between Ni toxicity and soil properties are helpful to develop soil-specific guidance on Ni toxicity thresholds for China.     

Effect of leaching and aging on the bioavailability of lead to the springtail Folsomia candida

Because it is unclear if leaching can account for differences in metal bioavailability observed between metal-spiked soils and historically contaminated field soils, we simultaneously assessed Pb toxicity to the springtail Folsomia candida in three transects of Pb-contaminated soils and in leached and unleached soils spiked at similar total Pb concentrations. Total Pb concentrations of 3,877 mg/kg dry weight and higher always caused significant effects on F. candida reproduction in the spiked soils. In the transects, only the soil with the highest Pb concentration of 14,436 mg/kg dry weight significantly affected reproduction. When expressed as pore-water concentrations, reproduction was never significantly affected at Pb concentrations of 0.539 mg/L, whereas reproduction was always significantly affected at Pb concentrations of 0.678 mg/L and higher, independent of the soil treatment. These results indicate that pore-water Pb concentrations can explain, at least in part, the observed differences in the toxicity data expressed as total Pb concentrations. Leaching after the spiking procedure only caused small differences in Pb toxicity and, therefore, cannot account for toxicity differences between laboratory-spiked soils and historically contaminated field soils.     

Copper inhibition of soil organic matter decomposition in a seventy-year field exposure

On a site contaminated decades ago with Cu from a wood treatment facility, we can observe that the decomposition of soil organic matter has been slowed. This represents an exceptional data set, and it allows us to address many challenges faced by regulators and risk assessors who are trying to derive appropriate soil quality criteria. These data are representative of a field study with a very well-equilibrated contamination and allow the derivation of chronic toxicity threshold values for the inhibition of microbial respiration. Soil respiration is the main determinant of the carbon balance, and it is assessed using the accumulation of soil organic matter (SOM) in this case. Using data derived from a 70-year-old field study also has the advantage of not being subject to risk assessment uncertainty factors arising from the potential aging effects of spiked soil or to the uncertainty caused by laboratory-to-field differences, both of which are very difficult to address. The resulting toxicity thresholds for an inhibition of SOM degradation are 154, 193, and 285 mg Cu/kg dry soil for inhibition levels of 10, 20, and 50%, respectively. Setting those thresholds correctly is critical for a proper risk assessment relative to sustainable development and agriculture.     

Avoidance of Cu- and Zn-contaminated soil by three ecologically different earthworm species

Earthworm avoidance response to soils contaminated with harmful substances has been proposed as a potential tool for assessing soil toxicity with low test effort. In the present study, the objective was to find out whether three ecologically different earthworm species, Aporrectodea tuberculata (Eisen), Lumbricus rubellus (Hoffmeister), and Dendrobaena octaedra (Savigny), avoid soils simultaneously spiked with Cu and Zn. In addition, metal-contaminated field soil taken close to a Cu-Ni smelter was tested with A. tuberculata using a two-section avoidance lest procedure. All three earthworm species clearly avoided Cu/Zn contaminated soil but differently: D. octaedra was the most sensitive species, responding to low metal concentrations, whereas L. rubellus responded only to the highest metal concentration tested, being the least sensitive species. Moreover, A. tuberculata showed clear avoidance response to the metal contaminated field soil. In conclusion, the results indicate that earthworm avoidance behavior is an ecologically relevant parameter for assessing harmfulness of metal contaminated soils, both spiked and field-contaminated soils. However, it is important to consider the specific species to be used in the earthworm avoidance test procedure.     

Effect of soil properties and aging on the toxicity of copper for Enchytraeus albidus, Enchytraeus luxuriosus, and Folsomia candida

In the present study, the effect of the heavy-metal salt copper chloride (CuCl2.2H2O) in soils freshly spiked (3 d) and aged (70 +/- 10 d; mean +/- SD) was studied in the test species Enchytraeus albidus, E. luxuriosus, and Folsomia candida. Up to nine soils were used: Besides the Organisation for Economic Co-operation and Development (OECD) artificial soil and the Agricultural Testing and Research Agency (Landwirtschaftliche Untersuchungs- und Forschungsanstalt, Speyer, Germany) 2.2 natural standard soils, the others were selected based on the EURO Soil approach, taking into account the effect of different soil parameters (pH, organic matter, grain size distribution, and carbon to nitrogen ratio). Additionally, the effect of the chloride ions was studied separately. The results revealed the following: First, a soil effect was observed; for example, in F. candida, median effective concentrations (EC50s) varied between 262 mg/kg in a sample from the same site as the original EURO Soil 5 soil and greater than 1,000 mg/kg in OECD soil. Second, an aging effect was observed, mainly in F. candida. For example, toxicity of offspring survival was increased twofold in the OECD soil and approximately eightfold with aging in the EURO Soil 7 soil, whereas the enchytraeid species did not react differently after aging. Third, an effect of chloride ions on reproduction of the animals was found; however, this effect was independent of the aging period. Fourth, species variation was seen in terms of sensitivity (EC50), decreasing in the following order: E. luxuriosus > E. albidus > F. candida. Differences in toxicity of offspring survival between enchytraeids and F. candida might be explained by the different routes of uptake.     

Phytotoxicity and bioaccumulation of copper and chromium using barley (Hordeum vulgare L.) in spiked artificial and natural forest soils

The toxicities of two heavy metals, copper (Cu2+) and chromium (Cr6+), to barley (Hordeum vulgare L.) were evaluated using two types of substrates: artificial and natural forest soils. Phytotoxicity was assessed using a standardized toxicity test. Endpoints included plant emergence and shoot and root growth. Shoot and root concentrations of Cu and Cr were also measured. Data indicated that the root biomass was the most sensitive endpoint. The results showed that toxicity of Cr to root growth (IC50=6.6 microg/g in artificial soil; IC50=61.8 microg/g in forest soil) was higher than that of Cu (IC50=13.7 microg/g in artificial soil; IC50>322 microg/g in forest soil). Data also indicated that the toxicity of Cu and Cr was significantly decreased in the spiked forest soil, suggesting lower metal bioavailability to barley in the natural soil. Analysis of tissue concentrations in barley showed that Cu and Cr were mainly accumulated in the roots. Toxicity was correlated with Cr residues in shoots (< or =11.2 microg Cr/g and < or =5.3 microg Cr/g for artificial and natural soils, respectively) and roots (< or =161 microg Cr/g and < or =51.7 microg Cr/g for artificial and natural soils, respectively) and Cu residues in roots (< or =61.8 microg Cu/g and < or =91.3 microg Cu/g for artificial and natural soils, respectively). Cu concentration in shoot tissues was < or =61.8 microg Cu/g. Since it may overestimate toxicity, effect and risk assessment using spiked soils, particularly in artificial soil, must be used with diligence.     

Ecotoxicological effects of hexahydro-1,3,5-trinitro-1,3,5-triazine on soil microbial activities

Although hexahydro-1,3,5-trinitro-1,3,5-triazine (also called RDX or hexogen) is a potentially toxic explosive compound that persists in soil, its ecotoxicological effects on soil organisms have rarely been assessed. In this study, two uncontaminated garden soils were spiked with 10 to 12,500 mg RDX/kg dry soil. Soil microbial activities, i.e., potential nitrification, nitrogen fixation, dehydrogenase, basal respiration, and substrate-induced respiration were chosen as bioindicators and were determined after 1-, 4-, and 12-weeks of exposure. Experimental results indicate that RDX showed significant inhibition (up to 36% of control) on indigenous soil microbial communities over the period of this study. All five bioindicators responded similarly to the RDX challenge. The length of exposure also affected the microbial toxicity of RDX, with 12-week exposure exerting more significant effects than the shorter exposure periods, suggesting that soil microorganisms might become more vulnerable to RDX when exposure is extended. The estimated lowest observable adverse effect concentration of RDX was 1,235 mg/kg. No biodegradation products of RDX were detected at all three sampling times. Compared with 2,4,6-trinitrotoluene (TNT), RDX is less toxic to microbes, probably because of its resistance to biodegradation under aerobic conditions, which precludes metabolic activation of nitro groups.     

Toxicity of copper to the collembolan Folsomia fimetaria in relation to the age of soil contamination

The toxicity of copper to the collembolan Folsomia fimetaria L. was studied in soil incubated with copper sulfate for different periods before the introduction of collembolans, to assess the effect of aging of contamination on the toxicity of copper. Adult survival, reproduction, and juvenile size were assessed. No clear influence of differences in contamination age was detected. The data were compared with results from a study performed in soil sampled at an old copper-contaminated site. Large differences in effects existed between spiked soil and field soil when concentrations were expressed on the basis of total soil copper concentrations. EC(10) and EC(50) values for reproduction in spiked soil were ca. 700 and 1400 mg Cu/kg soil, whereas no effects were found in field soil at copper concentrations up to 2500 mg/kg. Most of the differences disappeared when effects were expressed as a function of 0.01 M CaCl(2)-extractable soil copper. the lack of effects in field soil could be explained from the fact that in this field soil the CaCl(2)-extractable concentration was never higher than one-third of the EC(50) estimated for tests in the laboratory spiked soils.     

Studies on the effect of soil aging on the toxicity of pyrene and phenanthrene to a soil-dwelling springtail

Soil samples spiked with five concentrations of pyrene and phenanthrene were aged for 0, 10, 40, and 120 d before toxicity was investigated using a standardized bioassay with the soil-dwelling collembolan Folsomia fimetaria L. Toxic effects were measured as reductions in survival and reproductive output after 3 weeks of exposure. Both pyrene and phenanthrene were degraded in the test system during storage, phenanthrene to a higher degree than pyrene. However, when toxic effects of the tested substances were calculated on the basis of measured concentrations of the parent compounds, toxicity was unaffected by storage for up to 120 d. Many studies have shown a negative correlation between aging and the biodegradation of polycyclic aromatic hydrocarbons (PAHs), indicating a reduction in the bioavailability of PAHs to micro-organisms with time. Our results indicate that a reduction in toxicity, as related to the measured concentrations of the parent compounds in the soil, may not always be expected for aged soils. Provided that metabolites of pyrene and phenanthrene did not significantly contribute to the toxicity in aged soil samples, a possible explanation for the absence of aging effects is the high test concentrations used in combination with the low content of organic carbon (1.6%) in the test soil.     

Comparison of different microbial bioassays to assess metal-contaminated soils

These experiments compared the sensitivity of four different types of bioassay over time after five metals were added to a wide range of soils at the maximum concentrations in the European Union Sewage Sludge Directive. Three were chronic assays (most probable number of Rhizobium leguminosarum, soil microbial C and Biolog substrate utilization). The fourth bioassay, an acute biosensor, employed a lux-marked luminescent bacterium (Escherichia coli) in the soil pore water. Five metals were added to 23 different soils as a mixture at Zn = 300, Cd = 3, Pb = 300, Cu = 135, and Ni = 75 mg/kg as nitrate salts and compared with unamended controls. Zinc and Cu were the metals most likely to be toxic at the concentrations used here. In the case of Rhizobium, the number of cells in soil was not affected after 11 d; however, by 818 d the numbers had decreased by four orders of magnitude with increasing concentrations of Zn and Cu in soil solution. Microbial biomass also was not affected after 11 d, but significantly decreased with increased Zn (p < 0.001) and Cu (p < 0.01) in soil solution after 818 d. Toxicity to the soil microbial biomass increased with time, whereas the toxicity to the biosensor remained the same. Biolog substrate utilization profiles were not responsive to the concentrations used here.     

The influence of soil properties on the toxicity of molybdenum to three species of soil invertebrates

Mo toxicity to earthworms (Eisenia andrei), Collembola (Folsomia candida) and enchytraeids (Enchytraeus crypticus) was determined in 10 European soils and a standard artificial soil, freshly spiked with Na₂MoO₄, after 28 days exposure. Mo affected survival only in three low pH sandy soils; in all other soils LC50 was >3200 mg Mo/kg dry soil. EC50 values for the reproduction toxicity of Mo were 129-2378 mg/kg for earthworms, 72->3396 mg/kg for Collembola, and 301->2820 mg/kg for enchytraeids. Variation in toxicity among soils could not be explained by differences in available (pore water, water and 0.01 M CaCl₂ extractable) Mo concentrations. Clay content best predicted the EC50 for Mo toxicity to earthworms, while toxicity of Mo for enchytraeids was best described by soil pH. For Collembola no relationships could be derived due to the absence of toxicity in most soils. Soil properties had a strong but species-specific effect on Mo toxicity to soil invertebrates.     

Modeling the toxicity of copper and zinc salts to wheat in 14 soils

Interest is mounting in developing and utilizing soil-specific soil quality guidelines. This requires quantifying the effects that soil physicochemical properties have on various ecotoxicological endpoints, including phytotoxicity. To this end, 14 agricultural soils from Australia with differing soil properties were spiked with copper (Cu) and zinc (Zn) salts and used to conduct 21-d plant growth inhibition tests using wheat (Triticum aestivum L.) in pot trials. The toxicity of Cu and Zn was similar with 10% effect concentration (EC10) values ranging from 110 to 945 and from 235 to 965 mg/kg, respectively, while the corresponding median effect concentration (EC50) values ranged from 240 to 1,405 and 470 to 1,745 mg/kg, respectively. Copper toxicity values (EC10, EC20, and EC50) were best modeled by the logarithm of cation exchange capacity (CEC) and either soil pH or electrical conductivity. Zinc EC50 and EC20 values were best modeled using the logarithm of CEC, while the EC10 data were best modeled using soil pH and the logarithm of organic carbon. These models generally estimated toxicity within a factor of two of the measured values.