Bioassays for the ecotoxicological and genotoxicological assessment of contaminated soils (results of a round robin test)

For the assessment of contaminated or remediated soils, in addition to chemical analyses, ecotoxicological tests are performed which focus on the retention function of soils (determined by tests with aqueous soil extracts) and on the habitat function (determined by tests with soil). While numerous standardized test guidelines exist to investigate effects by chemical substances, respective test procedures for soil assessments are lacking. To close this gap, a round robin test was performed to support standardization. Four contaminated soils were tested using a total of fifteen test systems including ecotoxicological and genotoxicological tests with soil extracts and soil. The results obtained from the tests with soil extracts are presented in this article.     

Bioassays for the ecotoxicological and genotoxicological assessment of contaminated soils (Results of a round-robin test)

For the assessment of contaminated or remediated soils, aside from chemical analyses, ecotoxicological tests are performed which focus on the retention function of soils (determined by tests with aqueous soil extracts) and on the habitat function (determined by tests with soil). Whereas numerous tests exist as standardized guidelines for identifying the effect of chemicals, this is not the case for the assessment of soil quality. A round-robin test was performed to monitor the comparability of the results from ecotoxicological test methods on soils and to facilitate the standardization of corresponding test methods. Four contaminated soils were tested using a total number of fifteen test systems, including ecotoxicological and genotoxicological tests with soil extracts and soil. In the second part of this publication series, the results obtained from the tests with soil microorganisms and soil fauna are presented.     

Comparisons of terrestrial and aquatic bioassays for oil-contaminated soil toxicity

Background  Petroleum products are widely used in various sections of industry and they are one of the most abundant sources of environmental contamination. These products are classified by their physico-chemical properties such as boiling point, density and viscosity. Oil contamination in the environment is primarily evaluated by measuring the chemical concentrations of petroleum products in the solid or water phase. The results of chemical analyses do not correspond directly with the harmful environmental effects of petroleum products on the soil flora and fauna, because the interactions between oil compounds and the production of their methabolites in soil are not measured in chemical assessments. These kinds of effects of complex chemical mixtures in soil can be estimated by bioassays. Therefore, ecotoxicological tests are important for estimating soil quality in the risk assessment of oil-contaminated soil sites. Objectives  The objective of this study was to examine the oil-contaminated soil site of a closed petrol station with both chemical and ecotoxicological methods. The goals of this study were to compare the sensitivity of the terrestrial and aquatic bioassays and to compare the toxicity responses of aquatic bioassays determined from three different extraction procedures. In addition, our aim was to characterise a cost-effective battery of bioassays that could be applied to a comparison of oil-contaminated soils. It was in our interest to investigate oil-contaminated soil with oil concentrations of 2500–12000 mg/kg and to find out the possible differences between terrestrial and aquatic toxicity tests. Methods  Six soil samples from a closed petrol station were examined for toxicity with terrestrial and aquatic tests. Terrestrial tests includedEnchytraeus albidus survival and reproduction assays and seed germination assays using wheat, cress, lettuce, and red clover seeds and growth inhibition assays of onions. The toxicities of the water-extractable fractions of the soil samples obtained from three different extractions were tested with aquatic bioassays based on plants (onion and duckweed growth inhibition tests), microbes (luminescent bacteria test), and enzyme inhibition (reverse electron transport test, RET). Chemical analyses of the solid samples were carried out simultaneously. Results. Oil concentrations ranged from 2500 to 12000 mg/kg, BTEX varied from 300 to 2800 mg/kg, and fuel additives: MTBE and TAME from 0.0 1 to 260 mg/kg. Only the sample contain-ing 12000 mg/kg oil had a significant toxic impact on all test organisms. Soil samples with oil concentrations 2500–6200 mg/ kg had no or only slight adverse effects on the test organisms with one exception, theE. albidus reproduction test. TheE. albidus survival and reproduction tests were the most sensitive bioassays of the terrestrial tests, and the luminescent bacteria test of the aquatic tests.     

Application of Bioassays for Risk Characterisation and Remediation Control of Soils Polluted with Nitroaromatics and PAHs

Six soil samples taken from three contaminated sites have been assessed using chemical, ecotoxicological and genotoxicological methods. From two of these sites, samples were characterised after remediation. An overall assessment of the soil samples was done with a scheme consisting of two phases. Remediation of main pollutants in these samples, did in general not lead to a risk reduction of the water-extractable ecotoxic and genotoxic potential. It was concluded, that the assessment and remediation of contaminated sites without consideration of biological test systems is not sufficient. Focussing on single hazardous compounds does in many cases not lead to a reduction of the ecotoxic or genotoxic potential. Consequently, effects of accompanying hazardous compounds, which are not routinely quantified, might be underestimated. It was pointed out, that the assessment of contaminated soil samples on the basis of LID-values applying dual dilution series seems to be sufficient for the evaluation, if there is no need for a quantitative comparison of toxic potentials.     

Membrane Dialysis Extraction (MDE): A Novel Approach for Extracting Toxicologically Relevant Hydrophobic Organic Compounds from Soils and Sediments for Assessment in Biotests

Goal, Scope and Background   Organic solvents are routinely used to extract toxicants from polluted soils and sediments prior to chemical analysis or bioassay. Conventional extraction methods often require the use of heated organic solvents, in some cases under high pressure. These conditions can result in loss of volatile compounds from the sample and the degradation of thermally labile target analytes. Moreover, extracts of soils and sediments also frequently contain substantial quantities of organic macromolecules which can act as sorbing phases for target analytes and in doing so interfere with both chemical analysis and bioassays. Membrane dialysis extraction (MDE) is described as a simple, passive extraction method for selectively extracting toxicologically relevant hydrophobic organic compounds (HOCs) from polluted soils and sediments and anaylzed for its applicability in ecotoxicological investigations. Methods   Toxicologically relevant hydrophobic organic compounds were extracted from wet and dry sediments by sealing replicate samples in individual lengths of pre-cleaned low-density polyethylene (LD-PE) tubing and then dialysing in n-hexane. The efficacy of the MDE method for use in ecotoxicological investigations was assessed by testing the concentrated extracts in the neutral red assay for acute cytotoxicity, in the EROD assay for the presence of dioxin-like compounds and in the Danio rerio fish egg assay for embryotoxic and teratogenic effects. Conditions of the sediment sample (with or without water content), dialysis membrane length and duration of dialysis were analyzed with respect to their impact on three endpoints. Results of the MDE investigations were compared to data obtained in samples prepared using conventional Soxhlet extraction. Results and Discussion   The membrane dialysis extraction was found to be at least as efficient as Soxhlet methodology to extract toxicologically relevant HOCs from sediment samples. In most cases, MDE-derived extracts showed a higher toxicological potential than the Soxhlet extracts. Lack of any significant effects in any MDE controls indicated these differences were not caused by contamination of the LD-PE membrane used. The elevated toxicological potential of MDE extracts is most likely the result of enhanced bioavailability of toxic compounds in consequence of lower amounts of organic macromolecules (i.e. sorbing phases) in the MDE extracts. This effect is probably the result of a size-selective restriction by the LD-PE membrane. Conclusion   Membrane dialysis extraction was found to be a simple, efficient and cost-effective method for the extraction of sediment samples. MDE can be used to extract toxicologically relevant hydrophobic organic compounds from both wet and dry sediments without the risk of loosing volatile and thermally labile target analytes. The size-selectivity of the LD-PE membrane also appears to have the capacity to increase the bioavailablity of potential target analytes in the resulting extracts by retaining much of the organic macromolecules present in the sample. Thus, results suggest that MDE may be particularly useful for the extraction of toxicologically relevant hydrophobic organic compounds from soils and sediments for bioassays and other ecotoxicological investigations. Recommendation and Perspective   Further validation of MDE has been initiated and the applicability of the methodology to other sample types will be investigated. Of particular interest is the potential application of MDE to recover hydrophobic target analytes from biological samples such as muscle, other soft tissues and blood.     

Duration of the standard earthworm avoidance test: Are 48 h necessary?

Ecotoxicological tests are often recommended for the environmental risk assessment (ERA) of contaminated soils. In comparison to chemical residue analysis that focuses on individual contaminants, ecotoxicological tests are able to integrate the effects of the overall contamination, including interactions between individual contaminants, as well as between the contaminants and the various soil properties. However, their use is limited by the fact that the most sensitive tests are chronic tests such as the earthworm reproduction test which lasts 56 days. In order to promote the use of ecotoxicological tests for the routine assessment of contaminated test soils, the usage of short-term earthworm avoidance tests were investigated in the German project ERNTE. According to the International Organization for Standardization (ISO) guideline no. 17512-1, such a test has a duration of just 48 h which, although less than chronic tests, is still quite long in comparison to modern methods of chemical analysis whereby results are often available within 24 h (“on-site analysis”). Therefore, we assessed the effects of shortening the duration of the earthworm avoidance test from 48 to 24 h on the resulting ecotoxicological data. Eight uncontaminated natural soils and 22 contaminated natural soils containing a wide range of chemicals were tested concurrently for 24 and 48 h. Additionally, seven of the uncontaminated natural soils (one was excluded due to its low pH) plus Organization for Economic Co-operation and Development (OECD) artificial soil or LUFA (Landwirtschaftliche Untersuchungs- und Forschungsanstalt, Speyer, Germany) St. 2.2 soil were spiked with two model chemicals (zinc nitrate-tetrahydrate and tributyltin-oxide (TBT-O)) and tested accordingly. It was also investigated whether the results would differ when using either standard LUFA St. 2.2 or OECD artificial soil as a control when contaminated natural soils were tested. Statistical analysis of the data indicates that a decrease in test duration in general did not result in a different assessment of the test soils. In view of the fact that an ERA of contaminated soils is increasingly starting with an on-site analysis (i.e. data are available within 24 h) it is recommended to change the existing ISO guideline, i.e. to decrease the test duration from 48 to 24 h. In doing so, the use of a multi-concentration design increases the robustness of the test results. Both OECD and LUFA St. 2.2 soils are equally suitable as controls.     

Changes in the carbon concentrations and other soil properties of some Scottish agricultural soils: Evidence from a resampling campaign

The change in soil carbon (C) concentration, soil pH and major nutrients for approximately 1,000 topsoil sampled from on-farm experimental sites over a thirty-year period from 1950 to 1980 in north-east Scotland are summarized. This period coincided with increased agricultural intensification, which included regular liming and fertilizer additions. During 2017, 37 of these sites were resampled and reanlaysed. While pH and extractable phosphorus (P) and potassium (K) increased over this period, there was no detectable change in the percentage loss on ignition. Composite soil samples were taken by auger from a depth of 0–15 cm and compared with the corresponding archived samples collected at the initiation of each experiment. Analysis of these resampled soils indicated no significant change in soil carbon (C), although soil pH, extractable magnesium (Mg) and K and Nitrogen (N) concentrations were significantly greater (p < .001) but extractable soil P concentration was significantly less (p = .015) compared with the original samples. Even though measuring C concentration alone is a poor indicator of overall changes in soil C stocks, it does provide a relative quick “early warning” of C losses that would justify a more comprehensive measure of stocks.     

Experiences with Novel Approaches in Earthworm Testing Alternatives (7 pp)

Goal, Scope and Background   The earthworm avoidance test is a sensitive screening test. Currently, two test designs, a two-chamber system and a six-chamber system, are under standardization. In the scope of the present study, the two test systems are compared. To assess the results, two procedures are applied, which are based on a threshold value and a statistical method. Moreover, the sensitivity of the avoidance test is compared with the sensitivity of the reproduction test. Methods   The avoidance behaviour of E. fetida towards five chemicals (Cd, Cu, PCP, TBT, TNT) was tested in a sandy and a loamy soil. The ecotoxicological test was performed according to the draft guideline ISO/DIS 17512-1. The results were compared with the number of offspring determined in the reproduction test carried out according to ISO 11268-2. Results and Discussion   The results demonstrate that the avoidance behaviour towards organic chemicals and heavy metals is a suitable screening method showing first tendencies of a chemical's effects on the habitat function of soils. Effects caused by chemical substances become visible at low concentrations and within short test periods. The sensitivity of the reproduction test and the avoidance test is principally comparable; in some cases, the avoidance test showed more sensitive reactions. The dose-response-relationships were more pronounced in the two-chamber test than in the six-chamber-system. Recommendation and Outlook   The two-chamber-system proved to be more feasible than the six-chamber-system. As the sensitivity of the avoidance test and the reproduction test is comparable, the avoidance test can be considered as a suitable screening test as well. A possible field of application may be the selection of soil samples for which the reproduction assay seems necessary. To assess contaminated or remediated soils, the earthworm reproduction test is recommended as an indicator for the habitat function of the soils. To reduce costs, the labour intensive reproduction test could be largely replaced by the avoidance test in performing the earth-worm reproduction test only for relevant samples. Before the Draft International Standard of the guideline will be adopted as an ISO Guideline, there will be sufficient time left to gain further international experience required to finalize the method.     

Differentiation between bioavailable and total hazard potential of sediment-induced DNA fragmentation as measured by the comet assay with Zebrafish embryos

Goals, Scope and Background  While water quality strongly improved over decades in the Rhine River, sediments still reflect elapsed contaminations of organic pollutants and heavy metals. In comparing genotoxic effects induced by both sediment extracts and whole sediments, a ratio of bioavailable toxicity and total extractable toxicity is obtained. Since contaminated sites whose contaminants are toxic and as well bioavailable present an elevated risk to the ecosystem, such ratios may be used as a warning signal to identify sites of primary concern. Methods  Accordingly, two different exposure scenarios were compared to reveal the genotoxic potential of 18 sediment samples derived from 9 sample sites along the River Rhine. For assessment of effects on genome integrity, DNA fragmentation was measured using the comet assay with primary cells isolated from zebrafish embryos previously exposed to either organic sediment extracts or freeze-dried sediments at sublethal concentrations. Additionally, chemical data were used to determine responsible pollutants and correlate them with biological effects. Results  Whereas 17 out of 18 sediment extracts caused significant DNA damage to the embryo cells, only 4 native sediments showed a genotoxic potential. Thus, under field-like exposure conditions, a major part of potentially genotoxic compounds seem to remain particle-bound and ineffective, as shown for whole sediment exposure. Conversely, the organic extracts seem to contain enriched concentrations even of hardly soluble substances. Hence, organic extracts may be used as a screening tool to address potentially polluted sites, even though the relevance of these results for the field situation may be questionable. Investigations on native sediments determined few sites with bioavailable and therefore ecologically most relevant genotoxic sediment compounds. Discussion  However, these results may underestimate the total hazard potential of sample sites with hardly bioavailable substances. Chemical data revealed a variety of anthropogenic pollutants, ranging from PAHs to heavy metals. Nevertheless, chemical data on the measured priority pollutants did not fully explain the pollution pattern of the bioassays but clearly determined substances of concern (e.g., HCB, heavy metals) in particular sample sites. Conclusions  There is a striking advantage in assessing the genotoxicity by means of different exposure scenarios that focus on either bioavailable or extractable fractions, as the combination of the results allows obtaining information on specific properties of the genotoxicants and their bioavailability. An additional correlation with chemical data should be required to identify priority pollutants, as long as the responsible contaminant is known a priori. As many studies revealed inherent failures of such a correlation, an effect-driven analysis of pollutants is recommended as a promising tool to identify even non-priority pollutants by means of their ecotoxicological effectiveness.     

An initial study into the use of microwave remediation of hexachlorobenzene treated soil using selected oxidants and coated graphite rods

Background, Aims, and Scope  Microwave remediation has become an established method to treat contaminated soils, sediments, and sludges. Chlorinated contaminants are of particular interest due to their widespread industrial use in the past and current potential health hazards. Literature reports the use of microwave heating and oxidants for various reactions, but no work could be found combining microwave heating, persulfate, and coated graphite rods for remediation of halogenated hydrocarbon treated soil. Therefore, this research focused on combining microwave energy combined with oxidants, potassium hydroxide and potassium persulfate, and, coated and uncoated, graphite rods to remove hexachlorobenzene (HCB) from treated soil samples. Materials and Methods  Soil collected from a local pond was dried, sieved, and cleaned by extraction with methylene chloride. Samples were spiked with hexachlorobenzene, then depending on experimental parameters, heated in a microwave oven, had an oxidant added, and had a coated or uncoated graphite rod added. The samples were then extracted with methylene chloride using a Soxhlet extraction setup. The methylene chloride volume was then reduced and analyzed by gas chromatography/mass spectrometry (GC/MS). Additional analysis on the soil and rods was done with powder X-ray diffraction (XRD) and scanning electron microscopy (SEM). Results  The data show that heating alone remediated the soil, but the addition of graphite rods and oxidants enhanced the remediation amount. The oxidant alone removed 200% more HCB than just heating the sample. Using coated graphite rods caused the removal of 125% more than the uncoated graphite rod did. SEM and XRD data show no decomposition of the graphite rods. Discussion  Thermal and chemical effects are likely causes for the enhanced removal. The maximum particle size increased after heating due to vitrification. Conclusions  Although an initial study, this research shows that microwave heating and the addition of oxidants and coated or uncoated graphite rods removed HCB from the treated soil. The removal is due to thermal, microwave and graphite rods, and chemical, oxidants, effects. Recommendations and Perspectives  Hexachlorobenzene remediation of soil was enhanced by microwave heating, insertion of a coated or uncoated graphite rod, and addition of oxidants. Therefore, these procedures could provide a method for solving a widespread problem and returning once contaminated soil back to its natural environment. ESS-Submission Editor: Dr. Willie Peijnenburg (wjgm.peijnenburg@rivm.nl)     

Earthworm avoidance test for soil assessments

For ecotoxicological assessments of contaminated or remediated soils pointing to the habitat function of soils for biocenoses, standardized tests with earthworms (acute test, reproduction test) are available among others. Tests used for routine applications should be sensitive and indicate impacts on test organisms after short test periods. The usually applied earthworm tests do not satisfactorily fulfil these criteria. Therefore, in the present work, a behavioural test with earthworms (test criterion: avoidance) was investigated in detail using uncontaminated, artificially contaminated and originally contaminated soils. It was demonstrated that the avoidance behaviour is primarily determined by pollutants, and not by chemical-physical soil properties. The sensitivity of the presented test reaches the sensitivity of established tests. For waste sites, a considerably higher sensitivity was determined. An avoidance behaviour of at least 80% of the worms leaving the soil to be assessed is proposed as a criterion for toxicity.     

Copper Accumulations in Soils,Coffee, Banana,and Bean Plants Following Copper-Based Fungicides in Coffee Farms in Arusha and Kilimanjaro Regions,Tanzania

This study was carried out to investigate the levels of copper (Cu) contamination in coffee fields in Kilimanjaro and Arusha regions, Tanzania, to increase the database on the contamination of soils by Cu-based fungicides in coffee fields. Surface (0–20 cm deep) soil samples were collected from different farms in Kilimanjaro and Arusha regions. Coffee, banana, and bean plant samples were collected from the locations of soil sampling. Soil and plant samples were analyzed at the Department of Soil Science, Sokoine University of Agriculture, Morogoro, Tanzania. It was found that the calcium chloride (CaCl₂)–extractable Cu was less than the detection limit of flame atomic absorption spectrometry. Diethylenetriaminepentaacetic acid (DTPA)–extractable (24 to 366 mg Cu kg^?1 soil) and aqua regia–extractable (80 to 806 mg Cu kg^?1 soil) Cu levels were high enough to raise environmental alarm (based on European Union guidelines) in the Cu fungicide–treated soils as compared with natural Cu levels in untreated soils (1 to 12 mg Cu kg^?1 soil for DTPA and 22 to 32 mg Cu kg^?1 soil for aqua regia–extractable Cu). Coffee, banana, and bean plants grown on soils contaminated by Cu fungicides had varied concentrations of Cu that were greater than the concentrations of Cu in the plants collected from the uncontaminated soils. Stepwise regression analysis carried out to investigate the relationships between the soil properties and the concentrations of Cu in plants revealed a significant (P = 0.01) positive relationship (R² = 0.4) between organic carbon and the concentration of Cu in banana leaves. Aqua regia–extractable Cu was positively correlated (P = 0.03, R² = 0.4) with the concentrations of Cu in banana leaves. For bean leaves, electrical conductivity (EC) had a positive significant (P = 0.01) relationship (R² = 0.56) with the concentrations of Cu in the plants. It is recommended that further research be carried out to investigate the dynamics and bioavailability of Cu for the different crops interplanted in the coffee fields.     

用突变点检测方法预测木生境下污染土壤中的可提性镉、铅和锌

Accumulation of heavy metals in soils poses a potential risk to plant production, which is related to availability of the metals in soil. The phytoavailability of metals is usually evaluated using extracting solutions such as salts, acids or chelates. The purpose of this study was to identify the most significant soil parameters that can be used to predict the concentrations of acetic and citric acid-extractable cadmium (Cd), lead (Pb) and zinc (Zn) in contaminated woody habitat topsoils. Multiple linear regression models were established using two analysis strategies and three sets of variables based on a dataset of 260 soil samples. The performance of these models was evaluated using statistical parameters. Cation exchange capacity, CaCO3, organic matter, assimilated P, free Al oxide, sand and the total metal concentrations appeared to be the main soil parameters governing the solubility of Cd, Pb and Zn in acetic and citric acid solutions. The results strongly suggest that the metal solubility in extracting solutions is extractable concentration-dependent since models were overall improved by incorporating a change point. This change point detection method was a powerful tool for predicting extractable Cd, Pb and Zn. Suitable predictions of extractable Cd, Pb and Zn concentrations were obtained, with correlation coefficient (adjusted r) ranging from 0.80 to 0.99, given the high complexity of the woody habitat soils studied. Therefore, the predictive models can constitute a decision-making support tool for managing phytoremediation of contaminated soils, making recommendations to control the potential bioavailability of metals. The relationships between acetic and/or citric acid-extractable concentrations and the concentrations of metals into the aboveground parts of plants need to be predicted, in order to make their temporal monitoring easier.     

Optimisation of the Solid-Contact Test with Arthrobacter globiformis (7 pp)

Background, Aim and Scope   A number of biotests are available for the characterisation of solid matters such as soil or sediment. Among these, bacterial biotests using single test species often analyse the toxicity of water-soluble contaminants in aquatic extracts, but there is also a need for a fast and inexpensive bacterial solid-contact test. In this study, a solid contact test with added bacteria (Arthrobacter globiformis) was optimised (through miniaturization) for the development of a test kit with conserved bacteria. As in other tests, the results can be influenced by natural soil factors, often masking anthropogenic impacts. For this reason, a further goal of this study was the investigation of the influence of natural soil characteristics on the result of the solid contact test. The project is part of the joint research project 'Optimization of ecotoxicological test methods for routine use' (abbreviated as ERNTE-Forschungsvorhaben: 0330305). Materials and Methods: This method is based on an existing German standard (DIN 38412 L 48) using Arthrobacter globiformi for testing whole soils and sediments. The test principle is the measurement of the dehydrogenase activity of the test organism A. globiformis after an incubation time of two hours with the solid material. To attain the miniaturization in microplates, dye measurement was changed from spectrophotometrical determination of the substrate resazurine to the fluorimetric measurement of the product resorufin. A second step towards optimisation was the use of freeze-dried bacteria. Seven selected uncontaminated soils were tested in order to determine the influence of natural soil characteristics on the results of the solid contact test with A. globiformis. Freshly spiked and polluted field soils were analysed in order to obtain information about the sensitivity of the test. Results: It is possible to perform the contact test in microplates. The fluorimetric dye measurement can be carried out in the presence of the solid material, so the work-intensive step of centrifugation and filtration is no longer necessary. The measurement in the optimised contact test is based on the kinetics of the enzyme reaction. The investigation showed that conserved bacteria have the same activity and sensitivity as cultivated bacteria. Discussion: The study of the uncontaminated soils demonstrated the influence of various soil characteristics on the results of the solid contact test. This information is the basis for the selection of the control and reference soils and is crucial for setting the threshold value in toxicity testing. The investigation of freshly spiked and contaminated soils showed a different sensitivity dependent on the kind of the contamination. Conclusions: The solid contact test was successfully optimised using microplates, whereas now less than six hours are necessary for the analysis. The optimised test is rapid and sensitive, requiring small samples and no stock culture of the bacteria A. globiformis if using freeze-dried bacteria. In this study, the effect of natural soil factors such as pH-value was shown. This information is used to define the threshold value for toxicity. Therefore, the optimised contact test can be used for an efficient assessment of soil or soil substrates. Further studies will clear up if this optimisation is also valid for aquatic sediments and waste. Recommendations and Perspectives: Due to its short analysis time, the test is suitable for screening different kinds of solid matter and can be used for on-site analysis. - The optimised contact test with freeze-dried bacteria as part of a battery of tests is appropriate for the assessment of contaminated soils, sediments and waste.     

Recycling of Remediated Soil for Effective Composting Of Diesel-Contaminated Soil

Soil contaminated with diesel oil was remediated by the addition of remediated soil. Several mix ratios of contaminated soil to remediated soil were tested. Judging from TPH degradation rate and biochemical parameters, the optimum mix ratio (wet weight basis) was 1:1. In this mix ratio, the first order degradation rate constant of diesel oil based on TPH was 0.099/day. Degradation rate of TPH and total amount of CO₂ evolved in this condition were two times larger than those of contaminated soil without adding remediated soil. The addition of remediated soil was a very effective treatment option to facilitate the degradation rate of diesel oil in contaminated soil.     

Production of the forage halophyte Atriplex amnicola in metal‐contaminated soils

Clean‐up of contaminated soils is a costly and slow process that requires long periods of time to be effective. Therefore, direct use of contaminated sites with appropriate management is often likely to be a more efficient use of such land. Consequently, the production of safe animal forages from contaminated soils was the aim of this research. Field studies were conducted to evaluate the growth and elemental composition of river saltbush (Atriplex amnicola) grown on a metal‐contaminated soil. The soil was amended with compost at rates of 0, 15 and 30 t/ha to assess its role on plant growth and metal uptake. Compost application significantly (P < 0.05) increased biomass yield, crude protein (CP) and ash content of river saltbush; in contrast, it decreased the Zn and Pb concentrations in shoot tissues. When 30 t/ha of compost was added, the Pb concentrations in the stems and leaves decreased by 32 and 38%, respectively. Despite the large total and extractable content of metals in the studied soil, shoot concentrations of these metals in A. amnicola were always maintained below potentially toxic levels. The biomass material of A. amnicola had a high nutritive value compared to conventional forage crops and could safely be used as animal forage. This work demonstrates that an Atriplex spp, A. amnicola, has significant potential for use as a safe forage crop in the sustainable on‐site management of contaminated soils.     

Effects of azoxystrobin,chlorothalonil, and ethoprophos on the reproduction of three terrestrial invertebrates using a natural Mediterranean soil

The potential terrestrial toxicity of three pesticides, azoxystrobin, chlorothalonil, and ethoprophos was evaluated using reproduction ecotoxicological tests with different non-target species: the collembolan Folsomia candida, the earthworm Eisenia andrei, and the enchytraeid Enchytraeus crypticus. All reproduction tests were performed with natural soil from a Mediterranean agricultural area (with no pesticide residues) in order to improve the relevance of laboratory data to field conditions. Controls were performed with natural and standard artificial soil (OECD 10% OM). The fungicide azoxystrobin showed the highest toxicity to earthworms (EC₅₀ = 42.0 mg a.i. kg⁻¹ dw soil). Collembolans were the most sensitive taxa in terms of sublethal effects of chlorothalonil with an EC₅₀ of 31.1 mg a.i. kg⁻¹ dw soil followed by the earthworms with an EC₅₀ of 40.9 mg a.i. kg⁻¹ dw soil. The insecticide ethoprophos was the most toxic to collembolans affecting their reproduction with an EC₅₀ of 0.027 mg a.i. kg⁻¹ dw soil. Enchytraeids were generally the least sensitive of the three species tested for long-term effects. Earthworms were not always the most sensitive species, emphasizing the need to increase the number of mandatory assays with key non-target organisms in the environmental risk assessment of pesticides.     

Novel chelant‐based washing method for soil contaminated with Pb and other metals: A pilot‐scale study

Remediation with chelants can restore metal‐contaminated soils for use as a natural resource. Calcareous soil from Meza Valley, Slovenia, and acidic soils from Arnoldstein, Austria, and Pribram, Czech Republic (with 1,028, 862, and 926 mg ∙Pb∙kg⁻¹, respectively), were washed with 60–100 mmol EDTA per kilogram of air‐dried soil in series of 30 batches (50 kg soil batch⁻¹). The approach involves a novel reaction that incorporates alkaline substitution, precipitation and adsorption of toxic metals on polysaccharides, and chelant acidic precipitation via 83% EDTA (on average) and complete process water recycling (no wastewater was generated). The pH gradient was imposed by Ca(OH)₂ and H₂SO₄, and excess reagent was removed with the remediated soil as CaSO₄, thereby preventing the salification of the recycled waters. Remediation removed 60%, 78%, and 71% of the Pb from the Meza, Arnoldstein, and Pribram soils, respectively, and reduced the Pb bioaccessibility levels in the simulated human gastrointestinal phase by 5.0, 7.7, and 8.1 times. Residual emissions (EDTA, toxic metals) were reduced with soil aging and remediated soil deposition on a reactive permeable barrier. The solid waste generated from the process totaled 10.8 kg tons⁻¹ of the air‐dried soil, and the material/energy costs of remediation reached 20.6 € tons⁻¹. These results demonstrate the robustness, efficiency, and safety of this novel approach.     

Potential of Tamarix africana and other halophyte species for phytostabilisation of contaminated salt marsh soils

Purpose

Salt marsh plants are colonising wastes from a steel plant deposited on the Coina River Banks posing a potential contamination risk to the Tagus estuary ecosystem. The objectives of this study were to assess the uptake, accumulation and translocation of hazardous elements/nutrients in three spontaneous halophytic species, to evaluate the capacity of Tamarix africana to stabilise a contaminated salt marsh soil, and to evaluate the ecotoxicity of the pore water and elutriates from phytostabilised soils.

Materials and methods

The work comprises the following: fieldwork collection of soil samples from Coina River (an affluent of Tagus River) bank landfill, estuarine water and spontaneous plants (Aster tripolium, Halimione portulacoides and Sarcocornia sp.), and greenhouse studies (microcosm assay) with T. africana growing in one landfill salt marsh soil, for 97 days, and watered with estuarine water. Soils were analysed for pH, EC, C_organic, NPK, iron and manganese oxides. Soils total (acid digestion) elemental concentrations were determined by ICP/INAA. Estuarine waters, plants roots and shoots (acid digestion), soils available fraction (diluted organic acids extraction-RHIZO or pore water), and salts collected from the T. africana leaves surface were analysed for metals/metalloids (ICP-MS). Ecotoxicity assays were performed in T. africana soil elutriates and pore waters using Artemia franciscana and Brachionus plicatillis.

Results and discussion

Soils were contaminated, containing high total concentrations of arsenic, cadmium, chromium, copper, lead and zinc. However, their concentrations in the available fraction were <4 % of the total. The estuarine waters were contaminated with cadmium, but negligible ecotoxicological effect was observed. The spontaneous plants had significant uptake of the above elements, being mostly stored in the roots. Elemental concentrations in the shoots were within the normal range for plants. These species are not hazardous elements accumulators. Tamarix africana was well adapted to the contaminated saline soils, stored the contaminants in the roots, and had small concentrations of hazardous elements in the shoots. Excretion of hazardous elements by the salt glands was also observed. Elutriates from soils with and without plant did not show ecotoxicity.

Conclusions

The salt marsh species play an important role in the stabilisation of the soils in natural conditions. Tamarix africana showed potential for phytostabilisation of saline-contaminated soils. The low translocation of the elements from roots to shoots and/or active excretion of the elements by the salt glands was a tolerance mechanism in T. africana.

     

A Novel Particle Contact Assay with the Yeast Saccharomyces cerevisiae (8 pp)

Goal, Scope and Background   Numerous xenobiotics released into surface waters are transferred to suspended particulate matter and finally attached to sediments. Aquatic organisms may be exposed to them by direct particle feeding, by physical contact with contaminated surfaces as an exposure route, and by the uptake of dissolved contaminants after equilibration via the free water phase. In order to assess potential sediment toxicity, each of these exposure routes has to be addressed. This paper presents a newly developed particle contact assay that uses the fermentation performance of a specific Saccharomyces cerevisiae strain for the assessment of toxic effects in sediments. The test procedure is based on the characteristic feature of growing yeast cells to attach to sediment particles, which are also relevant for the accumulation of contaminants. The physical contact with lipophilic contaminants mirrors an exposition pathway for the direct uptake into the cells. In order to quantitatively characterize the toxic effects of particle attached pollutants on the fermentation performance, unpolluted native reference sediment was spiked with representatives for widely distributed anthropogenic contaminants. Methods   Saccharomyces cerevisiae was established as sensitive eukaryotic microorganism for the ecotoxicological assessment of particle attached anthropogenic contaminants in freshwater sediments. For this purpose, yeast cells were cultivated in sediment samples and the resulting fermentation performance was continuously measured. Sediments artifically spiked with HCB, PCB, g-HCH, DDT, and benzo(a)pyrene and solutions of each contaminant were comparatively investigated by means of their adverse effects on yeast fermentation performance. Additionally, four native river sediments characterized by increasing levels of pollution were assessed by the yeast particle contact assay, and simultaneously by standard aquatic tests with algae, daphniae, and luminescent bacteria using pore water and elutriates. Results of the bioassays were related to specific sediment contamination with respect to metals and organic priority pollutants. Results and Discussion   In sediments spiked with PCB and benzo(a)pyrene fermentation, performance was affected extensively below concentrations inhibiting fermentation in contaminant solutions. This suggests a high efficiency of the exposure route by physical contact. The fermentation performance was only slightly affected by single lipophilic pollutants, whereas mixtures of individually spiked sediments caused critically reduced fermentation performance suggesting additive synergistic effects. Native river sediments modestly to critically polluted by hazardous organic compounds lead to a slightly to dangerously reduced fermentation performance in the yeast contact assay. These inhibitory effects were much less pronounced in the standard bioassays conducted with algae, daphniae and luminescent bacteria, applying pore waters and elutriates as sample matrices. Using pore water, inhibition was measured only in the most polluted sediment, elutriates lead to a slight inhibition of the algal growth in the undiluted sample only. These results indicate an improved sensitivity of the yeast particle contact assay compared to the standard assays, due to uptake and physical cell contact as additional routes of exposure. Conclusion   The yeast particle contact assay is a valuable tool for the assessment of ecotoxicological potential in freshwater sediments. Since the assay addresses physical contact as an exposure route, it indicates bioavailability of lipophilic compounds in sediments. Outlook   The sensitive indication of bioavailable contaminants associated to sediment particles by the newly developed yeast particle contact assay recommends it as a complementary microbial bioassay in a test battery for assessing major pathways of contaminants in whole sediments.