Inter-laboratory evaluation of the ISO standard 11063 "Soil quality - Method to directly extract DNA from soil samples".

Extracting DNA directly from micro-organisms living in soil is a crucial step for the molecular analysis of soil microbial communities. However, the use of a plethora of different soil DNA extraction protocols, each with its own bias, makes accurate data comparison difficult. To overcome this problem, a method for soil DNA extraction was proposed to the International Organization for Standardization (ISO) in 2006. This method was evaluated by 13 independent European laboratories actively participating in national and international ring tests. The reproducibility of the standardized method for molecular analyses was evaluated by comparing the amount of DNA extracted, as well as the abundance and genetic structure of the total bacterial community in the DNA extracted from 12 different soils by the 13 laboratories. High quality DNA was successfully extracted from all 12 soils, despite different physical and chemical characteristics and a range of origins from arable soils, through forests to industrial sites. Quantification of the 16S rRNA gene abundances by real time PCR and analysis of the total bacterial community structure by automated ribosomal intergenic spacer analysis (A-RISA) showed acceptable to good levels of reproducibility. Based on the results of both ring-tests, the method was unanimously approved by the ISO as an international standard method and the normative protocol will now be disseminated within the scientific community. Standardization of a soil DNA extraction method will improve data comparison, facilitating our understanding of soil microbial diversity and soil quality monitoring.

Total concentrations and speciation of heavy metals in soils of the Shenyang Zhangshi Irrigation Area, China.

The Shenyang Zhangshi Irrigation Area (SZIA) was used for the spreading of municipal and industrial waste water, which is an economic way of irrigating crops, recycling nutrients and water treatment. Long-term irrigation resulted in a severe metal contamination of soils. To identify the soil phases implicated in retaining the metals, sequential extractions were performed. The most predominant metal was cadmium which was mainly associated with mobile, easily soluble and easily reducible fractions. Copper was mainly associated with the residual, EDTA extractable and moderately reducible fractions. Lead was bound to organic matter and poorly crystalline Fe-oxides. Nickel and zinc were mainly associated with the residual and strongly reducible fractions. Although copper, lead, nickel and zinc concentrations were of minor importance mobile metal concentrations of these metals as well as of cadmium exceeded German trigger values for plant production and plant growth.

Effects of vegetable oil residue after soil extraction on physical-chemical properties of sandy soil and plant growth.

Vegetable oil has the ability to extract polycyclic aromatic hydrocarbons (PAHs) from contaminated sandy soil for a remediation purpose, with some of the oil remaining in the soil. Although most of the PAHs were removed, the risk of residue oil in the soil was not known. The objective of this study was to evaluate the effects of the vegetable oil residue on higher plant growth and sandy soil properties after soil extraction for a better understanding of the soil remediation. Addition of sunflower oil and column experiment were performed on a PAH contaminated soil and/or a control soil, respectively. Soils were incubated for 90 d, and soil pH was measured during the soil incubation. Higher plant growth bioassays with Avena sativa L. (oat) and Brassica rapa L. (turnip) were performed after the incubation, and then soil organic carbon contents were measured. The results show that both the nutrient amendment and the sunflower oil degradation resulted in the decrease of soil pH. When these two process worked together, their effects were counteracted due to the consumption of the nutrients and oil removal, resulting in different pH profiles. Growth of A. sativa was adversely affected by the sunflower oil, and the nutrient amendments stimulated the A. sativa growth significantly. B. rapa was more sensitive to the sunflower oil than A. sativa. Only 1% sunflower oil addition plus nutrient amendment stimulated B. rapa growth. All the other treatments on B. rapa inhibited its growth significantly. The degradation of the sunflower oil in the soils was proved by the soil organic carbon content.

Ecotoxicological characterization of hazardous wastes.

In Europe hazardous wastes are classified by 14 criteria including ecotoxicity (H 14). Standardized methods originally developed for chemical and soil testing were adapted for the ecotoxicological characterization of wastes including leachate and solid phase tests. A consensus on which tests should be recommended as mandatory is still missing. Up to now, only a guidance on how to proceed with the preparation of waste materials has been standardized by CEN as EN 14735. In this study, tests including higher plants, earthworms, collembolans, microorganisms, duckweed and luminescent bacteria were selected to characterize the ecotoxicological potential of a boiler slag, a dried sewage sludge, a thin sludge and a waste petrol. In general, the instructions given in EN 14735 were suitable for all wastes used. The evaluation of the different test systems by determining the LC/EC(50) or NOEC-values revealed that the collembolan reproduction and the duckweed frond numbers were the most sensitive endpoints. For a final classification and ranking of wastes the Toxicity Classification System (TCS) using EC/LC(50) values seems to be appropriate.

Genotoxicity assessment of soils from wastewater irrigation areas and bioremediation sites using the Vicia faba root tip micronucleus assay.

Genotoxicity potential of soils taken from wastewater irrigation areas and bioremediation sites was assessed using the Vicia faba root tip micronucleus assay. Twenty five soils were tested, of which 8 were uncontaminated soils and taken as the control to examine the influence of soil properties; 6 soils were obtained from paddy rice fields with a history of long-term wastewater irrigation; 6 soils were obtained from bioremediation sites to examine effects of bioremediation; and 5 PAH-contaminated soils were used to examine methodological effects between direct soil exposure and exposure to aqueous soil extracts on micronuclei (MN) frequency ( per thousand) in the V. faba root tips. Results indicate that soil properties had no significant influences on MN frequencies (p > 0.05) when soil pH varied between 3.4 to 7.6 and organic carbon between 0.4% and 18.6%. The MN frequency measured in these control soils ranged from 1.6 per thousand to 5.8 per thousand. MN frequencies in soils from wastewater irrigation areas showed 2- to 48-fold increase as compared with the control. Soils from bioremediation sites showed a mixed picture: MN frequencies in some soils decreased after bioremediation, possibly due to detoxification; whereas in other cases remediated soils induced higher MN frequencies, suggesting that genotoxic substances might be produced during bioremediation. Exposure to aqueous soil extracts gave a higher MN frequency than direct exposure in 3 soils. However, the opposite was observed in the other two soils, suggesting that both exposure routes should be tested in case of negative results from one route. Data obtained from this study indicate that the MN assay is a sensitive assay suitable for evaluating genotoxicity of soils.

Polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and heavy metals (HMs) as well as their genotoxicity in soil after long-term wastewater irrigation.

A study was carried out on the residue level of major concern pollutants (PAHs, PCBs and HMs) and the assessment of their genotoxicity in soils obtained from Shenyang, northeast of China which had been subjected to wastewater irrigation for more than 40 years. Topsoils (0-20cm) in paddy fields were sampled along the upper, middle and lower reaches of the wastewater disposal channel. Sixteen USEPA PAHs were determined by High Performance Liquid Chromatography (HPLC) with fluorescence detector, eight PCBs were detected by Gas Chromatography (GC) with electron capture detector, and six heavy metals (Cd, Cu, Pb, Zn, Ni, and Cr) were measured by Atomic Absorption Spectrophotometer (AAS). The genotoxicity effect of soils was examined by Vicia faba micronucleus (MN) test. Archived soils that had undergone a similar history of wastewater irrigation provided by Technical University of Berlin, Germany were subjected to analysis of the above pollutants and Vicia faba/MN test for comparison. Results indicated elevated residues of the studied pollutants (PAHs, especially benzo (a) pyrene, the eight PCB congeners and heavy metals) in both tested and archived soils. The MN frequencies were 2.2-48.4 times higher compared with the control. However, there was no correlation between the MN frequencies and the concentration of pollutants detected. This investigation suggested a potential ecological risk even with a lower level of residual pollutants in soil matrix after long-term wastewater irrigation.

Removal of polycyclic aromatic hydrocarbons from manufactured gas plant-contaminated soils using sunflower oil: laboratory column experiments.

Laboratory column experiments were performed to remove PAHs (polycyclic aromatic hydrocarbons) from two contaminated soils using sunflower oil. Two liters of sunflower oil was added to the top of the columns (33 cm x 21 cm) packed with 1 kg of PAH-contaminated soil. The sunflower oil was applied sequentially in two different ways, i.e. five additions of 400 ml or two additions of 1l. The influence of PAH concentration and the volume of sunflower oil on PAH removal were examined. A soil respiration experiment was carried out and organic carbon contents of the soils were measured to determine degradability of remaining sunflower oil in the soils. Results showed that the sunflower oil was effective in removing PAHs from the two soils, more PAHs were removed by adding sunflower oil in two steps than in five steps, probably because of the slower flow rate in the former method. More than 90% of total PAHs was removed from a heavily contaminated soil (with a total 13 PAH concentration of 4721 mg kg(-1)) using 4 l of sunflower oil. A similar removal efficiency was obtained for another contaminated soil (with a total 13 PAH concentration of 724 mg kg(-1)), while only 2l was needed to give a similar efficiency. Approximately 4-5% of the sunflower oil remained in the soils. Soil respiration curves showed that remaining sunflower oil was degraded by allowing air exchange and supplying with nutrients. Organic carbon content of the soil was restored to original level after 180 d incubation. These results indicated that the sunflower oil had a great capacity to remove PAHs from contaminated soils, and sunflower oil solubilization can be an alternative technique for remediation of PAH contaminated soils.

Influence of soil moisture on sunflower oil extraction of polycyclic aromatic hydrocarbons from a manufactured gas plant soil.

The influence of soil moisture on efficiency of sunflower oil extraction of polycyclic aromatic hydrocarbons (PAHs) from contaminated soil was investigated. The PAH-contaminated soil was collected from a manufactured gas plant (MGP) site in Berlin, Germany. Half of the soil was air-dried, and the other half was kept as field-moist soil. Batch experiments were performed using air-dried and field-moist soils, and sunflower oil was used as extractant at oil/soil ratios of 2:1 and 1:1 (v/m). The experimental data were fitted to a first-order empirical model to describe mass-transfer profiles of the PAHs. Column extraction experiments were also conducted. Field-moist and air-dried soils in the column were extracted using sunflower oil at an oil/soil ratio of 2:1. In the batch experiments, PAHs were more rapidly extracted from air-dried soil than from field-moist soil. Removal rate of total PAH increased 23% at oil/soil ratio of 1:1 and 15.5% at oil/soil ratio of 2:1 after the soil was air dried. The most favorable conditions for batch extraction were air-dried soil, with an oil/soil ratio of 2:1. In the column experiments, the removal rate of total PAH from air-dried soil was 30.7% higher than that from field-moist soil. For field-moist soil, extraction efficiencies of the batch extraction (67.2% and 81.5%) were better than that for column extraction (65.6%). However, this difference between the two methods became less significant for the air-dried soil, with a total removal rate of 96.3% for column extraction and 90.2% and 97% for batch extractions. A mass-balance test was carried out for analytical quality assurance. The results of both batch and column experiments indicated that drying the soil increased efficiency of extraction of PAHs from the MGP soil.

Dissolution and removal of PAHs from a contaminated soil using sunflower oil.

This study reports on the feasibility of remediation of polycyclic aromatic hydrocarbon (PAH) contaminated soils using sunflower oil, an environmentally-friendly solvent. Batch experiments were performed to test the influence of oil/soil ratio on the remediation of PAH contaminated soil, and to test the mass transfer behaviors of PAHs from soil to oil. An empirical model was employed to describe the kinetics of PAH dissolution and to predict equilibrium concentrations of PAHs in oil. PAH containing oil was regenerated using active carbon. Results show that dissolution of PAHs from a Manufactured Gas Plant (MGP) soil at oil/soil ratios of one or two were almost the same. Nearly all PAHs (81-100%) could be removed by sunflower oil dissolution. Mass transfer coefficients for low molecular PAHs namely fluoranthene, phenanthrene and anthracene were one or two orders of magnitude higher than those for high molecular PAHs with 4-6 rings. Ninety milliliters of PAH containing oil could be regenerated by 10 g active carbon in a batch reactor. Such a remediation procedure indicates that sunflower oil is a promising agent for the removal of PAHs from MGP soils. However, further research is required before the method can be used for in situ remediation of contaminated sites.

Comparative study of extraction methods for the determination of PAHs from contaminated soils and sediments.

The following four methods were compared on the extraction efficiency of 16 EPA (US Environmental Protection Agency) polycyclic aromatic hydrocarbons (PAHs): German method of the Verband Deutscher Landwirtschaftlicher Untersuchungs und Forschungsanstalten (VDLUFA), two methods of the International Organization for Standardization using shaking (ISO A) and Soxhlet extraction (ISO B) and an ultrasonic method. Recovery rates of 16 PAHs were determined in two soils. Extraction efficiency was evaluated in five soils and three sediments. Effect of drying soils and sediments on extraction efficiency was tested using the VDLUFA and the ultrasonic methods. Our study shows that the number of aromatic rings, rather than extraction procedures, significantly influenced recovery rates of individual PAHs. No significant differences in extraction efficiency of the four methods were observed for less polluted samples. For highly polluted soils, extraction efficiency decreased in the following order: VDLUFA method > ISO A > ultrasonic method > ISO B. Influence of soil moisture on extraction efficiency depended to some extent on both solvent used and content of PAHs in samples. A mixture of dichloromethane/acetone (5:1) is recommended for PAH extraction from moist samples when the ultrasonic method is used.

Evaluation and refinement of a continuous seed germination and early seedling growth test for the use in the ecotoxicological assessment of soils.

Four higher plant species (Avena sativa L., oat; Lepidium sativum L., cress; Brassica rapa Metzg., turnip; and Phaseolus vulgaris L., bush bean) and 15 soils including five mineral oil-contaminated soils were tested to assess a soil-based continuous seed germination and early seedling growth bioassay. Seed germination was recorded and the shoot biomass of 14-day seedlings measured. An automatic self-watering system was compared with the daily hand watering. Results obtained from this study show that the self-watering system had minor effects on both seed germination and early seedling growth, and that soil nutrient status rather than soil texture significantly affected both seedling emergence and shoot biomass. Proportional dilution of suspicious samples from primary tests with an appropriate control soil is recommended for secondary tests. Shoot biomass is a more sensitive measurement endpoint than seed germination. Bush bean should not be recommended for use due to its low sensitivity to mineral oil and poor germination in soil.