Compost amendment in urban gardens: elemental and isotopic analysis of soils and vegetable tissues.

Urban horticulture poses a sustainable form of food production, fosters community engagement and mitigates the impacts of climate change on cities. Yet, it can also be tied to health challenges related to soil contamination. This work builds on a previous study conducted on eleven urban gardens in the city of Vienna, Austria. Following the findings of elevated Pb levels in some soil and plant samples within that project, the present study investigates the elemental composition of soil and plants from two affected gardens 1 year after compost amendment. Inductively coupled plasma mass spectrometry (ICP-MS) analysis of skin, pulp and seeds of tomato fruits revealed minor variations in elemental composition which are unlikely to have an impact on food safety. In turn, a tendency of contaminant accumulation in root tips and leaves of radishes was found. Washing of lettuce led to a significant reduction in the contents of potentially toxic elements such as Be, Al, V, Ni, Ga and Tl, underscoring the significance of washing garden products before consumption. Furthermore, compost amendments led to promising results, with reduced Zn, Cd and Pb levels in radish bulbs. Pb isotope ratios in soil and spinach leaf samples taken in the previous study were assessed by multi-collector (MC-) ICP-MS to trace Pb uptake from soils into food. A direct linkage between the Pb isotopic signatures in soil and those in spinach leaves was observed, underscoring their effectiveness as tracers of Pb sources in the environment.

Stable isotope analysis of atmospheric CO2 using a Gasbench II-Cold Trap-IRMS setting.

RATIONALE: The measurement of the stable carbon and oxygen isotope ratio of (atmospheric) carbon dioxide (CO2 ) is a useful technique for the investigation and identification of the sources and sinks of the most abundant greenhouse gases by far. For this reason, we are presenting a measuring system here that enables a wide range of users to carry out stable isotope analysis of atmospheric CO2 using off-the-bench hardware and software. METHODS: The fully automated system uses cryogenic and gas chromatographic separation to analyse CO2 from 12-mL whole air samples and consists of an autosampler, a Gasbench II (GB), a downstream cryo trap and a continuous flow gas interface feeding into a sector field mass spectrometer (GC Pal/GB/Cold Trap/ConFlo IV/DeltaV Plus). The evaluation of the system performance was based on the analysis of samples prepared from eight CO2 sources (four CO2 reference gases and four artificial air tanks). RESULTS: The overall measurement uncertainty (averaged single standard deviation (1σ) of measurement replicates from each CO2 source) in the determination of the carbon and oxygen isotope ratio was 0.04‰ and 0.09‰ (n = 24). Furthermore, we were able to show that the measurement data also allowed for the quantification of the CO2 mole fraction, with a precision of 1.2 µmol mol-1 in the analysis range of 400-500 µmol mol-1 . CONCLUSIONS: Our protocol provides a detailed description of the measurement set-up and the analysis procedure, how raw data should be evaluated and gives recommendations for sample preparation and sampling to enable a fully automated whole air sample analysis. The quantification limit of CO2 mole fractions and measurement precision for carbon and oxygen isotope ratios of CO2 should meet the requirements of a wide range of users.

Carbon isotope effects in the sorption of chlorinated ethenes on biochar and activated carbon.

As an alternative to activated carbon, biochar is a promising, environmentally friendly sorbent that can be used to remove organic groundwater pollutants, such as chlorinated ethenes (CEs). Stable isotope fractionation in biofilters is used to quantify pollutant degradation and to distinguish degradation from pollutant sorption on e.g. biochar. However, the sorption of CEs on biochar, and the potential abiotic fractionation processes remain to be tested. The sorption process of CEs and ethene on activated carbon and biochar was investigated with regard to the isotope effects for the differentiation from microbial degradation processes. Results from physical and chemical characterization of biochar indicated that biochar feedstock and pyrolysis conditions determined sorption performance depending on the surface chemistry and the pore size distribution of the coarse sorbent particles. The sorption capacity of the activated carbon was significantly higher with highly chlorinated ethenes, but similar to the biochars with low chlorination. Apparent carbon isotope fractionation factors (ε) of +0.1 to -4.4 ‰ were found above measurement uncertainties of GC/IRMS. The extent of isotope enrichment of the 13C bearing isotopologues in the residual aqueous phase (ε < 0) was characteristic for individual pairs of pollutant and sorbent material and could be related to pore-filling processes limited by the micropore size distribution of sorbent materials and the chemical properties of sorbed pollutants. Especially the large isotope fractionation during the sorption of ethene led to the assumption that diffusion processes within the pore matrix of the sorbent particles contributed to the observed isotope effects, but should still be considered a property of sorption. Concluding on the results indicated that sorption processes can have a significant contribution to carbon isotope fractionation in CEs and ethene. These should not be neglected in the evaluation of biofilters for groundwater purification, in which CEs are simultaneously degraded by microbes.

Plant invasion causes alterations in Darwin's finch feeding patterns in Galápagos cloud forests.

Invasive species pose a major threat to forest biodiversity, particularly on islands such as the Galapágos. Here, invasive plants are threatening the remnants of the unique cloud forest and its iconic Darwin's finches. We posit that food web disturbances caused by invasive Rubus niveus (blackberry), have contributed to the rapid decline of the insectivourous green warbler finch (Certhidae olivacea). We compared the birds' dietary changes in long-term management, short-term management and unmanaged areas. We measured C:N ratios, and δ15N­nitrogen and δ13C­carbon values in both consumer tissues (bird-blood) and food sources (arthropods), as indicators of resource use change, and collected mass abundance, and arthropod diversity data. We characterised the birds' diets using isotope mixing models. The results revealed that finches in (blackberry-invaded) unmanaged areas foraged more on abundant, yet lower quality, arthropods present in the invaded understory. This suggests that blackberry encroachment leads to a decrease in food source quality with physiological consequences for green warbler finch chicks. Results also implied that blackberry control has a short-term impact on food source quantity, which led to a decrease in chick recruitment that we observed in our previous studies; despite this, in the long-term, these managed systems show signs of recovery within three years of restoration.

Remediating Garden Soils: EDTA-Soil Washing and Safe Vegetable Production in Raised Bed Gardens.

Soil remediation is an important practice in the restoration of heavy metal-contaminated soils and reduce the heavy metal exposure of the local population. Here, we investigated the effect of an ex-situ soil washing technique, based on ethylenediaminetetraacetic acid (EDTA) as a chelating agent, on a contaminated Cambisol. Lead, Cd and Zn were investigated in different soil fractions, drainage water and four vegetables from August 2019 to March 2021. Three treatments consisting of (C) contaminated soil, (W) washed soil and (WA) washed soil amended with vermicompost and biochar were investigated in an outdoor raised bed set up. Our results showed that the total and bioavailable metal fractions were significantly reduced but failed to meet Austrian national guideline values. Initial concentrations in the soil leachate increased significantly, especially for Cd. Vegetables grown on the remediated soil took up significantly lower amounts of all heavy metals and were further reduced by the organic amendment, attaining acceptable values within EU guideline values for food safety. Only spinach exceeded the thresholds in all soil treatments. The increase in soil pH and nutrient availability led to significantly higher vegetable yields.

Effect of Chelant-Based Soil Washing and Post-Treatment on Pb, Cd, and Zn Bioavailability and Plant Uptake.

The remediation of Pb, Cd, and Zn contaminated soil by ex situ EDTA washing was investigated in two pot experiments. We tested the influence of (i) 0, 0.5, 1.0, and 1.5%wt zero-valent iron (ZVI) and (ii) a combination of 5%wt vermicompost, 2%wt biochar, and 1%wt ZVI on the metal availability in EDTA-washed soil using different soil extracts (Aqua regia, NH4NO3) and plant concentrations. We found that EDTA soil washing significantly reduced the total concentration of Pb, Cd, and Zn and significantly reduced the Cd and Zn plant uptake. Residual EDTA was detected in water extracts causing the formation of highly available Pb-EDTA complexes. While organic amendments had no significant effect on Pb behavior in washed soils, an amendment of ≥ 1%wt ZVI successfully reduced EDTA concentrations, Pb bioavailability, and plant uptake. Our results suggest that Pb-EDTA complexes adsorb to a Fe oxyhydroxide layer, quickly developing on the ZVI surface. The increase in ZVI application strongly decreases Zn concentrations in plant tissue, whereas the uptake of Cd was not reduced, but even slightly increased. Soil washing did not affect plant productivity and organic amendments improved biomass production. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11270-021-05356-0.

Unravelling the process of petroleum hydrocarbon biodegradation in different filter materials of constructed wetlands by stable isotope fractionation and labelling studies.

Maintaining and supporting complete biodegradation during remediation of petroleum hydrocarbon contaminated groundwater in constructed wetlands is vital for the final destruction and removal of contaminants. We aimed to compare and gain insight into biodegradation and explore possible limitations in different filter materials (sand, sand amended with biochar, expanded clay). These filters were collected from constructed wetlands after two years of operation and batch experiments were conducted using two stable isotope techniques; (i) carbon isotope labelling of hexadecane and (ii) hydrogen isotope fractionation of decane. Both hydrocarbon compounds hexadecane and decane were biodegraded. The mineralization rate of hexadecane was higher in the sandy filter material (3.6 µg CO2 g-1 day-1) than in the expanded clay (1.0 µg CO2 g-1 day-1). The microbial community of the constructed wetland microcosms was dominated by Gram negative bacteria and fungi and was specific for the different filter materials while hexadecane was primarily anabolized by bacteria. Adsorption / desorption of petroleum hydrocarbons in expanded clay was observed, which might not hinder but delay biodegradation. Very few cases of hydrogen isotope fractionation were recorded in expanded clay and sand & biochar filters during decane biodegradation. In sand filters, decane was biodegraded more slowly and hydrogen isotope fractionation was visible. Still, the range of observed apparent kinetic hydrogen isotope effects (AKIEH = 1.072-1.500) and apparent decane biodegradation rates (k = - 0.017 to - 0.067 day-1) of the sand filter were low. To conclude, low biodegradation rates, small hydrogen isotope fractionation, zero order mineralization kinetics and lack of microbial biomass growth indicated that mass transfer controlled biodegradation.

New Ag3 PO4 comparison material for stable oxygen isotope analysis.

RATIONALE: A silver phosphate reference material (Ag3 PO4 ) for the measurement of stable oxygen isotope compositions is much needed; however, it is not available from the authorities distributing reference materials. This study aims to fill this gap by calibrating a new Ag3 PO4 stable isotope comparison material produced by the University of Natural Resources and Life Sciences (BOKU). METHODS: Aliquots of Ag3 PO4 were distributed to four laboratories who frequently measure the δ18 O value in Ag3 PO4 ; the University of Natural Resources and Life Sciences (BOKU), the University of Western Australia (UWA), the University of Helsinki (UH), and the Helmholtz Centre for Environmental Research (UFZ). The instruments used to perform the measurements were high-temperature conversion elemental analysers coupled with continuous flow isotope ratio mass spectrometers. The working gas δ18 O value was set to 0‰ and the normalization was done by a three-point linear regression using the reference materials IAEA-601, IAEA-602, and NBS127. RESULTS: The mean δ18 O value of the new BOKU Ag3 PO4 comparison material on the VSMOW-SLAP scale is 13.71‰ and the combined uncertainty is estimated as ±0.34‰. This estimated uncertainty is within the range typical for comparison materials of phosphates and sulphates. Consistent results from the different laboratories probably derived from similar instrumentation, and use of the same reference materials and normalization procedure. The matrix effect of the different reference materials used in this study was deemed negligible. CONCLUSIONS: The BOKU Ag3 PO4 can be used as an alternative comparison material for stable oxygen isotope analysis and is available for stable isotope research laboratories to facilitate calibration.

Assessing adaptive and plastic responses in growth and functional traits in a 10-year-old common garden experiment with pedunculate oak (Quercus robur L.) suggests that directional selection can drive climatic adaptation.

Understanding how tree species will respond to a future climate requires reliable and quantitative estimates of intra-specific variation under current climate conditions. We studied three 10-year-old common garden experiments established across a rainfall and drought gradient planted with nearly 10,000 pedunculate oak (Quercus robur L.) trees from ten provenances with known family structure. We aimed at disentangling adaptive and plastic responses for growth (height and diameter at breast height) as well as for leaf and wood functional traits related to adaptation to dry environments. We used restricted maximum likelihood approaches to assess additive genetic variation expressed as narrow-sense heritability (h2), quantitative trait differentiation among provenances (QST), and genotype-by-environment interactions (GxE). We found strong and significant patterns of local adaptation in growth in all three common gardens, suggesting that transfer of seed material should not exceed a climatic distance of approximately 1°C under current climatic conditions, while transfer along precipitation gradients seems to be less stringent. Moreover, heritability reached 0.64 for tree height and 0.67 for dbh at the dry margin of the testing spectrum, suggesting significant additive genetic variation of potential use for future selection and tree breeding. GxE interactions in growth were significant and explained less phenotypic variation than origin of seed source (4% versus 10%). Functional trait variation among provenances was partly related to drought regimes at provenances origins but had moderate explanatory power for growth. We conclude that directional selection, either naturally or through breeding, is the most likely and feasible outcome for pedunculate oak to adapt to warmer and drier climate conditions in the future.

Successive and automated stable isotope analysis of CO2 , CH4 and N2 O paving the way for unmanned aerial vehicle-based sampling.

RATIONALE: Measurement of greenhouse gas (GHG) concentrations and isotopic compositions in the atmosphere is a valuable tool for predicting their sources and sinks, and ultimately how they affect Earth's climate. Easy access to unmanned aerial vehicles (UAVs) has opened up new opportunities for remote gas sampling and provides logistical and economic opportunities to improve GHG measurements. METHODS: This study presents synchronized gas chromatography/isotope ratio mass spectrometry (GC/IRMS) methods for the analysis of atmospheric gas samples (20-mL glass vessels) to determine the stable isotope ratios and concentrations of CO2 , CH4 and N2 O. To our knowledge there is no comprehensive GC/IRMS setup for successive measurement of CO2 , CH4 and N2 O analysis meshed with a UAV-based sampling system. The systems were built using off-the-shelf instruments augmented with minor modifications. RESULTS: The precision of working gas standards achieved for δ13 C and δ18 O values of CO2 was 0.2‰ and 0.3‰, respectively. The mid-term precision for δ13 C and δ15 N values of CH4 and N2 O working gas standards was 0.4‰ and 0.3‰, respectively. Injection quantities of working gas standards indicated a relative standard deviation of 1%, 5% and 5% for CO2 , CH4 and N2 O, respectively. Measurements of atmospheric air samples demonstrated a standard deviation of 0.3‰ and 0.4‰ for the δ13 C and δ18 O values, respectively, of CO2 , 0.5‰ for the δ13 C value of CH4 and 0.3‰ for the δ15 N value of N2 O. CONCLUSIONS: Results from internal calibration and field sample analysis, as well as comparisons with similar measurement techniques, suggest that the method is applicable for the stable isotope analysis of these three important GHGs. In contrast to previously reported findings, the presented method enables successive analysis of all three GHGs from a single ambient atmospheric gas sample.

Inhibition and stimulation of two perchloroethene degrading bacterial cultures by nano- and micro-scaled zero-valent iron particles.

The pollutant perchloroethene (PCE) can often be found at urban contaminated sites. Thus in-situ clean-up methods, like remediation using zero valent iron (ZVI) or bacterial dechlorination, are preferred. During the remediation with ZVI particles anaerobic corrosion occurs as an unwanted, particle consuming side reaction with water. However, in this reaction H2 is formed, which is usually scarce during anaerobic microbial dechlorination. Dehalococcoides needs H2 for cell growth using it as an electron donor to dechlorinate chlorinated hydrocarbons. Combining application of ZVI with bacterial dechlorination can turn ZVI in a H2 donor leading to a more controllable bacterial dechlorination, a smaller amount of ZVI suspension and decreased remediation costs. In this study nano- and micro scaled ZVI particles (nZVI, mZVI) were combined in microcosms with two dechlorinating bacterial cultures. The two cultures showed different dechlorination behaviors with ethene and cis-DCE as final products. Phospholipid fatty acids (PLFA) associated with Dehalococcoides (18:1w7, 18:1w7c, 10:Me16:0) and Geobacteriaceae (16,1w7c; 15:0; 16:0) have been found in both bacterial cultures, slight differences in their abundance could explain the different dechlorinating behaviors. The combination of both bacterial cultures with mZVI led to a stimulated dechlorination process leading to about two times higher kobs for PCE dechlorination (0.01-0.05 h-1). In the otherwise cis-DCE accumulating culture complete dechlorination to ethene was achieved. While addition of nZVI inhibited both cultures. Combined with nZVI the completely dechlorinating culture produced lower amounts of dechlorinated products (3.2 µmol) as compared to the single biotic treatment (5.1 µmol). Combining the incompletely dechlorinating culture with nZVI significantly reduced the kobs,PCE (single: 8 × 10-3 ± 3 × 10-4 h-1; combination: 5 × 10-3 ± 2 × 10-4 h-1). H2 produced by nZVI and mZVI was utilized by both bacterial cultures. The particle size, resulting specific surface areas, agglomeration tendencies and reactivity appears to be crucial for the effect on microbial cells.

Stable Isotope Probing of Microbial Phospholipid Fatty Acids in Environmental Samples.

Phospholipid fatty acid (PLFA) extracted from environmental samples describe the microbial community pattern and are sensitive to monitor and quantify shifts in the microbial community. Linkage with the stable isotope technique adds a functional perspective and is frequently used to quantify carbon turnover in microbial communities and detect physiological changes. Here we present a PLFA extraction method by using an organic solvent water mixture, followed by lipid separation based on solid-phase extraction and an alkaline methylation. Finally, we provide a protocol for the carbon stable isotope measurements of the extracted fatty acid methyl esters (FAMEs) by gas chromatograph-isotope ratio mass spectrometer (GC-IRMS) and calculation of concentration and δ13CVPDB values.

Degradation of polycyclic aromatic hydrocarbons in a mixed contaminated soil supported by phytostabilisation, organic and inorganic soil additives.

In soil, mixed contamination with potentially toxic trace elements and polycyclic aromatic hydrocarbons (PAHs) may persist for a long time due to strong adsorption to the soil matrix and to its toxicity to microorganism. We conducted an incubation batch experiment to test the effect of soil amendments (biochar, gravel sludge, iron oxides) on the immobilisation of trace elements. To monitor microbial degradation, a 13C-PHE (phenanthrene) label was introduced to soil for 13C-PLFA (phospholipid fatty acid) analysis. Soil amendments increased soil pH, reduced mobility of NH4NO3-extractable trace elements Cd and Zn, and increased mobile Cu. A small consortium of PHE degraders was identified mainly in the microbial groups of gram-negative bacteria and actinomycetes. The degradation process of PHE peaked 9days after incubation start. PAH concentrations remained constant in the soil within the 30-day incubation, except for the easily available 13C-PHE in the amended treatment. In order to test the effect of plants and soil amendments under more realistic conditions, we also conducted an outdoor pot experiment with black locust (Robinia pseudoacacia Nyirsegi). Furthermore, soil amendments increased the mobility of soil Cu and As and decreased the mobility of Cd, Pb and Sb. The uptake of trace elements to leaves was low. Σ 16 U.S. EPA PAHs were significantly reduced only in the combined treatment of black locust and soil amendments after 12months of plant growth. Soil amendment-assisted phytoremediation showed a high efficiency in PAH dissipation and may be a useful remediation technique for mixed contaminated soils.

Impact of sorption processes on PCE concentrations in organohalide-respiring aquifer sediment samples.

The evaluation of groundwater contaminant e.g. tetrachloroethene (PCE) degradation processes requires complete quantification of and pathway analysis of the groundwater contaminant under investigation. For example the reduction of PCE concentrations in the groundwater by unknown dissolution and/or sorption processes will impede interpretation of the fate and behaviour of such contaminants. In the present study PCE dissolution and sorption processes during anaerobic microbial degradation of chlorinated ethenes were investigated. For this purpose, microcosms were prepared using sediment samples from a PCE-contaminated aquifer, which in previous studies had demonstrated anaerobic organohalide respiration of PCE. Solid/water distribution coefficients (kd) of PCE were determined and validated by loss-on-ignition (LOI) and PCE sorption experiments. The determined kd magnitudes indicated methodological congruency, yielding values for sediment samples within a range of 1.15±0.02 to 5.93±0.34L·kg-1. The microcosm experiment showed lower PCE concentrations than expected, based on spiked PCE and observed anaerobic microbial degradation processes. Nevertheless the amount of PCE spike added was completely recovered albeit in the form of lower chlorinated metabolites. A delay due to dissolution processes was not responsible for this phenomenon. Sorption to sediments could only partially explain the reduction of PCE in the water phase. Accordingly, the results point to reversible sorption processes of PCE, possibly onto bacterial cell compartments and/or exopolymeric substances.

Investigations of microbial degradation of polycyclic aromatic hydrocarbons based on 13C-labeled phenanthrene in a soil co-contaminated with trace elements using a plant assisted approach.

Co-contaminations of soils with organic and inorganic pollutants are a frequent environmental problem. Due to their toxicity and recalcitrance, the heterogeneous pollutants may persist in soil. The hypothesis of this study was that degradation of polycyclic aromatic hydrocarbons (PAHs) is enhanced if heavy metals in soil are immobilized and their bioavailability reduced. For metal immobilization and enhanced biodegradation, distinct mineral and organic soil amendments (iron oxides, gravel sludge, biochar) were deployed in an incubation batch experiment. The second part of the experiment consisted of a greenhouse pot experiment applying fast-growing and pollution-tolerant woody plants (willow and black locust). Soil amendments initially immobilized NH4NO3-extractable zinc, cadmium, and lead; after 100 days of incubation, soil amendments showed reductions only for cadmium and a tendency to enhance arsenic mobility. In order to monitor the remediation success, a 13C-phenanthrene (PHE) label was applied. 13C-phospholipid fatty acid analysis (13C-PLFA) further enabled the identification of PHE-degrading soil microorganisms. Both experiments exhibited a similar PLFA profile. Gram-negative bacteria (esp. cy17:0, 16:1ω7 + 6, 18:1ω7c) were the most significant microbial group taking up 13C-PHE. Plants effectively increased the label uptake by gram-positive bacteria and increased the biomass of the fungal biomarker, although their contribution to the degradation process was minor. Plants tended to prolong PAH dissipation in soil; at the end of the experiment, however, all treatments showed equally low total PAH concentrations in soil. While black locust plants tended not to take up potentially toxic trace elements, willows accumulated them in their leaves. The results of this study show that the chosen treatments did not enhance the remediation of the experimental soil.

Isotopic effects of PCE induced by organohalide-respiring bacteria.

Reductive dechlorination performed by organohalide-respiring bacteria (OHRB) enables the complete detoxification of certain emerging groundwater pollutants such as perchloroethene (PCE). Environmental samples from a contaminated site incubated in a lab-scale microcosm (MC) study enable documentation of such reductive dechlorination processes. As compound-specific isotope analysis is used to monitor PCE degradation processes, nucleic acid analysis-like 16S-rDNA analysis-can be used to determine the key OHRB that are present. This study applied both methods to laboratory MCs prepared from environmental samples to investigate OHRB-specific isotope enrichment at PCE dechlorination. This method linkage can enhance the understanding of isotope enrichment patterns of distinct OHRB, which further contribute to more accurate evaluation, characterisation and prospection of natural attenuation processes. Results identified three known OHRB genera (Dehalogenimonas, Desulfuromonas, Geobacter) in diverse abundance within MCs. One species of Dehalogenimonas was potentially involved in complete reductive dechlorination of PCE to ethene. Furthermore, the isotopic effects of PCE degradation were clustered and two isotope enrichment factors (ε) (- 11.6‰, - 1.7‰) were obtained. Notably, ε values were independent of degradation rates and kinetics, but did reflect the genera of the dechlorinating OHRB.

Determination of carbon isotope enrichment factors of cis-dichloroethene after precursor amendment.

RATIONALE: Bacterial reductive dechlorination of the groundwater contaminant tetrachloroethene (PCE) involves the formation of lower chlorinated metabolites. Metabolites can be instantaneously formed and consumed in this sequential process; quantification and validation of their isotopic effects conventionally rely on separate laboratory microcosm studies. Here, we present an evaluation method enabling the determination of the carbon isotope enrichment factor (ε) for the intermediate cis-dichloroethene (cis-DCE) by a single laboratory microcosm study initially amending the precursor PCE only. METHODS: Environmental samples harboring organohalide-respiring bacteria were incubated under anaerobic conditions and then successively and repeatedly amended with PCE and cis-DCE in two separate laboratory microcosm studies. Reductive dechlorination was monitored by analyzing liquid samples using Purge-and-Trap gas chromatography isotope ratio mass spectrometry GC/MS-C/IRMS. The prerequisites of the presented evaluation method are mass and δ-value balancing. The evaluation method was validated by agglomerative hierarchical classification of Rayleigh plot data points. RESULTS: The sample-sensitive range of εcis-DCE extended from -10.6 ± 0.2‰ to -26.8 ± 0.6‰ (R2 ≥98%). The maximum standard deviations of εcis-DCE were ±1.8‰ for single microcosms, ±1.8‰ for replicates and ±1.0‰ for the compiled replicate data of PCE and cis-DCE amendments. A linear regression of the εcis-DCE for replicates obtained by each amendment study showed a slope of 95% (5 of the 7 data points are within a 95% confidence interval), demonstrating factor congruency and the practicability of the evaluation method. CONCLUSIONS: We found metabolite degradation and formation to be sequential but also stepwise during bacterial reductive dechlorination. The stepwise phases of the degradation of the intermediate eliminate the impact of instantaneous precursor degradation. These stepwise sections were used to determine εcis-DCE -values. Our results showed the validity of εcis-DCE -values over a wide range at initial precursor degradation (PCE). The presented evaluation method could substantially decrease lab costs for microcosm studies designed for εcis-DCE determinations. Moreover, the results indicated that the evaluation method can be applied to other PCE-metabolites.

Non-destructive soil amendment application techniques on heavy metal-contaminated grassland: Success and long-term immobilising efficiency.

Extensive contamination of grassland with cadmium (Cd), lead (Pb) and zinc (Zn) is a typical problem close to Pb/Zn smelter sites. The entry of Cd or Pb into the food chain is very likely, as are toxicity effects of Zn in plants. Previous promising results from pot and field experiments showed the high potential of using amendments for immobilisation to reduce metal input into the food chain via crops grown on smelter-contaminated soils at Arnoldstein (Austria) (Friesl et al., 2006). The aim of this study was to find a practical solution for large-scale contaminations in hilly regions that avoids erosion. Field application of amendments without destroying the vegetation cover (grassland) involved two approaches: (a) slurrying (Slu) the amendments into cut gaps in the vegetation cover and (b) injecting (Inj) the amendments through the vegetation cover. Here, we investigate the immobilising and long-term efficiency of treatments [gravel sludge (2.5%) + red mud (0.5%) (GS + RM)]. Risk assessment was based on soil, plant and water samples taken over a period of 10 years. Ammonium-nitrate-extractable Cd was reduced up to 50%, Pb up to 90%, and Zn over 90%. Plant uptake into the grass mixture and narrow leaf plantain was significantly reduced for Cd, Pb, and Zn. Harvesting early in vegetation period can further reduce uptake and meet the threshold for fodder crops. The reduction of these elements in the seepage water in 24 samplings within these 10 years reached 40%, 45% and 50%, respectively. Immobilisation increased microbial biomass and decreased human bioaccessibility for Pb. Our investigation of the long-term efficiency of GS + RM in all treatments shows that the Slu and Inj amendment application techniques have promising potential as a realistic and practical method for extensively contaminated hilly land. Slurrying performed best. We conclude that grassland remediation methods involving tillage are counterproductive from the viewpoint of bioaccessibility and soil protection and therefore should be avoided.

15N-nitrate and 34S-sulfate isotopic fractionation reflects electron acceptor 'recycling' during hydrocarbon biodegradation.

The analysis of stable carbon isotopes for the assessment of contaminant fate in the aquifer is impeded in the case of petroleum hydrocarbons (TPH) by their chain length. Alternatively, the coupled nitrogen-sulfur-carbon cycles involved into TPH biodegradation under sulfate- and nitrate reducing conditions can be investigated using nitrogen (δ15N) and sulfur (δ34S) isotopic shifts in terminal electron acceptors (TEA) involved in anaerobic TPH oxidation. Biodegradation of a paraffin-rich crude oil was studied in anaerobic aquifer microcosms with nitrate (NIT), sulfate (SUL), nitrate plus sulfate (MIX) and nitrate under sulfate reduction suppression by molybdate (MOL) as TEA. After 8 months, TPH biodegradation was not different (around 33%) in experiments receiving only nitrate (NIT, MOL) versus under mixed TEA-conditions (MIX), despite higher biodiversity under mixed conditions (H'NIT and H'MOL≈5.9, H'MIX=8.0). Molybdate addition effected higher nitrate depletion, possibly by increasing the production of nitrate reductase. Additional sulfate depletion under mixed conditions suggested bioconversion of polar intermediates. Microcosms only receiving sulfate (SUL) showed no significant TEA and TPH decrease. A Rayleigh kinetic isotope enrichment model for isotopic 15N/14N and 34S/32S shifts in residual TEA gave apparent enrichment factors ɛN,NIT and ɛN,MOL values of -16.7 to -18.0‰ for nitrate as sole TEA and ɛN,MIX of -6.0‰ and ɛS,MIX of -4.1‰ under mixed electron accepting conditions. The low isotopic fractionation under mixed terminal electron accepting conditions was attributed to lithotrophic, sulfide-dependent denitrification by Thiobacillus species, while it was hypothesized that Desulfovibrio replenished the reduced sulfur pool via oxidation of polar hydrocarbon metabolites. Concurrently, organotrophic denitrification was performed by Pseudomonas species, with isotopic fractionation expressed by ɛN,MIX representing the superposition of both denitrification processes. This is, to our knowledge, the first characterization of sulfur and nitrogen isotopic shifts associated to concurrent organotrophic and lithotrophic denitrification in a hydrocarbon-contaminated environment, and offers the prospect of improved understanding of biogeochemical cycles including in situ hydrocarbon biotransformation.

Transpiration and metabolisation of TCE by willow plants - a pot experiment.

Willows were grown in glass cylinders filled with compost above water-saturated quartz sand, to trace the fate of TCE in water and plant biomass. The experiment was repeated once with the same plants in two consecutive years. TCE was added in nominal concentrations of 0, 144, 288, and 721 mg l(-1). Unplanted cylinders were set-up and spiked with nominal concentrations of 721 mg l(-1) TCE in the second year. Additionally, (13)C-enriched TCE solution (δ(13)C = 110.3 ‰) was used. Periodically, TCE content and metabolites were analyzed in water and plant biomass. The presence of TCE-degrading microorganisms was monitored via the measurement of the isotopic ratio of carbon ((13)C/(12)C) in TCE, and the abundance of (13)C-labeled microbial PLFAs (phospholipid fatty acids). More than 98% of TCE was lost via evapotranspiration from the planted pots within one month after adding TCE. Transpiration accounted to 94 to 78% of the total evapotranspiration loss. Almost 1% of TCE was metabolized in the shoots, whereby trichloroacetic acid (TCAA) and dichloroacetic acid (DCAA) were dominant metabolites; less trichloroethanol (TCOH) and TCE accumulated in plant tissues. Microbial degradation was ruled out by δ(13)C measurements of water and PLFAs. TCE had no detected influence on plant stress status as determined by chlorophyll-fluorescence and gas exchange.