Environ. Sci. Technol. 2004, 38, 617-631
Combined Application of Stable Carbon Isotope Analysis and Specific Metabolites Determination for Assessing In Situ Degradation of Aromatic Hydrocarbons in a Tar Oil-Contaminated Aquifer C H R I S T I A N G R I E B L E R , * ,† M I C H A E L S A F I N O W S K I , †,‡ ANDREA VIETH,§ HANS H. RICHNOW,§ AND RAINER U. MECKENSTOCK‡ Environmental Mineralogy, Center for Applied Geosciences, Eberhard-Karls-University of Tu ¨ bingen, Wilhelmstrasse 56, D-72074 Tu ¨ bingen, Germany, Institute of Hydrology, Research Center for Environment and Health (GSF), Ingolsta¨dter Landstrasse 1, D-85764 Neuherberg, Germany, and Department of Remediation Research, Centre for Environmental Research (UFZ), Permoserstrasse 15, D-04318 Leipzig, Germany
To evaluate the intrinsic bioremediation potential in an anoxic tar oil-contaminated aquifer at a former gasworks site, groundwater samples were qualitatively and quantitatively analyzed by compound-specific isotope analysis (CSIA) and signature metabolites analysis (SMA). 13C/12C fractionation data revealed conclusive evidence for in situ biodegradation of benzene, toluene, o-xylene, m/p-xylene, naphthalene, and 1-methylnaphthalene. In laboratory growth studies, 13C/12C isotope enrichment factors for anaerobic degradation of naphthalene ( ) -1.1 ( 0.4) and 2-methylnaphthalene ( ) -0.9 ( 0.1) were determined with the sulfate-reducing enrichment culture N47, which was isolated from the investigated test site. On the basis of these and other laboratory-derived enrichment factors from the literature, in situ biodegradation could be quantified for toluene, o-xylene, m/p-xylene, and naphthalene. Stable carbon isotope fractionation in the field was also observed for ethylbenzene, 2-methylnaphthalene, and benzothiophene but without providing conclusive results. Further evidence for the in situ turnover of individual BTEX compounds was provided by the presence of acetophenone, o-toluic acid, and p-toluic acid, three intermediates in the anaerobic degradation of ethylbenzene, o-xylene, and p-xylene, respectively. A number of groundwater samples also contained naphthyl-2-methylsuccinic acid, a metabolite that is highly specific for the anaerobic degradation of 2-methylnaphthalene. Additional metabolites that provided evidence on the anaerobic in situ degradation of naphthalenes were 1-naphthoic acid, 2-naphthoic acid, 1,2,3,4-tetrahydronaphthoic acid, and 5,6,7,8* Corresponding author telephone: +49 (0) 7071 2973151; fax: +49 (0) 7071 295139; e-mail:
[email protected]. † Eberhard-Karls-University of Tu ¨ bingen. ‡ Research Center for Environment and Health (GSF). § Centre for Environmental Research (UFZ). 10.1021/es0344516 CCC: $27.50 Published on Web 11/25/2003
2004 American Chemical Society
tetrahydronaphthoic acid. 2-Carboxybenzothiophene, 5-carboxybenzothiophene, a putative further carboxybenzothiophene isomer, and the reduced derivative dihydrocarboxybenzothiophene indicated the anaerobic conversion of the heterocyclic aromatic hydrocarbon benzothiophene. The combined application of CSIA and SMA, as two reliable and independent tools to collect direct evidence on intrinsic bioremediation, leads to a substantially improved evaluation of natural attenuation in situ.
Introduction Since “natural attenuation” is on the way to become an important remediation strategy for organically polluted aquifers, methods are required to identify and monitor the degradation of individual pollutants in situ. Following the OSWER (Office of Solid Waste and Emergency Response) directive of the United States Environmental Protection Agency (U.S. EPA), three steps are suggested to “prove” in situ bioremediation: (i) groundwater chemistry data that demonstrate a clear and meaningful trend of decreasing contaminant mass and/or concentration over time, (ii) hydrogeologic and geochemical data that can be used to demonstrate indirectly the type(s) of natural attenuation processes active at the site, and (iii) data that provide direct evidence for degradation taking place in actually contaminated site media (microcosms) or in situ (aquifer) (1). The first two requirements can in most cases be achieved by physicochemical analysis of field samples and related mass balance calculations. Evidence for the degradation potential of the indigeneous microbial community at a contaminated site may be obtained by means of microcosm studies with field material. However, the assessment of an active biodegradation in situ (step iii) is most difficult to prove. Two recently introduced approaches [i.e., signature metabolites analysis (SMA) and compound-specific isotope analysis (CSIA)] seem to be serious candidates for the routine application in the evaluation of the natural attenuation potential of aquifers contaminated with petroleum-related hydrocarbons (2). Intermediates in the degradation pathways of contaminants (“metabolic biomarkers” or “signature metabolites”) have to fulfill a set of prerequisites to serve as indicators for in situ biodegradation: (i) an unequivocal and unique biochemical relationship to the parent hydrocarbon, (ii) release from the microbial cell to the extracellular medium, (iii) no commercial or industrial production or uses, (iv) biological and chemical stability, and (v) generation as an intermediate of mineralization rather than as a product of cometabolism (3). At best, the detection of a metabolite can be related to the degradation of only one compound, whereas other intermediates are indicative for several pathways (3). On the basis of the identification of specific metabolites, the in situ biodegradation could recently be shown for a number of aromatic hydrocarbons, such as toluene, ethylbenzene, xylenes, and naphthalene in laboratory and field studies (311). However, metabolites may be indicative for in situ biodegradation processes but probably fail in quantification of biodegradation because their actual presence is part of a continuous formation and further degradation, which probably depends on many factors. A second promising approach to detect bioremediation of hydrocarbons in situ is the compound-specific isotope analysis (CSIA). Lebedew et al. (12) first mentioned an isotope VOL. 38, NO. 2, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
617
effect along with the oxidation of hydrocarbons in soils and sediments resulting in a change of the carbon isotopic signatures in the residual organic compound fraction. Later, Stahl (13) reported a 13C/12C fractionation to occur during the aerobic degradation of hydrocarbons in a mixture of petroleum and seawater. Here, the degradation of alkanes was accompanied by a slight enrichment of the 13C content of the residual material. However, aerobic degradation of hydrocarbons is not necessarily accompanied by a stable carbon isotope fractionation. For toluene, no fractionation could be observed in degradation experiments with undefined enrichment cultures (14). In contrast, experiments with the aerobic culture Pseudomonas putida strain mt-2 yielded a significant carbon isoptope fractionation upon the degradation of toluene (15). A systematic study by Morasch et al. elucidated that isotope fractionation depends on the enzymatic pathways involved. Strong stable isotope fractionation was observed for a bond cleaving methyl-monooxygenase (P. putida mt2) but no fractionation was observed for dioxygenase reactions (16, 17). Therefore, under aerobic conditions, the extent of fractionation may strongly vary prohibiting quantification of biodegradation in oxic environments. Meckenstock et al. showed that anaerobic toluene degradation was accompanied by a similar 13C/12C fractionation for all terminal electron accepting processes (TEAP) tested (18). Growth experiments with various bacterial strains degrading toluene under denitrifying, iron-reducing, and sulfate-reducing conditions revealed similar enrichment factors (i.e., on average ) -1.7 ( 0.3‰). This was suggested to be due to the fact that every anaerobic culture or strain investigated so far degraded toluene via the benzylsuccinate synthase pathway (19-21). Ahad et al. (22) have determined slightly smaller enrichment factors for toluene under sulfatereducing ( ) -0.5‰) and methanogenic conditions ( ) -0.8‰). To date, several laboratory and field studies showed that anaerobic degradation of hydrocarbons such as BTEX and dicyclic aromatics induces 13C/12C fractionation. Qualitative evidence for in situ biodegradation was provided for benzene (23-25), toluene (16, 18, 26-28), ethylbenzene (24, 29), xylene isomers (28-30), ethyltoluene (29, 30), 1,2,4trimethylbenzene (29), and naphthalene (28). Besides qualitative evidence for in situ biodegradation of individual contaminants, a major interest is to apply stable carbon isotope fractionation in the environment to quantify in situ bioremediation of individual contaminants. The basic requirement for such a quantitative approach is a compoundspecific fractionation factor, which is representative for a range of environmental conditions as was shown for toluene (18). Today, a growing number of fractionation factors is available (e.g., for BTEX compounds and chlorinated solvents) that can be used to quantify in situ biodegradation. In groundwater environments, processes other than the biological hydrocarbon oxidation such as adsorption and dilution do not affect isotope ratios (18). Thus, for many compounds that are subject to isotope fractionation, a quantification of in situ bioremediation can be done. Here we report on laboratory experiments in which we determined stable carbon isotope fractionation factors for the anaerobic degradation of naphthalene and 2-methylnaphthalene by a sulfate-reducing enrichment culture. These and other laboratory-derived fractionation factors were used for a qualitative and quantitative evaluation of natural attenuation at a former gasworks site. Furthermore, field investigations focused on metabolites from anaerobic degradation of BTEX (benzene, toluene, ethylbenzene, and xylenes), naphthalenes, and benzothiophene as a representative of heterocyclic aromatic compounds. The combined application of CSIA and SMA led to conclusive qualitative and quantitative evidence regarding the in situ biodegradation of petroleum hydrocarbons. 618
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 2, 2004
Materials and Methods Organisms and Growth Conditions in Laboratory Experiments. Stable carbon isotope fractionation experiments were carried out with the sulfate-reducing culture N47, which was enriched from contaminated soil of the study site, Testfeld Su ¨ d (31). Precultures were grown with naphthalene as the sole carbon and energy source in the presence of the solid adsorber resin, Amberlite-XAD7 (Fluka, Switzerland), which functions as a substrate reservoir and maintains low but constant substrate concentrations in the liquid phase (31). The bicarbonate-buffered mineral medium (pH 7.2-7.4) was reduced with 1 mM sodium sulfide, and 10 mM sodium sulfate was added as an electron acceptor (32). The medium was carefully filled into 500-mL bottles, and either solid naphthalene or 2-methylnaphthalene was added in excess. The headspace of the bottles was subsequently flushed with N2-CO2 (80:20), and the bottles were closed with butyl rubber stoppers (Maag Technik, Switzerland). After 1 week of incubation at 30°C, the substrate-saturated medium was portioned in 50-mL aliquots into 100-mL serum bottles. Headspace of the bottles was again flushed with N2-CO2 (80: 20), and the bottles were closed with viton stoppers (Maag Technik, Switzerland). Serum bottles were inoculated in triplicates with sterile syringes through the stoppers and incubated at 30°C in the dark. Subsamples for concentration measurements and stable carbon isotope analysis were taken periodically with a syringe and extracted with cyclohexane (Acros Organics, USA) at ratios of 1:1 (v:v, water:cyclohexane) at the beginning and 20:1 at the end of the growth experiments. Biological activity in the subsamples was stopped with the amendment of NaOH (0.1 M final concentration). After 10 min of shaking, the cyclohexane phase was collected and stored for later isotope analysis. Bacterial activity was monitored by measuring sulfide concentrations (33). Field Investigation Site. The investigation site (Testfeld Su ¨ d) is a former gasworks site located in southwest Germany (Figure 1). The shallow Quaternary aquifer mainly consists of gravels and sands with a layer of loamy sediment embeddings and artificial fillings in the unsaturated zone. Local influx of mineral water from the deeper confined groundwater system into the alluvial aquifer and several drainage systems collecting the local infiltration result in a complex hydraulic situation (34). Multiple contaminant sources are distributed over the Testfeld Su ¨ d area, i.e., tar pits in the southern part as well as further BTEX and PAH sources in the central and western part close to and within the investigated area (Figure 1). Nonaqueous-phase liquids (NAPLs) are locally present in both the saturated and the unsaturated zones (35). The resulting contaminant plume has an estimated width of 120 m, and low molecular weight PAH compounds were detected at a distance of 280 m downstream of the NAPL zone (36). The overall length of the plume is unknown so far as no monitoring wells are available further downstream. The actual investigation took place in July 2001 and focused on a 7500 m2 section within the area of the proposed PAH plume (Figure 1). In this area, more than 20 monitoring wells equipped with filter screens through the entire aquifer have been installed, and 15 wells have been selected for sampling and measurements. Within the investigated area, we detected two point sources: one southwest (termed source 1; S1) and another located in the central part of the section (termed source 2; S2; Figure 1). Sampling Procedures. The well water was replaced at least once by means of a submersible pump (MP1, Grundfos Corp.) before water was sampled and filled into precleaned brown glass bottles, which were closed without headspace by screw caps with Teflon-coated silicon septa. Sample bottles (1 L) for PAH analysis already contained an internal standard
FIGURE 1. Schematic overview of the former gasworks site “Testfeld Su1 d”. The arrow depicts the main direction of the groundwater flow. The insert shows the investigation area and the positions of selected observation wells, the two source zones S1 and S2, and the transect chosen for detailed quantitative investigations. Isolines show the hydraulic heads at the time of sampling. mixture and 10 mL of cyclohexane for later extraction. Samples for stable carbon isotope analysis were filled in 500mL brown glass bottles, and a volume of 3 mL from filled bottles was replaced by pentane (Merck, Germany). Biological activity in the samples was prevented by the amendment of NaOH (0.1 M final concentration). Samples for BTEX analysis and the analysis of major anions and cations were filled without pretreatment into 100-mL flasks avoiding headspace. Samples were transported and stored under cooled conditions (4 °C) until extraction. For extraction, the solvent phase (pentane or cyclohexane) was collected after 60 min of intensive shaking and stored at 4°C for subsequent isotope analysis. Sample Analysis. To evaluate the occurrence and availability of individual electron acceptors and nutrients, analysis of major anions (NO3-, SO42-, PO43-) was performed by ion chromatography (Dionex DX-120). Concentrations of BTEX compounds were analyzed directly from untreated samples by purge-and-trap GC-MS. The Teckmar 3000 purge-and-trap autosampler device operated with helium as a carrier (gas flux, 50 mL min-1; purge time, 11 min) and was connected to a GC-MS system (GC, HP 6890; MS, HP 5973). A VOCARB 3000 (Supelco,
USA) trap was used at a desorption temperature of 225°C and a desorption time of 4 min. A DB-624 GC column (60 m length; 0.25 mm i.d.; 1.4 µm film thickness; J&W Scientific, USA) was used for the separation of the target compounds. A split ratio of 10:1 was used, and the flow rate of the carrier gas was 1 mL min-1. The oven temperature was held at 44 °C for 5 min, then increased at a rate of 8 °C min-1 to 90 °C and held for 4 min, then increased further with a rate of 6 °C min-1 to 200 °C and held for 5 min, and increased finally with a rate of 20 °C min-1 to 230 °C and held for 3 min. The GC-MS transfer line was held at 315 °C resulting in a temperature of 175 °C in the ion source. The MS was operated in the selected ion mode (SIM). A stock solution of fluorobenzene and 1,2-dichlorobenzene-d4 in methanol, 200 µg mL-1 each (U.S. EPA Standard 524, Supelco), was used as internal standard for calibration. The limit for quantification for each of the BTEX compounds was between 0.1 and 1 µg L-1 depending on the total load of contaminants and required sample dilution. Naphthalene, methylnaphthalenes, and benzothiophene were extracted from the pretreated and weighted water samples (see above) and analyzed with a GC-MS (GC, HP 6890; MS, HP 5973). The GC column for separation of target VOL. 38, NO. 2, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
619
compounds was a DB-5MS (0.25 µm film thickness, 0.2 mm i.d., 30 m length; J&W Scientific). Samples were injected splitless (front inlet was maintained in the pulsed splitless mode) with a flow rate of the carrier gas (helium) of 1 mL min-1. The oven temperature was held at 65 °C for 4 min, then increased at a rate of 10 °C min-1 to 270 °C and held for 10 min, and then increased further with a rate of 10 °C min-1 to 310 °C and held for 6.5 min. The GC-MS transfer line was held at 315 °C, resulting in a temperature of 175 °C in the ion source. The MS was operated in the SIM. For quantification, an internal standard (Restek) was used, which contained 200 mg L-1 naphthalene-d8 and acenaphthened10 dissolved in cyclohexane. The limit for quantification of PAH compounds was between 0.01 and 0.1 µg L-1 depending on the total load of contaminants and required dilution of extracts. Samples for metabolite analysis (500 mL) received a NaOH amendment (0.1 M final concentration) and were stored at 4 °C until further processing. Detection and identification of polar metabolites in groundwater samples were performed by GC-MS. NaOH-amended samples (500 mL, pH g 12) were extracted twice with 20 mL of cyclohexane (Merck) to remove nonpolar aromatic hydrocarbons. Thereafter, the samples were acidified with HCl (37%, Merck) to pH e2 and extracted twice with 20 mL of dichloromethane (Merck). The combined dichloromethane (DCM) extracts were concentrated with a vacuum rotary evaporator to a final volume of about 1 mL and subsequently dried under a continuous nitrogen gas stream. The residue was redissolved in 1 mL of methanol (Merck), diluted with trimethylchlorosilane (TMCS) to a ratio of 9:1 (methanol:TMCS, v:v), and derivatized for 1 h at 75 °C. The solution containing the methylated aromatic carboxylic acids was again dried under a continuous nitrogen stream and resolved in 1 mL of ethyl acetate (Merck) for subsequent separation with a GC-MS (GC, HP 6890; MS, HP 5973). The GC column for separation of target compounds was a DB-5MS (0.25 µm film thickness, 0.2 mm i.d., 30 m length; J&W Scientific). Samples were injected splitless, and flow rate of the carrier gas (helium) was 0.9 mL min-1. The oven temperature was held at 43 °C for 5 min and then increased at a rate of 4 °C min-1 to 280 °C and held for 5 min. The MS was operated at 315 °C in the scan mode and acquired data from 29 to 400 mass units (m/z). The detection limit for methylated polar organic acids ranged from 0.01 µg L-1 (e.g., naphthoic acids) up to >1000 µg L-1 (e.g., benzoic acid). For identification of metabolites, instrumental library searches of the NIST (National Institute of Standard and Technology) database, comparison with published mass spectra, and coinjections with commercially available authentic reference compounds were used. Naphthyl-2-methylsuccinic acid, tetrahydro-2-naphthoic acids, and 5-carboxybenzothiophene were identified by comparison with synthesized reference compounds (37-39). For quantitative estimations, authentic standards were dissolved in 1 L of ultrapure water (Millipore) and treated similar to groundwater samples (i.e., liquidliquid extraction with cyclohexane and dichloromethane, derivatization, and GC-MS analysis) to obtain calibration curves. If no authentic standards were available (i.e., 5,6,7,8tetrahydro-2-naphthoic acid, carboxybenzothiophene isomers, and dihydrocarboxybenzothiophene), concentrations of compounds were estimated using calibration curves of substances with similar properties, particularly with similar retention time in the GC column. Additionally, duplicate samples of ultrapure water, groundwater from well B42 (less contaminated), and groundwater from well B54 (highly contaminated) were spiked with two compounds that have not been detected in samples from these wells [i.e., 1-naphthylacetic acid and naphthyl-2-methylensuccinic acid (NMeS)] to evaluate the recovery efficiency of the applied extractionderivatization protocol. Results showed that, for these two 620
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 2, 2004
compounds, the recovery efficiency varied between 50 and 150% in natural samples in relation to the contaminant-free reference sample (spiked ultrapure water). However, the presented data analysis for metabolites focuses on relative concentration trends rather than on absolute concentration values. Compound-Specific Isotope Analysis (CSIA). The carbon isotope composition of hydrocarbons was measured using a GC-C-IRMS (gas chromatography/combustion/isotope ratio mass spectrometry) system (Finnigan MAT, ThermoFinnigan, Germany). The GC-C-IRMS system consisted of a gas chromatograph (6890 series, Agilent Technology, USA) connected to a Finnigan MAT GC III combustion device with a 50 cm long water removal assembly hold at 0 °C (Nafion membrane). The Finnigan MAT 252 mass spectrometer was coupled via open split to the combustion device. Organic substances were combusted to CO2 in the furnace held at 940 °C on a CuO/Ni/Pt catalyst and transferred on-line into the MS to determine the isotope composition. Aliquots (0.5-4 µL) of extracts were injected using a hot injector held at 250 °C operated in split or splitless mode depending on the concentration of target compounds. A capillary column (ZB1 60 m × 0.32 mm, 1 µm film, Phenomenex Inc. Torrance, CA) was used for the chromatographic separation. The temperature program for field samples was 40 °C held for 3 min isothermally, then increased with a rate of 3 °C min-1 to 250 °C, and then increased to 300 °C with a rate of 20 °C min-1 and held for 5 min. Helium was used as carrier gas with a flow rate of 1.5 mL min-1. The temperature for the analysis of naphthalene and methylnaphthalene from batch experiments were 80 °C held for 2 min and then increased to 250 °C at a rate of 6°C min-1and held for 5 min. All samples were measured in at least five replicates. Systematic analytical error with respect to the linearity and precision of the instrument due to nonlinearity is 0.1 δ-units per decade in the signal interval between 45 and 3500 mV (29).
Calculations Calculations were based on the Rayleigh model for closed systems (40), expressed as ln(Rt/R0) ) (RC - 1) × ln(Ct/C0), where Rt is the isotopic composition of the substrate at a given time, R0 is the initial isotopic composition of the substrate, RC is the carbon isotope fractionation factor, and (Ct/C0) is the fraction of the substrate remaining. The fractionation factor (RC) was obtained by plotting ln(Ct/C0) versus ln(Rt/R0) and determining the slope of the linear regression (b), which is related to the fractionation factor (RC) by b ) (RC - 1). Subsequently, fractionation factors (RC) were converted into enrichment factors () according to ) (RC - 1) × 1000. All carbon isotope ratios are given in delta notation as δ13C values (‰), which are related to the ViennaPDB (Pee Dee Belemnite) standard. Growth experiments with bacterial strains and the respective hydrocarbon compound as the sole carbon and energy source were performed to obtain a compound-specific isotope fractionation factor (RC) and an isotope enrichment factor (). With knowledge of the initial substrate concentration (C0) and the laboratory-derived substrate-specific isotope fractionation factor (RC), the theoretical remaining substrate fraction (Ct) can be calculated according to
Ct ) C0
( ) Rt R0
(1/RC-1)
(1)
Here, an error propagation was considered for all measured input parameters. As only single measurements were performed for hydrocarbon concentrations from field samples, a standard deviation of 10% for the C0 values was considered.
TABLE 1. 13C/12C Isotope Fractionation Factors Used for Calculation of In Situ Biodegradation compound
13C/12C
fractionation factor (rC)
redox conditions
benzene toluene o-xylene m-xylene
0.9964 ( 0.0003 0.9983 ( 0.0003 0.9987 ( 0.0003 0.9972 ( 0.0002
BTEXs sulfate-reducing sulfate-reducing sulfate-reducing sulfate-reducing
naphthalene
0.9989 ( 0.0004
PAHs sulfate-reducing
Isotope fractionation factors used for the theoretical isotope fractionation are given in Table 1. The percentage of biodegradation (B) of the residual substrate fraction was calculated according to B ) (1 - (Ct/C0)) × 100 (%).
Results and Discussion Concentrations of Hydrocarbons and Physicochemical Characterization of the Aquifer. Highest concentrations among the selected compounds were found for naphthalene with 86 mg L-1 at B54, which is about 3 times the water solubility and indicates that the groundwater sample contained some NAPL phase. However, all contaminants showed a strong decline in their concentrations with distance from the sources along the groundwater flow path. Concentration of naphthalene, for example, dropped from 86 mg L-1 at B54 (S2) to 14 µg L-1 at B42, the least contaminated well which is located about 90 m downstream from source area two (S2). Within the same distance, concentrations of 1-methylnaphthalene and 2-methylnaphthalene declined from 1.9 and 3.9 mg L-1 to 2.5 and 1.5 µg L-1, respectively. Among the BTEX compounds, concentrations of benzene (max, 3.6 mg L-1; min, 46 µg L-1) showed the fastest decrease followed by toluene (max, 3.8 mg L-1; min, 7.4 µg L-1), m/p-xylene (max, 3.6 mg L-1; min, 5 µg L-1), ethylbenzene (max, 1.7 mg L-1; min, 10 µg L-1), and o-xylene (max, 1.6 mg L-1; min, 1.8 µg L-1) in relation to their source concentrations at S2. The physicochemical conditions in the investigated aquifer (i.e., redox potential, dissolved oxygen, sulfate, nitrate and iron) were mainly influenced by the distribution of the contaminants. With an exception in well B42 (least contaminated), oxygen was always below 1 mg L-1. This was in agreement with redox measurements and -values indicating anoxic conditions for the whole test site with lowest values at S1 and S2 (EH )