Using 2,6-dichlorobenzamide (BAM) degrading Aminobacter sp. MSH1 in flow through biofilters--initial adhesion and BAM degradation potentials.

Micropollutants in groundwater are given significant attention by water companies and authorities due to an increasing awareness that they might be present even above the legal threshold values. As part of our investigations of the possibility to remove the common groundwater pollutant 2,6-dichlorobenzamide (BAM) by introducing the efficient BAM degrader Aminobacter sp. MSH1 into biologically active sand filters, we investigated if the strain adheres to filters containing various filter materials and if the initial adherence and subsequent degradation of BAM could be optimized. We found that most of the inoculated MSH1 cells adhered fast and that parameters like pH and ionic strength had only a minor influence on the adhesion despite huge influence on cell surface hydrophobicity. At the given growth protocol, the MSH1 strain apparently developed a subpopulation that had lost its ability to adhere to the filter materials, which was supported by attempted reinoculation of non-adhered cells. Analysis by quantitative PCR showed that most cells adhered in the top of the filters and that some of these were lost from the filters during initial operation, while insignificant losses occurred after 1 day of operation. The inoculated filters were found to degrade 2.7 µg/L BAM to below 0.1 µg/L at a 1.1-h residence time with insignificant formation of known degradation products. In conclusion, most filter materials and water types should be feasible for inoculation with the MSH1 strain, while more research into degradation at low concentrations and temperatures is needed before this technology is ready for use at actual waterworks.

Demonstrating a natural origin of chloroform in groundwater using stable carbon isotopes.

Chloroform has been for a long time considered only as an anthropogenic contaminant. The presence of chloroform in forest soil and groundwater has been widely demonstrated. The frequent detection of chloroform in groundwater in absence of other contaminants suggests that chloroform is likely produced naturally. Compound-specific isotope analysis of chloroform was performed on soil-gas and groundwater samples to elucidate whether its source is natural or anthropogenic. The δ(13)C values of chloroform (-22.8 to -26.2‰) present in soil gas collected in a forested area are within the same range as the soil organic matter (-22.6 to -28.2‰) but are more enriched in (13)C compared to industrial chloroform (-43.2 to -63.6‰). The δ(13)C values of chloroform at the water table (-22.0‰) corresponded well to the δ(13)C of soil gas chloroform, demonstrating that the isotope signature of chloroform is maintained during transport through the unsaturated zone. Generally, the isotope signature of chloroform is conserved also during longer range transport in the aquifer. These δ(13)C data support the hypothesis that chloroform is naturally formed in some forest soils. These results may be particularly relevant for authorities' regulation of chloroform which in the case of Denmark was very strict for groundwater (<1 µg/L).

Reliable test methods for the determination of a natural production of chloroform in soils.

Chloroform is one of the most frequently found anthropogenic groundwater contaminants. Recent investigations, however, suggested that chloroform in groundwater may also originate from a natural production in soils. As societies response to the occurrence of chloroform in groundwater may depend upon its origin as anthropogenic or naturally produced, test methods are needed to measure the potential of natural soil chloroform production. Field measurements of ambient air and soil air, and field and laboratory incubation studies were evaluated for measurement of relative soil chloroform production at a site with four different vegetation types (spruce forest, beech forest, grassland, and grain field) on comparable geological soil. All test methods showed varying soil production of chloroform with spruce forest soil being most productive and grain field soil being least productive. Field measurements of the ratio of soil air to ambient air chloroform concentrations exhibited the smallest difference between high production and low production areas, whereas laboratory incubation studies showed the largest difference. Thus, laboratory incubation studies are suggested as most efficient for estimating relative chloroform production in soil. The study indicated that soil samples should be tested not more than 14 days after sampling. Furthermore, it was found that potentially limiting compounds, such as chloride or nitrate, are not needed to be added in spike experiments to obtain reliable production results. However, it should be recognized that the processes of soil chloroform production are not known yet in all details. Other factors than those studied here may affect the test methods for soil chloroform production too.

Leaching of Cryptosporidium parvum oocysts, Escherichia coli, and a Salmonella enterica serovar Typhimurium bacteriophage through intact soil cores following surface application and injection of slurry.

Increasing amounts of livestock manure are being applied to agricultural soil, but it is unknown to what extent this may be associated with contamination of aquatic recipients and groundwater if microorganisms are transported through the soil under natural weather conditions. The objective of this study was therefore to evaluate how injection and surface application of pig slurry on intact sandy clay loam soil cores influenced the leaching of Salmonella enterica serovar Typhimurium bacteriophage 28B, Escherichia coli, and Cryptosporidium parvum oocysts. All three microbial tracers were detected in the leachate on day 1, and the highest relative concentration was detected on the fourth day (0.1 pore volume). Although the concentration of the phage 28B declined over time, the phage was still found in leachate at day 148. C. parvum oocysts and chloride had an additional rise in the relative concentration at a 0.5 pore volume, corresponding to the exchange of the total pore volume. The leaching of E. coli was delayed compared with that of the added microbial tracers, indicating a stronger attachment to slurry particles, but E. coli could be detected up to 3 months. Significantly enhanced leaching of phage 28B and oocysts by the injection method was seen, whereas leaching of the indigenous E. coli was not affected by the application method. Preferential flow was the primary transport vehicle, and the diameter of the fractures in the intact soil cores facilitated transport of all sizes of microbial tracers under natural weather conditions.

Methodological problems in determining TCAA in soils-the discovery of novel natural trichloroacetyl containing compounds and their interference with a common method for determining TCAA in soil and vegetation.

Trichloroacetic acid (TCAA) is a pollutant with several sources and is also formed naturally in soil. We show that almost all investigated environmental compartments (soil, soil water, groundwater, spruce needles and throughfall, but not rain) contain compounds, which make false positives in the thermal decarboxylation method often used for determination of TCAA in environmental samples. The compounds are dominating quantitatively over TCAA in soil, soil water and groundwater, while TCAA is dominating in needle and throughfall samples. The compounds behave differently from TCAA with regard to the velocity and the pH-dependence of the chloroform release. We did not manage to reveal the whole chemical structure of the compounds, but a trichloroacetyl group seems to be the only plausible structure giving rise to CHCl(3) both upon heating and under alkaline conditions. Besides the trichloroacetyl group, the compounds did in general contain a carboxylic acid group, although in needle and throughfall samples, trichloroacetyl compounds with a neutral charge at pH 7.5 seemed to co-exist with the carboxylic acids. Trichloroacetyl groups in humic substances and possibly other macromolecular structures contribute to the major portion of the total trichloroacetyl-CHCl(3) in topsoil, but smaller molecules with less UV-VIS absorption seem to constitute the major part of trichloroacetyl-CHCl(3) in soil water and groundwater. The trichloroacetyl containing compounds are most likely naturally occurring compounds formed in the natural chlorination processes in soil, but additional studies are needed to substantiate this hypothesis.

Leaching of herbicidal residues from gravel surfaces - A lysimeter-based study comparing gravels with agricultural topsoil.

Evidence from the past shows that pesticide use in populated areas may impact groundwater quality. The approval of herbicides such as diflufenican and glyphosate for use on paved and unpaved gravel surfaces in the European Union is based on their behaviour and fate in agricultural soils. However, this might be very different from their fate in gravel surfaces. We therefore conducted an outdoor study with 21 small lysimeters containing different gravel types and a sandy arable topsoil as control. The lysimeters were sprayed with a commercial product for gardening, containing diflufenican and glyphosate. The concentrations of the herbicides and their relevant degradation products in the outlet was followed for 19 months. Diflufenican, glyphosate and AMPA did not leach from any of the lysimeters. However, one diflufenican degradation product (AE-0) leached from two of the gravel types for more than a year and a second degradation product (AE-B) leached from all gravels for up to one year. Concentrations in the leachate peaked at 0.5-3 µg/L, with highest concentrations over the longest periods observed with rock chippings on top of the gravel. We conclude, that the different properties of gravel compared to those of agricultural soils may lead to very different herbicide leaching patterns but also that the leaching depends highly on the type of gravel and type of herbicide.

Method for rapid screening of pesticide mineralization in soil.

A method has been developed for the analysis of (14)CO(2) evolution from the mineralization of (14)C-labelled organic compounds in soil samples. The new method is less space demanding and substantially cuts down laborious manual work compared to the traditional incubation bottle method used. Furthermore, the use of scintillation cocktail is largely reduced with the new method. In the new method, (14)CO(2) is trapped in filter paper held in the lid of a 20 ml glass vial by surface tension. The trapping solution used is Ca(OH)(2), which fixates CO(2) in the filter paper and the analysis of trapped (14)CO(2) is done using the Cyclone trade mark Storage Phosphor system. The lids are placed in a 32 well holder and exposed to a phosphor screen prior to scanning in a Cyclone trade mark scanner. The new filter method has been tested and compared to results obtained using the traditional method. The results show good agreement but due to a smaller capacity for CO(2) with the filter method compared to the traditional method, the interval between sampling has to be shorter using the filter method when the CO(2) development is high. The detection limits for the filter method is higher compared to the traditional method. With the filter method, the level of radioactivity has to exceed 300 dpm before detection is possible, while the same limit for the traditional method is around 30 dpm. On the other hand, the gas trapping faster and the efficiency is higher with the filter method.

Leaching of Salmonella enterica in clay columns comparing two manure application methods.

Transfer of zoonotic bacterial pathogens through intact soil columns was monitored in an outdoor lysimeter over 36 d. Manure spiked with Salmonella enterica serovar Senftenberg was applied to either the soil surface or injected 0.08 m into the soil to compare leaching associated with the two manure application methods. The highest concentrations of S. enterica (up to 60,000 S. enterica CFU/mL) were detected on Day 1 in the first drainage samples, with measurable but declining concentrations persisting for 10 to 36 d depending on replicate columns. The total recovery of leached S. enterica in drainage samples ranged from 0.08% to 13.8%. When comparing the two application methods, there was no statistically significant difference in the leaching concentration of S. enterica at each sampling time during the study period. In addition, comparison of enumerations by selective plating and real-time polymerase chain reaction yielded similar concentrations of S. enterica, indicating that mainly viable and culturable cells were leached from the columns. When the experiment was terminated, the fluorescent dye Acid Yellow was applied to four selected columns and the distribution of dye and size of active (dye-stained) pores were measured with a digital camera and visualization software. The profiles showed that the area covered by active pores ranged from 0.1% to 3.6%. The relatively small fraction of active pores in the soil profile was consistent with the evidence of rapid transport of S. enterica and chloride in the columns.

Trichloromethyl compounds--natural background concentrations and fates within and below coniferous forests.

Pollution with organochlorines has received major attention due to various environmental effects, but it is now increasingly recognized, that they also take part in biogeochemical cycles and that natural background concentrations exist for several chlorinated compounds. We here report the natural occurrence and cycling of organic compounds with a trichloromethyl moiety in common. The study areas are temperate coniferous forests. Trichloromethyl compounds can be found in all compartments of the forests (groundwater, soil, vegetation and throughfall), but not all compounds in all compartments. The atmospheric input of trichloromethyl compounds is found to be minor, with significant contributions for trichloroacetic acid (TCAA), only. In top soil, where the formation of the compounds is expected to occur, there is a clear positive relationship between chloroform and trichloroacetyl containing compounds. Other positive relations occur, which in combination with chlorination experiments performed in the laboratory, point to the fact that all the trichloromethyl compounds may be formed concurrently in the soil, and their subsequent fates then differ due to different physical, chemical and biological properties. TCAA cannot be detected in soil and groundwater, but sorption and mineralization experiments performed in the laboratory in combination with analyses of vegetation, show that TCAA is probably formed in the top soil and then partly taken up by the vegetation and partly mineralized in the soil. Based on this and previous studies, a conceptual model for the natural cycling of trichloromethyl compounds in forests is proposed.

Microbial oxidation of pyrite coupled to nitrate reduction in anoxic groundwater sediment.

Although many areas in Denmark are intensively agricultured, the discharge of nitrate from groundwater aquifers to surface water is often lower than expected. In this study it is experimentally demonstrated that anoxic nitrate reduction in sandy sediment containing pyrite is a microbially mediated denitrification process with pyrite as the primary electron donor. The process demonstrates a temperature dependency (Q10) of 1.8 and could be completely inhibited by addition of a bactericide (NaN3). Experimentally determined denitrification rates show that more than 50% of the observed nitrate reduction can be ascribed to pyrite oxidation. The apparent zero-order denitrification rate in anoxic pyrite containing sediment at groundwater temperature has been determined to be 2-3 micromol NO3- kg(-1) day(-1). The in situ groundwater chemistry at the boundary between the redoxcline and the anoxic zone reveals that between 65 and 80% of nitrate reduction in the lower part of the redoxcline is due to anoxic oxidation of pyrite by nitrate with resulting release of sulfate. It is concluded that microbes can control groundwater nitrate concentrations by denitrification using primarily pyrite as electron donor at the oxic-anoxic boundary in sandy aquifers thus determining the position and downward progression of the redox boundary between nitrate-containing and nitrate-free groundwater.

Degradation of the potato glycoalkaloid alpha-solanine in three agricultural soils.

The toxic glycoalkaloids produced by the potato plant (Solanum tuberosum L.) have previously been found in upper soil from a potato field during several months. Further insight into the fate of the glycoalkaloids is needed, as only little information about their degradation in soil is available. Degradation of the glycoalkaloid, alpha-solanine, has been followed for 42d in three agricultural soils with common texture and carbon contents. A similar degradation pattern was found in all soils, and the kinetics was well described by a sum of two first-order equations. Overall, degradation rates for the initial first reaction were in the range 0.22-1.64d(-1). Estimated half-lives were in the range 1.8-4.1d for the three top soils at 15 degrees C; the fastest degradation was observed in the sandy soil. The major proportion of alpha-solanine in the sandy soil was degraded by the fast process, while the proportion was lower for the two other soils. Fast degradation appeared to be related to the presence of low amount of sorbents. Additionally, degradation was followed at 5 degrees C in A- and C-horizon soil from the sandy location, and for both horizons the half-lives were of similar length (4.7-8.7d). For the slow process, degradation rates were in the range 0.000-0.123d(-1), and residuals were still present in all soils and all temperatures at the end of the experiment (d 42). Overall, fast degradation was found in both top- and subsoil even at low temperatures, and the risk for alpha-solanine leaching to the groundwater appears to be low.

Metabolites of the phenylurea herbicides chlorotoluron, diuron, isoproturon and linuron produced by the soil fungus Mortierella sp.

Phenylurea herbicides are used worldwide, and often pollute surface- and groundwater in concentrations exceeding the limit value for drinking water (0.1 microg l(-1)). Bacteria degrade phenylurea herbicides by successive N-dealkylation to substituted aniline products. Little is known about the corresponding fungal pathways, however. We here report degradation of chlorotoluron, diuron, isoproturon and linuron by the soil fungus Mortierella sp. Gr4. Degradation was fastest with linuron and resulted in successively dealkylated metabolites and 3,4-dichloroaniline. A major new metabolite was detected that has not yet been fully identified. Thin layer chromatography and nuclear magnetic resonance spectroscopy indicate that it is a non-aromatic diol. Degradation of isoproturon, chlorotoluron and diuron involved successive N-demethylation and, in the case of isoproturon and chlorotoluron, additional hydroxylation. A new hydroxylated isoproturon metabolite was detected. The study thus shows that the fungal pathways differ from the bacterial pathways and yield new metabolites of possible environmental concern.

Fate of toxic potato glycoalkaloids in a potato field.

The toxic glycoalkaloids, alpha-solanine and alpha-chaconine, are present in all parts of the potato plant and are possibly transferred to the terrestrial environment. The amounts of glycoalkaloids in plant, soil, and groundwater were followed in a potato field to investigate their distribution and fate during the season. The amount of glycoalkaloids in the plants was up to 25 kg/ha during maturity and decreased to below 0.63 kg/ha during plant senescence. The glycoalkaloids were detected in the upper soil (up to 0.6 kg/ha); this amount accounted only for a minor fraction of the amount present in the plants. Maximum glycoalkaloid concentration of 2.8 mg/kg dry weight soil was detected in September. Dissipation during winter appeared to be slow because glycoalkaloids were still present in the soil in March. No traces of glycoalkaloids were detected in the groundwater (detection limit 0.2 microg/L). From these results, the leaching potential of the glycoalkaloids is evaluated to be small.