Compound-Specific Chlorine Isotope Analysis of the Herbicides Atrazine, Acetochlor, and Metolachlor

A gas chromatography-single quadrupole mass spectrometry method was developed and validated for compound-specific chlorine isotope analysis (Cl-CSIA) of three chlorinated herbicides, atrazine, acetochlor, and metolachlor, which are widespread contaminants in the environment. For each compound, the two most abundant ions containing chlorine (202/200 for atrazine, 225/223 for acetochlor, and 240/238 for metolachlor) and a dwell time of 30 ms were determined as optimized MS parameters. A limit of precise isotope analysis for ethyl acetate solutions of 10 mg/L atrazine, 10 mg/L acetochlor, and 5 mg/L metolachlor could be reached with an associated uncertainty between 0.5 and 1 . To this end, samples were measured 10-fold and bracketed with two calibration standards that covered a wide range of δ37Cl values and for which amplitudes matched those of the samples within 20% tolerance. The method was applied to investigate chlorine isotope fractionation during alkaline hydrolysis of metolachlor, which showed a shift in δ37Cl of +46 after 98% degradation, demonstrating that chlorine isotope fractionation could be a sensitive indicator of transformation processes even when limited degradation occurs. This method, combined with large-volume solid-phase extraction (SPE), allowed application of Cl-CSIA to environmentally relevant concentrations of widespread herbicides (i.e., 0.5-5 μg/L in water before extraction). Therefore, the combination of large-volume SPE and Cl-CSIA is a promising tool for assessing the transformation processes of these pollutants in the environment

Characterization of Attachment and Growth of Thiobacillus denitrificans on Pyrite Surfaces

Anaerobic growth and attachment of the autotrophic denitrifying bacterium Thiobacillus denitrificans on pyrite surfaces were studied. Polished pyrite slabs were exposed to T. denitrificans for 1 to 9 weeks. The reacted pyrite surfaces were imaged with scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Cells were observed as isolated attached cells, cells in division and cells forming microcolonies embedded in organic films. Bacteria began to colonize pyrite surfaces after 1 week, forming microcolonies after 3 weeks. The rate of colonization of the pyrite surface was around 35 cells mm−2 h−1 for the 3-week period. After 9 weeks, larger areas of the pyrite surface were covered by organic films. Bacterial enumeration on the pyrite surface and in solution showed that most of the cells were not attached to the mineral surface. Nevertheless, both attached and free-living bacteria probably contributed to pyrite-driven denitrification. The results may be applied to the natural environment to better understand pyrite-driven denitrification in aquifers and to improve the long-term performance of bioremediation processes using pyrite

C and Cl-CSIA for Elucidating Chlorinated Methanes Biotic and Abiotic Degradation at a Polluted Bedrock Aquifer

AbstractIn this study,concentration and δ13C and δ37Cl values of chloroform (CF) and carbon tetrachloride (CT) measured in groundwater samples are related to redox conditions to elucidate natural attenuation processes in a polluted site. Shifts in δ13C and δ37Cl of CF and CT were detected over time.δ13CCF values found in the potential sources were -34.5 ± 0.6 ‰ (tank near S3 well), -37.3 ± 0.6 ‰ (in a fracture near S1 well) and -46.2± 0.4 ‰ (barrels) in 2004, whereas in later sampling campaigns, δ13CCF enriched values such as -25.2 ± 0.5 ‰ were measured in S3 in 2013. 2D plots (δ13CCFvs δ37ClCF) showed different isotopic patterns in different wells and depths. δ37ClCF values let distinguish that the former sources evolved towards completely different isotopic signatures, whilst δ37ClCF values of +1.3 ± 0.3 ‰ were reached in 2013 for S3,S1 moved towards more depleted δ37ClCF values (down to -3.9 ± 0.6‰). These data indicate that differentreductive dechlorination processes might occur

Denitrification of groundwater with pyrite and Thiobacillus denitrificans

Anaerobic batch and flow-through experiments were performed to confirm the role of pyrite as electron donor in bacterial denitrification and to look into the feasibility of pyrite-driven denitrification of nitrate- contaminated groundwater. Nitrate reduction was satisfactorily accomplished in experiments with pyrite as the sole electron donor, in presence of the autotrophic denitrifying bacterium Thiobacillus denitrificans and at nitrate concentrations comparable to those observed in contaminated groundwater. The experimental results corroborated field studies in which the reaction occurred in aquifers. Nitrate reduction rates and nitrate removal efficiencies were dependent on pyrite grain size, initial nitrate concentration, nitrate-loading rate and pH. The N and O isotopic enrichment factors (εN and εO) obtained experimentally for pyrite-driven nitrate reduction by T. denitrificans ranged from − 13.5¿ to − 15.0¿ and from − 19.0¿ to − 22.9¿, respectively. These values indicated the magnitude of the isotope fractionation that occurs in nitrate- contaminated aquifers dominated by autotrophic denitrification

Monitoring groundwater nitrate attenuation in a regional system coupling hydrogeology with multi-isotopic methods: the case of Plana de Vic (Osona, Spain)

This paper describes an integrated application of classical hydrogeological methods and multi-isotopic methods (d15N, d18ONO3, d34S, d18OSO4, d13C) to assess the fate of groundwater nitrate in the Osona area, declared vulnerable to nitrate pollution by the Catalan Government in 1998, where nitrate is derived from intensive pig farming activities. Previous studies, involving a small area, indicated the occurrence of denitrification processes and their relationship with pyrite oxidation [Vitòria, L., Soler, A., Canals, A., Otero, N., 2008. Environmental isotopes (N, S, C, O, D) to determine natural attenuation processes in nitrate contaminated waters: example of Osona (NE Spain). Appl. Geochem. 23, 3597-3611]. For the present study, groundwater samples were collected at 60 production wells at three different periods between April 2005 and May 2006 to confirm that denitrification takes place in a larger area than that studied by Vitòria et al. [Vitòria, L., Soler, A., Canals, A., Otero, N., 2008. Environmental isotopes (N, S, C, O, D) to determine natural attenuation processes in nitrate contaminated waters: example of Osona (NE Spain). Appl. Geochem. 23, 3597-3611]. The aim of the study was to characterize the denitrification processes that control natural attenuation and to study their spatial and temporal variations. Nitrate concentration ranged from 10 to 529 mg/l, with 82% of the wells above the drinking water threshold of 50 mg NO3/l. Nitrate isotopic composition ranged from +5.3% to +35.3% for d15N and from +0.4% to +17.6% for d18ONO3 , and the samples showed a positive correlation between d15N and d18ONO3 , with a eN/eO ratio of 1.8, consistent with denitrification processes. The link between denitrification and pyrite oxidation is demonstrated by coupling chemical data with nitrate and sulfate isotopes. Furthermore, a spatial distribution of samples with significant denitrification was observed, allowing us to determine two main hydrogeological zones where natural attenuation was most effective. In several of the studied points, denitrification processes related to pyrite oxidation predominated and an estimation of the isotopic enrichment factors was performed using the temporal variations of nitrate concentration and the isotopic composition of dissolved nitrate (d15NNO3 and d18ONO3). Finally, using estimated isotopic enrichment factors, an approximation of the degree of natural attenuation of nitrate was performed on those samples showing clear denitrification, and a median value of 30% of contaminant diminution was obtained

Isotopic fractionation associated to nitrate reduction by zero valent iron

One of the methodologies developed in recent years to induce nitrate attenuation is the use of permeable reactive barriers (PRB). This in situ remediation technique involves the interception of groundwater flow to remove contaminants by physical, chemical or biological processes. In the case of nitrate pollution, heterotrophic or autotrophic denitrification can be induced by using organic or inorganic substrates, respectively. On the other hand, several PRB filled with Zero-Valent Iron (ZVI) have been installed to remediate groundwater polluted with chlorinated solvents (Wilkin et al., 2014) or nitrate (Gu et al., 2002)

Solid-phase extraction method for stable isotope analysis of pesticides from large volume environmental water samples

Compound-specific isotope analysis (CSIA) is a valuable tool for assessing the fate of organic pollutants in the environment. However, the requirement of sufficient analyte mass for precise isotope ratio mass spectrometry combined with prevailing low environmental concentrations currently limits comprehensive applications to many micropollutants. Here, we evaluate the upscaling of solid-phase extraction (SPE) approaches for routine CSIA of herbicides. To cover a wide range of polarity, a SPE method with two sorbents (a hydrophobic hypercrosslinked sorbent and a hydrophilic sorbent) was developed. Extraction conditions, including the nature and volume of the elution solvent, the amount of sorbent and the solution pH, were optimized. Extractions of up to 10 L of agricultural drainage water (corresponding to up to 200 000-fold pre-concentration) were successfully performed for precise and sensitive carbon and nitrogen CSIA of the target herbicides atrazine, acetochlor, metolachlor and chloridazon, and metabolites desethylatrazine, desphenylchloridazon and 2,6-dichlorobenzamide in the sub-μg L−1-range. 13C/12C and 15N/14N ratios were measured by gas chromatography-isotope ratio mass spectrometry (GC/IRMS), except for desphenylchloridazon, for which liquid chromatography (LC/IRMS) and derivatization-GC/IRMS were used, respectively. The method validated in this study is an important step towards analyzing isotope ratios of pesticide mixtures in aquatic systems and holds great potential for multi-element CSIA applications to trace pesticide degradation in complex environments

Isotopic evidence of nitrate degradation by a zero-valent iron permeable reactive barrier: Batch experiments and a field scale study

Permeable reactive barriers (PRBs) filled with zero-valent iron (ZVI) are a well-known remediation approach to treat groundwater plumes of chlorinated volatile organic compounds as well as other contaminants. In field implementations of ZVI-PRBs designed to treat these contaminants, nitrate consumption has been reported and has been attributed to direct abiotic nitrate reduction by ZVI or to denitrification by autochthonous microorganisms using the dissolved hydrogen produced from ZVI corrosion. Isotope tools have proven to be useful for monitoring the performance of nitrate remediation actions. In this study, we evaluate the use of isotope tools to assess the effect of ZVI-PRBs on the nitrate fate for the further optimization of full-scale applications. Laboratory batch experiments were performed using granular cast ZVI and synthetic nitrate solutions at pH 4-5.5 or nitrate-containing groundwater (pH = 7.0) from a field site where a ZVI-PRB was installed. The experimental results revealed nitrate attenuation and ammonium production for both types of experiments. In the field site, the chemical and isotopic data demonstrated the occurrence of ZVI-induced abiotic nitrate reduction and denitrification in wells located close to the ZVI-PRB. The isotopic characterization of the laboratory experiments allowed us to monitor the efficiency of the ZVI-PRB at removing nitrate. The results show the limited effect of the barrier (nitrate reduction of less than 15-20%), probably related to its non-optimal design. Isotope tools were therefore proven to be useful tools for determining the efficacy of nitrate removal by ZVI-PRBs at the field scale

Compound-specific chlorine isotope fractionation in biodegradation of atrazine

Atrazine is a frequently detected groundwater contaminant. It can be microbially degraded by oxidative dealkylation or by hydrolytic dechlorination. Compound-specific isotope analysis is a powerful tool to assess its transformation. In previous work, carbon and nitrogen isotope effects were found to reflect these different transformation pathways. However, chlorine isotope fractionation could be a particularly sensitive indicator of natural transformation since chlorine isotope effects are fully represented in the molecular average while carbon and nitrogen isotope effects are diluted by non-reacting atoms. Therefore, this study explored chlorine isotope effects during atrazine hydrolysis with Arthrobacter aurescens TC1 and oxidative dealkylation with Rhodococcus sp. NI86/21. Dual element isotope slopes of chlorine vs. carbon isotope fractionation (ΛArthroCl/C = 1.7 ± 0.9 vs. ΛRhodoCl/C = 0.6 ± 0.1) and chlorine vs. nitrogen isotope fractionation (ΛArthroCl/N = −1.2 ± 0.7 vs. ΛRhodoCl/N = 0.4 ± 0.2) provided reliable indicators of different pathways. Observed chlorine isotope effects in oxidative dealkylation (εCl = −4.3 ± 1.8 ) were surprisingly large, whereas in hydrolysis (εCl = −1.4 ± 0.6 ) they were small, indicating that C-Cl bond cleavage was not the rate-determining step. This demonstrates the importance of constraining expected isotope effects of new elements before using the approach in the field. Overall, the triple element isotope information brought forward here enables a more reliable identification of atrazine sources and degradation pathways

Enhanced denitrification in groundwater and sediments from a nitrate-contaminated aquifer after addition of pyrite

Using chemical, isotopic and microbiologic techniques we tested in laboratory experiments the extent to which the addition of pyrite to groundwater and sediments from a nitrate-contaminated aquifer could stimulate denitrification by indigenous bacteria. In addition to this biostimulated approach, a combined biostimulated and bioaugmented treatment was also evaluated by inoculating the well-known autotrophic denitrifying bacterium Thiobacillus denitrificans. Results showed that the addition of pyrite enhanced nitrate removal and that denitrifying bacteria existing in the aquifer material were able to reduce nitrate using pyrite as the electron donor, obviating the need for the inoculation of T. denitrificans. The results of the 16S rRNA and nosZ gene-based DGGE and the quantitative PCR (qPCR) showed that the addition of pyrite led to an increase in the proportion of denitrifying bacteria and that bacterial populations closely related to the Xanthomonadaceae might probably be the autotrophic denitrifiers that used pyrite as the electron donor. Not only autotrophic but also heterotrophic denitrifying bacteria were stimulated through pyrite addition and both populations probably contributed to nitrate removal. Isotopic analyses (δ15N and δ18ONO3) were used to monitor enhanced denitrification and the N and O isotopic enrichment factors (−26.3±1.8¿ and −20.4± 1.3¿, respectively) allowed to calculate the degree of natural nitrate attenuation in the aquifer. Furthermore, flow-through experiments amended with pyrite confirmed the long-term efficiency of the process under the study conditions. Further research under field conditions is needed to determine whether stimulation of denitrification by pyrite addition constitutes a feasible bioremediation strategy for nitrate-contaminated aquifers