Nitrogen and oxygen isotopes of dissolved nitrate to evaluate the efficiency of induced groundwater denitrification at field-scale

Nitrate, pollutants, denitrification, nitrate vulnerable zones, stable isotopes

Impact of fertilization with pig slurry on the isotopic composition of nitrate retained in soil and leached to groundwater in agricultural areas

The isotopic composition of N and O of nitrate (NO3−) is usually employed to trace its sources of pollution in groundwater. In agricultural areas, the amount of NO3− that reaches the aquifers after fertilization is controlled by different transformation processes that can affect the nitrogen species isotopic composition. Aiming to address the reliability of using isotope tools to trace sources of groundwater NO3−, the goal of this study was to check the effect of fertilization on the isotopic composition of N compounds retained and leached from soils. The concentration and isotopic composition (δ15N and δ18O) of ammonium (NH4+), NO3− and nitrite (NO2−) was characterized after the application of pig slurry in lysimeters containing either soil under fallow (LF) or the same soil continuously cropped and fertilized (LC) during the previous six years. Results showed that the leached NO3− isotopic signature did not directly reflect the isotopic composition of the applied pig slurry. Just after fertilization, nitrification led to lower δ15NNO3 values in soil extracts and leachates (e.g. from +5.9 ± 0.9¿ to +3.8 ± 3.1¿ in soil extracts of LF lysimeters). These values increased after complete nitrification (+11.5 ± 1.3¿) towards the δ15Nbulk of pig slurry (+19.6 ± 0.5¿). Later on, due to soil organic matter and plant debris mineralization and subsequent nitrification, values decreased towards the initial δ15NNO3 of soil but remained above them (+8.6 ± 1.0¿). Both LF and LC experiments showed a similar trend and the latter ones allowed to reinforce that long-term fertilization with pig slurry can increase the soil δ15NNO3. Concerning the δ18O of NO3− from soil extracts and leachates, it mainly depended on the δ18O of irrigation water and oxygen, after nitrification of NH4+ from pig slurry. Therefore, studies aiming to trace groundwater NO3− pollution sources in rural areas by using an isotopic approach should consider the fertilization history of each setting. Also, analyzing the δ15Nbulk of soil is recommended, since it could mask the isotopic signature of the N applied through fertilization

Optimization of induced denitrification strategies in polluted water bodies from agricultural sources

[eng] The worldwide persistence of nitrate (NO -) in groundwater is worrying since this compound has been related to human illnesses and to eutrophication of aquatic ecosystems. The main sources of pollution are linked to intensive use of fertilizers and septic system leakage. Since 1991, European directives have been applied to mitigate NO - pollution by limiting N application in agricultural lands. However, due to the long residence time of N in the soil organic matter pool, the outcome of the implemented management practices can be delayed for decades. Consequently, remediation strategies must be developed and optimized. The NO3- reduction to innocuous dinitrogen gas (N2), can occur intrinsically in many environments due to the ubiquity of denitrifying microorganisms. However, the electron donor presence is usually a limiting factor in the contaminated aquifers. Hence, one feasible treatment involves inducing the denitrification by application of an external electron donor. During the implementation of bioremediation strategies, the contaminant removal can be estimated by monitoring its concentrations before and after the treatment. However, this method does not reveal the specific processes involved in the attenuation, making it challenging to focus on the improvement of the remediation approach. Isotopic analyses have proved to be a powerful tool in identifying the sources and transformation processes of groundwater contaminants. During the enzymatic NO3- reduction, the unreacted residual substrate becomes enriched in the heavy isotopes 15N and 18O, allowing to distinguish the biotic reduction from processes such as dilution with non-polluted water that could also lead to a concentration decrease without influencing the isotopic signature. The present thesis focusses on investigating the use of low-cost electron donors to promote the denitrification, and on using isotopic tools to evaluate the denitrification efficiency at field- scale. The tested materials in batch and flow-through experiments were: whey, corn stubble, wheat hay, animal compost, magnetite, siderite and olivine. Different parameters that could affect the biotic NO3- reduction efficiency were evaluated (e.g., temperature, ratio between electron donor and acceptor, harmful by-product accumulation, abiotic reactivity or coexistence of other contaminants) and the isotopic fractionation values (ε15N and ε18O) were determined for all tested conditions. At field-scale, three different polluted water bodies were studied, in which the determined ε15N and ε18O at the laboratory were applied to quantify the natural and/or induced denitrification. In a polluted aquifer in Spain, the NO3- attenuation was evaluated during a long-term induced denitrification strategy by acetic acid injections. In a polluted aquifer in Argentina, the natural NO3- attenuation was evaluated considering changes in the reactivity and isotopic fractionation due to the simultaneous presence of NO - and Cr6+. Finally, in a constructed wetland (CW) treating agricultural runoff water, the NO - attenuation was evaluated before and after application of an electron donor both in the autumn-winter and spring-summer seasons. The laboratory experiments demonstrated that magnetite nanoparticles, corn stubble, wheat hay, animal compost and whey could efficiently promote the denitrification in polluted water bodies. In these biotic experiments, the complete NO3- reduction to N2 was demonstrated by transient or negligible accumulation of other nitrogen compounds such as nitrite (NO2-), ammonium (NH +) or nitrous oxide (N O). However, the N O was found to be the end- product of the abiotic NO - reduction, which can be mediated by ferrous iron, if present. The ε15NNO3/N2 and ε18ONO3/N2 values were calculated for all the batch experiments and for the periods of the flow-through experiment that allowed complete denitrification. For the non- complete denitrification periods, the NO - isotopic characterization showed a mix of denitrified and non-denitrified water at the outflow. The isotopic characterization of NO - and N2O allowed to distinguish the biotic from the abiotic NO - reduction by ferrous iron at the laboratory-scale. A two-stage isotopic fractionation pattern was found for Cr6+, which can be reduced simultaneously to NO -. Also, the carbon compounds isotopic analysis allowed to assess the fate of the studied organic carbon materials to be used as electron donors. In the field-scale studies, the chemical and isotopic characterization allowed to trace the extent of the natural or induced denitrification and to evaluate the safety of the treatments. In a pilot plant to remediate groundwater NO3- pollution (Spain), acetic acid was injected by pulses to an alluvial aquifer for 22 months. According to the isotopic results, the induced denitrification achieved at least 50 % NO3- attenuation. The isotopic analyses also allowed to identify the reoxidation of NO2- to NO3- during the treatment and to recognize a mixture between the denitrified and partially or non-denitrified groundwater in one of the sampling points. In a polluted aquifer with both NO3- and Cr6+ (Argentina), the calculated natural attenuation was 20 % for NO3- and 60 % for Cr6+. For this calculation, the two stage trend observed for the ε53Cr was considered. The attenuation of Cr6+ in a few samples was found to be due by both reduction and dilution. In a CW treating agricultural runoff water, a slight natural NO3- attenuation was only observed when the flow was below 5.5 L/s. According to the isotopic results, after the biostimulation by stubble application, at least 60 % NO3- was removed at 16 L/s. The biostimulation treatment in autumn lasted in one month, while in spring the attenuation remained for three months.[cat] La persistència del nitrat (NO -) en aigües subterrànies és preocupant ja que aquest, pot provocar malalties en humans i eutrofització d’ecosistemes aquàtics. Els principals orígens de contaminació són l’ús intensiu de fertilitzants i les pèrdues dels sistemes sèptics. Tot i que els darrers anys s’ha limitat la quantitat de N aplicat en zones agrícoles, degut al llarg temps de residència del N en la matèria orgànica del sòl, el resultat de les pràctiques implementades, es pot ajornar fins dècades. Per tant, és necessari desenvolupar i optimitzar estratègies de remediació. La reducció del NO - a dinitrogen gas (N ), que és un gas innocu, es dona intrínsecament en molts ambients degut a la ubiqüitat dels microorganismes amb capacitat de desnitrificar. Malauradament, la presència de donadors d’electrons sol ser un factor limitant en aqüífers contaminats per NO -. Per això, un possible tractament consisteix en induir la desnitrificació gràcies a l’aplicació d’un donador d’electrons extern. Durant la implementació d’estratègies de bioremediació, l’eliminació del contaminant es pot determinar mitjançant la monitorització de les seves concentracions abans i després del tractament. Però aquest mètode no mostra el procés específic involucrat en l’atenuació i això dificulta l’optimització de l’estratègia de remediació. Els anàlisis isotòpics resulten útils per identificar fonts i processos de transformació de diversos contaminants en aigües subterrànies. Durant la reducció enzimàtica del NO3-, el substrat residual es va enriquint en els isòtops pesats 15N i 18O. Això permet distingir la reducció biòtica d’altres processos com la dilució amb aigua no contaminada que també podria donar lloc a una disminució de la concentració del NO3- però sense influenciar la seva signatura isotòpica. Aquesta tesi es centra en investigar l’ús de donadors d’electrons de baix cost (sèrum làctic, restes vegetals (blat i panís), compost animal, magnetita, siderita i olivina) per induir la desnitrificació i l’ús d’eines isotòpiques per avaluar l’eficiència de desnitrificació a escala de camp. Durant els experiments al laboratori s’han avaluat diferents paràmetres que poden afectar l’eficiència de la reducció biòtica del NO3- (ex. temperatura, ràtio entre el donador i acceptor d’electrons, acumulació de productes intermedis tòxics, reactivitat abiòtica o coexistència d’altres contaminants) i s’han calculat els valors de fraccionament isotòpic (ε15N i ε18O) per totes les condicions investigades. A escala de camp, s’han estudiat tres masses d’aigua contaminades en les que s’han aplicat els valors de ε15N i ε18O determinats al laboratori per quantificar la desnitrificació natural o induïda. En un aqüífer contaminat a Espanya, l’atenuació del NO3- s’ha avaluat durant una estratègia de desnitrificació induïda mitjançant la injecció d’àcid acètic. En un aqüífer contaminat a Argentina, l’atenuació natural del NO3- s’ha avaluat considerant canvis en la reactivitat i el fraccionament isotòpic degut a la presencia simultània de NO - i Cr6+. En un aiguamoll construït en el qual es tracta aigua d’escorrentia agrícola, l’atenuació del NO - s’ha avaluat abans i després de l’aplicació d’un donador d’electrons tant a la tardor-hivern com a la primavera-estiu. Els experiments de laboratori han demostrat que les nanopartícules de magnetita, les restes vegetals (blat i panís), el compost animal i el sèrum làctic poden induir la desnitrificació en aigües contaminades. En aquests experiments biòtics, la reducció completa del NO - a N ha estat demostrada per una acumulació transient o negligible d’altres compostos nitrogenats com el nitrit (NO -), l’amoni (NH +) o l’òxid nitrós (N O). Tot i això, s’ha vist que el N2O és el producte final de la reducció abiòtica del NO - provocada per l’oxidació de Fe2+, si és present en l’aigua. Els valors de ε15NNO3/N2 i ε18ONO3/N2 s’han calculat pels experiments de tipus batch i pels períodes d’un experiment de tipus flux continu durant els que es va assolir una desnitrificació completa. La caracterització isotòpica del NO - i el N O ha permès distingir la reducció del NO2- biòtica de l’abiòtica per oxidació de Fe2+ al laboratori. Per al Cr6+, un contaminant que es pot reduir simultàniament al NO3-, s’ha observat un fraccionament isotòpic en dos estadis. A més, l’anàlisi isotòpic dels compostos de carboni ha permès avaluar el consum dels donadors d’electrons de carboni orgànic estudiats. En els estudis a escala de camp, la caracterització química i isotòpica ha permès traçar l’eficiència de la desnitrificació natural i/o induïda i avaluar la seguretat dels tractaments. En una planta pilot per remeiar la contaminació de NO3- d’aigües subterrànies (Espanya), s’ha injectat àcid acètic a l’aqüífer durant 22 mesos. D’acord amb els resultats isotòpics, la desnitrificació induïda ha assolit almenys un 50% d’atenuació del NO3-. La caracterització isotòpica també ha permès identificar la reoxidació de NO2- a NO3- durant el tractament i reconèixer una barreja entre aigua desnitrificada i aigua parcialment o no desnitrifricada en un dels punts de mostreig. En un altre aqüífer contaminat amb NO3- i Cr6+ (Argentina), l’atenuació natural calculada ha estat del 20% per al NO3- i del 60 % per al Cr6+. Per a aquest càlcul s’ha tingut en compte el fraccionament isotòpic en dos estadis observat pel Cr6+ en els experiments de laboratori. L’atenuació del Cr6+ en algunes mostres ha estat deguda en part a dilució i en part a reducció. En l’aiguamoll construït, l’atenuació natural del NO3- només es dona quan el flux és inferior a 5.5 L/s. D’acord amb els resultats isotòpics, després de la bioestimulació per aplicació de restes vegetals (panís), s’ha aconseguit una reducció del 60 % del NO3-, a un flux de 16 L/s. El tractament de bioestimulació a l’octubre-hivern ha durat un mes, mentre que a la primavera-estiu s’ha mantingut durant tres mesos

Isotopic fractionation associated to nitrate attenuation by ferrous iron containing minerals

Biotic and abiotic laboratory experiments of nitrate and nitrite reduction by Fe-containing minerals were performed and the isotopic fractionation of the different reactions was calculated in order to determine whether it is possible to distinguish biotic and abiotic reactions involving N compounds. Results of biotic experiments showed nitrate reduction up to 96 % with transient NO2- accumulation and no significant N2O production. No significant nitrate attenuation was observed in abiotic nitrate reduction experiments. Abiotic experiments of nitrite reduction showed a rapid decrease in nitrite concentrations in those experiments with added Fe2+ coupled with a significant N2O production. Preliminary results of the N and O isotopic fractionation of the biotic experiments of nitrate reduction show differences in the ε15NNO3 and ε18ONO3 when different minerals were added. The abiotic experiments of nitrite reduction contrarily, showed similar ε15NNO2 in all the experiments

Characterisation of the natural attenuation of chromium contamination in the presence of nitrate using isotopic methods. A case study from the Matanza-Riachuelo River basin, Argentina

The groundwater contamination by hexavalent chromium (Cr(VI)) in a site of the Matanza-Riachuelo River basin (MRB), Argentina, has been evaluated by determining the processes that control the natural mobility and attenuation of Cr(VI) in the presence of high nitrate (NO ) contents. The groundwater Cr(VI) concentrations ranged between 1.9E-5 mM and 0.04 mM, while the NO concentrations ranged between 0.5 mM and 3.9 mM. In order to evaluate the natural attenuation of Cr(VI) and NO in the MRB groundwater, Cr and N isotopes were measured in these contaminants. In addition, laboratory batch experiments were performed to determine the isotope fractionation (ε) during the reduction of Cr(VI) under denitrifying conditions. While the Cr(VI) reduction rate is not affected by the presence of NO , the NO attenuation is slower in the presence of Cr(VI). Nevertheless, no significant differences on ε values were observed when testing the absence or presence of each contaminant. The εCr determined in the batch experiments describe a two- stage trend, in which Stage I is characterized by εCr ~−1.8‰ and Stage II by εCr ~−0.9‰. The respective εN obtained is −23.9‰ whereas εO amount to −25.7‰. Using these ε values and a Rayleigh fractionation model we estimate that an average of 60% of the original Cr(VI) is removed from the groundwater of the contaminated site. Moreover, the average degree of NO attenuation by denitrification is found to be about 20%. This study provides valuable information about the dynamics of a complex system that can serve as a basis for efficient management of contaminated groundwater in the most populated and industrialized basin of Argentina.This research was supported by the Instituto de Hidrología de Llanura “Dr. Eduardo J. Usunoff” (IHLLA) and by IDAEA-CSIC as part of the programme EMHE “Enhancing Mobility between Latin-American and Caribbean countries and Europe”. We thank the IHLLA technic staff for their assistance in water sampling and Ms. M. F. Altolaguirre, B.S., and Ms. O. Floriani, B.S., for assisting in the logistics of manipulation and conservation of samples. This research is also supported by the project PACE-ISOTEC (CGL2017-87216-C4-1-R), financed by the Spanish Government and AEI/FEDER from the UE, and the project MAG (2017 SGR 1733) from the Catalan Government. We also thank the Centres Científics i Tecnològics of the Universitat de Barcelona for its analytical support

Nitrate and nitrite reduction by ferrous iron minerals in polluted groundwater: Isotopic characterization of batch experiments

Since nitrate (NO3-) has been related to human health and environmental problems, safe and sustainable strategies to remediate polluted water bodies must be investigated. This work aims to assess the feasibility of using ferrous iron (Fe(II))-containing minerals to stimulate microbial denitrification while avoiding pollution swapping (e.g. accumulation of the by-products nitrite (NO2-) or nitrous oxide (N2O)). To accomplish the objective, samples obtained from several batch experiments were characterized chemically and isotopically. Magnetite, siderite and olivine were tested micro-sized and magnetite was also tested nano-sized. In microbial experiments, NO3- polluted groundwater was employed as inoculum. In these experiments, NO3- reduction to nitrogen gas (N2) was only completed in microcosms containing magnetite nanoparticles, suggesting an increased Fe(II) availability from nano-sized compared to micro-sized magnetite. In abiotic experiments, no reactivity was observed between NO3- or NO2- and micro-sized magnetite, siderite or olivine, while NO2- was rapidly reduced when dissolved Fe2+ was added. These results point to the need of a certain amount of dissolved Fe2+ to stimulate the abiotic NO2- reduction by Fe(II) oxidation. For the microbial NO3- reduction by magnetite nanoparticles, the calculated e15NNO3 was -33.1‰ (R2 = 0.86), e18ONO3 was -10.7‰ (R2 = 0.74) and e15NNO3/e18ONO3 was 3.1. For the abiotic NO2- reduction by Fe2+, the e15NNO2 ranged from -14.1 to -17.8‰ (R2 > 0.89). Considering the wide range of e15NNO2 reported in the literature, it is not likely that NO2- isotopic characterization can be useful at field-scale to distinguish abiotic from microbial NO2- reduction. Nevertheless, the measured d15N for N2O in microbial and abiotic tests, allowed determining if it was an intermediate or a final product of the reactions by comparing these results with the modelled isotopic composition calculated using the e15N values determined for the substrates. Hence, isotopic data confirmed that the product of the microbial NO3- reduction was innocuous N2 while the product of the abiotic NO2- reduction was N2O. The latter reaction would be advantageous to avoid NO2- accumulation during denitrification only if the generated N2O is further reduced by microorganisms.Peer ReviewedPostprint (author's final draft

Upside down sulphate dynamics in a saline inland lake

International audienceThe sulphur cycle has a key role on the fate of nutrients through its several interconnected reactions. Although sulphur cycling in aquatic ecosystems has been thoroughly studied since the early 70's, its characterisation in saline endorheic lakes still deserves further exploration. Gallocanta Lake (NE Spain) is an ephemeral saline inland lake whose main sulphate source is found on the lake bed minerals and leads to dissolved sulphate concentrations higher than those of seawater. An integrative study including geochemical and isotopic characterization of surface water, porewater and sediment has been performed to address how sulphur cycling is constrained by the geological background. In freshwater and marine environments, sulphate concentration decreases with depth are commonly associated with bacterial sulphate reduction (BSR). However, in Gallocanta Lake sulphate concentrations in porewater increase from 60 mM at the water-sediment interface to 230 mM at 25 cm depth. This extreme increase could be caused by dissolution of the sulphate rich mineral epsomite (MgSO 4 •7H 2 O). Sulphur isotopic data was used to validate this hypothesis and demonstrate the occurrence of BSR near the water-sediment interface. This dynamic prevents methane production and release from the anoxic sediment, which is advantageous in the current context of global warming. These results underline that geological context should be considered in future biogeochemical studies of inland lakes with higher potential availability of electron acceptors in the lake bed compared to the water column