Biotic nitrosation of diclofenac in a soil aquifer system (Katari watershed, Bolivia)

Highlights

• Formation of NO-DCF, NO-DPA and NO₂-DCF during soil aquifer water transfer

• Occurrence of NO₂-DCF and NO-DPA in groundwater samples at tens of ng/L levels

• Codenitrification was probably responsible for DCF and DPA N-nitrosation

• NO-DCF evolved into NO₂-DCF while NO-DPA remained stable

• Formation of NO₂-DCF was probably due to spontaneous N-NO bond cleavage

Abstract

Up till now, the diclofenac (DCF) transformation into its nitrogen-derivatives, N-nitroso-DCF (NO-DCF) and 5-nitro-DCF (NO₂-DCF), has been mainly investigated in wastewater treatment plant under nitrification or denitrification processes. This work reports, for the first time, an additional DCF microbial mediated nitrosation pathway of DCF in soil under strictly anoxic conditions probably involving codenitrification processes and fungal activities. This transformation pathway was investigated by using field observations data at a soil aquifer system (Katari watershed, Bolivia) and by carrying out soil slurry batch experiments. It was also observed for diphenylamine (DPA). Field measurements revealed the occurrence of NO-DCF, NO₂-DCF and NO-DPA in groundwater samples at concentration levels in the 6–68 s/L range. These concentration levels are more significant than those previously reported in wastewater treatment plant effluents taking into account dilution processes in soil. Interestingly, the p-benzoquinone imine of 5-OH-DCF was also found to be rather stable in surface water. In laboratory batch experiments under strictly anoxic conditions, the transformation of DCF and DPA into their corresponding N-nitroso derivatives was well correlated to denitrification processes. It was also observed that NO-DCF evolved into NO₂-DCF while NO-DPA was stable. In vitro experiments showed that the Fisher-Hepp rearrangement could not account for NO₂-DCF formation. One possible mechanism might be that NO-DCF underwent spontaneous NO loss to give the resulting intermediates diphenylaminyl radical or nitrenium cation which might evolve into NO₂-DCF in presence of NO₂ radical or nitrite ion, respectively.

Introduction

Diclofenac (DCF) is one of the most widely prescribed non-steroidal anti-inflammatory drugs (NSAID). DCF has been recently included in the EU Commission watch list of organic pollutants in surface water (SW) (Directive 2013/39/EU) as the consequence of its high frequency of detection rate in SW and its potential ecotoxicological impact. A lot is known about DCF fate, both in the human body and in the environment. The majority of DCF is metabolized in the human body and only 1% of the orally administered dose is excreted as un-metabolized DCF. The main human metabolites of DCF are 4′-OH-DCF and 5-OH-DCF. Both of them are excreted mainly in conjugated form and only < 1% is excreted unchanged. Important metabolites are also 3′-OH-DCF and 4′,5-diOH-DCF (Vieno and Sillanpää, 2014). DCF has been deeply investigated during aerobic biological wastewater treatment. DCF transformation leads to the formation of 4′-OH-DCF and to 1-(2,6-dichlorophenyl)-1,3-dihydro-2H-indol-2-one (DCF-lactam) through a dehydration reaction (Bouju et al., 2016). The biodegradation of 4′-OH-DCF was much quicker than that of DCF leading to 4′-OH-DCF-lactam. Formation of dichlorobenzoic acid has also been reported (Pérez and Barcelo, 2008). In a fixed-bed column bioreactor filled with river sediment under aerobic conditions, the p-benzoquinone imine derivative of 5-OH-DCF was identified as the main transformation product (TP) of DCF (Gröning et al., 2007). Extensive transformation of DCF was also evidenced in soil after 112 d of incubation with half-lives in the 1.4–4.3 d range, where 5-OH-DCF was identified as a major intermediate and 2,6-dichlorobenzoic acid as a minor intermediate (Dodgen et al., 2014). Further recent investigations concluded that the improved degradation of DCF was strongly dependent on a high activity of nitrifiers under nitrifying activated sludge (NAS). In these conditions, DCF bio-transformed additionally into its nitrogen-derivatives, N-nitroso-DCF (NO-DCF) and 5-nitro-DCF (NO₂-DCF, Osorio et al., 2014). It was anticipated that nitric oxide (NO) was responsible for such reactions to occur (Osorio et al., 2016). NO is a very reactive specie in aqueous media originating as a by-product of nitrification and denitrification processes and quickly evolves in NO-derived reactive species, mainly dinitrogen trioxide (N₂O₃) and peroxynitrite (ONOO⁻), both being strong nitrosating species. N₂O₃ is likely formed in presence of oxygen or under acidic conditions through the decomposition of nitrous acid (HNO₂), while ONOO⁻ is likely formed under neutral/basic conditions through the reaction of NO with superoxide (O₂⁻, k = 0.4–1.9 × 10⁹ M^− 1 s^− 1, Toledo and Augusto, 2012). N₂O₃ and ONOO⁻ are able to react with phenols (Jewell et al., 2014) and secondary amines (e.g., DCF) to give C-nitroso and N-nitroso derivatives, respectively but are also known to react with primary aromatic amines such as sulfamethoxazole (SMX) to give different TPs including 4-nitro-SMX and desamino-SMX (Nödler et al., 2012). The formation of appreciable NO-DCF and NO₂-DCF concentrations was found to be unlikely (in the ng/L range in WWWTP effluents, Osorio et al., 2014) and the presence of oxygen is requested for the reaction of NO with amines to proceed (Itoh et al., 1997). However, DCF degradation has also been reported in the hyporheic zone underlying streams under strictly anoxic conditions (Lewandowski et al., 2011). DCF was also highly removed in manage aquifer recharge under anoxic conditions (Rauch-Williams et al., 2010). Similarly to SMX, DCF degradation was also found to be influenced by denitrifying conditions in a laboratory scale column experiment during the reduction of nitrate (Branzhaf et al., 2012) at near neutral pH. But, up till now, the formation of nitro derivatives was only observed in case of SMX using water/sediment batch experiments (Barbieri et al., 2012). Since, at near neutral pH, the formation of N₂O₃ can be ruled out due to a pKa (HNO₂/NO₂⁻) of 3.3 and ONOO⁻ cannot be produced in absence of oxygen, these results probably call for nitrosation/nitration mechanisms under denitrifying conditions different from those reported under aerobic conditions. One assumption is codenitrification which is a relevant microbial denitrification pathway in soil and that leads to N-nitrosation of secondary amines and desamination reaction for primary amines (Spott et al., 2011). Checking for this assumption will be the main contribution of this work. Specific objectives include: (i) to investigate the relevance of the formation of NO-DCF and NO₂-DCF in a soil aquifer system thanks to a monitoring survey of SW and groundwater (GW) samples, (ii) to better understand the correlation between denitrification and the NO-DCF and NO₂-DCF formation in batch soil slurry experiments and (iii) to get new insights into the mechanisms of NO₂-DCF formation. Diphenylamine (DPA) was also included in this work as an additional secondary aromatic amine due to its high occurrence at our sampling site and due to the availability of N-nitroso-DPA (NO-DPA) and 4-nitro-DPA (NO₂-DPA) as analytical standards.