Study of the photolytic and photocatalytic transformation of amiloride in water

Abstract

The diffusion of drug residues in wastewaters and surface waters as rivers and streams may constitute a problem for the environment, with consequences on the ecosystem and also on the human health. This paper deals with the study of the photo-induced transformation of amiloride, an orally administered diuretic agent, under simulated solar light. Direct photolysis and photocatalyzed degradation processes, using titanium dioxide as a photocatalyst, were investigated. The study involved the monitoring of the drug decomposition, the identification of intermediate compounds of the decomposition, the assessment of mineralization, as well as the evaluation of the toxicity associated to the degradation products.

Amiloride underwent complete degradation within 30 min of irradiation (heterogeneous photocatalysis) or 4 h (homogeneous photolysis). HPLC coupled to HRMS, via ESI interface, demonstrated to be a powerful tool to identify and measure degradation products of the studied drug. By considering the photocatalytic process, the identified intermediates are formed through: (1) dechlorination and hydroxylation of the heteroaromatic ring; (2) the detachment of the guanidinic moiety; (3) cleavage of the heteroaromatic ring. The drug photomineralization was a rather slow process and after 4 h of irradiation 25% of the total organic carbon (TOC) was still present. Chlorine was stoichiometrically released as chloride ions within the considered irradiation times (4 h), while nitrogen was only partially converted into ammonium ions. This was due to the formation of guanidine, known to be hardly mineralized photocatalytically, and some other small molecules still containing the nitrogen. Acute toxicity, measured with a Vibrio fischery assay, showed that amiloride transformation proceeded through the formation of toxic compounds.

Introduction

The presence in the environment of new xenobiotic compounds as a consequence of the massive use of chemicals in different productive fields constitutes a ticklish and complex emerging subject-matter. Many of these substances are now considered “persistent organic pollutants” (POPs) and their effects on the ecosystem and on the living organisms are becoming target of various studies [1], [2], [3].

In recent years, pharmaceutical residues have appeared as a new class of pollutants, for which public and scientific concern has progressively increased, due to their potential impact on the human health and on the environment [4], [5], [6], [7], [8]. Human and veterinary drugs can be released in the environment mainly as a consequence of manufacturing processes, disposal of unused or expired products and accidental spills during manufacturing and distribution or excreta by humans and animals. These substances and their metabolites may accumulate in soils and sediments and contaminate groundwater, or be discharged into sewers through urine and faeces and then enter sewage-treatment plants (STPs), prior to entering rivers and streams, lakes and sea [4], [9], [10], [11], [12]. Sometimes, not only the parent compound may arouse adverse effects on ecosystem and human health but also its metabolites. For most medical substances and their metabolites the transformation pathways in the aquatic system are largely unknown and investigations into their occurrence in environmental compartments are still rare [13], [14]. Pharmaceuticals can undergo both abiotic and biotic processes of transformation. Abiotic reactions in surface waters may occur via hydrolysis or direct and indirect photolysis [15], [16], [17].

Some compounds may be unaffected by sewage-treatment and remain in the water effluents or be transformed into breakdown products. The presence of pharmaceuticals in the environment was considered a consequence of a combination of a partial removal in STP and of refractoriness in natural (biotic or abiotic) transformations. Heterogeneous photocatalysis represents an example of advanced oxidation processes able to achieve a complete oxidation of organic and inorganic species, including also pharmaceutical substances [18], [19], [20], [21], [22]. It takes advantage of some semiconductor solids, which can be used as photocatalysts suspended in the water effluent to be treated, or immobilised on various types of supports. Among them TiO₂ is widely used because it is non-toxic, inexpensive, as well as biologically and chemically inert. The electron/hole pair (e⁻/h⁺) generated under light illumination of energy greater than 3.2 eV reacts with the molecules, objects of degradation, or water molecules oxidized by the photoholes (h⁺) and gives rise to the generation of hydroxyl radicals, responsible for the complete decomposition of the chemical substances. Moreover, intermediates coming from an artificial photocatalytic process can be identical to those found in the metabolic system of living organisms [23] and in the environment, as a consequence of naturally occurring reactions [24]. The target drug of this study is amiloride (3,5-diamino-N-diaminomethylene)-6-chloropyrazine-carboxamide monohydrochloride), an orally administered diuretic agent, which acts as a sodium channel inhibitor and is excreted 50% in urine and 40% in faeces [25]. Amiloride showed photosensitization properties, related to the adverse clinical photobiological responses observed in patients exposed to sunlight [26].

The main objectives of this research were to assess the degradation of the pollutant through the identification of possible intermediate products as well as the determination of the final products by employing liquid chromatography coupled to mass spectrometry and ion chromatography.

Identification of intermediate compounds formed during the degradation is necessary to verify the real ability of the oxidation technology in reducing toxicity of water and to inspect the possible formation of dangerous substances. Toxic effects of drugs have been tested on microorganisms [3], [27], [28], on phytoplankton [29] and on insects [30]. In recent years, the attention has been focused also on genotoxicological effects connected to the spreading of some types of drugs in the aquatic environment, in particular in drinking water, wastewater and sludge [31], [32]. Toxicity was also demonstrated at concentrations at which pharmaceuticals are normally found in the aquatic environment in the range of μg/l to ng/l [4], [33], [34], [35]. Some drugs are suspected to be able to affect endocrine system of living organisms such as fish [2] and, even if in trace quantities, they may cause endocrine disruption also in humans, with consequent alterations in reproduction or development [9], [36]. In the present study, the acute toxicity of the irradiated solutions was also evaluated; a bacterial assay based on the bioluminescence reduction of the marine bacterium Vibrio fischeri was carried out.