Investigation of the microbial degradation of phenazone-type drugs and their metabolites by natural biofilms derived from river water using liquid chromatography/tandem mass spectrometry (LC-MS/MS)

Abstract

The degradation of the pharmaceuticals phenazone and metamizole, two pyrazolone-derivates in widespread use, using biofilms created by natural organisms from the national park Unteres Odertal, Germany, were investigated. An analytical method based on LC-MS/MS was optimised to determine the substances phenazone and methylaminoantipyrine (MAA), the hydrolysis product of metamizole (also known as dipyrone), as well as their metabolites 1,5-dimethyl-1,2-dehydro-3-pyrazolone (DP), acetaminoantipyrine (AAA), formylaminoantipyrine (FAA) and 4-aminoantipyrine (AA). Performance characteristics of the method were evaluated in terms of recovery, standard deviation, coefficient of variation, method detection limits (MDL) and method quantification limits (MQL). Degradation studies of phenazone and MAA were conducted using a laboratory-scale continuous flow biofilm reactor fed with different nutrient media and with variable hydraulic retention times of 24 and 32 h. MAA was degraded rapidly to FAA and AA, while phenazone was not degraded under the prevailing conditions even after 32 h. By operating the bioreactor in batch mode to study the phenazone degradation potential of the biofilm under limiting nutrient conditions, an elimination rate of 85% phenazone was observed, but because of the slow elimination rate and aerobic conditions, the metabolite DP was not detected. In additional batch experiments using bacterial isolates from the natural biofilm to decompose phenazone, some bacterial strains were able to form DP from phenazone in marginal concentrations over the sampling period of eight weeks. Obviously, the microorganisms need a reasonably long time to adapt their metabolisms to enable the removal of phenazone from water samples.

Introduction

The occurrence of pharmaceutical residues in sewage, surface and groundwater has been investigated and confirmed in many previous studies. In the aquatic environment, more than 50 compounds from various groups of pharmaceuticals such as analgetics, antibiotics, anti-epileptics, anti-rheumatics, beta blockers, chemotherapeutics, steroid hormones, and X-ray contrast media have been found (Heberer, 2002). Most of these compounds derive either from domestic sewage or from hospital or industrial discharges. The results of many studies confirm that normal operation of sewage treatment plants (STPs) results in the incomplete removal of pharmaceuticals, so that as much as 80% of the total load of pharmaceuticals entering STPs may be discharged into surface waters (Ternes, 1998, Ternes et al., 1999). Detected concentrations of pharmaceutical residues are generally higher in STP effluents than in rivers, therefore municipal STP discharge is seen as the main source for the river contamination. The concentration ranges in surface water sampled downstream of sewage treatment plant discharges are typically in the tens of nanograms per litre range, although concentrations up to the μg L⁻¹ range have been observed (Ternes, 1998, Ternes et al., 2001, Cahill et al., 2004). Although the individual residual compounds often occur at low concentrations, pharmaceutical activity can accumulate if several compounds are present; the possible risks have not yet been well examined (Kolpin et al., 2002). Therefore there is a need to investigate both the behaviour and the fate of drug residues in the environment and to apply suitable techniques for their elimination. In several studies, the efficiency of sewage treatment plants in eliminating pharmaceutical residues has been investigated. For polar compounds such as acidic pharmaceuticals, microbial degradation is the most important removal process in activated sludge wastewater treatment. According to Carballa et al. (2004), the efficiencies of STPs in terms of the elimination rate of selected pharmaceutical residues are between 30% and 75% mainly based on activated sludge treatment. Comparing the removal efficiency of a conventional activated sludge plant and a membrane bioreactor, which is a relatively newly developed technology using higher biomass concentrations with longer retention times, Clara et al. (2004) found no significant differences especially for polar compounds. In contrast, Zuehlke et al. (2006) confirmed an observable increased removal rate of phenazone-type pharmaceuticals by membrane bioreactor pilot plants. Antibiotics, analgetics and other pharmaceuticals were often detected at high concentrations in treated effluents and were therefore not completely eliminated by activated sludge treatment (Andreozzi et al., 2003, Kasprzyk-Hordern et al., 2009).

In the present study, the possibility of microbial degradation of phenazone and metamizole, two frequently prescribed analgetics, has been investigated. Phenazone is a very polar substance, hardly biodegradable and known to be persistent through activated sludge STPs but not persistent in membrane bioreactors (Zuehlke et al., 2006). Residues of phenazone, metamizole and other pyrazolone-derivates and metabolites are often detected in rivers, wastewater and sometimes also in ground and drinking water (Ternes et al., 2001, Reddersen et al., 2002, Schmidt and Brockmeyer, 2002, Wiegel et al., 2004). According to Zuehlke et al. (2006), a conventional wastewater treatment plant removes only up to 30% of phenazone. The behaviour of metabolites derived from metamizole such as acetaminoantipyrine (AAA) (removal rate 40%) and formylaminoantipyrine (FAA) (removal rate 15%) were also investigated. In that study, their removal could be increased by membrane bioreactor pilot plants. In another study (Massmann et al., 2008), phenazone and its microbial metabolite 1,5-dimethyl-1,2-dehydro-3-pyrazolone (DP) and metabolites of metamizole could be eliminated at higher rates during bank filtration under oxic conditions, but the degradation efficiency was strongly dependent on seasonal variations and was optimum only in wintertime. Clearly, further studies are required to exploit the degradation of phenazone-type pharmaceuticals in order to develop strategies for improved removal of such residues from aqueous systems. While transformations of pharmaceuticals in the human body and in other mammals have been studied extensively, the microbial degradation of such compounds, its degradation pathways and end products have rarely been investigated and are largely unknown (Quintana et al., 2005).

Because of their species-rich communities and the possibility to adapt their metabolisms to occurring pollutants, sediment bacteria provide a great potential for eliminating environmental contaminants from water/sediment systems. Several studies have explored the degradation pathways of pollutants by microbial communities taken from river or marine sediments or sewage sludge and confirmed their important role in elimination process of pharmaceutical or other chemical residues (Diaz-Cruz et al., 2003, Shi et al., 2004, Loeffler et al., 2005, Urase and Kikuta, 2005, Vargha et al., 2005, Kolar et al., 2007). Bacteria organized in biofilms offer different advantages in contrast to bacterial isolates. The degradation potential of biofilms derived from river waters has hardly been examined. Winkler et al. (2001) investigated the degradation of two pharmaceuticals (ibuprofen and clofibric acid) by river derived biofilms and confirmed the microbial elimination of ibuprofen.

Our investigation aimed to examine the potential of microbial degradation, relating to phenazone and metamizole, using biofilms derived from the national park Unteres Odertal, Germany.

Numerous unknown and unexamined microorganisms inhabit the national park Unteres Odertal. The flood plain areas of the national park are intensely flooded by the river Oder during winter times causing exposure of local microorganisms to river pollutants. While passing through these areas, contaminants can be adsorbed or metabolised by local microorganisms living in biofilms of the river systems. These natural microbial communities can be expected to provide yet until now hardly explored potentials for the degradation of persistent organic compounds and hence potentially support wastewater treatment.

In order to study the biodegradability of phenazone and metamizole and its metabolites DP, methylaminoantipyrine (MAA), AAA, FAA and 4-aminoantipyrine (AA) in aqueous matrices, the suitability of the sample clean-up procedure and the determination step using LC-MS/MS had first to be optimised and confirmed. Subsequently, biofilm reactors were constructed for laboratory use to investigate the behaviour of target analytes exposed to biofilms in continuous-flow mode and batch mode. Additional tests with isolated monospecific bacterial colonies derived from naturally formed biofilms from the national park Unteres Odertal were undertaken to investigate their degradation potential.