Assessment of the stability of the ethanol metabolite ethyl sulfate in standardised degradation tests

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Abstract

Ethyl sulfate (EtS) is a non-oxidative metabolite of ethanol, used for forensic purposes as an ethanol consumption marker in addition to the ethanol metabolite ethyl glucuronide (EtG) which after certain scientific publications is prone to biological degradation. As ethanol is widely consumed in many western cultures, knowledge about the stability of ethyl sulfate against biodegradation is of importance for forensic investigations—where EtS until now was thought to be stable against bacterial degradation. Using standardized test methods from the panel of OECD tests, the stability of EtS against bacterial degradation was assessed in this study. These experiments showed that EtS was stable in the closed bottle test (CBT) (OECD 301 D), but not in the manometric respiratory test (MRT) (OECD 301 F) with higher bacterial density. With respect to forensic investigations the assumption of EtS stability could be disproved and the possibility of bacterial degradation of EtS should be taken into account when alcohol uptake some hours prior to death needs to be ruled out by determination of alcohol consumption markers in putrefied corpses, where ethanol concentration could have been generated post-mortem by fermentation processes.

Introduction

Alcohol metabolism not only comprises the oxidative pathway via acetaldehyde to acetic acid, but also minor non-oxidative pathways. Ethyl sulfate (EtS), besides ethyl glucuronide (EtG), is a minor metabolite of ethanol. It is formed by sulfotransferase enzymes when ethanol is consumed. Both substances are used as a proof for alcohol consumption if found in urine, blood, other body fluids or hair of a person suspected of drinking [1], [2], [3], [4], [5], [6], [7], [8], [9]. There is evidence, that EtG can be produced and degraded by bacteria present in the sample matrix, putting into question the reliability of EtG as an alcohol consumption marker [10], [11], [12], [13].

In post-mortem cases, putrefaction and fermentation can result in generation of ethanol in a corpse. As a consequence, EtG and EtS could then be used as markers for differentiation of ante mortem ethanol consumption or post-mortem generation of ethanol [14], [15]. However, the absence of EtG can result from bacterial degradation (as well as its presence might be a result from bacterial formation from ethanol) – while until now, EtS was described as stable against bacterial degradation [12], [13], [16] – and therefore – if detected – would be useful in those cases, to prove alcohol consumption prior to death.

Another issue concerning the biodegradability of EtS is the substance's behaviour in wastewater treatment plants as EtG and EtS are excreted into the wastewater in large amounts. If EtG and EtS were not biodegradable under these circumstances, accumulation effects could occur. Assuming that 0.032% of the ethanol consumed is excreted as EtG and 0.022% as EtS [17], an annual amount of about 550 t EtG and 350 t EtS would be excreted into Germany's wastewaters as the estimated ethanol consumption in 2006 was 10.1 L pure ethanol per person (older than 15) [18] and year and the estimated number of inhabitants of 71 million people above the age of 15 [19]. Until now, bacterial stability of EtG has been tested in several studies, but there was no data for the stability of EtS under conditions in wastewater treatment [13], [16].

The closed bottle test (CBT, OECD 301 D [20]) is a first, simple test for the assessment of the biodegradability of an organic compound in the presence of low bacterial density and a low content of nutrients. It is a routine method in ecotoxicological laboratories performed using highly standardised procedures. In contrast, the manometric respiratory test (MRT) (OECD 301 F [20]) gives information about degradation in the presence of higher bacterial density and nutrient content, compared to the CBT. Both tests are batch tests.

The assessment of EtS stability in OECD test procedures can show whether EtS in principle can be decomposed bacterially and thus on the one hand yield information on the usefulness and/or advantages of EtS against EtG as an alcohol consumption marker, on the other hand on the whereabouts of this alcohol metabolite.

Section snippets

Materials

HPLC-grade acetonitrile and formic acid (analytical grade) were obtained from Merck (Darmstadt, Germany). Sodium ethyl sulfate was obtained from ABCR (Karlsruhe, Germany). Deionized water was prepared with a cartridge-deionizer from Memtech (Moorenweis, Germany). Deuterated ethyl sulfate was prepared by an in-house procedure [2].

The solutions for the OECD test were prepared from chemicals with analytical grade and comprised potassium dihydrogen phosphate, dipotassium hydrogen phosphate,

Results

The results show clearly, that the manometric respiratory test with the higher inoculum density (compared to the CBT) provides evidence for the possibility of bacterial degradation or decay of EtS, while no degradation has been detected in the CBT – with lower inoculum density – and has never been reported before in studies on the stability of EtS in urine samples.

EtS was not degraded in CBT within 28 days (see Fig. 1). At each of the four measuring dates (7, 14, 21, 28 days), the reduction of

Conclusions

EtS was stable for 28 days under CBT conditions with a relatively low density of test substance and bacteria from a wastewater treatment plant's effluent but for only 6 days under the conditions of the manometric respiratory test with higher concentrations of inoculum and test substance. After 6 days, gradual decay of the analyte substance was observed.

With respect to post-mortem toxicology, our test results show that EtS is biodegradable using bacteria available in the environment, depending

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