Anaerobic inhibition and biodegradation of antibiotics in ISO test schemes.

Municipal sewage is the main exposure route for antibiotics that are used in human medical care. Antibiotics that adsorb to the primary sludge and/or sur-plus activated sludge will enter the anaerobic digesters of municipal sewage treatment plants. Here anaerobic biodegradation or inhibition of anaerobic bacteria resulting in a disturbance of the process might occur. ISO standards 13641 (2003) and 11734 (1999) were used for assessing the anaerobic inhibition of 16 and the anaerobic biodegradability of 9 antibiotics respectively. Digestion sludge from a municipal sewage treatment plant (1g/l d.s.) was used as inoculum in both tests. In ISO 13641 (2003) most antibiotics showed only moderate inhibition effects after a 7 day incubation period, with EC50 values between 24 mg/l and more than 1000 mg/l (equal to mg/g d.s.). In contrast, metronidazol was decisively toxic to anaerobic bacteria with an EC50 of 0.7 mg/l. In the anaerobic degradation tests according to ISO standard 11734 (1995), only benzylpenicillin showed certain ultimate biodegradation after 60 days and most antibiotics inhibited the digesting sludge in the respective parallel tested inhibition controls. Thus the inhibition of anaerobic bacteria by antibiotics observed in the degradation tests was higher than expected from the results of the inhibition tests. The possible explanations are that distinct substrates are used (yeast extract versus sodium benzoate), that the digestion sludge loses activity during the washing steps performed for the degradation tests and that the exposure time in the degradation tests was 8 times longer than in the inhibition test.

Ultimate biodegradation and elimination of antibiotics in inherent tests.

The biodegradation and elimination of antibiotics in municipal wastewater treatment plants is of particular concern because sewage is the main exposure route for antibiotics used in human medicine. The inherent biodegradability of 17 antibiotics was determined in a combined test design based on the Zahn-Wellens test (OECD 302 B, 1992) and the CO2-evolution test (OECD 301 B, 1992). CO2 Evolution test (Modified Sturm test). OECD Guideline for the Testing of Chemicals, Paris). Only benzylpenicillin sodium salt (Penicillin G) proved to be ultimately biodegradable, reaching ThCO2 degradation extents of 78-87%. Among the others, only amoxicillin, imipenem and nystatin showed certain ultimate biodegradation in few of the parallel flasks and can be regarded as partially biodegradable with formation of stable metabolites. The DOC-elimination of tetracycline-HCl showed a typical degradation curve starting with 18% and reaching the plateau phase at 80% after 21 days. Nevertheless, the CO2-evolution measured in parallel did not support the data, indicating that the time needed for reaching the adsorption equilibrium was underestimated. Several other antibiotics showed considerable DOC-elimination in the inherent test while only minor incidences of ultimate biodegradation were observed. The combination of CO2-evolution and DOC-elimination is a suitable instrument for assessing the behaviour of chemicals within one test. It enables one to assess both inherent ultimate biodegradability and DOC-elimination by sorption. The applicability of the test is limited to substances with a moderate toxicity.

Standardized tests fail to assess the effects of antibiotics on environmental bacteria.

Pharmaceuticals are designed and used because of their specific biological effects. Over the past decade, compounds from various classes of pharmaceuticals have been detected in the environment. Concern has grown about the adverse effects pharmaceuticals in the environment might potentially have on human and ecological health. A sound risk assessment is therefore urgently needed for pharmaceuticals. Standardized tests for assessing the effects of chemicals on environmental organisms are widely used for this purpose. However it is questionable whether classical standardized tests give reliable data needed for environmental risk assessment. In this study we investigated the suitability of the respiration inhibition test OECD 209 for the assessment of the effects of antibiotics, disinfectants and cytotoxics on sewage sludge bacteria. We found that inhibition concentrations can strongly depend on the test period and the type of compound. We conclude that tests to assess the effects of pharmaceuticals on environmental organisms such as bacteria have to be evaluated before their results can be used in environmental risk assessment.

Assessment of degradation of 18 antibiotics in the Closed Bottle Test.

Large quantities of antibiotics are used in health care. After administration, they are discharged into the effluent and reach sewage treatment plants (STPs); if they are not degraded, they will eventually enter the environment. Antibiotics can affect bacteria in the environment and thus disturb natural elemental cycles. For this reason, it is necessary to take a closer look at the fate and effects of these substances in the environment. The biodegradability of 18 clinically important antibiotics and their effects on environmental bacteria was studied using the Closed Bottle Test (CBT) (OECD 301 D 1992). In addition, a toxicity control was performed in the CBT and the colony forming units (CFUs) were monitored. Disappearance of some of the 18 antibiotics was monitored by HPLC (high performance liquid chromatography) analysis. The antibiotics were used in two concentrations: (a) according to OECD 301 D in the mg/l-range and (b) on the basis of calculated concentrations in the influent of STPs in the microg/l-range. None of the 18 antibiotics were readily biodegradable. The HPLC analysis showed that some substances were partially or even completely disappeared by a non-biotic mechanism. In the case of some antibiotics, partial biological removal took place in test vessels containing readily biodegradable sodium acetate and the test compound. However, in the toxicity control, toxicity had not been eliminated.

Aquivalence revisited--new model formulation and application to assess environmental fate of ionic pharmaceuticals in Hamilton Harbour, Lake Ontario.

A model formulation based on "aquivalence", as defined in terms of activity is presented to estimate the multimedia fate of ionizing chemicals. The aquivalence approach is analogous to fugacity but aquivalence is applicable to neutral and ionizing compounds, and has been applied previously to speciating chemicals, notably metals. The new aquivalence-based mass-balance model treats ionizing organic compounds that exist as interconverting neutral and ionic species which are subject to fate processes at differing rates. The model is illustrated by application to four ionizing pharmaceuticals in Hamilton Harbour, Lake Ontario. At the system pH of 7.9-8.5, ibuprofen, gemfibrozil, and naproxen are expected to be almost entirely ionic and triclosan split between ionic and neutral forms. Measured seasonal surface water concentrations, which were 2-10 times lower in the late summer and fall than during spring, were used to solve for unknown values of chemical half-life in the water column due to degradation (photo- and bio-) of the ionizing and neutral forms and secondarily, ionic sorption coefficients of the ionizing forms. Model estimates of half-lives in the habour's water ranged from 11 to 77, 11 to 147 and 10 to 37 for ionic ibuprofen, gemfibrozil, and naproxen, respectively; and 4-22 days and 2-9 days for ionic and neutral triclosan, respectively, with the shortest half-lives in spring and the longest in summer.

Deactivation and transformation products in biodegradability testing of beta-lactams amoxicillin and piperacillin.

Antibiotics have increasingly been detected in effluents and the environment. However, information on the degree of deactivation and mineralization, and the nature of possible formed dead-end transformation products is scarce but desirable for proper risk assessment. An important group of antibiotics is the beta-lactams. We studied the transformation of the closely structurally related beta-lactams piperacillin and amoxicillin in two OECD biodegradability batch tests. None of the antibiotics was biodegraded in the closed bottle test (CBT). However, primary abiotic elimination as monitored by HPLC-UV-VIS was 20% and 100% in the CBT within 14 days, respectively. With HPLC-UV-VIS and ion trap LC-MS/MS primary elimination was shown to be more than 94% for both antibiotics within seven days in the Zahn-Wellens test (ZWT). Both compounds were deactivated by hydrolysis. For piperacillin, a dead-end transformation product resulted after hydrolysis of the beta-lactam ring. For amoxicillin full mineralization of the transformation products was observed.

Biodegradability of 14C-labeled antibiotics in a modified laboratory scale sewage treatment plant at environmentally relevant concentrations.

For all new pharmaceuticals, an environmental risk assessment (ERA) has to be performed according to guidelines developed by the European Medicines Evaluation Agency. An important factor of this procedure is the assessment of the predicted environmental concentration in the aquatic environment, which is significantly influenced by the biodegradability of pharmaceuticals in sewage treatment plants. Established standardized methods for determining biodegradation under laboratory conditions apply to substance concentrations, which are much higherthan those expected in reality. Against this background, the laboratory scale sewage treatment plant (LSSTP), as described by the Organisation for Economic Cooperation and Development (OECD) Guideline No. 303A, was modified to construct a lossless system, which allows laboratory testing at realistic concentrations. To verify the experimental setup, the antibiotics benzylpenicillin, ceftriaxone, and trimethoprim were tested at low concentrations (microg/L) using 14C-labeled compounds. The results show that approximately 25% of benzylpenicillin was mineralized, whereas ceftriaxone and trimethoprim were not mineralized at all. Due to the high total recoveries of added radioactivity (> or =95%) and the fact that the findings comply with available literature data, the lossless operation of the test system could be proved. Consequently, the modified LSSTP is a suitable tool to determine more realistic biodegradation data required for the exposure assessment within the scope of an ERA for pharmaceuticals.