Antibiotics are designed to inhibit the growth of or kill bacteria. Of the many classes of antibiotics that have been synthesized two particular classes of antibiotic have been frequently detected in the natural environment, namely the tetracyclines and sulfonamides. Following use, these compounds can enter the environment via the application of animal manures or domestic seawage sludge to land as a fertiliser or from wastewater treatment plant effluents. Once in the environment the compounds can persist and, depending on their properties, are distributed around the different environmental media (i.e. surface waters, groundwaters, soils and sediments). Several studies have suggested that tetracyclines and sulfonamides may have significant impacts on microbial function in the natural environment. However most of these studies have used existing standardised test procedures and/or unrealistic exposure conditions. The aim of the current study therefore was to develop a more environmentally realistic test system for assessing the effects of antibiotics on aquatic microbial communities and to apply this to assess the potential impacts of a sulphonamide and a tetracycline antibiotic on aquatic microbial communities. The developed system, which was an aquatic microcosm composed of fresh river water, return line treated sewage and OECD synthetic sewage, was initially used to assess the effects of 3, 5-dichlorophenol, an OECD reference toxicant. Effects of DCP were seen within the concentration range that is considered valid by other standard microbial toxicity tests, such as OECD method 209 (3.2-32 mg/L). Since the developed system was able to quantify effects on aquatic microbial communities, it can be seen as being more environmentally relevant than existing standard tests, which rely on testing single microbial species or sludge communities. The test system was then used to test the effects of chlortetracycline (CTC) and sulfamethoxazole (SMX) on multisubstrate utilization. Following addition of the study antibiotics, effects were observed for both antibiotics on total substrate utilization and on the utilization of specific, ecologically relevant end-points (such as nutrient cycling and polymer degradation) also. Effects were also observed on the structure of microbial communities and on the functional diversity of substrate utilization (especially in the case of SMX exposure) and these effects generally persisted for up to 3 d after addition of the study compounds at a lower concentration of 0.1 mg/L for both compounds. The results indicate that both antibiotics were inhibiting various aspects of substrate utilization, most of which were related to ecologically relevant processes that occur in the aquatic environment. The results also suggest that SMX and CTC exposure result in changes in the community structure of ecologically relevant groups of microbes (such as bacteria involved in the nitrogen and carbon cycle bacteria). Microbial communities that were exposed to CTC and SMX showed a varying degree of recovery, although more long term effects were seen in SMX exposures; functional effects were observed for the duration of the exposure. It was hypothesized that the observed recovery may have been caused by the selection of antibiotic resistance in the exposed communities. Culture based resistance studies showed that there was a significant temporal rise in CTC resistance at all CTC concentrations, but not SMX resistance, across the dose range. In addition, a greater number of distinct microbial morphotypes could be isolated from combined microcosms on day 7 compared with day 1. An increase in CTC and SMX resistance in control exposures was also observed. In addition, distinct resistant morphotypes developed in both dosed and undosed microcosms on day 7 of the study. Furthermore, the results of a series of different culture based experiments suggested that multidrug resistance was present in morphotypes that were isolated from dosed and control microcosms. These data therefore suggest that multidrug resistance was present at the start of the exposure and may also have been co-selected by exposure to CTC and SMX. In general, CTC and SMX show signs that they may pose a risk to the environment and possibly the wider health of humans and animals.
