Toxicity of five antibiotics and their mixtures towards photosynthetic aquatic organisms: Implications for environmental risk assessment

Abstract

The individual and combined toxicities of amoxicillin, erythromycin, levofloxacin, norfloxacin and tetracycline have been examined in two organisms representative of the aquatic environment, the cyanobacterium Anabaena CPB4337 as a target organism and the green alga Pseudokirchneriella subcapitata as a non-target organism. The cyanobacterium was more sensitive than the green alga to the toxic effect of antibiotics. Erythromycin was highly toxic for both organisms; tetracycline was more toxic to the green algae whereas the quinolones levofloxacin and norfloxacin were more toxic to the cyanobacterium than to the green alga. Amoxicillin also displayed toxicity to the cyanobacterium but showed no toxicity to the green alga. The toxicological interactions of antibiotics in the whole range of effect levels either in binary or multicomponent mixtures were analyzed using the Combination Index (CI) method. In most cases, synergism clearly predominated both for the green alga and the cyanobacterium. The CI method was compared with the classical models of additivity Concentration Addition (CA) and Independent Action (IA) finding that CI could accurately predict deviations from additivity. Risk assessment was performed by calculating the ratio between Measured Environmental Concentration (MEC) and the Predicted No Effect Concentration (PNEC). A MEC/PNEC ratio higher than 1 was found for the binary erythromycin and tetracycline mixture in wastewater effluents, a combination which showed a strong synergism at low effect levels in both organisms. From the tested antibiotic mixtures, it can be concluded that certain specific combinations may pose a potential ecological risk for aquatic ecosystems with the present environmentally measured concentrations.

Introduction

Antibiotics are biologically active molecules with an increasing use in both human and veterinary medicine. These pharmaceuticals play a major role in livestock industries and modern agriculture, which use them as therapeutics and growth promoters in livestock production, as feed additives in fish farms and to prevent crop damage induced by bacteria (Sarmah et al., 2006); although since 2006 antibiotics have been forbidden as growth promoters in animal feed in the EU. From the antibiotics administered to animals, a large proportion is excreted without metabolizing and is dispersed with the manure used for soil amendment, eventually reaching surface waters (Elmund et al., 1971). A large portion of the antibiotics administered in fish farms (70–80%) has been reported to reach the environment (Schneider, 1994; Hektoen et al., 1995).

The major pathway whereby residual antibiotics from human use enter the environment is the effluent of wastewater treatment plants (WWTP) as conventional plants are relatively inefficient in completely removing pharmaceuticals (McArdell et al., 2003; Göbel et al., 2005; Xu et al., 2007; Rosal et al., 2010b). Antibiotic contamination of the natural environment has been reported in many countries in Europe, North America and Asia, all classes of antibiotics having been detected in river water (Kolpin et al., 2002, 2004; Batt et al., 2006; Xu et al., 2007; Yang et al., 2008; Ginebreda et al., 2009; Tamtam et al., 2009; Watkinson et al., 2009), seawater (Gulkowska et al., 2007; Xu et al., 2007), groundwater (Hirsch et al., 1999; Lindsey et al., 2001; Sacher et al., 2001), drinking water (Zuccato et al., 2000), sediments (Kerry et al., 1996; Kim and Carlson, 2007), agricultural soils due to dispersion of sewage sludge and manure (Sarmah et al., 2006), biota (Dolliver et al., 2007; Kong et al., 2007),and WWTP effluents (Costanzo et al., 2005; Batt et al., 2006; Watkinson et al., 2009; Gros et al., 2010; Rosal et al., 2010b). Although recorded environmental levels are usually low, at ng L⁻¹ to μg L⁻¹ in waters (see Table S1 under Supplementary Information) and μg kg⁻¹ to mg kg⁻¹ in soils and sediments, antibiotics are considered to be “pseudopersistant” contaminants due to their continued release into the environment and permanent presence (Daughton and Ternes, 1999; Hernando et al., 2006).

Although the major concern of antibiotics is associated with the development of resistance mechanisms by bacteria and its implications in human health, their sustained release to different environmental compartments and their bioactive properties also raise serious concerns about the toxicity of antibiotics to non-target organisms (Migliore et al., 1997Halling-Sørensen et al., 1998, 2000; Baguer et al., 2000). Algae and cyanobacteria play a crucial role in aquatic ecosystems. They are primary producers which supply nutrients for the rest of the aquatic biota (Greenberg et al., 1992). Cyanobacteria, in addition, are prokaryotes and are thus considered as sensitive organisms to antibiotics (Maul et al., 2006). In fact, the European Medicines Evaluation Agency (EMEA) explicitly recommends the use of cyanobacteria for effect testing of antimicrobials due to their sensitivity (EMEA, 2006).

Different classes of antibiotics have been detected simultaneously in environmental compartments (Kolpin et al., 2004; Li et al., 2009; Watkinson et al., 2009; Suzuki and Hoa, 2012). Therefore, aquatic organisms may be exposed to mixtures of antibiotics. While the individual concentrations of antibiotics in aquatic environments may be low, the combined concentrations could result in significant toxicity to aquatic organisms. Besides, as chemicals in a mixture may either not interact or interact synergistically or antagonistically, it is essential to investigate the potential interactions in mixtures of antibiotics of different classes (Cleuvers, 2003; Teuschler, 2007; Rodea-Palomares et al., 2010). This issue has important implications in terms of environmental toxicity and risk assessment strategies, which are often carried out considering the individual effect and additive behavior. This may severely underestimate the risk associated with antibiotic mixtures as well as mixtures of antibiotics with other pharmaceuticals and anthropogenic contaminants (Kolpin et al., 2002).

The aim of the present study was to evaluate the individual and combined toxicity to the green alga Pseudokirchneriella subcapitata and the recombinant bioluminescent filamentous cyanobacterium Anabaena CPB4337 of five antibiotics from different classes: amoxicillin (β-lactam), erythromycin (macrolide), tetracycline (tetracycline) and the quinolones norfloxacin and levofloxacin. All of them have a variety of uses in human and veterinary medicine (Table S1). In order to identify and quantify the nature of the interactions between the antibiotics, binary and multicomponent mixtures of them were prepared and tested. Results were analyzed by the method of the Combination Index (CI)-isobologram equation which we have previously used to study pollutant interactions (Rodea-Palomares et al., 2010; Rosal et al., 2010a; Boltes et al., 2012; Rodea-Palomares et al., 2012). In this paper, we also report a first approach of risk assessment of the antibiotic mixtures for the aquatic environment based on currently available exposure data (Table S1) and our toxicity results.