Selective serotonin reuptake inhibitors (SSRIs) are used as antidepressant medications, primarily in the treatment of clinical depression. They are among the pharmaceuticals most often prescribed in the industrialized countries. Selective serotonin reuptake inhibitors are compounds with an identical mechanism of action in mammals (inhibit reuptake of serotonin), and they have been found in different aqueous as well as biological samples collected in the environment. In the present study, we tested the toxicities of five SSRIs (citalopram, fluoxetine, fluvoxamine, paroxetine, and sertraline) as single substances and of citalopram, fluoxetine, and sertraline in binary mixtures in two standardized bioassays. Test organisms were the freshwater algae Pseudokirchneriella subcapitata and the freshwater crustacean Daphnia magna. In algae, test median effect concentrations (EC50s) ranged from 0.027 to 1.6 mg/L, and in daphnids, test EC50s ranged from 0.92 to 20 mg/L, with sertraline being one of the most toxic compounds. The test design and statistical analysis of results from mixture tests were based on isobole analysis. It was demonstrated that the mixture toxicity of the SSRIs in the two bioassays is predictable by the model of concentration addition. Therefore, in risk assessment based on chemical analysis of environmental samples, it is important to include the effect of all SSRIs that are present at low concentrations, and the model of concentration addition may be used to predict the combined effect of the mixture of SSRIs.

From a theoretical point of view, it has often been argued that the model of independent action (IA) is the most correct reference model to use for predicting the joint effect of mixtures of chemicals with different molecular target sites. The theory of IA, however, relies on a number of assumptions that are rarely fulfilled in practice. It has even been argued that, theoretically, the concentration addition (CA) model could be just as correct. In the present study, we tested the accuracy of both IA and CA in describing binary dose-response surfaces of chemicals with different molecular targets using statistical software. We compared the two models to determine which best describes data for 158 data sets. The data sets represented 98 different mixtures of, primarily, pesticides and pharmaceuticals tested on one or several of seven test systems containing one of the following: Vibrio fischeri, activated sludge microorganisms, Daphnia magna, Pseudokirchneriella subcapitata, Lemna minor, Tripleurospermum inodorum, or Stellaria media. The analyses showed that approximately 20% of the mixtures were adequately predicted only by IA, 10% were adequately predicted only by CA, and both models could predict the outcome of another 20% of the experiment. Half of the experiments could not be correctly described with either of the two models. When quantifying the maximal difference between modeled synergy or antagonism and the reference model predictions at a 50% effect concentration, neither of the models proved significantly better than the other. Thus, neither model can be selected over the other on the basis of accuracy alone.

More or less well-defined mixtures of antibiotics used in aquacultures may be distributed in the aquatic environment. Therefore, a systematic mixture ecotoxicity study was performed with the aquaculture antibiotics oxytetracycline, oxolinic acid, erythromycin, florfenicol, and flumequine. Test organisms were freshwater algae (Pseudokirchneriella subcapitata), activated sludge microorganisms, and luminescent bacteria (Vibrio fischeri). Design and statistical analysis of test results were based on isobolographic analysis. Synergistic effects were observed when combinations of erythromycin and oxytetracycline were tested on activated sludge microorganisms, and in these cases model predictions indicate independent action on the different bacterial species in the sludge. As predicted from the modes of action, concentration addition was evident when flumequine and oxolinic acid were mixed and tested on sludge bacteria. In the algae test, the combined toxicity of antibiotics could not be predicted based on knowledge of the modes of action of the individual compounds. Independent of the test species, our results gave examples of combined effects that were higher than predicted based on the assumption of concentration addition. This result underlines the need to consider the effects of mixtures of antibiotics on environmental organisms. The isobolographic method appears to be a suitable tool for this purpose, particularly for well-defined mixtures with few substances.

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