Elsevier

Toxicology Letters

Volume 142, Issue 3, 15 May 2003, Pages 185-194
Toxicology Letters

Short communication
Aquatic ecotoxicity of pharmaceuticals including the assessment of combination effects

https://doi.org/10.1016/S0378-4274(03)00068-7Get rights and content

Abstract

To evaluate the ecotoxicological potential of ten prescription drugs against aquatic organisms from different taxonornical classes, a set of biotests were performed using the cladoceran Daphnia magna, the chlorophyte Desmodesmus subspicatus and the macrophyte Lemna minor. Endpoints were immobilisation for Daphnia and inhibition of the average growth rate for Desmodesmus and Lemna. For most of the substances, toxicities were moderate, with EC50s in the range from 10 to 100 mg l−1 or even far above, whereas Lemna was the most sensitive test species in the majority of all tested compounds. Tests with combinations of various pharmaceuticals revealed stronger effects than expected from the effects measured singly. Clofibrinic acid and Carbamazepine have been found to act by a non-specific mode of action (non-polar narcosis), and with Daphnia the combination effect of these substances followed the concept of concentration addition, while in the algae test the concept of independent action could be used to calculate the mixture toxicity. The anti-inflammatory drugs Diclofenac and Ibuprofen have also been found to act unspecific by non-polar narcosis and to follow the concept of concentration addition in the algal test as well as in the Daphnia test. The measured toxicities of the tested pharmaceuticals shows that acute effect of single substances in the aquatic environment are very unlikely. But we should keep in mind that considerable combination effects can occur and that toxicity data from chronic studies are needed to assess the environmental risk of drug residues.

Introduction

In recent years reports about residues of pharmaceuticals in surface and drinking waters have increased in scientific publications (Ternes, 1998, Daughton and Ternes, 1999, Kümmerer, 2001, Daughton and Jones-Lepp, 2001, Ternes et al., 2001). Among the detected substances in rivers were e.g. beta blocker (e.g. Metoprolol up to 1.54 μg l−1) and beta-sympathomimetics (Hirsch et al., 1996, Sedlak and Pinkston, 2001), analgesic and anti-inflammatory drugs (e.g. Diclofenac up to 1.2 μg l−1; Ternes, 1998, Stumpf et al., 1998, Buser et al., 1998a, Buser et al., 1999), estrogens (e.g. 17β-estradiol up to 0.013 μg l−1; Kuch and Ballschmitter, 2000, Adler et al., 2001, Huang and Sedlak, 2001) and also antibiotics (e.g. Erythromycin up to 1.7 μg l−1; Hirsch et al., 1999, Lindsey et al., 2001, Adler et al., 2001) as well as lipid lowering agents (e.g. Clofibrinic acid up to 0.2 μg l−1; Stan et al., 1994, Buser et al., 1998b, Ahrer et al., 2001, Öllers et al., 2001) and anti-epileptic drugs (e.g. Carbamazepine up to 2.1 μg l−1; Ternes, 1998, Möhle et al., 1999, Seiler et al., 1999). Particularly, some small streams receiving a relatively large amount of their water from sewage water treatment plants are found to be considerably polluted, with peak concentrations of several pharmaceuticals of more than 1 μg l−1. Due to their specific mode of action and the fact that these compounds are intentionally designed to exert an effect on humans, mammals or other vertebrates, residues of pharmaceuticals could be as or even more important for human health than those of pesticides, which are created to affect weeds, fungi and invertebrate varmints. However, until now the mode of action of pharmaceuticals is not well enough understood to make general statements about potential environmental effects caused by these substances. Drinking water treatment diminishes residues, but is not able to remove these substances completely. Thus, even in tap water some pharmaceuticals like Clofibrinic acid could be detected in concentrations up to 270 ng l−1 (Heberer et al., 1997, Heberer et al., 2001a, Heberer et al., 2001b, Ternes et al., 2001). In contrast to the amount of analytical data, information about the ecotoxicological effects of drug residues are sparse (Webb, 2001, Cleuvers, 2002). To create a broader basis for the evaluation of the ecotoxicological relevance of pharmaceutical compounds and mixtures, biotests with Desmodesmus subspicatus, Daphnia magna and Lemna minor were performed.

Drug residues found in the aquatic environment usually occur as mixtures, not as single contaminants. Thus, scientific assessment of risk to aquatic life should consider this complex exposure situation. By analyzing combination effects ecotoxicologists try to elucidate the problem of risk assessment for complex mixtures of various substances since more than 20 years and a lot of excellent studies have been performed in this topic (e.g. Altenburger et al., 2000, Backhaus et al., 2000, Faust et al., 2001). Basically, two different concepts are in use for the prediction of mixture toxicity, and generally they are termed concentration addition and independent action.

The concept of concentration addition can be traced back to the early works of the pharmacologists, Loewe and Muischnek (1926) (Loewe, 1927, Loewe, 1953) and it can be described mathematically for a mixture of n substances by Eq. (1) (Berenbaum, 1985):i=1nciECxi=1In this equation, ci are the individual concentrations of the single substances present in a mixture with a total effect of x%, and ECxi are those concentrations of the single substances that would alone cause the same effect x as observed for the mixture. As an important point, concentration addition means, that substances applied below their individual no effect concentration (NOEC) can nevertheless contribute to the total effect of the mixture. Concentration addition is based on the idea of a similar action of chemicals, whereas interpretations of this term can differ considerably. From a mechanistic point of view, similar action means in a strict sense that substances should have the same specific interaction with a molecular target site in the observed test organism (Pöch, 1993). In contrast, used in a more common sense, a similar action could be observed for all substances which are able to cause the same toxicological response under consideration, e.g. death of the test organisms. For example, concentration addition is able to predict mixture toxicities of inert chemicals, i.e. chemicals that are not reactive when considering overall acute effects, and that do not interact with specific receptors in the organism (Broderius et al., 1995, Van Loon et al., 1997). The mode of action of such compounds is called narcosis (Van Leeuwen et al., 1992, Verhaar et al., 1992). Narcosis type toxicity is considered to be caused by an absolutely non-specific mode of action, in that the potency of a chemical to induce narcosis is entirely dependent on its hydrophobicity, which is generally expressed by its n-octanol–water partition coefficient (log Kow). As a result, in the absence of any specific mechanism of toxicity, a chemical will, within certain boundaries, always be as toxic as its log Kow indicates. Thus, the narcosis type of action is also called baseline toxicity (Verhaar et al., 1992).

The alternative concept of independent action was already formulated by Bliss (1939). It is based on the idea of dissimilar action of compounds in a mixture, i.e. that the compounds have different molecular target sites and modes of action. As a result of such a dissimilar action, the relative effect of one of the toxicants in a mixture should remain unchanged in the presence of another one. For a binary mixture the combination effect can be calculated by the equationEcmix=1−1−Ec11−Ec2or in generalEcmix=1−i=1n1−Eciin which E(c1), E(c2), are the effects of the single substances and E(cmix) is the total effect of the mixture. Following this equation, a substance applied in a concentration below its individual NOEC will not contribute to the total effect of the mixture, i.e., there will be no mixture toxicity if the concentrations of all used single substances are below their NOEC.

At given concentrations of the single compounds in a mixture, the combination effect will in general be higher if the substances follow the concept of concentration addition. Thus, misleadingly the different concepts were sometimes brought in correlation to the terms synergism and antagonism. But synergisms or antagonisms between the used substances and their effects can occur independently of a similar or dissimilar mode of action.

Section snippets

Daphnia acute immobilisation test

Daphnia tests were conducted following the European Guideline: ‘Methods for determination of ecotoxicity; Annex V, C.2, Daphnia acute immobilisation test’ (Commission of the European Communities, 1992) using the water flea D. magna Strauss. Daphnids were bred in ADaM, a culture medium imitating natural fresh water (Klüttgen et al., 1994). Experiments were run at temperatures of 20±1 °C and photoperiods of LD 16:8 (∼20 μEs−1 m−2). Twenty daphnids younger than 24 h were used for the controls and

Results

The EU-Directive 93/67/EEC (Commission of the European Communities, 1996) classifies substances according to their EC50-value in different classes; <1 mg l−1 (very toxic to aquatic organisms); 1–10 mg l−1 (toxic to aquatic organisms) and 10–100 mg l−1 (harmful to aquatic organisms). Substances with an EC50 above 100 mg l−1 would not be classified. The toxicity of the tested pharmaceuticals was very heterogeneous, with EC50 values ranging from 7.5 mg l−1 (Propranolol) to 174 mg l−1 (Naproxen) in the

Discussion

The measured toxicity of the substances was heterogeneous and for most substances moderate with EC50s between 10 and 100 mg l−1 or even above. In the acute Daphnia and algal test, solely the tested β-blockers showed EC50s below 10 mg l−1; Propranolol in both tests and Metoprolol in the algal test only. Bearing in mind that environmental concentrations of pharmaceuticals are in most cases below or only little above 1 μg l−1, acute effects of such compounds in the aquatic environment are very

Conclusions

The toxicity of the tested pharmaceuticals was very heterogeneous. In the majority of cases, based upon the obtained EC50s, Lemna was the most sensitive species, and it is recommended to perform such tests routinely in addition to the other standard tests. Regarding the assessment of the environmental risk of pharmaceuticals, acute effects seems to be unlikely, but we need to bear in mind that considerably combination effects of substances can occur, even if the toxicity of the single substance

Acknowledgements

The author wants to thank Stephanie Esser for technical support and Arnd Weyers for critically reading the manuscript.

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