**3. Basic concepts of chemical mixture toxicology**

Therefore, the main aim of this chapter was to review the existing knowledge on the chronic and mixture toxicity of the residues of sulfonamides in the environment, since it has not been done yet. This will be achieved by: (1) presenting current approaches for Environmental Risk Assessment (ERA) for pharmaceuticals with respect to the evaluation of chronic and mixture toxicity of these compounds; (2) introducing the reader to basic concepts of chemical mixture toxicology; and finally (3) by discussing detailed available information on chronic and mixture

**2. Environmental risk assessment of pharmaceuticals vs. chronic and**

The approaches currently being used to assess the potential environmental effects of human and veterinary drugs in the U.S. and in the European Union are in some respects dissimilar [34-39]. The Environmental Risk Assessment (ERA) process usually starts with an initial exposure assessment (Phase I). But with some exceptions, a fate and effects analysis (Phase II) is only required when exposure-based threshold values, the so-called action limits, are exceeded in different environmental compartments. Thus risk assessment, described by Risk Quotient (RQ), is performed by the calculation the ratio of the predicted (or measured) environmental concentration (PEC or MEC respectively) and predicted biological noneffective concentrations (PNEC) on non-target organisms. If RQ is less than one it indicates that no further testing is recommended. Calculations of environmental concentrations rely e.g. on information on treatment dosage and intensity along with default values for standard husbandry practices, and are based on a total residue approach reflecting worst-case assump‐ tions. For example, the recently introduced European guidance on assessing the risks of human drugs excludes the testing of pharmaceuticals whose PECsurface water is below an action limit of 0.01 μg L-1; in the U.S. this threshold value is 0.1 μg L-1. Moreover, there are two different action limits for veterinary pharmaceuticals, one each for the terrestrial and the aquatic compart‐ ments. No fate and effect analysis is required for veterinary pharmaceuticals used to treat animals if the PECsoil is < 100 μg kg-1 dry weight of soil. However, a Phase II assessment is not required for veterinary medicines used in an aquaculture facility if the estimated concentration of the compound is < 1 μg L-1 [40-41]. If the PECsurface water of a pharmaceutical is above the action limit, effects on algae, crustaceans and fish are investigated. However, if PECsoil is higher than the action limit, then Phase II, divided into two parts: Tier A, in which the possible fate of the pharmaceutical or its metabolites and its effects on earthworms (mortality) and plants (germination and growth) as well as the effects of the test compound on the rate of nitrate mineralization in soil are determined; and Tier B in which only effect studies are recommended

The main problem associated with this approach is the fact that the no actual sales figures or measured environmental concentrations are at hand when a risk assessment is conducted. Therefore, only crude PEC calculations are performed [42]. Moreover, the (eco)toxicity tests included in Phase II focus on acute toxicity of only single compounds. Chronic and mixture toxicity is not obligatory. As the risk of an acute toxic effect from pharmaceuticals in the

toxicity of the residues of sulfonamides in the environment.

**mixture toxicity of pharmaceuticals**

64 Organic Pollutants - Monitoring, Risk and Treatment

for affected taxonomic levels [34-39].

To predict the toxicity of mixtures, ecotoxicologists use concepts originally developed by pharmacologists in the first half of the 20th century [43-48]. Since more than 20 years, they have been trying to elucidate the problem of risk assessment for complex mixtures of various substances. As a result a lot of excellent studies have been performed in this topic [49-51]. One of the main interests of scientists in the field of combination toxicology is to find out whether the toxicity of a mixture is different from the sum of the toxicities of the single compounds; in other words, will the toxic effect of a mixture be determined by additivity of dose or effect or by supra-additivity (synergism - an effect stronger than expected on the basis of additivity) or by infra-additivity (antagonism - an effect lower than the sum of the toxicities of the single compounds) The toxic effect of a mixture appears to be highly dependent on the dose (exposure level), the mechanism of action, and the target (recep‐ tor) of each of the mixture constituents. Thus, information on these aspects is a prerequi‐ site for predicting the toxic effect of a mixture [46-47, 52].

Generally, three basic concepts for the description of the toxicological action of constituents of a mixture have been defined by Bliss and are still valid half a century later: (1) simple similar action (concentration addition, CA), (2) simple dissimilar action (independent action, IA), (3) interactions (synergism, potentiation, antagonism) [45].

Concentration addition (CA), also known as 'simple joint action', is based on the idea of a similar action of single compounds, whereas interpretations of this term can differ consider‐ ably. From mechanistic point of view, similar action means in a strict sense that single substance should show the same specific interaction with a molecular target site in the observed organisms. This is a nonintereactive process, which means that the chemicals in the mixture do not affect the toxicity of one another. Each of the chemicals in the mixture contrib‐ utes to the toxicity of the mixture in proportion to its dose, expressed as the percentage of the dose of that chemical alone that would be required to obtain the given effect of the mixture. All chemicals of concern in a mixture act in the same way, by the same mechanisms, and differ only in their potencies [46-47, 52].

It has been shown that the concept of concentration addition is also applicable to nonreac‐ tive, nonionized organic chemicals, which show no specific mode of action but whose toxicity toward aquatic species is governed be hydrophobicity. The mode of action of such compounds is called narcosis or baseline toxicity [53-54]. The potency of a chemical to induce narcosis is entirely dependent on its hydrophobicity, generally expressed by its octanol-water partition coefficient logKow. As a result, in the absence of any specific mechanism of toxicity, a chemical will, within certain boundaries, always be as toxic as its logKow indicates. Mathematically, the concept of concentration addition for a mixture of *n* substances is described by [48]:

$$\sum\_{i=1}^{n} \frac{\mathbb{C}\_{i}}{\text{ECx}\_{i}} = \mathbf{1} \tag{1}$$

For these reasons, prediction of the effect of a mixture based on the knowledge of each of the constituents requires detailed information on the composition of the mixture, expo‐ sure level, mechanism of action, and receptor of the individual compounds. However, often such information is not or is only partially available and additional studies are needed. In addition to considering which of these concepts should be used to evaluate combined toxic effects, the design of the study is important in quantifying the combined effects. Most of such studies are based on a comparison of observed values with those predicted by a reference mode (IA or CA). An important aspect of toxicity studies of mixtures is the impracticability of 'complete' testing. If all combinations are to be studied at different dose levels, an increasing number of chemicals in a mixture results in an exponential increase in number of test groups: to test all possible combinations (in a complete experimental design) at only one dose level of each chemical in a mixture consisting of 4 or 6 chemi‐

What Do We Know About the Chronic and Mixture Toxicity of the Residues of Sulfonamides in the Environment?

are time consuming and impossible from a practical and economical point of view. To reduce the number of test groups without losing too much information about possible interactions between chemicals, several test scenario's (statistical designs) have been proposed [52]. The study design largely depends on the number of compounds of a mixture and on the question whether it is desirable to assess possible existing interactions be‐

**•** One approach is to test the toxicity of the mixture without assessing the type of interactions. This is the simplest way to study effects of mixtures by comparing the effect of a mixture with the effects of all its constituents at comparable concentrations and duration of exposure at one dose level without testing all possible combinations of two or more chemicals. This approach requires a minimum number of experimental groups (n + 1, the number of compounds in a mixture plus the mixture itself). If there are no dose-effect curves of each of the single compounds it is impossible to describe the effect of the mixture in terms of synergism, potentiation, antagonism, etc. This strategy would be of interest for a first

**•** The second approach is based on assessment of interactive effects between two or three compounds which can be identified by physiologically based toxicokinetic modeling, isobolographic or dose-effect surface analysis, or comparison of dose-effect curves. How‐ ever, interactive effects of compounds in mixtures with more than three compounds can be best ascertained with the help of statistical designs such as (fractionated) factorial designs, ray designs or dose-effect surface analysis. Here we would like to described shortly only the isobole methods as so far they are mainly used in the studies concerning the determi‐

An isobole, originally developed by Loewe and Muischnek [44], is a contour line that represents equi-effective quantities of two agents or their mixtures [52]. The theoretical line of additivity is the straight line connecting the individual doses of each of the single agents that produce the fixed effect alone. The method requires a number of mixtures to be tested and is used for a graphical representation to find out if mixtures of two compounds behave in a dose-additive manner and subsequently can be regarded as chemicals with a similar

–1) test groups, respectively, would be required. Such *in vivo* studies

http://dx.doi.org/10.5772/53732

67

cals, 16 (24

–1) or 64 (26

tween chemicals in a mixture [52]:

screening of adverse effects of a mixture.

nation of pharmaceutical mixture toxicity [25-26,46-47].

where *ci* represents 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. According to Eq. (1), the effect of the mixture remains constant when one component is replaced by an equal fraction of an equally effective concentration of another. As an important point, concentration addition means that substances applied at less than their individual "no observable effect concentrations" (NOECs) can nevertheless contribute to the total mixture effect [46-47].

The alternative concept of independent action (IA), also known as 'independent joint action' was already formulated by Bliss [45]. IA is when toxicants act independently and have different modes of toxic action [43, 46-47]. In this case the agents of a mixture do not affect each other's toxic effect. As a result of such a dissimiliar action, the relative effect of one of the toxicants in a mixture should remain unchanged in the presence of another one. For binary mixture the combination effect can be calculated by the equitation [46]:

$$E\left(c\_{mix}\right) = 1 \cdot \left[ (1 - E\left(c\_1\right))(1 - E\left(c\_2\right)) \right] \tag{2}$$

In which *E(c1), E(c2)* are the effect of single substances and *E(cmix)* is the total effect of the mixture. Following this equitation, 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 [45-47, 52].

Additionally, compounds may interact with one another, modifying the magnitude and sometimes the nature of the toxic effect. This modification may make the composite effect stronger or weaker. An interaction might occur in the toxicokinetic phase (processes of uptake, distribution, metabolism, and excretion) or in the toxicodynamic phase (effects of chemicals on the receptor, cellular target, or organ).These include terms such as synergism and poten‐ tiation (i.e., resulting in a more than additive effect), or antagonism (i.e., resulting in a less than additive effect) [52]. It must be highlighted that at given concentrations of the single com‐ pounds 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 some‐ times brought in correlation to the term synergism and antagonism. But synergism or antag‐ onisms between the used substances and their effects can occur independently of a similar or dissimilar mode of action [46].

induce narcosis is entirely dependent on its hydrophobicity, generally expressed by its octanol-water partition coefficient logKow. As a result, in the absence of any specific mechanism of toxicity, a chemical will, within certain boundaries, always be as toxic as its logKow indicates. Mathematically, the concept of concentration addition for a mixture of *n*

represents the individual concentrations of the single substances present in a mixture

alone cause the same effect *x* as observed for the mixture. According to Eq. (1), the effect of the mixture remains constant when one component is replaced by an equal fraction of an equally effective concentration of another. As an important point, concentration addition means that substances applied at less than their individual "no observable effect concentrations" (NOECs)

The alternative concept of independent action (IA), also known as 'independent joint action' was already formulated by Bliss [45]. IA is when toxicants act independently and have different modes of toxic action [43, 46-47]. In this case the agents of a mixture do not affect each other's toxic effect. As a result of such a dissimiliar action, the relative effect of one of the toxicants in a mixture should remain unchanged in the presence of another one. For binary mixture the

In which *E(c1), E(c2)* are the effect of single substances and *E(cmix)* is the total effect of the mixture. Following this equitation, 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

Additionally, compounds may interact with one another, modifying the magnitude and sometimes the nature of the toxic effect. This modification may make the composite effect stronger or weaker. An interaction might occur in the toxicokinetic phase (processes of uptake, distribution, metabolism, and excretion) or in the toxicodynamic phase (effects of chemicals on the receptor, cellular target, or organ).These include terms such as synergism and poten‐ tiation (i.e., resulting in a more than additive effect), or antagonism (i.e., resulting in a less than additive effect) [52]. It must be highlighted that at given concentrations of the single com‐ pounds 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 some‐ times brought in correlation to the term synergism and antagonism. But synergism or antag‐ onisms between the used substances and their effects can occur independently of a similar or

concentrations of all used single substances are below their NOEC [45-47, 52].

*ECxi* =1 (1)

are those concentrations of the single substances that would

*E*(*cmix*) =1 - (1 - *E*(*c*1))(1 - *E*(*c*2)) (2)

∑ *i*=1 *<sup>n</sup> Ci*

can nevertheless contribute to the total mixture effect [46-47].

combination effect can be calculated by the equitation [46]:

substances is described by [48]:

66 Organic Pollutants - Monitoring, Risk and Treatment

with a total effect of *x%,* and *ECxi*

dissimilar mode of action [46].

where *ci*

For these reasons, prediction of the effect of a mixture based on the knowledge of each of the constituents requires detailed information on the composition of the mixture, expo‐ sure level, mechanism of action, and receptor of the individual compounds. However, often such information is not or is only partially available and additional studies are needed. In addition to considering which of these concepts should be used to evaluate combined toxic effects, the design of the study is important in quantifying the combined effects. Most of such studies are based on a comparison of observed values with those predicted by a reference mode (IA or CA). An important aspect of toxicity studies of mixtures is the impracticability of 'complete' testing. If all combinations are to be studied at different dose levels, an increasing number of chemicals in a mixture results in an exponential increase in number of test groups: to test all possible combinations (in a complete experimental design) at only one dose level of each chemical in a mixture consisting of 4 or 6 chemi‐ cals, 16 (24 –1) or 64 (26 –1) test groups, respectively, would be required. Such *in vivo* studies are time consuming and impossible from a practical and economical point of view. To reduce the number of test groups without losing too much information about possible interactions between chemicals, several test scenario's (statistical designs) have been proposed [52]. The study design largely depends on the number of compounds of a mixture and on the question whether it is desirable to assess possible existing interactions be‐ tween chemicals in a mixture [52]:


An isobole, originally developed by Loewe and Muischnek [44], is a contour line that represents equi-effective quantities of two agents or their mixtures [52]. The theoretical line of additivity is the straight line connecting the individual doses of each of the single agents that produce the fixed effect alone. The method requires a number of mixtures to be tested and is used for a graphical representation to find out if mixtures of two compounds behave in a dose-additive manner and subsequently can be regarded as chemicals with a similar mode of action. When all equi-effect concentrations are connected by a downward concave line, the effect of the combinations is antagonistic, and a concave upward curve indicates synergism. The use of the isobole procedure to evaluate the effects of binary mixtures is widely used, but is very laborious and requires large data sets in order to produce sufficiently reliable results [52].

potential effects of SAs in the environment appear to indicate a possible negative impact on different ecosystems and imply a threat to public health. The most sensitive assays for the presence of SAs are bioindicators containing chlorophyll (algea and duckweed) [3, 22-23]. A highly toxic effect of SMX on duckweed (*Lemna gibba*) was observed. This was also supported by the results of one of our studies, where we evaluated the ecotoxicity potential of twelve sulfonamides (sulfaguanidine, sulfadiazine, sulfathiazole, sulfamerazine, sulfamethiazole, sulfachloropyridazine, sulfamethoxypyridazine, sulfamethoxazole, sulfisoxazole, sulfadime‐ thoxine, sulfapyridine, sulfadimidine) to enzymes (acetylcholinesterase and glutathione reductase), luminescent marine bacteria (*Vibrio fischeri*), soil bacteria (*Arthrobacter globiformis)*, limnic unicellular green algae (*Scenedesmus vacuolatus*) and duckweed (*Lemna minor*). We found that SAs were not only toxic towards green algae (EC50 = 1.54 – 32.25 mg/L) but were even more strongly so towards duckweed (EC50 = 0.02 – 4.89 mg/L) than atrazine, a herbicide (EC50 = 2.59 mg/L) [33]. This indicates that even low concentrations of SAs may significantly affect the

What Do We Know About the Chronic and Mixture Toxicity of the Residues of Sulfonamides in the Environment?

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However, data relating to the long-term exposure of non-target organisms, and especially how continuous exposure for several generations may affect a whole population is very limited. Most chronic toxicity data for sulfonamides, is available for invertebrates, probably because these are the briefest and therefore least expensive chronic toxicity tests to run. Available

The major concern over the effects of all antimicrobials (including sulfonamides) on microbial assemblages is the development of antimicrobial resistance and the effect of this on public health. Recently, Baran et al. [3] has reviewed the papers concerning the influence of presence of SAs in the environment to antimicrobial resistance. They concluded that SAs in the envi‐ ronment increase the antimicrobial resistance of microorganisms and the number of bacterial strains resistant to SAs increases systematically in recent years. Resistant bacterial species commonly carried single genes, but in recent years, an increased number of pathogens that possess three SAs-resistant genes have been observed. Moreover, they have also highlighted that these drugs have shown the highest drug resistance, almost twice as high as tetracyclines and many times higher than other antibiotics. Most often, bacterial resistance to SAs has been described in *Escherichia coli*, *Salmonella enterica* and *Shigella spp*. from the manure of farm animals, from meat and from wastewater [3]. The implications of antimicrobial resistance for aquatic ecosystem structure and function remain unknown, but the human health implications

Additionaly, Heuer and Smalla [58] investigated the effects of pig manure and sulfadiazine on bacterial communities in soil microcosms using two soil types. In both soils, manure and sulfadiazine positively affected the quotients of total and sulfadiazine-resistant culturable bacteria after two months. The results suggest that manure from treated pigs enhances spread of antibiotic resistances in soil bacterial communities. Monteiro and Boxall [59] have recently examined the indirect effects of sulfamethoxazole on the degradation of a range of human medicines in soils. It was observed that the addition of SMX significantly reduce the rate of degradation of human non-steroidal anti-inflammatory drugs, naproxen. This observation

chronic toxicity data for sulfonamides is summarized in Table 3 and discussed below.

growth and development of plants.

of widespread resistance are of clear concern [55].
