**1. Introduction**

Thanks to their low cost and their broad spectrum of activity in preventing or treating bacterial infections, sulfonamides (SAs) are one of the oldest groups of veterinary chemo‐ therapeutics, having been used for more than fifty years. To a lesser extent they are also applied in human medicine. After tetracyclines, they are the most commonly consumed veterinary antibiotics in the European Union. As these compounds are not completely metabolized, a high proportion of them are excreted unchanged in feces and urine. Therefore, both the unmetabolized antibiotics as well as their metabolites are released either directly to the environment in aquacultures and by grazing animals or indirectly during the application of manure or slurry [1-3].

Physico-chemical properties and chemical structures of selected SAs are presented in Table 1. They are fairly water-soluble polar compounds, the ionization of which depends on the matrix pH. All the sulfonamides, apart from sulfaguanidine, are compounds with two basic and one acidic functional group. The basic functional groups are the amine group of aniline (all the SAs) and the respective heterocyclic base, specific to each SA. The acidic functional group in the SAs is the sulfonamide group. With such an SA structure, these compounds may be described by the *pKa1, pKa2* and *pKa3* values corresponding to the double protonated, once protonated and neutral forms of SA (Table 1) [3-7].

© 2013 Białk-Bielińska et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Białk-Bielińska et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**Substance [CAS] Abbreviation**

Sulfisoxazole [127-69-5] SSX

Sulfamethiazole [144-82-1] SMTZ

Sulfadimidine (sulfamethazine) [57-68-1] SDMD (SMZ)

Sulfamethoxypyridazine

Sulfachloropyridazine

Sulfadimethoxine [122-11-2]

SDM 2NH S

[80-35-3] SMP

[80-32-0] SCP

**Chemical structure Selected physico-chemical**

<sup>O</sup> <sup>N</sup> M = 267.3 g mol-1

<sup>2</sup>NH S

<sup>2</sup>NH S

<sup>2</sup>NH S

<sup>2</sup>NH S

<sup>2</sup>NH S

O O

O

O O

O

O O

NH

O

**Table 1.** Structures and physico-chemical properties of selected sulfonamides (according to [1,6-13])

O

HN

HN

O

HN N

N

N N

N N

N

N

OMe

OMe

OMe

Cl

HN

O

<sup>N</sup> <sup>N</sup>

S

HN

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

**properties**

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61

pKa2 = 2.15 pKa3= 5.00 logP = 1.01

M = 270.3 g mol-1 pKa2 = 2.24 pKa3 = 5.30 logP = 0.47

M = 278.3 g mol-1 pKa2 = 2.46 pKa3 = 7.45 logP = 0.27

M = 280.3 g mol-1 pKa2 = 2.20 pKa3 = 7.20 logP = 0.32

M = 284.7 g mol-1 pKa2 = 1.72 pKa3 = 6.39 logP = 0.71

M = 310.3 g mol-1 pKa2 = 2.5 pKa3 = 6.0 logP = 1.63

**Substance [CAS] Abbreviation**

Sulfaguanidine [57-67-0]

Sulfapyridine [144-83-2] SPY

Sulfadiazine [68-35-9] SDZ

Sulfamethoxazole [723-46-6] SMX

Sulfathiazole [72-14-0] STZ

Sulfamerazine [127-79-7] SMR

SGD <sup>2</sup>NH S

60 Organic Pollutants - Monitoring, Risk and Treatment

O

O

O

O

O O

O

O

HN N

N

HN

O

HN <sup>N</sup>

O

HN N

N

HN

O

<sup>2</sup>NH S

<sup>2</sup>NH S

<sup>2</sup>NH S

2NH S

<sup>2</sup>NH S

O

NH C

N

**Chemical structure Selected physico-chemical**

NH

NH2

**properties**

M = 214.2 g mol-1 pKa2 = 2.8 pKa3 = 12.0 logP = -1.22

M = 249.2 g mol-1 pKa2 = 2.37 pKa3 = 7.48 logP = 0.03

M = 250.3 g mol-1 pKa2 = 1.98 pKa3 = 6.01 logP = -0.09

<sup>N</sup> <sup>O</sup> M = 253.3 g mol-1

<sup>S</sup> M = 255.3 g mol-1

pKa2 = 1.81 pKa3 = 5.46 logP = 0.89

pKa2 = 2.06 pKa3= 7.07 logP = -0.04

M = 264.3 g mol-1 pKa2 = 2.16 pKa3 = 6.80 logP = 0.11


**Table 1.** Structures and physico-chemical properties of selected sulfonamides (according to [1,6-13])

Due to their properties, after disposal in soils, these compounds may enter surface run-off or be leached into the groundwater. Moreover, they are also quite persistent, non-biodegradable and hydrolytically stable, which explains why in the last ten years they have been regularly detect‐ ed not only in aquatic but also in terrestrial environments [1-3,7,14]. Although SAs concentra‐ tions in environmental samples are quite low (at the μg L-1 or ng L-1 level), they are continuously beingreleased[3,15].Therefore,thekindofexposureorganismsmaybesubjectedtowillresemble that of traditional pollutants (e.g. pesticides, detergents), even those of limited persistence. Consequently, SAs as well as other pharmaceuticals may be considered pseudo-persistent.

**Substance Bacteria**

*Vibrio fischeri* **Green algae / Cyanobacteria/ Diatom\***

**SMR** >50(30 min) 11.90(24h, S.vacuolatus) 0.68(7d, L. minor) **SSX** >50(30 min) 18.98(24h, S.vacuolatus) 0.62(7d, L. minor) **SMTZ** >100(30 min) 24.94(24h, S.vacuolatus) 2.54(7d, L. minor)

**SMP** >100(30 min) 3.82(24h, S.vacuolatus) 1.51(7d, L. minor)

2.83(96h, S. leopoliensis)

\*\* duckweed *Lemna gibba*, *Lemna minor*, carrot *Daucus carota*;

\*\*\*\* fish: *Oryzias latipes*, rainbow trout *Onchorhynchus mykiss*;

**Plants\*\* Invertebrates\*\*\* Vertebrates\*\*\*\***

135.7/78.9(48h/96h, D. magna)

111/311(48h/24h, M. macrocopa) 185.3/147.5(48h/96h, D. magna)

391/430(48h/24h, M. macrocopa) >100(48h, O. myskiss) <sup>a</sup>

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63

in mg L-1) estimated for different

1.54(24h, S.vacuolatus) 0.21(7d, L. minor) 100(48h, C. dubia) 35.36(24h, T.platyurus) <sup>a</sup>

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

>50(30 min) 4.89(7d, L. minor) 616.7(24h, D. magna) >500(96h, O. latipes) <sup>a</sup>

>100(30 min) 1.74(7d, L. minor) 215.9/506.3(48h/24h, D. magna) >500(96h, O. latipes) <sup>a</sup>

>50(30 min) 2.48(7d, L. minor) 535.7(96h, O. latipes) <sup>a</sup>

>500(5 min) 11.2 (72h, C. vulgaris) 0.248(7d, L.gibba) 270/639.8(48h/24h, D. magna) >100 (96h O. latipes) <sup>a</sup>

**STZ** >1000(15min) 13.10(24h, S.vacuolatus) 3.552(7d, L.gibba) 149.3/85.4(48h/96h, D. magna) >500(48h, O. latipes) <sup>a</sup>

**SDMD** 344.7(15 min) 19.52(24h, S.vacuolatus) 1.277(7d, L. gibba) 174.4/158.8(48h/96h, D. magna) >500(48h, O. latipes) <sup>a</sup>

**SCP** 26.4(15 min) 32.25(24h, S.vacuolatus) 2.33(7d, L. minor) 375.3/233.5(48h/96h, D. magna) 589.3(48h, O. latipes) <sup>a</sup>

**SDM** >500(15 min) 2.30 (72h, P. subcapitata) 0.445(7d, L.gibba) 248.0/204.5(48h/96h, D. magna) >100(48h, O. latipes) <sup>a</sup>

\* green algae: *Pseudokirchneriella subcapitata* (previously *Scenedesmus capricornutum*), *Scenedesmus dimorphus, Chlorella vulgaris*; cya‐

This demonstrates the lack of data relating to the long-term exposure of non-target organisms, and especially how continuous exposure for several generations may affect a whole popula‐ tion. Moreover, as these compounds occur in natural media not as a single, isolated drug but usually together with other compounds of the same family or the same type, accumulated concentrations or synergistic-antagonistic effects can be also observed. The simultaneous presence of several pharmaceuticals in the environment may result in a higher level of toxicity

towards non-target organisms than that predicted for individual active substances.

\*\*\* crustacean: *Moina macrocopa, Clathrina dubia, Thamnocephalus platyurus, Daphnia magna*; rotifer: *Brachionus calyciflorus*;

>50(30 min) 9.85(24h, S.vacuolatus) 0.02(7d, L. minor) 184/297(48h/24h, M. macrocopa)

**SQO** <sup>b</sup> 0.25(96h, P. subcapitata) 13.55(7d, L.gibba) 3.47(48h, D. magna) 0.45(96h, S. dimorphus) 2.33(7d, L. minor)

nobacteria *Synechococcus leopoliensis, Microcystis aeruginosa*; diatom *Cyclotella meneghiniana*;

**Table 2.** Summary of the ecotoxicological risk (described by EC50 or LC50a

sulfonamides (data obtained from [16,19-20,22-33]); *<sup>b</sup>* sulfaquinoxaline

SAs are designed to target specific metabolic pathways (they competitively inhibit the conver‐ sionof*p*-aminobenzoicacid,PABA)byinhibitingthebiosyntheticpathwayoffolate(anessential moleculerequiredbyalllivingorganisms), sotheynotonlyaffectbacteria(targetorganisms)but can also have unknown effects on environmentally relevant non-target organisms, such as unicellularalgae,invertebrates,fishandplants[16-18].Belongingtodifferenttrophiclevels,these taxonomic groups may be exposed to by SAs to various extents [15-16,19-20].

However, knowledge of the potential effects of SAs on the environment is very limited. Recently, a few review papers have been published that summarize the available ecotoxicity data of pharmaceuticals, including some sulfonamides [16-17,19-21]. Such data as are available on the potential effects of pharmaceuticals in the environment appear to indicate a possible negative impact on different ecosystems and imply a threat to public health. However, if we look just at the sulfonamides, most current studies have investigated acute effects mainly of single compounds and mostly with reference to sulfamethoxazole (SMX), one of the most common SAs, used in both veterinary and human medicine [16-17,20]. Available information on the ecotoxicity of selected sulfonamides has been review and is presented in Table 2.


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\* green algae: *Pseudokirchneriella subcapitata* (previously *Scenedesmus capricornutum*), *Scenedesmus dimorphus, Chlorella vulgaris*; cya‐ nobacteria *Synechococcus leopoliensis, Microcystis aeruginosa*; diatom *Cyclotella meneghiniana*;

\*\* duckweed *Lemna gibba*, *Lemna minor*, carrot *Daucus carota*;

Due to their properties, after disposal in soils, these compounds may enter surface run-off or be leached into the groundwater. Moreover, they are also quite persistent, non-biodegradable and hydrolytically stable, which explains why in the last ten years they have been regularly detect‐ ed not only in aquatic but also in terrestrial environments [1-3,7,14]. Although SAs concentra‐ tions in environmental samples are quite low (at the μg L-1 or ng L-1 level), they are continuously beingreleased[3,15].Therefore,thekindofexposureorganismsmaybesubjectedtowillresemble that of traditional pollutants (e.g. pesticides, detergents), even those of limited persistence. Consequently, SAs as well as other pharmaceuticals may be considered pseudo-persistent.

SAs are designed to target specific metabolic pathways (they competitively inhibit the conver‐ sionof*p*-aminobenzoicacid,PABA)byinhibitingthebiosyntheticpathwayoffolate(anessential moleculerequiredbyalllivingorganisms), sotheynotonlyaffectbacteria(targetorganisms)but can also have unknown effects on environmentally relevant non-target organisms, such as unicellularalgae,invertebrates,fishandplants[16-18].Belongingtodifferenttrophiclevels,these

However, knowledge of the potential effects of SAs on the environment is very limited. Recently, a few review papers have been published that summarize the available ecotoxicity data of pharmaceuticals, including some sulfonamides [16-17,19-21]. Such data as are available on the potential effects of pharmaceuticals in the environment appear to indicate a possible negative impact on different ecosystems and imply a threat to public health. However, if we look just at the sulfonamides, most current studies have investigated acute effects mainly of single compounds and mostly with reference to sulfamethoxazole (SMX), one of the most common SAs, used in both veterinary and human medicine [16-17,20]. Available information on the ecotoxicity of selected sulfonamides has been review and is presented in Table 2.

**Plants\*\* Invertebrates\*\*\* Vertebrates\*\*\*\***

taxonomic groups may be exposed to by SAs to various extents [15-16,19-20].

**Substance Bacteria**

*Vibrio fischeri*

62 Organic Pollutants - Monitoring, Risk and Treatment

**Green algae / Cyanobacteria/ Diatom\***

16.59(96h, S. leopoliensis) 3.42(24h, S.vacuolatus) **SPY** >50(30 min) 5.28(24h, S.vacuolatus) 0.46(7d, L. minor)

2.22(24h, S.vacuolatus)

**SGD** >50(30 min) 43.56(96h, P. subcapitata) 30.30(7d, L.gibba) 0.87(48h, D. magna) 3.40(96h, S. dimorphus) 0.22(7d, L. minor)

**SD**Z >25(30 min) 7.80(72h, P. subcapitata) 0.07(7d, L. minor) 221(48h, D. magna)

2.19(72h, P. subcapitata) 13.7(21d, D. magna) 0.135(72h, M. aeruginosa) 212(48h, D. magna)

**SMX** 23.3(30 min) 1.53(72h, P. subcapitata) 0.081(7d, L.gibba) 189.2(48h, D.magna) 123.1(48h, D.magna) >750(48h, O. latipes) <sup>a</sup>

74.2(5 min) 2.4(96h, C. meneghiniana) 0.0612(21d, D.carota) 15.51(48h, C. dubia) 84.9(24h, M.macrocopa) >100(30 min) 0.0268(96h, S. leopoliensis) 0.0454(28d, D.carota) 0.21(7d, C. dubia) 9.63(48h, B.calyciflorus)

>84(30 min) 0.15(96h, P. subcapitata) 0.132(7d, L.gibba) 177.3(96h, D.magna) 205.1(48h, D.magna) 562.5(96h, O. latipes) <sup>a</sup> 78.1(15 min) 0.52(72h, P. subcapitata) 0.0627(14d, D.carota) 25.2(24h, D. magna) 70.4(48h, M.macrocopa) 27.36(24h, O. myskiss)

\*\*\* crustacean: *Moina macrocopa, Clathrina dubia, Thamnocephalus platyurus, Daphnia magna*; rotifer: *Brachionus calyciflorus*;

\*\*\*\* fish: *Oryzias latipes*, rainbow trout *Onchorhynchus mykiss*;

**Table 2.** Summary of the ecotoxicological risk (described by EC50 or LC50a in mg L-1) estimated for different sulfonamides (data obtained from [16,19-20,22-33]); *<sup>b</sup>* sulfaquinoxaline

This demonstrates the lack of data relating to the long-term exposure of non-target organisms, and especially how continuous exposure for several generations may affect a whole popula‐ tion. Moreover, as these compounds occur in natural media not as a single, isolated drug but usually together with other compounds of the same family or the same type, accumulated concentrations or synergistic-antagonistic effects can be also observed. The simultaneous presence of several pharmaceuticals in the environment may result in a higher level of toxicity towards non-target organisms than that predicted for individual active substances.

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 toxicity of the residues of sulfonamides in the environment.

environment is unlikely and organisms in the environment are exposed to mixtures of pharmaceuticals, such limited focus results in important uncertainties. Additionally, same drugs (like sulfonamides) are used to treat both humans and animals. Although the exposures may differ, their potential effects on non-target organisms will be the same, and so the effecttesting approaches should be similar. For these reasons, many scientists have already pointed out the need for more reliable PEC and PNEC calculations for more realistic ERA of pharma‐

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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‐

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)

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

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

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

site for predicting the toxic effect of a mixture [46-47, 52].

interactions (synergism, potentiation, antagonism) [45].

only in their potencies [46-47, 52].

ceutical [40-42].
