**4. State of the knowledge concerning mixture and chronic toxicity of the residues of sulfonamides in the environment**

#### **4.1. What do we know about the long-term effects of the presence of the residues of sulfonamides in the environment?**

Chronic toxicity tests are studies in which organisms are exposed to different concentrations of a chemical and observed over a long period, or a substantial part of their lifespan. In contrast to acute toxicity tests, which often use mortality as the only measured effect, chronic tests usually include additional measures of effect such as growth rates, reproduction or changes in organism behavior [55-56]. Therefore, the standard acute toxicity tests do not seem appro‐ priate for risk assessment of pharmaceuticals, because of the nature of these compounds. The use of chronic tests over the life-cycle of organisms for different trophic levels could be more appropriate [57]. However, there is still an ongoing debate between ecotoxicologists over the determination which tests should be considered to be chronic or acute (based on their dura‐ tion). This applies not only to aquatic animal testing with invertebrates and fish, but also to standard 96-h algal and 7-d higher plant test methods.

Molander et al. [19] reviewed the data published in the *Wikipharma* database – a freely available, interactive and comprehensive database on the environmental effects of pharmaceuticals that provides an overview of effects caused by these compounds on non-target organisms identi‐ fied in acute, sub-chronic and chronic ecotoxicity tests. Looking at the data set as a whole, they concluded that crustaceans like *Daphnia magna* and *Ceriodaphnia dubia* were the species most commonly used (29% of all tests performed); this is hardly surprising since they are abundant and widespread, easy to keep in the laboratory, and sensitive towards a broad range of environmental contaminants. Less commonly, such tests were performed on marine bacteria *Vibrio fischeri* (12%), algae *Pseudokirchneriella subcapitata* (9.5%) and fish *Poeciliopsis lucida* (9%) and *Oncorhynchus mykiss* (8%) [19]. They have also estimated that acute tests based on microorganisms (exposure time ≤ 30 min), algae (exposure time ≤ 72 h), invertebrates (exposure time ≤ 48 h) and vertebrates (exposure time ≤ 96 h) constitute 55% of all the data compiled [19]. This information was corroborated by Santos et al. [20], who estimated that acute effects in organisms belonging to different trophic levels predominate over chronic ones in more than 60% of all the tests performed. This also concerns the available information on the ecotoxicity of sulfonamides (see Table 2).

Looking at the available acute toxicity data, it can be concluded that SAs are practically nontoxic to most microorganisms tested including selected strains of bacteria, such as *Vibrio fischeri* and *Pseudomonas aeruginosa*. However, data as are available from acute tests on the 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 growth and development of plants.

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

**4. State of the knowledge concerning mixture and chronic toxicity of the**

Chronic toxicity tests are studies in which organisms are exposed to different concentrations of a chemical and observed over a long period, or a substantial part of their lifespan. In contrast to acute toxicity tests, which often use mortality as the only measured effect, chronic tests usually include additional measures of effect such as growth rates, reproduction or changes in organism behavior [55-56]. Therefore, the standard acute toxicity tests do not seem appro‐ priate for risk assessment of pharmaceuticals, because of the nature of these compounds. The use of chronic tests over the life-cycle of organisms for different trophic levels could be more appropriate [57]. However, there is still an ongoing debate between ecotoxicologists over the determination which tests should be considered to be chronic or acute (based on their dura‐ tion). This applies not only to aquatic animal testing with invertebrates and fish, but also to

Molander et al. [19] reviewed the data published in the *Wikipharma* database – a freely available, interactive and comprehensive database on the environmental effects of pharmaceuticals that provides an overview of effects caused by these compounds on non-target organisms identi‐ fied in acute, sub-chronic and chronic ecotoxicity tests. Looking at the data set as a whole, they concluded that crustaceans like *Daphnia magna* and *Ceriodaphnia dubia* were the species most commonly used (29% of all tests performed); this is hardly surprising since they are abundant and widespread, easy to keep in the laboratory, and sensitive towards a broad range of environmental contaminants. Less commonly, such tests were performed on marine bacteria *Vibrio fischeri* (12%), algae *Pseudokirchneriella subcapitata* (9.5%) and fish *Poeciliopsis lucida* (9%) and *Oncorhynchus mykiss* (8%) [19]. They have also estimated that acute tests based on microorganisms (exposure time ≤ 30 min), algae (exposure time ≤ 72 h), invertebrates (exposure time ≤ 48 h) and vertebrates (exposure time ≤ 96 h) constitute 55% of all the data compiled [19]. This information was corroborated by Santos et al. [20], who estimated that acute effects in organisms belonging to different trophic levels predominate over chronic ones in more than 60% of all the tests performed. This also concerns the available information on the ecotoxicity

Looking at the available acute toxicity data, it can be concluded that SAs are practically nontoxic to most microorganisms tested including selected strains of bacteria, such as *Vibrio fischeri* and *Pseudomonas aeruginosa*. However, data as are available from acute tests on the

**4.1. What do we know about the long-term effects of the presence of the residues of**

sufficiently reliable results [52].

68 Organic Pollutants - Monitoring, Risk and Treatment

**sulfonamides in the environment?**

of sulfonamides (see Table 2).

**residues of sulfonamides in the environment**

standard 96-h algal and 7-d higher plant test methods.

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

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 of widespread resistance are of clear concern [55].

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 may have serious implications for the risks of other compounds that are applied to the soil environment such as pesticides.


Only few studies have also explored effects of SAs on aquatic microbes. It must be highlighted that it was already proved that the effects of antibiotics like SAs on bacteria should not be determined using acute tests. These compounds possess specific mode of action and impacts frequently became evident upon extending the incubation period. Most of the toxicity data available for *Vibrio fischeri* using short exposure times (between 5 and 30 min) rather than a 24 h exposure show that SAs have a low toxic potential in this respect, because these compounds interfere only slightly with biosynthetic pathways. Toxicity tests with bacteria have shown that chronic exposure to antibiotics is crucial rather than acute [14,50, 62-63]. This is also supported by the results of [30,61,64]. The toxicity of sulfadimi‐ dine (sulfamethazine) in standard 15 min acute test with this luminescent bacteria ob‐ tained EC50 was 344.7 mg L-1 [30] but in 18 h test its toxicity was in the range of 3.68 – 4.57 mg L-1 depending on the type of strain of these marine bacteria [64]. Also Zou et al. [61] determine the chronic (24 h exposure) and acute (15 min exposure) toxicity to *Photobacteri‐ um phosphoreum* for seven SAs. These experiments revealed that sulfachloropyridazine (SCP) was more toxic than other SAs, whereas sulfapyridine (SPY) was relatively less toxic than other SAs (see Table 3). The order of acute toxicity was as follows: SCP > SSX > SMX > SMM > SDZ > SDMD > SPY. However, the order of chronic toxicity was different: SMM > SCP > SDZ > SMX > SSX > SDMD > SPY. Clearly, different order of toxicity between the acute and chronic exposure indicated a different toxicity mechanism (see Fig. 1). It has been reported that the acute toxic effects of pollutants to *P. phosphoreum* are caused by interfer‐ ing LUC-catalyzed bioluminescent reaction and therefore LUC was found to be the receptor protein for the antibiotics. In contrast, the receptor for the antibiotics in the chronic toxicity

**Figure 1.** (A) Scheme of SA ionization in equilibrium, (B) Mechanism for synergistic effect between SA and TMP in bac‐

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71

Studies conducted on the toxicity mechanism of single SAa indicated that the pKa played a vital role in the toxic effect of SAs or their antibacterial activity [32]. Because LUC (Lucyferase) is an endoenzyme, and SAs have to be transported into the cell before bind with LUC, it was clear that the antibiotic toxicity included both LUC-binding and a toxic transportation effect

test was dihydropterinic acid synthetase (DHPS).

terium (adopted from Reference [61])

**Table 3.** An overview of the available information on the chronic toxicity of sulfonamides to different organisms

What Do We Know About the Chronic and Mixture Toxicity of the Residues of Sulfonamides in the Environment? http://dx.doi.org/10.5772/53732 71

may have serious implications for the risks of other compounds that are applied to the soil

**Substance name Type of organism Acute toxicity Chronic toxicity Ref.**

EC50, 48h = 221 mg L-1 EC50, 21d = 13.7 mg L-1

(166 – 568 mg L-1) (12.2 – 15.3 mg L-1)

EC50, 24h = 26.27 mg L-1 EC50, 48h = 9.63 mg L-1

(16.32 – 42.28 mg L-1) (7.00 – 13.25 mg L-1)

EC50, 48h = 15.51 mg L-1 EC50, 7d = 0.21 mg L-1 (12.97 – 18.55 mg L-1 (0.14 – 0.39 mg L-1)

EC50, 48h = 131 mg L-1 EC50, 21d = 3.466 mg L-1

(119 – 143 mg L-1) (2.642 – 4.469 mg L-1)

EC50, 48h = 3.86 mg L-1 EC50, 21d = 0.869 mg L-1 (3.19 – 5.08 mg L-1) (0.630 – 1.097 mg L-1)

EC50, 48h = 202 mg L-1 EC50, 21d = 4.25 mg L-1 (179 – 223 mg L-1) (3.84 – 4.62 mg L-1)

(169.6 – 274.9 mg L-1) no effect up to 30 mg L-1

(89.5 – 136.9 mg L-1) no effect up to 35 mg L-1

EC50, 48h = 391.1 mg L-1 no effect up to 30 mg L-1

EC50, 48h = 616.9 mg L-1 LOEC = 35 mg L-1

EC50, 48h = 215.9 mg L-1 EC50, 21d

EC50, 48h = 110.7 mg L-1 EC50, 8d

(291.7 – 1303.6 mg L-1)

(341.9 – 440.3 mg L-1)

**Table 3.** An overview of the available information on the chronic toxicity of sulfonamides to different organisms

4.08 (± 0.06) M 3.84 (± 0.04) M 4.45 (± 0.05) M 4.50 (± 0.06) M 4.43 (± 0.03) M 5.05 (± 0.05) M 4.78 (± 0.04) M


3.12 (± 0.04) M 2.92 (± 0.05) M 3.32 (± 0.04) M 3.32 (± 0.02) M 3.81 (± 0.02) M 3.67 (± 0.03) M 4.30 (± 0.04) M [60]

[29]

[61]

[25-26]

[16]

environment such as pesticides.

70 Organic Pollutants - Monitoring, Risk and Treatment

**SDZ** *Daphnia magna*

*Brachionus calyciflorus*

*Photobacterium phosphoreum*

*Clathrina dubia*

*Daphnia magna*

**SDMD** *Daphnia magna*

**SDMD** *Moina macrocopa*

**SMX**

**SDMD SPY SMX SDZ SSX SMMa SCP**

**SQO**

**SGD**

**SDMD**

**STZ**

**STZ**

*a*

Sulfamonometoxine

**Figure 1.** (A) Scheme of SA ionization in equilibrium, (B) Mechanism for synergistic effect between SA and TMP in bac‐ terium (adopted from Reference [61])

Only few studies have also explored effects of SAs on aquatic microbes. It must be highlighted that it was already proved that the effects of antibiotics like SAs on bacteria should not be determined using acute tests. These compounds possess specific mode of action and impacts frequently became evident upon extending the incubation period. Most of the toxicity data available for *Vibrio fischeri* using short exposure times (between 5 and 30 min) rather than a 24 h exposure show that SAs have a low toxic potential in this respect, because these compounds interfere only slightly with biosynthetic pathways. Toxicity tests with bacteria have shown that chronic exposure to antibiotics is crucial rather than acute [14,50, 62-63]. This is also supported by the results of [30,61,64]. The toxicity of sulfadimi‐ dine (sulfamethazine) in standard 15 min acute test with this luminescent bacteria ob‐ tained EC50 was 344.7 mg L-1 [30] but in 18 h test its toxicity was in the range of 3.68 – 4.57 mg L-1 depending on the type of strain of these marine bacteria [64]. Also Zou et al. [61] determine the chronic (24 h exposure) and acute (15 min exposure) toxicity to *Photobacteri‐ um phosphoreum* for seven SAs. These experiments revealed that sulfachloropyridazine (SCP) was more toxic than other SAs, whereas sulfapyridine (SPY) was relatively less toxic than other SAs (see Table 3). The order of acute toxicity was as follows: SCP > SSX > SMX > SMM > SDZ > SDMD > SPY. However, the order of chronic toxicity was different: SMM > SCP > SDZ > SMX > SSX > SDMD > SPY. Clearly, different order of toxicity between the acute and chronic exposure indicated a different toxicity mechanism (see Fig. 1). It has been reported that the acute toxic effects of pollutants to *P. phosphoreum* are caused by interfer‐ ing LUC-catalyzed bioluminescent reaction and therefore LUC was found to be the receptor protein for the antibiotics. In contrast, the receptor for the antibiotics in the chronic toxicity test was dihydropterinic acid synthetase (DHPS).

Studies conducted on the toxicity mechanism of single SAa indicated that the pKa played a vital role in the toxic effect of SAs or their antibacterial activity [32]. Because LUC (Lucyferase) is an endoenzyme, and SAs have to be transported into the cell before bind with LUC, it was clear that the antibiotic toxicity included both LUC-binding and a toxic transportation effect (which can be described using pKa). pKa is a decisive factor in transporting SAs into the cell. Three species (neutral, cationic and anionic) of SAs depend on the pKa and surrounding pH values. The neutral species have higher cell membrane permeability than anionic species. Therefore, pKa was the key parameter of sulfonamides toxic effects. Some similarity in acute and chronic toxicity mechanisms was observed. However, in conclusion the distinct receptor proteins of SAs in acute toxicity and chronic toxicity led to the different toxicity mechanisms of single antibiotics [61]. A comparison of the results of short and long term bioassays with *Vibrio fischeri* demonstrates the risk of underestimating the severe effects of substances with delayed toxicity in acute tests.

**4.2. What do we know about mixture toxicity of the residues of sulfonamides in the**

As SAs occur in natural environment 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 need to be considered. Sulfonamides are widely used in combination therapy together with their potentiator (mostly trimethoprim, TMP) in human and veterinary medicine [1-3]; thus, the occurrence of TMP together with other antibiotics has

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

Santos et al. [20] pointed out that, ecotoxicological data show that the effects of mixtures may differ from those of single compounds. For example, Cleuvers [46] showed that a mixture of diclofenac and ibuprofen exhibited a greater than predicted toxicity to *D. magna*, and that the addition of two more drugs increased the toxicity towards the test species even further. Available mixture toxicity data for sulfonamides is summarized in Table 4 and discussed

> **Toxicity of single compounds (EC50single)**

2.30 mg L-1

**Mixture toxicity (EC50mixture)**

0.275 mg L-1

212 mg L-1 Antagonistic

**Conclusions Ref.**

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73

Synergistic growth inhibition between SAs and TMP or PMT and

SMX:AcSMX:TMP mixture.

interaction for mixtures: SMZ + SDZ SMZ + SGD SMZ + SMR

[28]

[26]

for

**environment?**

below.

SDM

Trimethoprim

SMX:AcSMX:TMP

SDM:AcSDM:TMP

SDZ:AcSDZ:TMP

SDZ

**Substance name/ Mixture composition**

been commonly detected [3].

**Test scenario (statistical design)**

Evaluation of the toxicity of the mixture of selected compounds based on concentration addition concept.

SMZ + TMP according to

Assessment of interactive effects between two compounds identified by

SGD 3.86 mg L-1 SMR 277 mg L-1 SDM 202 mg L-1 SDMD (SMZ) 270 mg L-1

SMX 1.50 mg L-1 SDZ 2.19 mg L-1

(TMP) 80.8 mg L-1 Pyrimethanine (PMT) 5.06 mg L-1 AcSMX >100mg L-1 AcSDM >100mg L-1 AcSDZ >100mg L-1

**Test description (organism, test duration)**

*Selenastrum capricornutum*,

OECD 201

SDA + TMP 0.465 mg L-1 SDM + PMT 2.36 mg L-1

(20:105:3) 0.784 mg L-1

(176:8:1) 2.17 mg L-1

(42:24:1) 2.08 mg L-1

*Daphnia magna*,

48 h

Similar conclusions can be obtained if only acute toxicity of SAs to invertebrates is taken into consideration. Detailed information is presented in Table 3. No acute effects on *D. magna* were observed in the investigation of Wollenberger et al. [60]. However, reproduc‐ tive effects (EC50) were observed for sulfadiazine in the range of 5 to 50 mg L-1. This drug caused mortality in the parent generation during the long-term (3 weeks) exposure. Such results suggest that crustacean reproduction test should be included in the test strategy [60]. Similar correlation was also found by Isidori et al. [29] who investigated the acute and chronic toxicity of SMX to *B. calciflorus* (24 h and 48 h exposure) and *C. dubia* (48 h and 7 d exposure time). As expected chronic tests showed higher toxicity that acute tests. Also Park and Choi [16] evaluated the acute and chronic aquatic toxicities of four SAa using standard tests with *D. magna* and *M. macrocopa*. The results from the chronic toxicity tests in this study showed that sensitivity of *M. macrocopa* was similar to that of *D. magna.* However, the exposure duration for *M. macrocopa* was only 8 days whereas for D. magna was 21 days. *Moina* shares many characterisitcs with *D. magna* (e.g. large population densities, high population growth rates, short generation time, and easiness of culture) and is often preferred for hazard evaluation because of its relatively short life span and wide geographical distribution. However, Park and Choi found no significant effects on reproduction of *D. magna* at concentrations of SMZ up to 30 mg L-1. In contrast De Liguoro et al. [25-26] observed strong inhibitory effect of SMZ on reproduction of *D. magna* (nearly 100% inhibition with SMZ at a concentration of 12.5 mg L-1). This could be explained by that fact that in the Park and Choi study, daphnids were fed daily not only with algae, but also with the EPA recommended YCT that contains yeast, a known good natural source of folate [16]. Eguchi et al. [28] have shown that SAs interfere with folate synthesis in green algae. Therefore, this supplement of folic acid may well have protected the reproduction of the test organisms by compensating for the deficiencies caused by SMZ. Generally, when testing antibacterials on *D. magna*, effects on the reproductive output occur at concentra‐ tions which are at least one order of magnitude below the acute toxic levels [60].

Unfortunately, there is no information about long-term effects of the residues of these compounds to higher plants and other aquatic as well as terrestrial organisms. Therefore, it seems to be necessary for researchers to study the chronic toxicity of antibiotic [46-47, 55] because of their widespread use and continuous emissions into the environment [14].

#### **4.2. What do we know about mixture toxicity of the residues of sulfonamides in the environment?**

(which can be described using pKa). pKa is a decisive factor in transporting SAs into the cell. Three species (neutral, cationic and anionic) of SAs depend on the pKa and surrounding pH values. The neutral species have higher cell membrane permeability than anionic species. Therefore, pKa was the key parameter of sulfonamides toxic effects. Some similarity in acute and chronic toxicity mechanisms was observed. However, in conclusion the distinct receptor proteins of SAs in acute toxicity and chronic toxicity led to the different toxicity mechanisms of single antibiotics [61]. A comparison of the results of short and long term bioassays with *Vibrio fischeri* demonstrates the risk of underestimating the severe effects of substances with

Similar conclusions can be obtained if only acute toxicity of SAs to invertebrates is taken into consideration. Detailed information is presented in Table 3. No acute effects on *D. magna* were observed in the investigation of Wollenberger et al. [60]. However, reproduc‐ tive effects (EC50) were observed for sulfadiazine in the range of 5 to 50 mg L-1. This drug caused mortality in the parent generation during the long-term (3 weeks) exposure. Such results suggest that crustacean reproduction test should be included in the test strategy [60]. Similar correlation was also found by Isidori et al. [29] who investigated the acute and chronic toxicity of SMX to *B. calciflorus* (24 h and 48 h exposure) and *C. dubia* (48 h and 7 d exposure time). As expected chronic tests showed higher toxicity that acute tests. Also Park and Choi [16] evaluated the acute and chronic aquatic toxicities of four SAa using standard tests with *D. magna* and *M. macrocopa*. The results from the chronic toxicity tests in this study showed that sensitivity of *M. macrocopa* was similar to that of *D. magna.* However, the exposure duration for *M. macrocopa* was only 8 days whereas for D. magna was 21 days. *Moina* shares many characterisitcs with *D. magna* (e.g. large population densities, high population growth rates, short generation time, and easiness of culture) and is often preferred for hazard evaluation because of its relatively short life span and wide geographical distribution. However, Park and Choi found no significant effects on reproduction of *D. magna* at concentrations of SMZ up to 30 mg L-1. In contrast De Liguoro et al. [25-26] observed strong inhibitory effect of SMZ on reproduction of *D. magna* (nearly 100% inhibition with SMZ at a concentration of 12.5 mg L-1). This could be explained by that fact that in the Park and Choi study, daphnids were fed daily not only with algae, but also with the EPA recommended YCT that contains yeast, a known good natural source of folate [16]. Eguchi et al. [28] have shown that SAs interfere with folate synthesis in green algae. Therefore, this supplement of folic acid may well have protected the reproduction of the test organisms by compensating for the deficiencies caused by SMZ. Generally, when testing antibacterials on *D. magna*, effects on the reproductive output occur at concentra‐

tions which are at least one order of magnitude below the acute toxic levels [60].

because of their widespread use and continuous emissions into the environment [14].

Unfortunately, there is no information about long-term effects of the residues of these compounds to higher plants and other aquatic as well as terrestrial organisms. Therefore, it seems to be necessary for researchers to study the chronic toxicity of antibiotic [46-47, 55]

delayed toxicity in acute tests.

72 Organic Pollutants - Monitoring, Risk and Treatment

As SAs occur in natural environment 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 need to be considered. Sulfonamides are widely used in combination therapy together with their potentiator (mostly trimethoprim, TMP) in human and veterinary medicine [1-3]; thus, the occurrence of TMP together with other antibiotics has been commonly detected [3].

Santos et al. [20] pointed out that, ecotoxicological data show that the effects of mixtures may differ from those of single compounds. For example, Cleuvers [46] showed that a mixture of diclofenac and ibuprofen exhibited a greater than predicted toxicity to *D. magna*, and that the addition of two more drugs increased the toxicity towards the test species even further. Available mixture toxicity data for sulfonamides is summarized in Table 4 and discussed below.



most commonly used pharmaceuticals belonging to different groups (atorvastatin, acetami‐ nophen, caffeine, sulfamethoxazole, carbamazepine, levofloxacin, sertraline and trimetho‐ prim) to the aquatic macrophytes *Lemna gibba* and *Myriophyllum sibircu*m. Given the diversity in mode of action of these compounds, the toxicity of the mixture in the microcosms was likely via response addition. Generally, both species displayed similar sensitivity to the pharma‐

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On the other hand, Eguchi et al. [28] found that a mixture of trimethoprim or pyrimethamine (pyrimethamine is often used as a substitute for trimethoprim), sulfamethoxazole and sulfadiazine significantly increased growth inhibition (synergistic effect of the mixture was observed) in the algae *S. capricornutum*. To investigate the synergistic influence of combined drugs on the growth of green algae, SAs and TMP or PMT (TMPs) were simultaneously added to *S. capricornutum* culture. In this experiment, the concentration of TMPs was fixed at the no observed effect concentration (NOEC) and the concentration of the SAs were altered. These combined drugs are frequently used in the veterinary field in many countries. Combination of SMZ and SDA with TMP rendered the growth inhibitory activity significantly increased in comparison with their individual activities (see Table 4 and Fig. 2(A)). On the contrary, combination of SDM with PYR did not show such an effect. Moreover, as SAs are thought to be partly metabolized to AcSAs in the bodies of animals, Eguchi et al. [28] have also tested the toxicity of the mixture of SAs their metabolites and TMP. Therefore the test of combined drugs was done by using the combinations corresponding AcSAs at a ratio according to the concen‐ trations detected in the urine of pigs fed with SAs. The ratio was SMZ:AcSMZ:TMP = 20:105:3, SDM:AcSDM:PMT = 167:8:1 and SDA:AcSDA:TMP = 42:24:1. A similar synergistic effect to that described above was observed with combinations of SMZ, TMP, and AcSMZ (see Table 4). However, combination of SDM or SDA with their acetylate and PMT or TMP did not show a synergistic effect on growth in excretion ratio. A reason must be that the concentration of TMP used was not enough to express synergistic influence in combination with SDM or SDA. These results indicate that several combined drugs that show a synergistic effect *in vitro* may have an actual synergistic effect on algae in ecosystem although excretion ratio can vary in animal condition or other factors. The synergistic effect observed by the combination of SAs and TMPs in this study indicates that the simultaneous release of several antimicrobial agents may result in greater toxicity to microorganisms in the environment than the release of the same agents individually. Furthermore, the rate of growth inhibition by SAs by addition of folic acid was investigated in this study. It was observed that the growth inhibitory activity of the combination of SDA and TMP was significantly reduced by the addition of 20 ng/l of folic acid to the medium. Significantly, folic acid exhibited a similar effect when SDA was tested

alone, but not when TMP was tested alone (see Fig. 2(B)) [28].

Both SAs and TMPs inhibit the folate synthesis pathway in bacteria, but their inhibition sites are different. SAs inhibit dihydropterinic acid synthetase (DHPS), thereby inhibiting the synthesis of folic acid. On the other hand, TMPs inhibits dihydrofolic acid reductase (DHFR), which converts folic acid to 7,8-dihydrofolic acid (7,8- DHF) and 5,6,7,8-tetrahydrofolic acid (5,6,7,8-THF), both active forms of folic acid suitable for utilization. Therefore, the synergistic effect of the combination of SAs and TMPs is likely to be due to the cumulative effect of their

ceutical mixture [22].

**Table 4.** An overview of the available information on the mixture toxicity of sulfonamides to different organisms

The toxicity of mixture of sulfonamides to non-target organisms was firstly reported by Brain et al. [22] and Eguchi et al. [28]. Brain et al. investigated the toxicity of the mixture of eight most commonly used pharmaceuticals belonging to different groups (atorvastatin, acetami‐ nophen, caffeine, sulfamethoxazole, carbamazepine, levofloxacin, sertraline and trimetho‐ prim) to the aquatic macrophytes *Lemna gibba* and *Myriophyllum sibircu*m. Given the diversity in mode of action of these compounds, the toxicity of the mixture in the microcosms was likely via response addition. Generally, both species displayed similar sensitivity to the pharma‐ ceutical mixture [22].

SQO

SDMD

binary mixtures of SMZ +6 compounds

SGD Assessment of

SPY Evaluation of the

isobologram method.

74 Organic Pollutants - Monitoring, Risk and Treatment

interactive effects between two compounds identified by isobologram method.

SGD *Pseudokirchneriella*

toxicity of the mixture of selected compounds based on concentration addition concept

TMP 149 mg L-1

*Daphnia magna*,

*subcapitata*, 96 h

*Photobacterium phosphoreu*m, 15 min and 24 h

SDMD 4.08 (± 0.06) M 5.08 (± 0.05) M

SPY 3.84 (± 0.04) M 4.85 (± 0.07) M SMX 4.45 (± 0.05) M 5.50 (± 0.07) M SDZ 4.50 (± 0.06) M 5.42 (± 0.03) M SSX 4.43 (± 0.03) M 5.45 (± 0.03) M SMM 5.05 (± 0.05) M 6.01 (± 0.05) M SCP 4.78 (± 0.04) M 5.73 (± 0.05) M

**Table 4.** An overview of the available information on the mixture toxicity of sulfonamides to different organisms

The toxicity of mixture of sulfonamides to non-target organisms was firstly reported by Brain et al. [22] and Eguchi et al. [28]. Brain et al. investigated the toxicity of the mixture of eight

SMX 3.32 (± 0.04) M 2.79 (± 0.01) M SDZ 3.32 (± 0.02) M 2.76 (± 0.03) M SSX 3.81 (± 0.02) M 2.79 (± 0.07) M SMM 3.67 (± 0.03) M 2.73 (± 0.02) M SCP 4.30 (± 0.04) M 3.00 (± 0.03) M

21 d

SQO 3.466 mg L-1

SQO 0.246 mg L-1

TMP 3.22 (± 0.07) M

TMP 5.37 (± 0.02) M

131 mg L-1

0.896 mg L-1

43.559 mg L-1

Toxicity of single compound (-logEC50, 15 min)

Toxicity of single compound (-logEC50, 24h)

3.12 (± 0.04) M 2.78 (± 0.02) M

2.92 (± 0.05) M 2.89 (± 0.02) M

Toxicity of binary mixture SAs and TMP(-logEC50, mixture)

Toxicity of binary mixture SAs and TMP (-logEC50, mixture)

SMT + SDM Complex interaction (synergism additivity and antagonism) for mixture of SMT +

SQO

Additive (antagonistic) interaction between SQO and SGD.

Antagonistic interaction between SAs and TMP in acute toxicity test.

Synergistic interaction between SAs and TMP in chronic toxicity test.

Simple additivity for SMZ + TMP

[25]

[61]

On the other hand, Eguchi et al. [28] found that a mixture of trimethoprim or pyrimethamine (pyrimethamine is often used as a substitute for trimethoprim), sulfamethoxazole and sulfadiazine significantly increased growth inhibition (synergistic effect of the mixture was observed) in the algae *S. capricornutum*. To investigate the synergistic influence of combined drugs on the growth of green algae, SAs and TMP or PMT (TMPs) were simultaneously added to *S. capricornutum* culture. In this experiment, the concentration of TMPs was fixed at the no observed effect concentration (NOEC) and the concentration of the SAs were altered. These combined drugs are frequently used in the veterinary field in many countries. Combination of SMZ and SDA with TMP rendered the growth inhibitory activity significantly increased in comparison with their individual activities (see Table 4 and Fig. 2(A)). On the contrary, combination of SDM with PYR did not show such an effect. Moreover, as SAs are thought to be partly metabolized to AcSAs in the bodies of animals, Eguchi et al. [28] have also tested the toxicity of the mixture of SAs their metabolites and TMP. Therefore the test of combined drugs was done by using the combinations corresponding AcSAs at a ratio according to the concen‐ trations detected in the urine of pigs fed with SAs. The ratio was SMZ:AcSMZ:TMP = 20:105:3, SDM:AcSDM:PMT = 167:8:1 and SDA:AcSDA:TMP = 42:24:1. A similar synergistic effect to that described above was observed with combinations of SMZ, TMP, and AcSMZ (see Table 4). However, combination of SDM or SDA with their acetylate and PMT or TMP did not show a synergistic effect on growth in excretion ratio. A reason must be that the concentration of TMP used was not enough to express synergistic influence in combination with SDM or SDA. These results indicate that several combined drugs that show a synergistic effect *in vitro* may have an actual synergistic effect on algae in ecosystem although excretion ratio can vary in animal condition or other factors. The synergistic effect observed by the combination of SAs and TMPs in this study indicates that the simultaneous release of several antimicrobial agents may result in greater toxicity to microorganisms in the environment than the release of the same agents individually. Furthermore, the rate of growth inhibition by SAs by addition of folic acid was investigated in this study. It was observed that the growth inhibitory activity of the combination of SDA and TMP was significantly reduced by the addition of 20 ng/l of folic acid to the medium. Significantly, folic acid exhibited a similar effect when SDA was tested alone, but not when TMP was tested alone (see Fig. 2(B)) [28].

Both SAs and TMPs inhibit the folate synthesis pathway in bacteria, but their inhibition sites are different. SAs inhibit dihydropterinic acid synthetase (DHPS), thereby inhibiting the synthesis of folic acid. On the other hand, TMPs inhibits dihydrofolic acid reductase (DHFR), which converts folic acid to 7,8-dihydrofolic acid (7,8- DHF) and 5,6,7,8-tetrahydrofolic acid (5,6,7,8-THF), both active forms of folic acid suitable for utilization. Therefore, the synergistic effect of the combination of SAs and TMPs is likely to be due to the cumulative effect of their

also have a similar folate synthesis pathway, the growth inhibitory effect of SAs on these organisms is likely to be the result of the same inhibitory mechanism. Therefore, algal cells could survive in the presence of SAs, but not TMP, when folic acid was added to the medium. De Liguoro et al. [25-26] evaluated the acute mixture toxicity of combining sulfamethazine with TMP towards *D. magna* and effects of different mixtures of sulfaquinoloxine (SQO) and sulfaguanidine (SGD) on *D. magna* and *P. subcapitata* (see Table 4). The additive toxicity of these compounds was evaluated using the isobologram method. In Fig. 3A, the isoboles showing the different type of combination effects are presented. Taking into account confidence intervals SMZ showed infra-additivity when paired with SDZ, SGD, SMA or SDM. When SMZ was paired with SQO the interaction was more complex, as each type of combination effects (supra-additivity, additivity and infra-additivity) was observed at the three different combi‐ nation ratios. Simple additivity was recorded when SMZ was combined with the sulfonamidepotentiator TMP (Fig. 3A). Tests with paired SQO and SGD were based on the individual EC50 (for *D. magna* see [25]). In each paired test, the concentration–response relationship was analyzed for three selected combination ratios equidistantly distributed on the additivity line. In Fig. 3B, the isoboles based on the effects of different mixtures of SQO and SGD on *D. magna* and *P. subcapitata* are depicted. Only in one test, where relatively low concentrations of SQO were combined with relatively high concentrations of SGD on *P. subcapitata*, the two paired compounds showed simple additivity. In all the other tests a less than additive (antagonistic) interaction was detected. In this study, binary tests confirmed the tendency of SAs mixtures to act less than additively. So, in general terms, it seems sufficiently precaution‐ ary to consider their environmental toxicity as additive. However, when combining SQO and SGD on P. *subcapitata*, the obtained asymmetric isobologram shows that the interaction is mixture-ratio dependent, a phenomenon already observed when mixtures of SQO and

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Zou et al. [61] have recently highlighted that these results cannot represent the mixture toxicity between the SAs and TMP in an actual environment because non-target organisms (microlage and *D. magna*) were used in these studies. Bacterium is typically the target-organism of an antibiotic, and thus in their opinion, a bioassay with *Photobacterium phosphoreum* is a more reliable tool to determine the toxicity of various antibiotics. Moreover, most studies focus on the acute mixture toxicity. Therefore, in their study they have: determined not only the acute (15 min exposure) but also chronic (24 h exposure) toxicity *to P. phosphoreum* for single SA and their potentiator, and for their mixtures (SA with TMP); evaluated the differences between chronic and acute mixture toxicity; and revealed the difference between their toxicity mecha‐ nisms by using QSAR models. A comparison of chronic vs. acute mixture toxicity revealed the presence of an interesting phenomenon, that is, that the joint effects vary with the duration of exposure; the acute mixture toxicity was antagonistic, whereas the chronic mixture toxicity was synergistic. Based on the approach of QSARs and molecular docking, this phenomenon was proved to be caused by the presence of two points of dissimilarity between the acute and chronic mixture toxicity mechanism: (1) the receptor protein of SAs in acute toxicity was LUC, while in chronic toxicity it was DHPS, and (2) there is a difference between actual concentration of binding-LUC in acute toxicity and individual binding-DHPS in chronic toxicity (see Fig. 1). The existence of these differences poses a challenge for the assessment of routine combi‐ nations in medicine, risk assessment, and mixture pollutant control, in which, previously, only

sulfamethazine were tested on *D. magna* [26].

**Figure 2. (A)** The dose-response curve of SAs (SMT – sufamethzine, SDA – sulfadiazine, SDM – sulfadimethoxine) com‐ bined with TMP (trimethoprim) or PMT (pyrimethamine); **(B)** Recovery of growth inhibition be addition of folic acid (\*observed siginifcant difference to negative control – without folic acid, 1) concentration of SDA (sulfadiazine) in com‐ bination, TMP was used at the NOEC, 2) used at the EC50 concentration) (adopted from References [28])

actions on two different sites in the folate biosynthesis pathway. Since SAs block the synthesis of folate, the growth inhibitory effect of this compound can be reversed by the addition of folate. In contrast, TMP blocks enzymes downstream of folate in the synthesis pathway, thus addition of folate will not reverse the growth-inhibiting effect of this compound. Since algea also have a similar folate synthesis pathway, the growth inhibitory effect of SAs on these organisms is likely to be the result of the same inhibitory mechanism. Therefore, algal cells could survive in the presence of SAs, but not TMP, when folic acid was added to the medium.

De Liguoro et al. [25-26] evaluated the acute mixture toxicity of combining sulfamethazine with TMP towards *D. magna* and effects of different mixtures of sulfaquinoloxine (SQO) and sulfaguanidine (SGD) on *D. magna* and *P. subcapitata* (see Table 4). The additive toxicity of these compounds was evaluated using the isobologram method. In Fig. 3A, the isoboles showing the different type of combination effects are presented. Taking into account confidence intervals SMZ showed infra-additivity when paired with SDZ, SGD, SMA or SDM. When SMZ was paired with SQO the interaction was more complex, as each type of combination effects (supra-additivity, additivity and infra-additivity) was observed at the three different combi‐ nation ratios. Simple additivity was recorded when SMZ was combined with the sulfonamidepotentiator TMP (Fig. 3A). Tests with paired SQO and SGD were based on the individual EC50 (for *D. magna* see [25]). In each paired test, the concentration–response relationship was analyzed for three selected combination ratios equidistantly distributed on the additivity line. In Fig. 3B, the isoboles based on the effects of different mixtures of SQO and SGD on *D. magna* and *P. subcapitata* are depicted. Only in one test, where relatively low concentrations of SQO were combined with relatively high concentrations of SGD on *P. subcapitata*, the two paired compounds showed simple additivity. In all the other tests a less than additive (antagonistic) interaction was detected. In this study, binary tests confirmed the tendency of SAs mixtures to act less than additively. So, in general terms, it seems sufficiently precaution‐ ary to consider their environmental toxicity as additive. However, when combining SQO and SGD on P. *subcapitata*, the obtained asymmetric isobologram shows that the interaction is mixture-ratio dependent, a phenomenon already observed when mixtures of SQO and sulfamethazine were tested on *D. magna* [26].

Zou et al. [61] have recently highlighted that these results cannot represent the mixture toxicity between the SAs and TMP in an actual environment because non-target organisms (microlage and *D. magna*) were used in these studies. Bacterium is typically the target-organism of an antibiotic, and thus in their opinion, a bioassay with *Photobacterium phosphoreum* is a more reliable tool to determine the toxicity of various antibiotics. Moreover, most studies focus on the acute mixture toxicity. Therefore, in their study they have: determined not only the acute (15 min exposure) but also chronic (24 h exposure) toxicity *to P. phosphoreum* for single SA and their potentiator, and for their mixtures (SA with TMP); evaluated the differences between chronic and acute mixture toxicity; and revealed the difference between their toxicity mecha‐ nisms by using QSAR models. A comparison of chronic vs. acute mixture toxicity revealed the presence of an interesting phenomenon, that is, that the joint effects vary with the duration of exposure; the acute mixture toxicity was antagonistic, whereas the chronic mixture toxicity was synergistic. Based on the approach of QSARs and molecular docking, this phenomenon was proved to be caused by the presence of two points of dissimilarity between the acute and chronic mixture toxicity mechanism: (1) the receptor protein of SAs in acute toxicity was LUC, while in chronic toxicity it was DHPS, and (2) there is a difference between actual concentration of binding-LUC in acute toxicity and individual binding-DHPS in chronic toxicity (see Fig. 1). The existence of these differences poses a challenge for the assessment of routine combi‐ nations in medicine, risk assessment, and mixture pollutant control, in which, previously, only

actions on two different sites in the folate biosynthesis pathway. Since SAs block the synthesis of folate, the growth inhibitory effect of this compound can be reversed by the addition of folate. In contrast, TMP blocks enzymes downstream of folate in the synthesis pathway, thus addition of folate will not reverse the growth-inhibiting effect of this compound. Since algea

bination, TMP was used at the NOEC, 2) used at the EC50 concentration) (adopted from References [28])

(B)

**Figure 2. (A)** The dose-response curve of SAs (SMT – sufamethzine, SDA – sulfadiazine, SDM – sulfadimethoxine) com‐ bined with TMP (trimethoprim) or PMT (pyrimethamine); **(B)** Recovery of growth inhibition be addition of folic acid (\*observed siginifcant difference to negative control – without folic acid, 1) concentration of SDA (sulfadiazine) in com‐

(A)

76 Organic Pollutants - Monitoring, Risk and Treatment

acute toxicity mechanism of single antibiotics it can be concluded that the transportation toxic effect is highly related to pKa values and the interaction toxic effect can be described by LUC-SAs binding or LUC-TMP binding. The synergistic effect between SAs with TMP has also been observed in the field of medicine and proved to be caused by the blocking of synthesis of folic acid. First SAs inhibit DHPS, which catalyzes the formation of dihydropteroic acid. Then TMP inhibits DHFR, which catalyzes the formation of tetrahydrofolic acid from dihydrofolic acid. In an acute mixture toxicity there were more SAs-binding-LUC and TMP-binding-LUC. However, in chronic mixture toxicity, the concentration of SAs-binding-DHPS was less compere to TMP-binding-DHFR. It can therefore be concluded that the dissimilarities in the concentrations of individual chemical-binding receptor proteins also lead to the different joint

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These examples highlight the fact that 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. More ecotoxicological studies should therefore be done to evaluate the impact of different mixtures of pharmaceuticals in non-target organisms.

The reason for concern regarding risks of mixtures is obvious. Man is always exposed to more than one chemical at a time. This dictates the necessity of exposure assessment, hazard identification, and risk assessment of chemical mixtures. However, for most chemical mixtures data on exposure and toxicity are fragmentary, and roughly over 95% of the resources in toxicology is still devoted to studies of single chemicals. Moreover, organisms are typically exposed to mixtures of chemicals over long periods of time; thus, chronic mixture toxicity

However, testing of all kinds of (complex) mixtures of chemicals existing in the real world or of all possiblecombinationsofchemicalsofasimple(defined)mixtureatdifferentdoselevelsisvirtually impossible. Moreover, even if toxicity data on individual compounds are available, we are still facing the immense problem of extrapolation of findings obtained at relatively high exposure concentration in laboratory animals to man being exposed to (much) lower concentrations.

As stated by several authors, it is essential to investigate if mixtures of pharmaceuticals interact, leading to a larger effect in the environment than would be predicted when each compound is considered individually. Mixtures with antibiotics in the environment may be very complex (e.g. wastewater effluent) but they also may be simple. Although the latter may be more easily studied experimentally, in both cases the identification and quantitative description of

Over past 10 years there has been increasing interest in the impacts of SAs and other veterinary medicinesintheenvironmentandthereisnowamuchbetterunderstandingabouttheirenviron‐ mental fate and their impacts on aquatic and terrestrial organisms. However, there are still a number of uncertainties that require addressing before there can be a full understanding of the environmental risks of these compounds. Areas requiring further research are presented below.

analysis is the best way to perform risk assessment in regards to organisms.

synergism caused by specific substances is crucial.

effect (SA with TMP) in acute and chronic mixture toxicity [61].

**5. Conclusions**

**Figure 3. (A)***D. magna* immobilization test: 48 h EC50 isobolograms of SMZ paired with other SAs and TMP at three selected combination ratios; **(B)** isobolograms of paired SQO and SGD *in D. magna* immobilization test (adopted from References [25-26])

a synergistic effect has been observed between SA and their potentiator. The toxicity effect of mixtures is associated with the transportation of toxic effects of individual chemicals into cells, the interaction toxic effects of individual chemical-binding-receptor proteins. According to acute toxicity mechanism of single antibiotics it can be concluded that the transportation toxic effect is highly related to pKa values and the interaction toxic effect can be described by LUC-SAs binding or LUC-TMP binding. The synergistic effect between SAs with TMP has also been observed in the field of medicine and proved to be caused by the blocking of synthesis of folic acid. First SAs inhibit DHPS, which catalyzes the formation of dihydropteroic acid. Then TMP inhibits DHFR, which catalyzes the formation of tetrahydrofolic acid from dihydrofolic acid. In an acute mixture toxicity there were more SAs-binding-LUC and TMP-binding-LUC. However, in chronic mixture toxicity, the concentration of SAs-binding-DHPS was less compere to TMP-binding-DHFR. It can therefore be concluded that the dissimilarities in the concentrations of individual chemical-binding receptor proteins also lead to the different joint effect (SA with TMP) in acute and chronic mixture toxicity [61].

These examples highlight the fact that 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. More ecotoxicological studies should therefore be done to evaluate the impact of different mixtures of pharmaceuticals in non-target organisms.
