**4. Antibiotic effects on the environment**

Veterinary ATBs are designed to affect microorganisms found in animals. However, as discussed above, they are rapidly eliminated in its active form or as by-products, contaminat‐ ing the environment. After contaminating the environment, such drugs have the potential to cause adverse effects to the aquatic and terrestrial biota of different trophic levels and also to humans, through consumption of contaminated food derived from aquaculture or through contact with contaminated water. ATBs transference in the body is determined by its ability to move through the lipid bilayer of epithelial cells. The most important properties affecting their permeation across biological membranes are lipophilicity, hydrogen bonding capacity, size, and charge [58].

To demonstrate the negative effects of these compounds, several authors have performed toxicity tests using a wide range of test organisms under controlled conditions [59–70].

Toxicity tests are divided into acute and chronic. The acute toxicity test is designed to evaluate the effects on organisms in a short period of exposure, with the goal of determining the concentration of a studied substance that produces deleterious effects in controlled conditions. When the test organism is fish, lethal effect is observed in most of the times, from which the concentration of the toxic agent that causes 50% mortality (LC50) is determined. On the other hand, for microcrustaceans, the observed effect can be lethality and also mobility, and in the latter case, the average effective concentration (EC50) that causes 50% immobility is calculated [71]. In chronic toxicity tests, organisms are continually exposed to the evaluated substance for a significant period of time of their life cycle, which can range from half to two thirds of the cycle [71]. Depending on the tested substance characteristics, due to the long test period, it may be necessary to the test solutions to be renewed. In this test, sublethal effects, such as changes in growth and reproduction, changes in behavior (such as movement difficulty and increased lid opening frequency, in the latter case to fish), physiological, biochemical, and tissue alterations [72, 73], among others are evaluated. The chronic toxicity test depends directly on the results of the acute test, once sublethal concentrations are calculated from CL50 and CE50.

**Antibiotics Concentration (µg Kg-1) Location References**

**Table 5.** Antibiotic concentrations reported in sediments of aquatic environments in several regions of the world.

Veterinary ATBs are designed to affect microorganisms found in animals. However, as discussed above, they are rapidly eliminated in its active form or as by-products, contaminat‐ ing the environment. After contaminating the environment, such drugs have the potential to cause adverse effects to the aquatic and terrestrial biota of different trophic levels and also to humans, through consumption of contaminated food derived from aquaculture or through contact with contaminated water. ATBs transference in the body is determined by its ability to move through the lipid bilayer of epithelial cells. The most important properties affecting their permeation across biological membranes are lipophilicity, hydrogen bonding capacity,

To demonstrate the negative effects of these compounds, several authors have performed toxicity tests using a wide range of test organisms under controlled conditions [59–70].

Toxicity tests are divided into acute and chronic. The acute toxicity test is designed to evaluate the effects on organisms in a short period of exposure, with the goal of determining the concentration of a studied substance that produces deleterious effects in controlled conditions. When the test organism is fish, lethal effect is observed in most of the times, from which the concentration of the toxic agent that causes 50% mortality (LC50) is determined. On the other hand, for microcrustaceans, the observed effect can be lethality and also mobility, and in the latter case, the average effective concentration (EC50) that causes 50% immobility is calculated [71]. In chronic toxicity tests, organisms are continually exposed to the evaluated substance for a significant period of time of their life cycle, which can range from half to two thirds of

11.5–7342.7

118 Emerging Pollutants in the Environment - Current and Further Implications

**Tetracyclines**

Tetracycline

Oxytetracycline

Chlortetracycline

size, and charge [58].

Adapted from Fata-Kassinos et al. (2011).

**4. Antibiotic effects on the environment**

1.3 Huangpu River, China [52]

21.7 Huangpu River, China [52] 10.4–22.0 Ilha Solteira Reservoir, Brazil [46] 17.9 Cache La Poudre River, USA [56]

0.6–18.6 Huangpu River, China [52]

Reservatório de Ilha Solteira, Br

14.8 Cache La Poudre River, USA [56]

6.3 Huangpu River, China [52] 16.1 Ilha Solteira Reservoir, Brazil [46] 10.8 Cache La Poudre River, USA [56]

Ilha Solteira Reservoir, Brazil [46]

For choosing the test organism, the following selection criteria are often used: abundance and availability, significant ecological representation, cosmopolitanism, knowledge of its biology, physiology and diet, genetic stability and uniformity of their populations, sensitivity, com‐ mercial importance, ease of cultivation in the laboratory and, if possible, the species should be native, to a better representation of ecosystems [71].

The sensitivity of algae to ATBs varies widely. In a performed toxicity test, it was shown that the green alga *Selenastrum capricornutum* was less sensitive than *Microcystis aeruginosa* microalgae for most of the tested molecules. The growth of *M. aeruginosa* was inhibited when concentrations of less than 0.1 mg L-1 were exposed [66]. Blue-green algae (cyanobacteria) were also sensitive to several ATBs, such as amoxicillin, penicillin benzyl, spiramycin, tetracycline, among others. All these results are very worrying, once that algae are located at the base of the food chain, and a drop in the population of these organisms can disrupt aquatic ecosystems.

Reproductive effects have also been observed in aquatic organisms, such as *Artemia* sp. and *Daphnia magna* when exposed to ATBs [55, 61, 66, 74, 75]. It is important to consider that reproductive effects in any population of organisms can cause considerable damage to the natural balance since the organisms are directly related to each other in the trophic chain.

Numerous studies have evaluated the acute toxicity of ATBs for different aquatic organisms. For example, Wollenberger et al. [75] studied the acute toxicity of nine commonly used veterinary ATBs and reported lower acute toxicity (CE5048h, mg L-1) of the oxolinic acid (4.6) and higher toxicity to oxytetracycline (~1000). Previously, Dojmi di Delupis et al. [76] had reported moderate toxicity to aminosidine, bacitracin, erythromycin, and moderate lincomy‐ cin ATBs to *D. magna* microcrustacean, with CE5048h value of between 30 and 500 mg L-1, with bacitracin being the most toxic. In another study, Kolodziejska et al. [77] determined the toxicity of four veterinary ATBs for different aquatic organisms. In this study, oxytetracycline and florfenicol had stronger effects on *Lemna minor* (CE50 = 3.26 and 2.96 mg L-1, respectively) and on green alga *Scenedesmus vacuolatus* (CE50 = 40.4 and 18.0 mg L-1) than on the marine bacterium *Vibrio fischeri* (CE50 = 108 and 29.4 mg L-1) and on microcrustacean *D. magna* (CE50 = 114 and 337 mg L-1).

The chronic effects of ATBs to aquatic organisms were also studied. Kin et al. [78] evaluated the chronic toxicity of acetaminophen and lincomycin ATBs for two crustaceans species (*D. magna* and *Moina macrocopa*) and for the fish *Oryzias latipes*. To *D. magna*, acetaminophen ATB caused no significant effect on reproduction when exposed to the concentration of 5.72 mg L-1. Similar results were observed for survival and growth when microcrustaceans were exposed to the highest concentration of lincomycin (153 mg L-1). For fish, a significant reduction in survival was observed 30 days after hatching, when exposed to 95 mg L-1 of acetaminophen and 0.42 mg L-1 of lincomycin. Several other studies were conducted to evaluate ATBs acute and chronic toxicity of different classes, using organisms of different trophic levels, as can be seen in Table 6.



survival was observed 30 days after hatching, when exposed to 95 mg L-1 of acetaminophen and 0.42 mg L-1 of lincomycin. Several other studies were conducted to evaluate ATBs acute and chronic toxicity of different classes, using organisms of different trophic levels, as can be

inhibition EC50

inhibition EC50

subcapitata 72 h, growth EC50 3.1 [66]

subcapitata 72 h, growth EC50 1.8 [81]

Alga Microcystis aeruginosa 7 days, growth EC50 0.05 [66]

Duckweed Lemna gibba 7 days, wet weight EC50 0.219 [82] Duckweed Lemna gibba 7 days, frond number EC50 0.318 [82] Duckweed Lemna gibba 7 days, chlorophyll a EC50 0.630 [82] Duckweed Lemna gibba 7 days, chlorophyll b EC50 0.650 [82] Duckweed Lemna gibba 7 days, carotenoids EC50 1.620 [82] Invertebrate Daphnia magna 24 h, immobilization EC50 380.1 [79] Invertebrate Daphnia magna 48 h, immobilization EC50 225.0 [79] Invertebrate Moina macrocopa 24 h, immobilization EC50 515.0 [79] Invertebrate Moina macrocopa 48 h, immobilization EC50 272.0 [79] Fish Oryzias latipes 48 h, survival LC50 88.4 [79] Fish Oryzias latipes 96 h, survival LC50 78.9 [79]

inhibition EC50

inhibition EC50

inhibition EC50

inhibition EC50

Rotifer Brachionus calyciflorus 24 h, survival LC50 34.21 [83] Rotifer Brachionus calyciflorus 48 h, growth EC50 1.87 [83] Algae Chlorella vulgaris 48 h, growth EC50 6.4 [83]

**Concentration (mg L-1)**

>20.0 [80]

13.0 [80]

235.4 [79]

87.0 [79]

64.50 [83]

121.01 [84]

**References**

seen in Table 6.

**Antibiotic**

Chlortetra-cycline

Algae

Algae

**Oxytetra-cycline**

**taxonomic group Species Test duration/end point**

120 Emerging Pollutants in the Environment - Current and Further Implications

Bacteria Vibrio fischeri 5 min, luminescence

Bacteria Vibrio fischeri 15 min, luminescence

Bacteria Vibrio fischeri 5 min, luminescence

Bacteria Vibrio fischeri 15 min, luminescence

Bacteria Vibrio fischeri 30 min, luminescence

Bacteria Vibrio fischeri 30 min, luminescence

Pseudokirchneriella

Pseudokirchneriella



**Antibiotic**

Algae

Sulfathiazole

**taxonomic group Species Test duration/end point**

122 Emerging Pollutants in the Environment - Current and Further Implications

Pseudokirchneriella

Bacteria Vibrio fischeri 15 min, luminescence

Bacteria Vibrio fischeri 5 min, luminescence

Bacteria Vibrio fischeri 15 min, luminescence

inhibition EC50

inhibition EC50

Invertebrate Daphnia magna 24 h, immobilization EC50 616.7 [79] Invertebrate Daphnia magna 48 h, immobilization EC50 149.3 [91] Invertebrate Daphnia magna 48 h, immobilization EC50 135.7 [92]

inhibition EC50

Duckweed Lemna gibba 7 days, wet weight EC50 1.277 [82] Duckweed Lemna gibba 7 days, frond number EC50 >1.000 [82] Duckweed Lemna gibba 7 days, chlorophyll a EC50 >1.000 [82] Duckweed Lemna gibba 7 days, chlorophyll b EC50 >1.000 [82] Duckweed Lemna gibba 7 days, carotenoids EC50 >1.000 [83] Invertebrate Daphnia magna 24 h, immobilization EC50 133 [89] Invertebrate Daphnia magna 24 h, immobilization EC50 506.3 [79] Invertebrate Daphnia magna 48 h, immobilization EC50 174.4 [91] Invertebrate Daphnia magna 48 h, immobilization EC50 105 [89] Invertebrate Daphnia magna 48 h, immobilization EC50 185.3 [92] Invertebrate Daphnia magna 48 h, immobilization EC50 215.9 [79] Invertebrate Daphnia magna 48 h, immobilization EC50 202 [88] Invertebrate Daphnia magna 96 h, immobilization EC50 158.8 [91] Invertebrate Daphnia magna 96 h, immobilization EC50 147.5 [92] Invertebrate Daphnia magna 21 days, survival NOEC 30 [79] Invertebrate Daphnia magna 21 days, reproduction NOEC 30 [79] Invertebrate Daphnia magna 21 days, reproduction NOEC 1.563 [88] Invertebrate Daphnia magna 21 days, growth NOEC 1.563 [88] Invertebrate Moina macrocopa 24 h, immobilization EC50 310.9 [79] Invertebrate Moina macrocopa 48 h, immobilization EC50 110.7 [79] Invertebrate Moina macrocopa 7 days, survival NOEC 30 [79] Invertebrate Moina macrocopa 7 days, reproduction NOEC 30 [79] Fish Oryzias latipes 48 h, survival LC50 >100 [91] Fish Oryzias latipes 96 h, survival LC50 >100 [91] Fish Oryzias latipes 48 h, survival LC50 >500 [79] Fish Oryzias latipes 96 h, survival LC50 >500 [79]

subcapitata 72 h, growth EC50 8.7 [81]

**Concentration (mg L-1)**

344.7 [91]

>1000 [91]

>1000 [91]

**References**

**Table 6.** Acute and chronic effects of antibiotics to aquatic organisms. Adapted from Ji et al. [79].

Genotoxic and enzymatic effects on aquatic organisms exposed to ATBs were also observed by several authors. For example, Botelho et al. (submitted manuscript) reported genotoxic effects of oxytetracycline and florfenicol ATBs in concentrations found in the water of a major Brazilian reservoir where fish farming activity is practiced with *Oreochromis niloticus* fish species. In this study, DNA damage was observed using the comet test when exposed to concentrations of 425 and 4000 ng L-1 of florfenicol and oxytetracycline, respectively.

Oliveira et al. [98] observed the inhibition of catalase activity in adult brain and gills of *Danio rerio* fish when exposed to higher concentrations of amoxicillin (50 and 100 mg L-1). There was also a tendency for the induction of glutathione S-transferase (GST) enzyme at all concentra‐ tions of the same ATB. In this same study, a dose-dependent catalase was observed in the brain of *D. rerio* adults after oxytetracycline exposure, while GST activity increased after exposure to concentrations higher than 1 mg L-1 of oxytetracycline in muscle and liver samples.

Most of the studies related to ATBs effects on aquatic organisms refer to acute effects (mainly lethality) in a short period of time. Note that in the aquatic environment, due to the phenom‐ enon of dilution, the concentrations of chemicals in general, including ATBs, are found at the levels of µg L-1 and ng-1. Thus, the observed effects will be chronic, i.e., at a considerably longer period than that observed for acute effects. Thus, in toxicity evaluations, especially to aquatic organisms, the use of environmentally relevant concentrations should be taken into account since this way the effects will be more realistic and will portray in a more real way what happens in the environment if such chemical agents are present.

Soil plays important roles in ecosystems since it is the basis of nutrients and the animal and plants habitat, in addition to functioning as an immense bioreactor, where the degradation of pollutants and nutrients transformation occurs. However, as already seen in this chapter, the soil may also be the final destination of ATBs used in veterinary medicine originating from manure and sewage mud used to fertilize vegetables [99] or from package disposal. Due to the ecological importance of soil for the ecosystem, it is important to know whether or not ATBs have negative effects on the fauna.

As shown earlier in this chapter, once in the soil, depending on the physical and chemical characteristics of the ATBs and the soil, they may follow different pathways, such as being leached or carried superficially by rain, contaminating aquatic environments (low *K*d values) or persisting in the soil (high *K*d values).

In general, the effects of ATBs to aquatic organisms are higher than those of soil fauna, and thus little is known about the toxicity of these drugs for these organisms. According to Ding and He [100], once in the soil, ATBs can change the structure of the microbial community because even to those which have a broad spectrum of action, selective effects on several microorganism groups may occur. As a result, the relative abundance of microorganisms is changed, interfering with the interactions between different species.

The sorption of pollutants in general in the soil is one of the major mechanisms controlling toxicity, by reducing its availability [101]. Thus, in toxicity studies with chemical agents, the choice of a molecule with low *K*d is recommended. In addition, toxicity to organisms in the soil decreases over time due to transformations the molecule undergoes over time through less toxic secondary compounds and due to tolerance of some soil microorganisms to ATBs [102], such as some *Pseudomonas* species, for example [103].

Girard et al. [104] studied the effects of ciprofloxacin ATB on soil microbial communities and observed a reduction in soil microbial activity in the first 25 days of experiments, when exposed to concentrations ranging from 0.2 to 20 mg kg-1. According to the authors, this behavior is due to this molecule being bacteriostatic. From this result, according to the authors, ciprofloxacin could interfere with the recycling of nutrients in the soil. In Table 7, some studies that were conducted with oxytetracycline ATB for three organisms that live in the soil can be observed.


**Table 7.** Chronic effect of oxytetracycline antibiotic on organisms that represent the soil fauna.

Generally, veterinary ATBs can suffer abiotic or biotic degradation on soil–water compart‐ ment. However, some degradation products have similar toxicity to the parent compound [106]. Degradation can be affected by environmental conditions, such as temperature, humid‐ ity, season, soil type, pH, and characteristics of the molecule, such as size, among others. With respect to the season, for example, in winter, the degradation half-life of ivermectin is six times higher than in summer, and degradation was faster in sandy soil than in sandy loam soil [107, 108].
