Impact of ampicillin and oxytetracycline on microbial respiratory activity in the Coreaú River, Brazil: evidence of hormesis and ecological resilience

Impacto da ampicilina e da oxitetraciclina na atividade respiratória microbiana no Rio Coreaú, Brasil: evidências de hormese e resiliência ecológica

Abstract

Antibiotic contamination in freshwater ecosystems is a growing concern. This study assessed the effects of ampicillin and oxytetracycline, individually and in combination, on microbial respiratory activity in water samples from the Coreaú River, Ceará, Brazil. Respirometric microcosms were incubated for 28 days under exposures of 5, 10, and 25 mg.L^-1. The lowest concentrations of antibiotics stimulated microbial respiration, indicating possible microbial adaptation and metabolism of the compounds. Conversely, the highest concentration of oxytetracycline suppressed microbial activity. In contrast, the same concentration of ampicillin enhanced it by 88%, suggesting the presence of resistant strains. Mixtures of the antibiotics generally produced antagonistic effects, often stimulating microbial activity beyond the levels observed in individual treatments. The A5 × O25 treatment (5 mg.L^-1 ampicillin + 25 mg.L^-1 oxytetracycline) yielded a 262% increase in CO² capture compared to the control, reflecting a strong hormetic response. These findings demonstrate that even sublethal concentrations of antibiotics can influence microbial dynamics and resilience. Although the concentrations tested were higher than those usually reported in surface waters, the results help understand potential microbial responses to antibiotic exposure and highlight the complexity of dose-response interactions in aquatic environments.

Keywords:
antibiotics; aquatic ecosystems; environmental impact; resistance.

Resumo

A contaminação por antibióticos em ecossistemas de água doce é uma preocupação crescente. Este estudo avaliou os efeitos da ampicilina e da oxitetraciclina, individualmente e em combinação, na atividade respiratória microbiana em amostras de água do Rio Coreaú, Ceará, Brasil. Microcosmos respirométricos foram incubados por 28 dias sob exposições de 5, 10 e 25 mg.L^-1. Baixas concentrações de antibióticos estimularam a respiração microbiana, indicando uma possível adaptação microbiana e metabolização dos compostos. Em contrapartida, a concentração mais alta de oxitetraciclina suprimiu a atividade microbiana. Em contraste, a mesma concentração de ampicilina aumentou em 88%, sugerindo a presença de cepas resistentes. Misturas dos antibióticos produziram majoritariamente efeitos antagônicos, com frequência estimulando a atividade microbiana além dos níveis observados nos tratamentos individuais. O tratamento A5 × O25 (5 mg.L^-1 de ampicilina + 25 mg.L^-1 de oxitetraciclina) resultou em um aumento de 262% na captura de CO² em comparação com o controle, refletindo uma forte resposta hormética. Esses achados demonstram que mesmo concentrações subletais de antibióticos podem influenciar a dinâmica e a resiliência microbiana. Embora as concentrações testadas tenham sido mais altas do que as normalmente reportadas em águas superficiais, os resultados contribuem para a compreensão das potenciais respostas microbianas à exposição a antibióticos e destacam a complexidade das interações dose-resposta em ambientes aquáticos.

Palavras-chave:
antibióticos; ecossistemas aquáticos; impacto ambiental; resistência.

INTRODUCTION

Human activities such as urban expansion, mining, pesticide use, and effluent discharge significantly alter the biological communities and functioning of limnic ecosystems, which act as ecotones and therefore, concentrate high biodiversity (GRENNI et al., 2018; NA et al., 2018; Hassaan; El Nemr, 2020; ADLA et al., 2022). Lotic environments are susceptible to both direct and indirect exposure to a range of harmful chemicals that are used in human activities. These compounds, which exhibit a wide range of chemical structures, endanger the environment, aquatic organisms, and humans. Scientific studies have confirmed that conventional biological treatment systems, such as activated sludge and trickling filters, are often ineffective at fully removing persistent micropollutants like antibiotics (HAMZA et al., 2016; BELETE et al., 2023; LOGANATHAN et al., 2023). Such inefficiency highlights a broader environmental concern regarding emerging pollutants, which include pharmaceuticals, personal care products, surfactants, and disinfectants, with antibiotics, psychiatric drugs, and anti-inflammatory agents posing the greatest environmental risks (VERLICCHI et al., 2012; KUMAR et al., 2023; WANG et al., 2024).

Antibiotics, widely used in human and veterinary medicine, frequently enter aquatic ecosystems through excretion, hospital and agricultural effluents, and incomplete removal by wastewater treatment plants. Even at low concentrations, they can persist and interfere with microbial community dynamics, altering nutrient cycles and trophic structures (YANG et al., 2021). Moreover, their continuous release exerts selective pressure that favors the proliferation of antibiotic-resistant bacteria and the dissemination of resistance genes, posing long-term risks to aquatic biodiversity and public health (Kümmerer, 2009; ZHAO et al., 2015; CHIESA et al., 2018; KRAUPNER et al., 2021; MELLO et al., 2022).

In aquatic environments, antibiotic molecules can be transformed by a variety of abiotic factors, such as pH, temperature, oxidation, and photooxidation, among others (Gothwal; Shashidhar, 2015), as well as microbial metabolism (Gothwal; Shashidhar, 2015; BENGTSSON-PALME et al., 2018). Molecules can be degraded completely or converted into bioactive compounds with varying toxicity levels. They can also enter the trophic chain, affecting aquatic and terrestrial organisms (WANG et al., 2009; Huang; Yang et al., 2024; LI et al., 2024). The presence of antibiotics in natural environments drives the selection of antibiotic-resistant bacterial strains, a major concern for the scientific community (BARTHOLIN et al., 2023). Antibiotic-resistant bacteria pose a significant public health challenge, increasing the prevalence of infections in humans and animals that are difficult to treat. Global antibiotic consumption increased by 16.3% between 2016 and 2023, with estimates suggesting a further 52% growth between 2023 and 2030 (KLEIN et al., 2024).

Considering the use of these pharmaceutical agents in animals, the World Organization for Animal Health (WOAH) has documented the prevalence of the administration of tetracyclines, penicillins, and macrolides in terrestrial animals, as well as amphenicols, tetracyclines, and fluoroquinolones in food-producing aquatic animals (WOAH, 2021). The World Health Organization (2018) identifies β-lactams, macrolides, tetracyclines, quinolones, and sulfonamides as the most commonly used antibiotics in Brazil. The introduction of antibiotics into aquatic environments reduces species diversity and microbial species abundance, causing a loss of approximately 75% of microbial populations (KERGOAT et al., 2021). The findings of Robson et al. (2020) indicated a decline in both gross primary production and community respiration, which was attributed to the absence of a protective extracellular polysaccharide layer resulting from the combined use of fluoxetine, diphenhydramine, and ciprofloxacin. Modifications in the structure of microbial communities give rise to changes in nutrient cycling within aquatic ecosystems (YERGEAU et al., 2012; MARTIN-LAURENT et al., 2019; CHEN et al., 2021).

Ampicillin is a broad-spectrum, β-lactam antibiotic widely used in human medicine that inhibits bacterial cell wall synthesis. It is moderately soluble in water (3.4 mg.mL^-1 at 25°C), has a logK_ow of -1.35, and a reported half-life in surface water of approximately 27 days at neutral pH and 25°C (MITCHELL et al., 2014). Oxytetracycline, in contrast, is a tetracycline-class antibiotic frequently employed in veterinary medicine. It inhibits protein synthesis by binding to the 30S ribosomal subunit. It is moderately soluble in water (100 mg.mL^-1 at 25°C), shows a higher affinity for organic matter (Kd values ranging 200–800 L.kg^-1 depending on sediment type), and its half-life in water can vary from 2 to 7 days depending on light and pH conditions (LI et al., 2019; ZHONG et al., 2022).

The effects of varying antibiotic doses on microbial communities in aquatic environments remain insufficiently explored, particularly considering the concomitant presence of multiple antibiotic concentrations in these ecosystems. Some studies have indicated that antibiotics can reduce microbial respiratory activity (Schallenberg; Armstrong, 2004; KATIPOGLU-YAZAN et al., 2013; SHIBATA et al., 2014; GRENNI et al., 2018), while others have demonstrated that antibiotics can stimulate microbial respiration in natural environments (WILSON et al., 2004; PARENTE et al., 2018; Gonia; Talarczyk, 2020). Therefore, microbial community structure in aquatic environments is directly altered by the mixture and concentration of these antimicrobials, even at sublethal concentrations (TIAN et al., 2023). Microbial cell respiration, measured by CO₂ release, serves as an indicator of heterotrophic catabolism of organic matter in natural environments (Surger; Blank, 2022). Thus, exposing microbial communities to lethal and sublethal antibiotic concentrations helps assess their resistance to the compound across different concentrations.

The Coreaú River, located in the state of Ceará, has its origin in the confluence of the Jatobá and Caiçara streams in the lower part of the Ibiapaba Mountain Range. The river flows a total distance of 167.5 km to its confluence with the Atlantic Ocean. It is situated within the Coreaú Hydrographic Basin, which encompasses 24 municipalities and has a total drainage area of 10,633.66 km², representing 7% of the state of Ceará’s total territory (Ceará, 2024). Although the Coreaú River has significant importance to the regional economy, there is a lack of scientific studies evaluating anthropogenic impacts along its course. To address this gap, the present study tested the hypothesis that different concentrations of ampicillin and/or oxytetracycline alter the respiratory activity of the river’s microbial community. This is the first investigation to assess the influence of antibiotics on the microbiota of the Coreaú River, providing novel insights into microbial responses to pharmaceutical contaminants in a tropical semiarid ecosystem.

MATERIALS AND METHODS

Study area and sampling

Sampling was carried out a few kilometers upstream from the estuary of the Coreaú River at the Lima Brandão Dam (Figure 1) (3°07’18.1”S, 40°49’17.9”W) in the municipality of Granja/CE, where tidal level variations do not influence the river yet. The water sample was immediately cooled to 4°C until the respirometric experiments were set up on the same day.

Figure 1
Geographical localization of (a) Ceará, Brazil; (b) municipality of Granja; and (c) geographical location of the sampling site.

Sample characterization and preparation of respirometric microcosms

pH, conductivity, and turbidity of the Coreaú River water sample were analyzed using a pH Meter LUCA-210 (Lucadema), a Microprocessor-Based Conductivity Meter AT-255 (Alfakit), and a 2020we Portable Turbidity Meter (LaMotte).

The active substances tested in the experiments were ampicillin (Prati Donaduzzi, Toledo, PR, Brazil) and oxytetracycline (Terramycin^® L/A, Zoetis, Campinas, SP, Brazil). Stock solutions were prepared in sterile distilled water at 2,000 mg L^-1, and no additional solvent was required. Therefore, no solvent control was necessary. Each microcosm contained 100 mL of river water (filtered at 8 μm) in a 500 mL glass flask (hermetically sealed). To capture CO₂, 10 mL of 1 M NaOH were placed in suspended cups. As NaOH was physically separated from the water column, no interference with microbial activity was expected.

Preliminary assays were conducted with concentrations of 1 to 50 mg.L^-1. At 1 mg.L^-1, no detectable variation in microbial respiration was observed, while at 50 mg.L^-1, microbial activity was completely inhibited within one week. Based on these results, concentrations of 5, 10, and 25 mg.L^-1 were selected as a range that produced measurable responses without immediate total inhibition. Although these levels are above typical environmental concentrations (ng.L^-1–μg.L^-1), they were chosen to allow the detection of non-linear dose–response effects in controlled microcosms.

The treatments were conducted with three concentrations (5.0, 10.0, and 25.0 mg L^-1) of ampicillin (A5, A10, and A25) or oxytetracycline (O5, O10, and O25). Both antibiotics were also combined at all concentrations tested, resulting in nine mixture treatments (e.g., A5 × O5, A5 × O10, A25 × O25). Two types of controls were included: an experimental control (sealed flask without water) and an environmental control (river water without antibiotics). All treatments were performed in triplicate.

Incubation and evaluation of microbial respiration

The flasks were incubated in the dark for 28 days at 25°C in B.O.D. At seven-day intervals, the flasks were opened to obtain the residual NaOH solution and subsequent titration using HCl. This was done to determine the quantity of CO₂ released by each microcosm and captured by the NaOH solution. The contents present in the cups were transferred to Erlenmeyer flasks, where 2 mL of 10% m/v BaCl₂ were added, and titration was performed using a standardized solution of 0.5 mol/L HCl. The equation below was used to calculate the degree of microbial activity (Equation 1):

(1) $${\text{mg CO}}_{\text{2}}=\left({\text{V}}_{\text{B}}-{\text{ V}}_{\text{A}}\right)*{\text{ M}}_{\text{HCl}}*\text{ 22}$$

Where:

VB: the volume of HCl used to titrate the environmental control (mL);

VA: the volume of HCl used to titrate each vial (mL);

M_HCl the molarity of the HCl used in the titration (mol.L^-1);

22: the equivalent weight of CO₂.

Following each titration event, a fresh 0.5 M NaOH solution was introduced to the plastic cups to maintain the capture of CO₂ produced by microbial activity and facilitate the subsequent synthesis of Na₂CO₃ (SILVA et al., 2007).

Evaluation of antibiotic interaction using the Bliss Independence Model

To quantitatively determine the interaction between ampicillin and oxytetracycline in the microcosms, the Bliss Independence Model was employed. This model assumes that the combined effect of two agents acting independently can be predicted from their individual effects (Duarte; Vale, 2022). The expected fractional effect of the mixture (E_exp) was calculated as Equation 2:

(2) $$\text{E}\_\text{exp }=\text{ E}\_\text{A }+\text{ E}\_\text{O }-\left(\text{E}\_\text{A }\times \text{ E}\_\text{O}\right)$$

Where:

E_A and E_O: the normalized effects of ampicillin and oxytetracycline, respectively, expressed as the relative variation in microbial respiration compared to the control. The observed combined effect (E_obs) was determined from the mean CO₂ capture measured in each antibiotic combination. The deviation between E_obs and E_exp was expressed as the percentage difference [(E_obs − E_exp) × 100 / E_exp], which was used to classify the type of interaction: differences within ± 5% were considered additive, values above this range indicated synergistic, and below -5% indicated antagonistic effects. This analysis provided a quantitative assessment of the interactive dynamics between antibiotics, complementing the qualitative interpretation of microbial responses in the respirometric assays.

Statistical analysis

The assumptions of ANOVA were verified by testing data normality (Shapiro–Wilk) and homogeneity of variances (Levene’s test, p > 0.05) and a one-way analysis of variance (ANOVA) was performed. A Tukey test was conducted to determine statistically significant differences between the treatments, with a 5% significance level. All statistical analyses were conducted using the SigmaPlot v12.0 software package.

RESULTS AND DISCUSSION

Despite its distance from major Brazilian urban centers, the Coreaú River Basin experiences anthropogenic impacts, including agricultural pesticide use and sewage discharge from some municipalities upstream of the water collection site used in this study (MIOLA et al., 2016; AQUINO et al., 2021; DE OLIVEIRA et al., 2021; OLIVEIRA et al., 2021). Given the influence of physicochemical parameters on the action of antibiotics (JAFARI OZUMCHELOUEI et al., 2020; MARRONE et al., 2024), pH, conductivity, and turbidity were evaluated, and the data are shown in Table 1. Of the three parameters evaluated, pH, appears to exert the greatest influence on the action of antibiotics. This is because it can influence the solubility, adsorption, light sensitivity, and other characteristics (JAFARI OZUMCHELOUEI et al., 2020) of antibiotic molecules, especially at values further from neutrality.

Table 1
Analysis of physicochemical parameters for Rio Coreaú.

Physicochemical parameter	Coreaú River
pH	7.51 ± 0.01
Conductivity	643.2 ± 0.1 μS/cm
Turbidity	3.65 ± 0.04 NTU

• Source: Elaborated by the authors.

Ampicillin was employed at concentrations of 5, 10, and 25 mg L^-1. It is important to note that the concentrations tested here (5–25 mg.L^-1) exceed environmentally relevant levels. They were selected after preliminary trials showed no measurable microbial response at 1 mg.L^-1 and complete inhibition at 50 mg.L^-1. Thus, the chosen range provided intermediate conditions under which microbial adaptation, stimulation, or inhibition could be detected. Although these values are higher than those typically found in the environment, determining the thresholds at which microbial inhibition occurs remains essential for understanding dose–response relationships, resistance mechanisms, and potential risks under accidental or extreme contamination events.

During the initial three-week period, no statistically significant differences were observed in the quantity of CO₂ captured between the various treatments, indicating that there was no variation in microbial activity between them (Figure 2). This lack of response may be related to the resilience of the microbial community during the first weeks of exposure, as well as to the availability of organic matter in the microcosms, which could have masked the immediate effects of the antibiotics. In addition, the stability and gradual degradation of the molecules in the aquatic environment may have delayed their impact on microbial respiration. After 28 days of incubation, a significant variation was observed. Based on the values found, we concluded that the lowest concentration of ampicillin used resulted in greater microbial activity, while the concentrations of 10 and 25 mg.L^-1 resulted in intermediate microbial activity values, in comparison to the control (Figure 2). Inhibition is observed during the initial stages due to the action of the antibiotic. However, after this, the microbial community may have used these compounds as a carbon source, potentially undergoing respiration and CO₂ release (GUAN et al., 2017).

Figure 2
Microbial activity in the water of the Coreaú River over 28 days of incubation determined by the release and capture of CO₂ in the microcosms. (a) Representation in columns with statistical evaluation by Tukey at 5% probability. Treatments followed by the same letter do not have statistically significant differences. Times in which letters were not inserted did not have significant differences. The bars correspond to the standard deviation.

Falkowski et al. (2008) and Ward et al. (2017) report that in natural environments like rivers, bacteria experience variations in environmental factors such as nutrients, oxygen, temperature, and interspecies competition. At concentrations below the lethal threshold, ampicillin has been observed to stimulate microbial metabolic activity, thereby driving competition and the search for nutrients.

The tests conducted with oxytetracycline revealed limited variation in microbial activity. During the first two weeks, no significant differences were observed, but in the third and fourth weeks, the highest concentration (25 mg.L^-1) reduced activity compared to the control. The stress-induced acceleration of microbial metabolism, a complex response to factors like chemical pollutants or nutrient scarcity, likely generated the variations observed over the first three weeks (ZHANG et al., 2021). This phenomenon can induce adaptations that alter catabolic pathways to enhance metabolic activity and biodegradation as a survival strategy, leading to a period of dynamic physiological adjustment within the community (AMARNATH et al., 2023).

As was observed with ampicillin, the lowest concentration of the antibiotic (5 mg.L^-1) demonstrated elevated microbial activity compared to the other concentrations (Figure 3), including the control. During the third and fourth weeks of incubation, there was a marked increase in microbial respiratory activity (Figure 3), which may be attributed to the adaptation of the microbial community to the low concentration of the antibiotic. Low-dose exposure may stimulate biological adaptation processes, leading to selection of resistant bacteria at low doses and negative reactions at higher doses (Calabrese; Mattson, 2017). In recent years, the discussion surrounding dose-response effects, commonly known as hormesis, has proliferated (Agathokleous; Calabrese, 2019; IAVICOLI et al., 2021; LI et al., 2022).

Figure 3
Microbial activity in the water of the Coreaú River over 28 days of incubation determined by the release and capture of CO₂ in the microcosms. (a) Representation in columns with statistical evaluation by Tukey at 5% probability. Treatments followed by the same letter do not have statistically significant differences. Times in which letters were not inserted did not have significant differences. The bars correspond to the standard deviation.

The term ‘hormesis’ is used to describe the capacity of living systems to adapt in response to environmental stressors, thereby stimulating them and enabling the generation of tolerance in the face of a challenge. This process is thought to enhance resilience (Calabrese; Agathokleous, 2020; CALABRESE et al., 2023). In microbiology, the hormetic response has been observed to enhance the resilience of bacterial communities in environmental settings when they are exposed to sublethal doses of antimicrobial compounds, including antibiotics (CALABRESE et al., 2010). The indiscriminate use of drugs and other chemicals that promote the selection of resistant bacteria has been shown to generate hormetic responses, representing a significant risk to human health and the environment (SHEN et al., 2021; SUN et al., 2023).

In comparison, the same concentrations of ampicillin or oxytetracycline when introduced to the water of the Coreaú River yield statistically different microbial activities across all treatments. Ampicillin consistently enhances microbial activity in the microcosms more significantly than oxytetracycline treatments, with the effect being particularly pronounced at a concentration of 25 mg L^-1. Mitchell et al. (2014) demonstrated that the hydrolysis half-life in water at pH 7 and 25°C was 27 days for ampicillin, while Zhong et al. (2022) showed that the half-life was 66 h for oxytetracycline at the same conditions. Ampicillin and other β-lactam antibiotics are commonly used in hospital settings and by the general population to treat recurrent infections (Huang; Qin et al., 2024). In contrast, oxytetracycline is more commonly used in veterinary medicine to treat animal infections (LI et al., 2019). The region is devoid of large herds or large animal breeders. Given that the majority of antibiotics reach aquatic environments through domestic sewage (MELLO et al., 2022) and that there are cities upstream of the Coreaú River collection point, it is plausible that the region is subject to a greater selective pressure for bacteria resistant to ampicillin than to oxytetracycline. This inferred pressure is consistent with the microbial community’s response observed in Figure 4.

Figure 4
Microbial activity in the water of the Coreaú River after 28 days of incubation determined by the release and capture of CO₂ in the microcosms with ampicillin or oxytetracycline. The three antibiotic concentrations tested resulted in significant variation between treatments with ampicillin and oxytetracycline by Tukey at 5% probability.

To evaluate the combined action of ampicillin and oxytetracycline, mixtures of concentrations of 5, 10, and 25 mg.L^-1 were prepared for both drugs, resulting in nine antibiotic mixtures. Aquatic environments often exhibit the simultaneous presence of multiple classes of antibiotics (Enick; Moore, 2007; Huang et al., 2023). Consequently, local organisms are exposed to these drugs, even at very low concentrations, where synergistic or antagonistic interactions occur between antibiotic molecules (FELIS et al., 2020).

The interactions between ampicillin and oxytetracycline in the mixed treatments were quantitatively assessed using the Bliss Independence Model. Most combinations exhibited antagonistic effects (Table 2), indicating that the concurrent presence of both antibiotics reduced their individual inhibitory impacts on microbial respiration. In such cases, respiration rates were observed to be higher than expected from independent action, suggesting that the antibiotics may have interfered with each other’s effects on the microbial community.

Table 2
Interaction analysis between ampicillin (A) and oxytetracycline (O) on microbial respiration based on the Bliss Independence Model.

Combination	E_A %	E_O %	E_exp %	CO2 expected (mg)	CO2 observed (mg)	E_obs %	Dif (E_obs − E_exp) %	Interpretation
A5 × O5	-182.83	-79.81	-408.55	69.010	27.13	-99.93	308.62	Synergism
A5 × O10	-182.83	32.42	-91.12	25.37	43.99	-224.18	-133.06	Antagonism
A5 × O25	-182.83	59.47	-221.46	43.55	49.13	-262.15	-40.69	Antagonism
A10 × O5	-90.05	-79.81	-95.08	26.46	31.78	-134.43	-39.35	Antagonism
A10 × O10	-90.05	32.42	-7.28	14.57	14.29	-5.31	1.97	Additive
A10 × O25	-90.05	59.47	-40.91	19.07	34.83	-156.76	-115.85	Antagonism
A25 × O5	-88.21	-79.81	-4.77	14.24	24.08	-77.52	-72.75	Antagonism
A25 × O10	-88.21	32.42	-27.18	17.26	31.17	-129.70	-102.52	Antagonism
A25 × O25	-88.21	59.47	23.72	10.35	10.63	21.67	-2.05	Additive

• E_A and E_O: the normalized effects of each antibiotic relative to the control; E_exp: the expected combined effect; E_obs: the observed effect in the combined treatments.

Only one combination (A5 × O5) displayed a synergistic interaction, while two combinations (A10 × O10 and A25 × O25) showed additive behavior, with observed respiration values closely matching the expected ones. Overall, microbial respiration in the mixed treatments remained generally higher than or similar to that in the corresponding single-antibiotic treatments, and only the combinations classified as additive exhibited respiration levels comparable to the control (Figure 5). The outcomes of this investigation suggest that the interaction between ampicillin and oxytetracycline was predominantly antagonistic, with limited occurrences of additive and synergistic responses. The present findings are consistent with the observations of Marx et al. (2015), who emphasized that combining a bactericidal agent with a bacteriostatic one (e.g., penicillins with tetracyclines) is not recommended in human therapies, as such associations are expected to produce antagonistic effects.

Figure 5
Microbial activity in the water of the Coreaú River after 28 days of incubation determined by the release and capture of CO₂ in the microcosms with mixtures of ampicillin and oxytetracycline concentrations. Treatments followed by the same letter do not have statistically significant differences. The bars correspond to the standard deviation.

The increased CO₂ release observed in some mixtures may reflect microbial adaptation or co-metabolic processes. However, since antibiotic degradation was not directly quantified, these interpretations should be considered preliminary. This is evident from the significantly higher CO₂ capture values observed in the combined treatments compared to the highest values recorded in treatments with isolated antibiotics and the control (Table 3). The A5 x O25 treatment showed a 262.07% increase compared to the control, while the highest value in treatments with isolated antibiotics was 182.81% higher than the control. The magnitude of microbial activity reached 49.13 mg CO₂ in the A5 x O25 treatment, in stark contrast to the 5.5 mg CO₂ observed in the O25 treatment. The metabolism of antibiotics can occur in the environment in the form of co-metabolism, whereby labile substrates present in the environment are utilized as a carbon and energy source (ZHAO et al., 2024). As Fischer and Majewsky (2014) have observed, organic nutrients can promote microbial growth and development and induce the activity of non-specific enzymes that assist in antibiotic co-metabolism. Despite the antagonistic effects observed between antibiotic molecules, no discernible pattern of average stimulation of microbial activity emerges according to the mixtures of antibiotic concentrations. This can be explained by the differing adaptation mechanisms of the microbiome to each treatment, which gives rise to varying hormetic responses and, consequently, different levels of respiration by the microbial community. In recent literature, studies have started to investigate the hormetic effects of antibiotic mixtures in natural environments (IAVICOLI et al., 2021). This is because the antibiotic mixtures, even at low doses, have been shown to result in the selection of multidrug-resistant microorganisms (Sanz-García et al., 2022).

Table 3
Mean values and variation of microbial activity between control and antibiotic treatments after 28 days of incubation in the water of the Coreaú River.

Treatment	Microbial activity (mg CO2 captured)	Statistical analysis*	Variation in relation to the Control (%)
Control	13.57	Hi	---
A5	38.38	abc	182.81
A10	25.79	defgh	90.04
A25	25.54	defgh	88.24
O5	24.4	defghi	80.13
O10	9.17	j	-32.44
O25	5.5	j	-59.47
A5 x O5	27.13	cdefg	99.95
A5 x O10	43.99	ab	224.24
A5 x O25	49.13	a	262.07
A10 x O5	31.78	cde	134.18
A10 x O10	14.29	hij	5.38
A10 x O25	34.83	bcd	156.69
A25 x O5	24.08	defghi	77.43
A25 x O10	31.17	cdef	129.67
A25 x O25	10.63	j	-21.64

• *Averages followed by the same letter comparing each treatment do not differ by the Tukey test at 5% probability.

Limitations and future perspectives

This study was limited by the controlled microcosm conditions, which do not fully reproduce the complexity of natural freshwater systems. The use of commercial antibiotic formulations instead of analytical-grade standards may have introduced excipients that affected solubility and bioavailability, possibly explaining why measurable microbial responses occurred only at higher concentrations. In addition, degradation products were not quantified, and the Bliss Independence Model was applied solely to respiration data. Future investigations should combine this quantitative framework with molecular tools, such as metagenomics, to elucidate the community-level mechanisms driving antagonistic or synergistic interactions and to better predict ecosystem resilience under realistic contamination scenarios.

CONCLUSION

This study used ampicillin and oxytetracycline at concentrations of 5, 10, and 25 mg L^-1, individually and in mixtures, in microcosms containing Coreaú River water. Following a 28-day incubation period, the lowest concentrations of the antibiotics used alone resulted in the highest values of microbial respiratory activity. At the highest concentration tested (25 mg L^-1), oxytetracycline significantly reduced microbial activity, while ampicillin increased it by 88%, indicating resistance to β-lactams among the local microbiota. The antagonistic effect, which lacked a discernible response pattern according to the combinations of antibiotic concentrations in the mixtures, suggests that the microbial community may adapt through shifts in community composition, selection of resistant or tolerant taxa, and activation of stress-response or detoxification mechanisms. Notably, the tolerance generated by hormesis enhances the resilience of the microbial community, thereby facilitating the selection of multidrug-resistant microorganisms. The hormetic effect is evident when comparing the inhibition observed in the O25 treatment and the stimulation of microbial activity in the A5 x O25 treatment, which exhibited an 8.9-fold variation in CO₂ capture. The study offered a novel insight into the impact of two broad-spectrum antibiotics on the microbiology of a natural setting, with no influence from large urban centers. It emphasized the importance of the hormetic effect in risk assessment for human health and the environment.

ACKNOWLEDGMENTS

We thank Cassiano Ricardo de Souza for providing Figure 1.

DATA AVAILABILITY STATEMENT

Data will be made available on request.

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Edited by

• Editor:
  Davi Gasparini Fernandes Cunha, Universidade de São Paulo (USP), São Carlos, São Paulo/SP, Brasil. http://orcid.org/0000-0003-1876-3623

Publication Dates

• Publication in this collection
  01 May 2026
• Date of issue
  2026

History

• Received
  13 May 2025
• Accepted
  24 Nov 2025