Individual and combined effects of ionophore antibiotics monensin and lasalocid on growth, oxidative stress and antioxidant responses of Microcystis aeruginosa Open Access

During the digestive process of ruminants, methane (CH₄) gas is generated as a by-product of the metabolism of anaerobic microbes (Tseten et al. 2022). Globally, CH₄ emissions from enteric fermentation have accounted for 43% of the total greenhouse gas emissions from livestock (Herrero et al. 2016). To enhance production efficiency and minimize CH₄ emissions from livestock, scientists have started to investigate the functionality of various forms of feed additives (Sun et al. 2021). Methane inhibitors are included in ruminant feed to alter energy loss pathways, with the goal of minimizing methane emissions arising from enteric fermentation (Palangi & Lackner 2022).

Ionophore antibiotics represent a category of feed additives that alter the rumen fermentation pattern of cattle and this is widely used in cattle diets to increase feed efficiency, body weight gain, and reduce environmental emissions (Hersom & Thrift 2018). Ionophores prevent methanogenesis by reducing hydrogen and formate availability, which are key substrates for methanogenesis (Azzaz et al. 2015). Monensin supplementation in cattle feed can reduce methane emissions by 27–30% over 2–4 weeks, depending on feed energy content (Guan et al. 2006).

Ionophores could be defined as a group of natural and synthetic compounds forming lipid soluble cation complexes, by allowing cations to transport across low polar barriers such as organic solvents and lipids (Pressman 1976). These compounds are lipid soluble and have a reversible relationship with cations and this enables a mechanism to transport ions across biological membranes (Meliton 2012). Since the outer surfaces of the ionophores are hydrophobic, they are able to form complexes with hydrophobic membranes, allowing them to pass across (Novilla et al. 2017). Monensin, lasalocid, salinomycin, laidlomycin, and narasin are commercially available ionophores with similar modes of action, enabling dose-reliant animal performances (Bretschneider et al. 2008; Duffield et al. 2012). In gram-negative bacteria, the outer membrane is not permeable to larger hydrophobic molecules. Consequently, it exhibits a reduced response to ionophores, whereas gram-positive bacteria and animal cells tend to be more sensitive (Decad & Nikaido 1976). Among the rumen bacteria, in many cases, gram-positive bacteria are more sensitive to ionophores than gram-negative bacteria but this resistance is not always clear-cut (Houlihan & Russell 2003).

Nearly 50–60% of monensin will be excreted as parent form by animals and it has a relatively short half-life when it is mixed into the soils (Hillis et al. 2007). According to Arikan et al. (2018), monensin has been detected in environment manure, soil, surface water, groundwater, and sediments as 0.3–4.5 mg/L, 0.0004 μg/kg, 0.01–0.05 μg/L, 0.04–0.39 μg/L, and 1.5–31.5 μg/kg, respectively. The EC₅₀ (half maximal effective concentration) of monensin is highly species-dependent and it is ranging from 0.2 to 7.3 mg/L (Hansen et al. 2009). In 75% of surface water samples taken from streams in El Pantanoso, Argentina, monensin was detected at concentrations of 0.4 and 4.7 μg/L as median and maximum values, respectively (Pérez et al. 2021). The recent study by Sandoz et al. (2018) reported a maximum monensin concentration of 84.37 μg/L in water samples taken near a beef cattle feedyard system. Furthermore, monensin detection levels at a beef lagoon are reported as 40 μg/L (Sassman & Lee 2007). In a dairy wastewater system, the theoretically estimated maximum concentration of monensin in a nearby lagoon is recorded as 246 μg/L, assuming no degradation occurs (Watanabe et al. 2008).

The effects of monensin on freshwater ecosystems are largely unknown. The experimental results on monensin treatment in algae revealed both negative and positive effects on algae growth, with some algae showing no response to the antibiotics (Winkworth et al. 2015). However, negative effects of monensin on the zooplankton communities and species richness are reported (Hillis et al. 2007). Antibiotic toxicity in freshwater does not consistently inhibit the growth of green algae and cyanobacteria (Sharma et al. 2021). In a range-finding test, Anabaena flos-aquae exposed to monensin sodium for 72 h showed a 72-h EC₅₀ of 6.8 mg/L and a 72-h no observed effect concentration of 0.2 mg/L (Rychen et al. 2017).

Harmful algal blooms (HABs) pose a significant environmental concern and are prevalent across freshwater, brackish water, and marine ecosystems (Hallegraeff 1993). With the environmental degradations that have occurred over the past centuries, there is a clear increase in the severity and global distribution of HABs (Kling et al. 2011; Paerl & Paul 2012). HABs are influencing ecosystem function and structure, resulting in both present and future impacts. The formation of toxins, carcinogens, irritants, taste and odor compounds, and poor aesthetics has brought several social and ecological issues (Watson & Molot 2013). Among many Cyanobacterial species, Microcystis aeruginosa is the most common and extensively dispersed species found in tropical to the sub-frigid zone. The blooms of M. aeruginosa have created numerous environmental issues such as forming bad odors and toxic compounds (Harke et al. 2016).

The primary objective of administrating ionophore antibiotics in ruminant feeds is to improve digestibility, thereby reducing environmental emissions. However, excessive and frequent antibiotic use in livestock feed can lead to their accumulation in nearby aquatic environments. Consequently, this study aimed to identify one of the secondary environmental impacts associated with the use of monensin, lasalocid, and their combination. In this study, a series of laboratory experiments were conducted with predetermined concentrations of monensin and lasalocid, as well as their 1:1 (w/w) mixture, to assess the potential effects of these antibiotics on the growth, photosynthetic pigment production, oxidative stress, and antioxidant enzyme activities of M. aeruginosa. The primary expected outcome of this study was to evaluate the potential risks posed by monensin and lasalocid as environmental contaminants with regard to the proliferation of M. aeruginosa blooms in aquatic environments near cattle farming areas.

M. aeruginosa (strain NIES – 111) samples were ordered from the National Institute of Environmental Studies (NIES, Tsukuba, Japan) and cultured in BG – 11 media. The cultures were incubated in an incubator (MIR554PJ, Panasonic, Osaka, Japan) at a temperature of 20 ± 1 °C, following a 12-h light:12-h dark cycle controlled by an electric timer, with light intensity set at PAR 30 ± 4 μmol/m² s (Supplementary data 01). The samples were shaken two times per day manually at specific intervals. Monensin sodium salt Thin Layer Chromatography ((TLC) 90–95%) and lasalocid A sodium salt (analytical standard) were obtained from Sigma Aldrich (Tokyo, Japan). Monensin and lasalocid antibiotics were dissolved in 70% ethanol (2 and 1 mg from each monensin and lasalocid dissolved in 1,000 and 500 μL of 70% ethanol, respectively). The mixtures were left undisturbed on a glass Petry dish for approximately 2–3 h to allow for the extensive evaporation of ethanol, to avoid potential collateral effects on the treatments. The dissolved antibiotics were diluted into a 40 mg/L stock solution by diluting with prepared BG – 11 media.

Experiments were planned in three steps after conducting a few early trials; Test I – Monensin (T_M) test, Test II – lasalocid (T_L) test, and Test III – T_M:T_L (w/w) – 1:1 test. Based on the previous literature and after a few early trials, for lower concentrations (approximately ≤ 500 μg/L), antibiotic concentrations on treatments were decided as L₁ to L₂ range; higher concentrations possible in the environment, M, H₁ and H₂ range (500–2,000 μg/L) concentrations determined to test the further impact. Five treatments excluding the control were arranged with three replicates. The same concentrations were arranged for T_M test, T_L test, and T_M:T_L – 1:1 (w/w) test. In the 1:1 test, the concentrations were arranged 50% (w/w) from each antibiotic to create the predetermined concentration (Table 1).

The initial OD₇₃₀ (absorption at 730 nm in spectrophotometer) of cell cultures for each treatment was adjusted to 0.5119, 0.5322, and 0.5458 for T_M, T_L, and T_M:T_L (w/w) 1:1, respectively, by mixing the main cultures with BG – 11 media. Antibiotic treatments were introduced on the same day for each treatment, emulating the natural scenario where antibiotics are commonly added to ponds along with the nutrition source.

All experiments were maintained in the same incubator at 20 ± 1°C temperature and lighting (12 h:12 h light-dark cycle) 30 ± 4 μmol/m² s for 7 days and samples were shaken two times per day manually with constant intervals. Optical density (OD₇₃₀) of each replicate was measured daily and on day 7 cells samples were collected into 1 mL centrifuge tubes and pelleted through centrifuging 12,000 G at 4 °C for 15 min (Tomy MX – 105, Digital Biology, Tokyo, Japan) and kept preserved at −80 °C freezer. Three samples from each replicate of every treatment were collected to assess the respective parameters. The same procedure was repeated for all three tests (Test I, Test II, and Test III) separately by providing equivalent conditions.

The optical density of all replicates for each treatment was recorded daily for 7 days using the spectrophotometer (Shimadzu UV mini – 1280, Tokyo, Japan) at a wavelength of 730 nm. Three measurements from each replicate were measured and recorded. The flasks were properly shaken before taking the samples, to disperse cyanobacteria cells equally throughout the media (Oginni et al. 2021).

The protein content was measured by following Bradford assay methods (Bradford 1976) with minor alterations. Cell samples, from which chlorophyll was extracted, were washed with 750 μL of 95% ethanol. This step aimed to mitigate chlorophyll interferences and prevent potential distortions in colorimetric measurements. Later, samples were kept 24 h in 25 °C in an oven to evaporate ethanol. In measuring cellular protein, each sample was kept in a water bath for 10 min at 70 °C after the addition of 0.5 M, 0.5 ml NaOH. After that samples were centrifuged at 7,000G for 10 min under 4 °C. The absorptions were measured spectrophotometrically (Shimadzu UV mini – 1280, Tokyo, Japan) under 595 nm wavelength, after 10 min precise incubation period of mixture with formulated protein assay Bradford reagent (Fujifilm Wako Chemicals, Osaka Japan). Protein levels were determined using a pre-prepared standard curve.

The objective is measuring H₂O₂ content is to identify the oxidative stress levels of the cyanobacteria. Preserved cell pellets were kept at room temperature (20–25°C) for 15 min for thawing and pellets were homogenized in 1 mL of 0.1 M phosphate buffer. After that, samples were centrifuged at 10,000G for 10 min at 4 °C. The 1 mL of reaction mixture for one sample was prepared using 1:3 ratio of sample supernatant and 0.1M Ti (SO₄)₂ in 20% (v/v) H₂SO₄, respectively. The absorbance of each sample was measured at 410 nm wavelength using a spectrophotometer (Shimadzu UV mini – 1280, Tokyo, Japan) and H₂O₂ concentrations were determined using a pre-prepared standard curve for known concentrations (Patterson et al. 1984).

CAT activity was measured according to Aebi (1984) with fewer modifications. The enzymes from preserved cell pellets were extracted into 1 mL of extraction solution prepared with 6.057, 0.37, 0.177, 0.155, 0.308, 2.171 mg/mL of tris, 2Na(EDTA.2Na), ascorbic acid, dithiothreitol, glutathione (reduced form), MgCl₂.6H₂O, respectively and 45 μL/mL of 1 M HCl. The mixture was centrifuged at 12,000G for 20 min under 4 °C. The reaction solution was prepared with 5.606 and 0.37 mg/mL of Tris, 2Na(EDTA.2Na) and 45 μL/mL of 1 M HCl. The testing mix was prepared with, a 23:1:1 ratio of reaction solution, 750 mM H₂O₂ solution and enzyme samples, respectively. The samples were measured under 240 nm wavelength for 3 min with 10 s continuous intervals under the spectrophotometer. CAT activity was calculated as the reduction of absorption units per second per milliliter of the enzyme sample and divided from the cell protein content of the same treatment (μg/mL) to interpret the CAT activity on cellular basis.

GPX was measured by following the method by Ewing & Janero (1995), with slight modifications. Enzymes were extracted into an extraction solution (CAT method) from preserved cell pellets. The mixture was centrifuged at 12,000G for 20 min under 4 °C. The reaction solution was prepared with Tris, 2Na(EDTA.2Na), HCl, H₂O₂, 2.22% guaiacol in ethanol. The testing mix was prepared with 24:1 reaction solution and sample solution, respectively. The absorbance of the test mixture was measured using a spectrophotometer under 470 nm wavelength for 3 min with 10 s continuous intervals. GPX activity was calculated as the increase in absorption units per second per milliliter of the enzyme sample and later divided from cellular protein content of the same treatment (μg/mL) to interpret the GPX activity on a cellar basis.

The initial pH measurements of the BG – 11 media and the pH measurements of the monensin and lasalocid 2,000 μg/L solutions were taken (AS ONE – AS 800, Osaka, Japan) at the beginning of the test. The pH values of each treatment flask were not measured during the test period to avoid cross-contamination of the culture flasks. At the end of each test, three pH measurements of the culture media were taken from each replicate of each treatment.

All data were recorded manually and later transferred into MS Excel (version 16.0) 2016. Descriptive statistical analysis was performed using MS Excel (version 16.0), and all other statistical analyses were conducted using IBM SPSS (Version 25. IBM Corp., Armonk, NY, USA). The three tests monensin, lasalocid, and T_M:T_L (w/w) 1:1 were conducted separately following a completely randomized design model, therefore statistical analyses were performed separately for each test. All treatments in each of the three tests were conducted with three replicates, and three samples from each replicate were collected. Hence, nine independent analyses were performed for each parameter of each treatment. The normality of data for each parameter was tested using the Shapiro–Wilk test and Kolmogorov–Smirnov tests. Significance between control and treatments, and between treatments, was tested using one-way analysis of variance (ANOVA) with post-hoc Tukey HSD (honestly significant difference) test (for normally distributed data) for levels of significance p < (0.05)* and p < (0.01)** and Kruskal–Wallis one-way ANOVA with pairwise comparison (for data not normally distributed) for levels of significance p < (0.05)*. Pearson correlation analysis was performed to check the relationship between interrelated parameters.

M. aeruginosa shows varied responses to monensin, lasalocid, and a T_M:T_L 1:1 (w/w) mix during a one-week treatment. In all treatments, visible changes in the cultures appeared in the latter part of the week. When observing the cultures on the day – 07, all replicates of the monensin test, L1, L2, and M treatments exhibited a gradual increase in green color intensity compared to the control. The H1 and H2 treatments displayed a sequential increase in brownish color, accompanied by a corresponding sequential decrease in green color intensity, respectively. In the H2 treatment, a microscopic examination of the culture media revealed the presence of ruptured cells of M. aeruginosa. In the lasalocid treatment, all experimental groups, including the control, exhibited vigorous growth of M. aeruginosa. However, the L1, L2, M, H1, and H2 treatments, respectively, exhibited a subtle increasing trend in the intensity of green color compared to the control, which was not readily discernible to the naked eye. The L1, L2, M, and H1 treatments of the T_M:T_L (w/w) 1:1 exhibited a consistent pattern of color change, aligning with the trend observed in the monensin treatment, nevertheless with a lower variation in color intensity with adjacent treatments (Supplementary data 2–4).

According to Pearson correlation analysis between the H₂O₂/cell protein ratio and the CAT activity/cell protein ratio, the monensin treatment and the lasalocid treatment showed Pearson correlation values of 0.655 and 0.335, respectively, with p-values of <0.01 and 0.013, respectively, indicating a significant correlation between the two parameters.

The mean initial pH value of the media (BG – 11) at the beginning of the treatment was recorded as (8.55 ± 0.02) for the media prepared for all three tests, and at the end of the treatment period, pH values of the test media have changed as showed (Table 2). The mean pH of 2,000 μg/L monensin in BG – 11 (n = 3) is recorded (7.85 ± 0.38) indicating mixing of monensin has caused to reduction in the pH of BG – 11. All three controls showed similar increased pH values compared to the pH value of BG – 11 after the 7-day treatment period. In the monensin treatment, the M, H1, and H2 treatments exhibit a statistically significant (Kruskal–Wallis one-way ANOVA, p < 0.05) lower pH compared to the control. In contrast, in the lasalocid treatment, the pH values of each treatment remain unchanged compared to the control. But in the T_M:T_L (w/w) 1:1 test, a similar trend as to the monensin treatment is observed, where M and H1 treatments exhibit a statistically significant (p < 0.05) low pH compared to the control.

Nutrient influxes into aquatic ecosystems are modulated by various environmental determinants. Fertilizers used in agriculture contribute to nutrient heterogeneity in soils, with cattle manure being a significant source, varying depending on animal-specific factors and dietary composition (William et al. 2007). Acid rains increase the leaching of minerals and metal ions, including Cu²⁺, Mn²⁺, and Al³⁺ (Koptsik et al. 2003). The composition of runoff from areas near cattle farms is highly variable and unpredictable. Therefore, to ensure consistency, all experiments in this study utilized the standard BG – 11 media to minimize potential variability arising from changes in the growth medium.

Cyanobacteria exhibit diverse responses and defence mechanisms against biotic and abiotic stressors. Cyanobacteria adapt to diverse stress conditions through modifications in their physiological, biochemical, and molecular pathways (Singh & Montgomery 2011). Increased toxicity might result in the generation of reactive oxygen species (ROS), which interact with macromolecules such as proteins, DNA, and lipids. This interaction disturbs the membrane structure and leads to oxidative stress conditions. The antioxidant defence systems such as CAT, GPX, APX (ascorbate peroxidase), SOD (superoxide dismutase), and GR (glutathione reductase) are enabled to counteract these stress conditions (Fujita & Hasanuzzaman 2022).

The results of the current study revealed that the stress responses of the same cyanobacterial species vary depending on individual contaminants as well as their combinations. The results of the monensin test of our study were partially consistent with the intrinsic biochemical properties of ionophores, including their antimicrobial effects and their distinctive modulation of membrane activity, as well as the complex cellular responses demonstrated by cyanobacteria under various stress conditions. Monensin possesses three reactive functional groups, a carboxyl group is situated at the C₁ position and two hydroxyl groups are located at the C₇ and C₂₆ positions. Among these, the two hydroxyl groups are pivotal in the formation of metal ion complexes (Sakakibara et al. 1988). The capability of monensin to form complexes with monovalent cations follows the order: Na⁺ > K⁺ > Li⁺ > Rb⁺ > Cs⁺ (Lutz et al. 1970).

An experiment on Synechococcus UTEX 625’ has revealed that Na⁺ stimulates the transport of HCO₃⁻ ion which is a dissolved inorganic carbon source for photosynthesis. The results of the research determined that the transportation of Cl⁻ ions and PO₄³⁻ require μM levels of Na⁺ while, NO₃⁻ and HCO₃⁻ require mM levels of Na⁺ ions for the maximum transport activity. The Na⁺ binding ability of monensin inhibited the ion transportation mechanisms of cyanobacteria (Espie & Kandasamy 1994). This is one of the fundamental factors that drives the antibacterial action of ionophore antibiotics.

Similarly, monensin catalyses the exchange of Na⁺ and H⁺ ions across biological membranes (Sandeaux et al. 1982). Monensin is able to act as an antiporter of metal ions and protons, that are involved in exchanging the H⁺ either for K⁺ or Na⁺ (Pressman 1976; Russell & Strobel 1989). Once monensin is introduced, it exchanges the intracellular K⁺ for extracellular H⁺ or extracellular Na⁺ for intracellular H⁺ (Russell 1987). These processes resulted in blocking the nutrition transport across the membrane and maintaining a pH difference across the membrane. The unbalanced Na⁺ and K⁺ distribution tends to activate the ATP utilizing primary pumps to re-establish the ion balance in between the cell membrane. Prolonged ATP utilization under conditions of malnutrition depletes cellular energy reserves, ultimately leading to cell death.

In the current study, the cellular mortality observed at 1,000 and 2,000 μg/L concentrations of monensin can be ascribed to the ionophore's sodium ion (Na⁺) chelation mechanism. This process obstructs the transmembrane transport of vital anions, including bicarbonate (HCO₃⁻), nitrate (NO₃⁻), and phosphate (PO₄³⁻), which are indispensable for the photosynthetic process. At lower concentrations of monensin (100, 200, and 500 μg/L), M. aeruginosa exhibited an increased optical density, which was statistically significant compared to the control on the day – 05 of the treatment (Figure 2(a)). This trend persisted on day – 07 (Figure 3(a)), with only the 200 and 500 μg/L concentrations showing statistical significance. This observation reflected a stress response induced by the presence of an exogenous substance.

Previous studies indicated that the decrease in photosynthetic pigments resulting from environmental stress may be attributed to the replacement of Mg²⁺ ions, inhibition of chlorophyll biosynthesis enzymes, or an elevation in ROS (Chittora et al. 2020). In the monensin test, Chl. ‘a’ concentration (Figure 5(a)) significantly reduced compared to the control with increasing monensin concentration (100–2,000 μg/L). This is attributed to stress-induced inhibition of Chl. ‘a’ synthesis. This standpoint is further strengthened by the increase in H₂O₂/cell protein ratio, which was observed with increasing monensin treatment concentration ranging from 100 to 2,000 μg/L. The catalase enzyme functioned as a protective antioxidant enzyme, decomposing H₂O₂ generated under oxidative stress conditions to safeguard cells from oxidative damage. The distribution of CAT activity/cell protein ratio (Figure 6(a)) in the monensin test followed the same trend as the H₂O₂/cell protein ratio (Figure 5(a)), indicating the catalase activity of the cells, which enables them to defend against the H₂O₂ produced under oxidative stress conditions induced by monensin.

Guaiacol peroxidase performs the same comparable protective function against hydrogen peroxide, generated by stressed cells. Our study revealed that monensin induced a comparable trend in oxidative stress (Figure 6(a)), CAT activity (Figure 7(a)), and GPX activity (Figure 8(a)) curves, demonstrating the activation of cellular defence mechanisms in M. aeruginosa in response to monensin-induced oxidative stress. Monensin activity increases the concentration of H⁺ ions in the media as a result of its ion transportation mechanism. Most of the time cyanobacteria uphold an acidified cytoplasm during the growth at high pH, which is maintained by the sodium cycle (Buck & Smith 1995; Miller et al. 1984). The pH distribution on day – 07 (Table 2) in the monensin treatment showed a significantly lower increase in pH (9.73 ± 0.22) in 1,000 and 2,000 μg/L (9.63 ± 0.027) treatments compared to the control (11.76 ± 0.02). This could be a result of the ion antiport mechanism of monensin through M. aeruginosa plasm membranes, which causes the accumulation of H⁺ ions outside the membrane, that has been significantly enabled at comparatively higher concentrations of monensin. Monensin effects underscore the typical antibacterial mechanism of ionophore antibiotics, particularly at concentrations of 1,000 and 2,000 μg/L. This is illustrated by the OD₇₃₀ distribution on day – 7 (Figure 3(a)) and the cellular protein distribution curve (Figure 4(a)). Consequently, for all monensin treatments at concentrations ≤500 μg/L, an enhanced growth response (Figure 4(a)) and decreased Chl ‘a’ measurement (Figure 5(a)) should be attributed to the stress conditions induced by monensin.

Lasalocid treatment showed a deviation from the monensin results. On the day – 03 curve (Figure 2(b)), concentrations of 200, 500, 1,000, and 2,000 μg/L showed a statistically significant increase in optical density values. However, by day – 07 (Figure 3(b)), although there was a continuing increasing trend in optical density, no significant difference was observed. During the 100, 200, and 500 μg/L concentrations, M. aeruginosa cells showed a decrease in cellular protein contents (Figure 4(b)) but an increase in OD₇₃₀ of the day – 07 (Figure 3(b)). This may be due to higher Chl. ‘a’ production by cells under lasalocid action. In the latter two treatments (1,000 and 2,000 μg/L), protein content decreased while OD₇₃₀ values increased. This increase might have been caused by the increase in cell number rather than an increase in Chl. ‘a’ content. Since OD₇₃₀ is influenced by the turbidity or cloudiness of the cyanobacteria culture, it is difficult to make a precise assumption.

Lasalocid has higher Chl. ‘a’ production for lower concentrations (100, 200, and 500 μg/L) and inhibition for higher concentrations (1,000 and 2,000 μg/L) (Figure 5(b)). This might be a stress response of M. aeruginosa for lasalocid action. Based on the cellular protein results, it can be assumed that lasalocid has a negative impact on the growth of M. aeruginosa, even though the OD₇₃₀ (Figure 3(b)) values showed a progressive increase with the increasing treatment concentration. The oxidative stress curve of lasalocid (Figure 6(b)) showed a significant progressive increase up to 1,000 μg/L treatment and a noticeable reduction in 2,000 μg/L treatment. Verifying the oxidative stress status, CAT activity distribution (Figure 7(b)), and the GPX distribution (Figure 8(b)) followed the same trend by showing the lowered enzyme activities at 2,000 μg/L concentration. The action of lasalocid on M. aeruginosa did not show the typical antibacterial actions of ionophore antibiotics.

In the T_M:T_L (w/w) 1:1 test, the behavior of OD₇₃₀ on the day – 07 (Figure 3(c)), cellular protein curve (Figure 4(c)), and Chl. ‘a’ curve (Figure 5(c)) exhibited a trend similar to that observed in the monensin treatment. The activity of the monensin and lasalocid combination seems to have brought the individual impact of each antibiotic into an intermediate state in the T_M:T_L (w/w) 1:1 test. Based on the OD₇₃₀ data on day – 05 and day – 07 (Figures 2(c) and 3(c)), treatments did not show any significant difference compared to the control. Initially, a significant impact could be observed for the M and H1 treatments on day – 03 (Figure 2(c)). According to the cellular protein content, it can be assumed that the T_M:T_L (w/w) 1:1 test has a positive impact on the growth of M. aeruginosa, up to the M treatment.

With the outcomes of our study, we can make an argument that the selected environmentally possible ranges of monensin (100, 200, and 500 μg/L) have a positive impact on the growth of M. aeruginosa, potentially leading to the formation of HABs caused by M. aeruginosa. When considering environmentally relevant concentrations (100, 200, and 500 μg/L) of lasalocid, it can be inferred that lasalocid exerts a negative impact on the formation of HABs caused by M. aeruginosa. The synergistic effect of the two antibiotics has produced an intermediate outcome when compared to the individual effectiveness of each antibiotic, simultaneously underscoring the notable activity of monensin. According to the T_M:T_L (w/w) 1:1 ratio, the combination of the two antibiotics still has a positive impact on the growth of M. aeruginosa-based HABs, with the impact of monensin becoming more prominent. This situation may depend on the concentration ratio of monensin to lasalocid.

It is recommended to introduce two or more ionophore antibiotics into cattle feed rotationally to maintain their continuous effect over a considerable period. Consequently, there is a possibility of accumulating more than one ionophore antibiotic in the same livestock ponds. Therefore, based on our experimental results, within the environmental concentration ranges of 100–500 μg/L, the accumulation of monensin is comparatively more harmful in creating M. aeruginosa blooms than accumulating either lasalocid or a 1:1 (w/w) mixture of monensin and lasalocid.

Monensin treatment demonstrated a concentration-dependent effect on the growth of M. aeruginosa, with lower concentrations showing a positive impact and higher concentrations leading to growth reduction or cell death. Additionally, monensin reduces the Chl. ‘a’ content, induces oxidative stress and increased catalase activity in the cyanobacteria. Lasalocid showed a growth reduction of M. aeruginosa for lower concentrations and growth promotion for higher concentrations, necessitating further investigation. The combined treatment of monensin and lasalocid yielded intermediary responses compared to individual treatments, suggesting potential interactive effects between the two antibiotics. Overall, within environmentally relevant concentrations, monensin may promote the formation of M. aeruginosa-based HABs, while lasalocid might have a negative impact. This study could be further expanded by incorporating additional variables and parameters.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.