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Article

Synergistic Removal of Ciprofloxacin and Sulfamethoxazole by Lemna minor and Salvinia molesta in Mixed Culture: Implications for Phytoremediation of Antibiotic-Contaminated Water

by
Leticia Yoshie Kochi
1,*,
Rafael Shinji Akiyama Kitamura
1,
Camila Silva Rocha
1,
Julio Cesar Moreira Brito
2,
Philippe Juneau
3 and
Marcelo Pedrosa Gomes
1,*
1
Laboratório de Fisiologia de Plantas sob Estresse, Departamento de Botânica, Setor de Ciências Biológicas, Universidade Federal do Paraná, Avenida Coronel Francisco H. dos Santos, 100, Centro Politécnico Jardim das Américas, C.P. 19031, Curitiba 81531-980, Paraná, Brazil
2
Fundação Ezequiel Dias, Rua Conde Pereira Carneiro, 80, Belo Horizonte 30510-010, Minas Gerais, Brazil
3
Ecotoxicology of Aquatic Microorganisms Laboratory, GRIL, EcotoQ, TOXEN, Department of Biological Sciences, Université du Québec à Montréal, Montréal, Succ. Centre-Ville, Montréal, QC H3C 3P8, Canada
*
Authors to whom correspondence should be addressed.
Water 2023, 15(10), 1899; https://doi.org/10.3390/w15101899
Submission received: 19 April 2023 / Revised: 7 May 2023 / Accepted: 11 May 2023 / Published: 17 May 2023
(This article belongs to the Special Issue Environmental Feedbacks between Global Changes, Contaminants and Aquatic Plants)
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Abstract

:
Aquatic macrophytes have been used for the removal of antibiotics from contaminated water. Here, we have studied the capacity of Lemna minor and Salvinia molesta to reclaim ciprofloxacin (1.5 µg Cipro L−1) and/or sulfamethoxazole (0.3 µg Sulfa L−1) from artificially contaminated waters when plants were exposed in monoculture (L. minor or S. molesta) or in mixed culture (L. minor + S. molesta). Neither Cipro nor Sulfa alone induced negative effects on plants. As shown by the Abbot modelling, Cipro and Sulfa displayed antagonistic effects on plants. In both species, increased H2O2 concentrations and antioxidant enzyme activities were observed in plants when grown together. Although the antibiotics showed natural degradation, their concentration in water from treatments with plants was significantly lower, indicating the ability of the plants to uptake the compounds. When in co-culture, greater growth rates were observed for both plant species, which resulted in greater Cipro and Sulfa removal in the mixed system when compared with those with plants in monoculture. Both plants showed tolerance to the studied concentrations of antibiotics, with greater antibiotic uptake being reported for S. molesta. Although at the tested concentrations the antibiotics did not induce negative effects on plants, exposure to Cipro changed the relative yield of S. molesta, which may result in effects on community structure. The use of both L. minor and S. molesta in artificial wetlands may increase the phytoremediation capacity of systems.

1. Introduction

Several physical and chemical methodologies, such as reverse osmosis, adsorption, photolysis/photocatalysis, ionizing radiation, and electrochemical oxidation, have been indicated as effective for the removal of antibiotic residues in wastewaters [1]. However, these approaches are expensive, demand high amounts of electrical energy, and produce large amounts of sludge [2,3]. In this context, the use of plants (phytoremediation) has emerged as a green strategy, coupling low cost and the excessive removal of antibiotics from contaminated water.
Artificial wetlands are an efficient and accessible remediation method based on the natural capacity of plants to remove and/or metabolize water contaminants [4,5]. Although the most common plants used in wetlands are emerging plants [5], floating plants have shown great efficiency on the removal of xenobiotics, such as antibiotics. For instance, Lemna minuta showed great ability to remove levofloxacin and diclofenac [6]; L. gibba was effective in the removal of tetracycline [7]; and L. minor efficiently removed amoxicillin, enrofloxacin, oxytetracycline [8], erythromycin [9], ciprofloxacin, and sulfamethoxazole [10] from contaminated waters. Similarly, Salvinia molesta has shown the capacity to remove erythromycin [9] and ciprofloxacin [11], and the Salvinia species has been indicated as effective for phytoremediation programs. Due to their rapid and fruitful growth and easy management, the use of floating macrophytes in artificial wetlands have been indicated as useful [4].
Most investigations on the contaminant removal capacity of plants have been focused on laboratory bioassays or conducted in constructed wetlands composed of a single plant species. However, the bio-accumulating capacity of aquatic macrophytes is influenced by external factors, such as climate conditions and the presence of different plant species in the system—which will therefore maximize or minimize the decontamination and removal efficiency of the system [4]. Apparently, systems using aquatic macrophyte polycultures tend to be more efficient at removing contaminants than monoculture systems [4]. Similarly, the co-occurrences and interactions between different types of chemicals alter plant phytoremediation capacity [8,12]. For instance, the competitive uptake of amoxicillin, oxytetracycline, and enrofloxacin was observed when L. minor plants were submitted to binary or tertiary mixtures of those antibiotics [8]. Likewise, the co-occurrence of glyphosate and its by-product aminomethylphosphonic acid (AMPA) interferes with the capacity of S. molesta to remove these contaminants from water [12,13]. Therefore, studies focusing on polyculture systems, as well as mixtures of contaminants, are urgently needed to represent more realistic situations of exposed plants in artificial wetlands.
Among antibiotics, particular attention has been given to fluoroquinolones and sulfonamides classes, which include ciprofloxacin (Cipro) and sulfamethoxazole (Sulfa), since these antibiotics are widely used worldwide to treat bacterial infections [14,15]. Due to the fact that only part of these antibiotics is absorbed by the organism, these substances or their metabolites are excreted and are ultimately found in waterbodies [10]. Cipro concentrations up to 15 mg l−1 have been observed in surface waters in South Africa [16], and up to 44 mg of Cipro l−1 was detected in hospital wastewater [17]. Similarly, Sulfa has been detected in concentrations up to 40 mg l−1 in surface water in Kenya [18]. Once in the environment, residues of antibiotics can promote the appearance and spread of resistance in microorganisms, including pathogenic ones, constituting a global health problem [19]. Therefore, plants constitute a powerful tool against antibiotic resistance, and increasing our understanding of their tolerance and antibiotic phytoremediation potential is of great importance. Recently, Gomes and collaborators [20] have demonstrated the importance of hydrogen peroxide (H2O2)-scavenging enzymes in the acquired tolerance of plants to different antibiotics. According to these authors, the activity of ascorbate peroxidase (APX) and catalase (CAT) is intrinsically related to the capacity of plants to tolerate and reclaim antibiotics from contaminated waters; therefore, evaluations of plant oxidative stress markers are essential in studies involving antibiotic phytoremediation.
In this study, we evaluated the capacity of L. minor and S. molesta to reclaim antibiotics (Cipro and/or Sulfa) from artificially contaminated waters. The phytoremediation capacity of plants was determined when plants were exposed in monoculture (L. minor or S. molesta) or co-culture (L. minor + S. molesta). In addition, to identify if polycultures are better indicated than monocultures for phytoremediation purposes, our results have a direct impact on plant biology and toxicology fields by bringing new knowledge on plant physiological responses to a mixture of different contaminants.

2. Materials and Methods

2.1. Plant Material and Bioassays

The free-floating macrophyte Lemna minor L. (Araceae) was obtained from cultures held at the Laboratory of Plant Stress Physiology (UFPR, Curitiba, Brazil), while Salvinia molesta DS. Mitch. (Salviniaceae) specimens were collected in Barigui Park, Curitiba, Paraná State, Brazil (25°25′18″ S; 49°18′22″ W). Before initiating the experiments, the macrophytes were acclimated and depurated in biological oxygen demand (B.O.D.) culture chambers for 25 days in aquariums (30 L) containing sterile ASM−1 medium [21,22,23] (pH 7.0 ± 0.5) at 22 ± 3 °C, under a 12 h photoperiod (100 µmol photons m−2 s−1). For the bioassays, the plants were transferred to 250 mL Erlenmeyer flasks (each constituting one replicate) containing 100 mL of sterile ASM−1 with appropriate concentrations of antibiotics (with pH corrected to 7.0 ± 0.5, when necessary). Four flasks were used in each of the treatments (as described below). The bioassays were conducted under the same temperature and illumination conditions as the acclimatization. The density of cultures was of 15 g plant L−1 being 0.75 g per flask of each plant species used in mix cultures (L. minor + S. molesta). Before use, plant surfaces were disinfected in hypochlorite solution (0.5%) for 3 min [12].
The plants were exposed for seven days to environmental representative concentrations of ciprofloxacin (Cipro; 0 and 1.5 µg L−1) and sulfamethoxazole (Sulfa; 0 and 0.3 µg L−1), under completely randomized experimental design with treatments separated in a factorial scheme with four replications, totaling 48 experimental units. These concentrations were based on those observed in rivers of Curitiba (Paraná, Brazil). In addition to the antibiotics alone, the plants were also exposed to combined concentrations of Cipro and Sulfa (Cipro + Sulfa). Analytical-grade antibiotics obtained from Sigma-Aldrich (São Paulo, Brazil) were used in all experiments. Stock solutions (10 mg L−1) were prepared in ultra-pure water and then diluted to achieve the respective nominal concentration used in the study. Parallel bioassays were conducted in flasks without plants under the same conditions to evaluate the degradation of chemicals by light, temperature, and hydrolysis.

2.2. Physiological Evaluations

At harvesting, plants were centrifuged at 3000 rpm for 10 min at room temperature (in centrifuge tubes with small holes to remove surface water) and weighed to determine their fresh weights, and the relative growth rate (RGR) was calculated according to OECD [24]. Then, plants were flash-frozen in liquid nitrogen and then stored at −80 °C in aluminum foil until further analysis.
For photosynthetic pigments, 0.1 g of fresh leaves were extracted in 80% acetone, and the concentration of total chlorophylls and pheophytins was assessed following Lichtenthaler and Wellburn [25] and Vernon [26], respectively. Antioxidant enzyme activities, as well as H2O2 [27] and malondialdehyde (MDA) concentrations [28], were determined using 0.1 g of plants (leaves + roots). The enzymes were extracted in 1 mL of phosphate buffer (100 mM, pH 7.8) containing 100 mM EDTA, 1 mL of L-ascorbic acid, and a 2% polyvinylpyrrolidone solution (PVP m/v) [13]. The activities of superoxide dismutase (SOD; EC 1.15.1.1) [29], APX (EC 1.11.1.11) [30], and CAT (EC 1.11.1.6) [31] were assessed after determining the total protein concentrations by the Bradford’s method [32].

2.3. Combined Toxicity Evaluation

The Abbot’s model was used to evaluate the combined toxicity of Cipro and Sulfa in the studied physiological parameters. The Abbot’s model uses a ratio of the concentration of each substance in combination to the concentration of that substance alone that would produce the same effect. The expected inhibitions (Cexp) were predicted according to Iswarya et al. [33] as follows:
C e x p = A + B − A B 100
where A and B represent the inhibition caused by Cipro and Sulfa, respectively.
The ratio of inhibition (RI) for each index was calculated as follows:
R a t i o   o f   i n h i b i t i o n R I = O b s e r v e d   i n h i b i t i o n   ( C o b s ) C e x p
where Cobs is the inhibition observed under all of the treatments, which was accessed by the following formula:
C o b s = T x − C x C x
where Tx is each indicator value of treatments and Cx is each index value of control. The interactive effects of Cipro and Sulfa were evaluated by comparing RI with 1. A ratio of inhibition (RI) value of 1 indicates additive effects, where the combined effect of the substances is equal to the sum of their individual effects. An RI value less than 1 indicates synergistic effects, where the combined effect of the substances is greater than the sum of their individual effects. An RI value greater than 1 indicates antagonistic effects, where the combined effect of the substances is less than the sum of their individual effects. The RI was calculated for each of the four replicates for each treatment, and the mean and standard deviation were determined. Only if the mean RI was greater than one standard deviation from 1 (1 + SD) was the interactive effect assumed to be significantly different from additivity [34].

2.4. Relative Yield and Competitive Balance Index

To calculate the resource competitiveness of L. minor and S. molesta, the relative yield (RY) [35] and competitive balance index (CBI) [36] were estimated as follows:
R e l a t i v e   y i e l d   R Y = f r e s h   b i o m a s s   i n   m i x t u r e f r e s h   b i o m a s s   i n   m o n o c u l t u r e
C o m p e t i t i v e   b a l a n c e   i n d e x   C B I   L . m i n o r = ln ⁡ R Y   L . m i n o r R Y   S . m o l e s t a

2.5. Chemical Analyses

Antibiotic concentrations in the growth media were quantified at the beginning of the treatments and at harvesting. The concentrations of the antibiotics in the growth media and plants were determined by using LC-MS/MS. For the growth media assays, filtered solution (50 mL) was concentrated via solid-phase extraction (SPE) using a Visiprep TM SPE Vacuum manifold (Sigma-Aldrish, São Paulo, Brazil) with 200 mg and 3 mL−1 Phenomenex Strata-X® cartridges (Torrance, CA, USA). The extraction of antibiotics from leaves was performed according to Palmada et al. [37], with modifications by Migliore et al. [38], using 0.1 g of fresh tissue. After homogenization with 1.5 mL of acetonitrile containing 1% acetic acid, samples were kept in ultrasonic bath for 5 min, vortexed for 1 min, and then centrifuged for 10 min at 3000× g. The collected pellet was resuspended in 5 mL in phosphate buffer (pH 7.4). After extraction, plant samples were dried in a SpeedVac machine (RC1010, Thermo, Waltham, MA, USA), and the residues suspended in acidified water (pH 4.5) and concentrated via SPE as for growth media samples. SPE conditions were according to Boeger et al. [39]. The cartridges were conditioned with 4 mL of methanol followed by 6 mL of ultrapure water, and analytes were eluted in 4 mL of methanol. The cartridges containing the samples were eluted with 3 mL of 50% methanol in water (v/v). The eluate was dried in a SpeedVac machine (RC1010, Thermo), and the residues were resuspended in the mobile phase (water and acetonitrile at 50:50 v/v containing 0.1% formic acid and 5 µM ammonium formate).
The analyses were carried out using an LC-MS/MS system composed of a Xevo TQD triple-quadrupole (Waters, Milford, MA, USA) mass spectrometer equipped with an electrospray (ESI) ionization source coupled to an HPLC Varian SYS-LC-240-E equipped with an autosampler. Drugs were evaluated following Böger et al. [39]. The chromatographic separations were performed with a Zorbax Eclipse XDB-C8 column with 4.6 mm × 150 mm, with 5 µm particle size (Agilente, Milford, CT, USA) using water as phase A and acetonitrile/water (95:5 v/v) as phase B, both containing 0.1% formic acid and 5 mM of ammonium formate. Mass spectrometry analyses were operated in a positive ion mode. Analytical-grade Cipro and Sulfa (Sigma-Aldrich, Markham, ON, Canada) were used to prepare the calibration curves. Standard stock solutions (1000 μg mL−1) of these compounds were prepared using different compositions of methanol and water with formic acid and ammonium formate depending on solubility. The six-point calibration curves showed good linearity for the analytes (r2 ≥ 0.95; p < 0.0001). Each sample batch included three blanks, three standards, and three fortified samples (for quality control). The recoveries for all compounds were greater than 87%. The limit of detection (LOD) and limit of quantification (LOQ) of each analyte were as follows: 10 and 20 ng Cipro L−1, and 10 and 20 ng Sulfa L−1, respectively.

2.6. Statistical Analysis

Statistical analyses were performed using JMP 13.0 software (SAS Institute Inc., Cary, NC, USA). Data were tested for normality (Shapiro–Wilk) and homogeneity (Bartlett), and then statistically evaluated. Initial and final concentrations of antibiotics in the growth media were compared via Student’s t test, while the other data were evaluated using two-way ANOVA, with the interaction between Cipro and Sulfa included in the model. When differences were detected by ANOVA, the means were compared using the post hoc Tukey test (significance at p < 0.05).

3. Results

3.1. Antibiotic Concentrations in Growth Media at T0 and Antibiotic Degradation

As shown in Table 1, no antibiotics were detected in the control treatments. We did not observe inter-contamination between treatments with different antibiotics, nor did the concentration of any given antibiotic significantly differ between treatments alone or in combination (p > 0.05). In systems without plants, mean Cipro and Sulfa degradation in growth media was 26.5 ± 3.69% and 50.5 ± 4.20%, respectively, when chemicals were applied alone. When combined, mean Cipro and Sulfa degradation was 26.21 ± 4.63% and 50.75 ± 4.57%, respectively.

3.2. Physiological Responses of L. minor When Exposed Alone or Together with S. molesta to Antibiotics

The relative growth rate (RGR) (Figure 1A), H2O2, and malondialdehyde (MDA, a marker of oxidative damages) concentration (Figure 2A,C) and ascorbate peroxidase (APX) activity (Figure 3A) in L. minor plants were not affected by antibiotics when this species was grown in monoculture or together with S. molesta (p > 0.05). In L. minor plants exposed in monoculture, the total chlorophylls/total pheophytins ratio was increased in plants treated with only Cipro and decreased in plants treated with Cipro + Sulfa in relation to control (Figure 1C). The greatest catalase (CAT) activity was observed in plants treated with Cipro + Sulfa when L. minor was in monoculture (Figure 3C), while lower superoxide dismutase (SOD) activity was observed in plants treated with only Cipro in relation to control (Figure 3E).
With exception of plants treated with only Sulfa, the total chlorophylls/total pheophytins ratio was greater in L. minor plants grown in co-culture with S. molesta in relation to those grown in monoculture (Figure 1E). Regardless of the treatment, RGR (Figure 1A), H2O2, and MDA concentrations (Figure 2A,C), as well as the activity of APX, CAT, and SOD (with exception of plants treated with only Sulfa for SOD activity), in L. minor plants were greater when plants were cultivated with S. molesta than when they were in monoculture (Figure 3A,C,E).

3.3. Physiological Responses of S. molesta When Exposed Alone or Together with L. minor to Antibiotics

In relation to control, the RGR, total chlorophylls/total pheophytins ratio (Figure 1B,D), H2O2 concentrations (Figure 2B), and SOD activity (Figure 3F) in S. molesta plants was not affected by antibiotics when plants were cultivated in monoculture (p > 0.05). Moreover, MDA concentrations were lower in S. molesta plants exposed to Cipro + Sulfa in relation to control (Figure 2D). Similarly, APX activity was lower in plants treated with Cipro + Sulfa in relation to those treated with only Cipro or Sulfa (Figure 3B). On the other hand, CAT activity was greater in plants treated with Cipro + Sulfa in relation to those of control and only Cipro treatments (Figure 3D).
When together with L. minor, the RGR of S. molesta plants exposed to only Sulfa was lower than that for plants exposed to Cipro + Sulfa (Figure 1B), while their H2O2 concentrations were the lowest in relation to all the other treatments (Figure 2B). Moreover, the total chlorophylls/total pheophytins ratio was increased (Figure 1F), and the MDA concentration (Figure 2D) and APX and SOD activities (Figure 3B,F) decreased in S. molesta plants treated with only Cipro in relation to control. Decreased total chlorophylls/total pheophytins ratio was also observed in plants treated with Cipro + Sulfa in relation to control (Figure 1F). Similarly, lower MDA concentrations were observed in plants exposed to only Cipro, while increased MDA concentrations were observed in those plants treated with only Sulfa and Cipro + Sulfa in relation to control (Figure 2D).
With the exception of plants treated with only Sulfa, greater RGR was observed in plants growing together with L. minor in relation to plants exposed in monoculture (Figure 1B). Higher total chlorophylls/total pheophytins ratio (Figure 1F), H2O2 concentrations (Figure 2B), and APX, CAT, and SOD activities (Figure 3B,D,F), as well as lower MDA concentrations (Figure 2D), were also observed in S. molesta plants grown together with L. minor in relation to those grown in monoculture, regardless of the treatment.

3.4. Combined Toxicity Evaluation

For almost all the physiological parameters investigated in the present study (RGR, MDA, APX, CAT, and SOD), Cipro and Sulfa showed antagonistic effects on plants, regardless the culture system (monoculture or co-cultures) (Table 2). In L. minor plants, the antibiotics showed antagonistic and synergistic effects on total chlorophyll and H2O2 concentrations, respectively; however, when in co-culture, their interactive effects were additive and antagonistic, respectively. Similarly, in S. molesta plants, the antibiotics showed antagonistic and additive effects on RGR when plants were grown in monoculture or in co-culture, respectively, while antagonistic and synergistic interactions were observed on chlorophyll concentration under these conditions.

3.5. Relative Yield and Competitive Balance Index

While the relative yield (RY) of L. minor was not affected by the treatments, the RY of S. molesta was greater in plants exposed to Cipro and to Cipro + Sulfa treatments (Table 3). For all the treatments, the RY of L. minor was greater than 1, which indicates that the competitive effect of plants under a mixed-culture system was higher than under the monoculture. Similar results were observed for S. molesta; however, when exposed to Sulfa only, the RY of those plants was roughly 1, which indicates that the competitiveness of these plants was similar under two culture systems (Table 3).
The competitive balance index (CBI) values for L. minor were only higher than 1 in the treatment with Sulfa only, indicating that, except for that treatment, the competitiveness of L. minor is lower than that of S. molesta (Table 3).

3.6. Antibiotic Removal

The amount of Cipro or Sulfa removed by L. minor and S. molesta plants from water did not significantly differ (p > 0.05) when plants were treated with Cipro or with Cipro + Sulfa when plants were in monoculture or together (Figure 4A,B). Regardless of the treatment, the Cipro removal was greatest when plants were grown together (Figure 4A). When grown in monoculture, greater Cipro removal was observed in S. molesta in relation to L. minor plants, regardless of the treatment (Figure 4A). Sulfa removal was higher in co-culture in relation to L. minor grown alone, but it did not differ between plants of S. molesta grown in monoculture and together with L. minor (Figure 4B). Moreover, when grown in monoculture and treated with only Sulfa, greater Sulfa removal was observed in S. molesta plants in relation to L. minor plants; however, this was not observed when plants were treated with Cipro + Sulfa (Figure 4B).

3.7. Antibiotic Concentration in Plants

Cipro and Sulfa concentrations in plant tissues did not differ in plants treated with the antibiotics alone (Cipro or Sulfa) or in mixture (Cipro + Sulfa), regardless of the culture system (Figure 5A,B). When cultivated in monoculture, greater Cipro concentration was found in S. molesta in relation to L. minor plants, regardless of the treatment. However, when grown together, Cipro concentrations in plant tissues did not significantly differ (p > 0.05) between the species (Figure 5A). In contrast, higher Sulfa concentration was found in S. molesta in relation to L. minor when plants were grown together, regardless of the treatment (Figure 5B). Moreover, when growing together, the concentration of Sulfa in L. minor plants was lower than those observed when this species was grown in monoculture, regardless of the treatment (Figure 5B).

4. Discussion

For the first time, we examined the isolated and interactive effects of Cipro and Sulfa on the physiological parameters and phytoremediation capacities of L. minor and S. molesta plants grown in monoculture or in co-culture. In addition, to attest to the phytoremediation capacity of both species, we observed that when cultivated together, plants had their growth stimulated, which increased antibiotic removal efficiency.
Neither Cipro or Sulfa alone induced negative effects on L. minor or S. molesta plant physiology. Although the studied concentrations of antibiotics are environmentally relevant, they are lower than those shown to induce phytoxicity. For instance, toxic responses were only seen in L. minor and Azolla filiculoides when plants were exposed to Cipro concentrations ≥1.05 mg L−1 [40,41], and no effect was reported in Spirodela polyrhiza when plants were exposed to 2.0 mg Cipro L−1 [42]. Similarly, in concentrations up to 100 µg L−1, Cipro was not toxic to S. molesta and Egeria densa plants [11]. Likewise, Sulfa toxicity to plants has been reported only when the antibiotic concentration is in the range of mg L−1 [43,44]. This demonstrates the tolerance of both studied macrophytes to environmentally representative concentrations of Cipro and Sulfa, allowing them to be considered for phytoremediation purposes.
In general, when plants are exposed to co-contaminants, strong toxicity is expected. For instance, when Elodea canadensis plants were simultaneously exposed to the fluoroquinolone enrofloxacin and the herbicide Roundup, synergistic effects of the contaminants were observed in plant physiology. Photosynthesis and APX activity were decreased, and the H2O2 concentrations increased in these plants when compared with those exposed to each contaminant alone [45]. Similarly, when exposed to glyphosate and Cipro together, synergistic effects of the contaminants were observed on photosynthesis, yield, and oxidative stress markers (H2O2 and MDA concentrations) [46]. Antiretroviral drugs, such as tenofovir, lamivudine, and efavirenz, also exhibited synergistic effects on the growth and enzyme activities of L. minor, as well as photosynthetic microorganisms, such as Synechococcus elongatus and Chlorococcum infusionum [47]. The observed enzyme activities include cytochrome P450 and CAT. The synergistic and additive effects of polystyrene nanoplastics and copper were also evidenced by using the Abbot’s model on Platymonas helgolandica plants, with an increased growth inhibition rate, SOD activity, and reduced glutathione concentrations being observed in plants exposed to the combination of the contaminants [48,49]. Although we have hypothesized that co-contaminants would have strong toxicity on plants, our results did not support this hypothesis. As shown by the Abbot modelling, in almost all the studied physiological parameters, Cipro and Sulfa showed antagonistic effects. For instance, in L. minor growing in monoculture, Cipro induced increases, and Sulfa decreases in pigments and MDA concentrations, and no effect was observed when both antibiotics were together (Figure 1B and Figure 2C). One can argue that antibiotics could interfere with or compete for plant uptake, resulting in lower exposure and, thus, evidencing the antagonistic effects of Cipro and Sulfa on plant physiology. However, the concentrations of each antibiotic have not differed in plants grown with each antibiotic alone or in mixture (Figure 5). A possible explanation would be that when faced with the presence of both contaminants in their tissues, plants activate more intense protective responses, allowing their survival. For instance, high CAT activity was observed in L. minor plants exposed to both Cipro and Sulfa together (Figure 3C). High activity of CAT was also observed by Gomes et al. [8] in L. minor exposed to amoxicillin and enrofloxacin and was related to the plant’s tolerance to antibiotics. The increase in CAT activity may prevent the overaccumulation of reactive oxygen species (ROS), protecting plants from oxidative damages due to the interference of antibiotics in metabolism related to ROS generation, such as photosynthesis and respiration [50].
Interestingly, some integrative responses have changed when comparing same plant species under different culture systems (monoculture and co-culture) (Table 2), which may be a result of the differential physiological responses of plants when growing in monoculture or together. For instance, when not exposed to antibiotics and in co-culture, both plant species have shown oxidative responses. In both species, increased H2O2 concentrations and antioxidant enzyme activities were observed in plants when grown together (Figure 2). H2O2 is an important plant signaler, being intrinsically related to plant responses to several stresses [51]. Below a physiological threshold from which H2O2 becomes toxic, its accumulation in plant tissues is known to stimulate photosynthesis, resulting in increased plant growth [52,53]. MDA concentrations have also increased in L. minor plants grown together with S. molesta, which could be interpreted as increased oxidative damage (Figure 2C). However, as growth reduction or any phytotoxicity indicator (such as chlorophyll decrease or pheophytin increase) were not seen, we may advance the hypothesis that the increase in MDA concentrations was not related to severe cell damages. Although excess accumulation of aldehydes (such as MDA) leads to cell damage, recent evidence suggests that lipid-derived aldehydes play important signaling roles in plant growth and stress responses [54,55]. In addition, to activate antioxidant defenses [54,55,56], aldehydes can stimulate plant growth and elicit appropriate responses, such as the regulation of gene expression at appropriate concentrations [57]. Therefore, the MDA accumulation could be associated with growth induction in L. minor plants grown in the presence of S. molesta.
An RY of plants greater than 1 (except for S. molesta exposed to Sulfa alone) indicates that the competitive effect of plants under a co-culture system was higher than under monoculture. The increase in the competitive pressure when in the presence of a potential competitive species could indeed result in increased plant biomass production [35], as also observed here. However, it is expected that once in co-culture, the increase in RY of one species may be followed by a decreased RY of its competitor. This was not the case in our study. However, since greater biomass production was observed for both L. minor and S. molesta when in co-culture, a possible explanation could be the presence of a substance with an allelopathic effect in the growth media, which can limit or stimulate the development, reproduction, and survival of target organisms [58,59]. Studies have reported the allelopathic potential of L. minor plants, showing, however, the negative effects of the allelochemicals produced by these plants [59,60]. Similarly, negative allelopathic effects have been described for S. molesta plants [61,62]. To the best of our knowledge, it is the first time that positive effects on plant growth have been described for co-cultures of L. minor and S. molesta. This topic merits more attention if some or both species are used to compose artificial wetlands, since their allelochemicals can prevent the growth of other plants of interest used for phytoremediation.
Since the co-culture of L. minor and S. molesta increased plant biomass production, we expected great antibiotic removal by plants in a co-culture system. Indeed, Cipro removal from water was greater when plants were grown together (Figure 4A). This result must be related to the increased RGR experienced by plants when they are grown in co-culture. Interestingly, greater Cipro removal was observed in S. molesta in relation to L. minor (Figure 4A). The CBI values for L. minor lower than 1 in treatments with Cipro also indicate the greater competitiveness of S. molesta in relation to L. minor (Table 3). Greater competitiveness may imply the higher uptake of nutrients and, consequently, of antibiotics. Fluoroquinolones uptake and translocation in plants were related to plant transpiration [63], and since the RGR of the plant species did not differ (regardless of the treatment, p < 0.05), we can assume that the transpiration rate in S. molesta must be greater than that of L. minor, assuring greater uptake of the antibiotic (and nutrient as well). Reinforcing this hypothesis, greater Cipro concentrations were observed in S. molesta in relation to L. minor when plants were grown in monoculture (Figure 5A).
Although the removal efficiency and plant concentrations of Sulfa did not differ in S. molesta and L. minor growing in monoculture (Figure 4B and Figure 5B), greater Sulfa removal was observed when plants were growing together (Figure 4B). As indicated by an RY = 1, the competitiveness of S. molesta was similar under the two different culture conditions when exposed to Sulfa only. Moreover, the CBI of L. minor when exposed to Sulfa only was greater than 1, indicating the greater competitiveness of L. minor in relation to S. molesta. Therefore, we expected greater concentrations of Sulfa in L. minor plants, which was not the case. As greater concentrations of Sulfa were observed in plants of S. molesta in relation to L. minor when plants were in co-culture, we can advance the hypothesis that the Sulfa uptake was greater in S. molesta, probably due to its greater respiration rates. Similarly to Cipro, Sulfa uptake and translocation are also related to plant transpiration [63]. Since no Sulfa was detected in the water of plants growing together at harvesting (Table 1), the high uptake of Sulfa by S. molesta plants reduces the antibiotic availability for L. minor, explaining the lower concentration of Sulfa observed in those plants.

5. Conclusions

Overall, both L. minor and S. molesta plants showed tolerance and great removal capacity for environmentally relevant concentrations of Cipro and/or Sulfa in water. The co-culture of plants increased the antibiotic removal capacity of the system and must be considered for phytoremediation projects. Although negative effects of the antibiotics were not seen in plant physiology, the exposure to Cipro increased the RY of S. molesta. This result indicates that by influencing interactions between the two plant species over time, Cipro may affect community structure [42]. Therefore, it is urgent to evaluate the effect of this antibiotic in aquatic systems as well as to develop natural-based solutions to remediate antibiotic-contaminated waters.

Author Contributions

Conceptualization, L.Y.K. and M.P.G.; methodology, L.Y.K., R.S.A.K., and M.P.G.; validation, L.Y.K. and M.P.G.; formal analysis, L.Y.K., R.S.A.K., C.S.R., and J.C.M.B.; investigation, L.Y.K., R.S.A.K., C.S.R., and J.C.M.B.; resources, P.J. and M.P.G.; data curation, P.J. and M.P.G.; writing—original draft preparation, L.Y.K., P.J., and M.P.G.; writing—review and editing, J.C.M.B., P.J., and M.P.G.; visualization, L.Y.K.; supervision, M.P.G.; project administration, M.P.G.; funding acquisition, P.J. and M.P.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–Brazil (CAPES)—Finance Code 001; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil)—Finance Code 406190/2018-6; and the Natural Science and Engineering Research Council of Canada (NSERC)—Finance Code RGPIN-2017-06210. M.P. Gomes received research productivity grants from the CNPq—Finance Code 302226/2022-2.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

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Water 15 01899 g001 550
Figure 1. Relative growth rate (RGR) (A,B), total chlorophyll concentration (C,D), and total chlorophyll/total pheophytin ratio (E,F) in L. minor and S. molesta plants growing in monoculture or in co-culture (L. minor + S. molesta) in growth media contaminated with antibiotics. Bars represent the means ± SD of four replicates. Lowercase letters indicate significant differences between treatments for the same isolated or co-culture; uppercase letters indicate significant differences between isolated or co-cultures for the same treatment. Cipro = 1.5 µg L−1; Sulfa = 0.3 µg L−1.
Figure 1. Relative growth rate (RGR) (A,B), total chlorophyll concentration (C,D), and total chlorophyll/total pheophytin ratio (E,F) in L. minor and S. molesta plants growing in monoculture or in co-culture (L. minor + S. molesta) in growth media contaminated with antibiotics. Bars represent the means ± SD of four replicates. Lowercase letters indicate significant differences between treatments for the same isolated or co-culture; uppercase letters indicate significant differences between isolated or co-cultures for the same treatment. Cipro = 1.5 µg L−1; Sulfa = 0.3 µg L−1.
Water 15 01899 g001
Water 15 01899 g002 550
Figure 2. Hydrogen peroxide (H2O2) (A,B) and malondialdehyde (MDA) concentration (C,D) in L. minor and S. molesta plants growing isolated or in mix (L. minor + S. molesta) in growth media contaminated with antibiotics. Bars represent the means ± SD of four replicates. Lowercase letters indicate significant differences between treatments for the same isolated or co-culture; uppercase letters indicate significant differences between isolated or co-cultures for the same treatment. Cipro = 1.5 µg L−1; Sulfa = 0.3 µg L−1.
Figure 2. Hydrogen peroxide (H2O2) (A,B) and malondialdehyde (MDA) concentration (C,D) in L. minor and S. molesta plants growing isolated or in mix (L. minor + S. molesta) in growth media contaminated with antibiotics. Bars represent the means ± SD of four replicates. Lowercase letters indicate significant differences between treatments for the same isolated or co-culture; uppercase letters indicate significant differences between isolated or co-cultures for the same treatment. Cipro = 1.5 µg L−1; Sulfa = 0.3 µg L−1.
Water 15 01899 g002
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Figure 3. Ascorbate peroxidase (APX) (A,B), catalase (CAT) (C,D), and superoxide dismutase (SOD) (E,F) activities in L. minor and S. molesta plants growing isolated or in mix (L. minor + S. molesta) in growth media contaminated with antibiotics. Bars represent the means ± SD of four replicates. Lowercase letters indicate significant differences between treatments for the same isolated or co-culture; uppercase letters indicate significant differences between isolated or co-cultures for the same treatment. Cipro = 1.5 µg L−1; Sulfa = 0.3 µg L−1.
Figure 3. Ascorbate peroxidase (APX) (A,B), catalase (CAT) (C,D), and superoxide dismutase (SOD) (E,F) activities in L. minor and S. molesta plants growing isolated or in mix (L. minor + S. molesta) in growth media contaminated with antibiotics. Bars represent the means ± SD of four replicates. Lowercase letters indicate significant differences between treatments for the same isolated or co-culture; uppercase letters indicate significant differences between isolated or co-cultures for the same treatment. Cipro = 1.5 µg L−1; Sulfa = 0.3 µg L−1.
Water 15 01899 g003
Water 15 01899 g004 550
Figure 4. Ciprofloxacin (A) and sulfamethoxazole (B) removal from water by L. minor and/or S. molesta plants grown in monoculture or in co-culture in contaminated water. Bars represent the means ± SD of four replicates. Lowercase letters indicate significant differences between treatments for the same isolated or co-culture; uppercase letters indicate significant differences between isolated or co-cultures for the same treatment. Cipro = 1.5 µg L−1; Sulfa = 0.3 µg L−1.
Figure 4. Ciprofloxacin (A) and sulfamethoxazole (B) removal from water by L. minor and/or S. molesta plants grown in monoculture or in co-culture in contaminated water. Bars represent the means ± SD of four replicates. Lowercase letters indicate significant differences between treatments for the same isolated or co-culture; uppercase letters indicate significant differences between isolated or co-cultures for the same treatment. Cipro = 1.5 µg L−1; Sulfa = 0.3 µg L−1.
Water 15 01899 g004
Water 15 01899 g005 550
Figure 5. Ciprofloxacin (A) and sulfamethoxazole (B) concentration in plant tissues of L. minor and/or S. molesta plants grown in monoculture or in co-culture in contaminated water. Bars represent the means ± SD of four replicates. Lowercase letters indicate significant differences between treatments for the same isolated or co-culture; uppercase letters indicate significant differences between isolated or co-cultures for the same treatment. Cipro = 1.5 µg L−1; Sulfa = 0.3 µg L−1; Mix (L. minor) = concentration in L. minor tissue when in co-culture with S. molesta; Mix (S. molesta) = concentration in S. molesta tissue when in co-culture with L. minor. n.d.—not detected (below detection limit).
Figure 5. Ciprofloxacin (A) and sulfamethoxazole (B) concentration in plant tissues of L. minor and/or S. molesta plants grown in monoculture or in co-culture in contaminated water. Bars represent the means ± SD of four replicates. Lowercase letters indicate significant differences between treatments for the same isolated or co-culture; uppercase letters indicate significant differences between isolated or co-cultures for the same treatment. Cipro = 1.5 µg L−1; Sulfa = 0.3 µg L−1; Mix (L. minor) = concentration in L. minor tissue when in co-culture with S. molesta; Mix (S. molesta) = concentration in S. molesta tissue when in co-culture with L. minor. n.d.—not detected (below detection limit).
Water 15 01899 g005
Table
Table 1. Ciprofloxacin (Cipro) and sulfamethoxazole (Sulfa) in the growth media at the beginning (T0) and after seven days (T7) in flasks of L. minor and S. molesta plants growing in monoculture or in co-culture (L. minor + S. molesta).
Table 1. Ciprofloxacin (Cipro) and sulfamethoxazole (Sulfa) in the growth media at the beginning (T0) and after seven days (T7) in flasks of L. minor and S. molesta plants growing in monoculture or in co-culture (L. minor + S. molesta).
TreatmentsMonocultureCo-Culture
Cipro (µg L−1)Sulfa (µg L−1)Cipro (µg L−1)Sulfa (µg L−1)
T0T7T0T7T0T7T0T7
Controln.d.n.dn.d.n.d.n.d.n.d.n.d.n.d.
Cipro1.480 ±
0.066 a		1.087 ±
0.061 b		n.d.n.d.1.550 ±
0.086 a		1.137 ±
0.128 b		n.d.n.d.
Sulfan.d.n.d.0.285 ±
0.050 a		0.142 ±
0.029 b		n.d.n.d.0.373 ±
0.064 a		0.191 ±
0.020 b
Cipro + Sulfa1.473 ±
0.061 a		1.088 ±
0.094 b		0.283 ±
0.051 a		0.130 ±
0.020 b		1.498 ±
0.050 a		1.103 ±
0.095 b		0.383 ±
0.033 a		0.117 ±
0.019 b
Notes: n.d.—not detected (below detection limit). Means that have the same letter on the line did not differ statistically (p > 0.05) by Student’s t test.
Table
Table 2. Abbot modelling to evaluate the combined toxicity of ciprofloxacin and sulfamethoxazole in L. minor and S. molesta plants growing in monoculture (monoculture) or in co-culture.
Table 2. Abbot modelling to evaluate the combined toxicity of ciprofloxacin and sulfamethoxazole in L. minor and S. molesta plants growing in monoculture (monoculture) or in co-culture.
IndexCultureSpeciesObserved
Inhibition (%)		Expected
Inhibition (%)		RIInteraction
Growth rateMonocultureL. minor78.00 ± 52.40−87.20−0.89 ± 0.60antagonistic
S. molesta60.46 ± 1.3482.42−0.44 ± 0.20antagonistic
Co-cultureL. minor88.43 ± 43.31−116.22−0.76 ± 0.37antagonistic
S. molesta47.28 ± 15.4876.880.28 ± 0.70additive
ChlorophyllsMonocultureL. minor−15.57 ± 5.394.43−1.21 ± 0.19antagonistic
S. molesta36.56 ± 6.08−58.01−0.63 ± 0.10antagonistic
Co-cultureL. minor−19.11 ± 3.1415.760.10 ± 1.04additive
S. molesta58.05 ± 10.0523.862.43 ± 0.42synergistic
H2O2MonocultureL. minor14.61 ± 15.486.932.10 ± 1.06synergistic
S. molesta−36.57 ± 5.8137.72−0.96 ± 0.15antagonistic
Co-cultureL. minor57.24 ± 42.74−51.85−1.10 ± 0.82antagonistic
S. molesta−8.68 ± 1.4015.68−0.55 ± 0.89antagonistic
MDAMonocultureL. minor2.20 ± 13.2310.780.20 ± 1.22antagonistic
S. molesta−34.04 ± 6.1029.44−1.15 ± 0.20antagonistic
Co-cultureL. minor−1.28 ± 1.180.52−2.45 ± 2.26antagonistic
S. molesta32.12 ± 6.56−1.32−24.27 ± 4.94antagonistic
APXMonocultureL. minor10.00 ± 3.28−0.18−54.57 ± 179.07antagonistic
S. molesta−44.29 ± 39.95−82.190.53 ± 0.44antagonistic
Co-cultureL. minor−74.83 ± 6.5795.96−0.77 ± 0.06antagonistic
S. molesta31.40 ± 17.25−47.90−0.62 ± 0.36antagonistic
CATMonocultureL. minor57.39 ± 10.9765.420.87 ± 0.16antagonistic
S. molesta64.57 ± 30.37−51.30−1.25 ± 0.59antagonistic
Co-cultureL. minor60.64 ± 8.2577.95−0.77 ± 0.11antagonistic
S. molesta−23.85 ± 6.8835.55−0.67 ± 0.19antagonistic
SODMonocultureL. minor−1.32 ± 6.885.22−0.25 ± 0.82antagonistic
S. molesta25.42 ± 14.24−68.55−0.37 ± 0.20antagonistic
Co-cultureL. minor−63.23 ± 1.9683.36−0.75 ± 0.02antagonistic
S. molesta−17.89 ± 2.94−31.820.55 ± 0.09antagonistic
Notes: Expected inhibition and ratio of inhibition (RI) were calculated according to Abbot’s formula. Values represented the mean ± SD (n = 4).
Table
Table 3. Abbot modelling to evaluate the combined toxicity of ciprofloxacin and sulfamethoxazole in L. minor and S. molesta plants growing in monoculture or in co-culture.
Table 3. Abbot modelling to evaluate the combined toxicity of ciprofloxacin and sulfamethoxazole in L. minor and S. molesta plants growing in monoculture or in co-culture.
TreatmentsRY L. minorRY S. molestaCBI L. minor
Control3.58 a1.59 b0.81 b
Cipro3.47 a3.20 a0.08 d
Sulfa4.13 a1.00 b1.41 a
Cipro + Sulfa3.68 a3.05 a0.18 c
Notes: Values represented the mean (n = 4). Letters indicate significant differences between treatments for the same plant species. Cipro = 1.5 µg L−1; Sulfa = 0.3 µg L−1.
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MDPI and ACS Style

Kochi, L.Y.; Kitamura, R.S.A.; Rocha, C.S.; Brito, J.C.M.; Juneau, P.; Gomes, M.P. Synergistic Removal of Ciprofloxacin and Sulfamethoxazole by Lemna minor and Salvinia molesta in Mixed Culture: Implications for Phytoremediation of Antibiotic-Contaminated Water. Water 2023, 15, 1899. https://doi.org/10.3390/w15101899

AMA Style

Kochi LY, Kitamura RSA, Rocha CS, Brito JCM, Juneau P, Gomes MP. Synergistic Removal of Ciprofloxacin and Sulfamethoxazole by Lemna minor and Salvinia molesta in Mixed Culture: Implications for Phytoremediation of Antibiotic-Contaminated Water. Water. 2023; 15(10):1899. https://doi.org/10.3390/w15101899

Chicago/Turabian Style

Kochi, Leticia Yoshie, Rafael Shinji Akiyama Kitamura, Camila Silva Rocha, Julio Cesar Moreira Brito, Philippe Juneau, and Marcelo Pedrosa Gomes. 2023. "Synergistic Removal of Ciprofloxacin and Sulfamethoxazole by Lemna minor and Salvinia molesta in Mixed Culture: Implications for Phytoremediation of Antibiotic-Contaminated Water" Water 15, no. 10: 1899. https://doi.org/10.3390/w15101899

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