Physiological Responses and Accumulation of the Emerging Contaminant Ivermectin Using Salvinia Minima

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After administration, it is partially metabolized by the animal and therefore excreted in its original form. IVM can enter water bodies through groundwater, runoff, soil erosion, and direct deposition. Once in aquatic and wetland environments, due to its chemical characteristics, can persist for a long time, increasing its environmental risk. Macrophytes are in frequent contact with this drug, resulting in chronic exposure and leading to an accumulation process. The objective of this study is to evaluate the uptake of IVM in S. minima, and its phytotoxicity potential. Bioassays were performed to expose S. minima to different concentrations of IVM, 5 mg/L, 10 mg/L, and 30 mg/L. After 10 days, the accumulation of the compound in fronds, roots, and effluent was measured. Morphological parameters and photosynthetic pigments were evaluated. IVM was found in fronds and roots of S. minima after exposure. The percentage of remotion of the drug in effluent were significantly, up to 66%. The highest concentration evaluated showed phytotoxic effects. S. minima proved to be a promising species for IVM removal processes and early toxicity marker physiological parameters, especially in wetlands subject to intensive livestock farming activities. Of interest for its applicability in wetlands subjected to intensive livestock farming. Emerging pollutant Ivermectin phytoremediation phytotoxicity macrophytes Figures Figure 1 Figure 2 Highlights Physiological parameters were assessed to determine the phytotoxicity of the emerging pollutant Ivermectin (IVM) in S. minima . Bioassays were performed in order to determine the concentration of IVM in effluent, fronds, and roots of S. minima . Negative effects were shown in fronds, roots, and the content of photosynthetic pigments. IVM was accumulated in fronds and roots of the species, the removal process of the drug was more effective at the highest concentrations. S. minima is promising for its use as a phytoremediation species for IVM removal as morphological parameters are early markers of phytotoxicity. Especially for its applicability in wetlands subjected to intensive livestock farming. 1. Introduction Livestock farming in wetlands is a common practice in the American, European, and African continents (Ballut-Dajud et al. 2022 ), with the intensification of animal production directly associated with the occurrence of parasitic diseases (Malan et al. 1997 ). Inappropriate control of endo- and ectoparasites is one of the most important causes of weight loss in livestock, hence the impact of parasitic diseases can lead to economic losses (Bianchin et al. 2007 ; Fiel and Steffan 2016 ). In this context, currently, Ivermectin (IVM) is the most widely used antiparasitic drug worldwide (Shoop and Soll 2002 ) due to it being highly effective against a wide range of nematodes and arthropods (Ottesen and Campbell 1994 ). It is a macrocyclic lactone that belongs to the avermectin family (Halley et al. 1989 ). Abamectin (ABA) is a precursor to IVM, the latter consists of a mixture of a minimum of 80% avermectin B1a, and a maximum of 20% avermectin B1b components (Fisher and Mrozik 1989 ). This veterinary drug is marketed in 3.15% and 1% formulations (Lifschitz et al. 2007 ). There are different routes of IVM administration in cattle, such as oral, topical (pour-on), and injectable (Leathwick et al. 2020 ), but the conventional injectable formulation has now been considerably replaced for the topical in livestock practices (Laffont et al. 2003 ). After administration, IVM is partially metabolized by livestock (Chiu et al. 1990 ), regardless of the route of administration, the drug is mainly eliminated via feces, and to a lesser extent, less than 2%, in urine (Gonzalez Canga et al. 2009), and up to 5% excretion has been reported during the lactation period in milk (Chicoine et al. 2007 ). Between 80 and 98% of the drug non metabolized was found in feces (Alvinerie et al. 1999 ; Carrillo Heredero et al. 2022). IVM moves from feces to the underlying soil, where is relatively persistent in dung, manure, and soil with a reported half-life of 7 days to several months (Boxall et al. 2004). Due to the high lipophilicity of this molecule, it tends to bind to soil particles, and it can bind strongly to organic materials and sediment (Halley et al. 1989 ). Consequently, IVM can enter water bodies through four routes: groundwater, runoff, soil erosion, and direct deposition, and it can persist for a long time (Mancini, 2020), which further increases its environmental risk. Several studies have demonstrated the presence of IVM in different environmental compartments, such as sediments, feces, water, amphibians, invertebrates, and plant species (Iglesias et al. 2018 ; Mesa et al. 2020 ; Iglesias et al. 2022 ). Recently, Peluso et al. ( 2023 ) found 8.03 mg/kg IVM in sediments of the Parana River lower Basin. Great interest has been lately aroused by IVM biomagnification process (Mesa et al. 2020 ), and the impact of this drug in aquatic environments (Liebig et al. 2010 ), due to the increasing livestock activity carried out in wetlands (FAO, 2020). Hence, it has been defined as an emerging pollutant (Horvat et al. 2012 ). On one hand, studies reported the presence of this drug in terrestrial and aquatic plants (Wang et al. 2019 ; Mesa et al. 2020 ). Mesa et al. ( 2017 ) carried out a study to evaluate the toxicity of IVM in cattle dung to freshwater invertebrates using mesocosm with Salvinia Sp., a common aquatic plant species in wetlands. On the other hand, the effects of abiotic stress caused by the presence of this drug on plant species have also been studied (Syslová et al. 2019 ; Navrátilová et al. 2020 ). Aquatic plants are in frequent contact with this compound, resulting in chronic exposure and leading to an accumulation process. Due to the ability of the genus Salvinia to remove a variety of organic pollutants (Mendes et al. 2021 ; Gomes et al. 2023 ), the aim of this study is to evaluate the phytotoxicity of IVM in Salvinia minima and the accumulation process of the drug performed by bioassays. 2. Materials and Methods 2.1 Chemicals and reagents Ivermectin standard (CAS-No. 70288-86-7; 94% ivermectin B1a, 2.8% ivermectin B1b; PubChem 24278497) and standard solution Abamectin Pestanal® (CAS n° 71751-41-2; PubChem 329753906) used as internal standard were acquired from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Stock solutions of IVM and ABA were prepared by serial dilution in acetone (HPLC-MS quality) (Schweitzer et al. 2010 ). They were stored at -18°C and protected from light to avoid photodegradation of the drugs. Acetonitrile (CAS-No. 75-05-08; PubChem 329755061) and Ammonium acetate (CAS-No. 631-61-8 PubChem 329765068) LiChrosolv® isocratic grade for liquid chromatography (analytical grade quality) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Ivermectin 1% FACyT® was used to perform the bioassays, the drug was supplied by FACYT Laboratorio (FACYT S.R.L., Santa Fe, Argentina). 2.2 Obtaining and acclimatization of plants Specimens of Salvinia minima were obtained from the Instituto de Technology Argentina (INTA), Buenos Aires, Argentina. Uniform and similar size macrophytes with fully expanded fronds, and no signs of chlorosis or necrosis were taken and individually washed with plenty of distilled water (Rosa et al. 2017 ). Afterwadr, plants were placed in glass containers (40 x 20 x 28 cm and 24 L capacity). The acclimatization of S. minima was carried out for 15 days in containers with nutrient medium (0.588 mg CaCl 2 /L, 0.246 mg MgSO 4 /L, 0.126 mg NaHCO 3 /L, 0.055 mg KCl/L) (Mendes et al. 2021 ), and under controlled photoperiod conditions with 12/12 h light/dark cycles and light intensity of 200 µmol m − 2 s − 1 , and temperature 25 ± 2°C. (Prado et al. 2016 ). 2.3 Exposure protocols Toxicity and accumulation of IVM to S. minima bioassays were carried out in glass mesocosm (10 x 9 x9 cm and 0.5 L capacity). Different IVM concentrations were tested, 5 mg/L; 10 mg/L, and 30 mg/L, based on experiments carried out on organic and inorganic contaminants (Liu et al. 2018 ; Polechońska et al. 2019 ; Yu et al. 2021 ; Loureiro et al. 2023 ). Test concentrations were obtained by adding the required volume of Ivermectin 1%, FACyT® in a nutrient medium. The 10 days assay was performed in triplicate, with controls of nutrient solution (Emiliani et al. 2021 ). 2.4 Measurement of physiological parameters Morphological parameters were evaluated at initial and final time of the experiment. Total floating leaf number (frond), submerged leaf (root) length (Jampeetong and Brix 2019), and fresh weight (FW) were measured. After visual assessment, leaves showing chlorosis or necrosis effects were counted (Sitarska et al. 2023 ). Morphological parameters were individually evaluated, and data were expressed as the difference between final and initial time of each system. 2.5 Determination of photosynthetic pigments Photosynthetic pigments (chlorophyll a , b , and carotenoids) were measured according to the method of Jampeetong and Brix ( 2009 ) at the end of the experiment. A sample of 0.1 g fresh fronds (FW) was homogenized with 96% ethanol and stored in darkness for 24h at 25°C. Photosynthetic pigment concentrations were calculated according to Lichtenthaler and Wellburn ( 1983 ). Concentrations of chlorophyll and carotenoids were expressed as µg/g FW. 2.6 Measure of Ivermectin concentration in mesocosms At the end of the 10-day exposure assay to increasing concentrations of IVM, the systems underwent processing to assess the drug concentration in S. minima (fronds and roots) as well as in the aqueous solution. S. minima individuals were removed and washed with abundant distilled water. Tissue samples were separated into fronds and roots. Subsequently, samples were macerated in liquid nitrogen. Liquid and solid phase extraction was performed according to the methodology proposed by Peluso et al. ( 2023 ). Biomass samples were extracted in liquid phase and then subjected to solid-phase extraction (SPE). On the other hand, 15 mL aliquots from effluent were taken, filtered, and proceeded with solid extraction. SPE was performed using C18 columns (Strata C18-E, 200 mg/ 3mL, 150 x 3 mm, Phenomenex, CA, USA) with ABA as the internal standard (Sanderson et al. 2007 ). IVM determinations were performed using an HPLC-MS system (ThermoScientific Ultimate 3000 - Thermo LTQ XL). IVM concentration in fronds and roots are expressed as mg IVM/g biomass (fronds or roots), and effluent concentrations were shown as mg IVM/L nutrient medium. According to Lazo et al. ( 2022 ) the removal efficiency (RE) of IVM was calculated using Eq. 1. \(RE=\frac{\left({C}_{i} {- C}_{f}\right)}{{\text{C}}_{i}}\) x 100 (1) The results of RE are shown as a percentage (%). 2.7 Statistics Statistical analyses were performed using the GraphPad Prism version 8.0. Program (GraphPad Software, San Diego, California, USA). The analysis of variance (One-way ANOVA) test was used to compare the raw data between control (nutrient medium) and IVM-treated samples in triplicate. It was followed by Dunnett’s multiple comparisons test at p < 0.05. To analyze the relationship between healthy and injured fronds of control, and treatments, a two-way repeated-measures ANOVA was performed, followed by a Turkey test ( p < 0.05). Differences were considered statistically significant. The results are shown as the mean of the three experimental units, and standard deviation (SD). 3. Results 3.1 Effects of Ivermectin on physiological parameters After 10 days of treatment, differences in the morphological parameters of S. minima individuals were observed compared to the control system (Table 1 ). Even though the fresh weight (FW) and the number of total fronds (NTF) were not significantly different between the control and the IVM treatments, the root length (RL) of the treatment 30 mg/L IVM was significantly different from the control (Table 1 ). At the highest IVM concentration used, the roots of S. minima were brittle. Table 1 Comparison of mean ± SD among root length (RL), fresh weight (FW) and the number of total fronds (NTF) of Salvinia minima across treatments and control ( n = 3). Different letter superscripts between columns indicate significant differences ( p < 0.05) between treatments. IVM concentration RL (cm) FW (g) NTF 0 mg/L (control) 3.1 ± 0.2 a 4.0 ± 0.3 11 ± 1 5 mg/L 3.1 ± 0.3 a 4.1 ± 0.3 12 ± 2 10 mg/L 3.2 ± 0.2 a 4.1 ± 0.1 14 ± 4 30 mg/L 2.7 ± 0.1 b 4.1 ± 0.1 10 ± 2 Significant differences were observed in the number of healthy and injured fronds (presence of chlorosis and/or necrosis) between the 30 mg/L IVM treatment and the control (Fig. 1 ). 3.2 Effects of Ivermectin on pigments Physiological parameters, such as Chlorophyll a , b , and carotenoids were not significantly different between control and 5 mg/L, or 10 mg/L IVM treatments. However, compared to the control, S. minima specimens exposed to 30 mg/L IVM have expressed significantly lower photosynthetic pigment values compared to the control (Table 2 ). Table 2 Comparison of mean ± SD of photosynthetic pigments of Salvinia minima among treatments and control ( n = 3). Different letter superscripts between columns indicate significant differences ( p < 0.05) between treatments. IVM concentration Photosynthetic pigments Chlorophyll a (µg/g FW) Chlorophyll b (µg/g FW) Carotenoids (µg/g FW) 0 mg/L (control) 526 ± 64 a 245 ± 11 a 1636 ± 159 a 5 mg/L 455 ± 101 a 238 ± 62 a 1440 ± 281 a 10 mg/L 547 ± 160 a 281 ± 92 a 1952 ± 179 a 30 mg/L 397 ± 35 b 210 ± 23 b 1378 ± 169 b 3.3 Accumulation of Ivermectin in mesocosm At the end of the 10-day exposure assay, the amount of IVM accumulated was similar in both fronds and roots. Additionally, the amount of IVM removed from the aqueous medium was close to 0.2 mg- IVM /g- biomass for all IVM treatments (Fig. 2 a and b ). On the other hand, 3.4 ± 0.8 mg IVM/L aqueous medium, 4.8 ± 0.2 mg IVM/L aqueous medium and 10.1 ± 2.2 mg IVM/L aqueous medium were observed for treatments 5, 10, and 30 mg/L respectively (Fig. 2 c). The concentration of IVM in the aqueous media decreased considerably in the 10 mg/L and 30 mg/L treatments (52 ± 2%, and 66 ± 7%, respectively; Fig. 2 d). However, the 5 mg/L treatment showed a high percentage of drug removal (Fig. 2 d). 4. Discussion Several studies have shown that IVM is a compound with the ability to persist for long periods in the environment (Halley et al. 1989 ; Boxall et al. 2004). Close contact between this contaminant and plant species has been reported (Iglesias et al. 2018 ; Wang et al. 2019 ; Mesa et al. 2020 ). However, the study of IVM toxicity to plant species is a recent field of research (Vokřál et al. 2018) and there were no studies reporting the use of plants as biomarkers for IVM contamination. The evaluation of morphological parameters was used as an indicator of the phytotoxicity in the genus Salviniaceae (Loureiro et al. 2023 ). The fact that no significant differences were found between roots length and FW was also observed in other studies. Prado et al. ( 2016 ) found no loss in FW for S. rotundifolia after exposure to chromium. Even though effects on RL in the genus Salviniaceae by exposure to contaminants such as copper (Liu et al. 2018 ) and high NaCl concentrations (Jampeetong and Brix, 2009 ) was affected, in our study no significant differences were observed, but at high IVM concentrations the roots were brittle. Although the NTF did not vary significantly, the ratio between healthy and injured fronds was increased significantly for the 30 mg/L IVM treatment. The increase in necrotic and chlorotic areas was observed from the edge toward the center of the fronds (Emiliani et al. 2021 ), indicating the presence of phytotoxic effects of IVM on S. minima . Several studies that have shown that the drug can negatively impact the physiology and yield of plants due to drug-induced changes in the transcriptome (Syslová et al. 2019 ; Navrátilová et al. 2020 ). Chlorophyll a and b play a crucial role in the photosynthesis process and are often used to assess stress in plants (Xiao-Dong et al. 2004 ) while carotenoids are light collector pigments and serve as regulators of plant growth (Sun et al. 2022 ). In our study, photosynthetic pigment concentration was significantly decreased in the 30 mg/L treatment, suggesting that IVM could cause an increase in chloride ion uptake (Syslová et al. 2019 ), consequently affecting the photosynthesis process. This inference could be supported by the positive correlation between the concentration of photosynthetic pigments and the rate of photosynthesis observed by Nichols et al. ( 2000 ). Chlorosis and necrosis are common responses to stress (Yadav 2010 ) and can lead to a limitation of photosynthetic efficiency (Jampeetong and Brix 2009 ). Therefore, the negative effects of IVM on the photosynthetic pigment production of S. minima correlated with the increase in the healthy-injured frond ratio for the 30 mg/L treatment. Although FW was not significantly affected, morphological parameters were sensitive to IVM exposure at the highest concentration. The removal capacity of organic pollutants by the genus Salviniaceae has been previously reported (da Silva Santos et al. 2020 ; Mendes et al. 2021 ; Loureiro et al. 2023 ). Mesa et al. ( 2017 ) suggest that this genus could play a role as an accumulator of IVM due to the high concentration of the drug evidenced in roots. Our study showed that S. minima exposed to high concentrations of IVM are able to remove between 30 and 70% approximately, in agreement with other studies where a similar efficiency in the removal of organic pollutants has been demonstrated (da Silva Santos et al. 2020 ; Mendes et al. 2021 ). However, we observed that S. minima has shown being more effective in the removal at high concentrations of IVM. Wang et al. ( 2019 ) found a higher concentration of IVM in leaves of Echinodorus amazonicus compared to the roots. Conversely, our study showed an elevated concentration of the compound in roots at the highest concentration, which could be explained due to root exudates that allow microbial population for enhanced degradation of organics pollutants (Jatav et al. 2015). 5. Conclusions Our study has shown that S. minima is promising for its use as a phytoremediation species for IVM, particularly in wetlands experiencing livestock intensification, presenting a range of contaminant removal between 30 and 70%, approximately. Although phytotoxic effects were observed after 10 days of exposure in the 30 mg/L treatment, these did not affect the removal capacity of the drug. Based on the physiological responses and RE% of IVM, our study suggests that S. minima is optimal to remove the drug to the range of 5 to 30 mg/L. On the other hand, the evaluation of morphological parameters and photosynthetic pigments proved to be an early marker of the health status of S. minima due to IVM exposure. Declarations 7.1 Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. 7.2 Funding This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (MSO: PICT2017 N°2982 and PICT2020 N°1073), MINCyT-ANPCyT-FONCyT for financial support (MSO: PIP Olivelli Nº0106), Program “Corredor Azul: Connecting people, nature and economies along the Paraná-Paraguay river system” from Fundación Humedales/Wetlands International, DOB Ecology and Universidad Argentina de la Empresa (UADE) (D20T02). 7.3 Competing Interests The authors have no relevant financial or non-financial interests to disclose. 7.4 Author Contributions Study conception and design were performed by Melisa Soledad Olivelli, Julieta Peluso, Carolina Mariel Aronzon, and Judith Elizabeth Lacava. Material collection was carried out by Melisa Soledad Olivelli. Bioassays and data collection were performed by Judith Elizabeth Lacava. Data analyses were performed by Melisa Soledad Olivelli and Judith Elizabeth Lacava. The first draft of the manuscript was written by Judith Elizabeth Lacava. Funding acquisition was carried out by Rubén Darío Quintana. All authors contributed to the review and editing of the manuscript. All authors read an approved the final manuscript. References Alvinerie M, Sutra JF, Galtier P, Lifschitz AL, Virkel G, Sallovitz J, Lanusse C (1999) Persistence of ivermectin in plasma and feces following administration of a sustained-release bolus to cattle. Res Vet Sci 66(1):57–61. https://doi.org/10.1053/rvsc.1998.0240 Ballut-Dajud GA, Sandoval Herazo LC, Fernández-Lambert G, Marín-Muñiz JL, López Méndez MC, Betanzo-Torres EA (2022) Factors Affecting Wetland Loss: A Review. 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Mendes EJ, Malage L, Rocha DC, Kitamura RSA, Gomes SMA, Navarro-Silva MA, Gomes MP (2021) Isolated and combined effects of glyphosate and its by-product aminomethylphosphonic acid on the physiology and water remediation capacity of Salvinia molesta . Journal of Hazardous Materials, 417. https://doi.org/10.1016/j.jhazmat.2021.125694 Mesa LM, Gutiérrez MF, Montalto L, Perez V, Lifschitz, AL (2020) Concentration and environmental fate of ivermectin in floodplain wetlands: An ecosystem approach. Science of The Total Environment, 706. https://doi.org/10.1016/j.scitotenv.2019.135692 Mesa LM, Lindt I, Negro L, Gutierrez MF, Mayora G, Montalto L, Ballent M, Lifschitz AL (2017) Aquatic toxicity of ivermectin in cattle dung assessed using microcosms. Ecotoxicol Environ Saf. 144:422-429. https://doi.org/10.1016/j.ecoenv.2017.06.016. Navrátilová M, Raisová Stuchlíková L, Skálová L, Szotáková B, Langhansová L, Podlipná R (2020) Pharmaceuticals in environment: the effect of ivermectin on ribwort plantain ( Plantago lanceolata L .). Environ Sci Pollut Res 27:31202–31210. https://doi.org/10.1007/s11356-020-09442-4 Nichols PB, Couch JD, Al-Hamdani SH (2000) Selected physiological responses of Salvinia minima to different chromium concentrations. Aquatic Botany, 68(4):313-319. https://doi.org/10.1016/S0304-3770(00)00128-5 Ottesen EA and Campbell W (1994) Ivermectin in human medicine. Journal of Antimicrobial Chemotherapy 34(2): 195–203. https://doi.org/10.1093/jac/34.2.195 Peluso J, Chehda AM, Olivelli MS, Ivanic FM, Pérez Coll CS, Gonzalez F, Valenzuela L, Rojas D, Cristos D, Butler M, Candal RJ, Aronzon CM (2023) Metals, pesticides, and emerging contaminants on water bodies from agricultural areas and the effects on a native amphibian. Environ. Res. 226(1). https://doi. org/10.1016/j.envres.2023.115692. Polechońska L, Klink A, Dambiec M (2019) Trace element accumulation in Salvinia natans from areas of various land use types. Environmental Science and Pollution Research, 26:30242–30251. https://doi.org/10.1007/s11356-019-06189-5 Prado C, Chocobar Ponce S, Pagano E, Prado FE, Rosa M (2016) Differential physiological responses of two Salvinia species to hexavalent chromium at a glance. Aquatic Toxicology, 175, 213–221. https://doi.org/10.1016/j.aquatox.2016.03.027 Rosa M, Prado C, Chocobar-Ponce S, Pagano E, Prado FE (2017) Effect of seasonality and Cr(VI) on starch-sucrose partitioning and related enzymes in floating leaves of Salvinia minima . Plant Physiology and Biochemistry, 118:1–10. https://doi.org/10.1016/j.plaphy.2017.05.014 Sanderson H, Laird B, Pope L, Brain R, Wilson C, Johnson D, Bryning G, Peregrine AS, Boxall A, Solomon K (2007) Assessment of the environmental fate and effects of ivermectin in aquatic mesocosms. Aquat Toxicol 85(4):229-240. 10.1016/j.aquatox.2007.08.011 Schweitzer N, Fink G, Ternes TA, Duis K, (2010) Effects of ivermectin-spiked cattle dung on a water–sediment system with the aquatic invertebrates Daphnia magna and Chironomus riparius . Aquatic Toxicology, 97(4):304–313. https://doi.org/10.1016/j.aquatox.2009.12.017 Shoop W and Soll M (2002) International, Chemistry, pharmacology and safety of the macrocyclic lactones: ivermectin, abamectin and eprinomectin. In: Vercruysse J and Rew RW (ed) Macrocyclic Lactones in Antiparasitic Therapy, 1st edn. USA, pp 1-29 Sitarska M, Traczewska T, Hołtra A, Zamorska-Wojdyła D, Filarowska W, Hanus-Lorenz B (2023) Removal of mercury from water by phytoremediation process with Salvinia natans (L.) All.. Environ Sci Pollut Res 30:85494–85507. https://doi.org/10.1007/s11356-023-27533-w Sun T, Rao S, Zhou X, Li L (2022) Plant carotenoids: recent advances and future perspectives. Mol Horticulture 2, 3. https://doi.org/10.1186/s43897-022-00023-2 Syslová E, Landa P, Navrátilová M, Stuchlíková LR, Matoušková P, Skálová L, Szotakov B, Vaněk T, Podlipná R (2019) Ivermectin biotransformation and impact on transcriptome in Arabidopsis thaliana . Chemosphere, 234:528–535. http://doi.org/10.1016/j.chemosphere.2019.06.102 Vocal I, Šadibolová M, Podlipná R, Lamka J, Prchal L, Sobotová D, Lokvencová K, Szotáková B, Skálová L (2019) Ivermectin environmental impact: Excretion profile in sheep and phytotoxic effect in Sinapis alba . Ecotoxicology and Environmental Safety, 169:944–949. http://doi.org/10.1016/j.ecoenv.2018.11.097 Wang D, Han B, Li S, Cao Y, Du X, Lu T (2019) Environmental fate of the anti-parasitic ivermectin in an aquatic micro-ecological system after a single oral administration. PeerJ 7:e7805. http://doi.org/10.7717/peerj.7805 Xiao-Dong H, El-Alawi Y, Penrose DM, Glick BR, Greenberg BM (2004) Responses of three grass species to creosote during phytoremediation. Environmental Pollution 130(3):453–463 . https://doi.org/10.1016/j.envpol.2003.12.018 Yadav SK (2010) Cold stress tolerance mechanisms in plants. A review. Agronomy for Sustainable Development, 30:515–527. https://doi.org/10.1051/agro/2009050 Yu H, Peng J, Cao X, Wang Y, Zhang Z, Xu Y, Qi W (2021) Effects of microplastics and glyphosate on growth rate, morphological plasticity, photosynthesis, and oxidative stress in the aquatic species Salvinia cucullata . Environmental Pollution, 279. https://doi.org/10.1016/j.envpol.2021.116900 Cite Share Download PDF Status: Published Journal Publication published 07 Oct, 2024 Read the published version in Wetlands → Version 1 posted Reviewers agreed at journal 13 May, 2024 Reviewers invited by journal 11 May, 2024 Editor invited by journal 10 May, 2024 Editor assigned by journal 10 May, 2024 First submitted to journal 09 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4384154","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":301427341,"identity":"31144d4e-f07c-4a2a-8710-43292a0ac8a8","order_by":0,"name":"Judith Elizabeth Lacava","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYBACxnYILQcXaCCopRlCGxOvhYEZQiXCVBLWwtzM/PDDzza79A232x9+usFgI7vhAPOzD/gdxmYs2duWnLvhzhlj6RyGNOMNB9iMZ+DXwmDGwHOGOXfDjRwGoJbDiRsOIDyGQwv7N8Y/Z+rTDW6kP/6dw/AfqIX9MwEtPGbMPBWHEwxuJJgBbTkA1MJDyBaeYmmZiuOGM2/kmFnnGCQbzzzMU4xXi2F7+8aPbwyq5fmADrudU2En23e8fTN+LQ0oXAMGeEzhBPIE5EfBKBgFo2AUMDAAAKefSBtchDltAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4047-7618","institution":"UNSAM: Universidad Nacional de San Martin","correspondingAuthor":true,"prefix":"","firstName":"Judith","middleName":"Elizabeth","lastName":"Lacava","suffix":""},{"id":301427342,"identity":"ec30ad98-c601-415f-92d0-2dd49fc93301","order_by":1,"name":"Melisa Soledad Olivelli","email":"","orcid":"","institution":"National University of San Martin: Universidad Nacional de San Martin","correspondingAuthor":false,"prefix":"","firstName":"Melisa","middleName":"Soledad","lastName":"Olivelli","suffix":""},{"id":301427343,"identity":"f9ab26de-cae2-4700-95a2-1b63c5879705","order_by":2,"name":"Julieta Peluso","email":"","orcid":"","institution":"National University of San Martin: Universidad Nacional de San Martin","correspondingAuthor":false,"prefix":"","firstName":"Julieta","middleName":"","lastName":"Peluso","suffix":""},{"id":301427344,"identity":"4e8cf851-3339-4a3e-a0d5-7b9800eac25b","order_by":3,"name":"Carolina Mariel Aronzon","email":"","orcid":"","institution":"National University of San Martin: Universidad Nacional de San Martin","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"Mariel","lastName":"Aronzon","suffix":""},{"id":301427345,"identity":"bb3d7bb3-b8bf-488c-bd69-bb3c9ea91154","order_by":4,"name":"Rubén Darío Quintana","email":"","orcid":"","institution":"National University of San Martin: Universidad Nacional de San Martin","correspondingAuthor":false,"prefix":"","firstName":"Rubén","middleName":"Darío","lastName":"Quintana","suffix":""}],"badges":[],"createdAt":"2024-05-07 16:02:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4384154/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4384154/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13157-024-01864-x","type":"published","date":"2024-10-07T15:56:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56940268,"identity":"15a01fc4-8f05-4397-99f0-1803eed32343","added_by":"auto","created_at":"2024-05-22 12:04:00","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":117524,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of mean ± SD of \u003cem\u003eSalvinia minima\u003c/em\u003e fronds with and without symptoms of damage in fronds among treatments and control are shown (\u003cem\u003en \u003c/em\u003e= 3). Different letter superscripts between columns indicate significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) between treatments.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4384154/v1/db9f9842d1a539e4feed3f60.jpg"},{"id":56940269,"identity":"693e732a-1b6c-4637-8a41-a29ac18fda4f","added_by":"auto","created_at":"2024-05-22 12:04:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":115300,"visible":true,"origin":"","legend":"\u003cp\u003eAccumulation of Ivermectin (shown as mean ± SD; \u003cem\u003en \u003c/em\u003e= 3) in the mesocosms, a) accumulation in fronds of \u003cem\u003eSalvinia minima\u003c/em\u003e; b) accumulation in roots of \u003cem\u003eS. minima;\u003c/em\u003e c) concentration of IVM in the aqueous media; d) Removal efficiency percentage (RE%). Different letter superscripts between columns indicate significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) between treatments.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4384154/v1/dfcc3b328b40b056b3dc8700.png"},{"id":66597105,"identity":"44f78ba0-3cd6-49c1-b0a1-23db427b70e1","added_by":"auto","created_at":"2024-10-14 16:07:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":745523,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4384154/v1/d80ca6b3-3649-4506-9ede-1d2304aa8547.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003ePhysiological Responses and Accumulation of the Emerging Contaminant Ivermectin Using Salvinia Minima\u003c/p\u003e","fulltext":[{"header":"Highlights","content":"\u003cp\u003ePhysiological parameters were assessed to determine the phytotoxicity of the emerging pollutant Ivermectin (IVM) in \u003cem\u003eS. minima\u003c/em\u003e. Bioassays were performed in order to determine the concentration of IVM in effluent, fronds, and roots of \u003cem\u003eS. minima\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eNegative effects were shown in fronds, roots, and the content of photosynthetic pigments. IVM was accumulated in fronds and roots of the species, the removal process of the drug was more effective at the highest concentrations.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. minima\u003c/em\u003e is promising for its use as a phytoremediation species for IVM removal as morphological parameters are early markers of phytotoxicity. Especially for its applicability in wetlands subjected to intensive livestock farming.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eLivestock farming in wetlands is a common practice in the American, European, and African continents (Ballut-Dajud et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), with the intensification of animal production directly associated with the occurrence of parasitic diseases (Malan et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Inappropriate control of endo- and ectoparasites is one of the most important causes of weight loss in livestock, hence the impact of parasitic diseases can lead to economic losses (Bianchin et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Fiel and Steffan \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In this context, currently, Ivermectin (IVM) is the most widely used antiparasitic drug worldwide (Shoop and Soll \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) due to it being highly effective against a wide range of nematodes and arthropods (Ottesen and Campbell \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). It is a macrocyclic lactone that belongs to the avermectin family (Halley et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Abamectin (ABA) is a precursor to IVM, the latter consists of a mixture of a minimum of 80% avermectin B1a, and a maximum of 20% avermectin B1b components (Fisher and Mrozik \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). This veterinary drug is marketed in 3.15% and 1% formulations (Lifschitz et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are different routes of IVM administration in cattle, such as oral, topical (pour-on), and injectable (Leathwick et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), but the conventional injectable formulation has now been considerably replaced for the topical in livestock practices (Laffont et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). After administration, IVM is partially metabolized by livestock (Chiu et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), regardless of the route of administration, the drug is mainly eliminated via feces, and to a lesser extent, less than 2%, in urine (Gonzalez Canga et al. 2009), and up to 5% excretion has been reported during the lactation period in milk (Chicoine et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Between 80 and 98% of the drug non metabolized was found in feces (Alvinerie et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Carrillo Heredero et al. 2022). IVM moves from feces to the underlying soil, where is relatively persistent in dung, manure, and soil with a reported half-life of 7 days to several months (Boxall et al. 2004). Due to the high lipophilicity of this molecule, it tends to bind to soil particles, and it can bind strongly to organic materials and sediment (Halley et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Consequently, IVM can enter water bodies through four routes: groundwater, runoff, soil erosion, and direct deposition, and it can persist for a long time (Mancini, 2020), which further increases its environmental risk. Several studies have demonstrated the presence of IVM in different environmental compartments, such as sediments, feces, water, amphibians, invertebrates, and plant species (Iglesias et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mesa et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Iglesias et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Recently, Peluso et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) found 8.03 mg/kg IVM in sediments of the Parana River lower Basin. Great interest has been lately aroused by IVM biomagnification process (Mesa et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and the impact of this drug in aquatic environments (Liebig et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), due to the increasing livestock activity carried out in wetlands (FAO, 2020). Hence, it has been defined as an emerging pollutant (Horvat et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn one hand, studies reported the presence of this drug in terrestrial and aquatic plants (Wang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mesa et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Mesa et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) carried out a study to evaluate the toxicity of IVM in cattle dung to freshwater invertebrates using mesocosm with \u003cem\u003eSalvinia\u003c/em\u003e Sp., a common aquatic plant species in wetlands. On the other hand, the effects of abiotic stress caused by the presence of this drug on plant species have also been studied (Syslov\u0026aacute; et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Navr\u0026aacute;tilov\u0026aacute; et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Aquatic plants are in frequent contact with this compound, resulting in chronic exposure and leading to an accumulation process. Due to the ability of the genus \u003cem\u003eSalvinia\u003c/em\u003e to remove a variety of organic pollutants (Mendes et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gomes et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the aim of this study is to evaluate the phytotoxicity of IVM in \u003cem\u003eSalvinia minima\u003c/em\u003e and the accumulation process of the drug performed by bioassays.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals and reagents\u003c/h2\u003e \u003cp\u003eIvermectin standard (CAS-No. 70288-86-7; 94% ivermectin B1a, 2.8% ivermectin B1b; PubChem 24278497) and standard solution Abamectin Pestanal\u0026reg; (CAS n\u0026deg; 71751-41-2; PubChem 329753906) used as internal standard were acquired from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).\u003c/p\u003e \u003cp\u003eStock solutions of IVM and ABA were prepared by serial dilution in acetone (HPLC-MS quality) (Schweitzer et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). They were stored at -18\u0026deg;C and protected from light to avoid photodegradation of the drugs.\u003c/p\u003e \u003cp\u003eAcetonitrile (CAS-No. 75-05-08; PubChem 329755061) and Ammonium acetate (CAS-No. 631-61-8 PubChem 329765068) LiChrosolv\u0026reg; isocratic grade for liquid chromatography (analytical grade quality) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).\u003c/p\u003e \u003cp\u003eIvermectin 1% FACyT\u0026reg; was used to perform the bioassays, the drug was supplied by FACYT Laboratorio (FACYT S.R.L., Santa Fe, Argentina).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Obtaining and acclimatization of plants\u003c/h2\u003e \u003cp\u003eSpecimens of \u003cem\u003eSalvinia minima\u003c/em\u003e were obtained from the Instituto de Technology Argentina (INTA), Buenos Aires, Argentina. Uniform and similar size macrophytes with fully expanded fronds, and no signs of chlorosis or necrosis were taken and individually washed with plenty of distilled water (Rosa et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Afterwadr, plants were placed in glass containers (40 x 20 x 28 cm and 24 L capacity). The acclimatization of \u003cem\u003eS. minima\u003c/em\u003e was carried out for 15 days in containers with nutrient medium (0.588 mg CaCl\u003csub\u003e2\u003c/sub\u003e/L, 0.246 mg MgSO\u003csub\u003e4\u003c/sub\u003e/L, 0.126 mg NaHCO\u003csub\u003e3\u003c/sub\u003e/L, 0.055 mg KCl/L) (Mendes et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and under controlled photoperiod conditions with 12/12 h light/dark cycles and light intensity of 200 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and temperature 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. (Prado et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Exposure protocols\u003c/h2\u003e \u003cp\u003eToxicity and accumulation of IVM to \u003cem\u003eS. minima\u003c/em\u003e bioassays were carried out in glass mesocosm (10 x 9 x9 cm and 0.5 L capacity). Different IVM concentrations were tested, 5 mg/L; 10 mg/L, and 30 mg/L, based on experiments carried out on organic and inorganic contaminants (Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Polechońska et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Loureiro et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Test concentrations were obtained by adding the required volume of Ivermectin 1%, FACyT\u0026reg; in a nutrient medium. The 10 days assay was performed in triplicate, with controls of nutrient solution (Emiliani et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Measurement of physiological parameters\u003c/h2\u003e \u003cp\u003eMorphological parameters were evaluated at initial and final time of the experiment. Total floating leaf number (frond), submerged leaf (root) length (Jampeetong and Brix 2019), and fresh weight (FW) were measured. After visual assessment, leaves showing chlorosis or necrosis effects were counted (Sitarska et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Morphological parameters were individually evaluated, and data were expressed as the difference between final and initial time of each system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Determination of photosynthetic pigments\u003c/h2\u003e \u003cp\u003ePhotosynthetic pigments (chlorophyll \u003cem\u003ea\u003c/em\u003e, \u003cem\u003eb\u003c/em\u003e, and carotenoids) were measured according to the method of Jampeetong and Brix (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) at the end of the experiment. A sample of 0.1 g fresh fronds (FW) was homogenized with 96% ethanol and stored in darkness for 24h at 25\u0026deg;C. Photosynthetic pigment concentrations were calculated according to Lichtenthaler and Wellburn (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). Concentrations of chlorophyll and carotenoids were expressed as \u0026micro;g/g FW.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.6 Measure of Ivermectin concentration in mesocosms\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eAt the end of the 10-day exposure assay to increasing concentrations of IVM, the systems underwent processing to assess the drug concentration in \u003cem\u003eS. minima\u003c/em\u003e (fronds and roots) as well as in the aqueous solution. \u003cem\u003eS. minima\u003c/em\u003e individuals were removed and washed with abundant distilled water. Tissue samples were separated into fronds and roots. Subsequently, samples were macerated in liquid nitrogen. Liquid and solid phase extraction was performed according to the methodology proposed by Peluso et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Biomass samples were extracted in liquid phase and then subjected to solid-phase extraction (SPE). On the other hand, 15 mL aliquots from effluent were taken, filtered, and proceeded with solid extraction. SPE was performed using C18 columns (Strata C18-E, 200 mg/ 3mL, 150 x 3 mm, Phenomenex, CA, USA) with ABA as the internal standard (Sanderson et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). IVM determinations were performed using an HPLC-MS system (ThermoScientific Ultimate 3000 - Thermo LTQ XL). IVM concentration in fronds and roots are expressed as mg IVM/g biomass (fronds or roots), and effluent concentrations were shown as mg IVM/L nutrient medium. According to Lazo et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) the removal efficiency (RE) of IVM was calculated using Eq.\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(RE=\\frac{\\left({C}_{i} {- C}_{f}\\right)}{{\\text{C}}_{i}}\\)\u003c/span\u003e \u003c/span\u003e \u003cem\u003ex\u003c/em\u003e 100 (1)\u003c/p\u003e \u003cp\u003eThe results of RE are shown as a percentage (%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistics\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using the GraphPad Prism version 8.0. Program (GraphPad Software, San Diego, California, USA). The analysis of variance (One-way ANOVA) test was used to compare the raw data between control (nutrient medium) and IVM-treated samples in triplicate. It was followed by Dunnett\u0026rsquo;s multiple comparisons test at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. To analyze the relationship between healthy and injured fronds of control, and treatments, a two-way repeated-measures ANOVA was performed, followed by a Turkey test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Differences were considered statistically significant. The results are shown as the mean of the three experimental units, and standard deviation (SD).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effects of Ivermectin on physiological parameters\u003c/h2\u003e \u003cp\u003eAfter 10 days of treatment, differences in the morphological parameters of \u003cem\u003eS. minima\u003c/em\u003e individuals were observed compared to the control system (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Even though the fresh weight (FW) and the number of total fronds (NTF) were not significantly different between the control and the IVM treatments, the root length (RL) of the treatment 30 mg/L IVM was significantly different from the control (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). At the highest IVM concentration used, the roots of \u003cem\u003eS. minima\u003c/em\u003e were brittle.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD among root length (RL), fresh weight (FW) and the number of total fronds (NTF) of \u003cem\u003eSalvinia minima\u003c/em\u003e across treatments and control (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3). Different letter superscripts between columns indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between treatments.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIVM concentration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRL (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFW (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNTF\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0 mg/L (control)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5 mg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 mg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30 mg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSignificant differences were observed in the number of healthy and injured fronds (presence of chlorosis and/or necrosis) between the 30 mg/L IVM treatment and the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effects of Ivermectin on pigments\u003c/h2\u003e \u003cp\u003ePhysiological parameters, such as Chlorophyll \u003cem\u003ea\u003c/em\u003e, \u003cem\u003eb\u003c/em\u003e, and carotenoids were not significantly different between control and 5 mg/L, or 10 mg/L IVM treatments. However, compared to the control, \u003cem\u003eS. minima\u003c/em\u003e specimens exposed to 30 mg/L IVM have expressed significantly lower photosynthetic pigment values compared to the control (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of photosynthetic pigments of \u003cem\u003eSalvinia minima\u003c/em\u003e among treatments and control (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3). Different letter superscripts between columns indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between treatments.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIVM concentration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003ePhotosynthetic pigments\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChlorophyll \u003cem\u003ea\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(\u0026micro;g/g FW)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChlorophyll \u003cem\u003eb\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(\u0026micro;g/g FW)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCarotenoids\u003c/p\u003e \u003cp\u003e(\u0026micro;g/g FW)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0 mg/L (control)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e526\u0026thinsp;\u0026plusmn;\u0026thinsp;64 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e245\u0026thinsp;\u0026plusmn;\u0026thinsp;11 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1636\u0026thinsp;\u0026plusmn;\u0026thinsp;159 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5 mg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e455\u0026thinsp;\u0026plusmn;\u0026thinsp;101 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e238\u0026thinsp;\u0026plusmn;\u0026thinsp;62 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1440\u0026thinsp;\u0026plusmn;\u0026thinsp;281 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 mg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e547\u0026thinsp;\u0026plusmn;\u0026thinsp;160 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e281\u0026thinsp;\u0026plusmn;\u0026thinsp;92 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1952\u0026thinsp;\u0026plusmn;\u0026thinsp;179 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30 mg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e397\u0026thinsp;\u0026plusmn;\u0026thinsp;35 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e210\u0026thinsp;\u0026plusmn;\u0026thinsp;23 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1378\u0026thinsp;\u0026plusmn;\u0026thinsp;169 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Accumulation of Ivermectin in mesocosm\u003c/h2\u003e \u003cp\u003eAt the end of the 10-day exposure assay, the amount of IVM accumulated was similar in both fronds and roots. Additionally, the amount of IVM removed from the aqueous medium was close to 0.2 mg-\u003cem\u003eIVM\u003c/em\u003e/g-\u003cem\u003ebiomass\u003c/em\u003e for all IVM treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cb\u003eb\u003c/b\u003e). On the other hand, 3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 mg IVM/L aqueous medium, 4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mg IVM/L aqueous medium and 10.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 mg IVM/L aqueous medium were observed for treatments 5, 10, and 30 mg/L respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The concentration of IVM in the aqueous media decreased considerably in the 10 mg/L and 30 mg/L treatments (52\u0026thinsp;\u0026plusmn;\u0026thinsp;2%, and 66\u0026thinsp;\u0026plusmn;\u0026thinsp;7%, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). However, the 5 mg/L treatment showed a high percentage of drug removal (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eSeveral studies have shown that IVM is a compound with the ability to persist for long periods in the environment (Halley et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Boxall et al. 2004). Close contact between this contaminant and plant species has been reported (Iglesias et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mesa et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the study of IVM toxicity to plant species is a recent field of research (Vokř\u0026aacute;l et al. 2018) and there were no studies reporting the use of plants as biomarkers for IVM contamination.\u003c/p\u003e \u003cp\u003eThe evaluation of morphological parameters was used as an indicator of the phytotoxicity in the genus Salviniaceae (Loureiro et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The fact that no significant differences were found between roots length and FW was also observed in other studies. Prado et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) found no loss in FW for \u003cem\u003eS. rotundifolia\u003c/em\u003e after exposure to chromium. Even though effects on RL in the genus Salviniaceae by exposure to contaminants such as copper (Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and high NaCl concentrations (Jampeetong and Brix, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) was affected, in our study no significant differences were observed, but at high IVM concentrations the roots were brittle. Although the NTF did not vary significantly, the ratio between healthy and injured fronds was increased significantly for the 30 mg/L IVM treatment. The increase in necrotic and chlorotic areas was observed from the edge toward the center of the fronds (Emiliani et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), indicating the presence of phytotoxic effects of IVM on \u003cem\u003eS. minima\u003c/em\u003e. Several studies that have shown that the drug can negatively impact the physiology and yield of plants due to drug-induced changes in the transcriptome (Syslov\u0026aacute; et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Navr\u0026aacute;tilov\u0026aacute; et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChlorophyll \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e play a crucial role in the photosynthesis process and are often used to assess stress in plants (Xiao-Dong et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) while carotenoids are light collector pigments and serve as regulators of plant growth (Sun et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In our study, photosynthetic pigment concentration was significantly decreased in the 30 mg/L treatment, suggesting that IVM could cause an increase in chloride ion uptake (Syslov\u0026aacute; et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), consequently affecting the photosynthesis process. This inference could be supported by the positive correlation between the concentration of photosynthetic pigments and the rate of photosynthesis observed by Nichols et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Chlorosis and necrosis are common responses to stress (Yadav \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and can lead to a limitation of photosynthetic efficiency (Jampeetong and Brix \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Therefore, the negative effects of IVM on the photosynthetic pigment production of \u003cem\u003eS. minima\u003c/em\u003e correlated with the increase in the healthy-injured frond ratio for the 30 mg/L treatment. Although FW was not significantly affected, morphological parameters were sensitive to IVM exposure at the highest concentration.\u003c/p\u003e \u003cp\u003eThe removal capacity of organic pollutants by the genus Salviniaceae has been previously reported (da Silva Santos et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mendes et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Loureiro et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Mesa et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) suggest that this genus could play a role as an accumulator of IVM due to the high concentration of the drug evidenced in roots. Our study showed that \u003cem\u003eS. minima\u003c/em\u003e exposed to high concentrations of IVM are able to remove between 30 and 70% approximately, in agreement with other studies where a similar efficiency in the removal of organic pollutants has been demonstrated (da Silva Santos et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mendes et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, we observed that \u003cem\u003eS. minima\u003c/em\u003e has shown being more effective in the removal at high concentrations of IVM.\u003c/p\u003e \u003cp\u003eWang et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) found a higher concentration of IVM in leaves of \u003cem\u003eEchinodorus amazonicus\u003c/em\u003e compared to the roots. Conversely, our study showed an elevated concentration of the compound in roots at the highest concentration, which could be explained due to root exudates that allow microbial population for enhanced degradation of organics pollutants (Jatav et al. 2015).\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eOur study has shown that \u003cem\u003eS. minima\u003c/em\u003e is promising for its use as a phytoremediation species for IVM, particularly in wetlands experiencing livestock intensification, presenting a range of contaminant removal between 30 and 70%, approximately. Although phytotoxic effects were observed after 10 days of exposure in the 30 mg/L treatment, these did not affect the removal capacity of the drug. Based on the physiological responses and RE% of IVM, our study suggests that \u003cem\u003eS. minima\u003c/em\u003e is optimal to remove the drug to the range of 5 to 30 mg/L. On the other hand, the evaluation of morphological parameters and photosynthetic pigments proved to be an early marker of the health status of \u003cem\u003eS. minima\u003c/em\u003e due to IVM exposure.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e7.1 Data Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7.2 Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Consejo Nacional de Investigaciones Cient\u0026iacute;ficas y T\u0026eacute;cnicas (CONICET), Agencia Nacional de Promoci\u0026oacute;n Cient\u0026iacute;fica y Tecnol\u0026oacute;gica (MSO: PICT2017 N\u0026deg;2982 and PICT2020 N\u0026deg;1073), MINCyT-ANPCyT-FONCyT for financial support (MSO: PIP Olivelli N\u0026ordm;0106), Program \u0026ldquo;Corredor Azul: Connecting people, nature and economies along the Paran\u0026aacute;-Paraguay river system\u0026rdquo; from Fundaci\u0026oacute;n Humedales/Wetlands International, DOB Ecology and Universidad Argentina de la Empresa (UADE) (D20T02).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7.3 Competing Interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7.4 Author Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy conception and design were performed by Melisa Soledad Olivelli, Julieta Peluso, Carolina Mariel Aronzon, and Judith Elizabeth Lacava. Material collection was carried out by Melisa Soledad Olivelli. Bioassays and data collection were performed by Judith Elizabeth Lacava. Data analyses were performed by Melisa Soledad Olivelli and Judith Elizabeth Lacava. The first draft of the manuscript was written by Judith Elizabeth Lacava. Funding acquisition was carried out by Rub\u0026eacute;n Dar\u0026iacute;o Quintana. All authors contributed to the review and editing of the manuscript. All authors read an approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlvinerie M, Sutra JF, Galtier P, Lifschitz AL, Virkel G, Sallovitz J, Lanusse C (1999) Persistence of ivermectin in plasma and feces following administration of a sustained-release bolus to cattle. 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Environmental Pollution, 279. https://doi.org/10.1016/j.envpol.2021.116900\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"wetlands","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wela","sideBox":"Learn more about [Wetlands](https://www.springer.com/journal/13157)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/wela/default.aspx","title":"Wetlands","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Emerging pollutant, Ivermectin, phytoremediation, phytotoxicity, macrophytes","lastPublishedDoi":"10.21203/rs.3.rs-4384154/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4384154/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIvermectin (IVM) is a macrocyclic lactone widely used to control endo- and ectoparasites in livestock. After administration, it is partially metabolized by the animal and therefore excreted in its original form. IVM can enter water bodies through groundwater, runoff, soil erosion, and direct deposition. Once in aquatic and wetland environments, due to its chemical characteristics, can persist for a long time, increasing its environmental risk. Macrophytes are in frequent contact with this drug, resulting in chronic exposure and leading to an accumulation process. The objective of this study is to evaluate the uptake of IVM in \u003cem\u003eS. minima,\u003c/em\u003e and its phytotoxicity potential. Bioassays were performed to expose \u003cem\u003eS. minima\u003c/em\u003e to different concentrations of IVM, 5 mg/L, 10 mg/L, and 30 mg/L. After 10 days, the accumulation of the compound in fronds, roots, and effluent was measured. Morphological parameters and photosynthetic pigments were evaluated. IVM was found in fronds and roots of \u003cem\u003eS. minima\u003c/em\u003e after exposure. The percentage of remotion of the drug in effluent were significantly, up to 66%. The highest concentration evaluated showed phytotoxic effects. \u003cem\u003eS. minima\u003c/em\u003e proved to be a promising species for IVM removal processes and early toxicity marker physiological parameters, especially in wetlands subject to intensive livestock farming activities. Of interest for its applicability in wetlands subjected to intensive livestock farming.\u003c/p\u003e","manuscriptTitle":"Physiological Responses and Accumulation of the Emerging Contaminant Ivermectin Using Salvinia Minima","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-22 12:03:55","doi":"10.21203/rs.3.rs-4384154/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-05-13T12:19:33+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-11T23:26:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Wetlands","date":"2024-05-10T15:21:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-10T07:16:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Wetlands","date":"2024-05-09T12:12:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"wetlands","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wela","sideBox":"Learn more about [Wetlands](https://www.springer.com/journal/13157)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/wela/default.aspx","title":"Wetlands","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3a3679cb-91fa-4f33-926b-1e36f1a4340e","owner":[],"postedDate":"May 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-14T16:00:16+00:00","versionOfRecord":{"articleIdentity":"rs-4384154","link":"https://doi.org/10.1007/s13157-024-01864-x","journal":{"identity":"wetlands","isVorOnly":false,"title":"Wetlands"},"publishedOn":"2024-10-07 15:56:56","publishedOnDateReadable":"October 7th, 2024"},"versionCreatedAt":"2024-05-22 12:03:55","video":"","vorDoi":"10.1007/s13157-024-01864-x","vorDoiUrl":"https://doi.org/10.1007/s13157-024-01864-x","workflowStages":[]},"version":"v1","identity":"rs-4384154","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4384154","identity":"rs-4384154","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}