The impact of phytoremediation conditions on the CECs removal efficiencyThe efficiency of phytoremediation depends to the greatest extent on the structure and chemical properties of the pollutants removed and the dynamics and nature of the plant’s metabolism. The well-being of the plant used, and thus the purification results, can also be influenced by the conditions of the process. In the case of the system using a floating plant, the pH of the culture medium, the daily light exposure time, and the amount of the plant per volume of purified water were considered important for the course of the process. The experimental design chemometric approach was applied to assess the impact of selected parameters on the treatment efficiency. Such an approach reduces the number of experiments necessary for selecting optimal conditions. It saves both time and energy, which meets the assumptions of green chemistry. The characteristics of the course of experiments and the values of removal efficiency of TRC, DES, and E2 obtained for successive sets of conditions are listed in Supplementary Table S3. Based on this data, separate mathematical models were developed to characterize the relationships between the RE% and the pH, the mass of plants, and the time of exposure to the light. The statistical analysis (ANOVA) results of regression models generated for TRC, DES, and E2 are included in Supplementary Tables S4–S6.The ANOVA results indicate that the obtained regression models are characterized by determination coefficients (R2) values of 0.88, 0.76, and 0.63 for DES, TRC, and E2, respectively. These values indicate that 88% of the variations in DES removal efficiency can be explained by changes in the independent variables selected for the construction of the model, and the model does not explain 12% of the observed changes. In the case of TRC and E2, the applied model explains 76% and 63% of the differences in RE%, respectively. The adjusted R2 values, which equal between 0.50 and 0.77, prove the high significance of the models. The optimization process was carried out for a mixture of pollutants, which causes more errors in the models but reflects the real conditions where pollutants do not occur individually.The Pareto analysis shows the importance of independent variables and interactions in the developed models50. Pareto charts obtained based on statistical analysis for analyzed EDCs are shown in Fig. 1.Figure 1The Pareto charts for TRC, DES, and E2 (x1 = pH; x2 = light exposure per day (h); x3 = amount of plant (g)).In Fig. 1, only those parameters whose impact on the efficiency of removing is statistically significant (p < 0.05) were included. In the case of each CEC, all three considered parameters, i.e. lighting time, pH, and amount of plant, were significant. The obtained data indicate that the removal efficiency depends on linear values of the independent variables and quadratic values and interactions between them. In the case of DES and TRC, the greatest influence on the obtained RE% value is the weight of the plant per volume unit of the purified solution. The daily light exposure time was indicated as a factor whose significance is second in the case of TRC. pH of purified solution is the second most important factor in removing DES. The RE% of E2 depends the most on pH value, followed by light exposure time.Based on CCD models that only considered statistically significant data, response surface plots (RSP) were generated. The RSPs show the influence of selecting two optimized parameters on the predicted removal efficiency. Figure 2 presents the predicted RE% values from the CCD models as a function of daily light exposure and pH.Figure 2Effects of pH and light exposure time on CECs removal efficiency (mass of the plant 2 g).The simultaneous impact of pH and plant density, as well as the plant density and light exposure time on CECs removal rates, are included in Supplementary Figs. S3 and S4.Effect of the mass of plantPlant organisms, together with microorganisms coexisting with them, are, to the greatest extent, responsible for removing pollutants during phytoremediation. Organic micropollutants in water are adsorbed on the surface of floating plants and then collected inside the plant organism, where they undergo bioaccumulation or biodegradation. Therefore, it can be expected that the greater the mass of plants in the studied system, the greater the efficiency of the purification processes51. It was observed that the RE value increases as the plant density increases from 0.5 to 2.1 g per 100 ml of purified solution. Increasing plant density above these values does not increase or even decrease the observed degree of removal. This is related to the fact that density affects the growth and quality of plants52. Too many neighboring plants disturb reproduction, biomass accumulation, and morphology due to competition between individuals53. Plant density equal to 2 g per 100 mL of the purified solution was selected as the most optimal.Effect of the light exposure timeThe length of the light period in the diurnal cycle of 24 h is an important environmental signal for plants. They have developed mechanisms to measure the length of the photoperiod. This mechanism enables plants to synchronize developmental processes, such as the onset of flowering, with a specific time of the year. It is important in regulating responses to abiotic and biotic stresses as well as the redox state of plants54. During the conducted research, the influence of light exposure time in the range of 10–16 h a day on the results was examined. It turned out that extending the light exposure time improves the efficiency of CECs removal, but only to the level of 13 h a day. Further extending the duration of light action, although it promotes the intensive course of life processes, not only does it not bring benefits but also reduces the effectiveness of processes. It can be assumed that excessive exposure to light causes abiotic stress, which disturbs metabolic processes and thus reduces the removal potential of CECs by floating plants. Finally, the photoperiod equal to 13 h per day should be considered optimal from the point of view of the system’s efficiency.Effect of the pH of purified solutionpH affects all chemical and biological processes that occur in water. This is one of the most important factors limiting the distribution and welfare of plants in aquatic habitats. Different species thrive in different pH ranges, with the optimum value for most aquatic plants being 6.5–8. Various environmental and anthropogenic factors can contribute to lowering or upgrading the pH of water outside the optimal range. The acidifying factors are acid-generating soils and rocks, industrial and agricultural wastewater, landfill leachate, and atmospheric acid precursors. High pH is less common than low pH in natural waters, as anthropogenic sources are more often acidic than alkaline. Alkalization of water may occur under the influence of alkaline rocks and soils and runoff from the production and use of asphalt, lime, cement, and soap. The influence of pH on the results of EDC removal was assessed in the range of 5–9. In the case of the considered substances, changes in pH impacted the RE values obtained, which turned out to be the highest when the pH of the culture solution was closest to neutral. It can be concluded that such conditions are optimal for small floating plants. Both too-acidic and too-alkaline living environments negatively affect aquatic plants, causing problems with osmoregulation, tissue damage, reduced growth and reproduction55. Based on the tests, a pH equal to 7 was chosen as optimal. Literature data confirm the correctness of the choice. Various studies indicate that plants from the Lemnaceae family can function well at a pH of 3.5 and 10, but the optimal pH, including growth rate and protein content, is from 6.5 to 7.556,57.Effectiveness and kinetics of CECs removal by floating plantsThe course of W. arrhiza and L. minor in removing BPA, DEET, DES, TRC, E1, and E2 from the laboratory mineral culture medium was examined under the established optimal conditions. For this purpose, the floating plant cultures were set up according to the procedure in the experimental section. The initial concentration of each CEC was 100 µg/L in the first research cycle. In addition, a second test cycle was carried out in the case of TRC, BPA, DES, and E1, in which the concentrations were 500 µg/L. Current concentrations of CECs were monitored using USAEME-GC/MS method sequentially after 6 h and 1, 2, 3, 5, 7, and 14 days of W. arrhiza or L. minor cultivation in the first cycle of research and after 1, 3, 5, and 7 days of experiment in the second cycle of research. The obtained average reduction profiles are shown in Fig. 3. A gradual loss of each CEC’s concentration was observed during the cultivation of both plants.Figure 3CECs removal efficiency observed during experiments with W. arrhiza and L. minor; first research cycle: C0 = 100 µg/L, second research cycle: C0 = 500 µg/L.The results obtained in the first research cycle (upper graphs in Fig. 3, C0 = 100 µg/L) indicate that a quite intensive reduction of the pollutants concentrations occurred already during the first hours of the experiment, especially in vessels with W. arrhiza. After 6 h of contact with the medium solution with plants, the concentration of the tested compounds decreased by 11–68% in the case of W. arrhiza and 5–32% in the case of L. minor. The reduction in concentration after 1 day ranged from 45 to 81% in the system with W. arrhiza and from 19 to 70% in the system with L. minor. Seven days of contact of the solution with the plants removes 88–98% of the initial amount of CECs in the case of W. arrhiza and 87–97% in the case of L. minor. After 14 days of the experiment, these values are 93–99.6% and 89–98%, respectively. The results obtained after the first day of the second experiment cycle (lower graphs in Fig. 3, C0 = 500 µg/L) indicate a similar or even higher reduction in concentrations compared to the first cycle in the case of W. arrhiza (62–76%) and slightly lower in the case of L. minor (12–57%). After 7 days, the degree of concentration reduction is similar to the results obtained during the first experiment (94–96% W. arrhiza; 84–93% L. minor). The analysis of literature data indicates that the efficiency of removal of organic micropollutants by floating plants depends significantly on the plant species as well as the structure and properties of the removed compound45,58,59. Studies conducted with W. arrhiza have shown that the removal efficiency of phthalates by this plant ranges from 78 to over 99%38. In the case of benzotriazoles RE is from 23 to 100%35 while for benzotriazole ultraviolet stabilizers is in the range 65–92%. More data are available in the literature regarding the removal of CECs by L. minor. Sucralose and fluoxetine are removed by this plant by 56 and 32%, respectively60; cefadroxil, metronidazole, trimethoprim, and sulfamethoxazole in 100, 96, 59 and 73%, respectively40; diclofenac, naproxen, caffeine, ibuprofen, and clofibric acid at 99, 40, 99, 44 and 16%, respectively61; for benzotriazoles the RE value ranges from 20 to 81%63. Reinhold and coworkers studied the removal of DEET and TRC by duckweed communities consisted predominantly of L. minor and Lemna punctata62. The results of their research indicate that TRC is removed by plants in 97%, which is in good agreement with the results obtained by us. Similar results of TRC removal by L. minor are presented in the paper61. However, no effect of the presence of living Lemna plants on DEET concentration was observed compared to cultures without live plants62. More than 95% reduction in the concentration of E1 and E2 (initial concentration 1 µg/L) upon contact with a plants of Lemna species was observed after 6 days in batch experiments63.By comparing the graphs ln C = f(t) and 1/C = f(t) it was determined that the rate of reduction meets the model of the pseudo-first-order kinetics. Therefore, the CECs removal rate constant (k) was determined based on the formula:$$C_{t} = \, C_{0} e^{ – kt} ,$$
(4)
where Ct and C0 are the pollutants concentrations at time t and t = 0, respectively, (µg/L). The k values (day−1) calculated for subsequent time intervals of the conducting experiments are summarized in Supplementary Tables S7 and S8. The relevant half-life values are reported in Supplementary Table S9. In the case of experiments with W. arrhiza, the highest CECs removal rate is observed during the first day of contact of the plant with the solution, and the observed k1 values range from 0.59 to 1.67 day−1. The k1 values for experiments with L. minor are in the range of 0.12–1.20 day−1, and the highest removal rate is observed for most analytes on the second day of contact with the plant. The removal rate of CECs by W. arrhiza on days 0–7 ranges from 0.30 to 0.57 day−1 in the case of a lower concentration of compounds (100 µg/L) and from 0.40 to 0.46 day−1 in the case of a higher concentration (500 µg/L). In the case of L. minor, the observed ranges are as follows: 0.29–0.98 day−1 and 0.26–0.38 day−1 for lower and higher concentrations, respectively. The results indicate that W. arrhiza immediately and without problems adapts to the presence of CECs in the medium at concentrations of 100 and 500 µg/L. In the case of L. minor, the increase in pollutant concentrations initially reduces the intensity of CECs removal. Still, the plant adapts well to the prevailing conditions in the following days. Similar behavior of both plants was observed during previous research21,22.Removal mechanism investigationRemoval of CECs during the contact of polluted waters with plants in the applied conditions of the experiment, as well as real conditions prevailing in nature, is the sum of biological and abiotic processes. Apart from uptake by the plant followed by bioconcentration and/or biodegradation, the removal of pollutants may also be caused by hydrolysis, photolysis, and sorption (evaporation can be omitted due to the low volatility of the tested compounds)40,64. Additional experiments were performed under different conditions to determine the contribution of individual mechanisms to the removal of CECs. Placing the tested solutions in the dark without plants allows the evaluation of the kinetics of the hydrolysis process. When the solution is placed under lighting conditions but without plants, the removal of CECs is the sum of hydrolysis and photolysis processes. Determination of the total share of sorption, hydrolysis, and photolysis is possible when experiments are conducted with a dead plant with access to light. In studies conducted with live plants in the light, hydrolysis, photolysis, sorption, and uptake into the plant is responsible for removing CECs. The course of experiments to determine the share of individual processes in the total effect is presented in Table 2.Table 2 Mechanisms responsible for the removal of CECs and the course of their determination.The rate constants of individual processes were determined based on the following relationships65: kuptake = kI–kII; ksorption = kII–kIII; kphotodegradation = kIII–kIV; khydrolysis = kIV (where kI, kII, kIII, kIV are the rate constants determined for experiments I, II, III, and IV). The detailed data of the rate constants for all obtained removal mechanisms are gathered in Table 3.Table 3 The values of the plant uptake, sorption, photodegradation, and hydrolysis rate constants.Based on the averaging of the determined values, it can be concluded that the largest contribution to the removal of CECs is the plant uptake, with the mean values of kuptake equal to 0.299 day−1 and 0.277 day−1 for W. arrhiza and L. minor, respectively. Interestingly, despite the similarity of both plants, the uptake of DEET by W. arrhiza is characterized by high intensity, while it has a small share in the removal of this compound by L. minor. The obtained results confirm the observations made by Reinhold and colleagues, who also did not observe a significant impact of biological processes on the efficiency of DEET removal62. Sorption is the dominant mechanism for removing BPA, E1, and E2 by W. arrhiza and DEET and E2 by L. minor. It should be emphasized that the processes of sorption and plant uptake are closely related because the pollutant must first be adsorbed on the surface of roots or leaves to be introduced into the plant organism39. The average ksorption values are about half that of kuptake and are 0.164 day−1 and 0.123 day−1 for W. arrhiza and L. minor, respectively. Hydrolysis’s contribution in removing CECs is similar to the contribution of sorption; the average value of khydrolysis for the tested compounds is 0.131 day−1. Studies have shown that DEET, TRC, BPA, DES, E1, and E2 are very slightly susceptible to photodegradation; the rate constant of this process (kphotodegradation) equals only 0.026 day−1.As part of the mechanism assessment of CECs removal, the concentrations of the tested compounds in the plant material were determined after a 7-day contact with the enriched nutrient solution containing 100 µg of each compound per 1 g of W. arrhiza or L. minor, following the procedure described in “Extraction, detection, and quantification of CECs in culture media” section. The experiment conducted in this way allows for the assessment of what part of the tested compound that has been adsorbed and taken up by the plant remains accumulated in the plant. The results of the experiment are shown in Fig. 4.Figure 4The concentration of CECs in W. arrhiza and L. minor tissues after 7-days cultivation in culture medium enriched with DEET, TRI, BPA, DES, E1, E2 (100 µg per 1 g of the plant).As can be seen in Fig. 4, no DES was detected in the plants, while the content of the remaining ECs ranged from 0.15 µg/g (BPA) to 8.17 µg/g (E2) in the case of W. arrhiza and from 0.19 µg/g (TRI) to 12.23 µg/g (E2) in the case of L. minor. Based on the rate constant data listed in Table 3, it can be calculated that, under the conditions of the experiment, the total amount of individual CECs that were adsorbed and uptake by plants ranges from 58 µg/g (E2) to 82 µg/g (DES) in the case of W. arrhiza and from 52 µg/g (DEET) to 82 µg/g (DES) in the case of L. minor (because the sum of ksorption and kuptake constitutes from 58 to 82% and from 52 to 82% of the sum of the rate constants of all processes, for W. arrhiza and L. minor, respectively). The obtained results indicate that the plant takes up the vast majority of CECs after adsorption and that only a small part of the collected organic compounds is accumulated in their original form. Organic compounds introduced into a plant cell may be immobilized there unchanged and/or undergo enzymatic modifications, enzymatic degradation, or conjugation with other compounds, mainly glucose and glutathione39. The uptake of organic compounds into plants and their further fate is affected by many factors, such as chemical hydrophobicity, molecular ionization, and the tendency of the compound to undergo sorption, which precedes the introduction of the compound into the plant65,66. The values of the logarithm of the tested CECs partition coefficients in the n-octanol/water system (log Kow) are 2.02, 4.76, 3.32, 5.07, 3.43, and 3.94. The logarithm values of the soil organic carbon adsorption coefficient (log Koc) are 1.97, 4.28, 3.18, 4.68, 3.16, and 3.52 for DEET, TRC, BPA, DES, E1, and E2, respectively67,69,70,70. In the studied group, DES is the compound with the highest log Kow and log Koc values. This compound is characterized by the highest level of sorption and uptake by plants (82% of total removal), and the most intense transformations in the plant (no presence in unchanged form in the tissues of W. arrhiza and L. minor). DEET, characterized by the lowest hydrophobicity and tendency to sorption, is much less subject to both uptake by the plant and changes inside it. However, there is no strict correlation between the log Kow and log Koc values and the fate of CECs because other factors, such as molecular volume, spatial structure, etc., also influence the processes. Ionization has also been shown to influence the plant’s uptake and modifications of organic compounds because charged molecules have a reduced ability to permeate cell membranes71. The values of the negative logarithm of the dissociation constants (pKa) of the tested compounds are 7.9, 9.6, 9.1, 10.8, and 10.7, respectively, for TRC, BPA, DES, E1, and E2 (no data for DEET)67,68. Under the experimental conditions (pH 7), the tested compounds are in a non-ionized form, which is beneficial for phytoremediation.Influence of the matrix on the course of phytoremediationExperiments were carried out using raw municipal wastewater, treated municipal wastewater, and raw landfill leachate enriched with a mixture of TRC, BPA, DES, and E1 at 100 µg/L each as a culture medium, maintaining optimal conditions used in previous cultures. The study lasted 7 days; the concentrations of the tested compounds were determined before the start of the experiment and after 1, 3, 5, and 7 days of contact of the plant with the solution. The CECs removal efficiency obtained using real matrices is shown in Fig. 5.Figure 5The removal efficiency of CECs registered after 1, 3, 5 and 7 days of contact of raw municipal wastewater, treated municipal wastewater and landfill leachates with W. arrhiza (a), L. minor (b).In the case of raw wastewater, the average removal efficiency of the tested compounds after 7 days of treatment was 84% and 75%; in the case of treated wastewater, 93% and 89%, and in the case of landfill leachate, 59% and 56%, for W. arrhiza and L. minor, respectively. The observed effectiveness of W. arrhiza and L. minor is similar for each matrix used, with a slight advantage for W. arrhiza. A worse removal efficiency by L. minor is visible mainly in the first days of the experiment. This may indicate a more difficult acclimatization of this plant to the presence of a highly contaminated matrix. The high efficiency of CECs removal from raw wastewater indicates the resistance of the tested plants to the presence of pollutants. Raw municipal wastewater contains certain amounts of nutrients (N and P, see Table S1, Supplementary Material) that support the growth and development of plants. Additionally, there are high concentrations of easily digestible organic compounds, including carbohydrates, fats, and proteins, which may also positively affect the functioning of plants from the Lemnaceae family, given their well-documented ability to provide mixotrophic nutrition71. The observed ability of floating plants to remove CECs from landfill leachates is significantly lower than in the case of municipal wastewater. Leachate is a liquid that is a toxic cocktail containing high concentrations of pollutants such as soluble organic matter, inorganic components, heavy metals, and xenobiotic organic compounds72. Leachates are characterized by high phytotoxicity73, so it can be assumed that the removal of the tested compounds during the experiment is carried out to a small extent by biological processes, mainly by sorption, hydrolysis, and photolysis.
Removal of contaminants of emerging concern by Wolffia arrhiza and Lemna minor depending on the process conditions, pollutants concentration, and matrix type
