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Relationship of Photosynthetic Activity of Polygonum acuminatum and Ludwigia lagunae with Physicochemical Aspects of Greywater in a Zero-Liquid Discharge System
Relationship of Photosynthetic Activity of Polygonum acuminatum and Ludwigia lagunae with...
Takahashi, Karen;Araújo, Gabriela;Pott, Vali;Yoshida, Nídia;Lima, Liana;Caires, Anderson;Paulo, Paula
2022-09-27 00:00:00
resources Article Relationship of Photosynthetic Activity of Polygonum acuminatum and Ludwigia lagunae with Physicochemical Aspects of Greywater in a Zero-Liquid Discharge System 1 1 2 3 2 4 Karen Takahashi , Gabriela Araújo , Vali Pott , Nídia Yoshida , Liana Lima , Anderson Caires 1 , and Paula Paulo * Faculty of Engineering, Architecture and Urbanism, and Geography, Federal University of Mato Grosso do Sul, Campo Grande 79090-900, MS, Brazil Bioscience Institute, Federal University of Mato Grosso do Sul, Campo Grande 79090-900, MS, Brazil Chemistry Institute, Federal University of Mato Grosso do Sul, Campo Grande 79090-900, MS, Brazil Physics Institute, Federal University of Mato Grosso do Sul, Campo Grande 79090-900, MS, Brazil * Correspondence: paula.paulo@ufms.br Abstract: Landscape harmony is a key factor in the application of nature-based solutions to provide green areas. The search for plants that meet this requirement is crucial in this context. We evaluated the adaptation, resistance, and performance of Polygonum acuminatum and Ludwigia lagunae, macrophytes from the Pantanal biome, in greywater-fed mesocosms simulating zero-liquid discharge systems. Four irrigation solutions were tested for 212 d. Neither species exhibited stress conditions in the adaptation phase, with photosynthetic activity (Fv/Fm) close to that obtained in Pantanal. However, over time, the mesocosms irrigated with greywater (GW) without nutrient supplementation exhibited stress according to correlation analyses of photosystem PSII and physicochemical parameters; L. lagunae Citation: Takahashi, K.; Araújo, G.; for dissolved oxygen below 3 mg L and P. acuminatum for water temperatures above 27 C. Pott, V.; Yoshida, N.; Lima, L.; Caires, Supplementation of GW with nutrients resulted in good growth and performance. Both species were A.; Paulo, P. Relationship of 2 1 able to receive high chemical oxygen demand (COD) loads, averaging 34 g m day for L. lagunae Photosynthetic Activity of Polygonum 2 1 and 11 g m day for P. acuminatum, with an average removal of 85% by both. L. lagunae had better acuminatum and Ludwigia lagunae evapotranspiration capacity, with greater potential for use in cooling islands, whereas P. acuminatum with Physicochemical Aspects of showed a more resistant metabolism without nutrient supplementation. Greywater in a Zero-Liquid Discharge System. Resources 2022, 11, Keywords: constructed wetlands; domestic sewage; heat islands; photosynthetic activity; water reuse 84. https://doi.org/10.3390/ resources11100084 Academic Editor: Farshid Aram Received: 23 August 2022 1. Introduction Accepted: 20 September 2022 Greywater (GW) is the fraction of domestic wastewater generated during personal and Published: 27 September 2022 home care activities (namely, laundries, sinks, showers, toilets, kitchens, and dishwashers) but excluding water from toilets, and can account for about 85% of domestic wastewater [1]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in It is a potential resource for reuse, as following low-resource-intensive treatment, greywater published maps and institutional affil- can be recycled for non-potable purposes (toilet flushing, irrigation, landscaping, and iations. recharging groundwater aquifers). The use of nature-based solutions (NBSs) to treat GW in urban areas can convert a significant fraction of wastewater into a water source to mitigate the impact of urban heat islands, flooding, and food supply, offering various ecosystem services beneficial to the Copyright: © 2022 by the authors. environment [2–4]. NBSs are techniques that mimic natural processes in urban landscapes Licensee MDPI, Basel, Switzerland. with low inputs of energy and chemicals [2]. Among the different NBSs, constructed (or This article is an open access article treatment) wetlands (CW) have been studied and applied since the 1950s and have become distributed under the terms and a consolidated and reliable technology [5], widely applied for decentralised greywater conditions of the Creative Commons treatment [4,6]. Attribution (CC BY) license (https:// Plants may perform several roles in CWs, but their direct roles can be underesti- creativecommons.org/licenses/by/ mated [7]. A recent review [8] summarised the most direct roles of the direct uptake of 4.0/). Resources 2022, 11, 84. https://doi.org/10.3390/resources11100084 https://www.mdpi.com/journal/resources Resources 2022, 11, 84 2 of 16 nutrients, such as nitrogen, phosphorous, and sulphur, and aeration by plant roots. Plants, such as aquatic macrophytes, are potential options for CW applications because of their ability to grow in wetlands and provide structures from their root systems that assist in pollutant removal processes throughout aquatic macrophyte metabolism [9–11]. Adapta- tion to greywater is of concern, as greywater might be rich in diluted chemicals used for personal and house care [2,12]. Ornamental plants are usually utilised for secondary or tertiary treatments because of the reported toxic effects of high organic/inorganic loading on plants in systems that use them for primary treatment [13], whereas our research group is pursuing the use of ornamental plants in CW units without primary treatment. By finding proper species, both resistant and ornamental, it is more probable to increase the availability of cooling islands in urban areas by treating GW in decentralised CWs. According to a literature review from Sandoval et al. (2019) [13], a literature sur- vey of 87 CWs from 21 countries showed that the four most commonly used flowering ornamental vegetation genera were Canna, Iris, Heliconia and Zantedeschia. Looking for resistant plants with an ornamental appeal, our research group has already tested Heliconia psittacorum, Cyperus isocladus, Arundina bambusifolia, Alpinia purpurata, Canna x generalis, Equisetum giganteum, Caladium Hortulanum and Hedychium coronarium [6,14,15]. Among them, Canna generalis is the most promising in terms of adaptation, growth, and survival. Recently, we started a trial with plant species from the Brazilian Pantanal [16,17], which is recognised worldwide as an important ecosystem representing the largest floodplain on the planet [18] and which accounts for a rich diversity of macrophytes with at least 280 identified species [19]. In a preliminary study, P. acuminatum, L. lagunae, and Sesbania virgata presented some bioactive compounds with antimicrobial properties (unpublished data), where the first the species showed more promising results, deserving further investi- gation. Therefore, the objective of this study was to assess the use of P. acuminatum and L. lagunae in greywater-fed mesocosms simulating zero-liquid discharge systems, focusing on the adaptation and resistance of these two species, in addition to their performance in greywater treatment. 2. Materials and Methods 2.1. Plant Species Aquatic macrophyte seedlings Polygonum acuminatum Kunth (Polygonaceae) and Ludwigia lagunae (Morong) H. Hara (Onagraceae) were collected in December 2017 in the 0 0 South Pantanal biome (19 34 37” S and 57 00 42” W). Registration number in the Brazilian national genetic patrimony management system: A60E575. Seedlings were cultivated in a 0 0 greenhouse (20 30 09” S and 54 36 44” W) in Campo Grande, Mato Grosso do Sul, Brazil. The aquatic plants were acclimatised in the greenhouse, and the species were cultivated in 12 L volume pots filled with fine gravel and irrigated with tap water. 2.1.1. Irrigation Solutions The experiment was carried out using four types of irrigation solutions: tap water (TW); tap water plus nutrients (TW*); laundry greywater (GW ) and laundry greywa- ter plus nutrients (GW *). A modified Hoagland solution was used when the irrigation solution was provided with nutrient [20]. Eleven stock solutions (SS) were prepared and different volumes were added to the irrigation solution when required. The vol- ume added of each solution and their chemical composition (g L ) were as follows: 1 1 1 SS-A (5 mL L ) Ca(NO ) 4H O: 236; SS-B (5 mL L ) KNO : 101; SS-C (2 mL L ) 3 2 2 3 1 1 MgSO 7H O: 246.5; SS-D (1 mL L ) KH PO : 136; SS-E (1 mL L ) EDTA: 13.0; SS-F 4 2 2 4 1 1 1 (1 mL L ) FeCl 6H O: 7.80; SS-G (1 mL L ) MnCl 4H O: 1.810; SS-H (1 mL L ) H BO : 3 2 2 2 3 3 1 1 2.860; SS-I (1 mL L ) ZnSO 7H O: 0.220; SS-J (1 mL L ) CuSO 5H O: 0.080 and; SS-K 4 2 4 2 (1 mL L ) H MoO : 0.020. 2 4 To provide a greywater with more stable characteristics throughout the experiment, the GW was produced in a laundry machine (WD9102RNW/XAZ Samsung 10.4 kg model) always washing two men’s blue jeans and four women’s blue jeans in room-temperature Resources 2022, 11, x FOR PEER REVIEW 3 of 17 −1 −1 −1 L ) Ca(NO3)2∙4H2O: 236; SS-B (5 mL L ) KNO3: 101; SS-C (2 mL L ) MgSO4∙7H2O: 246.5; −1 −1 −1 SS-D (1 mL L ) KH2PO4: 136; SS-E (1 mL L ) EDTA: 13.0; SS-F (1 mL L ) FeCl3∙6H2O: 7.80; −1 −1 −1 SS-G (1 mL L ) MnCl2∙4H2O: 1.810; SS-H (1 mL L ) H3BO3: 2.860; SS-I (1 mL L ) −1 −1 ZnSO4∙7H2O: 0.220; SS-J (1 mL L ) CuSO4∙5H2O: 0.080 and; SS-K (1 mL L ) H2MoO4: 0.020. To provide a greywater with more stable characteristics throughout the experiment, the GWL was produced in a laundry machine (WD9102RNW/XAZ Samsung 10.4 kg Resources 2022, 11, 84 3 of 16 model) always washing two men’s blue jeans and four women’s blue jeans in room- temperature water, using 60 mL of concentrated laundry detergent and 80 mL of softener, using the program type “cotton”. The volume produced per batch was around 85.5 L. water, using 60 mL of concentrated laundry detergent and 80 mL of softener, using the program type “cotton”. The volume produced per batch was around 85.5 L. 2.1.2. Experimental Set-Up 2.1.2.After the Experimental acclim Set-Up atization period, the seedlings were individually planted in non- transparent plastic pots of 21 cm height and top diameter and a total volume of 5.5 L. The After the acclimatization period, the seedlings were individually planted in non- pots were filled with 6 kg of fine gravel (porosity 0.44; particle size d10 = 13 mm, d30 = 11 transparent plastic pots of 21 cm height and top diameter and a total volume of 5.5 L. The mm, and d60 = 10 mm). A polyvinyl chloride tub of 20 mm diameter and 20 cm height was pots were filled with 6 kg of fine gravel (porosity 0.44; particle size d = 13 mm, d = 11 mm, 10 30 installed 5 cm from the edge. The tube (Sampling port 1—SP1) was used as a piezometer and d = 10 mm). A polyvinyl chloride tub of 20 mm diameter and 20 cm height was for measuring parameters using a probe and was also used as an irrigation water inlet installed 5 cm from the edge. The tube (Sampling port 1—SP1) was used as a piezometer point. In addition, a hose was connected to the bottom of the pot and extended externally for measuring parameters using a probe and was also used as an irrigation water inlet −1 to surface height (Figure 1) for determining evapotranspiration (for 1.5 L mesocosm ), point. In addition, a hose was connected to the bottom of the pot and extended externally and collecting effluent samples (Sampling port 2—SP2). The pots were randomly to surface height (Figure 1) for determining evapotranspiration (for 1.5 L mesocosm ), arranged and distributed into groups of four pots. and collecting effluent samples (Sampling port 2—SP2). The pots were randomly arranged and distributed into groups of four pots. Figure 1. Schematic drawing of the mesocosms. SP1: sampling port 1, piezometer located inside the Figure 1. Schematic drawing of the mesocosms. SP1: sampling port 1, piezometer located inside the mesocosm; SP2: sampling port 2, located in an external hose used to keep the mesocosm liquid level, mesocosm; SP2: sampling port 2, located in an external hose used to keep the mesocosm liquid level, for emptying and for sampling. for emptying and for sampling. The experiment was conducted in a greenhouse, situated at the university campus, The experiment was conducted in a greenhouse, situated at the university campus, to provide a controlled environment free of external influences such as wind and artificial to provide a controlled environment free of external influences such as wind and artificial lighting. A total of 24 mesocosms were used, distributed in triplicate for each type of lighting. A total of 24 mesocosms were used, distributed in triplicate for each type of irrigation and plant. The experiment was conducted in two phases: the experimental irrigation and plant. The experiment was conducted in two phases: the experimental phase totalled 212 days, with the adaptation phase occurring in the first 35 days. The adaptation phase allowed the plants to adapt to the greywater, by its gradual addition. Initially, 1.5 L of TW were added to all the mesocosms. Every 7 days, the liquid medium was depleted, and new irrigation water was added. At each water renewal, the type of irrigation water (Table 1), diluted in tap water, was gradually added, with the concentration increasing by increments of 25% each week. The mesocosms in the control group received 100% TW at every change. Resources 2022, 11, 84 4 of 16 Table 1. Composition of irrigation solutions used during Stage 1 of the experiment. Irrigation Solution Experimental Day TW TW* GW GW * L L Day 0 (0%) 100% TW 100% TW 100% TW 100% TW Day 7 (25%) 100% TW 25% TW* + 75% TW 25% GW + 75% TW 25% GW * + 75% TW L L Day 14 (50%) 100% TW 50% TW* + 50% TW 50% GW + 50% TW 50% GW * + 50% TW L L Day 21 (75%) 100% TW 75% TW* + 25% TW 75% GW + 25% TW 75% GW * + 25% TW L L Day 28 (100%) 100% TW 100% TW* 100% GW 100% GW * L L TW: tap water; TW*: tap water + nutrient; GW : laundry greywater; GW *: laundry greywater + nutrient. L L After the adaptation phase, irrigation was continued once a week, always after sample collection, with the addition of sufficient irrigation solution to make up the 1.5 L volume. Throughout the experiment, especially for plants that received nutrients, it was necessary to add irrigation water more frequently. 2.2. Measurements of Plant Growth Non-destructive measures of the height and width of stems and the number of leaves were obtained for plants irrigated with the different types of irrigation solutions (TW, TW*, GW , and GW *). The height and width (neck) of the stems were measured using L L a pachymeter. The leaves considered for counting had a length of more than 7 mm. The measurements started one month before the experiment and were carried out until the 7th day after the addition of the last concentration (35th day) of the adaptation phase, with measurements taken once a week. 2.3. Photosynthetic Activity The performance of the plants was evaluated using photosynthesis analysis. First, the photosynthetic metabolic pathway was determined to identify the metabolic type. Slides with transversally cut leaves of P. acuminatum and L. lagunae were prepared and submitted for analysis of leaf anatomy using a 100 m lens electron microscope by the Botany/Plant Anatomy Laboratory of the Federal University of Mato Grosso do Sul. Fluorescence analy- sis was performed in vivo using a portable fluorometer (FluorPen FP 100, Photo Systems Instruments, Drásov, Czech Republic) to assess the photosynthetic activity of photosystem II (PSII) and chlorophyll a. Fluorescence analysis can determine the photosynthetic perfor- mance of plants in a non-destructive manner. Measurements were made before starting the experiments (day 0) to evaluate the behaviour of the species under natural conditions in the greenhouse. After day zero, photosynthetic activity was measured daily during the first 49 days of experiment and, subsequently, measurements were made daily except during weekends. In addition, triplicate measurements were made on P. acuminatum and L. lagunae on site in the Pantanal, in the same region where the seedlings used in the experiment had been collected. Daily readings of the photosynthetic analysis were performed between 9:00 a.m. and 11:00 a.m., the time of the highest photosynthetic rates of the plants. The most visually healthy and fully developed leaves were selected for analysis. Measurements were always made on a single leaf, changing only when aging was observed or when the leaf had fallen. The leaves were subjected to dark adaptation for 20 min prior to fluorescence measure- ments. Then, the initial fluorescence (F ) was determined using low-intensity modulated 2 1 light (less than 1 mol m s ) for 50 s. The maximum fluorescence (F ) was deter- 2 1 mined with a pulse of saturating light (6000 mol m s ) of 0.8 s [21,22]. The Fv/Fm ratio was obtained from F and F (F = F F ); the ratio represents an important plant m v m 0 0 physiological parameter: the maximum quantum yield of PSII [23,24]. Resources 2022, 11, 84 5 of 16 2.4. Physicochemical Monitoring Measurements were performed weekly in two sampling stages. In the piezometer of sampling port 1, data on pH, temperature, dissolved oxygen (DO), redox potential (ORP), electrical conductivity (EC), salinity (SAL), and total dissolved solids (TDS) were analysed ® ® on site using a Hanna Edge HI2002 for pH and temperature, a Hanna Edge HI2040 for DO, an ORP meter ICEL OR-2300, and a Hanna Instruments HI2300 ET/TDS/NaCl meter (Hanna Instruments, Smithfield, RI, USA). For the second sampling stage, 200 mL samples were collected after discarding the first 30 mL to analyse chemical oxygen demand (COD) and total solids (TS). All analyses were performed according to standard methods for the examination of water and wastewater [25]. 2.5. Statistical Analysis The experimental data (plant growth measurements and photosynthetic activity (Fv/Fm) from the adaptation phase were statistically analysed to evaluate possible differ- ences in the vigour of plants irrigated with different types of irrigation solutions. Addi- tionally, the COD and TS data were analysed to determine the differences in their removal by species and types of irrigation. For this, normal distribution was verified, and variance analysis (ANOVA) followed by Tukey post hoc tests at 5% (p < 0.05) were applied. Pearson’s correlations between Fv/Fm and physicochemical parameters measured for irrigation water (inlet) and inside the mesocosms (SP1) were calculated using Minitab version 18.1. 3. Results 3.1. Plant Performance with Greywater Irrigation 3.1.1. Plant Growth Growth of the aquatic macrophyte P. acuminatum (Figure 2a–c) did not differ among the different types of irrigation water, and the greywater did not interfere with growth. In Resources 2022, 11, x FOR PEER addition, REVIEW nutrient supplementation made no difference to plant growth in6 of terms 17 of neck diameter, height, and numbers of leaves. P. acuminatum 6 400 25 a a a 20 a a 300 a a 4 a a a 15 3 200 0 0 0 TW TW* GWL GWL* TW TW* GWL GWL* TW TW* GWL GWL* (a) (b) (c) L. lagunae 6 500 50 400 40 a a ab 300 30 ab 3 b 200 20 a b 100 10 0 0 0 TW TW* GWL GWL* TW TW* GWL GWL* TW TW* GWL GWL* (d) (e) (f) Figure 2. Mean growth of P. acuminatum (a–c) and L. lagunae (d–f) plants. Data: (a,d) neck diameter Figure 2. Mean growth of P. acuminatum (a–c) and L. lagunae (d–f) plants. Data: (a,d) neck diameter (mm); (b,e) stem height (mm); and (c,f) number of leaves. TW: tap water; TW*: tap water + nutrients; (mm); GW (b L,: g e) rstem eywater; an heightd GW (mm); L*: g and reywater + (c,f) number nutrients. The lowercase of leaves. TW: tap letwater; ters indi TW*: cate sttap atisti water cal + nutri- groupings based on ANOVA and Tukey tests. Values followed by the same lowercase letter are not ents; GW : greywater; and GW *: greywater + nutrients. The lowercase letters indicate statistical L L significantly different at p < 0.05. Bars indicate the standard error of the mean. groupings based on ANOVA and Tukey tests. Values followed by the same lowercase letter are not significantly different at p < 0.05. Bars indicate the standard error of the mean. In L. lagunae, the neck (Figure 2d) was greater in plants irrigated with greywater with nutrient supplementation and did not differ from the diameters of plants irrigated with TW and TW*. The number of leaves of L. lagunae plants irrigated with TW* and GWL* was greater than that of plants where no nutrients were added to the irrigation solution (Figure 2e). The results indicated that the nutrients were important for the diameter and number of leaves of L. lagunae; they were, respectively, 59% and (at least) 40% higher than those of plants irrigated with tap water without the addition of nutrients. The evapotranspiration of P. acuminatum and L. lagunae reached maximum values in the mesocosms irrigated with −1 TW* and GWL*, reaching 0.69 (TW*) and 0.56 (GWL*) L day for P. acuminatum and 1.32 −1 (TW*) and 1.33 (GWL*) L day for L. lagunae. These values were reached between the 117th and 160th days of the experiment (Figure 3) when a high mean maximum temperature of 39.9 °C and a minimum temperature of 22.3 °C were reached in the greenhouse, with high relative humidity (93.6%). Diameter (mm) Diameter (mm) Stem (mm) Stem (mm) Leaf (number) Leaf (number) Resources 2022, 11, 84 6 of 16 In L. lagunae, the neck (Figure 2d) was greater in plants irrigated with greywater with nutrient supplementation and did not differ from the diameters of plants irrigated with TW and TW*. The number of leaves of L. lagunae plants irrigated with TW* and GW * was greater than that of plants where no nutrients were added to the irrigation solution (Figure 2e). The results indicated that the nutrients were important for the diameter and number of leaves of L. lagunae; they were, respectively, 59% and (at least) 40% higher than those of plants irrigated with tap water without the addition of nutrients. The evapotranspiration of P. acuminatum and L. lagunae reached maximum values in the mesocosms irrigated with TW* and GW *, reaching 0.69 (TW*) and 0.56 (GW *) L day for P. acuminatum and 1.32 L L (TW*) and 1.33 (GW *) L day for L. lagunae. These values were reached between the 117th and 160th days of the experiment (Figure 3) when a high mean maximum temperature of Resources 2022, 11, x FOR PEER REVIEW 7 of 17 39.9 C and a minimum temperature of 22.3 C were reached in the greenhouse, with high relative humidity (93.6%). (a) (b) (c) (d) Figure 3. Images of the plant species at the beginning of the experiment, day 7 (a,c), and day 140 Figure 3. Images of the plant species at the beginning of the experiment, day 7 (a,c), and day 140 (b,d). (b,d). Images (a,c) refer to P. acuminatum, and images b and d refer to L. lagunae, both irrigated with Images (a,c) refer to P. acuminatum, and images b and d refer to L. lagunae, both irrigated with TW*. TW*. 3.1.2. Photosynthetic Activity The leaves of P. acuminatum (Figure 4a) and L. lagunae (Figure 4b) did not show the The leaves of P. acuminatum (Figure 4a) and L. lagunae (Figure 4b) did not show the anatomy of Kranz; therefore, they were classified as C3 photosynthesis type [26]. anatomy of Kranz; therefore, they were classified as C3 photosynthesis type [26]. (a) (b) Resources 2022, 11, x FOR PEER REVIEW 7 of 17 (a) (b) (c) (d) Figure 3. Images of the plant species at the beginning of the experiment, day 7 (a,c), and day 140 (b,d). Images (a,c) refer to P. acuminatum, and images b and d refer to L. lagunae, both irrigated with TW*. Resources 2022, 11, 84 7 of 16 The leaves of P. acuminatum (Figure 4a) and L. lagunae (Figure 4b) did not show the anatomy of Kranz; therefore, they were classified as C3 photosynthesis type [26]. (a) (b) Figure 4. Microscopic image of leaf transverse section of P. acuminatum (a) and L. lagunae (b), with indication of the xylem (x) and phloem (p). The quantum efficiency of the PSII system (Fv/Fm) for P. acuminatum during the first 51 d was constant for all treatments, with Fv/Fm values of approximately 0.80. Although the average Fv/Fm during this period was below the average value obtained in situ in the Pantanal biome (0.84 0.01; Table 2), a normal function of PSII was indicated. After this period, a reduction in Fv/Fm was noted, indicating that the decrease in Fv/Fm over time was dependent on irrigation type (Figure 5a). A reduction in Fv/Fm was first detected in the GW treatment (day 54), followed by detection in the control group (TW) on day 169. It took a little longer for a drop in Fv/Fm to appear in the treatments with extra nutrients supplied; the drop was observed in the TW* treatment on day 184, and in the GW * treatment on day 202. Table 2. Average values for photosynthetic activity measured as Fv/Fm of P. acuminatum and L. lagunae plants irrigated with tap water (TW), tap water + nutrients (TW*), laundry greywater (GW ), and laundry greywater + nutrients (GW *) during the first 51 days of the experiment (number between brackets are the standard deviation of at least three replicates). Natural Habitat Tap Water Tap Water + Greywater Greywater + Plant Specie Day 0 (Pantanal) (TW) Nutrients (TW*) (GW ) Nutrients (GW *) L L P. acuminatum 0.84 0.01 0.80 0.77 0.06 0.79 0.04 0.79 0.02 0.81 0.02 L. lagunae 0.83 0.01 0.80 0.72 0.04 0.78 0.05 0.65 0.12 0.78 0.04 At the end of the experiment, the lowest photosynthetic activity was observed in plants irrigated with GW only, with the drop in Fv/Fm reaching 0.28. This indicated that irrigation with GW in zero-discharge systems was phytotoxic to the functioning of the photosystem and hence energy production, and the plant could not recover, indicating that this was due to the lack of nutrients. The same condition of irrigation with the addition of nutrients (GW *) was the one with the best performance, confirming the need for additional nutrients for the adaptation of plants to the wetland environment with zero discharge. Resources 2022, 11, x FOR PEER REVIEW 9 of 17 Resources 2022, 11, 84 8 of 16 P. acuminatum 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 TW TW* GWL GWL* 0.10 Pantanal 0.00 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 100 110 120 130 140 150 160 170 180 190 200 210 Time (days) (a) L. lagunae 0.90 0.80 0.70 0.60 0.50 0.40 0.30 TW TW* 0.20 GWL GWL* 0.10 Pantanal 0.00 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 Time (days) (b) Figure 5. Fv/Fm over 212 days of experiments for P. acuminatum (a) and L. lagunae (b). Irrigation with TW (tap water—control); TW* (tap water + nutrients); GWL Figure 5. Fv/Fm over 212 days of experiments for P. acuminatum (a) and L. lagunae (b). Irrigation with TW (tap water—control); TW* (tap water + nutrients); GW (laundry greywater); and GWL* (laundry greywater + nutrients). (laundry greywater); and GW * (laundry greywater + nutrients). Fv/Fm Fv/Fm Resources 2022, 11, 84 9 of 16 The quantum efficiency of the PSII system of L. lagunae in the control and nutrient- irrigated groups showed similar behaviours throughout the experiment (Figure 5b). Two periods were observed, each with an approximate duration of 90 days. The beginning of each period took approximately 50 days when high values of Fv/Fm were observed, followed by a fall. The first 51 days of the experiment (Table 2) showed that the three treatments with nutrients and the control had average Fv/Fm values below those obtained in-situ in the Pantanal biome. Nevertheless, the conditions with nutrients presented normal functioning of PSII. Phytotoxicity to PSII was also observed in L. lagunae irrigated with greywater without nutrient supplementation. During the first 51 days, the lowest Fv/Fm was that of the GW group, with an average of 0.73 0.04, starting to drop on day 22, and reducing over time until reaching zero on day 84. When comparing the photosystems of the groups irrigated with GW and GW *, a difference was clear, indicating that when nutrients were added, L L irrigation was sufficient, and even showed better responses than in the control and TW*. 3.1.3. Photosynthetic Activity and Physicochemical Characteristics During the 212 days of the experiment, physicochemical analyses were carried out for samples taken inside the mesocosms (piezometer, SP1) and in the outlet (SP2) every time a new batch of irrigation water was added. Table 3 shows the average values of the analysed parameters for the different irrigation water types. Table 3. Median values with standard deviation for the different solutions used to irrigate the mesocosms (the number of analysed samples are between brackets). Parameter TW TW* GW GW * L L pH 6.69 0.62 (70) 5.63 0.38 (20) 6.67 0.58 (77) 5.87 0.48 (16) Temp ( C) 26.73 3.07 (70) 24.26 3.96 (10) 22.44 4.48 (77) 24.39 2.80 (16) EC (S cm ) 56.15 8.75 (66) 2636.35 769.55 (20) 179.92 30.49 (62) 2109.40 1084.13 (16) ORP (mV) 349.53 178.36 (65) 431.00 88.11 (19) 227.04 173.64 (77) 318.87 27.49 (15) DO (mg L ) 6.12 0.67 (70) 6.59 0.73 (19) 4.93 1.44 (76) 5.78 0.70 (16) 9.43 3.21 (6) 19.87 13.74 (14) 325.72 71.37 (37) 342.79 116.12 (16) COD (mg L ) 1 na TS (mg L ) - 1926.71 413.28 (14) 240.00 75.09 (28) 1940.77 718.02 (13) TW: tap water; TW*: tap water + nutrients; GW : laundry greywater; GW : laundry greywater + nutrients; Temp: L L temperature; EC: electrical conductivity; ORP: oxy-reduction potential; DO: dissolved oxygen; COD: chemical na oxygen demand; TS: total solids. —not analysed. To better understand the influence of the variables studied, correlation analysis was performed, and only values of Spearman’s r that were above 0.70 were considered. A table with the correlation values is presented in the Supplementary Materials (Table S1). Data of L. lagunae irrigated with GW that had null Fv/Fm were not taken into account in the correlation analysis. The most significant correlation with Fv/Fm was obtained for ORP for P. acuminatum irrigated with GW (Spearman’s r 0.80). Initially, the ORP was positive (Figure 6a) and above 300 mV until day 61, when the Fv/Fm was above 0.75, and the oxy-reduction potential decreased to values below 300 mV; the Fv/Fm decreased accordingly. This drop could be related to the increase of the temperature of the GW irrigation solution (Spearman’s r 0.71), which began to increase on day 117, from 21.45 2.20 C, and continued to 27.56 2.28 C, when the ORP was below 200 mV and kept on decreasing, reaching 288.67 mV. From day 117 onwards, the temperature of all irrigation solutions increased owing to the temperature in the greenhouse, where the average minimum and maximum temperatures increased by 5 C. Even with the non-significant correlation between DO and Fv/Fm of the mesocosms irrigated with GW (Figure 6b), a decrease in DO was observed (Figure 6c) when the ORP reached negative values (146th day). Until then, the mean DO values were around 4.21 1.13 mg L , suffering a decrease around 50%, making the recovery of the photosynthesis of the plant difficult. Resources 2022, 11, x FOR PEER REVIEW 11 of 17 Resources 2022, 11, 84 10 of 16 P. acuminatum 400 1.0 40 1.0 8 1.0 100 1.0 0.8 0.8 0.8 80 0.8 200 30 6 0.6 0.6 60 0.6 0.6 0 20 4 40 0.4 0.4 0.4 0.4 0 100 200 -200 10 2 20 0.2 0.2 0.2 0.2 0 0.0 0 0.0 0 0.0 -400 0.0 0 100 200 Time (days) 0 100 200 0 100 200 Time (days) Time (days) ORP Fv/Fm-GWL TEMP Fv/Fm-I GWL DO Fv/Fm-GWL COD Fv/Fm-GWL (a) (b) (c) (d) Figure 6. Relationships of Fv/Fm of mesocosms planted with P. acuminatum irrigated with laundry greywater without nutrient supplementation (GWL), with (a) Figure 6. Relationships of Fv/Fm of mesocosms planted with P. acuminatum irrigated with laundry greywater without nutrient supplementation (GW ), with ORP; (b) temperature; (c) DO and; (d) COD. ORP: oxi-reduction potential; DO: dissolved oxygen; and, COD: chemical oxygen demand. Samples collected at the (a) ORP; (b) temperature; (c) DO and; (d) COD. ORP: oxi-reduction potential; DO: dissolved oxygen; and, COD: chemical oxygen demand. Samples collected at the piezometers (SP1) except for (b) where I: irrigation solution. piezometers (SP1) except for (b) where I: irrigation solution. ORP (mV) Temperature (ºC) -1 DO (mg L ) Fv/Fm -1 COD (mg L ) Fv/Fm Resour Resources ces 2022 2022 , 11 , 11 , 84 , x FOR PEER REVIEW 12 of 17 11 of 16 For L. lagunae, the EC and salinity parameters showed an influence on the drop in For L. lagunae, the EC and salinity parameters showed an influence on the drop in Fv/Fm of plants irrigated with GW (Figure 7a), and the most significant correlation was Fv/Fm of plants irrigated with GWL (Figure 7a), and the most significant correlation was with EC ( 0.85). with EC (−0.85). L. lagunae 800 1.0 8 1.0 0.8 0.8 600 6 0.6 0.6 400 4 0.4 0.4 200 2 0.2 0.2 0 0.0 0 0.0 0 100 200 0 100 200 Time (days) Time (days) EC Fv/Fm-GWL DO Fv/Fm-GWL (a) (b) Figure 7. Relationships of Fv/Fm of mesocosms planted with L. lagunae then irrigated with laundry Figure 7. Relationships of Fv/Fm of mesocosms planted with L. lagunae then irrigated with laun- greywater without nutrient supplementation (GWL), with (a) electrical conductivity (EC) and (b) dry greywater without nutrient supplementation (GW ), with (a) electrical conductivity (EC) and dissolved oxygen (DO). All samples collected at the piezometers (SP1). (b) dissolved oxygen (DO). All samples collected at the piezometers (SP1). −1 For GWL, initially the EC in the mesocosms were below 200 µS cm , until day 24, a For GW , initially the EC in the mesocosms were below 200 S cm , until day 24, a period when the Fv/Fm of L. lagunae varied by up to 0.70. After this period, the Fv/Fm period when the Fv/Fm of L. lagunae varied by up to 0.70. After this period, the Fv/Fm −1 decreased and the EC, reaching values of 591 µS.cm on day 83, in the same week that the decreased and the EC, reaching values of 591 S.cm on day 83, in the same week that the Fv/Fm reached zero. Fv/Fm reached zero. A correlation of Fv/Fm with dissolved oxygen was also observed in the mesocosms A correlation of Fv/Fm with dissolved oxygen was also observed in the mesocosms (Spearman’s ρ 0.78). Figure 7b shows that the decrease in DO was accompanied by a (Spearman’s r 0.78). Figure 7b shows that the decrease in DO was accompanied by a −1 decrease in Fv/Fm, and values below 5 mg L (on the 48th day of the experiment) were decrease in Fv/Fm, and values below 5 mg L (on the 48th day of the experiment) were stressful conditions for L. lagunae. From this date onwards, the DO remained at an average stressful conditions−1 for L. lagunae. From this date onwards, the DO remained at an average of 2.93 ± 0.97 mg L . Even with the low concentrations of DO, the absence of nutrients of 2.93 0.97 mg L . Even with the low concentrations of DO, the absence of nutrients was the factor that most limited the recovery of L. lagunae, since the plants irrigated with was the factor that most limited the recovery −1 of L. lagunae, since the plants irrigated with GWL* presented DO values above 3.00 mg L until the 48th day and after this period; even −1 GW * presented DO values above 3.00 mg L until the 48th day and after this period; with the average value of 1.56 ± 0.95 mg L , the Fv/Fm ratio remained similar to those in even with the average value of 1.56 0.95 mg L , the Fv/Fm ratio remained similar to the other treatments. those in the other treatments. 3.1.4. Performance of the Mesocosms for the Treatment of Greywater 3.1.4. Performance of the Mesocosms for the Treatment of Greywater Figure 8 shows the average COD (a) and TS (b) loads applied to the mesocosms and removed fro Figure 8m show the liquid s the average phase in CODth(a) e groups and TSirr (b)igated with loads applied GW to L and G the mesocosms WL* and and rcom emoved paring from the two sp the liquid ecies phase . Both p in the lan gr tsoups presented satisfacto irrigated with GW ry results and GW for COD * and and TS comparing L L removal. The groups irrigated with GWL* exhibited better removal. The highest applied the two species. Both plants presented satisfactory results for COD and TS removal. The −2 −1 COD loads occurred for the GWL* group, mainly for L. lagunae, 34.22 ± 19.91 g m day , groups irrigated with GW * exhibited better removal. The highest applied COD loads −2 −1 2 1 while P. acuminatum received 11.06 ± 4.05 g m day . The load for L. lagunae was higher occurred for the GW * group, mainly for L. lagunae, 34.22 19.91 g m day , while 2 1 because the plants that received nutrients grew more, and consequently evapotranspired P. acuminatum received 11.06 4.05 g m day . The load for L. lagunae was higher more, demanding greater frequency and volume of irrigation solution. because the plants that received nutrients grew more, and consequently evapotranspired more, demanding greater frequency and volume of irrigation solution. The trend of high loadings for L. lagunae irrigated with GW * was also observed 2 1 for TS, with values of 94.57 52.99 g m day , while that of P. acuminatum was 2 1 46.20 25.58 g m day . Regarding the removal, L. lagunae showed the best responses (Table 4), with 55% for the mesocosms irrigated with GW and 58% for the mesocosms irrigated with GW *. The set of data used for calculations are available in Table S2 as supplementary material. -1 EC (uS cm ) -1 DO (mg L ) Fv/Fm Resources 2022, 11, x FOR PEER REVIEW 13 of 17 Resources 2022, 11, 84 12 of 16 60 160 GW GW GW GW L L L L 94.57 34.22 29.94a 100 66.07a 30 80 46.20 11.06 9.38b 19.64b 6.61 5.10b 16.49 5.65 10 4.29b 12.64b 9.98 4.08b 0 0 AL RL AL RL AL RL AL RL AL RL AL RL AL RL AL RL P. acuminatum L. lagunae P. acuminatum L. lagunae P. acuminatum L. lagunae P. acuminatum L. lagunae (a) (b) 2 1 −2 −1 Figure 8. Average applied and removed loads of COD and TS (g m day ) during 212 days of Figure 8. Average applied and removed loads of COD and TS (g m day ) during 212 days of irrigation of P. acuminatum and L. lagunae with laundry greywater supplied with nutrients (GWL*) irrigation of P. acuminatum and L. lagunae with laundry greywater supplied with nutrients (GW *) and laundry greywater without nutrients (GWL). AL (applied load) based on the irrigation solution and laundry greywater without nutrients (GW ). AL (applied load) based on the irrigation solution concentration; and RL (removed load): results calculated from samples collected in the sampling concentration; and RL (removed load): results calculated from samples collected in the sampling port port 2 (SP2) representing effluent samples. Lowercase letters (a,b) indicate statistical groupings 2 (SP2) representing effluent samples. Lowercase letters (a,b) indicate statistical groupings based on based on ANOVA and Tukey tests. Values followed by the same lowercase letter are not ANOVA and Tukey tests. Values followed by the same lowercase letter are not significantly different significantly different at p < 0.05. Bars indicate standard errors. at p < 0.05. Bars indicate standard errors. The trend of high loadings for L. lagunae irrigated with GWL* was also observed for Table 4. Average percentage removal of COD and total solids load in the mesocosms irrigated with −2 −1 TS, with values of 94.57 ± 52.99 g m day , while that of P. acuminatum was 46.20 ± 25.58 laundry greywater with (GW *) and without (GW ) nutrient supplementation. L L −2 −1 g m day . Regarding the removal, L. lagunae showed the best responses (Table 4), with 55% for the mesocosms irrigated with GWL and 58% for the mesocosms irrigated with GW GW * L L GWL*. The set of data used for calculations are av a ailable in Table S2 as supplementary P. acuminatum L. lagunae P. acuminatum L. lagunae material. COD (%) 74.15 15.38 75.17 16.09 82.06 18.29 88.64 6.55 TS (%) 24.82 35.56 55.13 18.37 41.96 24.72 57.77 30.70 Table 4. Average percentage removal of COD and total solids load in the mesocosms irrigated with After day 51, the mesocosms of L. Lagunae did not contain the aerial parts of the plants. laundry greywater with (GWL*) and without (GWL) nutrient supplementation. GW 4. Discussion L GWL* P. acuminatum L. lagunae P. acuminatum L. lagunae The Pantanal aquatic macrophytes P. acuminatum and L. lagunae studied in the zero- COD (%) 74.15 ± 15.38 75.17 ± 16.09 82.06 ± 18.29 88.64 ± 6.55 liquid discharge system irrigated with greywater without nutrient supplementation did not TS (%) 24.82 ± 35.56 55.13 ± 18.37 41.96 ± 24.72 57.77 ± 30.70 exhibit stress during the adaptation phase. Both species showed strengths and weaknesses After day 51, the mesocosms of L. Lagunae did not contain the aerial parts of the plants. for use in zero-liquid discharge systems. Throughout the 212 days of the experiment, greywater without nutrient supplemen- 4. Discussion tation did not offer the minimum quantity of nutrients required to maintain the growth andThe P survival antan ofalL. aq lagunae uatic m , while acrophytes P. acuminatum P. acuminatum survived and at L. lagunae the same stconditions, udied in the z though ero- liquid discharge system irrigated with greywater without nutrient supplementation did presenting loss of vigour over time. However, with nutrient supplementation, both species not exhib showed the it sbest tressgr du owth ring and the a performance daptation p with hase. B greywater oth spinstead ecies sh ofowed st tap water ren .gths and weakne He sse alts for us hy plant e in s w zero ith C3 -liquid photo discharge s synthesis hav ye sta ems. n Fv/ Fm value of approximately 0.80 [27,28], and in plants in general it is possible to find values between 0.75 and 0.85 [29]. A decrease Throughout the 212 days of the experiment, greywater without nutrient in this ratio can be attributed to processes such as photoinhibition and UV-B stress [28]. The supplementation did not offer the minimum quantity of nutrients required to maintain correlation analysis showed that P. acuminatum was affected by the increase in temperature the growth and survival of L. lagunae, while P. acuminatum survived at the same of the irrigation water, which decreased the ORP value and decreased the availability of conditions, though presenting loss of vigour over time. However, with nutrient dissolved oxygen in the water that is necessary for root respiration, leading to a restriction in supplementation, both species showed the best growth and performance with greywater ATP production [27]. Despite these conditions, P. acuminatum remained alive, although with instead of tap water. a much lower growth rate compared to the same species in the GW nutrient-supplemented Healthy plants with C3 photosynthesis have an Fv/Fm value of approximately 0.80 mesocosms. While the temperature of the irrigation solutions with and without nutrients [27,28], and in plants in general it is possible to find values between 0.75 and 0.85 [29]. A was the same, the nutrient-supplied P. acuminatum had a greater capacity to face stress, decrease in this ratio can be attributed to processes such as photoinhibition and UV-B contributing to better vigour of the plants in the greenhouse environment and providing stress [28]. The correlation analysis showed that P. acuminatum was affected by the better conditions for metabolism. increase in temperature of the irrigation water, which decreased the ORP value and decreased the availability of dissolved oxygen in the water that is necessary for root -2 -1 COD (g.m dia ) -2 -1 TS (g.m dia ) Resources 2022, 11, 84 13 of 16 The literature indicates that for C3-type plants, the maintenance of nutritional condi- tion promotes improvements in thermal dissipation, reduction of the effective absorption of photons, and metabolic adaptations [30]. On the other hand, C3 species may be affected by stress conditions such as high temperature [30,31], water shortage [30,32], high salt con- centration [33,34] or nutritional problems [35,36]. Habermann et al. (2022) [37] identified smaller stomata and thinner mesophyll tissue in the leaves of Stylosanthes capitata (C3) and Megathyrsus maximus (C4) when exposed to elevation of temperature by 2 C. In the current study, the production of the greywater used for irrigation was uniform, using the same washing cycle, amount of clothes, and washing products; therefore, the elevation of water temperature was related to the ambient temperature. The parameter with the greatest influence on the decrease in photosynthetic activity for P. acuminatum was the ORP, but one of the greatest limitations was the temperature of the irrigation solution, meaning that the local environmental condition caused stress for P. acuminatum irrigated with GW . For L. lagunae, the main correlation of Fv/Fm was with electrical conductivity. The effects on EC of irrigating with greywater were observed by Al-Hamaiedeh and Bino (2010) [38], Pinto, Maheshwari, and Grewal (2010) [39], and Rodda et al. (2011) [40]. The increase in EC suggests that there was an accumulation of salts, particularly sodium chloride, over time. The accumulation of Na in the medium can hinder water uptake by the plant [40], affect plant function, and absorb and accumulate toxic ions in the plant [41]. For the mesocosms that received GW *, the salinity value was 9.9 5.5% NaCl. Even with salt accumulation, the presence of other nutrients (macro and micro) may have contributed to the better vigour of L. lagunae even under such conditions. For L. lagunae, the decrease in oxygen rate may have occurred because of the accumulation of Na, as it can cause sodicity in the soil [40,41], hindering plant growth and preventing soil infiltration, drainage, and diffuse atmospheric oxygen flow [41]. It is worth noting the inverse correlation of pH of the greywater irrigation with added nutrients (TW* and GW *) of 0.65 and 0.51, respectively. Although low, this correlation may reinforce the need for the addition of nutrients. The pH for both tap and laundry greywater were approximately 12% and 15% lower with nutrient supplementation, respectively. For both irrigation solutions, there was an inverse correlation, which might indicate that pH 5 is a suitable condition for L. lagunae. The literature indicates that the ideal pH to maintain available essential nutrients for the plant ranges between 5 and 6 [42]. This correlation was not observed for P. acuminatum, which presented more stable photosynthetic activity for both irrigation solutions. Both species were able to receive high COD loads, with an average of 2 1 2 1 34.22 19.91 g m day for L. lagunae and 11.06 g m day for P. acuminatum, with an average COD removal of approximately 85% for both. The mesocosms planted with L. lagunae and irrigated with GW * received a COD load higher than the range of a full-scale NBS. For instance, the recommended average load for a full-scale horizontal subsurface 2 1 flow CW is 23.71 g m day [43]. Despite the higher load, the COD removal by the mesocosms was satisfactory, with removal of 87% for the GW *-treated L. lagunae. The COD removal was also satisfactory for the other conditions, especially when compared to the full-scale process. Literature shows that, on average, a horizontal flow CW removes up to 75% COD, and a vertical flow CW removes values above 92% [43]. The lower removal rates of total solids in comparison to COD may be related to the production of mass particles in the mesocosm, such as divalent metallic sulphides produced under anaerobic conditions [11], which are favoured by the low ORP in the mesocosms. When comparing the two studied species, we can say that L. lagunae is more advanta- geous in terms of evapotranspiration capacity, with greater potential for use in NBS systems in urban areas to abate heat islands, because it presents a much larger number of leaves, whereas the leaves of P. acuminatum are thinner, longer, and fewer. The number of leaves is important for the rate of evapotranspiration and also for photosynthetic production. Evap- otranspiration can provide vegetation with the role of a natural temperature regulator [44], as blocking the sun’s rays and absorbing solar radiation can supply cooling [44,45]. Thus, Resources 2022, 11, 84 14 of 16 evapotranspiration is a relevant parameter for NBSs in local microclimates [46,47]. On the other hand, P. acuminatum had a more resistant metabolism and a higher photosynthetic capacity without nutrient supplementation, indicating that it is a more robust species under the regular greywater feeding system. We consider that the achieved results are promising, especially considering that the experiments were carried out under extreme conditions, such as non-nutrient supplementation and zero-liquid discharge systems where pollutants may accumulate. Experiments carried out with other species show that they adapt better, even flowering without nutrient supplementation, in open-air environments in traditional non-zero discharge systems, such as constructed wetlands [6,15]. The use of photosynthetic activity, a simple and low-cost method, proved to be very efficient in monitoring the plants and could be used during the maintenance of NBSs, for anticipating changes in the qualitative characteristics of the effluent due to the performance of the plants. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/resources11100084/s1, Table S1: Correlation data between Fv/Fm and physicochemical parameters for the four irrigation solutions used for L. lagunae and P. acumina- tum; Table S2. Author Contributions: Conceptualisation, K.T., P.P. and N.Y.; methodology, K.T., N.Y., L.L., A.C. and P.P.; validation, K.T. and P.P.; formal analysis, K.T.; investigation, K.T. and G.A.; resources, V.P., A.C. and P.P.; data curation, K.T. and G.A.; writing—original draft preparation, K.T. and P.P.; writing—review and editing, K.T., P.P., N.Y., L.L. and A.C.; visualisation, K.T.; supervision, P.P. and N.Y.; project administration, P.P.; funding acquisition, P.P. and N.Y. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by: National Foundation for Health-FUNASA (project No. 25100.015.578/2017-10); Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq; Fundação de Amparo à Pesquisa do Estado de Minas Gerais-FAPEMIG; Instituto Nacional de Ciência e Tecnologia em Estações Sustentáveis de Tratamento de Esgoto-INCT “ETEs Sustentáveis” (INCT “Sustainable Sewage Treatment Plants”); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES)-Finance Code 001 and PROBRAL Programme-CAPES/DAAD project No. 88881.371339/2019-01). APC was funded by the Federal University of Mato Grosso do Sul- UFMS/MEC-Brazil. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. References 1. Samayamanthula, D.R.; Sabarathinam, C.; Bhandary, H. Treatment and Effective Utilization of Greywater. Appl. Water Sci. 2019, 9, 90. [CrossRef] 2. 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