# A Cyclic Graywater Treatment Model for Sustainable Wastewater Management Applied in a Small Scale
## 2 Materials and Methods
### Sampling and Analyses  
To evaluate the phytoremediating potential of quinoa, water samples were taken during two trials: one with no plants, from 29 March to 25 May 2019, and another with a quinoa plant, from 29 May to 25 August 2019. These dates were selected based on quinoa's development cycle, which typically starts in the spring. Graywater collected from the [PERSON] household on the day of irrigation and drained water from each treatment level (GWT1, GWT2, and GWT3) made up the irrigation water. Graywater from the system's several cycle treatment levels (T1, T2, and T3) was subjected to quality checks. To ensure the reliability and representativeness of the results, a comprehensive sampling strategy was employed throughout the trial period. Sampling was conducted across two distinct experimental conditions (with and without plants), three treatment levels, and four different graywater qualities (one raw graywater and three water qualities from each treatment level). This led to a total of 112 samples collected over 14 irrigation events. Following sample collection, the samples were placed in a refrigerator for chemical, biological, and microbiological analysis. At each treatment level, the samples were gathered in sterile glass bottles with a capacity of one liter. pH, soluble salts (Ca\({}^{2+}\), Mg\({}^{2+}\), Na\({}^{+}\), K\({}^{+}\), HCO\({}_{3}\)\({}^{-}\), SO\({}_{4}\)\({}^{2-}\), Cl\({}^{-}\)), heavy metals (Cd, Cu, Co, Cr, Fe, Mn, Ni, Pb, Zn), two biochemical parameters (chemical oxygen demand (COD), biological oxygen demand (BOD), electrical conductivity (ECw), and microbiological characterization, (fecal coliforms (FC)), total coliforms (TC), _Escherichia coli_ (_E. coli_), and fecal streptococci (FS)) were among the analyses. Using a direct reading pH meter and the electrometric method, the pH was measured (NF ISO 10.390 2005). A pen-type conductivity meter (model 8361, Cond. & TDS) was used to measure ECw. Complexometry was used to detect calcium (Ca\({}^{2+}\)) and magnesium (Mg\({}^{2+}\)) while ethylenediaminetetraacetic acid (EDTA) was present. To measure sodium (Na\({}^{+}\)) and potassium (K\({}^{+}\)), a JENWAY PFP7 flame photometer (NF-A20-603) was used. Titration with sulfuric acid (H\({}_{2}\)SO\({}_{4}\)), in the presence of phenolphthalein and methyl orange, was used to detect bicarbonate (HCO\({}_{3}\)\({}^{-}\)). In the  
Figure 1: Cyclic Graywater Treatment System. presence of silver nitrate (AgNO\({}_{3}\)), precipitation titration was used to quantify chloride (Cl\({}^{-}\)). Using the UV-VIS spectrometer set to 650 nm, sulfate (SO\({}_{4}\)[2]-) was measured by nephelometry in the presence of 0.1 N hydrochloric acid. Atomic absorption spectrometry (Perkin Elmer) and flame emission spectrophotometry (Jenway, PFP7) were used to measure the levels of heavy metals. Using an OxiTop (Inductive Stirring System), BOD\({}_{5}\) and COD were measured in accordance with the May 1998 version of the NF EN 1899-1 standard. Bacteriological analyses were performed at the Tunisian private laboratory MULTILAB. The Multiple Tube Fermentation method (Most Probable Number, MPN) was employed to estimate microorganism concentrations based on the number of positive tubes showing turbidity. This method was used to quantify TC, FC, FS, and _E. coli_, which are critical indicators of water quality. The number of positive tubes observed was used to determine the MPN values according to [PERSON]'s table [31]. The specific culture media and incubation conditions used for each microorganism were as follows.