Experimental procedures

In carrying out the aims of this study, both above-ground (tomato) and below-ground (beetroot) crops were planted, and monitored. These crops were planted in white mead trays (40 litters) at the green house facility of Waainek Research Laboratory, Rhodes University, (see Figure 1). In each crop, four mead trays were used: two for tap water irrigation served as a negative control and the two for the effluent from fly ash-lime filter tower water as an experiment. The plants were purchased from the Grahamstown nursery and were irrigated for 3-4 months.

5.3.1. Greywater analysis

The greywater (i.e., the effluent from Fly-ash Lime Filter Tower with water hyacinth modification in Chapter 3 and Chapter 4) was analysed prior to using it for irrigation of the studied crops, and for physico-chemical and microbiological analysis. The analysis included:

Ammonium (NH4 ), Phosphate (PO4 ), Nitrate (NO3 ), Chemical oxygen demand (COD), Chlorides (Cf), Sulphates (SO⁴ "), faecal coliforms, and electrical conductivity (EC).3.4.3.

The analyses were conducted using methods described in Chapter 3.4 (see microbiological analysis and 3.4.4. physicochemical).

5.3.2. Plant analysis

The number of leaves per plant were randomly selected and counted once a week for measurements to assess plant growth and leaf area. The centimetres were measure using a ruler. For dry mass of the leaves (g) the leaves were washed thoroughly with distilled water;

oven dried at 65 0C for 72 hours; and the dry mass was determined. Plant growth was measured weekly. The measured parameters included stem height, size of leaves, number of leaves, number of fruits, and dry weight. Harvested crops were assessed for micro nutrients, fresh and dry weights. The selected microelements and metals assessed here, courtesy of Chemtech Laboratory Services (Pty) Ltd, included (Aluminum (Al), Calcium (Ca), Cadmium (Cd), Chromium (Cr), Copper (Cu), Iron (Fe), Mercury (Hg), Potassium (K), Magnesium (Mg), Sodium (Na), Lead (Pb), Silicon (Si) and Strontium (Sr). Metals were extracted from the soil and fruits. The analytical procedure followed EPA Method 200.7: Based on NIOSH Method 7300, while using an Inductively Coupled Argon Plasma Optical Emission Spectrometer (ICAP-OES). Although reported in this are metal analysis, dry weight and biomass were compared.

5.3.3. Soil analysis

Soil from the mead trays of the two plants was analysed for physico-chemical and microbiological parameters and nutrient content prior and after irrigated with greywater and tap water. The physico-chemical analysis included electrical conductivity (EC), organic matter (OM), phosphorus (P), particle density (PD), bulk density (BD), total Kjeldahl nitrogen, and pH (Miller, 2010; Janssen & Koopmann, 2005; Nelson & Sommers, 1996).

pH and electrical conductivity:The measurements were done using the method described by Sikora and Kissel (2000) as standard media for leaching protons from soil: calcium chloride (CaCl2) 0.01M and potassium chloride (KCl) 1M. The amount of the soil was weighed out on the Pioneer PA214 analytical balance (Ohaus Corporation, Pinebrook, New Jersey, USA) and different variations of water and buffers were measured. The pH was measured using a glass electrode which was immersed in soil solution (slurry) of water containing different

variations on the type of solution added in weight per volume (w: v); (1:3 [soil/ water]; 1:3 [soil/0.01 M CaCl2]; 1:3 [soil/ 1M KCl]; 1:6 [soil /water]. The soil and water solution were stirred vigorously at room temperature and the slurry was allowed to set for 15 minutes. The Cyberscan 5000 pH electrode was used to measure the pH of the soil slurry.

Bulk density: Samples were taken with a core sampler which consists of a metal cylinder, sharpened on one end, and a sampler head with a handle for pushing the cylinder into the soil.

The aluminium can with lid was weighed out on the Pioneer PA214 analytical balance;

recorded; and kept in the UFE 700 oven (Memmert, Schwabach, Germany) at 105 °C for at least one day after the soil was dried; cooled; weighed; and recorded. Samples were repeatedly kept in Gallenkamp Muffle Furnace, Thermo Scientific at 400 °C overnight, after the soil was dried; cooled; weighed; and recorded. Everything was recorded in a table (See Appendix 3). The bulk density was calculated using the formula, Bulk density = mass of dry soil (g)/ volume of solids (cm ) (Refer to Appendix 1B).

Particle size density: Water was slowly added to the soil and mixed thoroughly and vigorously shaken until the flask was full. The air bubbles were allowed to rise to the surface and float foam out of the flask by adding a few more drops of water. The flask was wiped outside; dried; and weighed out on the Pioneer PA214 analytical balance. The entire content was then descanted into a waste bucket and rinse flask. The flask was refilled with water;

wiped outside; dried; and weighed. Everything was recorded in a table (See Appendix 1).

Loss on ignition (LOI): Soil Organic content of air was dried; sieved using sieves ranging from 63 to 250 pm (Fluka Sieve Standard, Sigma-Aldrich, Johannesburg, RSA); and the soil samples were determined using Loss on ignition (LOI) method (Nelson & Sommers, 1996).

The soil samples were air-dried to remove moisture and each one screened through a 2-mm soil sieve. The crucibles were put at 105 °C in a drying oven for 24 hours to remove moisture and allowed to cool in a desiccator for 4 hours. Ten grams (g) soil sample (f) was placed in porcelain crucible and dried at 105 °C for 24 hours and then ignited at 400oC in muffle furnace for 24 hours, with the resultant mass weighed out on the Pioneer PA214 analytical balance. The LOI corresponds were calculated using the following equation:

Loss Of Ignition(%) = (“m g))')x 100 Equation 5.1

Am (g (Loss of mass after ignition) = Mass of soil dried at 105oC (ms) - mass of soil ignited at 400oC (mc)

ms Mass of soil dried at 105oC mc Mass of soil ignited at 400oC

The organic matter (OM) content of the samples was measured using the weight loss on ignition method.

Phosphorus: Total phosphorus was determined using (EPA/600/R-93/100, Method 365.1) by digesting sample (0.1g/ml) using ammonium persulphate digestion (Janssen & Koopmann, 2005). Phosphorus was quantified by a spectrophotometric method involving the formation of antimony-phosphate-molybdate, yellow complex that is reduced by ascorbic acid (⁰.¹mol/l) to a blue color. Calibration curve was done using 5mg/l phosphate stock solution of KH2PO4.