# Investigating the Effects of Fish Effluents as Organic Fertilisers on Basil (_Ocimum basilicum_)
## Appendix A
###### Abstract  
The samples were air-dried at a temperature not exceeding 30 \({}^{\circ}\)C and sieved through a 2 mm mesh. The available sulphate was extracted under controlled conditions using a phosphate buffer (ratio 1:2). The filtered extract was then analysed by Inductively Coupled Plasma Emission Spectroscopy. _DTPA-extractable manganese, iron, copper and zinc_  
The samples were air-dried at a temperature not exceeding 30 \({}^{\circ}\)C and sieved through a 2 mm mesh. Zinc, manganese, iron, and copper were extracted at 20 \({}^{\circ}\)C using a DTPA solution in a 1:2 ratio. In principle, the DTPA extraction allowed the metal in the samples to reach equilibrium with the chelating agent. A pH of 7.3 enabled DTPA to extract iron as well as other metals. _Not water-soluble boron_  
The samples were air-dried at a temperature not exceeding 30 \({}^{\circ}\)C and sieved through a 2 mm mesh. Boron availability was assessed by hot water extraction. The boron concentration in the extract was measured using ICP-OES (Inductively Coupled Plasma Optical Emission Spectroscopy). _Organic matter content by loss on ignition at 430 \({}^{\circ}\)C_  
The samples were air-dried at a temperature not exceeding 30 \({}^{\circ}\)C and sieved through a 2 mm mesh. The organic matter was destroyed by dry combustion at 430 \({}^{\circ}\)C, and the loss in weight of the samples was reported as the organic matter content (in %) on a dry matter basis. _mineral nitrogen (available N)_  
The samples were chopped and mixed to achieve a homogeneous sample. A portion was shaken with 2 M KCl to extract the mineral-N fractions, and a dry matter determination was performed. If mineralisable N was required, a second portion was incubated anaertically at 40 \({}^{\circ}\)C in water for one week to mineralise the nitrogen, followed by extraction of the mineralised fraction with KCl. Once in solution, nitrate-N, nitrite-N, and ammonium-N were measured colourimetrically. Nitrate-N and nitrite-N were determined based on the formation of a diazo compound between nitrite and sulphanilamide, which was then coupled with N-1-naphthylenediamine dihydrochloride to form a red azo dye. The colour was measured at 540 nm. In channel one, nitrate was fully reduced to nitrite by cadmium metal in an open tubular cadmium reactor (OTCR), so total oxidised nitrogen (TON) was measured as the sum of nitrite plus reduced nitrate. In channel two, only nitrite was measured. Nitrate-N was calculated by subtracting the nitrite value from the TON. For ammonium-N, in channel three, ammonium reacted with alkaline hypochlorite and phenol to produce indophenol blue. Sodium nitroprusside served as a catalyst in the formation of indophenol blue, which was measured at 640 nm. Precipitation of calcium and magnesium hydroxides was prevented by adding a combined potassium sodium tartrate/sodium citrate complexing reagent. _pH and line requirement_  
The samples were air-dried at a temperature not exceeding 30 \({}^{\circ}\)C and sieved through a 2 mm mesh. pH was defined as the pH, measured potentiometrically, of a suspension created by stirring the samples with water in a 1:2.5 ratio. As temperature influenced pH measurement, the measurement was performed in a temperature-controlled setting. _Appl. Sci._**2025**_, 1563_  
_Substrate--Olsen's extractable phosphorus_  
The samples were air-dried at a temperature not exceeding 30 \({}^{\circ}\)C and sieved through a 2 mm mesh. The available phosphorus was extracted at 20 \({}^{\circ}\)C by shaking the samples with 0.5 M sodium bicarbonate for 30 min. The phosphorus concentration was then determined by flow injection analysis/colourimetry, whereby acid ammonium molybdate formed the phosphomolybdate ion. This ion, when reduced with ascorbic acid, created a blue complex, measured spectrophotometrically at 880 nm. Calibration was carried out with commercial phosphate standards traceable to SI units.