Analytical procedures

Detailed analytical procedures for water, sediment, water hyacinth, and fish samples are pro- vided in the subsequent sub-sections. Irrespective of any ecosystem component, all analyses were performed in triplicates under controlled laboratory supervision.

▪ Water samples

Two pre-cleaned 1L HDPE plastic bottles were considered for the collection from each point;

one was utilized for heavy metal analyses; thus, it was acidified with concentrated HNO3 to render the pH below 2 to avoid any precipitation, and the other was non-acidified, which was utilized for various physico-chemical parameters. Specific parameters such as pH and DO were analyzed at the earliest (within 24 hours) after sampling was completed, and the re- maining samples were kept in a refrigerator at 4°C till further analyses of the remaining pa- rameters were carried out. A quality control procedure was maintained throughout. Standard solutions and reagent blanks were prepared under controlled laboratory conditions to cali- brate instruments prior to any analyses.

Table 4. 2. Different water quality parameters analyzed in the research, their units of measurement and make and model of the instruments used.

Parame- ters

Units of measure-

ment Analytical method [Make and model]

DO mg L^-1 Winkler's method

pH pH units Digital pH meter [Systronics - µ pH system 361]

EC µS cm^-1 Digital Conductivity meter [VSI-04-Deluxe]

Turbidity NTU Nephelometric Turbidimeter [Systronics]

TA mg L^-1 as CaCO3 APHA titrimetric method TH mg L^-1 as CaCO³ APHA titrimetric method

BOD5 mg L^-1 5-day BOD test

COD mg L^-1 Closed Reflux, Titrimetric Method

TDS mg L^-1

Oven Drying at 103-105˚C

TSS mg L^-1

F^- mg L^-1

Ion Chromatograph (IC) [792 Basic IC – Metrohm]

Cl^- mg L^-1 NO3- mg L^-1 PO43- mg L^-1 SO42- mg L^-1 Na⁺ mg L^-1

Flame Photometer [Systronics]

K⁺ mg L^-1

Ca²⁺ mg L^-1

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TKN mg L^-1 APHA Distillation Method [Pelican - Kelplus Distillation Ap- paratus]

Mg mg L^-1

Atomic Absorption Spectroscopy (AAS) [Varian Spectra 55B]

Cr mg L^-1

Cd mg L^-1

Fe mg L^-1

Mn mg L^-1

Cu mg L^-1

Pb mg L^-1

NH3- mg L^-1 Semi-automated colorimetric method

OrgN mg L^-1 Kjeldahl method

The reagents were prepared following the Standard Methods for the Examination of Wa- ter and Wastewater (APHA 2012). Analytical grade chemicals were used for analysis unless otherwise stated. Deionized water was used for dilutions done during the analysis. Standard solutions were prepared by diluting the stock solutions. For analyzing heavy metals, cations and anions, the samples were first subjected to micro-filtration using Whatman (No. 42) filter papers. Sample collection, transportation, preservation, and analyses adhered to Standard Methods (APHA 2012). The water samples were subjected to analyses for 28 different water quality parameters; detailed analytical procedure and the make and model of the instruments used for the analyses are shown in Table 4. 2.

▪ Sediment samples

For the analysis of sediment samples, the samples needed to be first dried completely, sub- jected to the oven-drying process. They were then sieved through a 2mm Indian Standard (IS) sieve, which helped remove debris. They were then converted to powdered forms after crush- ing, grinding, and subsequently sieving through a 75µ IS sieve. The powdered samples were then subjected to various analyses, i.e., heavy metals and nutrients. For heavy metal analyses, the powdered samples were digested by using a mixture of concentrated HNO3 (10 mL) + HF (2 mL) + HClO4 (2 mL) for a duration of 4h at a temperature of 400°C. The digested filtrates were then transferred to respective specimen tubes after diluting them to 100mL using milli- Q water and subsequently filtering them. The analyses for heavy metals were then performed through Atomic Absorption Spectroscopy (AAS). Sediment samples were also analyzed for different forms of nitrogen and phosphorus. Total Kjeldahl nitrogen, ammonia nitrogen and nitrate nitrogen in sediments were measured following the procedures described by Motsara and Roy (2008). While ammonia nitrogen was measured using the indophenol-blue method, nitrate-nitrogen was measured following the phenol-disulphonic acid method. Total phos- phorus in soil was measured following the stannous chloride method after acid digestion.

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▪ Water hyacinth samples

While collecting water hyacinth samples, care was taken to ensure that they had fewer possi- ble mobility options during sampling because of their location. This ensured that the plants' measured nutrient uptake was a proper representative of the plants' actual nutrient uptake.

The collected samples were then analyzed for different forms of nitrogen and phosphorus, the details of which are as follows:

a. Total Kjeldahl nitrogen

The Kjeldahl method was used to determine the total Kjeldahl nitrogen present in the plant.

The air-dried and finely ground samples were digested using potassium sulphate (K2SO4), copper sulphate (CuSO4) and sulphuric acid (H2SO4). The digested samples were then distilled using the APHA Distillation method. The distilled samples were then titrated using 0.02N H2SO4. Organic nitrogen was calculated by subtracting ammonia nitrogen from total Kjeldahl nitrogen.

b. Ammonia nitrogen

Ammonia nitrogen was determined following the Phenate method described in section 4500- NH3.F in Standard Methods for the Examination of Water and Wastewater (APHA 2012) after extraction from finely ground air-dried plant samples using potassium chloride (Motsara &

Roy 2008).

c. Nitrate nitrogen

Nitrate nitrogen was determined following the procedure described in section 4500-NO3-.B of Standard Methods for the Examination of Water and Wastewater (APHA 2012). Similar to the analysis of ammonia nitrogen, the air-dried and finely ground plant samples were first subjected to the potassium chloride extraction method (Motsara & Roy 2008) and subse- quently analyzed in the UV spectrophotometer at 220 nm and 275 nm wavelengths.

d. Total phosphorus

Total phosphorus in the plant samples was determined by first digesting the plant samples using a mixed acid of sulphuric acid (H2SO4) and perchloric acid (HClO4) followed by the stan- nous chloride method described in section 4500-P.D of the Standard Methods for Examina- tion of Water and Wastewater (APHA 2012).

▪ Fish samples

The fish samples collected were washed five times with distilled water, after which they were subjected to dissection using scissors and stainless steel to prevent any external contamina- tion. The muscle, liver, gill, and skin tissues were dissected separately for each sample and

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stored in HDPE bags at -20°C temperature till chemical analyses were performed (Fig. 4. 4).

Before the analyses, all the individual tissues of the samples were oven-dried at 40°C.

(a) Fish anatomy (image captured during the dissection process)

(b) Samples after dissection and oven-drying

Fig. 4. 4. Dissected and oven-dried fish parts specimens of (a) N. notopterus (b) C. batrachus and (c) C.

striata species. 1, 2, 3, and 4 indicate muscle, liver, gills and skin, respectively.

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Furthermore, they were subjected to grinding and crushing, followed by passing the sam- ples through a 150µm IS sieve to obtain homogeneity. 0.2g of each sample was considered for the acid digestion process (with ultra-pure nitric acid of 70% purity) for 35 minutes at 150°C.

The final filtrate from each sample comprised some oil and fat content, removed through the solvent extraction process.