MATERIALS AND METHODS. :- Waste water (effluent) samples from. Bhilai Steel Plant were collected in plastic cans, from the

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1 MATERIALS AND METHODS Sample collection :- Waste water (effluent) samples from Bhilai Steel Plant were collected in plastic cans, from the sites mentioned earlier with study area and sites. Analysis of physico-chemical characters of Bhilai Steel Plant liquid effluent. The analysis for physico-chemical characteristics of effluent were carried out both at the sampling sites as well as in the laboratory. Parameters like temperature, ph, electrical conductivity, total dissolved solids, dissolved oxygen, alkalinity, free-carbon dioxide and chloride were determined at the time of sampling while analyses for remaining parameters were made in the laboratory within about 30 minutes of sampling, in appropriately preserved samples. Preservations of sample were made as prescribed in Standard Methods. (APHA-AWWA-WPCF 1975). Temperature in oc, ph in ph units, oxidation reduction potential in m.v., total dissolved solids in mg.l, and electrical conductivity in m.mhos. em were determined with the help of a Century portable water analyser kit. 21

2 22 Osmotic pressure (OP) :- 0 P was calculated using the formula given by Tiwari and Ali (1988). o P (at m) = (~. mhos em ) Degradation ratio (DR) :- D R was calculated as per the formula of Pillsbury et al. (1969). DR = (7.5 - EC iw) 7.5 = ( IW ppm) 4800 EC iw = Electrical conductivity of irrigation water in m. IW ppm = mhos. cm-l Salt concentration of irrigation water in mg.l Specific electrical conductivity (SEC) :- SEC was calculated as per the formula of Trivedy and Goel (1984). SEC Jl mhos. cm-l at 25 c = observed conductance X cell constant X temperature factor. (Temperature factor was taken as given in Trivedy and Goel (1984) page 43 ). Alkalinity :- Following types of alkalinities were determined by titrimetric methods (APHA-AWWA-WPCF 1975). Phenolphthalein alkalinity (PA) :- 100 ml effluent sample was titrated against 0.02N HCl in the presence of phenolphthalein indicator.

3 23 P A mg. 1 Caco 3 = A X N X 50,000 A = ml titrant N = Normality of titrant Methyl orange alkalinity (MOA) :- 100 ml effluent sample was titrated against 0.02N HCl in the presence of methyl orange indicator. M 0 A mg.l Caco 3 = A X N X 50,000 A = ml titrant N = Normality of titrant. Total alkalinity (TA) :- T A was calculated as follows : T A mg.l CaC0 3 = P A mg.l Caco 3 + M 0 A mg.l Caco 3 Carbonate and bicarbonate alkalinities :- These were determined with the help of table given in APHA-AWWA-WPCF (1975) page 281. Free carbon dioxide (F co 2 ) :- F co 2 was determined titrimetrically following APHA-AWWA-WPCF (1975). 100 ml effluent sample was titrated with O.OlN NaOH solution in the presence of phenolphthalein indicator, to ph 8. 3.

4 24 F co 2 mg.l = A X N X 44,000 A = ml titrant N = Normality of titrant Total carbon dioxide (TC0 2 ) :- Tco 2 was computed from the concentrations of free carbon dioxide, alkalinity, and carbonate alkalinity, bicarbonate following APHA-AWWA-WPCF (1975). A = mg.l- 1 bicarbonate alkalinity B = mg.l carbonate alkalinity Acidity :- Acidity of all types were determined titrimetrically. (Trivedy and Goel 1984). 100 ml effluent sample was titrated with 0.05 N NaOH in the presence of phenolphthalein and methyl orange indicators. methyl orange acidity mg.l caco 3 = A x N x 50,000 Phenolphthalein acidity mg.l Caco 3 = B X N X 50,000 Total acidity mg.l caco 3 = (A+B) X N X 50,000

5 25 A = Volume of NaOH used with methyl orange indicator in titrating the sample to ph 3.7 B = Volume of NaOH used with phenolphthalein indicator in titrating the sample from ph 3.7 to 8.3 N = Normality of titrant. Chloride :- Chloride was determined by argentometric method (APHA - AWWA - WPCF 1975) 100 ml waster water sample was titrated with N silver nitrate solution using potassium chromate indicator Chloride mg.l = A X N x 35,450 A N = ml of titrant = Normality of titrant. Langelier calcium carbonate saturation index :- This was calculated as per the formula of Trivedy and Goel (1984), page 95. Langelier saturation index = ph - phs ph = Initial ph of water phs = (A+B) - (C+D)

6 26 Values of the factors A, B, C and D are derived from the values of temperature total dissolved solids, calcium hardness and total alkalinity respectively. Residual sodium carbonate (RSC) RSC was calculated as per the formula of Eaton(l950). RSC me.l = (C0 3 + HC0 3 ) - (Ca + Mg) Total phosphorus (P) :- Phosphorus was determined as phosphate by stannous chloride method (APHA - AWWA - WPCF 1975). 100 ml waste water sample was taken in a Kjeldahl flask. 5 ml strong acid solution (HN0 3 + H 2 so 4 1:75) was added to this and then sample was evaporated to a volume of about 1 ml. This digestion was continued to remove all the HN03" After digestion, sample was cooled and then 20 ml distilled water and 1 drop of phenolphthalein indicator were added. Then the solution was neutralized with ln NaOH till the formation of faint pink colour. Solution was filtered and volume was adjusted to 100 ml with distilled water. To this 4 ml ammonium molybdate and 10 drops of stannous chloride were added. Intensity of blue colour was measured at 690 nm. Distilled water was taken as blank. The amount of phosphate was determined with the help of a standard curve. p mg.l = mg P x 1000

7 Sulfate (so 4 l :- so 4 was determined by turbidimetric method (APHA-AWWA-WPCF 1975) ml waste water sample or portion diluted to 100 ml with distilled water was taken simultaneously with distilled water as blank. to this were added 5 ml conditioning reagent and 100 mg BaCl 2 crystals. This was stirred for l minute at a constant speed and then the intenstity of colour was measured at 420 nm. Amount of sulfate was calculated with the help of standard curve. so 4 mg.l = mg so 4 x 1000 Nitrite - nitrogen (N0 2 -N) :- N0 2 -N was determined by diazotization method (APHA-AWWA-WPCF 1975) 50 ml clear waste water sample or portion diluted to 50 ml was taken for nitrite estimation, simultaneously with distilled water taken as blank. l ml sulfanilamide solution was added, then after 2 minutes l ml 1-naphthyl-ethylenediamine solution was added. Intensity of ' pink colour was measured at 543 nm. Amount of N0 -N was 2 determined with the help of standard curve, and then computed in the formula. =

8 28 Nitrate-nitrogen (N0 3 -N) :- No 3 -N was determined by phenol disulphonic acid method (Jackson 1973). 25 ml waste water sample was taken and evaporated to dryness over an electric hot plate. Residue was dissolved in 2 ml phenoldisulphonic acid. Then 3.5 ml cone. ammonia solution was added. Distilled water blank was also run simultaneously. Intensity of yellow colour was measured at 420 nm. Amount of No -N 3 was determined with the help of standard curve. N0 3 - N mg.l Anunonia-nitrogen (NH 4 -N) - Ammania-N was determined by nesslerization method (APHA-AWWA-WPCF 1975). 50 ml waste water sample or portion diluted to 50 ml was taken. Distilled water blank was also run simultaneously. To this were added 1 ml each of zinc sulfate and sodium thiosulfate solutions. ph of this solution was adjusted to 10.5 with the help of ln NaOH solutionj flocculent precipitate was filtered. Then 1 drop of EDTA solution and 2 ml of nessler reagent were added. Intensity of yellow colour was measured at 430 nm. Amount of ammania-n was determined with the help of standard curve and ammonia concentration was calculated using the formula. Ammonia-N mg. 1-l = A A =~g N found calorimetrically.

9 29 Iron (Fe) :- (APHA-AWWA-WPCF 1975). Iron was determined by pheni'throline method. /' 100 ml waste water sample or portion diluted to 100 ml was acidified with 25 ml cone. HN0 3. This was then evaporated to dryness. Obtained residue was dissolved in 1+1 HCl. Then 1 ml hydroxylamine solution and 5 ml phenanthroline solution were added. ph was adjusted between 2. 9 to 3. 5, using dilute HCl or dilute NH 4 0H. Intensity of orange red colour was read at 510 nm. The concentration of iron in solution was calculated from the standard curve prepared from standard iron solutions. Fe mg.l = )lg Fe Silica (Sio 2 ) :- Sio 2 was estimated by molybdo - silicate method (APHA-AWWA-WPCF 1975). To 50 ml waste water sample lml 1+1 HCl and 2 ml ammonium molybdate were added. Then 1. 5 ml of 10% oxalic acid solution was added. Intensity of yellow colour was measured at 410 nm. Amount of silica was determined with the help of a standard curve and using the formula. sio 2 mg. 1 = ).lg Si0 2 Hardness :- Hardness values were determined by EDTA titrimetric method (APHA-AWWA-WPCF 1975).

10 30 Pretreatment of sample was done by acidifying 100 ml waste water sample with 5 ml cone HN0. After this a small 3 quantity of 30% H 2 o 2 was added till the formation of white residue; this residue was dissolved in warm (l+l) Hcl solution and was then neutralized with cone. NH 4 oh. final volume was adjusted to 100 ml. Distilled water blank was also run simultaneously. In this prepared sample following types of hardnesses were determined. Calcium hardness - 25 ml pretreated sample was taken and ln NaOH solution was added to raise the ph upto 13. This solution was titrated against the standard M EDTA in the presence of murexide indicator. Value was computed in the formula. Calcium hardness as mg.l-l Ca = Ax B x Calcium hardness as mg. 1-l caco 3 = A X B X 1000 A = ml titrant B = mg caco 3 equivalent to 1 ml of EDTA solution. Total hardness :- 25 ml pretreated solution was taken, to this 2 ml buffer solution (NH 4 Cl + NH 4 0H) was added to raise the ph between This solution was titrated against standard EDTA solution of 0.01 M, using eriochrome black T as indicator. Value was calculated as follows :

11 31 Total hardness as mg.l caco 3 = A X B X 1000 A = ml titrant B mg Caco 3 equivalent to 1 ml of EDTA. Magnesium (Mg) Magnesium content was calculated from the formula given by APHA-AWWA-WPCF (1975) page 223. Mg mg.l = Total hardness mg.l - Calcium hardness mg.l Caco 3 x Magnesium hazard index this was calculated as per formula of Paliwal (1972). Magnesium hazard index = Mg x 100 Ca + Mg Total dried residue :- Total residue was determined following APHA-AWWA-WPCF (1975). A well mixed sample was evaporated in a weighted dish and dried to constant weight in an oven at 103 to 105 C. The increase in weight over that of the empty dish was taken as total residue. Total residue mg.l = (A-B)X 1000 A = weight of sample + dish B = weight of dish.

12 32 Dissolved oxygen (DO) :- D 0 was determined by Azide modification method (APHA-AWWA-WPCF 1975). 300 ml waste water sample was collected in three B 0 D bottles, in each bottle 2 ml each of manganese sulfate and alkali-iodide-azide reagent were added. Bottle was shaken for about 15 minutes and then allowed the precipitate to settle down. Then 2 ml cone. H 2 so was added to dissolved 4 the precipitate. 203 ml of this solution was titrated against N sodium thiosulfate solution in the presence of starch indicator. Value was computed as follows : DO mg.l = ( 8 xlooo X N ) v v N = Normality of titrant v = Volume of sample (ml) v = volume of titrant used (ml) Percent oxygen saturation(%0 2 saturation) - This was calculated as per the formula of Adoni % o 2 saturation = 100 x observed D 0 Sx Sx = St x Cf Sx = solubility of oxygen St = solubility of oxygen at observed temperature Cf = Altitudinal correction factor. (The values forst and Cf were taken from Adoni 1985).

13 33 Biochemical oxygen demand (BOD) :- BOD was determined as given in APHA-AWWA-WPCF (1975). BOD mg.l = D D l - 2 = Initial Oxygen value in mg.l-l Oxygen value in mg.l after 5 days of incubation at 20 C. Chemical oxygen demand (COD) :- COD was determined by dichromate reflux method (APHA-AWWA-WPCF 1975). 50 ml waste water sample was taken with simultaneous running of distilled water blank. lg Hgso 4 was added to this then 25 ml 0.25 N dichromate solution and 75 ~l Cone. H so 2 4 were added in 1:3 ratio and was refluxed for 2 hrs. Excess dichromate was titrated against 0.1 N ferrous ammonium sulfate using ferroin indicator. COD mg.l-l = (a-b) x N X 8000 a = ml titrant used for blank b = ml titrant used for san.ple N = Normality of titrant. Phenol :- this was determined by chloroform extraction method (APHA-AWWA-WPCF 1975). 500 ml waste wnter sample was taken in a beaker. ph of this sample was adjusted at 4 using H 2 so 4 solution. Then 5 ml of 10% CuSO 4 was added. This was

14 34 transferred to the distillation apparatus. After collection of 450 ml distillate, distillation was stopped. Then 50 ml phenol-free distilled water was added to the distillation flask. Distillation was continued until a total of 500 ml of distillate, was collected. This distillate or a portion of it, diluted to 500 ml was taken. Distilled water blank was also simultaneously run. To this was added 10 ml 20% NH cl 4 solution and ph was adjusted with cone. NH 0H to This was then transferred to a separatory funnel, followed by addition of 3 ml of aminoantipyrine with shaking and then 3 ml of potassium ferricyanide solution. After this 25 ml chloroform was added with shaking the separatory funnel atleast for 10 times, then was decanted. Absorbance of phenol solution in chloroform was measured at 460 nm and concentration of phenol was estimated from the calibration curve. Phenol pg.l = A x 1000 B A =pg phenol in sample from calibration curve B = ml original sample. Treatment of soil and plant with effluent :- Local "Kanhar' soil was used for growing the experimental plants as well as for observing the effects of

15 35 effluent irrigation on soil. Abelmoschus esculentus, Crotalaria juncea, Cyamopsis tetragonoloba, Linum usitatissimum, Phaseolus vulgaris and Raphanus sativus were grown by seed sowing while Brassica oleracea var caulorapa and Capsicum annuum were grown by seedling transplantation. Experimental plots of 2 m x 2 m size were prepared in triplicate for control (tap water irrigation) as well as for treatment (effluent irrigation). Effluent irrigation was made at the rate of about 250 ml/plant, twice in a week. However, additional water requirement was fulfilled by irrigating with tap water. Collection of plant sample :- Leaf samples were taken periodically from atleast three plants from each treatment. Plants, after maturity were uprooted by digging and were separated into underground and aboveground parts. Underground parts (roots) were washed with tap water. Plant parts were then dried in hot air oven at 80 C, till constant weight. Analyses of plant samples were made in triplicate. Collection of soil sample :- Soil samples were collected from three places from each experimental plot, from the surface, up to 5 em depth, and were composited. Soil samples were dried in shade in ammonia free air and were then stored in polythene bags. All the analyses on soil were made in triplicate, on this air dry soil sample,

16 36 Sediment of the steel plant waste water were collected from the waste water collection sites. These were dried, stored and analysed similar to soil samples. Mechanical analysis of soil and sediment - Mechanical analysis of soil for sand silt and clay was done following the pipette method (Piper 1944). Preparation of soil/sediment suspension. - Soil/sediment suspension was prepared by taking 20 g (2-4 mm pore sieved) soil/sediment in a flask, to this 100 ml distilled water was added and was stirred for one hour. ph and electrical conductivity :- These were determined in freshly shaken suspension with the help of a Century portable water analyser kit. Osmotic pressure (OP) and Degradation ratio (DR) :- OP and DR were determined as described for waste water analysis. Total salt concentration (TSC) :- TSC concentration was determined by using formula given by Agarwal et al (1979). TSC me. 1-l = 10 E.C ( m. mhos. Cm- 1 ) Organic matter (OM) :- OM was determined by Walkley - Black rapid titration method (Jackson 1973). 0.5 g soil sample was taken in a flask followed by addition of 10 ml of 1 N K cr o sol uti on and 20 ml cone. H so with gentle stirring 4 for 1 minute. A standardization blank (without soil) was

17 37 also run simultaneously. This solution was diluted to 200 ml with distilled water and then 10 ml of 85% H 3 Po 4 was added. This solution was titrated against 0.5 N ferrous ammonium sulfate solution using diphenylamine as indicator. Organic matter content was determined using the equation. OM% = 10 ( l - T ) x 1.34 s Factor 1.34 is derived as follows : (ln) X 12 X 1.72 X 4000 o. 77 Readily Oxidizable OM% = 10 = 1.34 l -!) X 1.03 s Factor 1.03 is derived as follows (ln) X X l. 72 X = l. 03 S = Standardization blank titration, ml ferrous solution T = Sample titration, ml ferrous solution. Water holding capacity (WHC) :- WHC was determined following Piper (1944). WHC% = b - c-d x 100 c - a a = weight of porous pot with filter paper b = weight of porous pot + saturated soil c = weight of porous pot + oven dry soil d = water retained by one filter paper

18 38 Sulfatae, chloride and alkalinities :- These were determined in 1:5 soil suspension following the respective methods for effluent analysis. Exchangeable cations (calcium and magnesium) Ammonium acetate extract for exchangeable cations (Jackson 1973) was dried over a hot plate. The residue was dissolved in 10 ml of 6N Hcl, then calcium and magnesium were determined following the methods adopted for effluent analysis. Magnesium hazard index - It was also calculated as for the effluent. Available phosphorus or N H 2 so 4 extractable phosphorus :- 1 g soil sample was taken in a flask then 200 ml of N H 2 so solution was added. The suspension was 4 shaken for 30 minutes and filtered. Phosphorus was determined in appropriate portion of this suspension similarly as adopted for effluent analysis. Total elemental phosphorus This was determined after digesting the soil/sediment with perchloric acid. To 2 g sample, 20 rnl of HN0 and 30 ml of 60% HCl0 were added. 3 4 Digestion was carried on electric hot plate at about 130 C till the solution turned colourless and residue became white. After cooling, 50 ml distilled water was added and filtered. Final volume was adjusted with distilled water to 200 ml. In an appropriate portion of above solution ph was adjusted to

19 39 3 with the help of 4 N Na 2 co 3 and 2N HClo 4 using 2, 4 dinitrophenol indicator. Further procedures were same as adopted for waste water analysis. Total nitrogen :- Total nitrogen was determined by micro- Kjeldahl method followed by nesslerization. l g soil was taken in Kjeldahl flask. To this 10 ml cone. H so 2 4 and a pinch of selenium powder were added and heated at 360 C to 400 C, till solution turned colourless and residue became white. After cooling, it was filtered and volume was adjusted to 100 ml. In an appropriate portion of above solution nitrogen determination was made following the procedure as described for waste water analysis. Iron :- All forms of iron in soil were determined following Jackson (1973). Colour development was done following the method for water analysis. Dilute acid soluble iron - Soil iron extracted with dilute Hcl solution. Ferrous iron (Fe 2 +l :- Soil iron extracted with neutral ln ammonium acetate. Ferric iron (Fe 3 +) :- Soil iron extracted with ammonium acetate of 3 ph Water soluble iron :- Soil iron extracted with distilled water.

20 40 Analysis of pigments and protein in leaf Chlorophyll and carotenoids :- Quantitative estimation of chlorophylls was made following Arnon (1949) while carotenoids were determined following Duxbury and Yentsh (1956). 1 g fresh leaf material was homogenized with excess of 80% acetone and centrifuged at 3000 rpm. Supernatant volume was adjusted to 100 ml. The optical density of this extract was measured at 480, 510, 645 and 663 nm. From these readings concentration of various pigments were determined as follows Chlorophyll a mg.g Chlorophyll b mg.g Total Chlorophyll Carotenoids mg.g = 12.7 OD D645 x v a x 1000 X W = D D663 X V a x 1000 X W 20.2 OD D663 x V mg.g = a x 1000 X W = (7.6 0D X 0D510) X V w a = Length of light path in the cell ( 1 em) v = Volume of extract in ml w = Fresh weight of sample in g. Total soluble protein :- Total soluble protein was determined by folin-phenol method (Lowry et al 1951) 100 mg leaf material was homogenized with 80% ethanol and centrifuged at 3000 rpm, for about 30 minutes. Supernatant was discarded. To the residue 3 ml of 5% TCA was added and was again centrifuged at 3000 rpm. Supernatant was again

21 41 discarded and then 10% TCA was added. This process was repeated twice. After this, precipitate was dissolved in 2 ml of O.lN NaOH. From this solution 0.5 ml was taken in another tube to which 5 ml of alkaline CuSO and 0. 5 ml of 4 dilute olin-phenol reagent were added. This was incubated at 37 + l C for 30 minutes. Intensity of blue colour was read at 750 nm. Blank of O.lN NaOH was run as the test sample. Concentration of protein was determined with the help of a standard curve. Plant nutrient analysis :- Determination of plant nutrients was done after perchloric acid digestion following Jackson (1973). Perchloric acid digestion :- l g oven dried and powdered tissue was taken in a flask and then 5 ml of cone. HN0 3 was added. Flask was heated at l80 C C for 30 minutes. After cooling, 5 ml of ternary acid mixture (HN0 3 - H 2 so 4 HCl : 1 : 4) was added and digestion was continued till dense white fumes appeared as well as acid liquid was largely volatilized. Then suspension was centrifuged. Volume of decanted supernatant was made up to 10 ml with distilled water. Calcium, magnesium, phosphate and iron :- In appropriate v,:,lume of above supernatant analyses for Ca, Mg, P0 4 and Fe were done following the respective methods for their analyses in soil.

22 42 Germination percentage :- Germination of seeds was made in sterilized petriplates. These plates were lined with a thin layer of cotton then with a filter paper. 100 seeds were placed on filterpaper. This was then moistend either with the BSP effluent (treated) or with tap water (control) and allowed to germinate at room temperature in diffused light. Emergence of radicle was taken as evidence of germination. Germination was recorded daily at fixed time. Germination value (GV) :- GV was calculated following Boojh and Ramakrishnan (1981) GV = (MDG) = (PV) MDG = Mean daily germination PV = Maximum value of cumulatative germination percentage. Speed of germination index (SGI) :- SGI was calculated using formula of Kamal and Sinha (1974). e.g. if seeds were kept for 5 days so SGI = 2(5g + 4g + 3g + 2g + g) g = Number of seeds germinated after 24 hours. Germination relative index (GRI) : GRI was calculated using formula of Zur (1966).

23 43 GRI = xn (h-n) xn = Number of seed germinated h n = Total number of count = Count number Fresh weight of seedlings :- Seedlings were surface dried with blotting paper and weighed Dry weight of seedlings :- Seedlings were dried at 80 C to a constant weight. Moisture percentage of seedlings :- Moisture percentage of seedlings was determined using the formula. Moisture percentage of seedlings = Fresh weight of seedlings-dry weight of seedlings x 100 Fresh weight of seedlings Nutrient composition of seedling :- Nutrient concentration were determined in oven dried seedlings after digesting them with perchloric acid. Analysis for calcium, magnesium, phosphate and iron were made following the same methods as for plant nutrients analysis. Total nitrogen this was also determined in digested sample by nesslerization method as described for soil analysis. Crude protein :- Crude protein was calculated following Kaul and Saraf (1984). Crude protein% = % Total nitrogen x 6.25

24 44 Collection of algal samples :- Algae were collected from the soil surface mostly from the margin of the effluent channel. Effluent water being dark in colour, no algae were obtained growing submerged in it, below about 5 em effluent depth. Algae were preserved in 4% formalin solution. Percent change in treated over control :- This was calculated using the following formula. % change in treated over control = Value for treated - value for control x 100 value for control Statistical analysis Student "t" test : Student 't' test was computed following Sokal and Rahlf ( 19 73). t = Difference in mean S E D SED = JsE and SE = SD -IN N = number of observations. Coefficient of correlation Pearson's coefficient of correlation (r was calculated using the formula :- 2_XY - EX.'f..Y r = N 2 y -

25 45 X and y N = two variables. = number of observations ANOVA One way anaiysis of variance (ANOVA) was done following Sokal and Rahlf. (1973). Duncan's multiple range (DMR) test: DMR test was made following Duncan (1955). --/1--