2.5 Blood Chemistry Analysis
RBC and WBC count were performed following the methods of Rusia and Sood
[54]. Hb count was estimated by the cyanmethaemoglobin method, and Ht was
determined by the micro-Ht method of Nelson and Morris [55] using the diagnostic
reagent kit (Monozyme India, Ltd., India) at 540 nm using UV spectrophotometer.
Erythrocyte indices of fish, viz. MCV, MCH and MCHC, were calculated using
standard formulas:
MCV cubic micra
ð
Þ¼ Hct %
ð Þ=RBC millions  Cu  10
6
À
Á
Â
à  100
MCH picograms
ð
Þ¼ Hb g=dL
ð
Þ=RBC millions  Cu  10
6
À
Á
Â
à  100
MCHC g=dL
ð
Þ¼ Hb g=dL
ð
Þ=Hct %
ð Þ
½
 100
2.6 Biochemical Analysis
2.6.1 Estimation of Plasma Glucose and Protein
Plasma protein was estimated following the method of Lowry et al. [56] using
bovine serum albumin as standard. Briefly, 0.10 mL of plasma sample from control
and ASA-treated groups were added to the reaction mixture (0.90 mL of distilled
water and 5 mL of copper tartrate solution (5% copper sulphate, 10% sodium
potassium tartrate, 10% sodium sulphate in 0.5 M sodium hydroxide solution))
and kept at room temperature for 30 min. Subsequently, Folin-Ciocalteu phenol
reagent diluted in 0.1 N sodium hydroxide was added and incubated at room
temperature for 10 min, and the absorbance was read at 720 nm by using UV
spectrophotometer. For the preparation of ‘Standard’ (S) 1.0 mg of bovine serum
albumin was added to 10.0 ml of 1N NaOH and made up to 100.0 ml in a solution
standard flask. From this, 1.0 ml of solution was taken in ‘Standard’ tube and mixed
with 0.5 ml of Solution- C, kept for 10 min, and then 0.5 ml of Folin phenol reagent
was added. The optical density of the ‘Standard’ (S) was read as mentioned above.
Plasma glucose estimation was performed following the method of Cooper and
Mc Daniel [57]. In brief, after exposure, 0.1 mL of plasma samples from each
treatment were taken with 5 mL of O-toluidine reagent, and the aliquots were
incubated in boiling water bath for 10 min. After incubation, the aliquots were
cooled under running tap water. The absorbance was read against blank at 630 nm
using UV spectrophotometer.
2.6.2 Estimation of Enzymological Parameters
The enzyme activities of AST and, ALT, were determined by Diagnostic Reagent
Kits (Coral Clinical Systems, A Division of Tulip Diagnostics (P) Ltd., India)
following the manufacturers’ instructions.
Toxicity Assessment of Acetylsalicylic Acid to a Freshwater Fish Cyprinus. . .
197
RBC and WBC count were performed following the methods of Rusia and Sood
[54]. Hb count was estimated by the cyanmethaemoglobin method, and Ht was
determined by the micro-Ht method of Nelson and Morris [55] using the diagnostic
reagent kit (Monozyme India, Ltd., India) at 540 nm using UV spectrophotometer.
Erythrocyte indices of fish, viz. MCV, MCH and MCHC, were calculated using
standard formulas:
MCV cubic micra
ð
Þ¼ Hct %
ð Þ=RBC millions  Cu  10
6
À
Á
Â
à  100
MCH picograms
ð
Þ¼ Hb g=dL
ð
Þ=RBC millions  Cu  10
6
À
Á
Â
à  100
MCHC g=dL
ð
Þ¼ Hb g=dL
ð
Þ=Hct %
ð Þ
½
 100
2.6 Biochemical Analysis
2.6.1 Estimation of Plasma Glucose and Protein
Plasma protein was estimated following the method of Lowry et al. [56] using
bovine serum albumin as standard. Briefly, 0.10 mL of plasma sample from control
and ASA-treated groups were added to the reaction mixture (0.90 mL of distilled
water and 5 mL of copper tartrate solution (5% copper sulphate, 10% sodium
potassium tartrate, 10% sodium sulphate in 0.5 M sodium hydroxide solution))
and kept at room temperature for 30 min. Subsequently, Folin-Ciocalteu phenol
reagent diluted in 0.1 N sodium hydroxide was added and incubated at room
temperature for 10 min, and the absorbance was read at 720 nm by using UV
spectrophotometer. For the preparation of ‘Standard’ (S) 1.0 mg of bovine serum
albumin was added to 10.0 ml of 1N NaOH and made up to 100.0 ml in a solution
standard flask. From this, 1.0 ml of solution was taken in ‘Standard’ tube and mixed
with 0.5 ml of Solution- C, kept for 10 min, and then 0.5 ml of Folin phenol reagent
was added. The optical density of the ‘Standard’ (S) was read as mentioned above.
Plasma glucose estimation was performed following the method of Cooper and
Mc Daniel [57]. In brief, after exposure, 0.1 mL of plasma samples from each
treatment were taken with 5 mL of O-toluidine reagent, and the aliquots were
incubated in boiling water bath for 10 min. After incubation, the aliquots were
cooled under running tap water. The absorbance was read against blank at 630 nm
using UV spectrophotometer.
2.6.2 Estimation of Enzymological Parameters
The enzyme activities of AST and, ALT, were determined by Diagnostic Reagent
Kits (Coral Clinical Systems, A Division of Tulip Diagnostics (P) Ltd., India)
following the manufacturers’ instructions.
Toxicity Assessment of Acetylsalicylic Acid to a Freshwater Fish Cyprinus. . .
197