-26- MATERIALS AND METHODS

Transcription

1 -26- MATERIALS AND METHODS The pollutant : Sevin (1-naphthyl N-methyl carbamate) was used at a concentrations of 0.5 and 1.0 mg/l. At these concentrations, the insecticide was completely soluble in water. Unlike that reported by Haynes (1957), the above concentration were found experimentally to be less than LC 50 of this drug for the Oreochromis niloticus.. Experimental animls : The fish used in the present study is Oreochromis niloticus, family cichlidae, which are the most common fish in the Nile. The Oreochromis niloticus samples were obtained from Al-Kanater A1-Khayria fish Research center. Their weight was between grams. The individuals of the Oreochromis niloticus were carefully selected, so that they were all healthy, nearly of the same age and same stage of maturation. Fish samples were transported in well areated containers to 72 litre glass aquaria in the laboratory. The aquaria contained Nile water continuously areated using air pumps. The fish were kept for at least 48 hrs in these aquaria before transfer to the experimental aquaria. They were kept at stocking density of 24 fishes per aquarium to

2 -27- get them acclimatized,the temperature was kept at C. They were fed mainly on artifical powder (35% premix, 30% fish meal, 15% soy-bean and 20% Sorghum). Determination of the LC 50 of sevin : The areated aquarium of Nile water (72 L) containing 10,13 and 15 mg/l were used to verify the LC 50 of Sevin for Oreochromis niloticus. At 15 mg/l sevin all fishes died after 1 hr, at 13 mg/l sevin all fishes died after 4 hrs, while 50% of the fish survived for 48 hours at 10 mg/l Sevin. This last concentration was, therefore, considered the LC 50 for Oreochromis niloticus. Experimental design : The experimental animals were divided into three groups, each group comprised of 24 fishes. The first group : Fishes of the first group were kept in aquarium containing well areated chlorine free water. The second group : Fishes were kept in an aquarium of areated water containing 0.5 mg/l sevin.

3 - 28- The third group : Fishes were kept in an aguarium of areated water containing 1 mg/l sevin. Fish treated with either dose were captured by small hand net after 5,10 and 15 days, sacrificed and their organs were taken out. Histological and histochemical studies : Treated and untreated (control) fish were taken at 5,10 and 15 days. The ovaries, liver and muscle (dorsal fin position), were removed as soon as possible. The specimens obtained for study were sectioned into parts no larger than 1 Cm 3. The specimens were then placed in Buffered formalin fixative according to the following protocol : Buffered formalin fixative 24 hr 80% Alcohol 1/2 hr 95% Alcohol, 2 changes 1/2 hr each Absolute Alcohol, 3 changes 1/2 hr each Paraffin oil 24 hr Xylene 10 min Melted paraffin wax, 2 changes 1/2 hr each Embed in paraffin and cool

4 -29- Sectioning and staining : The paraffin blocks containing the organs were sectioned using Microtome. Sections of 5-7 microns thick were obtained and were mounted on chemically clean microscope slides, which were allowed to air dry in 37 C Oven overnight. The slides were stained according to the following protocol : 1) The ovaries : - Harris hematoxylin and Eosin stain (as a general stain). - Milligan Trichrome stain as a counting stain (Humason and Company, 1978). 2) The liver : - Harris hematoxylin and Eosin stain (as a general stain). 3) The muscle : - Harris hematoxylin and Eosin stain (as a general stain). Histochemical stains : 1- The ovaries : - PAS stain for glycogen. - Methyl green pyronin for DNA and RNA (Mattar, 1980). - Feulgen stain for DNA (Pearse, 1968). - Nin hydrin schiff's stain for Amino group containing proteins (Bancroft, 1967). - Performic acid alcian stain for SS. group containing proteins (Pears; 1968). - Performic acid schiff's stain for keratin (Pearse,1968).

5 The liver : - PAS stain for glycogen (Bancroft, 1967). - Methyl green pyronin for DNA and RNA (Mattar, 1980). - Feulgen stain for DNA (Pears, 1968). - Nin hydrin schiff's stain for Amino group containing proteins (Bancroft, 1967). - Performic acid schiff's stain for keratin (Pears, 1968). -Performic acid alcian blue stain for SS. group containing proteins (Pears, 1968). 3) The muscle : - PAS and methyl green for glycogen (Bancroft, 1967). - Acetylcholinestrase (Pears, 1968). - Ninhydrin schiff's stain for Amino group contaning protein (Bancroft, 1967). - Performic acid schiff's stain for keratin (Pears, 1968). - Performic acid alcian blue stain for SS.group containing proteins (Pears, 1968). Fixative : Buffered formalin : 10% totmarin ml Sodium acid phosphate (NaH 2 PO 4 H 2 0) Anhydrous disodium phosphate (NaH 2 PO 4 ) 4.0 g 6.5 g

6 -31- Milligan trichrome stain (1946) : Fixation : 10% formalin in normal saline Solutions : Mordant Solution A : Potassium dichromate Distilled water 3.0 g 10 0 ml Solution B : Hydrochloric acid, concentrated 10.0 ml 95% ethyl alcohol ml Mix 3 parts A with 1 part B; use within 4 hours. Acid fuchsin : Acid fuchsin (C.I ) Distilled water 0.1 g ml Phosphomolybdic acid : Phosphomolybdic acid Distilled water 2.0 g ml Use half of this solution for orange G solution below. Orange G : Orange G (C.I ) 2.0 g 1% Phosphomolybdic acid ml

7 -32 Fast green : Stock solution : Fast green FCF (C.I. 2053) 10.0 g 2% acetic acid (2 ml/98 ml distilled water) ml Work solution : Fast green stock solution 10.0 ml Distilled water 90.0 Procedure : 1- Deparaffinize and transfer slides through absolute alcohol into 95% alcohol (remove HgC1 2 if present). 2- Mordant in potassium dichromate-hydrochloric acid solution 5-7 minutes. 3- Rinse in distilled water. 4- Stain in acid fuchsin : 5-8 minutes. 5- Rinse in distilled water. 6- Fix stain in phosphomolybdic acid solution: 1-5 minutes 7- Stain in orange G : 5-10 minutes. 8- Rinse in distilled water. 9- Treat with 1% aqueous acetic acid (1 ml/99 ml distilled water) 2 minutes. 10- Stain in fast green : 5-10 minutes. 11- Treat with 1% acetic acid : 3'minutes. 12- Rinsein 95% alcohol,transfer to second 95% : 5 minutes 13- Finish dehydration in absolute alcohol, 2 changes : 3 minutes each. 14- Clear and mount.

8 Cytophotometric study : Section representing control and treated groups mounted on the same slide and stained for DNA, RNA, glycogen, Amino group, keratin and SS. group rich proteins were subjected to cytophotometric measurements using a Leitz MPV compact cytophotometer. The transmitted light values (I), obtained for stained specimens using suitable colour filter, were used to calculate the optical density (0.D) from the equation, O.D. = Log Io/I where Io = incident light = 100. Statistical analysis : The present investigations were statistically analysed using the computation laws given by Milton and Tsokos (1983). In order to test the differences between means of two sets of data whether significant or not, student t-test was applied. The calculated values obtained from evaluation of t-test were then checked in the student table in order to find out the level of significance (P value) the significance levels used were accepted only when P, < and P <