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The purpose of the study is to use the morphology of the …

Home » Biology Articles » Hydrobiology » Freshwater Biology » Effects of the organophosphorous methyl parathion on the branchial epithelium of a freshwater fish Metynnis roosevelti » Materials and Methods

Materials and Methods
- Effects of the organophosphorous methyl parathion on the branchial epithelium of a freshwater fish Metynnis roosevelti

Biological material

Metynnis roosevelti Eigenmann, 1915 (Serrasalmidae) was obtained from fish farms of the State of São Paulo. Fishes of total length 4.0 - 5.0 cm, mean weight of 3.0 g, were maintained in 30 litres aquaria, protected by green shields to avoid external stimuli of the fish (Fanta, 1995) and to improve their adaptation. The fishes were acclimated for two weeks to laboratory conditions (temperature 25°C; pH 7.0; photoperiod 8 hours light/16 hours dark). They were fed once a day, one hour after the start of the light period with commercial pellets.

Tested substance

Mentox 600 CE ® is an insecticide that contains 600 g/l of the active principle the organophosphorous methyl parathion. It is a colorless liquid, and has a strong scent. The product used in the experiments was supplied by the Mentox Industry.

Toxicity tests

Experiments were carried out in 30 liters aquaria, kept in the same conditions as during acclimation. Three tests were carried out: (i) control - 10 fishes were maintained in water without pollutant; (ii) and (iii) experimental - 10 fishes were kept in each of the aquaria contaminated with OP. Environmental conditions were the same in all three aquaria, with the exception of the contaminant.

Metynnis roosevelti was contaminated with 7µl of OP/litre and 1µl of OP/litre. These concentrations were lower than the LC50 for Brachydanio rerio (8.5µl OP/litre) in acute tests (96 hours). Seven ppm concentration caused the smallest number of dead individuals (1 fish in 96 hours of exposition). On the other hand, the concentration of 1 ppm was chosen because it was the smallest concentration used in toxicity tests with B. rerio. The results of these tests obtained by laboratories validated by IBAMA (Brazilian Environmental Institute), are used for the establishment the toxicity degree of the product.


A qualitative observation of some behavioural items was done with all fishes through on the experiment, mainly respiration, activity, balance, grouping, prefered region in the experimental aquaria. No numerical evaluation was done.


Gills of M. roosevelti were sampled for histology and for scanning electron microscopy. The 2nd right branchial arch was dissected out at 1, 4 and 8h after exposure to 7 ppm OP. From fishes contaminated with 1 ppm, the second right branchial arch was collected at 1, 4, 8, 24, 48, 72, and 96h after T0. At the same times, gills from control fish of both experiments were also extracted.

For histology, the gills were fixed in Bouin for 8h, routinely prepared, and the sections stained with hematoxylin and eosin (HE) (Culling et al., 1985). For scanning electron microscopy, 2nd left branchial arch was fixed in glutaraldehyde 2.5%, diluted in 0.2 M Cacodilate buffer pH 7.4 at 4 oC, overnight. The samples were washed in 0.5 M Cacodylate, pH 7.4, contrasted in 4% osmium tetroxide diluted in 0.2 M Cacodilate, pH 7.4 for 1h at 4 oC. The critical point was obtained in a Balzers CPD 030, and the metalization was done with gold in a Balzers SCD 050. Preparations were observed in a Jeol JSM 5300 scanning electron microscope.

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