EFFECTS OFLEAD NITRATE ON THE MORPHOLOGY OF THE EYES AND BODY OF THE ZEBRAFISH (Danio rerio)

Jonathan U. Doloque Peter Paul R. Pascual

Background Information
Lead Nitrate (Pb(NO3)2)
Commonly occurs as a colorless and white powder crystal.

Used in the manufacture of gasoline, batteries, paint as well as ammunition. Has been linked to several eye diseases and cardiovascular problems.

Background Information
Enters the aquatic habitat by mining, agricultural runoffs and oil spills. Must be handled and stored with appropriate safety precautions to prevent inhalation, skin contact and ingestion. Limited applications are in scrutiny by the International agency for research on cancer.

Background Information
Zebrafish (Danio rerio)
Characterized by its prominent stripes that extend lengthwise from head to tail. Grows for about 5cm (2in) in length and has a silver or golden with several bluishblack stripes. Main benefits of using zebrafish is with regard to their size, husbandry and morphology (Hill et. al, 2005).

Background Information
Morphometrics are a set of statistical procedures for analyzing variability in size and shapes of organisms (Reyment, 2009). Image J is a public domain java-based image program that was designed with an open architecture to allow its extensibility and efficient use.

Statement of the Problem

There are very few studies that are concerned with the effects of heavy metals in the condition of the adult organism.

Studies have only focused on developing embryo and have slightly neglected the presence of adult organisms in the aquatic habitat.

General objective of the Study

Aims to determine the effects of the different concentrations of Lead Nitrate (Pb(NO3)2) on the biometric landmarks of the adult zebrafish as well as its effect on the overall morphology of the eyes.

specific objectives of the Study

Aims to determine the specific concentration of the compound Lead nitrate(PbNO3) which has affected the: a. Morphology of the eye b. Biometric landmarks
Total length(LT) Fork length(LF) Length from head to tail(LA) Head length(LH) Pelvic fin(LP) Eye diameter(ED)

Significance of the Study

Gives a proper idea of the toxicological effects of Lead Nitrate that can affect the aquatic life present. Can help predict the morphological changes (damage) that has occurred on the morphology of the different aquatic life living in places with high concentrations.

Scope and Limitations

The study will only focus on the effects of the chemical on the biometric landmarks as well as the morphology of the eyes of the Zebrafish.

Will not deal with the physiology and mechanism of action of the Lead Nitrate. Will not deal with the effects of other biotic and abiotic factors present.

Flow chart of the methodology

Overnight acclimatization of zebrafish

, Exposure of zebrafish to four

concentration (Control, LC25,LC50,LC75) of lead nitrate(PbNO3) for 30 days

Sampling, biometric analysis, and histological analysis of four zebrafish for the 5th, 10th, 15th, 20th, 25th, and 30th day.

General Animal Husbandry

A mixed set of population of 300 Zebrafish
obtained will be obtained from the pet store. They will be placed in 4 aquaria with 10 liters of water and 1 aerator each.

The concentrations will be Control (pure water), LC25 (12.5mg), LC50 (25mg) and LC75 (37.5mg). There will be three replicates per concentration except for the control.
The fishes will be acclimatized for 24 hours and will be fed once every day.

General Animal Husbandry

Lead Nitrate will be homogenized for 30 minutes prior to the exposure of the fishes. They will be exposed to Lead Nitrate for 30 days with sampling of 4 fishes on the 5th, 10th, 15th, 20th, 25th and 30th day.

Biometrical Measurement

Fig. 1: Biometric landmarks that will be used in the study.

Microtome Section
The 4 fish sample sampling dates will be fixed (at each sampling point) for 24 h in Bouins solution. Fixed specimens will be dehydrated and subsequently embedded in paraffin. Sagittal serial sections will be cut at 5-7 m, stained with haematoxylin and eosin (gross histological). Stained sections will be studied using a simple microscope (Osman, 1970).

Statistical and Microtome Analysis

The data obtained from the Biometrics will be statistically computed using the SPSS 16.0 at the level of significance of = 0.05. Anova and HSD will be used to determine if there will be a significant correlation with the landmarks and the concentrations used. The microtome sections will be analyzed by giving a description of the changes that will occur during the period of exposure.