Effects of Zinc, a heavy metal, and Diazinon, a common pesticide, on the Embryonic Development of the
African Clawed Frog Xenopus laevis

Brett C. De Poister Fleetwood, Pennsylvania

Junior Water Prize Report

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Anurans (frogs and toads) are an indicating species, providing a measure of the health of the surrounding environment. Anurans are the highest life form that lay naked eggs in the water. These eggs are not protected by a shell, but rather by a permeable jelly, which allows substances such as herbicides, pesticides, fertilizers, and heavy metals to pass into the embryo. These substances can have harmful or deadly effects on frog eggs and tadpoles at various stages of development.

These chemicals are also harmful to juvenile and adult anurans. Anurans breathe and drink through their permeable skin; therefore, they absorb pollutants directly, possibly causing illness or death depending on the concentration and toxicity. Pollutants are suspected of causing high mortality rates and a high percentage of abnormalities in the eggs, tadpoles, and adult anurans. When adult anurans are abnormal, their chance of survival decreases which could eventually kill off their population. The decline of anuran populations has been increasing in many environments worldwide.

The cause for these population declines and abnormalities is not known, however, there are a large number of theories. One theory is that over-exposure to the pollution of heavy metals and pesticides causes population declines. This project determined how exposure to zinc (heavy metal) and diazinon (pesticide) affected the African clawed frogÕs (Xenopus laevis) embryonic development. Zinc was chosen as a heavy metal due to its many uses in industry and its high probability of being present in the environment. Concern has been raised about the possible risks associated with the presence of zinc in the aquatic environment. This concern has triggered scientific debate about risk assessment methodology for essential elements such as zinc.

Zinc is an essential element that is required for human health and for all other living organisms. Organisms absorb zinc from their environment, when their cellular requirements for zinc are satisfied, growth and development are normal, however, excessive zinc intake can lead to toxicity and abnormal development. The dietary requirements of zinc increase the complexity of the interaction with the environment; a fact that should be taken into account when considering regulations aimed at governing the production and uses of zinc. Failure to do so may result in regulations that will not benefit the environment in the long run (Assche and Martin 1996).

Diazinon is an organophosphate insecticide commonly used in the United States on a wide variety of agricultural crops, lawns and gardens, and household pests. Diazinon kills insects by interfering with the action of important enzymes in the nervous system, leading to death of the insect. Because diazinon does not readily degrade, run-off from a treated area can contaminate water systems. The source of diazinon used for this project was Spectracideâ , a commonly used pesticide.

Xenopus laevis was chosen as the subject because of its hardiness in captivity and the ease of obtaining embryos when needed for testing. It is commonly used for laboratory study. There is considerable information on its normal embryonic development.

The frog egg is divided into two regions. The top half is called the animal pole and the lower half is referred to as the vegital pole. The embryonic development of the egg consists of four major steps: cleavage, gastrulation, neurulation, and organogenesis (Burton, 1987).

Cleavage is a rapid cell division without growth that creates a hollow ball called the blastula. The cells produced by cleavage form the material bricks from which the basic shape of the future animal will be constructed in the next stage of development, which is gastrulation.

In the gastrulation stage tucking and folding of the blastula makes a three-layered structure called the gastrula. The three layers that make up the gastrula are the ectoderm, mesoderm, and endoderm. The ectoderm forms: the skin, lateral line system, brain, cephalic ganglia and nerves, spinal cord, spinal ganglia and nerves, olfactory organ, eyes and auditory organs. The middle layer is the mesoderm, which produces the skeleton of the head (including plectral apparatus), musculature of the head, axial skeleton, axial and abdominal musculature, skeleton of the shoulder girdle, skeleton of the forelimbs, musculature of the shoulder girdle, musculature of forelimbs, skeleton of pelvic girdle, skeleton of hind limbs, musculature of pelvic girdle, musculature of hind limbs, heart, arterial system, venous system, lymphatic system, pronephric system, mesonephric system, gonadal system, and finally the adrenals. The inner layer, or last layer, is the endoderm which produces the oro-pharyngeal cavity with visceral pouches (including external gills and operculum), lungs, thyroid, brachial derivatives (including middle ear and tympanic membrane), and lastly the intestinal tract and glands (Nieuwkoop and Faber, 1994).

These three layers are formed as the blastula turns into the gastrula. During gastrulation a curved slit called the blastopore forms just below the equator of the egg, and the outer layer of cells on the animal hemisphere (the hemisphere in which the animal pole is contained) actively moves into the slit and back under the surface. Eventually all the pale yolk cells are enclosed except for a white spot, the yolk-plug, now contrasting with the completely black embryo (Burton 1987).

In neurulation, a thickened strip of ectoderm, the neural plate, rises in two parallel folds and bends inward to form the neural tube, which will become the spinal cord. Three swellings at the head appear, which mark three main divisions of the brain.

In organogenesis, the final process, the formation of organs and growth of the embryo occurs. Organ creation consists of two processes: induction, in which the position of each cell determines how it will develop because it receives a series of chemical messages from the cells around it. When a cell divides, each daughter cell receives identical genes; however, the new cells receive a signal that activates only certain genes.

The second process involves the movements of a cell in gastrulation, and subsequent events, which are controlled by changes in the "stickiness" of contact between a cell and its neighbors. When moving, the cells slide freely over each other, eventually sticking to form a solid mass of tissue when they have reached the right place. Contact with a particular type of cell increases stickiness and the joining of the cells becomes permanent (Burton, 1987).

Through these processes (cleavage, gastrulation, neurulation, organogenesis), the organs are formed until the embryo is half formed. At this point, it leaves the egg as a tadpole and continues formation through metamorphosis.

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Embryos from stage four to thirty-four, according to Nieuwkoop and Faber, were divided into five different groups: the control group, two groups to which different concentrations of zinc (0.5ppm and 0.1ppm) were added, and two groups with different concentrations of diazinon (2.6ppm and 1.27ppm). Samples from embryo stages 9-12, 13-16, 17-20, 21-24, 25-28 and 29-34 were removed from the solutions and preserved using Karnovsky's fixative and Bouin's 2000 for later histological analysis. The histology tests were accomplished by preserving the eggs, embedding them in a plastic resin, sectioning the embryos and then staining the slides. The various stages of internal development of the embryo were observable in the stained sections.

When all testing was complete, the embryos were embedded into JB-4. A microtome was used to cut sections of the embryos 5-10µm thick. The slides were stained with GillÕs Hematoxylin #3, treated with sodium bicarbonate, and stained with Eosin so the embryos and their various parts could be observed easily under the microscope. Approximately seven hundred slides were prepared for analysis. Pictures were taken of slides showing normal and abnormal development.

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In the embryos exposed to 1.27ppm of diazinon undergoing gastrulation, the cells had problems involuting properly (Figure 1, Gastrulation Photos, ). During gastrulation, there is the possibility that the diazinon is affecting the behavior of the cells. This means that while the cells were involuting, it is possible the diazinon affected the enzymes which controlled the movement of the cells. With the enzymes functioning abnormally, the resulting tissue layers were poorly organized.

Neurulation is the process in which a thickened strip of ectoderm, the neural plate, rises in two parallel folds and bends inward to form the neural tube. The neural plate of many of the embryos exposed to zinc and diazinon had poor formation. It is interesting to note that diazinon kills insects by interfering with the action of important enzymes in the nervous system. With the development of the neural plate, archenteron and axial structures, the diazinon and zinc may interfere with the enzymes forming the neural plate causing poor organization of the notochord and somatic tissue while blastocoel was still present in the embryos and there was an abundance of mesoderm. In some embryos, there were strange cavities present. The archenteron was abnormally formed in numerous embryos, and the axial structures, the beginning of the notochord and somatic tissue, were weakly developed (Figure 2, Neurulation Photos). During organogenesis, the neural tissue of the anterior region was poorly organized and the forebrain was poorly developed. In the posterior region, the layers of tissue were poorly organized and one embryo had a mass of tissue protruding from its end.

In the group exposed to 2.6ppm of diazinon, the embryos had difficulty gastrulating and the cells had problems involuting. In neurulation, common abnormalities observed were weak development of the neural plate, the archenteron, segmental plate mesoderm and axial structures. The blastocoel was still present and the blastopore did not close. There was poor organization of the ectodermal layer, there was an abundance of mesoderm, and tissue was forming in the archenteron. During organogenesis, the neural tissue of the anterior region was poorly organized, and there was inadequate development of the forebrain, notochord, somites and gut. Endodermal yolk cells were present in the gut of one of the embryos. The posterior end had poor organization of tissue layers with a mass of tissue protruding from the end of some embryos (Figure 3,Organogenesis Photos ).

Embryos exposed to 0.1ppm of zinc had problems gastrulating normally. During neurulation, common abnormalities observed were the blastopore not closed, presence of blastocoel, abundance of mesoderm, poor development of the archenteron, the neural plate and axial structures. During organogenesis, the anterior end had poor organization of neural tissue and inadequate development of the forebrain. Some embryos had improper formation of somites and abnormal digestive systems. The posterior end had inferior organization of tissue layers. The embryos were stunted in length and had masses of tissues protruding from the anterior end.

Embryos exposed to 0.5pp of zinc undergoing blastulation had poor formation of micromeres and macromeres. During gastrulation, embryos had problems involuting. During neurulation, there was an abundance of mesoderm, poor formation of archenteron, improper closing of the blastopore, poor organization of endoderm and ectoderm, and inferior development of the axial structures. During organogenesis, the anterior region had poor organization of the neural tissues and poor development of the forebrain. The embryo had improper development of the notochord. In addition, there was poor organization of somatic tissues and inadequate development of the digestive tract. The posterior region had poor organization of posterior tissue. In many later staged embryos, there was a mass of tissue protruding from the posterior end.

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Embryos exposed to the two different concentrations of zinc and diazinon developed similar abnormalities and statistically the degree of the abnormalities was the same. As the hypothesis stated, abnormalities were seen in gastrulation and neurulation. The abnormalities observed started to occur in later gastrulation. The majority of problems appearing dealt with gastrulation and the blastopore not closing completely, or not at all.

Gastrulation is the tucking and folding of the blastula to make a three-layer structure called a gastrula. This process of tucking and folding is called involution. The embryos exposed to the pollutants had problems involuting, causing the three layers that make up the gastrula (ectoderm, mesoderm, and endoderm) to be poorly organized. In gastrulation, the pollutants may be causing two problems. First, there is the possibility that they are affecting the general health of the cells. Second, there is the possibility that they are affecting the behavior of the cells. This means that while the cells are involuting, it is possible that the pollutants affected the enzymes which told the cells where to go. With enzymes abnormal, the resulting tissue layers would be poorly organized.

After gastrulation, during neurulation and organogenesis, the anterior end begins to develop and then the posterior region begins to form. Almost all of the problems that occurred were due to problems during gastrulation, especially the anterior defects which led to posterior abnormalities. In the anterior region, the abnormalities seen caused a domino effect. The gastrula involuted improperly, resulting in poor organization of the ectoderm, mesoderm, and endoderm, leading to poor formation of the archenteron, causing poor neurulation - poor development of the neural plate, followed by poor formation of the notochord and somatic tissue, allowing poor organization of the neural tissue of the anterior region, the forebrain and the digestive tract (former archenteron); finally this led to poor development of the posterior end. This calamity of events seemed to take place whether the embryos were exposed to zinc or diazinon, but the embryos in the control group were not similarly affected.

A normality scale was developed to determine the severity of the abnormalities so a statistical analysis could be done. The normality scale ranged from one to four, one being normal and four being severely abnormal. A criterion was established for each of the three major groups of embryonic development: gastrulation, neurulation, and organogenesis.

A Student's T- test was performed for each concentration of the pollutants as compared to the control. The sample size consisted of 15 randomly selected embryos from a total of 750. In all of the concentrations there is a 95% probability that the data for each group is significantly different than the data from the control group. The degrees of freedom for both concentrations of zinc and both concentrations of diazinon were very similar. The type and degree of the observable abnormalities was similar for both chemicals. The statistical results support this conclusion.

This project supports the theory that elevated concentrations of heavy metals and pesticides in the environment are a possible cause for the recent increase of abnormalities in the anuran population. Zinc and diazinon were used for these studies, but they are only a small percentage of the chemicals in the environment. Since frogs are classified as bio-indicators, a disruption in anuran development in any habitat signals an ecological crisis. By recognizing the importance of saving frogs and acting to stop environmental contamination, we can possibly save other species, including ourselves.

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