Stephen Tinnin
Bartlesville High School
Bartlesville, Oklahoma 74008-8030.
The purpose of this experimentation was to investigate potential harm to the environment by pesticide contamination. Focus was placed on the embryos of sea urchins, which may serve as an indicator species for effect upon the marine ecosystem. The embryos of sea urchins may also function as a representative of a developing fetus in general. Vital functions of the embryo were examined, including the performance of the egg, sperm, and fertilization membrane, as well as overall development. Procedures included preparation of seawater, preparation of pesticide media, obtainment of gametes, exposure of gametes, and observation of gametes. Exposure entailed insecticide, herbicide, fungicide, and a mixture of the three at concentrations ranging from 0.01 ppm to 1000 ppm. A positive correlation was consistently found between increasing concentration of pesticide and harm presented to the embryo. Damage included decreased sperm activity, increase parthenogenesis, lowered rates of true fertilization, and hindrance upon development. Decreased sperm activity is seen as greatly lowering the possibility for fertilization; the extremely short life span of the parthenogenically activated eggs could urchin output. Further tests supported the idea that the toxicity of the pesticides caused membrane proteins to denature, allowing their direct passage through otherwise selective channels. This was supported with observations of the cytoplasm retracting from the hyaline layer. The current trend of pesticide use is seen as having a great potential for harm upon sea urchin development as well as other marine organisms and our ecosystem in general.
Pesticides create a public policy and regulatory dilemma because, in most cases, the toxicity that makes them useful in controlling pests also poses potential risks for humans and the environment (5,13,24). Pesticide use generally confers benefits in terms of improved quantity and quality of foods, disease prevention, structural protection, and nuisance control (5,24). At the same time, pesticide use may result in exposure of nontarget fish and wildlife, plants, farm-workers, and food consumers (13,24). There are currently about 21,000 pesticide products formulated from some 860 different active ingredients (30). EPA estimates that domestic users spent $8.5 billion for 1.1 billion pounds of active ingredients in 1993. Yet only 0.1% of the pesticide reaches the target pest (33,34). The remainder enters the environment.
The purpose of this experimentation was to investigate the severity of impact upon the environment by pesticide contamination. Focus was placed on the embryos of sea urchins, which may serve as an indicator species for effect upon the marine ecosystem. The embryos of sea urchins may also function as a representative of a developing fetus in general. Vital functions of the embryo were examined, including the performance of the egg, sperm, and fertilization membrane, as well as overall development. In light of the current scientific opinion that pesticides are not generally beneficial to the biological systems of organisms, it was felt that increasing concentrations of pesticides would increasingly harm the egg, sperm, and zygote of urchins.
Echinoid echinoderms, such as sea urchins, have been the subjects of many investigations of fertilization and early development, and much of our understanding of early developmental processes in animals has come from this research (12,16,26,27,28,37). Sea urchin gametes are readily obtained just before, and during, the breeding season and developing sea urchin embryos can be cultured in seawater or salt solutions that approximate the osmotic and ionic properties of seawater (12,16,26,27). Eggs and embryos of many sea urchin species are quite translucent, so it is possible to observe a number of cell activities during early development using a light microscope (16,27,28).
As in other echinoderms, the sexes are separate in sea urchins. In nature, gametes are discharged into the water and the sperm swim freely until they reach an egg (12,14,31). Since the sexes of sea urchins are difficult or impossible to distinguish by external features, sex of an individual animal must be determined by observing the gametes that it sheds. Injection of a small amount of potassium chloride into the coelom will induce an urchin to shed its gametes. The sex of the animal can then be determined by observing the color of gametes that are extruded from gonopores of the aboral (dorsal) surface of the animal within a few minutes after injection (12,16,27,28). The eggs of Lytechinus variegatus, the species of sea urchin used with this investigation, usually occur in a shade of pale yellow; sperm, when shed in mass, appear white or very light grey (16,27,37).
Developing sea urchins usually can be reared to the early pluteus larva stage in the laboratory, but they are difficult to culture successfully beyond this stage because the larvae usually will not develop further unless fed appropriate marine algae (14,26,28). Developing embryos should be maintained at a temperature appropriate for the particular species, which is room temperature for L. variegatus (14,31).
Cleavage for echinoid echinoderm embryos is holoblastic (12,26,31). That is, the entire cell is divided at cytokinesis during each cleavage division. The first cleavage, which is meridional, produces a two-cell embryo. The second cleavage division is also meridional and yields a four-cell embryo. In the third cleavage, the plane of division is at right angles to the first two cleavages and the product of the division is an eight-cell embryo with upper and lower quartets of cells. During the fourth cleavage, the four blastomeres of the upper (animal) quartet divide equally to form a single tier of eight medium-sized cells called mesomeres. However, the divisions of the other four (vegetal) blastomeres are very unequal, producing a middle tier of four larger macromeres and a lower tier of four much smaller micromeres that lie at the vegetal pole of the embryo. As cleavage proceeds, the embryo becomes organized as a single-layered, hollow ball of cells surrounding a cavity that is known as the blastocoel. The embryo at this stage of development is called a blastula (12,26,28).
Literally thousands of experimental investigations in developmental biology, as well as in cell and molecular biology, have been done using the gametes and embryos of echinoid echinoderms. The results of those and many studies have greatly expanded knowledge in several areas of biology.
Early in the twentieth century, F. R. Lillie studied sea urchin gametes extensively. Among his many observations was the discovery of a sperm clumping response induced by extracts from eggs' jelly (23). When unfertilized eggs are separated from the seawater in which they have been standing for an extended period, and the supernatant (or "egg water") is added to a sperm suspension, the sperm form dense clusters. Later, the clusters disperse, but sperm that have reacted in this way are no longer capable of participating in fertilization interactions (6,8,35). These observations became the basis for the fertilizin-antifertilizin hypothesis of sperm-egg interaction developed by Lillie and others. This hypothesis suggested that egg water contains a soluble agglutinating factor from the jelly surrounding mature sea urchin eggs that might play a role in fertilization, or possibly in polyspermy prevention (6,35).
The fertilizin-antifertilizin hypothesis was later re-examined when the responses of sperm in "egg-water" were analyzed further. It was observed that egg water induces sperm to undergo the acrosome reaction, including extrusion of the acrosomal process, and that the sperm clumping seen in egg water is not an agglutination process such as that caused by antibodies or other cross-linking factors (6,8). Rather, sperm actively swim into rosettelike clusters where their acrosomal processes adhere. It is now clear that the inability of sperm previously subjected to egg water to participate in fertilization is due to their acrosomal condition and not to an agglutinating factor from egg jelly coats that blocks binding sites (1). An acrosome surface protein called bindin that is involved in specific binding of sperm to eggs was discovered in the 1970's, and an egg membrane receptor that specifically binds to sperm was finally isolated nearly twenty years later (1,10).
The process of fertilization involves a complex set of cellular responses and interactions and many aspects of the egg cell's physiology change as a result of the activation occurring during and after sperm contact and entry. Some of these changes can be initiated artificially by various treatments. Egg activation without sperm contact, called parthenogenesis and artificial parthenogenesis, in sea urchin eggs has been investigated by many biologists since it was first studied by Oscar and Richard Hertwig during the 1880's. Many experimental treatments have been found to cause some, or even many, of the activation responses to occur in sea urchin eggs (4,9,20). One of the easiest to perform is the immersion in seawater made hypertonic to egg cells by addition of thirty grams of sodium chloride per liter. This procedure is one recommended by the great early twentieth century American developmental biologist, Ethel Browne Harvey, who assembled an extensive list of artificial parthenogenic agents (20).
Sven Horstadius, a Swedish embryologist using microsurgical techniques, made numerous artificial combinations of separated cells of 64-cell sea urchin embryos and studied the subsequent development of the embryos produced. Results of his and other experiments led to the development of the double gradient hypothesis of developmental regulation (21,17). The hypothesis proposed that two gradients of physiological activity exist, one with maximal activity at the animal pole and the other with maximal activity at the vegetal pole, and that normal development depends upon a properly balanced interaction between the two gradients. The double gradient hypothesis also was used to explain the disruptive effects that a number of chemical substances have on sea urchin development (18,22,30).
Other investigations of interactions among the blastomeres of the early sea urchin embryo have demonstrated that there are indeed influences that pass from vegetal cells to animal pole cells (15,19), but these results can be interpreted as inductive interactions and do not provide evidence for a physiological gradient. Furthermore, there is little or no experimental evidence supporting the existence of an animal influence that depends upon the existence of a gradient of an activity or a substance centered in the animal portion of the egg or embryo (37).
Finally, Ransick and Davidson reported experiments in which extra micromeres were transplanted to the animal pole of 8- or 16-cell embryos. Results of their experiments contradict expectations based on the double gradient hypothesis, which would predict that a recipient embryo in such an experiment would become strongly "vegetalized" (28). That is, the embryo would be expected to produce an enormous and probably exogastrulated archenteron. Instead, cells that normally are ectodermal precursors were induced to form vegetal plate cells. Invagination of this extra vegetal plate produced a second structurally normal archenteron that met the embryo's original archenteron and turned with it toward the prospective mouth area. The Endo-16 gene that codes for a gut-specific cell surface protein was expressed in an apparently normal temporal and spatial pattern in this induced second gut.
The effects of various chemicals on sea urchin development that have been interpreted in relation to the double gradient hypothesis may have other kinds of explanations. For example, the sometimes dramatic effects of lithium ions on developmental processes (17,25,35) probably can be explained on the basis of lithium's powerful interactions with cellular signaling systems (2,3). Lithium ions indeed cause changes in quantities of inositol phosphates in sea urchin embryos (7). It is not particularly surprising that lithium has significant effects on developmental processes that involve inductive interactions. The effects of other agents, such as the anesthetics procaine and tetracaine, may also be explainable in the contexts of basic cell biology and pharmacology (11).
I. Preparation of seawater
II. Preparation of media
Effects upon fertilization and development can be seen in results of sperm activity and parthenogenesis.
Insecticide: A positive correlation was seen between increasing interference with development and increasing concentration of Malathion, with higher concentrations multiplying the effects. Sperm activity was greatly affected even at low concentrations, and parthenogenesis was rampant at high concentrations. Data indicates that interference with fertilization should be common. In "Fertilization and Development" groups, though, the number of eggs fertilized seemed to be virtually unaffected. It is determined, then, that while the majority of sperm were quickly eliminated by the toxicity of Malathion, the eggs were parthenogenically activated. This, then, explains the high death rate between fertilization and cleavage one. Fertilization is therefore made difficult by the fact that sperm are killed before they get the chance to reach an egg, egg membranes and fertilization membranes are eroded by Malathion, the insecticide creates osmotic differences leading to deformation of the embryo, and if an egg gets through all that, it will likely be parthenogenically activated, rendering it useless to embryonic development.
Herbicide: Interruption of development by Dimethylamine salt was greater than that by Malathion, with the gradient between limited and extreme obstruction again lying between 1 ppm and 1000 ppm. Parthenogenesis in herbicide pollution proved to be a much smaller factor than in the insecticide, while sperm activity appeared to be moderately effected. Data from "Fertilization and Development" followed this trend, in that high concentrations provided few fertilized eggs, of which none developed further. Numbers of fertilized eggs in the 1000 ppm concentration were similar in "Fertilization and Development" and "Parthenogenesis", lending support to the idea that few, if any, eggs were truly fertilized, rather parthenogenically activated.
Fungicide: Impact upon pure development by Chlorothalonil was greater than that of any group, while obstruction of fertilization was minimal. Parthenogenesis caused by Cholorthalonil was apparent, though not predominant. The activity of sperm was relatively unaffected by concentrations of fungicide up to 1000 ppm; if anything, action seemed to have increased. The simple fact that activity increased, though, is not a true indication of the health of the sperm. Cessation of sperm life is recognized as a indicator of interference, but impediment of chemical interaction between egg and sperm can quickly void the ordeal of fertilization. Chemicals the jelly coat of an egg emit into the seawater may also be misinterpreted by a searching sperm. The notable difference between the number of eggs fertilized in the 1000 ppm concentrations of "Fertilization and Development" and "Parthenogenesis" supports the idea that these sperm were capable of suitable interaction with the egg to induce the formation of the fertilization membrane. Although hampering of development occurred to any eggs that were able to achieve fertilization.
Mixture of all: Rather than intensify the effect upon the embryo by an interaction of the different pesticides, impact upon development by the mixture appeared to be an average of previously observed phenomena. Characteristics of deformations from insecticides, herbicides, and fungicides all appeared upon embryos in this blend. An intensification of effects was apparent, though, in sperm activity. Diverse results of sperm exposure to the different pesticides provided a playing field for a deadly combination of chemicals. By overloading sperm with separate instructions, the entire system likely chose to shut down. The 1000 ppm concentration showed essentially no life on the part of the sperm, allowing little chance for insemination. Coupled with a high rate of parthenogenesis, few eggs provided an opportunity for fertilization. The proportionately high rate of fertilization in the 1000 ppm concentration likely stems from the high rate of parthenogenesis.
Overall Observations: Although 0.01 and 1 ppm concentrations are relatively acceptable according to EPA standards and present in many areas around the world, one of 1000 ppm is pragmatically unrealistic. This fact is accepted; but it must be realized that it serves as a large-scale representation as to the impact upon embryos exposed to lower concentrations. If zygotes at lower concentrations were allowed to fully develop, the minor effects would likely grow into major complications. The mixture used incorporated only a certain ppm of total pesticides. Since EPA regulations are based on specific chemicals, a mixture could contain 1 ppm of each pesticide and pass the same test as only 1 ppm of a particular chemical. Mixtures, therefore, have the potential to become much more dangerous than depicted in this experimentation, due to their additive nature and interaction with other chemicals. With 860 different active ingredients incorporated into 21,000 different pesticide products, regulations must take into account the compoundability of pesticides.
The altering of the marine environment has occurred too quickly. Pesticides present a serious threat toward sea urchins, along with marine life and the biosphere in general. Given sufficient time, species would most likely be able to adapt to these contaminated conditions. Humans can not expect decades, though, to be adequate for suitable genetic mutations to occur, and for the gene pool to fill with resistant characteristics. At the current pace, irreparable damage is likely in our marine environment. With an estimated one million cases of human pesticide over-exposure in the U.S. alone, resulting in twenty thousand deaths, the threat is not limited to simply marine organisms. The short half-life of most pesticides, though, provides us with a method to fix our mess. With a dramatic reduction in usage, irreversible damage can be avoided. This will require action by Congress; to overcome large "donations" by farming groups, the populous must force the issue, so that representatives' seats will depend upon action. Once again, environmental issues have fallen into the hands of the individual.