2001 US SJWP Winner's Abstracts

2001 U.S. National Competition
Stockholm Junior Water Prize Winner

Molecular Characterization of Potential Fish Pathogens in Waters Where Reported Pfiesteria piscicida Outbreaks Have Occurred

Brenda Goguen
Thomas Jefferson High School for Science and Technology
Alexandria, VA


Fish kills occurring along Atlantic slope waters, including in the Chesapeake Bay have been attributed to toxins released by the dinoflagellate Pfiesteria piscicida and to a proposed amoeboid form of P. piscicida. However, recent studies have indicated that the organism's presence is unrelated to fish kills and that its proposed amoeboid form does not exist. The purpose of this research, therefore, was to characterize protists and bacteria in waters where reported Pfiesteria outbreaks have occurred to molecularly determine if the organism is related to fish kills. DNA was extracted, amplified by polymerase chain reaction using fluorescently labeled protist and bacterial primers, and fingerprinted by amplicon length heterogeneity (ALH) from soil sediments taken from five rivers that empty into the Chesapeake Bay, from eighteen samples taken from an experimental fish tank from the Center of Marine Biotechnology (COMB), and from twenty-eight samples purported to be pure amoeboid Pfiesteria cultures (Tester samples). The fingerprints from the Chesapeake rivers found that P. piscicida was present in small amounts only in three of the five rivers, preliminarily indicating that P. piscicida may not have had a role in the fish kills. The fingerprints of the COMB samples also showed no direct correlation between the presence of P. piscicida and the occurrence of fish death. The fingerprints from the Tester samples revealed that many samples were not pure because other organisms were present in them. From this data, it is reasonable to question whether P. piscicida is a cause of fish kills. The data instead supports the hypothesis that other factors may be involved with the kills. Additionally, the bacterial ALH fingerprints may help determine if fish kills are the result of endosymbiotic bacteria associated with P. piscicida. This study demonstrates the importance of accurately studying all the possible causative factors in an epizootic event befor efinitively naming a cause.

Stockholm Junior Water Prize Finalists' Abstracts
2001 Competition

The Development of Tunable Biopolymers for the Remediation of Mercury Contaminated Water

Kate Gropp
Centennial High School
Corona, CA


Introduction: Current technologies for the remediation of mercury from water are not sufficient to reduce the concentrations of mercury to acceptable regulatory standards. There exists a need for the development of an alternative polishing process that will efficiently reduce mercury concentration levels to EPA standards.
Objective: To develop a highly specific tunable biopolymer. This bioploymer consists of a repeating elastin domain and a mercury-binding domain.
Materials and Methods: The MerR mercury-binding domain was selected for its 100 to 1000 times greater affinity for mercury than for other metals, mediating its specific and selective removal. The MerR domain was fused to the elastin domain which has been shown to undergo reversible, inverse phase transition a property particularly valuable in protein purification. Once the genes were fused through ligation the resulting plasmid was inserted into E. coli cells and expressed. The biopolymer was then purified, tested for the inverse phase transition, and tested for its mercury binding capacity.
Results: The biopolymer was purified with only minimal loss as shown by protein visualization gel. The biopolymer then demonstrated the inverse phase transition at the desirable range of 24-30° C. Also aggregation was determined to be a proportional function of both protein and NaCl concentrations. In the mercury-binding experiments, the protein bound a maximum of 76% of the mercury in solution.
Discussion: This project has demonstrated that the elastin protein will undergo the temperature transition while fused to another domain. It has also demonstrated that the binding capacity of the Mer-R domain is still functional while bound to the elastin. With these two properties functioning, the protein now has the potential to be applied to mercury remediation. This experimentation has not only developed a biopolymer for the remediation of mercury, but also has demonstrated the potential for the development of similarly tunable biopolymers.

Use of Daphnia magna as an Indicator of Water Toxicity

Avi Robbins and Jessica Chiafair
Lake Brantley High School
Altamonte Springs, FL


The purpose of this study was to exhibit the abilities of Daphnia magna as a bioindicator of toxicants present in freshwater through biochemiluminescence. The Daphnia fluoresce after ingestion of a detection substrate and exposure to long wave ultraviolet light. We hypothesize that subjection to toxicants will impair the Daphnia's abilities to fluoresce displaying toxicants in the water.
In order to show the impairment of fluorescence we cultivated many Daphnia magna in freshwater and starved them overnight prior to experimentation. The Daphnia were subjected to 0, 6.25, 12.5, 25, 50, and 100 percent toxicant solutions of CuSO4 in one half part per million. After forty-five minutes of exposure they were allowed to ingest a fluorescent detection substrate. The Daphnia cleave the bound sugar-marker molecule which is B-D-galactoside sugar with a 4-methylumbellifery fluorescent marker attached (C16H18O8). After subjection to toxic solutions the Daphnia will be adversely affected, and the fluorescent marker will not be cleaved. Detecting the fluorescence takes place by exposing the Daphnia to long wave ultraviolet light and comparing the amount of fluorescence of the affected Daphnia to that of the control.
When the Daphnia were proven to not cleave the fluorescent marker they were placed in freshwater river samples and shuttle drinking water. The Daphnia, once exposed to the water and then allowed to ingest the fluorescent detection substrate will exhibit the presence or lack of toxicants in the water. This will further be tested by chromatography or using a mass spectrometer with the same water samples the Daphnia were subjected to. Results are pending.

The Effects and Mechanisms of Action of Endocrine Disrupting Chemical Pollutants on Reproductive System Development

Jacqueline Ling
Midwood High School
Brooklyn, NY


Methoxychlor has replaced DDT as a pesticide and has found its way into the aquatic environment where it is behaving as an endocrine disrupting chemical (EDC) pollutant. EDCs can mimic the effects of natural hormones and, as such are causing severe problems in endocrine system development and function in vertebrates in a variety of ecosystems. As a result, life cycles and food chains are becoming severely impaired. In this study, we have subjected small freshwater fish (teleosts) to varying concentrations of methoxychlor in order to study the mechanisms and sites of action of this anthropogenic agent on the development of their reproductive systems. Behavioral, histological, and immunocytochemical methods were employed to study the brain, pituitary, and gonads (BPG axis). We have found that exposure of developing fish to methoxychlor: 1) retards growth, 2) retards development of sex steroid hormone-dependent secondary sex characters, 3) retards gonad development and gametogenesis, 4) retards gonadotropin hormone (GTH) producing cells in the pituitary gland and 5) does not seem to have any effect on the gonadotropin releasing hormone (GnRH) producing centers in the brain. Our results suggest, that although the effects of methoxychlor, as presented in this report, are seen only to retard reproductive system development, a failure of a concerted effort to eliminate such harmful pollutants from the environment may lead to a total "castration" of reproductive systems in subsequent generations.