2001 US SJWP Winner's Abstracts
2001 U.S. National Competition
Junior Water Prize Winner
Molecular Characterization of Potential Fish Pathogens in
Waters Where Reported Pfiesteria piscicida Outbreaks Have
Thomas Jefferson High School for
Science and Technology
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'
The Development of Tunable Biopolymers for the Remediation of Mercury
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.
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
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
Lake Brantley High School
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
The Effects and Mechanisms of Action of
Endocrine Disrupting Chemical Pollutants on Reproductive System
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.