2. Title
Analysis of the direct bacteriological contamination of the superficial and underground water
bodies of the San Salvador neighborhood in Caguas and its implications on the community water
systems.
Abstract
Most of the water used by the population of Puerto Rico (97%) comes from the Water
and Sewage Authority. However, the rest of the population (3%) acquires their water from
community water systems which obtain it from water bodies near the community and on which
the residents themselves build systems to distribute the water. Many of these community water
systems haven’t been examined by the agencies in charge of this and many of the ones that have
been inspected have shown deficiencies because of the lack of economic resources, the
disinformation of the communities on the established parameters for the conservation of tap
water, for the construction of water and septic systems and for the disposal of used waters, and
because of the lack of knowledge of the residents on the protocols for the good constructing,
processing and maintenance of water systems. Because of all these factors, this investigation
infers that the water from the water bodies that serve community water systems is contaminated
by human means, especially by fecal contamination developed from the percolation of septic
tanks. To determine with certainty all of this, the San Salvador neighborhood from Caguas,
Puerto Rico has been chosen to study the community water systems, which are known to be used
in the area, the septic systems and the presence of bacteriological pollution in the water bodies of
the zone. This inquire will also treat the health and legal implications of the construction and
3. maintenance of these water systems by analyzing bacteria in the water of the area and checking if
the results have any relationship with diseases derived from those bacteria and by researching the
established parameters for these water systems and comparing them with the qualitative evidence
on the systems of this community. This research also implies the participation of the community
in interviews and, lastly, in orientations about the results of the study. After this research, it is
expected to conclude that in the San Salvador neighborhood there are certain violations to the
federal and state regulations on tap and used water, which result from ignorance of the topic and
which cause the bacteriological contamination of water along with the spread of diseases related
to those bacteria.
4. Introduction
In Puerto Rico, 97% of the population receives their water resource from the Water and
Sewage Authority. The other 3% of the population (approximately 125, 000 persons) obtains
their water from community water systems, which, as their name says, are operated by the
residents of the community themselves (EPA, 2001). These community water systems can be
classified on superficial or underground, according to the source of the water. As studied by the
Water and Sewage Authority of Puerto Rico in 2004 (AAA, 2004) there are about 239 superficial
and underground community water systems in our Island. About 41% of these 239 community
water systems do not reach the bacteriological parameters established by the Department of
Health of Puerto Rico, the Environmental Quality Board of Puerto Rico, the Water and Sewage
Authority or by the Environmental Protection Agency (EPA) under the National Primary
Regulation on Tap Water (EPA, 2003). As several studies from these agencies expose, this may
be because of the insufficient amount of trustable epidemiologic information, the poor
economical resources invested on the construction and maintenance of these community water
systems, the scarce education of the people who administrate these systems, the lack of the
required filtration methods on most of them and by the construction deficiencies of old-
uninspected septic tanks which allow percolations. It can be said by this that this is a multisource
problem.
Because of all these, it has been decided to investigate the real conditions of the water of one
of these community water systems. The chosen community water system is located in the San
Salvador neighborhood in Caguas Puerto Rico. San Salvador neighborhood borders in the North
with Borinquen neighborhood and in the South with the Municipalities of Cayey and San
Lorenzo. This vicinity has a population of 3,272 habitants distributed in about 1,133 houses
5. (Census Bureau, 2000). The exact number of people of this area who use community water
systems is not certainly known, but that’s why it is one of the objectives of this research. Since
this is a rural community which was founded several decades ago, it is not so updated with the
required parameters and technology for the treatment of tap water and for the disposal of used
water. Many of the employed water of this area is disposed on septic tanks or flows through the
ditches, landing ultimately on the water bodies and the soil of the area as percolation. That way,
both the superficial and the underground water result affected. This can have a direct effect on
the population because it can be a medium of reproduction and transmition of microorganisms
some of which may be pathogenic. The diseases that these pathogenic agents cause are called
water diseases because they are mostly transmitted by the consumption of contaminated water.
According to the Health Department of Puerto Rico, (Departamento de Salud de Puerto Rico,
2008) the most common water diseases are: diarrhea, vomits, fever, respiratory infections,
urinary infections, meningitis, between others. Most of these are caused by protozoans such as
Cryptosporidium parvum, by bacteria such as Salmonella, Shigella, Klebsiella, Enterobacter,
Citrobacter, by viruses such as Group A Rotavirus or by parasites such as Giardias and
Amoebas. Most of these microorganisms are very hard to detect because of the small amounts of
them that are typically found on samples, but by detecting the presence of a specific bacteria we
can determine the presence of these. E. coli is a bacterium from the coliform group and it is used
as an indicator of fecal contamination because its incidence reveals the existence of other
microorganisms which can be pathogenic. It is also a well known and studied bacterium and
most of its commonly found strains are non-pathogenic. That’s why it is mostly used in the water
quality tests. However, the absence of these bacteria does not necessarily exclude contamination
of water with protozoa or virus.
6. Because of the geological and public health implications of the contamination of water in the
community water systems of the San Salvador neighborhood explained above, it has been
decided to propose the making of this investigation. By means of it, it is intended to investigate
about the established parameters for the conservation of tap water, and also about its concern and
observance in the area. On the other hand, it is deliberated to make some fieldwork to inspect
exactly how many community water systems are in the area and how many residents of the
neighborhood use water from them. It is also proposed to evaluate the condition, the given
permits and the maintenance of the community water and septic systems in the vicinity of San
Salvador to determine if they are a potential risk in the quality of both superficial and
underground water of the zone. To do this, it is planned to compare the construction of the
community’s septic tanks with the regulations of the Individual System of Residual Water
Disposition, as established by the Administration of Regulations and Permits of Puerto Rico
(ARPE) in the Edification Guideline.
After making this research, it is projected to determine if there are found bacteriological
pollutants from the coliform group in the underground and superficial water bodies of the San
Salvador neighborhood in Caguas. Because of the health issues related to the presence of such
microbes in water, it is also intended to analyze the existence and the recurrence, if any, of
diseases related to these bacteriological pollutants in the residents of the studied community.
Finally, after all this investigation and analysis has been made, the main objective of it is to raise
awareness about the regulations for the conservation of tap water by orienting the residents of the
area and publishing the results.
During this investigation it is expected to find out that the community water systems of
the San Salvador neighborhood from Caguas are being polluted by human means and, therefore,
7. have the presence of bacteriological pollutants which will have a direct effect on the health of
people who use the water from them without decontaminating it first.
8. Methodology
For this investigation, it is intended to study the community water systems of the San
Salvador neighborhood in Caguas because it is known that there are several families that live in
it and use the water from these kind of systems. These systems haven’t necessarily been
inspected or studied before and there is no certainty on the quality of the water in them because
of the percolations from septic tanks and from other human activities, because of the insufficient
amount of trustable epidemiologic information and because of the poor economical resources
invested on the construction and maintenance of them.
To reach the objectives of this research, there are several steps that have to be done. First, it
would be needed to research about the established parameters for the conservation of tap water in
Puerto Rico, mainly about the community water systems, the septic tanks and their use and
observance in the studied zone. This will be completed by visiting the agencies in charge of these
topics in Puerto Rico, such as the Environmental Quality Board (JCA), the Water and Sewage
Authority (AAA), the Environmental Protection Agency (EPA), the Health Department and the
Administration of Regulations and Permits of Puerto Rico (ARPE). It will also be done by
visiting and interviewing the residents of the San Salvador neighborhood to ask them about their
use of the community water systems, the construction of them and of the septic tanks. We will
also inquire about the permissions they have processed for them, if any. This way we will be
able to compare the existent evidence with the instituted regulations.
The next step on this investigation, in order to achieve the goals of it, would be to take
samples and analyze both the underground and superficial water bodies of the studied zone to
determine the presence of bacteriological pollutants. This will be done during a year by taking
9. about five samples monthly (of about 2ml each) of every of the water bodies found in the San
Salvador community (to have at least 60 samples of each water body after twelve months). After
taking each sample, they will be stored in a portable refrigerator until taken to a laboratory where
it should be analyzed. Then, the PCR (Polymerase Chain Reaction) will be used to detect the
existence of the most common fecal contamination indicator bacteria, called Escherichia coli. By
the PCR procedure trace amounts of DNA can be quickly and repeatedly copied to produce a
quantity sufficient to investigate using traditional laboratory methods (Reece, Urry, et. al., 2011).
This procedure will be used because it is a very sensitive method for the detection of genes from
these bacteria from the small quantities of them that will supposedly be found and because it will
help to analyze the samples in a relatively conventional way.
The PCR procedure implies several steps. First the samples must be purified by a few
protocols, which include centrifuging, adding Lysis solution and removing the cellular trash.
After all the protocol is done, we will keep only the chromosomal DNA that we want to study.
Then, we will prepare the final sample for the PCR by adding a primer solution specific for the
E. coli DNA and the processed sample to a PCR tube which has Taq polymerase, dNTPs
(nucleotides), Mg² and a buffer, which allow the amplification of the DNA to occur. Then, the
purified chromosomal DNA sample must be amplified by putting it in the Thermocycler and
adjusting it to make a 3 stage cycle which consists of: denaturation, which separates de DNA
strands and occurs at 95°C, annealing, which grips the primers to the DNA strands by allowing
them to form hydrogen bonds with the ends of the target sequence and occurs at 50°C, and
finally, elongation, where the DNA strands duplicate and elongate because the DNA polymerase
(Taq polymerase, in this case) adds nucleotides to the 3’ end of each primer at a temperature of
10. 72°C. This cycle is repeated for an average of 35 times. The whole amplification process can
take about 2 hours.
After the PCR procedure, electrophoresis must be done so that we will be able to perceive
if there is actually the presence of E. coli in the sample. The electrophoresis shows the amplified
genes by presenting bars. This process is made in an agarose matrix which has ethidium
bromide, a chemical compound that intertwines with the DNA strands and fluoresces. The matrix
also has a buffer (TBE or TAB) which provides the electrolytes (ions) so that the applied
electrical current (usually 90 to 100 Volts) can pass the matrix. There are also some wells in the
matrix on which the DNA samples are going to be put. Before putting the samples in the wells,
they have to be tinted with a loading dye which will also give weight to the sample, preventing it
from getting out of the course. Also, we have to make sure that the wells are aligned with the
negative charge source because that way, the DNA strands (which have a negative charge) will
be attracted to the opposite positive side of the matrix and, therefore will extend to it according
to their molecular weight, allowing us to determine the presence or absence of the studied DNA,
in this case the one from E. coli. Along with the strands, in the first two wells, two molecular
weight markers will be put to compare the molecular weight of the studied genes with the one of
a known gene. After carefully putting the markers and the DNA samples in the wells with the
help of a micropipette, the electrophoresis chamber must be turned on and watched for 45
minutes. After that, the agarose matrix should be put under an ultra violet light and observed for
the results of the presence of amplified genes. The markers will surely show constant bars which
indicate comparative amounts of molecular weight (in kb). If the samples result positive to the E.
coli gene they will show wide and consistently dark bars, if the sample results slightly positive to
the gene (have a little amount of the gene) they will show lighter and slimmer bars, finally, if the
11. samples show no bars at all it will mean that they have resulted to be negative to the presence of
the E. coli gene.
All of these simple laboratory methods will allow us to determine if the water is
contaminated with bacteriological pollutants because the presence of E. coli indicates the
presence of other prejudicial microorganisms.
After making sure of the presence or the absence of bacteriological pollutants, it is
proposed to investigate the relationship between the bacteriological pollutants present in the
water of San Salvador neighborhood and the occurrence or recurrence, if any, of infectious
diseases derived from these. This will be done by making interviews to the residents of the zone
and asking them about how they process and use their water, if it comes from a community water
system and if they have gotten any of the symptoms of the water diseases described in the
introduction after drinking water from the community water systems.
The last step on this investigation is one of the most important ones because it is the one
that allows the people of the community to know about the results of this research. It will consist
on orientations in the community centers, the handling of informative brochures and
personalized orientations in each of the houses of the neighborhood and on publications in the
regional newspapers. This way the residents of the San Salvador neighborhood will get educated
about the established parameter for the conservation of tap water, the regulations for the disposal
of used waters and for septic tanks and about the results of the analysis of the water they
consume. Therefore, they will get informed, will have the tools to correct any violations to the
regulations that they may have and will make sure that in the future the quality of the water they
are consuming is acceptable. On this step, it is also planned to publish the results in scientific
12. magazines as well as to report the outcomes to the Government’s Agencies in charge of water
conservation, such as the Water and Sewage Authority, so that they can help in the process of
keeping the quality of the water of the studied community. This will also increase consciousness
about the conditions of the community water systems and, as a result, promote the government’s
concern and actions about it.
13. Expected results and discussion
After making this investigation we will find that there are several community water
systems in the San Salvador neighborhood from Caguas either private or controlled by an
administrative board of residents. This research will show that approximately an 80% of the
families use the water from community water systems. It will also be found that there are quite a
few violations to the established parameters for the conservation of tap water, for the discard of
used waters, for the construction of community water systems and for septic tanks. These
violations, along with other human activities such as irresponsible waste disposal, are resulting in
a direct contamination to the water bodies of the studied area. This contamination will cause the
rapid and continuous reproduction of bacteria because it will mainly come from the percolations
of septic tanks. These results will be tested by the PCR which will show the presence of E. coli
genes in the water, which as a result will mean that there are other bacteriological pollutants in
the water of the zone and that it is not adequate for human consumption. This will also be shown
by the direct relationship that it will have with the recurrence of infectious diseases derived from
bacteria in the people who use the water without filtering and decontaminating it with some
chemical agent or with high temperatures before consuming it. This investigation will also result
in a better understanding and orientation of the residents on the water system’s topic, which will
also outcome a better preservation and care of the water of the area. This will raise the awareness
of the community about the importance of the conservation of tap water and all what it implies. It
is also be expected that people will still be satisfied with their community water systems even
though the study reveals the presence of bacteriological pollutants because most of the residents
have been in the neighborhood for decades and they must still feel comfortable and safe with
their water sources.
14. In case that the hypothesis doesn’t result as a fact, several other studies will be made on
other communities so that the conservation and quality of community water systems will be
promoted. This inquiry will therefore serve as a template for future studies in other several areas
of the Island where no previous investigations have been done. It can also lead to new researches
about how to improve the quality of the community water systems and about the specific bacteria
that are in them. When this study is completed, it can lead to an improvement in the used
techniques and therefore to an eventual repetition of it to compare the results. The outcome of
this research can also lead to resolve the economic problem that leads to the poor maintenance of
the community water systems. This can be done by making the government aware of the problem
and, therefore, helping in the promotion of the implementation of viable federal or state financial
aids so that these communities would be able to take correct care of their water systems without
monetary implications to hold them back.
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