A COMPARATIVE STUDY OF AMPHIBIAN POPULATIONS IN AGRICULTURAL
AND NON-AGRICULTURAL AQUATIC ECOSYSTEMS
BY
CHELSEY BATURIN
ERIC HOFFMASTER
JOSEPH BEVER
DIEDRA JOYNER
KEARRON BROWN
ROBERTO LEIVA
KRISTINE CARTER
JASMINE PULLEN
MARC-ANTHONY CHEVALIER
EBONY PRICE,
JULIAN COOK
GABRIEL RUIZ
ELIZABETH ENGLE
ANTOINETTE SOLOMON
JESSICA HARLIN
TESSA WALLS
GARRETT HARTMAN
DAVID YOW
August 30, 2007
TEACHER: MS. HEATHER SINCLAIR
TCS: PATRICK BURNS, WRIGHT CHANCE, AND HEATHER LANDER
INTRODUCTION
Amphibians are prophecies of ecosystems worldwide. Noteworthy for their semipermeable skin, these sensitive bio-indicators react drastically to environmental abnormalities.
All over the world amphibians are declining and a number of theories exist attempting to explain
these declines. Previous research indicates that agriculture is believed to contribute to the
declines. The salamander research group conducted a study to examine whether agriculture is
affecting the amphibian population in Western Maryland. The purpose of this study was to
observe whether there is a difference in amphibian populations between non-agricultural and
agricultural ponds. According to the Environmental Protection Agency (EPA), agriculture is a
major cause of poor water quality (2007). This study focused on amphibians because they are
known as bio-indicators. Bio-indicators are living organisms that are able to tell us whether our
environment is healthy or unhealthy (Water ~ Learning and Living, 2007). Because amphibians
use their semi-permeable skin to breathe they are extremely vulnerable to the environment.
The predecessors of amphibians were the first vertebrates to climb from the prehistoric
seas. These ancestors, rhipidistians, had imperfect lungs and sturdy rounded fins thought to have
been effective for locomotion on land. Over the years these creatures adapted their bone
structures, grew legs, developed more efficient lungs, improved the efficiency of their hearts, and
evolved their surface from scales to semi-permeable skin. Scientists believed this process was
completed somewhere around 370 million years ago, based upon the age of the oldest amphibian
fossils found, Ichtbyostega (Raven & Johnson, 1990).
Class Amphibia includes three different orders of amphibians. The order Anura consists
of frogs and toads. These amphibians have no tail. They have long hind legs, a great ability to
leap, and exceptional vocalization abilities. The order Caudata consists of salamanders and
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newts. These amphibians do have tails. Caudates do not leap to move from one place to the next,
but rather walk and sometimes run. They have elongated bodies, smooth moist skin, and range
in length from a few inches to a foot or more. The last order is Gymnophiona. This order consists
of amphibians with no legs. Amphibians such as caecilians are wormlike and typically blind.
These amphibians are mainly found in South America and are usually 30 centimeters long in
length (Raven & Johnson, 1990).
The name ‘amphibian’ comes from the Greek word ‘amphibia’ meaning “both lives” and
“dual lives.” “Dual lives” is a term that refers to the life cycle of amphibians (Raven & Johnson,
1990). The life cycle of an amphibian begins as a fertilized egg floating in a cluster in a pond.
Amphibians spend part of their lives living in water before undergoing the process of
metamorphosis and migrating to land to live as an adult (Raven & Johnson, 1990). During the
amphibian life cycle, the pond hydrates the egg because it lacks a protective outer layer. Inside
the egg, an embryo develops until the outer membrane of the egg dissolves, leaving a tadpole or
a nymph. The tadpole begins to undergo the process of metamorphosis during which it begins to
develop legs, arms and other distinctive characteristics. The adult becomes semi-terrestrial, but
remains near water and may remain within a ten-mile radius for most of its life. During mating
season, many amphibians return to their place of origin in order to give birth (Krogh, 2002).
Amphibians have the ability to live on both land and in water because of their semipermeable skin. This skin allows water, carbon dioxide and oxygen to flow in and out of
amphibian bodies through the process of diffusion. Diffusion is the process by which materials
move from an area of higher concentration to an area of lower concentration. Amphibians are
considered “environmental sponges” because their semi-permeable skin allows environmental
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toxins to be easily absorbed (SERC, 2007). Amphibians at all stages of their life cycle are
extremely vulnerable to toxins and acidity which make them even more susceptible to death.
There are several possible causes for the decline of amphibians, such as agriculture,
habitat destruction, exotic species, pollution, toxic substances, acidification and excess nutrients.
Scientists estimate that about 43% of amphibians or about 1,856 species are threatened and are
declining at a rapid rate worldwide (Stuart et al, 2004). Many studies indicate that this decline is
due to agriculture, including one by the U.S. Environmental Protection Agency (EPA) which
reported that “…agriculture ranks first as the leading source of water quality problems for lakes
and rivers” (Padgitt, M., Newton, D., Penn, R., Sandretto, C. 2007). Amphibians respond
negatively to poor water quality. This poor water quality comes from excess sediments and
nutrients, which are found on farms in large amounts. The excess sediments and nutrients later
run-off and seep into bodies of water, often creating other problems such as acidity and water
toxins. The results of this poor water quality can also be directly related to deformities such as
extra, missing, and deformed limbs in amphibians (Blaustein, 2007). Agricultural pollution,
because of run-off, is not found in just one region, but everywhere, and it can be linked to excess
nutrients, sediments, and other factors that harm amphibians (Ribaudo, M. and Johansson, R.
2006).
This study measured water quality to determine whether agriculture has a negative affect
on amphibian populations. This group studied three specific variables at each pond. The three
variables tested were TDS (total dissolved solids), nutrients (nitrates and phosphates), and
dissolved oxygen (DO). Temperature and pH were also tested because these variables also affect
amphibians. All variables are codependent on one another; if one is affected, others will also be
affected.
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TDS is a measure of the amount of sediments dissolved in water. High levels of TDS can
harm amphibians because of their semi-permeable skin, which absorbs these sediments and can
gradually kill them. The buildup of sediments reduces water clarity, which makes it difficult for
amphibians to find food and hide from other predators. Desiccation, or dehydration, is also likely
to occur when amphibians are exposed to excess salts. The agricultural impacts that can increase
TDS levels are organic sources, fertilizers, inorganic materials, and increased run-off due to
erosion, which often occurs because of cattle. For the purposes of this study, a healthy TDS
value is 50-250 ppm (parts per million), anything below or above this range is considered
unhealthy. The TDS ranges for the hypothesis were chosen based on EnviroSci Inquiry: Lehigh
River Watershed Explorations (Murphy, 2007). This study’s range was based on other aquatic
life since no study had been conducted with amphibians. If the pond has a TDS level lower than
50 ppm, amphibians would not receive enough nutrients. Similarly, if TDS was beyond 250 ppm,
the amphibian population would be affected because too much nutrients contain harmful toxins
which destroy the eggs and as a result, the population is affected as well. The TDS hypothesis is
if the agricultural ponds have TDS values lower than 50 ppm or higher than 250 ppm, there will
be fewer amphibians than in the non-agricultural ponds.
Nutrients, such as nitrogen and phosphorous are important to any ecosystem; they are a
necessity for life but too much can also cause problems. Excess nutrients, particularly nitrates,
can cause deformities in amphibians. High levels of phosphates and nitrates can trigger a process
called eutrophication. Crop farming adds fertilizers and cattle farming causes soil erosion, both
of which combine to add nutrients in ponds that accommodate amphibians. Based on research
about aquatic life by W.F. Sigler and J.M. Nuehold, a high level of nitrate is considered 2.0 ppm
or greater and a high level for phosphate is equal to or greater than 0.1 ppm. The nutrient
4
hypothesis is if the nutrients were above the set levels in the agricultural ponds, and below the set
levels in the non-agricultural ponds then there will be less amphibians in the agricultural ponds
than the non-agricultural ponds.
The third variable – dissolved oxygen (DO) – is related to the process of eutrophication.
Eutrophication is the process that occurs when excess algae grows because of an overabundance
of nutrients. The dissolved oxygen levels increase as the algae grows and decrease significantly
when the algae decay because the decomposing bacteria require large amounts of oxygen. This
process affects the entire aquatic ecosystem, leaving the water hypoxic, or with little or no
oxygen (Water Quality: Impacts of Agriculture, 2007). Studies have shown that a normal pond
would have a DO level of about 5-6 ppm (Water Chemistry, 2007). The dissolved oxygen ranges
were chosen from the Western U.P. Center for Science, Math, and Environmental Education at
Michigan Technological University. The DO hypothesis was that the two agricultural ponds
would have significantly lower dissolved oxygen levels and fewer amphibians than the nonagricultural ponds.
Examining these variables will help us to the answer the question, does agriculture affect
amphibian population? These variables are all interrelated. Cattle can increase soil erosion which
creates additional run-off, which leads to high levels of TDS. The presence of run-off might
increase nutrient levels, which could start eutrophication which ultimately causes a lower DO
level. So, by examining water chemistry and amphibian populations in non-agricultural and
agricultural ponds, this study may support whether or not agriculture is a reason for amphibian
decline.
5
METHODS
The salamander research group studied four ponds: two non-agricultural ponds and two
agricultural ponds highly impacted by cattle. The agricultural ponds were chosen because they
were highly impacted by nutrients and run-off. We planned to visit each of the four ponds on
four consecutive days from July 16th to July the 19th, at the same time (around ten thirty or eleven
a.m.) to limit daily variation in temperature, DO and pH. Temperature changes throughout the
course of the day, affecting the DO levels since warmer water holds less dissolved oxygen.
However, forecasted bad weather caused us to adjust the plan, and Ponds B and C were analyzed
on July 17, 2007.
After arriving at the ponds, the salamander research group made preliminary observations
of the physical attributes such as the color, size, and intake/outtake of the pond and its
surrounding land, taking note of anything that might hinder or affect our research, including the
presence of animals other than amphibians, vegetation, the weather, and any evidence of run-off.
The Salamander Group then split up into three groups: TDS, nutrients and DO. The TDS
group tested for total dissolved solids, pH and temperature by using a Testr 1 TDS meter and a
Hanna Instruments pH/temperature meter. The nutrients group measured the nitrate and
phosphate levels using the LaMotte classroom water and soil demonstration labs test kit. The DO
group measured the DO level using a Milwaukee DO meter. Each group took two readings per
pond, one at either sun or shade, or intake and outtake.
The nutrients group tested for two specific nutrients, nitrate and phosphate. After the
directions were followed we compared the color of the water to the color charts. The nitrate color
chart indicated possible values ranging from zero to forty ppm in increments of ten. If the color
did not change, then that meant the water had little or no nitrate. Next, the group tested for
6
phosphates. The color chart for phosphate ranged from zero to four ppm in increments of one.
The student had to compare the water in the test tube to the phosphate color chart. If there was no
color change we could assume that there was little to no phosphate in the water. We then took
another nitrate and phosphate sample from a different area of the pond.
Small groups of no more than three people sectioned off the perimeter of the pond into
randomly chosen five meter areas. Amphibians were collected from eight or nine sections at each
pond. Collecting from the entire perimeter of the pond would have skewed our data, because all
the ponds were not the same size.
After we sectioned off the perimeter we took a 15 minute break to allow the amphibians
to return to the edge of the pond. Each group was allowed five minutes to collect as much matter
from their five meter area as possible. In order to collect our amphibians, one person from each
group held the net into the water, while the other person held the bucket. The last person just
observed to make sure no one fell into the pond, while trying to catch the amphibians. After the
five minutes, each group sorted through their bucket to find amphibians and any other life forms.
The amphibians were then identified, counted and recorded according to species, then gently
returned back to the pond.
After we collected our results from each pond, we took our data back to Frostburg
State University for statistical analysis. We averaged our water chemistry values: temperature,
TDS, DO levels and nutrient levels (nitrate and phosphate). Then we compared the water quality
from agricultural ponds to that of the non-agricultural ponds. A graph was made to illustrate the
average amphibian population per five meter sections in non-agricultural ponds as compared to
agricultural ponds. The graph, including the confidence interval, was made using an alpha level
of 0.05 for chance. This statistical analysis illustrated the 95 percent confidence level of our
7
group that the actual amphibian population count would fall in the range illustrated in the graph.
Finally, we performed a two tailed t-test using an alpha level of 0.05 for chance to find the pvalue which would illustrate a statistically significant difference between the average amphibian
population counts at the agricultural and non-agricultural ponds.
RESULTS
Our first pond, Pond A, was a non-agricultural pond approximately 20-40 yards from a
forest and located at 544 Jacobs Road, Lonaconing, Maryland. A third of the pond was mostly
shallow, rocky and was shaded by trees. Vegetation grew throughout the water, which had a low
turbidity level, making it easy to see the bottom. Very little algae were present on the surface of
the water. A water pump was noticed at the deepest end of the pond.
Pond B shared similar characteristics with Pond A; both sites were non-agricultural
ponds, located near a house, surrounded by forests, and contained several species of insects.
Pond B, found at 1780 Swamp Road, Lonaconing, Maryland was a brownish color similar to that
of Pond A. However, less vegetation was present in Pond B. It also had numerous small fish.
Pond B was deeper, had a higher turbidity level and was exposed to more sun than Pond A
Our first agricultural pond, Pond C, was located on the same property as Pond B but
slightly down hill. The water had a high turbidity level and almost no grass was seen around the
pond. Pond C was accessible to thirty cows. It was surrounded by erosion and cow tracks were
around the bank of the pond. Some algae growth was present.
Pond D, located at 5204 Bittinger Road, Swanton, Maryland, was accessible to only 8
cows. Even though there were no cows present during the testing, there were signs that cows had
access to pond D. Some of these signs we observed were cow manure and cow tracks. We noted
8
that during the water chemistry testing it was drizzling. We observed fish, different types of
vegetation and muck. We noticed that there were a few water snakes (Nerodia erythrogasters).
The pond was a fairly small spring fed pond that was surrounded by trees. The trees did not
provide the pond with shade. The perimeter of the pond was surrounded by dirt and rocks and
had very little vegetation in or around the pond. Unlike the non-agricultural ponds there was not
a large amount of vegetation in the pond. All ponds tested were man-made, spring fed, and
contained a fair amount of insects.
The water chemistry results in the agricultural ponds differed from those of the nonagricultural ponds. The pH levels for the agricultural ponds were slightly higher with values
reading mostly in the range of 9.0 to 9.5. The average temperature for the agricultural ponds was
5°C higher than the non-agricultural ponds. The TDS measurements were drastically higher in
the agricultural ponds than in the non-agricultural ponds. The average TDS of the agricultural
ponds was 125 ppm, which is 92 ppm greater than the TDS of the non-agricultural ponds. The
average DO levels in the agricultural ponds were 4 ppm greater than in the non-agricultural
ponds. Nitrate, on average, in the agricultural ponds was 21 ppm, which is 20 ppm higher than
the 1 ppm measured in the non-agricultural ponds. Phosphate showed no difference between the
two types of ponds with an average of 2 ppm (Table 1).
TABLE 1: AVERAGES OF VARIABLES FOR ALL NON-AGRICULTURAL AND AGRICULTURAL PONDS
(Average)
TDS
ppm
(Average)
Nitrate
ppm
(Average)
Phosphate
ppm
(Average)
Dissolved
Oxygen
ppm
(Average)
7.7-8.7
22.5
33
1
2
8.1
7.1-9.6
27.3
125
20
2
12.0
pH
(range)
NonAgricultural
Ponds (A & B)
Agricultural
Ponds (C & D)
Temperature
˚
C
9
The results from the amphibian count showed an average of six amphibians per five
meter section at the non-agricultural ponds. The agricultural ponds had an average of one
amphibian per five meter section (See Figure 1). The amphibians most often found in both the
agricultural and non-agricultural ponds were the Red-Spotted Newts (Notophthalmus viridescens
viridescens). We also found a lot of Spring Peepers (Pseudacris crucifer), American Bullfrog
tadpoles (Rana catesbeiana) and a Pickerel frog (Rana palustris).
Figure 1. – Average Amphibian Population in Non-Agricultural vs. Agricultural Ponds
Figure 1: Average of Amphibian
Populations in Agricultural and NonAgricultural Ponds
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Amphibians
Average Number of
10
6
Non-Ag
4
Ag
2
0
Pond Type
We conducted a t-test between our average amphibian counts in non-agricultural ponds
and in agricultural ponds. We used an alpha level of 0.05. After we conducted a t-test we
received a p-value of 0.001. The t-test tells us that there is a statistically significant difference in
amphibian populations of the agricultural ponds as compared to the non-agricultural ponds. The
p-value shows us that there is a 99.9% chance that our data on amphibian counts was not by
chance.
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DISCUSSION
The purpose of our study was to determine how agriculture impacts amphibian
populations. Our t-test showed that there is a statistically significant difference between
agricultural and non-agricultural amphibian populations at our test sites. We found a notably
lower amphibian population in the agricultural ponds averaging one per five meter section
compared to an average of six in the non-agricultural ponds per five meter section. Figure 1
illustrates that the agriculture appears to effect amphibian populations. Even though there was a
significant difference in amphibian populations, our water chemistry results did not correspond
with our predictions, which led us to reject each group’s hypothesis.
The TDS group hypothesized that if the agricultural ponds have TDS values lower than
50 ppm or higher than 250 ppm, there will be fewer amphibians than in the non-agricultural
ponds. The results from our study forced the salamander group to reject the TDS group’s
hypothesis because, while the agricultural ponds had higher TDS levels and fewer amphibians,
the measured TDS values fell within the predicted range of healthy ponds. Additionally, the nonagricultural ponds actually fell below the predicted range, but there were significantly more
amphibians.
Although our hypothesis was rejected, the results suggest that there is a relationship
between TDS levels and amphibian populations. There were fewer amphibians in the agricultural
ponds when TDS values were higher, and there were more amphibians in the non-agricultural
ponds when TDS values were lower. The TDS range we used in this study was based on levels
required for fish. If we were to repeat this experiment we would change the TDS range in the
hypothesis.
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Our results imply that agriculture can increase TDS levels which seem to influence
amphibian populations in Western Maryland. The higher levels of TDS in the agricultural ponds
may have been caused by the cattle erosion, fertilizers and pesticides used in farming and crop
production. A higher amphibian count and lower TDS value in the non-agricultural ponds may
indicate a healthier pond ecosystem than in the agricultural ponds.
Based on research, the nutrient group found that a healthy level for nitrate is below 2 ppm
and below 0.1 ppm for phosphate. Therefore, the hypothesis was that if the nutrients were above
these set levels in the agricultural ponds, and below these set levels in the non-agricultural ponds
then there will be fewer amphibians in the agricultural ponds than in the non-agricultural ponds.
We rejected our nutrient group hypothesis because the results for nitrate were above 2 ppm in
only one of the agricultural ponds and the phosphate levels were above 0.1 ppm in both the
agricultural and non-agricultural ponds.
There was a considerable difference in nitrate levels between one of the agricultural
ponds and the non-agricultural ponds. Agricultural Pond C had a much higher nitrate level, 40
ppm, than all of the other ponds. Pond D, the other agricultural pond, had a level of 1 ppm. The
Ecological Society of America explains why there is a nitrate difference between the agricultural
ponds by stating that “the density of animals on the land is directly related to nutrient flows to
aquatic ecosystems…excess nutrients and manure production create a nitrate surplus on
agricultural lands” (Ecological Society of American, 2007) which means that if a farm contains
many cows the result will be an increase in nitrate levels. Pond C was home to approximately
thirty cows while Pond D was only home to eight cows.
The DO group’s hypothesis was that the two agricultural ponds would have significantly
lower DO levels, resulting in a lower salamander count at the agricultural ponds than the non-
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agricultural ponds. After conducting the study, we could not accept our hypothesis. The DO
levels were actually higher in the agricultural ponds than in the non-agricultural ponds. Despite
the very high DO levels, we found fewer amphibians.
The DO levels may also be explained by the excess nitrates which may have promoted
algae growth. We predicted that eutrophication would be at its end, but due to high levels of DO,
the process of eutrophication was at its climax. This may be the reason why the pH of the water
was also very basic.
Although these three variables were studied independently, they are interrelated.
Fertilizers and pesticides are found in agricultural soils which are eroded by grazing cattle,
because of this, high levels of TDS are found in agricultural bodies of water. This increased runoff could result in excess nitrates and phosphates. Nitrates come from cattle manure, which
explains why there was a high level of nitrate in Pond C. Phosphates come from fertilizers.
however, the agricultural ponds studied were effected by cattle farming, not by crop production.
A possible reason for the high DO levels in the agricultural ponds could be its small amount of
aquatic life. Since there was little life in the agricultural ponds, the DO from the algae growth
would just build up. Since there were high levels of TDS in the agricultural ponds, this would
explain why there was less life.
Even though the results of our experiment were educational, there were many limitations
to our study like time, weather, season of the year, and data size.
Time was a limitation for our study because we did not have enough time to collect a
larger variety of data from each pond. Another reason time was a limitation is because during our
testing, we were given five minutes to collect any matter from the edge of the pond and about
10-15 minutes for sorting through debris. If we had allotted ourselves more time to collect and
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sort through our findings, we may have found more amphibians, which would have better
supported our hypotheses.
Weather was a limitation because it was different each day, which most likely affected
our results. This was especially true for our experimentation of Pond D which had a lower
temperature and may have affected every other variable. In addition, we may have potentially
found more amphibian life if we would have done our testing in spring months which is the
height of their reproduction cycle.
The salamander group studied only four ponds – two agricultural and two nonagricultural; this became a problem when we began our data analysis. We were not able to draw
exceptionally accurate conclusions because our results came from only four ponds. If our study
was conducted through eight ponds, for example, our data would be fairly more illustrious of the
impacts of agriculture because four ponds would be analyzed and compared rather than two. This
would allow us to perform a chi-squared test to determine if there was a statistically significant
difference in the non-agricultural and agricultural pond water chemistry.
Unfortunately, there were a few sources of error that future scientists can avoid such as
testing devices, experimental bias, and predators. This includes any external disturbances
unrelated to the experiment such as human impact. A group member may have accidentally
misused or damaged a meter used for testing that would have altered the reading or given
inaccurate results. This could be resolved by using two devices to measure the same variable and
compare readings. The test kits that the nutrient group used were designed for streams, but
because of monetary limitations, these were the only test kits available. Within this test kit was
another limitation – the color chart – which others may have interpreted incorrectly. The
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intervals on the color chart made this difficult; it ranged from 0-40 with increments of ten for
nitrate and 0-4 with increments of one for phosphate.
Next, we wonder if our experimental bias may have played a role in catching amphibians
because we expected that there would be more amphibians in the non-agricultural ponds than the
agricultural ponds. Therefore, when catching amphibians in the agricultural ponds, we may have
subconsciously not put forth as much effort as when we were at the non-agricultural ponds.
Our study of Pond D had a major source of error derived from a dog that may have disturbed all
aquatic life in a section of the pond during the fifteen minute break. Precautions should have
been taken so that there would be no disturbances during our experiment. Lastly, at Pond B and
Pond D we noticed fish and plain-bellied water snakes. This is considered a source of error
because a major part of their diets is amphibians.
To better assist future scientists who plan to continue this study, our group has a few
suggestions to make. One general suggestion is to perform this study at different times of the
year as the climate changes. The changing weather would allow us to obtain more accurate
results. Another suggestion is visiting each pond at the same time and on consecutive days to get
more accurate results with temperature. Since not having enough time was a limitation, it would
also be helpful if there were more ponds to visit and extra time to visit them. This would be
beneficial because our results would be more accurate. We also suggest altering the ranges in the
hypotheses. More in depth research may also help in this process, though it may be difficult
because no study has been done in this area concerning amphibian life water criteria.
With so many suggestions and limitations, scientists must still have unanswered
questions. One question for further study is if we performed this study at different times of the
year, would the results be different, knowing that amphibians go through different life stages at
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different times? If we had done our study a few weeks after the original testing dates, or while
the weather was persistent, would there be any change in the data obtained? If the ponds were to
be tested a few weeks later would the dissolved oxygen levels be lower? We raised this question
because we would have observed the process of eutrophication in its later stage – all the algae
would have died and would be decomposing; therefore, the predicted levels of DO might be
present.
Amphibians are important for impending scientific studies regarding environmental
health. As noteworthy bio-indicators, amphibians can reflect the condition of their habitat. Their
semi-permeable skin makes amphibians more responsive to environmental alterations and their
populations continue to decline. Agriculture was just one probable cause of poor health and low
amphibian populations in the ecosystem investigated in this study; however, other determining
factors of amphibian populations were not studied. Because amphibians are considered prophetic
organisms, studies of amphibians are necessary to our awareness of other environmental issues
that can potentially impact more organisms, primarily humans.
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