1. I will attach a the research paper so you can use the fix and complete the original poster ( on powerpoint).
2.fix the instructor comments on poster on all part by using the research paper provided. you need to use in-text citation (APA format).
3. you need to paraphrase the sentences on you own word. do not take or copy from internet.
4. when you finish all parts, check the rubric attached for poster and check if you done everything on rubric.
5. do only : intro, method, result, and discussion parts. you do not need to do acknowledgment or reference (although you need to do in-text citation).
6. in the dissuasion, you need to discuss what the results mean to the hypnosis and what they mean on the physiological level; (do not just interpreter the results)
7. in intro you must add info on how frogs muscle physiology is effected by explaining physiological level.
8. the method, put the what has been tested on (eg; max force) then write a small definition of this and why we need to test it.
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The Physiological Effects of Wastewater Atorvastatin Concentration on Largemouth Bass
Muscle
2
Introduction
Medicinal drugs play a significant role in the therapeutic treatment and prevention of diseases in
both humans and animals. However, due to the toxic properties of medicines, there are several
unforeseen consequences on animals and other organisms in the environment. Although the focus
of research is always on the effects of pollution on humans, toxicological studies on animals and
other possible environmental impacts are not thoroughly investigated. According to Hoelzer et al.
(2017), most research topics are based on direct impacts of drug pollution on humans; and more
often, the indirect impact on humans through animals is overlooked. Some of the side effects of
drug components consumed by humans and animals from polluted water are outlined by Mala et
al. (2021). In Mala’s study, livestock anthelmintic drugs such as Ivermectin formed the largest
portion of drug pollutants in water. Other drugs included antibiotics and cholesterol suppressants
such as Atorvastatin.
The presence of many complex pollutants in water widens the scope of the study as each pollutant
has diverse effects on various organisms and at different concentrations. It is also noteworthy that
pharmaceutical products can cause adverse effects at concentrations far below those tested to be
safe and efficient in humans (Zaied et al., 2020). It is factual that most pharmaceutical products
are said to degrade upon prolonged exposure to the environment. This, however, does not translate
to decreased toxicity.
Instead, it is known that when different pharmaceutical products break down, they combine with
other broken components to form even more complex contaminants, which cause more unexpected
effects on the environment (Xin et al., 2021). These contaminants could later be passed on to
humans through the food chain or indirectly affect the ecosystem by inadvertently eradicating a
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species or altering its physiological responses. It is therefore unsafe to assume that the effect of
water pollution by these substances does not harm humans and the environment.
The magnitude of the significance of this topic is wider than it seems. For instance, data published
by Gemmill (2018) shows that more than ten million women in the United States are on
contraceptives at any given time. These contraceptives eventually end up in the environment and
accumulate. Other pharmaceutical drugs and cytotoxins used in cancer therapy are consumed in
large quantities annually, with some weighing thousands of tons. Due to the knowledge gap and
lack of prior research, it is increasingly difficult to get the number of drugs consumed globally.
However, a recent study conducted by Parra-Saldivar (2020) in Canada shows that naproxen,
acetaminophen, carbamazepine, acetylsalicylic acid, and ibuprofen are among the most commonly
consumed drugs.
Veterinary medications such as anti-helminthics, antifungals, antibacterials, and parasiticides also
cause environmental pollution when they are introduced into soils and surface water. They are then
deposited into major water sources during rainy seasons. In most cases, veterinary drug pollutions
cause long-term effects because they do not go through a water treatment plant like in the case of
human therapeutic drugs. The use of inorganic farming procedures alongside aquaculture and
irrigation hastens the effects. According to Bilal et al. (2019), the United States’ farming industry
is responsible for up to between 92,500 kg to 196,400 kg of antimicrobial residues that end up in
water sources annually.
Human and veterinary medicine residues are released into the environment in one or a combination
of the following ways: The main source of drug pollution is the pharmaceutical industry (Kreisberg
& DC, 2007). This occurs when industries and factories release drug contaminants and by-products
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into water sources. While most industries are compelled by government policies to abide by certain
water treatment standards, this intervention is not 100% effective (Sun et al., 2019). Although
toxicity levels have to be maintained at extremely low levels, they accumulate in the long term to
cause adverse physiological effects on vulnerable fish species such as largemouth bass. Drug
residues may also be released by humans during excretion.
After ingestion, absorption, and integration into the body’s metabolism, drug residues are
eliminated through the body by processes such as egestion, urination, and sweating. Ultimately,
the residues are carried through sewerage systems in the form of sludge and end up either in soil
or in water bodies. Pharmaceutical products used in aquaculture are released directly to the
environment through the pond’s outlet. Veterinary medicine used to treat livestock gets into the
environment in the same way. In areas where intense inorganic farming is practiced, these
medicines may enter the environment directly through the use of animal excrement as fertilizers.
Other relatively insignificant entry points include air released into the atmosphere and poor
disposal methods of unused pharmaceutical products or their containers.
Previous research has not been able to quantify the amount and concentration of drug residues in
the environment. However, it is undisputed that pharmaceuticals have all along been finding their
way into the environment (Miller et al., 2018). Current research illustrates how the amounts can
be quantified by obtaining information from different geographic locations and establishing their
usage patterns to identify the most likely drug residues to be released into the environment. In this
case, Atorvastatin concentration can be quantified by obtaining consumer data from the area of
study and establishing its usage patterns and other predisposing factors such as nearness to a water
body and climate. Geographical locations prone to rainy climates are more likely to show larger
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volumes of Atorvastatin residue deposits. A similar was used by Asghar et al. (2018) in an
investigation carried out in Wuhan, China, to quantify pharmaceutical residues in water surfaces.
Atorvastatin is a drug of the group statins, which are used to lower cholesterol in patients diagnosed
with unhealthily high levels of blood cholesterol (Stancu & Sima, 2001). In some cases, it is
prescribed to patients with underlying conditions that predispose them to heart attacks and strokes.
Factors that predispose a patient to the mentioned conditions include a medical history or family
history of heart diseases or diabetes. The drug is known to cause the following side effects in
humans: headaches, nausea, diarrhea, constipation, and other flu-like symptoms (Uses, 2020).
Exposure of Largemouth Bass Muscle fish to metabolites and residues of the drug leads to a
reduction in cholesterol levels, triglycerides, cortisol, estradiol, and testosterone in male fish.
Different sexes are affected in different ways. The rapid changes in cholesterol levels are also
associated with long-term alterations of main genes involved in lipid and cholesterol generation,
such as HMGCR1. Atorvastatin is quickly absorbed after ingestion and takes between one to two
hours to achieve maximum plasma concentration. This duration may vary depending on the
population being treated. It may take longer in adults compared to children because they have a
higher plasma capacity.
The drug’s metabolism occurs in the mucosa region of the gastrointestinal tract and within the
liver through a process known as first-pass metabolism. The presence of freshly ingested food in
the gut decreases the rate of Atorvastatin metabolism. However, this does not affect the clinical
features or excretion of the drug. It can therefore be taken with or without food (Bellosta et al.,
2018). Atorvastatin and its metabolites are excreted into the environment by a process known as
biliary secretion. Urine contains less than 2% of the drug, while the rest is eliminated in other
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ways. The aim of this experiment is to therefore test the impact of Atorvastatin on muscle
physiology of Large Bass fish. It is hypothesized that because the drug is involved in lipid
metabolism, animals affected by Atorvastatin will show high muscle fatigue, more tetany, and less
response due to muscle atrophy.
Methods
Environmental conditions
Largemouth Bass fish of male and female gender will be selected and housed at 700F under equal
12 hour light and dark cycles. The population will be divided into four groups, with the teach group
having three tanks, 20gallon tanks half filled with spring water. The four groups will be labeled
and tested under control; 1 mg/L ethanol 95% in spring water, low; 0.22 mg/L atorvastatin in
spring water, middle; 2.2 mg/L atorvastatin in spring water, and high; 22 mg/L atorvastatin in
spring water. The low drug range was based on the average values of Atorvastatin found in public
water systems, according to Ottmar et al. (2012) and Gracia-Lor et al. (2011). This value was
extrapolated to yield middle and high ranges.
The freshness of the water will be maintained by changing the water and its respective treatment
weekly on Fridays. A log will be immersed in each tank to act as a shelter for the fish. The Bass
fish will be kept on a daily meal of plankton, crushed fish pellets, and fish flakes.
The assessment of muscle physiology of the test subjects
Muscle physiology of the fish will be assessed by electromyography. This will be done by hooking
the bottom lip of the fish to a force transducer and the tail clamped using electric transducing
probes just behind the eye. There will be sensors on the anus, tail, and behind the fisheye to sense
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and record measurements that should represent the muscles of the whole body. The recording will
be done through Powelab physiography, and the minimum threshold will be determined through
the stimulation of the muscles to nil response. Measurements representing muscular response will
be taken thrice under different voltages, and the frequency of muscle tetany will be recorded as a
muscular response. The muscular latency, contraction, and relaxation will also be recorded.
Equipment Calibration
A force transducer will be connected to an amplifier calibrated to bridge mode x10 to filter 4Hz
and a force of 0. The transducer will also be connected to the power lab for power provision. The
setup will be tested for accuracy by adding a 10g weight to the transducer and allowing recording
to commence. Signals generated by weights of 0 and 10g will be highlighted and marked as points
1 and 2. Unit conversion set to grams before removing the pan from the transducer and connecting
the nerve stimulator to the BNC connecting to the banana converter and the power lab output.
After ensuring that the isolated stimulator is turned off, the settings will be switched to pulse mode
and manually to a frequency of 1Hz, 5-second duration, 1V amplitude, and to a marker channel
two, which will mark the end of the calibration process. During the experiment, nerve stimulation
will be commenced with subsequent responding until the muscle response range fills at least half
of the channel.
Data collection
The stimulator will be set to 5msec from 1V, and the voltage will increase gradually to 3V until
the nerves elicit a response. To determine the effects of the voltage, the stimulator will be set to
1mseconds, and the voltage varied to a sub-maximum range. Data of the threshold, force, and
EMG between 1V and 10V will be observed and recorded. Data on the twitch response and
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duration will also be collected by setting the similar in a way similar to the first part but with a
chart speed of 2000\seconds. The fish sample, in this case, will be moisturized with the ringer’s
lactate, and their nerves will be stimulated thrice. The period that the muscles take to relax will be
recorded, and the chart will return to its original speed, awaiting the next sample. For tetanus, the
duration will be set to 5millisconds, and the voltage at 1V to continuous mode from 20Hz t
1000Velocity /second. Samples moistened with ringer’s lactate will then be tested period before
tetanus and fatigue at five different frequencies.
Data analysis
Data analysis will be done using ANOVA, Dunnett’s test, mean determination, and standard
deviation. The aim of the analysis will be to determine how Atorvastatin affects muscle
physiology, with the reference being the control sample. This data should show how
pharmaceuticals that are disposed of in wastewater affect aquatic life and, in the long run, sensitize
people on proper drug disposal.
Results:
Source
F
P-value
Weight
0.7808
0.4678
Threshold
0.3344
0.7186
Maximum force
4.5984
0.0188*
Maximum EMG
0.7248
0.4933
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Frequency for tetanus
1.3627
0.273
Force at tetanus
1.6344
0.2145
Time to half fatigue
0.1459
0.8654
Maximum force corrected for weight
5.8737
0.0074**
Force at tetanus corrected for weight
1.8441
0.1783
Latency time
1.9645
0.1591
Contraction time
4.8625
0.0154*
Half Relaxation time
1.6615
0.208
Table 1: Results are shown for the differences that the results from the physiological test from the
fish. A T-test was done between the three different concentrations that the fish were kept in.
Significant differences (Bolded) were found in the maximum force, maximum force corrected for
weight and contraction time.
Max Force (grams) vs Concentrations
3
Max force (grams)
2.5
2
1.5
1
0.5
0
Control
Low
Concentrations
High
10
Figure 1: The mean max force corrected for weight is shown. The error bars show the standard
deviation calculated out for each concentration. The mean max force is less in the high
concentration shown against the low and control.
Contraction time (seconds)
Contraction Time (seconds) vs Concentrations
100
90
80
70
60
50
40
30
20
10
0
Control
Low
High
Atorvastatin Concentrations
Figure 2: The contraction time in seconds is shown. Error bars are added to show for the standard
deviation for each of the concentrations. The contraction time is much lower in the high
concentration compared to the low and control.
Discussion:
The purpose of this experiment is to characterize the effects of atorvastatin on largemouth
bass physiology and whether it has a degenerative affect. There was difficulty experienced due to
the fish dying within their tanks. Although the root to this problem was not determined, it could
have been due to the excess food in the tank causing algae to grow. This was observed in some
tanks that the water in the tank would appear to become cloudy and have a unpleasant smell. This
was then mitigated by feeding the bass a different food source and only feeding them what they
would be able to consume in three minutes time.
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Our study has shown that there was both insignificant and significant data between the
different concentration of atorvastatin between the bass. The significant data was the max force,
max force corrected by weight and time of contractions. These differences were determined by
using ANOVAs followed by Tukey Kramer analysis. A P-value under .05 showing significance
between the groups. The F-value shows significance between the difference controls with having
different sample sizes between each group.
The max force was corrected for weight to eliminate any possibility for it being due to the
fish having different weights. The max force corrected for weight showed that the bass in the high
treatment showed a significant less amount of force as compared to the low and control group. The
high group showed a third less force exhibited. Also, the different concentration groups were tested
for their max contraction times. The high group had a fourth less contraction time as the low and
control group.
The muscular exhaustion from the high group can be seen in other studies with atorvastatin
and exercise. This can be caused by the increase of exercise induced rise in muscle enzyme seen
in some studies (Noyes & Paul 2017). Overall, the group that received the highest concentration
of atorvastatin had significantly less muscle strength been exhausted much more quickly than those
that had a smaller concentration. This seems to support previous studies that atorvastatin does
cause muscle fatigue and weakness (Buettner 2012).
These effects from the wastewater Atorvastatin can cause detrimental effects on more than
just the largemouth bass. The bass muscle strength and endurance can be reduced due to the
wastewater drugs. This can lead to them to more predation from many different predators. This
can then also affect This can affect the predators of the largemouth bass also.
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This can lead us into further studies in the future about the possible effects of atorvastatin
and different pharmaceuticals in the waterways. First, a larger test sample can be made to give us
more data between the different concentrations of fish. Also, fish can be kept for a longer period
to see the long-term health effects that atorvastatin. This can more accurately depict the health
effects that are seen in bass that live their lives in these waterways containing atorvastatin. On top
of this more varying concentrations can be done. Where the concentrations used for this study are
from Spain are around 0.02 µg/L (Gracia-Lor 20110). There are concentrations in the United States
that are ten-fold that amount. In the waterways that wastewater treatment plants release into the
concentrations are 0.24 µg/L of atorvastatin (Ottmar 2012). This can be due to a variety of factors
due to the environmental laws in the differing countries and how their treatment plant processes
the waste. Overall, the effects of pharmaceutical products in wastewater are not well known and
future research will benefit both the environment and us.
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References
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