Acute Effect of Persia americana Mill.

(Avocado) leaves aqueous extract on

Pterygoplichthys ssp Gill. (Janitor Fish)
Roxane Charlaine F. Fallurin1,2,3, Harmony Gentle R. Gubatan1,2,3, Xien Jana D. Manalo1,2,3.,
Katrina Cassandra B. Manongsong1,2,3,, Maria Cristine B. Nuique1,2,3, Isolina Cleide S.
Pacheco1,2,3,, John Axl L. Torres1,2,3, Ernesto B. Guevarra, M.D, MPH, MSTM1,2,3,4
1
College of Arts and Sciences
2
Bachelors of Science in Biology
3
Our Lady of Fatima University

4
Research Development and Innovation Center

February 2018
ACUTE TOXIC EFFECT OF PERSIA AMERICANA MILL… i

Approval Sheet
This is to certify that this thesis entitled, “Acute effect of Persia americana Mill.
(Avocado) leaves aqueous extract on Pterygoplichthysssp Gill. (Janitor Fish),” prepared and
submitted by Roxane Charlaine F. Fallurin, Harmony Gentle R. Gubatan, Xien Jana D. Manalo,
Katrina Cassandra B. Manongsong, Maria Cristine B. Nuique, Isolina Cleide S. Pacheco and John
Axl L. Torres, in partial fulfillment of the requirements for the degree of Bachelor of Science in
Biology are hereby recommended for oral examination.

ERNESTO B. GUEVARRA, MD, MPH, MSTM

Research Adviser

PANEL OF EXAMINERS

Examined and approved by the Panel of Examiners in an Oral Examination with a grade of
1.75.

JOHN KENNETH L. PAGDANGANAN, RMT

Member

JHAYDEE ANN F. PASCUAL, MSC

Member

ARNEL ANTHONY R. RIOFLORIDO, RMT

Member

Accepted as partial fulfillment of the requirements for the degree of Bachelor of Science in
Biology.

ERNESTO R. LEUTERIO JR.

Dean of College of Arts and Sciences

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Certificate of Originality
We hereby certify that this thesis is our own work and that, to the best of our knowledge
and belief, it contains no material previously written or published by another person or
organization or any material which has been accepted for award of any other degree or diploma
from a university or institution of higher learning, except where due acknowledgement is made
thereof.

Furthermore, we declare that the intellectual content of this thesis is the product of our
work although we have received assistance from others on the manner of organization,
presentation, language and style.

Candidates:

MS. ROXANE CHARLEINE F. FALLURIN

MS.HARMONY GENTLE R. GUBATAN

MS. XIEN JANA D. MANALO

MS. KATRINA CASSANDRA B. MANONGSONG

MS. MARIA CRISTINE B. NUIQUE

MS. ISOLINA CLEIDE S. PACHECO

MR. JOHN AXL L. TORRES

Attested by:

ERNESTO B. GUEVARRA, MD, MPH, MSTM

Research Adviser

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Acknowledgements

We would like to like to thank first and foremost our heavenly Father for giving us the
strength and guidance that we needed in accomplishing this research because through Him all is
possible.
We show our outmost appreciation to our thesis writing teacher and adviser Dr. Ernesto
B. Guevarra for giving his utmost support, guidance and help by imparting his knowledge to us in
a way that assures that the study be done in the best manner possible. He was with us every step
of the way till this time of completion.
Also extending our thanks our thesis defense panel Mr. Roiflorido, Mr.Pagdanganan and
Ms. Pascual that offered their time and opinions that helped us improve our study.
Last but not least we would like to thank our parents that not only offered us financial
support but also the moral support that was much needed throughout this journey.
To all the people not mentioned but of help in any way, we extend our gratitude.

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Abstract
Pterygoplichthysdisjunctivus (Janitor fish) is an invasive species found in various
freshwater bodies all over the Philippines including Laguna de Bay, Marikina River and Agusan
marsh. These fish are considered invasive due to their disruptive behaviors like burrowing to the
ecosystem limiting survival of the native species. The invasiveness has caused the fish farmers a
less productive catch. The study was performed to find an alternative way of species control by
observing the acute toxic effect of the bioactive compounds that can be found in the aqueous
extract of the Persia americana Mill. (Avocado) leaves which in turn are said to be the most toxic
part of the avocado tree to different mammals. 24 fish were acclimated for more than 7 days in
the optimum pH and oxygen levels. After the acclimation period an aqueous extract was
introduced to each of the aquariums at concentrations of 2.5, 5.0, 7.5 and 100 g/L. The fish were
observed over a period of 72 to test the LC50 of the extract. Results showed that the extract had
no acute effect. In the first 5 minutes behavior responses occurred showing signs of sluggishness,
listlessness and decreased aggression but not limited to these. The study showed to be negative.

Key words: P. Americana, Acute toxicity, P. disjuctivus, Invasive species, Aqueous extract,
LC50, Bioactive compounds, Behavior response, Burrowing, Species control.

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Table of Contents
Approval Sheet...................................................................................................................i
Certificate of Originality....................................................................................................ii
Acknowledgement..............................................................................................................iii
Abstract..............................................................................................................................iv
Table of Contents...............................................................................................................v
List of tables.......................................................................................................................vii
List of Appendices..............................................................................................................viii
List of figures……………………………………………………………………………..ix
Chapter 1- The Problem and its Background

1.1. Introduction..........................................................................................1
1.2. Statement of the problem.....................................................................2
1.3. Hypothesis of the study.........................................................................2
1.4. Assumptions.........................................................................................
1.5. Significance of the study......................................................................2
1.6. Scope and limitations...........................................................................2
1.7. Definition of terms...............................................................................3

Chapter 2- Review of Related Literature

2.2 Related literature..............................................................................................4
2.3. Theoretical framework of the study................................................................6
2.4. Research paradigm..........................................................................................7
Chapter 3- Research Methodology
3.1. Research design...............................................................................................8
3.2. Locale of the study..........................................................................................8
3.3. Sample of the study.........................................................................................8
3.4. Ethical considerations......................................................................................8
3.5. Statistical analysis............................................................................................8
Chapter 4- Presentation, Analysis and Interpretation of Data.............................................9
Chapter 5- Summary of Findings, Conclusions and Recommendations.............................12

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Appendix.............................................................................................................................x
Bibliography........................................................................................................................xv
Curriculum vitae………………………………………………………………………….xvii

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List of Tables
Table 1 Control of LC50 of Avocado on Janitor fish.........................................................xi
Table 2 Control of LC50 of Avocado on Janitor fish.........................................................xi
Table 3 Toxicity behavioral responses from the avocado extract......................................9

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List of Appendices
Appendix A – Letter of Authentication of Avocado leaves……………………………… viii
Appendix B – Auxiliary tables…………………………………………………………….ix
Appendix C- Images of the Experiment…………………………………………………..xii

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List of Figures
Figure 1 Research paradigm of the study…………………………………………………7
Figure 2 Blending the dry avocado leaves.........................................................................xii
Figure 3 Weighing the powdered leaves............................................................................xii
Figure 4 Liter of water used for the extract........................................................................xii
Figure 5 Measuring the grams for the extract..................................…………………......xii

Figure 6 Maceration of the powdered leaves.....................................................................xii

Figure 7 Filtration of the macerated extract ready to use extracts....................................xii
Figure 8Preparation of the Tanks.......................................................................................xiii
Figure 9 Preparing the water for acclimation.....................................................................xiii
Figure 10 Measuring and weigh of the specimens.............................................................xiii

Figure 11Testing the pH of the water.................................................................................xiii

Figure 12Placing the fish in aquariums..............................................................................xiii
Figure 13 Starting of the acclimation period......................................................................xiii
Figure 14 Introducing 2.5 g/L extract................................................................................xiv

Figure 15 Introducing 5.0 g/L extract................................................................................xiv

Figure 16 Introducing 7.5 g/L extract................................................................................xiv
Figure 17Introducing 10 g/L..............................................................................................xiv

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Chapter 1- The Problem and its Background

Introduction
According to Almadin and Jumawa (2016) the popularity of Pterygoplichthysdisjunctivus
(janitor fish), as a ‘’clean-up crew’’, in domestic freshwater aquarium tanks is astonishing.The
janitor fish are very common, and documented in various freshwater bodies all over the
Philippines including Laguna de Bay, Marikina River and Agusan marsh. In 2002, Gurrero
(2014) mentioned that, the Laguna Lake Development Authority reported that the janitor fish
comprised 10-38% of the total fish catch in Siniloan Laguna, causing economic losses to the
fisher folk. Almadin & Jumawan (2016) indicated that, it is believed that the janitor fish
accidentally escaped from fish farms into the various bodies of fresh water, making them invasive
species. Studies have shown the janitor fish to be one of the three invasive freshwater fish species
in the Philippines. Invasive alien species are all those that are non-native micro- and macro-
species introduced into other countries. Invasive species have caused detrimental effects such as
the burrowing behavior leading to turbid water which can no longer be used as potable water.

Hubilla et al. (2007) stated that, the characteristic of sharp and hard fins of the janitor fish
allowed it to damage the fishing nets of the local fisher men in areas where they now dominate,
causing a huge problem not only for the ecological system but also for the fishing economies in
the area, particularly in sites like Marikina. The janitor fish has no natural predators in the areas
due to its hard scales. Local fishing communities were advised not to consume this type of fish
due to the possibility of toxic metal accumulation in its tissues. Delos Reyes et al. (2010)
mentioned that, as a temporary preventive way, the fishing communities employed ways to catch
and dispose this fish. Some were even turned to bags and other novelty items just to reduce the
rising numbers.

As of the present no contrite studies have been done about the effects avocado leaves on
the janitor fish. Most studies found are centered on mice and dogs giving very little information
on fish in general. For the past centuries, the use of avocado leaves as a folk medicine has been as
a decoction of dried leaves used to treat hypertension. Infusion of the young leaves were and are
used to treat coughs, aid digestion and decrease tension. In the study conducted by De Filips at al.
(1998) it was indicated that these treatments were not used with an accurate or known
concentration, leading it to be believed that avocado leaves are not toxic to human beings.

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Statement of the Problem

The principal problem of the study was to know if bioactive compounds present in
avocado leaves aqueous extract would have an acute toxic effect on the janitor fish.
Specifically, this study aimed to answer the following questions:
1.) Could this toxic effect be a feasible manner of species control?
2.) At what concentration would there be an acute effect at the fastest rate?

Hypothesis

The bioactive compounds present in avocado leaves aqueous extract was not effective as
an acute toxic substance for janitor fish.

Significance of the study:

Fishermen – to improve the conditions of the fishing process through reduction of the
janitor fish population that inauspicious the existence of indigenous species which is widely
profitable in the market.

Researchers – to enable further knowledge of the effects of avocado leaf to lower animals
specifically janitor fish which has not yet been tackled.

Government - to find new innovative, economical and environmentally safe techniques to

reduce the janitor fish population.

Scope and limitation:

The janitor fish were bought from a breeder. A total of 30 specimens with about the same
age and size were obtained. Proper apparatus, especially on temperature control, hardiness of the
water, pH level, aeration were used and measured at the beginning of the test and every 24 hours
in a 20 gallon tank. For the pH of freshwater testing, it remained greater than six but less than
eight. Acclimatization took about seven days. No fish were used for the test if any of them
appeared to be stressed or if more than 5 percent died during a period 72 hours of actual
implementation of the bioactive compound present in avocado leaves aqueous extract in
accordance of the Organization for Economic Cooperation and Development 423 protocol. Apart
from that, the avocado was obtained from Manila. The experiment was conducted for 2 weeks
and the experimentation was laboratory simulated, not mimicking the natural environment or

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habitat of the janitor fish, because performing the experiment in its natural habitat would have
caused detrimental effects to other organisms. The positive or negative result of the study was
due to the factors such as the water conditions, because it is lab simulated, and the components of
the avocado crude extract. Since janitor fish were widespread in the area particularly in rivers,
there was no major ecological effect due to the collection of species.

Definition of Terms

1. Acute Toxicity – lethal impact produced by Avocado leaves extract from single and little
exposure that range from 12-24 hours, resulting from morphological changes up to the
death of sample specimen used in research.
2. Pterygoplichthys spp. – a suckermouthsailfin armored catfish, well known as janitor fish.
It was the sample specimen used to test the effectiveness of avocado leaves extract.
3. Invasive species – when janitor fish were introduced to the Philippine freshwater
environment causing undesirably and unbalance ecosystem due to their rapid
reproduction that outnumbered those that are marketable and helpful native fish of the
country.
4. Persin – fungicidal composition found in Avocado leaves extract. It is highly toxic when
it comes in a large quantity against lower vertebrates, would have been a possible
contributor to the cause of acute effect.
5. Bioactive compound – was the substance present in Avocado leaves crude extract either
exhibits negative or positive effect against janitor fish.

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Chapter 2- Review of related literature

Related literature

P. americana: phytochemistry and composition

Rodriguez-Carpena et al. (2011), stated that when humans intake Perseaamericana it
gives positive results because of its several biological activities like antioxidant,
antihypersentensive, larvicidal, fungicidal, hypolipodemic, amoebicidal and gardicidal activities.
Likewise it causes negative impact on animals especially on lower vertebrates such as rats and
dogs. In the study of Wang, et al.,( 2012) it was stated that, the plant also contains glutathione
which is known as a potent antioxidant against formation of cancer cells. Moreover Lottenberg et
al., (2002) mentioned that, it also contains notable amount of phytosterols, which is noted to
lessen the total amount of cholesterol in an individual who is considering avocado as a part of
their diet. In addition to its composition, Dembitsky, et al., (2011) mentioned that, specifically of
the fatty acids, it also contains tecophenols, squalene, cycloartenol acetate and persin, In relation,
we were expecting that one or more these compounds would be associates with the effect either
positive or negative against janitor fish.

Pterygoplichthy ssp.(janitor fish): the invasive species

Species invasion was identified as one of the main factors in biodiversity loss, and it has
been the subject of multilateral agreements among countries under the Convention of Biological
Diversity to protect the natural environment. Devastating events of recent times in the Philippines
was caused by the species Pterygoplichthys or locally known as the janitor fish. According to
Chavez &Carandang, (2014), the Janitor fish species has been reported to have caused
interference in fishing-related activities in Marikina river Laguna de bay. Hubilla et al., (2007)
mentioned that it has also occurred at Candaba swamp in central Luzon and Agusan marsh in
Mindanao. Investigation of SAC (Sucker mouth catfish) in some areas visited outside of the
Laguna de bay were also conducted, areas in Taal lake batangas, Sta. Maria Bulacan, San Juan,
Batangas and Sariaya, Quezon, Angat, Bulacan, and Coloong Valenzuela city. Knowing this, it
had been expected to find quite a number of janitor fish in all of these respective places.
Determining the invasion level of SACs in visited areas, researchers used the suggested
framework by Coluatti & MacIsaac, (2004) as a guide, whereas stage 0- propagule in donor
region, stage I- survives transport and released, stage II- introduced, stage III- established,

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reproducing, stage IVA- widespread but rare, stage IVB- localized but dominant and stage V-
widespread and dominant by Chavez & Carandang, (2014).
Annual sex ratio of P. disjunctivus from Marikina river was slightly female biased (1.5
female: 1 male) but did not statistically deviate from 1:1 (p=0.50). Based on this we expected an
equal number of female and male specimen to be fished for the use of this study.

Acute toxicity of P. Americana

Acute toxicity refers to those adverse effects occurring following oral or dermal
administration of a single dose of a substance, or multiple doses given within 24 hours, or an
inhalation exposure of 4 hours. According to Padilla- Camberos, Martinez-Velasquez, Flores-
Fernandez, & Villanueva-Rodriguez, (2013), this is considering at what particular amount it is
lethal to at least 50% of the specimen. The acute toxicity tests were done on 30 mice, the result
showed that mortality had been observed in just 24 hours and showed a higher percentage with
higher doses. The mice given with the higher dosage of 500, 1000, 2000mg/kg showed higher
rates of mortality. Aside from mice, a study conducted by Craigmill et al., (1989) tested the
toxicity of avocado leaves implemented in goats and sheep. Raw leaves with an amount of 20g/kg
body weight, were given to lactating goats that resulted in the decrease of milk production.
Moreover in the study of Stadler et al., (1991) mentioned that, oral implementation of raw
avocado leaf was also fed up to the sheep that resulted to Cardiomyopathy and Myocardial lesion
due to the signs of having cardiac and respiratory stress that lead to their deaths Acute toxicity is
a primary topic in research, by fisheries research laboratories, for examining the effect of the
toxic substances in fishes. More than 400 chemicals have been tested in different samples of
invertebrates and fishes of different aquatic ecosystems. Acute toxicity estimates the
concentration of exposure by 50 percent or Lethal concentration 50 (LC50) to find out the impact
of the toxic substance to the organism at the certain period of time. LC50 is generally accepted by
some toxicologist and it is mostly used for testing possible unfavorable effects of chemical
substance to aquatic forms.
Winkaler et al.,(2006) conducted a study on the effects of neem leaf extract on
Prochiloduslineatus (streaked prochilod). The LC50 was tested for 24 hours at 2.5, 5.0 and 7.5
g/L or only clean water. In this research using the parameters of the above mentioned study, there
was 5 groups of 6 janitor fish separated into a control group and different concentrations to
observe if there were any visible differences in the result.

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Theoretical framework of the study

According to Rodriguez-Soana et al., (1998) persin has been responsible for poisoning
animals as early as 1942. Researchers had isolated persin from specialized cells found in the
avocado plant called idioblast oil cells. A high concentration of persin can be found in the leaves,
bark, seeds, skin and pits of the avocado, and are most toxic to lower animals like rats and birds.
Albeit with the information above the study was performed using the crude extract so it can be
inferred that persin may be a contributing factor to the acute effect the avocado leaves may have
had.
This was also done to the janitor fish, after placing them in the aquarium. The effect of
avocado leaves among fishes was not well noted, but it was clearly stated that persin only cause
negative effects on lower animals, which then again persin is a constituting substance in the
leaves.
The motive of this research was to test and know the toxic effect of P. americanaleaves
aqueous extract on Janitor fish,that cause ecological unbalance and economic problems to the
country. The procedure was conducted as soon as the sample specimens were obtained and the
extraction of the P. americana leaves was done.

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Research paradigm

Based on Kamaguete et al.

study, powdered P.
P.american leaves or seeds
americana seed mixed with
at certain dosages have had
water and alcohol solution
negative effects on animals.
has shown toxic effect at
500mg/kg dosage on rats.

Based on Winkaler et al. Aqueous extract of P.

study, concentrations of americana was administerd
neem leaf aqueos extract at to the janitor fishes at
2.5, 5.0 and 7.5 g/L on different dosages of those
streaked prochilod showed used in the neem leaf extract
negative effects. study.

The test showed if P.

americana will have an
effect on janitor fishes.

Figure 1 research paradigm of the study

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CHAPTER 3- Research methodology

Research design
The research design used in this study was intended to be purely experimental (because it
involved manipulation of the specimen in a controlled environment with the introduction of a
plant aqueous extract), but became a descriptive one because it involved the observation of the
behavior demonstrated by the fish.

Locale of the study

The fish was bought at from a respective breeder. The aqueous extraction along with the
experimentation was conducted in Monumento, Caloocan City.

Sample of the Study

Sampling of the fish was conducted in a way that the janitor fish were all of both sexes,
the average size of 15.21 cm, weight of 49.67 grams and at a juvenile age. The janitor fish were
divided into 5 groups of 6 in accordance to the Organization for Economic Cooperation and
Development 423 protocol and the study conducted by Winkaler et al., each with a different
dosage and concentration of the extract.
Proposed table for the tabulation of data can be seen in Table 1.

The leaves were dried and powdered. The powder then was dissolved in water at a
concentration of 2.5, 5.0, 7.5 and 10 g/L at room temperature, and then macerated for 24 hours.
The extract was administered in the water. The janitor fish were put into 5 aquariums with a
controlled environment of optimum pH and temperature. The fish were put in those 5 aquariums
7 days before the study was conducted for acclimatization purposes. Food pellets were given
during the acclimatization period at least once a day. The LC50 of every group were recorded at
appropriate times for the course of 72 hours. The introduction of the 10 g/L extract was done after
the testing of the other three extract.

Ethical Consideration
All fish were not mistreated and were handled with outmost care throughout the
acclimation period and experimentation.

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Chapter 4- Presentation, analysis and interpretation of data

Results

LC50 of the Janitor fish

Upon the 6th hour of the experiment no deaths had occurred in either tank A, B, C or D.
Tank E containing the control remained constant. After 24 hours of observing the fish, yet again
there were no deaths, this all in the same conditions as the acclimation period. At the 72 nd hour,
the results remained the same with zero deaths in all aquariums, this all showing a negative result
(Table 2).

Fish behavioral characteristics

Upon introduction of the extract the fish demonstrated different behaviors at the different
concentrations. This all happened in the span of 5 minutes. These were the observations basing on
Kane, Salierno, & Brewer (2005) Fish models in behavioral toxicology: Automated techniques,
updates and perspectives:

Table 3
Toxicity behavioral responses from the avocado extract
Behavior Tank A Tank B Tank C Tank D
Lethargy 2 6 0 0
Inappetence 2 6 0 0
Loss of orientation 4 0 6 6
Abnormal swimming 4 0 0 6
Flared opercula 4 0 6 6
Increased respiration 4 0 0 6
Hanging head up in the water 0 0 0 0
Corkscrew swimming 0 0 0 0
Sluggishness 2 6 6 0
Floating at the surface and sinking 0 0 0 0
at the bottom regularly
Listlessness 6 6 6 0
Sporadic hyperactivity 4 0 2 6
Tail chasing 0 0 0 0
Dec. aggression 2 6 6 0

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Social interaction 4 0
Dyspnea 0 0
Dec. swimming 6 6
Hyperactivity 6 0
Other response 4 (abnormal tail 0
movement)
0 1
6 2
4 1
0 6
4 (abnormal tail 0
movement)

Table 2 shows the behavioral response of the janitor fish from the extract by number of fish:
TANK A: 2 of the fish demonstrated lethargy, 2 inappetence, 4 loss of orientation, 4
abnormal swimming behavior, 4 flared opercula, 4 increased respiration, 2 sluggishness, 6
listlessness, 4 sporadic hyperactivity, 2 decreased aggression, 4 social interaction, 2 decreased
swimming behavior, 6 hyperactivity and 4 abnormal wiggling of tail.
TANK B: 6 demonstrated lethargy, 6 loss of orientation, sluggishness, 6 listlessness, 6
decreased aggression and 6 decreased swimming behavior.
TANK C: 6 demonstrated loss of orientation, 6 flared opercula, 6 sluggishness, 6
listlessness, 2 sporadic hyperactivity, 6 decreased aggression, 6 dyspnea, 4 decreased swimming
behavior and 4 with abnormal tail wiggling.
TANK D: 6 demonstrated loss of orientation, 6 abnormal swimming behavior, 6 flared
opercula, 6 increased respiration, 6 sporadic hyperactivity, 1 social interaction, 2 dyspnea, 1
decreased swimming behavior, 6 hyperactivity and they response was less than that of tank A.

Discussion
Fish are ideal specimen for testing the effects of various toxins because of the direct contact with
the aquatic environment where the toxin is introduced on their body surface.
The fish did not die after 6, 12, 16, 24, 48, 60, 72 hours of the introduction of the extract.
This means that the avocado leaf aqueous extract had no lethal effect upon the janitor fish, this all
monitored during the 24 hours of the experiment. Furthermore, upon the introduction of the
extract the fish demonstrated behaviors shown in Table 2 within 5 minutes. This could be an
indication of short term effects because behaviors were not demonstrated by the control after
introducing water as a mock extract. Seeing that according to Kane, Salierno, & Brewer, (2005)
behavior is not a random process, but rather a sequence of structured and predictable activities
designed to ensure the animals survival, behavioral endpoints are very good tools for determining
if a certain induced toxic material has an effect, be it lethal or not, on the organism.

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Due to the fact that the extract was a “crude” one, no element or rather, phytochemical
can be attributed to these responses.
The studies conducted by Kamagate, et al., (2016) showed that the avocado leaf extract
had negative effects on the rats. In the experiment that was conducted on the janitor fish, the
results were not the same, this can be explained by the fact that toxicants that may be of a harmful
affect to mammals my not have similar effect on aquatic animals and vice versa, this according to
Kane, Salierno, & Brewer, (2005). The introduction of the P. americana leaf aqueous extract did
not have any lethal effects deeming the research to have a negative outcome.

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Chapter 5- Summary of findings, conclusions and recommendations

Summary of findings
This study was conducted with the purpose of determining the acute toxic effect of
avocado leaf aqueous extract on janitor fish. The study was purely experimental because it
involved manipulation of the specimen in a controlled environment with the introduction of a
plant extract. A total of 24 fish were used and acclimated for more than 7 days. The whole study
was conducted over a period of 6 months.
Of the 24, none of the janitor fish died ant any concentration indicating that the aqueous extract
had no lethal effect at 2.5, 5.0, 7.5 or 10 g/L. Certain behavioral changes were observed upon the
introduction of the extract such as but not limited to sluggishness, decreased aggressive behavior
and listlessness.

Conclusion
P. americana (avocado) leaf aqueous extract bioactive compounds had no acute effect on
the janitor fish. This lead to no knowledge on what percentage the lethal dose was, at what
concentration would there be an acute effect and what concentration would yield a faster result.
Through the course of the experimentation only behavioral changes and responses were observed.
The study may not be used as a feasible manner of species control of the Janitor fish.

Recommendations
To ensure better outcomes to the study, the following recommendations are made to
future researchers if they will pursue the same study:
1. Isolate a specific compound from the avocado leaf. This is due to the fact that
the crude extract was not effective but other studies that used the
phytochemical persin were effective. This may yield better results.
2. Upon gathering the leaves it is best that the leaves are well separated and
sundried before oven drying, preventing any burning of the leaves.

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The following is recommended to researchers that seek to find alternative species control for the
invasive species; the Janitor fish:

1. An alternative plant may be used to test the acute toxicity also

isolating phytochemicals that may yield the best result.
2. During the acclimation period ensure that all behavioral characteristics
are observed this following through to the experimental period to be
able to make a complete behavioral assay study.
3. Gathering all information possible of the toxins that affect the Janitor
fish may be a good helper in deciding the best alternative species
control.

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Appendix A

Authentication of the avocado leaves used for the study

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Appendix B
Auxiliary Tables
Table 1.
Control of LC50 of Avocado on Janitor fish
Time
Concentratio
0-6 hrs. 7-12 hrs. 13-24 hrs. 25-36 hrs. 37-48 hrs. 49-60 hrs. 61-72 hrs.
n and dosage
2.5 g/L
(Tank A)
5.0 g/L
(Tank B)
7.5 g/L
(Tank C)
10 g/L
(Tank D)
No extract
(Tank E)

Table 2.
Control of LC50 of Avocado on Janitor fish
Time
Concentratio
0-6 hrs. 7-12 hrs. 13-24 hrs. 25-36 hrs. 37-48 hrs. 49-60 hrs. 61-72 hrs.
n and dosage
2.5 g/L
None None None None None None None
(Tank A)
5.0 g/L
None None None None None None None
(Tank B)
7.5 g/L
None None None None None None None
(Tank C)
10 g/L
None None None None None None None
(Tank D)
No extract
None None None None None None None
(Tank E)

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Appendix C
EXPERIMENT
Preparation of the avocado aqueous extract

Figure 4 Liter of water used for the extract

Figure 2Blending the dry avocado leaves

Figure 5 measuring the grams for the extract

Figure 6 Maceration of the powdered leaves

Figure 3Weighing the powdered leaves

Figure 7 Filtration of the macerated extract. ready to

use extracts

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Acclimation of the fish

Figure 8Preparition of the Tanks

Figure 11Testing the pH of the water

Figure 9Preparing the water for acclimation

Figure 12Placing the fish in aquariums

Figure 13 starting of the acclimation period

Figure 10 Measuring and weigh of the specimens

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Introducing the extract

Figure 14 introducing 2.5 g/L extract

Figure 17 Introducing 10 g/L

Figure 15 Introducing 5.0 g/L extract

Figure 16 Introducing 7.5 g/L extract

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Curriculum vitae

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