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

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.

Introduction aquarium tanks is astonishing. In 2002, Gurrero

According to Almadin and Jumawa (2014) mentioned that, the Laguna Lake
(2016) the popularity of Development Authority reported that the janitor
Pterygoplichthysdisjunctivus (janitor fish), as a fish comprised 10-38% of the total fish catch in
‘’clean-up crew’’, in domestic freshwater Siniloan Laguna, causing economic losses to the

1
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. 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.
Materials
An amount of 30 janitor fish of both sexes,
average size of 15.21 cm, weight of 49.67 grams
and at a juvenile age were used as subjects of
this study. Furthermore 5 aquariums of 20
gallons and pH, hardiness and temperature
testers were used. To keep the water quality
levels an oxygen pump was used. A blender,
oven and water were used to prepare the extract
from 85g (in powder form) of avocado leaves.
Methodology
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.
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 (Table 1). The introduction of
the 10 g/L extract was done after the testing of
the other three extract.
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
nd
the acclimation period. At the 72 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).
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,
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
2 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.
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.
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:
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.
Upon gathering the leaves it is best that the
leaves are well separated and sundried before
oven drying, preventing any burning of the
leaves.
The following is recommended to researchers
that seek to find alternative species control for
the invasive species; the Janitor fish:
An alternative plant may be used to test the
acute toxicity also isolating phytochemicals that
may yield the best result.
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.
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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