BRINE SHRIMP LETHALITY ASSAY OF BAAY (Dioscorea hispida) LEAVES,

STEMS, AND TUBERS

Rhea Cris Castillo, Ivan Dano, Eugene Manulat, Kc Reyna,

Elaine Sansait, and Arnel Tatoy
Graduate Studies, Philippine Normal University- Mindanao Campus, Prosperidad,
Agusan del Sur, Philippines

Abstract
The present study was conducted to determine the lethal concentrations of the different
parts of Dioscorea hispida through Brine Shrimp Lethality Assay. Cytotoxicity was
evaluated in terms of lethal concentration. Each part of the plant was powdered and 0.1
gram of it was added to the first of the five wells. One (1) mL of the mixture was added
to the vials with different concentrations. Fifteen brine shrimp nauplii were delivered into
each vial and after 24 hours, the surviving brine shrimp were counted and the lethal
concentrations were assessed. Results showed that all parts of Dioscorea hispida (leaves,
stems, and tubers) are all toxic with lethal concentrations of 28.28

g/ml, 218.21

g/ml, and 9.19 g/ml respectively.

Keywords: Dioscorea hispida, lethal concentration, cytotoxicity testing, Brine shrimp
Lethality Assay
INTRODUCTION
In times of food shortage, people find
food alternatives to satisfy their everyday
needs. Some people, especially the
natives, would sometimes resort to root
crops in lieu of rice as a primary source
of energy, an example of which are the
yams of the genus Dioscorea. Yam is the
leading form of staple food for millions
of people in the tropic and subtropical
countries. Many yams are of economic

importance as tuberous food crops. This

tuber contains the plant food reserves,
mainly starch, and it is often
incorporated in the human diet (Sheikh,
et.al., 2013). The underground tubers and
the aerial tubers of both wild and
cultivated varieties are harvested as a
starch source in West Africa, parts of
Southeast Asia, the Pacific Islands, India,
and Central America (Webster, et.al.,
1984). It contains reasonably substantial
amount of protein, starch and essential

amino acids relative to other root and

tuber crops (Cornago, et.al., 2011).
One of the species of the Dioscorea is
the Dioscorea hispida, locally known
here in the Philippines as kuyot or kot
(Richman, 2002). It is generally found in
the lowlands, jungle as well as in the thin
forest (Ashri, et.al., 2014). These tubers
are consumed by people especially
during rainy season in lean period
(Hudzari, et.al., 2011). Even the locals
from Assam, India use D. hispida as antiinfectious, hemostatic, anthelmintic,
antioxidant, anti-inflammatory, analgesic
and antitumerous (Goswami, et.al.,
2013). In the research of Ashri, et.al.
(2014), they identified that the starch
from this variety of yam has unique
properties which can be used as a new
starch source for various applications.
Though Dioscorea hispida has lots of
uses, the consumption of raw tuber can
be harmful since it contains toxic
alkaloids (Hudzari, et.al., 2011).
Hydroxybenzoic acid, hydroxycinnamic
acid and their derivatives are found in its
extracts (Theerasan, et.all. 2009). But in
spite of the high toxicity of this plant, it
is still eaten by some natives. For this to
be edible, the toxic substances have to be
removed. To completely remove toxic
substances, the tuber is peeled, sliced,
soaked in flowing water for up to 7 days,
or soaked in daily changed salted water
for up to 5 days and dried (Tattiyakul,
et.al., 2012).

In this present study, powdered samples

of the three parts of the plant were tested
for their cytotoxic effect against the
Brine shrimp nauplii. The findings of this
work will serve as basis in determining if
the plant can be safely consumed by the
local people and can be even used in
further
researches
regarding
its
importance in the field of medicine.
MATERIALS AND METHODS
PLANT SPECIES AND AREAS: The
species used in this study, Dioscorea
hispida commonly called baay", was
obtained in a mountainous area of Upper
Lucac, San Francisco, Agusan del Sur in
September 2014. Different parts were
taken from the plant for further
observation and experimentation.

Figure 1. Parts of Dioscorea hispida used

for cytotoxicity testing: (from left to
right) (a)leaves; (b)stems; and (c)tubers.
LETHAL CONCENTRATION: In
analyzing the lethal concentration of
Dioscorea hispida or baay" samples, the
Brine Shrimp Bioassay method was
used.
Preparation of Brine Shrimp: The
preparation of the artificial seawater was
done by dissolving 40 grams of NaCl

(analytical reagent) in one liter distilled

water.
Hatching the Shrimp Eggs: A shallow
oval-shape plastic container (35cm x
15cm x 10cm) was filled with artificial
sea water prepared earlier. The container
was sprinkled with minute brown shrimp
eggs covered with plastic cellophane and
punched with several holes and kept
illuminated by a fluorescent lamp for 48
hours. After 48 hours, hatched brownish
orange nauplii from the illuminated
container were pipette out and
transferred using the pipette to a petri
dish with shallow saline water for later
administration of the treatments.
Treatment of Brine Shrimp Nauplii:
The experimental set-up consisted of six
treatments namely: artificial seawater
(T1) as the negative control, and five
doses powdered samples: 1 ppm (T2), 10
ppm (T3), 100 ppm (T4), 1000 ppm
(T5), 10,000 ppm (T6). Each treatment
was done in 10 replicates and the
treatment period lasted for 24 hours
wherein 0.1gram of powdered sample
was added to the first well, shook by
inverting the test tube. One (1) ml of was
taken to be added to the succeeding test
tubes of different concentrations 1g/ml,
10 g/ml, 100 g/ml, 1000 g/ml and
10000g/ml. Fifteen brine shrimp nauplii
were delivered into each vial using the
pipette. The vials were kept illuminated
with fluorescent light during the
treatment period. The treated were

counted macroscopically in the stem of

the pipette against a well-lighted
background to determine the number of
survivors and by using a magnifying
glass.
Data Analysis: Toxicity was assessed by
counting the dead and live shrimps with
the aid of a 3x magnifying glass and by
computing the average percentage death
of nauplii for each treatment following
this formula (Lee et al., 1999) and the
Lethal concentration (LC50) is by using
Probit Statistical Analysis by linear
regression.
%Death= death in treated or control tube
x 100
No. of treated nauplii

In cases where control deaths occurred,

Abbots formula given below was used
to correct the data gathered:
% Death= death in treated tube death in control tube x
100
Total death

RESULTS AND DISCUSSIONS

Dioscorea hispida, a member of
Dioscoreaceae family, is commonly
known as Asiatic bitter yam or
Intoxicating
yam.
In
Agusan
(Philippines), where the plant sample
was derived for the present study, natives
call the plant baay and are much known
to them for developing in the superficial
part of the soil and becoming even more
evident in times of famine.

Cytotoxicity testing of Dioscorea

hispida revealed the following results:

LEAVES: As shown in Table 1, baay

leaves extract with the highest
concentration

of

10,000

g/ml

contributed to the highest percentage of

shrimp mortality. Nine shrimps survived

out of forty-five (45), with a corrected
percentage of sixty-nine percent (69%)
mortality since death did occur also in
the control group. On the other hand, the

lowest percentage of mortality, twentynine percent (29%), was recorded in the

well with the lowest concentration,
having eighteen (18) dead shrimps out of
forty-five (45). The table also clearly
shows that as the concentration of the
treatment in each well decrease, the
mortality
rate
also
decreases.
Furthermore, the leaf part of baay, as
revealed in the test, possesses a lethal
concentration of 28.28

g/ml, which

means that the aforementioned part is

highly toxic.
STEM: Table 2 shows that the treatment
with highest concentration of 10,000
g/ml contributed to the highest

mortality rate of shrimps of forty-nine

percent (49%). In this well, only eighteen
(18) shrimps survived out of forty-five
(45). On the contrary, the lowest rate of
mortality was noted from the well with
the treatment of lowest concentration,
having sixteen percent (16%) as the
percentage. The result also shows that
the concentration of the treatment is
directly proportional to the rate of shrimp
mortalitythe more concentrated a
treatment is, the greater number of dead
shrimps also will be recorded. Moreover,
the baay stem extract was recorded to
have a lethal concentration of 218.21
g/ml, having a descriptive meaning

of moderately toxic.
TUBER: Table 3 shows that the lowest
percentage of shrimp mortality, forty-

seven percent (47%), was recorded from

the well with the treatment of lowest
concentration. In this well, only five (5)
shrimps died out of forty-five (45). On
the other hand, the highest recorded
mortality rate was noted from the first
well, containing the treatment with
highest
concentration.
Seventy-six
percent (76%) corrected mortality rate
was observed, having thirty-nine (39)
dead shrimps out of forty-five (45). The
recorded results also show that the
relationship of the concentration of the
treatment and the mortality rate of
shrimps in each well is directly
proportional. The baay tuber extract, as
indicated in the results, is found out to be
highly
toxic,
having
a
lethal
concentration of 9.19 g/ml.
LETHAL CONCENTRATIONS OF
THE THREE PARTS OF Dioscorea
hispida: The three tables as discussed
above showed that the three parts which
were extracted for cytotoxicity testing
possess different lethal concentrations or
toxicity levels. The stem extract was
found to be moderately toxic, with a
lethal concentration of 218.21

g/ml.

Moreover, both the leaf extract and the

tuber extract were noted to be very
highly toxic, having lethal concentrations
of 28.28

g/ml and 9.19

g/ml,

respectively. This means that the tubers

are more toxic than leaf part, with a

lethal concentration difference of 19.09

g/ml.

Degras (1983) stated that the toxicity of

D. hispida can be greatly attributed to an
alkaloid called dioscorine, but in spite of
this, tubers are still consumed by the
people after removing the toxins
contained on it.
CONCLUSION
The leaf, stem, and tuber extracts of
Dioscorea hispida showed cytotoxicity
activity against the Brine Shrimp nauplii.
These can be clearly shown by their
lethal concentrations--28.28
218.21

g/ml, and 9.19

g/ml,
g/ml

respectively, which are all less than 1000

g/ml. Tubers of the plant possess the

highest toxicity level yet these are still

consumed by the local people after
undergoing detoxification processes.
Brine shrimp lethality assay method may
not be able to determine other substances
present in the plant but toxicity levels of
the different parts of the plant that the
method has revealed could be a great
help in future studies regarding the plants
other effects, especially in the fields of
medicine or pest-control.
ACKNOWLEDGEMENT
The researchers would like to extend
their heartfelt gratitude to Prof. Rose D.
Aquion, Economic Botany and Zoology

professor, for her guidance and support,

Prof. Levitah C. Mapatac, Caraga State
Univeristy Research Coordinator, for
conducting the Brine Shrimp Lethality
Assay, and to Almighty God who made
this research a success.

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