THAI NGUYEN UNIVERSITY
UNIVERSITY OF AGRICULTURE AND FORESTRY

FULL NAME
HOANG THI THUY DUONG

Topic title: THE EFFECTS OF CARBARYL ON GROWTH OF IPOMOEA
AQUATICA AND EICHHORNIA CRASSIPES

BACHELOR THESIS
Study mode: Full-time
Major

: Environmental Science and Management

Faculty

: International Programs Office

Batch

: 2014 – 2018
Thai Nguyen 15/09/2018


Thai Nguyen University of Agriculture and Forestry
Degree Program
Bachelor of Environmental Science and Management
Student name
Hoang Thi Thuy Duong
Student ID

DTN145429006
Thesis Title
The effects of carbaryl on growth of Ipomoea aquatica and
Supervisors
Supervisor’s signature

Eichhornia crassipes
Prof. Dang Van Minh

Abstract:
The purpose of this study was to assess the effects of carbaryl, which is one kind of
carbamate insecticide, on the growth of two aquatic plant species Ipomoea aquatica and
Eichhornia crassipes. Both of aquatic plant species were treated with four different
concentrations of carbaryl (0, 0.2, 0.5 and 1.0 g/L). Carbaryl levels in the water and
plants were tested before and after experiment by using a test kits. During the
experiment morphological changes of the plants were observed and recorded. To
determine the growth of plants, the fresh weight, height and root were recorded before
and after experiment. The plants were dried by using freeze dryer. Then dry weight was
measured, after that the chlorophyll in plants tissue were measured by using extraction
of dry leaves. The results show that the fresh weight, Relative Growth Rate and the
chlorophyll content of the treated plants decreased with increasing concentrations of
carbaryl. Carbaryl concentration in water decreased and then was no longer at the end of
experiment while cabaryl concentration of plant increased significantly. Moreover,
chlorosis and necrosis occurred at the leaf margin of the treated plants, and then they
extended into the inner portion of the leaf blade. Finally, the leaves decayed and the
plants died. These visible sign occurred in E.crassipes faster than those in I. aquatica.
Thus, Carbaryl can moderate the growth of these aquatic plants and E. crassipes is more
sensitive with carbaryl than I. aquatica.
Key words
Pesticide, carbaryl, growth, Ipomoea aquatica, Eichhornia

Number of

crassipes.
51 pages

pages
2


Date of

15/10/2018

Submission:

3


ACKNOWLEDGES
I would like to express my sincere gratitude to the teachers and staffs of the Faculty
of Environmental Science and Management - Thai Nguyen University of Agriculture and
Forestry. I would like to thank the International Education Program at Thai Nguyen
University as well as the International College of Digital Innovation at the Thai Royal for
giving me the opportunity and the extremely favorable conditions for me to do this thesis
in Thailand.
I would like to send special sincerely thanks to Prof. Dang Van Minh, my supervisor at
Thai Nguyen University, Vietnam, as well as Assoc. Prof. Arunothai Jampeetong, my
adviser at Department of Biology, Faculty of Science, Chiang Mai University, Thailand,
who are always willing to teach me a lot of knowledge, working skills, helping me in
study and follow me throughout the course of this thesis.

Nevertheless, I would like to acknowledge the head as well as graduate student of
Post-harvest laboratory of the Biology department, Chiang Mai University, Thailand for
technical equipment and their kind helps to run the process of this research work.
I would like to thank Mr. Pakawat Janyasupab and Ms. Munutsawan Manokieng for
helping me to collect plants, water sample and analysis data.
Hoang Thi Thuy Duong

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CONTENTS

ACKNOWLEDGES.........................................................................................................iv
LIST OF FIGURES..........................................................................................................vi
LIST OF TABLE.............................................................................................................vii
LIST OF ABBREVIATIONS..........................................................................................viii
1.1

Research rationale.................................................................................................1

1.2

Research’s objectives............................................................................................2

1.3

Research questions and hypotheses......................................................................3

1.4


Limitation.............................................................................................................3

1.5

Definition..............................................................................................................4

PART II: LITERATURE REVIEW....................................................................................5
2.1.

Pesticides..............................................................................................................5

2.2.

Carbaryl................................................................................................................8

2.3.

Effect of Carbaryl...............................................................................................10

2.4.

Aquatic plants.....................................................................................................13

PART III: MATERIALS AND METHOD........................................................................19
3.1.

MATERIALS......................................................................................................19

3.2.


METHODS.........................................................................................................21

PART IV: RESULT AND DISSCUSION.........................................................................24
4.1.

Results................................................................................................................24

4.2.

Discussion...........................................................................................................35

PART V: CONCLUSIONS...............................................................................................38
REFERENCES................................................................................................................39
APPENDICES.................................................................................................................42

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LIST OF FIGURES
Figure 1: Pathways of pesticide spread in the environment
Figure 2: UV spectra and structural formula of carbaryl
Figure 3: Ipomoea aquatica
Figure 4: Eichhornia crassipes
Figure 5: Average fresh weight changes of I. aquatica and E. crassipes
Figure 6: RGRs (relative growth rate) of I. aquatica (6a) and E. crassipes (6b).
Figure 7: SERs of I. aquatica.
Figure 8: Compare 4 plants of I. aquatic plants (8a); E. crasspes plants (8b) at 4
different treatments (1.control 0g/l carbaryl ; 2.low conecentration 0.2g/l carbaryl;
3.medium concentration 0.5g/l carbaryl and 4.high concentration 1.0g/l carbaryl )
Figure 9: Chloropyll in leaves tissue of I.aquatica (9a) and E. crassipes (9b)

Figure 10: Carbaryl concentration in water and plants before and after experiment

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LIST OF TABLE
Table 1: Leaves injury of I.aquatica and E. crassipes plants after experiment
Table 2: Roots of I.aquatica and E.crassipes after experiment
Table 3: Standard color
Table 4: The roots of I.aquatica and E.crassipes after experiment

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LIST OF ABBREVIATIONS

RGRs: The Relative Growth Rate
SERs: Shoot Elongation Rate
SE: Standard Error

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PART I: INTRODUCTION
1.1 Research rationale
Thailand is an agricultural country, with hot and humid tropical climates, which is a
favorable condition to the development of agricultural crops, but it is also conducive
to the emergence and growth of pests, weeds damage crops. Therefore, along with
chemical fertilizers, the use of pesticides for pest and disease control, preservation
of national food security remains an important and essential measure. Chemical

pesticides have been used extensively since the early 1958 to kill pests, insect pests
and crop protection. Since then, pesticides are still associated with the progress of
industrial production, scale, number and type are increasing. But the presence of
pesticide residues and metabolites in food, water and soil currently represents one of
the major issues for the environmental chemistry. Pesticides are the most important
environmental pollutants because of their increasing use in agriculture (Food and
Agricuture Organization, 1993). Among the pesticides, organophosphate and
carbamate species have been used extensively because of their high insecticidal
activity and relatively low persistence (American Public Health Association, 1999).
As an example, carbamate insecticides are widely used for the protection of fruits,
crops, forests and livestock. In particular, carbaryl (SEVIN, lnaphthyl-Nmethylcarbamate) has been extensively used against many agricultural pest. It has
been commonly judged to have moderate to low mammalian toxicity. Carbaryl is
highly susceptible to chemical hydrolysis and biodegradation, leading to several
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metabolites, mainly 1-naphthol, which does not accumulate in the body. However,
some adverse effects have also been reported including alterations of liver
microsomal enzymes, sub-chronic neurotoxicity after long term exposure and
changes in the immunological function in in vitro culture (Geneva, 1994). For this
reason, a number of analytical procedures have been employed to monitor
carbamate pesticides and to determine their concentrations. Many of these
determination methods are though accurate and selective, but require relatively
expensive instrumentation, relatively more time and make use of higher toxic
organic reagents. So, a problem is how can people know that the water is
contaminated by carbaryl without advance equipment? The aquatic plants was
consider as an alternative way because they have various levels of resistance to
insecticides and can be used as bio-indicators that can indicate the quality of water
resources. For this reason, “The effects of carbaryl on growth of Ipomoea aquatica
and Eichhornia crassipes’’ were investigated.


1.2 Research’s objectives
The main goal of the study is to investigate the influence of Carbaryl on I. aquatica
and E. crassipes in each stage of development and compare the respond ability with
Carbaryl of these two aquatic plants. Research’s results might provide valuable
information to the protection of the agricultural environment and public health.

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1.3 Research questions and hypotheses
1.3.1. Questions
 How carbaryl impact on the growth of aquatic plants?
 How carbaryl effect on morphology of aquatic plants?
 Which kinds of plants have more sensitive with carbaryl?
1.3.2. Hypothesis
Carbaryl could decrease the growth of I. aquatica and E. crassipes. Beside, E.
crassipes is more sensitive with carbaryl than I. aquatica

1.4 Limitation
Although this research was carefully prepared, I am still aware of its limitations
and shortcomings.
First of all, the research was conducted in the two types of aquatic plants which
have lasted for four months. Four months is not enough for the researcher to observe
all of the growth of these plants. It would be better if it was done in a longer time.
Second, the population of the experimental group is small, only 100 I. aquatica and
20 E. crassipes plants and might not represent the majority of the plants.
In addition, since the assessment of the pre toxic and post toxic was conducted
by observer, it is unavoidable that in this study, certain degree of subjectivity can be
found.

1.5 Definition

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Pesticide is any substance or mixture of substances intended for preventing,
destroying, repelling or mitigating any pest
Carbaryl is one type of carbamate compound. Its structure comprises of ary1 Nmethylcarbamate esters of phenols.
Aquatic plants are plants that have adapted to living in aquatic environments
(saltwater or freshwater)

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PART II: LITERATURE REVIEW
2.1.

Pesticides
According to Phillip L. Spector Regulations of Pesticides by the Environmental

Protection Agency (1976), the government body that regulates pesticides in the
U.S., a pesticide is any substance or mixture of substances intended for preventing,
destroying, repelling or mitigating any pest. Though often misunderstood to refer
only to insecticides, the term pesticide also applies to herbicides, fungicides, and
various other substances used to control pests. Pesticides also include plant
regulators, defoliants and desiccants.
Pesticides are often grouped into "families" because they share similar chemical
properties, or they act on the pest in the same way. A pesticide product may have
active ingredients from more than one chemical family. Some common families
include:

 Organophosphates are usually made from phosphoric acid. Most
organophosphates are insecticides. They control pests by acting on the
nervous system (for example, the pesticide interferes with nerve-impulse
transmissions by disrupting the enzyme (cholinesterase) that regulates
acetylcholine (a neurotransmitter)).With a few exceptions, most are highly
toxic. Organophosphates are used because they are less persistent
(breakdown faster) in soil, food or feed for animals than other families,
such as organochlorine pesticides. However many are being phased-out or

13


used only in critical applications. For examples chlorpyrifos, dimethoate,
fenthion, malathion, naled, temephos and trichlorfon
 Organochlorines (Chlorinated Hydrocarbons): Controls pests by disrupting
nerve-impulse transmission (disrupts ion flow at the axon/synapse
level).Generally persistent in soil, food, and in human and animal bodies
(does not breakdown quickly).They can accumulate in fatty tissues.
Traditionally used for insect and mite control, but many are no longer used
due to their ability to remain in the environment for a long time.
 Carbamates and Thiocarbamates are made from carbamic acid. Control
pests by acting on the nervous system (interfere with nerve-impulse
transmission by disrupting the enzyme (cholinesterase) that regulates
acetylcholine (a neurotransmitter).In general, are less persistent in the
environment than the organochlorine family. Includes insecticides,
herbicides and fungicides. The health hazard to humans and animals is mild
with herbicides and fungicides, while greater with insecticides. For instance
are Carbaryl (Banned in European countries, only certain uses will be
cancelled in Canada due to concerns of health risks), Propoxur (some uses
of this insecticide, such as control of mosquitoes, black flies, all indoor use

except bait trays, are to be phased out in Canada), Methomyl (in Canada
some uses are proposed to be cancelled), Carbofuran (limited use in
Canada) (accoding to Pest Control Products Act (PCPA) and Regulations).
Pesticides play an important role in ensuring good crop yields, but also carry a risk
of toxic compounds entering the surrounding environment. Pesticides are often
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found in rivers or streams but also in the groundwater. The risk of unwanted
spread of pesticides to the environment is mainly related to cropping and spraying
techniques and also to natural conditions and processes (climate, soil properties,
etc.). Some of the pesticides sprayed on fields can be transported to the
surrounding land and water. Pesticides can be transported away in the air through
wind drift and evaporation and then carried back to soil or water by rainfall.
Surface run-off and percolation of water to the drainage system can carry
pesticides out into rivers and lakes. Leaching of water through the soil can carry
pesticides down to the groundwater. Various spillages during handling can also
lead to pesticides going astray into the environment (Fig.1).

Fig.1 Pathways of pesticide spread in the environment
(Source: Lennart Torstensson, 2016)
2.2.

Carbaryl
Carbaryl is one type of carbamate compound. Its structure comprises of ary1

N- methylcarbamate esters of phenols (fig 2). It is used on a variety of crops

15



including soy, corn, and citrus, and is also applied to livestock and home garden
uses (Hassall, KA. 1990). Carbaryl is a Cholinesterase Inhibitor, and commonly
causes acute symptoms in both laboratory studies and in occupational human
exposures. It may also cause mutagenic effects in humans. As with many other
insecticides, carbaryl negatively impacts several aquatic organisms and poses a
threat to populations of beneficial insects such as bees (World Health
Organization, 2007).

Fig.2. UV spectra and structural formula of carbaryl.
Carbaryl is commonly sold under the trade name Sevin, and goes by trade
names such as Adios, Carbamec, Denapon, Hexavin, and Panam. In addition to
also being effective as a molluscicide and acaricide, carbaryl can also be used to
treat pest infestations, such as that due to species of lice, on livestock, poultry,
pets, and in the case of head lice, on people (Bond, C.; Hallman, A.; Buhl, K.;
Stone, D. 2016). However, carbaryl is utilized in settings other than farmland. It is
also heavily used in suburban yard and garden settings, and is frequently used to
raise and maintain ornamental and shade trees.

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2.3. Effect of Carbaryl
2.3.1. Effect on human
As a cholinesterase inhibitor, however, carbaryl can have serious acute
health impacts on mammals, despite the rapid metabolism process. According to
the Enviromment Protection Agency, occupational exposure to carbaryl can result
in nausea, vomiting, blurred vision, coma, and difficulty breathing. Likewise,
laboratory tests in mice, rabbits, and guinea pigs indicates that the chemical can
have a severe acute impact on some mammalian organisms (Bond, C.; Hallman,

A.; Buhl, K.; Stone, D. 2016).The oral LD50 in rats can range from 250 mg/kg to
850 mg/kg, and dermal values are somewhat similar (Kesavachandran, 2011). A
recent study reports that carbaryl is a structural mimic of the neurohormone
melatonin and directly binds to MT2 melatonin receptor (Ki = 70 nM).This could
significantly impact circadian rhythms and increase risk for diabetes and metabolic
disorders (Insecticide Exprosure, 2017).
Like other mammals, humans are affected by carbaryl via skin contact,
ingestion, and inhalation. Acute human symptoms, given a large enough exposure,
can also include burns, irritation, and stomach cramps; however, as noted above,
the severity of the symptoms is dependent on the route of transmission. According
to Extension Toxicology Network, the only human fatality from direct exposure to
carbaryl was through intentional ingestion (Extension Toxicology Network, 1993).
As pertains to these acute effects, carbaryl is officially regarded as a Type II
Moderately Hazardous Pesticide. Likewise, regulatory organizations and agencies
such as the Enviroment Protect Agency list carbaryl as a suspected carcinogen,
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endocrine disruptor, reproductive toxicant, and developmental toxicant. And
although evidence is inconclusive, symptoms such as lasting headaches, memory
loss, and muscle weakness resulting from prolonged low-level carbaryl exposure,
suggesting some potential for the chemical to cause chronic effects.
One conclusive influence of the chemical, however, is in its ability to
cause mutagenic effects in some animals. In laboratory studies of rats, carbaryl has
been shown to affect cell division and growth. There is a possibility that this same
effect may occur in the human stomach upon ingestion of the chemical; however,
this has not been studied conclusively. The same amount of evidence also exists
for carbaryl's potential to affect the immune system in animals and insects
(Caroline Cox, 1993).
2.3.2. Effect on metabolism

Carbaryl affects its insect and mollusk targets through both ingestion
and dermal contact. The chemical is a Cholinesterase Inhibitor, which inhibits the
cholinesterase enzyme from degrading acetylcholin, eventually leading to
interference in the host's nervous system. Although typically fatal in insects,
carbaryl is detoxified, metabolized, and excreted by vertebrate hosts, especially
humans and other mammals. In particular, the chemical does not seem to
concentrate in fats or milks. Mammals that ingest carbaryl metabolize up to 85%
of the chemical within 24 hours (Extension Toxicology Network, 2007).
2.3.3. Effect on ecological
Like other insecticides, carbaryl poses some ecological and
environmental risks as well, which can both directly and indirectly impact on
human’s health. Carbaryl has low persistence in soil, and is rapidly broken down
18


by microbial communities in surface water. However, the chemical can pose a
threat to certain aquatic organisms. Carbaryl is somewhat toxic to fish, with some
LC50s ranging from 1 to 10 mg/L. Because fish too rapidly metabolize the
chemical, it does not pose a significant threat to bioaccumulation in the aquatic
environment, but some bottom-feeders, such as catfish or crawfish, can
accumulate the chemical in their tissues. Out of water, carbaryl is relatively nontoxic to most animal life (Baron& Ronald, L. (1991). For instance, the chemical is
practically non-toxic to birds. However, as it affects insects in general, carbaryl
can inflict a heavy blow to beneficial insect species, such as bees and other
pollinators. In the previous study “The Effect of Carbamate Insecticide on the
Growth of Three Aquatic Plant Species: Ipomoea aquatica, Pistia stratiotes
anorrect itd Hydrocharis dubia” show that carbaryl have a strong effect on the
growth of these three aquatic plants (Boonyawanich et al., 2000).

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2.4. Aquatic plants
2.4.1. Definition and classification
According to Hickey and King, aquatic plants are plants that have adapted
to living in aquatic environments (saltwater or freshwater). They are also referred
to as hydrophytes or macrophytes. A macrophyte is an aquatic plant that grows in
or near water and is emergent, submerging or floating, and includes helophytes a
plant that grows in marsh, partly submerged in water, so that it regrows from buds
below the water surface (Hickey & King, 2001). In lakes and rivers macrophytes
provide cover for fish and substrate for aquatic invertebrates, produce oxygen, and
act as food for some fish and wildlife (Kesavachandran, C. 2011) Aquatic plants
are classified by conditions fresh and location of water. There are three main types
of aquatic plants are: Single-celled phytoplankton; Periphyton (algae growing
attached to substrates) and Multicellular macrophytes (Abowei & J.F.N. 2012).
Aquatic plants provide many ecological benefits and are essential in promoting the
diversity and function of aquatic systems (Carpenter & Lodge. 1986). Aquatic
habitats, both freshwater and marine, are some of the most productive areas
worldwide. Firstly is using of aquatic plants as food , for example ,Marine plants –
One of the primary and oldest uses of marine macro-algae has been for human
consumption. Species of algae are consumed by people throughout the world, with
Eastern Asian countries consuming more than any other country worldwide. In
Asia, macro-algae have served as a vegetable since ancient times (Burtin, 2003)
.In Japan, people consume on average 1.4 kg of macro-algae per person every year

20


(Burtin, 2003) Besides nutritional benefits, macro-algae are used for their
antibiotic, antiviral, antifouling, anti-inflammatory, cytotoxic, and antimitotic
activities; some of which have been pursued in pharmaceutical industries. Aquatic

plants, both marine and freshwater, are used extensively worldwide as livestock
fodder, fertilizer, compost, mulch and bioremediation (Wersal &Jonhn D. Madsen,
2012). Furthermore, the aquatic plants have various levels of resistance to
insecticides, heavy mental and they can be used as bio-indicators that can indicate
the quality of water resources.
2.4.2. Ipomoea aquatica
Ipomoea aquatica is known as water spinach, river spinach, water
morning glory, water convolvulus, or by the more ambiguous names Chinese
spinach, Chinese Watercress, Chinese convolvulus, swamp cabbage or kangkong
in Southeast Asians. It is an herbaceous, trailing vine with milky sap and hollow
stems that can reach 10 feet in length. The stems produce roots at the nodes and
float in aquatic habitats. The leaves are simple and alternate, usually slender and
arrowhead-shaped, but they vary to triangular or cordate (heart-shaped). They
have long petioles, and the blades range from 2 to 7 inches long (figure 3).The
flowers are trumpet-shaped, like those of morning glories, white or pale lavender,
and about 2 inches across; they are borne singly or few together in the axils of the
leaves. The fruits are spherical capsules that are woody at maturity and about 1/2
inch in diameter (Richard & Ramey, 2007).

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Figure3. Ipomoea aquatica
(Source: Taxonomy - Ipomoea aquatica)
The species is found in marshy habitats and ditches, muddy stream banks,
ponds, lakes, rice paddies and waste areas. It can't tolerate frost. But it is widely
naturalized in tropical and subtropical areas around the world. At present, water
spinach is established in ditches, canals and a few natural lakes in Thailand. It also
grows terrestrially on the banks of streams and canals. It is not popularly used as a
green vegetable, especially in East and Southeast Asian cuisines, it also wellknown with pharmacological effects such as diabetes and stomach diseases.


22


2.4.3. Eichhornia crassipes
Eichhornia crassipes also have some other names such as vetch, vetiver,
water hyacinth, or Japanese waterloo. It is an herbaceous aquatic plant that lives in
water but can also survive on land in moist soils. E. crassipes grows about 30 cm
high, leaves are blue, slightly rounded like flowers petals, smooth leaves. Thin leaf
bulbs, in the petioles foamed up like bubbles to help the E. crassipes floating on
the water. The roots are about one meter long, black, feather-like, dropping into
the water (figure 4).

Figure4. Eichhornia crassipes
(Source: Hồ Đình Hải, 2012)

The plant blooms in the summer E. crassipes is light purple, blue dots,
petals on a yellow, there are six indentured, top three cells contain ovule, capsule.
The hyphae reproduce very fast; one mother can lay seedlings, doubling every two
weeks. Body and leaf of E. crassipes is used as an antidote to inflammation of the
skin due to the sweet taste, cool non-toxic. Besides E. crassipes has a sedative
23


effect, diuretic, except for heat, blood pressure .... E. crassipes is also used as food
in many regions, present in the special dishes bring nuanced nuance of the river.
Stir fried tofu is very delicious, sprouts and petioles used to cook shrimp,
snakehead fish, dried shrimp, boiled sauteed fish stock or fried meat or ... E.
crassipes can be processed to be braided, woven into the mat in the industry
(Nguyen Van Duong, 1993).

In natural form, the E. crassipes is capable of absorbing heavy metals such
as lead, mercury, strontium and organic substances that have not been decomposed
and treated in water. This effect of the E. crassipes is in the long roots. The rate of
treatment and suction, filtration wastewater of the E. crassipes are very well. The
researchers conducted the study and made impressive figures on the working
efficiency of E. crassipes in the treatment of wastewater as follows: 1ha of water
drop, E. crassipes can clean up to 3 tons of waste water each day. Specifically,
34kg of sodium, 22kg of calcium, 17kg of phosphorus, 4kg of magnesium, 2.1kg of
phenol, 89g of mercury, 104g of aluminum, 297g of alkali, 321g of strontium.
Another experiment dropped E. crassipes in a dirty water pot containing 10mg zinc
per 1 liter, in 38 days the amount of zinc accumulated in the E. crassipes was higher
than normal vegetable by 133%. E. crassipes also has the effect of degrading
phenol and cyanide, which are very dangerous substances a small amount from
0.15 to 0.2g can cause of death (According to the Pharmacology by University Ho
Chi Minh City). E. crassipes can be considered as a cost-effective, user-friendly
and inexpensive waste disposal solution that we can take advantage of. This aquatic
24


plant is very popular in aquariums, artificial ponds to keep the water clean, as it is
known to cleanse the water of the E. crassipes. Investigations confirmed that the
lake system had water E. crassipes and the ornamental fishes or creatures in general
were more vigorously cultured than those without E. crassipes. A couple of jobs,
people use the E. crassipes to clean the water environment for the lake, both can
decorate the scene more lively and vibrant, flowers of the E. crassipes is also very
beautiful. In addition to plants, other plants are also used to exploit and utilize the
effect of water filtration, water treatment as well as shrubs, reeds, and some other
types.

25