CITRIC ACID AS CHELATING AGENT IN THE PHYTOREMEDIATION OF

LEAD(PB) AND ARSENIC(AS) FROM MINE TAILINGS USING WATER HYACINTH

(EICHHORNIA CRASSIPES)

Faculty Promoter:
Engr. Katelyn G. Gabon, ChE
Saint Louis University Faculty
Contact Information: Mobile No.: 09480832261
Email: katelyngabon@gmail.com

Researchers:
Baccay, April Ellen B.
Student Researcher
Contact Information: Mobile No.: 09260259511
Email: aprilellen81@gmail.com

Baculo, Joana Marie M.

Student Researcher
Contact Information: Mobile No.: 09152800801
Email: joanamariebaculo@gmail.com

Benabise, Philip S.
Student Researcher
Contact Information: Mobile No.: 09568595958
Email: philbenabise1415@gmail.com

Cabuslay, Berna Josephine M.

Student Researcher
Contact Information: Mobile No.: 09973784788
Email: berna010513@gmail.com

Crisostomo, Christopher B.
Student Researcher
Contact Information: Mobile No.: 09568869000
Email: chrstphrcrsstm@gmail.com

Dulay, Ruby Ann Joy P.

Student Researcher
Contact Information: Mobile No.: 09484971286
Email: rubyannjoydulay@yahoo.com

Habon, Marjun A.
Student Researcher
Contact Information: Mobile No.: 09951480572
Email: coninobah@yahoo.com
Lungao, Louie K.
Student Researcher
Contact Information: Mobile No.: 09174201789
Email: slungao.sl@gmail.com

Thomas, Jonalyn B.
Student Researcher
Contact Information: Mobile No.: 09157093320
Email: thomasjonalyn@yahoo.com
 1

CITRIC ACID AS CHELATING AGENT IN THE PHYTOREMEDIATION OF

LEAD(PB) AND ARSENIC(AS) FROM MINE TAILINGS USING WATER HYACINTH
(EICHHORNIA CRASSIPES)

ABSTRACT

Heavy metal pollution is a serious problem that is caused by mine tailings. Mine tailings
pose a great risk in the environment, if not handled or stored properly, as they are rich in heavy
metals. The mine tailings produce by the different mining companies contains different types of
chemicals and heavy metals at different concentrations. Methods for the removal of
contaminants such as heavy metals are available nowadays, however, they are found to be costly
and lacks efficiency. An emerging green technology and a proven remedy to this problem is
phytoremediation which have been extensively studied. A limitation of phytoremediation is the
long periods of absorption that it needs to detoxify the contaminated area which discourages its
large scale application. This research was then focused to the investigation of the effectiveness of
adding citric acid, an organic chelating agent, in enhancing the phytoremediation of Pb and As
from mine tailings using Water Hyacinth. The chelating agents make the heavy metals available
for the plants to absorb or adsorb thus enhancing the heavy metal uptake of the plant. The
parameters varied were the amount of citric acid added (15, 20 and 50 mg) and the contact time
(4, 8 and12 days). From the statistical results, it was found that the removal of arsenic is only
affected by the amount of citric acid. Both time and interaction of citric acid and time has no
effect in the removal of arsenic and lead. Furthermore, from the experimental results, it was
found that the citric acid apart from its chelating effect plays a role in the regulation of growth of
the water hyacinth.

Keywords: phytoremediation, chelating agent, citric acid, water hyacinth, lead, arsenic, heavy
metal, mine tailings

INTRODUCTION

Mine tailings are wastes from mines. They are usually liquid in state but contain some
solid matter such as soil and small rock particles. Mine tailings pose a great risk in the
environment, if not handled or stored properly, as they are highly acidic and metal-rich solution
(Coumans, 2000).The mine tailings produce by the different mining companies contains different
types of chemicals and heavy metals at different concentrations because it depends on how they
process their minerals, on what mining method they use and on what valuable materials they
extract.
The tailings produced by the mining companies were stored in a so-called tailings dam.
The tailings were stored for a long period of time. The problem in Canada, US, and elsewhere is
that no one knows exactly what to do with these tailings (Dupare, D.B., 2015). Much of the
sludge they contain is too toxic to remediate and let back into the environment. The issue extends
far beyond Canada where there are estimated 3,500 tailings ponds worldwide.
An estimated 39 percent of tailings-pond dam failures happen in the states—a rate higher
than anywhere in the world (Moskowitz, 2014). In the Philippines, from year 1982 up to the
present, several tailing dam failures were reported causing great damages to living organisms
 2

within the area. One of which was the “Marcopper Mining Disaster” that happened in
Marinduque on year 1996 where 2-3 million tons of mine waste leak into 26-kilometer Boac
River causing a lot of damage to the surrounding area (Coumans, 2000). This shows that it is not
enough to isolate the mine tailings from the environment. These tailings can leak and
contaminate any time while at storage therefore, there must be some kind treatment that will
make them less hazardous in a fast-paced method. One of the methods that were studied for
several times to treat wastewater and soil that contains heavy metals was phytoremediation
(Wuana, 2011).

Phytoremediation is a process of removing pollutants such as heavy metals with the use
of plants. Phytoremediation encompasses five processes of metal removal from soil or water.
These processes include: rhizofilteration, phytostabilisation, phytoextraction, phytovolatilization
and phytodegradation. Rhizofilteration is the use of plants to absorb, concentrate and precipitate
organic and inorganic pollutants from aqueous sources (Dushenkov et al., 1995; Salt et al., 1995;
Flathman and Lanza, 1998; Zhu et al., 1999). Phytostabilisation involves the use of plants (roots)
to immobilize the inorganic contaminant through the process of sorption, precipitation,
complexation or metal valence reduction in the soil or aqueous environment (Berti and
Cunningham, 2000; Ghosh and Singh, 2005). Phytoextraction which is also known as
phytoaccumulation is the removal or absorption, concentration and precipitation of the elemental
pollutant into the plant material (Salt et al., 1995; Salt et al., 1997; Rulkens et al., 1998).
Phytovolatilization involves the extraction, transformation of the pollutant into a volatile and less
toxic form which is then transpired into the atmosphere (Ghosh and Singh, 2005).
Phytodegradation is the uptake and breakdown of organic molecules to simpler forms by plants
using plant enzymes such as the dehalogenases, oxygenases and reductases (Black, 1995;
Chaudhry et al., 1998).

Phytoremediation is the use of plants as absorbing medium for contaminants in soil and
water bodies. Hyperaccumulators, plants that have a phytoremediation capability, are directly
placed in the contaminated area and are left to absorb the contaminants. This process is a proven
remedy but it takes a long period of time for the plants to detoxify the contaminated
environment. Many plants have been proven to be very effective in phytoremediation but are not
always used as a solution in chemical spills and other instances of contamination. Water
Hyacinth is one of the hyperaccumulators proven to absorb heavy metals in wastewater (Rai,
2009).

Hyperaccumulators are plants that have high tolerance to the heavy metals. At present,
there are nearly 400 known hyperaccumulators but most are not appropriate for phytoextraction
because of their slow growth and size. Several researches have screened fast growing, high
biomass accumulating plant, including agronomic crops, for their ability to tolerate and
accumulate metals in their shoots (Mellem et al., 2012).

In India, researchers investigated the bioaccumulation potential of water hyacinth can

phytoremediate metals such as Potassium, Sodium, Zinc, Lead, Iron, Cadmium, Magnesium,
Copper and Calcium (Upadhyay and Tripathi, 2007).

In this study, water hyacinth (Eichhornia crassipes) was chosen because of its growth rate
 3

which is among the highest for any plant and the populations can double in as little as 12 days by
sending off short runner stems which develop into new plants (Mellem et al., 2012). A typical
biomass from land plants is composed of 30–50% cellulose, 20–40% hemicellulose and 15–30%
lignin. Its enormous biomass production rate, its high tolerance to pollution, and its heavy metal
and nutrient absorption capacities (Chanakya et al., 1993; Singhal and Rai, 2003; Ingole and
Bhole, 2003; Liao and Chang, 2004; Jayaweera and Kasturiarachchi, 2004) qualify it for use in
mine tailings treatment.

The long periods of absorption lead the researchers to use chelating agents such as citric
acid in order to compensate for this disadvantage brought by phytoremediation. Chelating agents
are compounds that help in the digestion processes of either plants or animals. The chelating
agents make the heavy metals available for the plants to absorb or adsorb. Chelating agents are
very specific with the heavy metals it can bond to make it readily available. There are numerous
chemicals proven to be very effective as chelating agents, one of which is the citric acid. Citric
acid is an organic and weak acid, because in its solution it has very small quantities of H + ions. It
is an excellent chelating agent and binding metals used to remove heavy metals, by chelating the
metals in hard water. Due to its small size molecule, it can easily penetrate into the roots of
plants and adsorbed at high speed (Kuèanda, 2014). Solubilisation of heavy metals arsenic,
cadmium, cobalt, copper and zinc was observed for citric acid (Dupare, D.B., 2015).

Chelating agents are capable of binding to toxic metal ions to form complex structures
which are easily excreted from the body removing them from intercellular or extracellular
spaces. Chelating agents possess ligand binding atoms that form either two covalent linkages or
one covalent and one co-ordinate or two co-ordinate linkages in the case of bidentate chelates.
Bidentate or multidentate form ring structures that include the metal ion and two ligand atoms
attached to the metal. They help transport nutrients throughout plants and can be very useful any
time, heavy metals need to be broken down, isolated or moved. Proteins, polysaccharides and
poly nucleic acids are excellent polydentate ligands for many metal ions. Organic compounds
such as malate, organic diacids such as malate, polypeptides such as phytochelation also act as
chelators (Kaushik, 2015).

Based from a study, it was reported that the addition of citric acid increases the
accumulation in Indian mustard (B. juncea) tissues. The citric acid chelates the water, thereby
enhancing its solubility and availability in the water solution. The addition of citric acid causes a
1000-fold increase of uranium in the shoots of B. juncea compared to accumulation in the control
(no citric acid addition). Despite the promise of some acidifying agents for use in
phytoextraction, little research is reported on this subject (Brown et al. 1994; Salt et al. 1995a;
Blaylock and Huang, 2000).

However, the enhancement of plant uptake varies greatly, depending on the specific
metal, chelate, plant combination and on soil conditions. Uncustomized addition of chelating
agent results in unsuccessful phytoextraction meanwhile causing negative effects on the eco-
environment. Further research is needed to screen environment-friendly and cost-effective
chelates and optimize processes involved in chelate induced phytoextraction to site specific
conditions (Song, 2015).
 4

Chelating agents are used to enhance the phytoextraction of a number of metal

contaminants including As, Cd, Cu, Ni, Pb, and Zn (Blaylock et al. 1997; Huang et al. 1997a;
Huang et al. 1997b). It is suggested that the phytoextraction process is enhanced when metal
availability to plant roots is facilitated through the addition of acidifying agents to the water or
soil (Brown et al. 1994; Salt et al. 1995a; Blaylock and Huang, 2000). The figure below shows
an example of how chelation works.

Figure 1.1 Iron combines with EDTA to form chelate

EDTA is acting as a hexadentate ligand or all six sites on the EDTA bind to the iron,
forming chelate which is a complex in which a ligand forms a ring that includes the metal atom.
Chelating agents help transport metal ions throughout plants and can be very useful any time,
heavy metals need to be broken down, isolated or moved.

The controversy surrounding the use of chelates deals with the fate of the residual chelate
in the soil after metal absorption occurs (Brooks, 1998a). Thus, the citric acid was the chelating
agent used due to its high biodegradability, availability, low cost and low toxicity compared to
EDTA (Kaushik, P., 2015) which is the most commonly used chelating agent. Citric acid is a
naturally occurring chelating agent. The chelation process is water activated. Citric acid chelates
the water, thereby enhancing its solubility and availability in the water solution.

Chelate-induced hyperaccumulation is in contrast to the normal practice of

phytoextraction where plants are given a gradual exposure to non-toxic quantities of metal in
solution, and accumulation occurs gradually over time as the plants grow.

This research study focused on the ability of the water hyacinth to remove heavy metals,
specifically lead and arsenic, present in the mine tailings with the aid of citric acid. The
extractive ability of the plants with and without citric acid was determined.

Samples of tailings from the tailings pond of Benguet Mining Corporation located in Balatoc, Benguet
was collected and subjected to test for heavy metals content using XRF (X-ray Fluorescence). The
tailings was obtained on March 2018, thus the effect on the absorption by hyperaccumulators was limited
to the characteristics of the mine tailings obtained on the specific date.
 5

RESEARCH METHODOLOGY

Gathering of Materials
Samples of tailings in slurry form were collected from the tailings pond of Balatoc. The
samples were analyzed for heavy metal content using XRF (X-ray Fluorescence). For the Water
Hyacinth, they were harvested, washed and transplanted into clean tap water for 1 week.

The Citric acid used in the study was available at the Environmental Research
Laboratory, Saint Louis University. The crystalline form with a 99% laboratory grade was used
to prepare the Citric acid solutions.

Preparation of the Growing Medium

The mine tailings which had been gathered were in slurry form. Fresh samples of the
tailings collected were transferred to one large container and were thoroughly mixed. Necessary
adjustments by dilution with deionized water was made to produce a medium with a liquid to
solid composition suitable for the aquatic plants.

Preparation of the Citric Acid Solutions

Three different amounts of citric acid in terms of mass were prepared. The different
amounts of citric acid used in the study were 50 milligrams, 20 milligrams and 15 milligrams.
They were diluted in a deionized water before mixing it to the tailings.

Mixing of Citric Acid with the Mine Tailings and Dosing

In a clean plastic container with a wide mouth of about 1.5 feet diameter and 6-8 inches
depth, a constant volume of 500 mL of deionized water was added to 1.5 L of the prepared mine
tailings. The citric acid solution was then added for each respective container. It was mixed
thoroughly for 5 minutes. Every after 4 days contact time, same amount of citric acid will again
be added to the growing medium.

Growing plants in mixture

The stabilized plants were transplanted to their respective container. The plants were
being grown in the mixture and all set-ups were placed in an improvised greenhouse. Water
Hyacinth of the same size, same age group and within the range of 100–150 grams were used for
the study. One plant was placed for each container. The weight was the basis for the size of the
plant. The plants were weighed after removing excess water.

Data Gathering Tools

The instrument used in the study was the XRF (X-ray fluorescence) found in the Mining
Department Laboratory of Saint Louis University. Using XRF, the elemental composition of the
tailing samples was determined.

Data Gathering Procedure

Collection of Sample

Samples of the treated tailings were collected every after 4 days. In the collection of
 6

sample, the growing medium were stirred for 5 minutes before collecting a sample.
Approximately 20 mL sample were collected and stored in a properly labeled test tube. The
collection of sample was at Day 0 (initial), Day 4, Day 8 and Day 12 of contact time.

Determination of Pb and As Present in Tailings Sample

Samples to be analyzed were gathered after the addition of the citric acid. The samples
were stored in test tube and were analyzed the following day using XRF. The arsenic and lead
content of the tailings every after 4 days of contact time were recorded.

RESULTS/FINDINGS

A. PERCENTAGE REMOVAL OF LEAD (PB)

Table 4.1 below presents the percentage removal of lead for every trial at different
amounts of citric acid upon varying contact time.

Table 4.1. Removal of Lead at Different Amounts of Citric acid upon varying Contact
Time

Amount of Citric Acid Trial 4 days 8 days 12 days

1 4.41 30.88 25.00
15 mg 2 18.84 24.64 11.59
3 6.56 18.03 -42.62
1 27.63 23.68 9.21
20 mg 2 -17.39 -13.04 -28.26
3 21.33 21.33 20.00
1 10.96 -2.74 28.77
50 mg 2 14.67 9.33 2.67
3 1.28 33.33 26.92
1 10.81 17.57 14.86
Control (0 mg) 2 4.92 -6.56 11.48
3 20.93 23.26 27.91

For treatment 1 with 15 mg citric acid, percentage removal is highest after 8 days of
contact time for all the trials then a drop in the removal occurred after 12 days of contact time
which means that the amount of lead in the mine tailings increased compared to the amount
recorded at day 8. This observation was also evident in other experimental set-ups with 20 mg
citric acid trials 1 and 3 and for 50 mg citric acid trials 2 and 3 as well as for trial 1 of the control
which has no citric acid. The trend generated by table 4.1 is presented below.
 7

40.00

% Percentage Removal (ppm)

30.00
20.00
10.00
0.00
1 2 3 1 2 3 1 2 3 1 2 3
-10.00
15 mg 20 mg 50 mg Control
-20.00
-30.00
-40.00
-50.00

4 days 8 days 12 days

Figure 4.1. Percentage Removal of Pb at Varying Amounts of Citric Acid and

Contact Time

It was observed that the water hyacinth exhibited symptoms of toxicity such as browning
of leaves and wrinkling of stem after 8 days of contact. From a review on lead toxicity in plants
conducted by Sharma and Dubey (2005), it was revealed that Pb inhibits photosynthesis, upsets
mineral nutrition and water balance and with excess Pb uptake, plants may experience stunted
growth, chlorosis and blackening of root system. The decreased ability of the water hyacinth to
absorb the heavy metal with progress of time may be traced back to the absorption of Pb where
the amount of Pb is phytotoxic to the plant.

Comparison on the Percentage Removal of Lead at different amounts of citric acid upon
varying contact time

Two- way analysis of variance (ANOVA) with replication was applied for the comparison of
the percentage removal of Pb at different amounts of citric acid upon varying contact time.

Table 4.2. Summary of F values between Pb percentage removals at different amounts of

citric acid upon varying contact time

Source of
MS F P-value F crit Decision
Variation
Treatment 69.40622 0.275211 0.842829 2.90112 Accept Null
Time 346.7462 1.374925 0.268126 2.90112 Accept Null
Treatment x
149.8755 0.594289 0.791953 2.188766 Accept Null
Time

From table 4.2, since F critical of 2.90112 is greater than the F computed, the null
hypothesis is accepted which means that there no significant difference on the Pb percentage
removals as affected by the amount of citric acid. Statistical analysis also show that there is no
significant difference on the Pb percentage removals as affected by time. Consequently, there is
 8

no significant difference on the percentage removals of lead as affected by the interaction of

citric acid and contact time.

B. PERCENTAGE REMOVAL OF ARSENIC (AS)

Table 4.3 below presents the percentage removal of arsenic at different amounts of citric
acid upon varying contact time. Results for arsenic reflect intermittent behavior particularly at
treatment 2, treatment 1 trial 2 and treatment 3 trial 1 where there was an erroneous increase in
As concentration as time progress. These observations are described to be erroneous because the
increase in concentration surpassed the initial amount of As detected. These observations may
have been due to uncontrolled behavior of plant towards the amount of citric acid added and this
deviation may have been due to differences in the absorption capacity of the plant and contact
time as well as the plants used in these set-ups may have shorter roots which affects the
absorption capacity.

Table 4.3. Percentage Removal of As at Different Amounts of Citric acid upon varying
Contact Time

Amount of Citric
Trial 4 days 8 days 12 days
acid
1 19.05 15.48 36.90
15 mg 2 -23.33 -5.00 -1.67
3 -10.77 6.15 38.46
1 -29.58 -9.86 8.45
20 mg 2 -4.44 24.44 -1.11
3 -20.90 -20.90 -22.39
1 -4.17 11.11 6.94
50 mg 2 22.22 24.69 20.99
3 23.75 23.75 35.00
1 21.74 7.61 15.22
Control (0 mg) 2 34.41 21.51 25.81
3 36.78 28.74 17.24

The trend generated for the data in table 4.3 is presented in figure 4.2 below. A
progressive behavior was observed for treatment 3 having 50 mg citric acid where increase in
percentage removal was observed as time progresses especially for trial 3. Meanwhile for trials 1
and 2, a decrease in percentage removal occurred after 12 days of contact. Comparing the
percentage removal at 50 mg citric acid and control, the removal of arsenic is observed to be
higher for the control at 4 days contact time. However, the percentage removal decreases as the
contact time increase. On the other hand, the percentage removal for arsenic at 50 mg citric acid
generally increases with contact time.
 9

50.00

40.00

30.00

20.00
% Removal

10.00 4 days
8 days
0.00 12 days
1 2 3 1 2 3 1 2 3 1 2 3
-10.00 15 20 50 Control

-20.00

-30.00

-40.00

Figure 4.2. Percentage Removal of As at Varying Amounts of Citric

Acid and Contact Time

Comparison on the Percentage Removal of Arsenic at different amounts of citric acid upon
varying contact time

Table 4.4 shows the statistical results for arsenic. Results show that there is a significant
difference between the percentage removals of arsenic at different amounts of citric acid upon
varying contact time which reveals that the amount of citric acid has an effect to the removal of
arsenic from the mine tailings. Meanwhile, there is no significant difference between the
percentage removals of arsenic as affected by time and as affected by the interaction of citric
acid and time. These results then show that the changes in the remediation of arsenic are brought
about by the citric acid introduced.

Table 4.4. Summary of F values between As Percentage Removals at different amounts of

citric acid upon varying contact time

Source of Variation MS F P-value F crit Decision

Treatment 1319.92 7.586334 0.000573 2.90112 Reject Null
Time 393.2946 2.260489 0.100327 2.90112 Accept Null
Treatment x Time 324.3171 1.864035 0.094465 2.188766 Accept Null

From a study conducted by Farid et al. (2016) on phytoremediation of As assisted by

citric acid, it was revealed that citric acid significantly enhances As concentration in different
parts of the plant used as well as it regulates the plant growth. Another study by Min et al. (2011)
revealed that citric acid is highly effective extracting agent for arsenic. This supports the results
in this study that removal of As is affected by the amount of citric acid. Furthermore, the citric
 10

acid helped regulate the growth of the plant thus improving the absorption ability of the plant as
manifested by the increasing removal with increasing contact time at 50 mg citric acid as
compared to the control. Also, it was observed that plants on the set ups with citric acid showed
higher tolerance to the metals based from the change in color of leaves of the hyacinth. Chlorosis
and stunted growth were observed for the control set ups earlier than those in experimental set-
ups.

CONCLUSIONS

Basing from the experimental and statistical results, it is concluded that:

1. There is a significant difference on the percentage removals of arsenic while there is no
significant difference on the percentage removals of lead as affected by the amount of citric acid.
2. There is no significant difference on the percentage removals of Pb and As as affected by
time.
3. There is no significant difference on the percentage removals of Pb and As as affected by
time and the interaction between the contact time and amount of citric acid.
4. Citric acid helped regulate growth of the Water Hyacinth which improved heavy metal
absorption ability of plants.

ACKNOWLEDGEMENTS

The researchers would like to acknowledge the contributions of the following groups and
individuals in the development of this research paper:
To Engr. Katelyn G. Gabon, the researchers’ adviser and primary consultant, for her
suggestions, knowledge and experience that aided the researchers throughout the conception and
eventual realization of this paper. She is the foremost reason for making this research possible;
To Engr. Jonalyn Kimpay and Engr. Benedict Marzan for invaluable support and
encouragement that allowed for the improvement of this study;
To Engr. Joseph Marie S. Kitma III, Engr. Daphne C. Leal and Engr. Melissa May M.
Boado, for their constructive recommendations and insights that served to solidify the
foundations behind this paper;
To Sir Francisco D. Pascua Jr. and Ma’am Jennelyn Amon, our Environmental and
Research Laboratory Technicians, for their unwavering assistance, supervisions and generously
providing the apparatuses necessitated in the execution of this report;
To Engr. Romeo M. Santos and Engr. Raymond Masliyan of Mining Engineering
Department of Saint Louis University, for allowing the researchers to use the XRF analyzer
available in their laboratory;
To Ms. Freda Kate D. Samuel of Benguet State University, for providing assistance in the
statistics part of the research;
To the Chemical Engineering Faculty for their unwavering support, insights, knowledge,
expertise and inspiration for the completion of our research work;
To the researchers’ parents and families from their infinite care, patience, support,
financial and moral support and unwavering love towards the accomplishment of this study.
To the researchers’ friends, schoolmates, and loved ones for showing care and support
which helped in the completion of this endeavor.
 11

Finally, our greatest gratitude to the Almighty Father, for his guidance, blessings, grace
and love, for his gift of ingenuity, creativity, capability and patience to carry out this project;
Ad Majorem Dei Gloriam.

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