METHODOLOGY

This study will follow seven (7) phases, as shown below in Figure 4, namely: Phase

I– Collection of Samples; Phase II– Arsenic Content Testing; Phase III– Collection and

Preparation; Phase IV– Ex Vivo Testing; Phase V– In Vivo Quantitative Testing; Phase

VI– Statistical Analysis; and Phase VII– Disposal, Risk, and Safety. The In Vivo arsenic

content testing will be done at the selected laboratories in the Philippines and the Ex Vivo

testing will be done in a controlled setting in the Municipality of Carmen, and if possible,

with the assistance of agriculturists and biosystems engineers.

Figure 5. The Seven Phases of the Study

PHASE I: COLLECTION OF SAMPLES

Collection of the Soil and Water Samples in the Rice Field

The soil and water samples used in the control and experimental groups will be

collected from a rice field in the vicinity of Barangay Salvacion, Municipality of Carmen.

The soil will be collected from an actual rice field, while the water will be extracted from

the irrigation surrounding the rice plant using a mechanical plunger.

Collection of Rice Samples

Actual rice plants will be gathered from a rice field in Salvacion, Carmen. Six (6)

rice stalks, alongside a few packets of seedlings, will be collected and purchased from the

vicinity. An agriculturist will review the plants to ensure that the rice plants are of similar

age and appropriate to be transplanted. After the verification by the agriculturist, the rice

stalks will then be transplanted into a pot with a flooded condition to imitate the paddy

field setting as much as possible. During this process, The researchers will follow a readily

accessible local farmer on how to correctly cultivate the young rice plants so that there are

few to no fatalities of rice plants. Meanwhile, a few rice stalks and a few seedlings will be

tested for their arsenic content through a fluorescence spectrometer to determine the

baseline and the initial amount of arsenic in our samples to set the statistical basis for our

study.

Collection of Oxalic Acid

500g of pure oxalic acid (COOH2) will be purchased from Chemi-Source Unlimited

Corporation (Sasa, Davao City) or a pharmacy/laboratory that sells this product. If certain

circumstances make both sources impossible, oxalic acid will be purchased online instead.
 Collection of Citric Acid

Although it may be less costly to produce citric acid through manual extraction, it is

deemed time-consuming and inefficient because only a few grams will be produced in such

a lengthy process (7-14 days on average). 1 The most practical option would be to purchase

citric acid from Chemi-Source Unlimited Corporation (Sasa, Davao City) or a local

pharmacy/laboratory. 250g of anhydrous citric acid will be purchased for the

administration of soil, while 250g of monohydrate citric acid will be for the water samples.

Citric acid sourced at an online store will be the last option.

PHASE II: ARSENIC CONTENT TESTING

Testing the Arsenic Content in Soil Samples

The researchers will test the soil samples for its arsenic content and its

concentration to determine the range of arsenic content, and to determine the amount of As

in statistically accurate and unbiased calculations through the staged sequential extraction

method.

Sequential extraction is a common analytical method used to identify elements in

solid materials. This method goes through a 5-step process, based on the exact procedures

followed by one Korean study.2

1 Nile Red, “Extracting the citric acid from lemons,” NileRed, January 15, 2017, YouTube video, 16:20, https://www.youtube.com/watch?
v=FMtayizdFiw
2 Lee, Myeong Eun, Eun-Ki Jeon, Jong-Gook Kim, and Kitae Baek. "Oxalic Acid-based Remediation of Arsenic-contaminated Soil." Journal of Soil and
Groundwater Environment 23, no. 1 (2018): 85-91.
 Figu

re 6.

Steps

for

sequential extraction method. (Lee et al., 2018; see Table 3.)

After collecting 2g of soil sample for this process, non-specific adsorption will

follow. Ammonium sulfate [(NH4)2SO4)] will be used as an extractant for this process. The

resultant solution will be shaken using an overhead shaker for four (4) hours before going

through specific adsorption, which takes sixteen (16) hours of shaking.

The next step is the conversion of amorphous hydrous oxide of iron (Fe) and

aluminum (Al) to crystal form by utilizing an ammonium-oxalate buffer through four (4)

hours of shaking in the absence of visible light. The residual chemical component will be

treated with 1% aqua regia (HNO₃+3 HCl) while being heated in a water bath at 80

degrees Celsius for 1.5 hours.

Before submission, soil samples will be sun-dried at temperatures of more than

28°C for twenty (20) hours (which takes 2 days considering the sunlight), crushed, and then

sieved considering homogeneity, where only fine particles (specifically at sizes of less than

or equal to 0.2 mm) will be collected.3

The soil samples will then be analyzed through atomic fluorescence spectrometry

3 Ullah, Aman, Yibing Ma, Jumei Li, Nazia Tahir, and Babar Hussain. "Effective amendments on cadmium, arsenic, chromium and lead contaminated
paddy soil for rice safety." Agronomy 10, no. 3 (2020): 359.
using an x-ray fluorescence spectrometer. The procedure will be done at the Philippine

Nuclear Research Institute in Quezon City.

The soil samples to be used in the experiment should result as arsenic-positive, but

the amount of arsenic is to be determined by the quantitative analysis from the physicists at

PNRI.

Testing the Arsenic Content in Water Samples

The testing of the arsenic content level in the water samples will be done through a

wastewater analysis using the nitric acid leaching method with the help of professional

scientists at the University of the Immaculate Conception Science Resource Center (UIC-

SRC) in Davao City. The water samples tested should result as arsenic-positive to be used

in the study.

Initial Screening for Arsenic Content in Rice

To determine the statistical baseline for the arsenic content in rice, an untreated rice

stalk will be tested. Three segments from the rice plant (root, stem, grain/husk) will be

pruned out of a fresh plant sample, and three samples of each segment will be sent to the

Philippine Nuclear Research Institute (Quezon City) to be analyzed through a fluorescence

spectrometer after the sequential extraction process (see fig. 6) to accurately determine the

arsenic content of the rice, as well as the type of arsenic. The samples will be tested for

arsenic content before and after the treatments.

 PHASE III: COLLECTION AND PREPARATION

Documents and Forms

Before the experimental phase begins, the researchers will gather documents and

papers necessary for the execution of specific experiment methods, as some can be

hazardous when done without caution. Based on the templates released by the Society for

Science and the Public, the following forms will be produced, disseminated, and signed

according to the appropriate personnel:

● Adult Sponsor and Safety Assessment Form (for the parents of the research

proponents and solicitors of the project) 4

● Approval Form (for the research adviser, the research proponents, and their

parents) 5

● Human Informed Consent Form (for the research proponents signifying that

everyone understands the potential risks, terms, and conditions that come

with conducting this study) 6

Preparation of Citric Acid and Oxalic Acid Solutions

In preparation for the mixture to be administered to the soil, the anhydrous citric acid

and oxalic acid components will be mixed heterozygously into one solution without

interrupting its chemical composition. Both acids have similar color and look, so it would

be challenging to discern. The oxalic and citric acid mixture that will be prepared will

4 Society for Science (2022). Retrieved from:https://sspcdn.blob.core.windows.net/files/Documents/SEP/ISEF/2023/Forms/1-Checklist-for-Adult-

Sponsor.pdf
5 Society for Science (2022). Retrieved from:https://sspcdn.blob.core.windows.net/files/Documents/SEP/ISEF/2023/Forms/1B-Approval-Form.pdf
6 Society for Science (2022). Retrieved from: https://sspcdn.blob.core.windows.net/files/Documents/SEP/ISEF/2023/Forms/4-Sample-Informed-
Consent.pdf
 contain varying concentrations of 0.1 mol/L, 0.5 mol/L, and 1 mol/L into a liquid boron

fertilizer at a constant amount and will be utilized to the different experimental samples.7

Oxalic acid and citric acid have pH levels of less than 2 and can potentially alter the

pH levels of the rice soil and water, making them more acidic. This is why boron fertilizer

will be used, which will counteract the solution’s acidity without affecting the chelating

properties. Not only will this help control the pH level of the soil, but this will also help

reduce boron deficiency among rice plants.8

Meanwhile, 250 mL of oxalic acid and monohydrate citric acid solution will be

prepared for the water samples.9

Preparation of the Experimental and Control Groups

The researchers will set up four (4) sets of soil and water samples. The first one will

be the control group (T1), where no treatment will be applied; the second one (T2) will be

treated with 0.1 mol/L of the solution; the third batch (T3) will be treated with 0.5 mol/L of

the solution; while the fourth batch (T4) will be treated with 1 mol/L of the solution (citric

acid and oxalic acid mixture). Before treatment, the soil must surpass the arsenic content of

18 ppm until 20 ppm. The threshold set by the European Union for arsenic concentration

level in the soil is 20 ppm.10 18 ppm of As will give us a good measure of the fluctuation of

arsenic post-treatment while still enabling the rice to survive in the controlled condition

7 Pa, Faizul Che, and Wong Kok Kein. "Removal of iron in rice husk via oxalic acid leaching process." In IOP Conference Series: Materials Science
and Engineering, vol. 701, no. 1, p. 012021. IOP Publishing, 2019.
8 Prashanth, K. M., H. M. Chidanandappa, D. Ravikumar, Parashuram Chandra Vamshi, and Basavaraj Naik. "Effect of different levels of borax
application on growth and yield of rice (Oryza sativa L) at Bhadra command, Karnataka." Journal of Pharmacognosy and Phytochemistry 7, no. 3
(2018): 3028-3031.
9 Pa et al., “Removal of iron in rice…”(see 2.2- Leaching process).
10 European Union, “Arsenic,” Food Safety (European Union), accessed November 28, 2022,
https://food.ec.europa.eu/safety/chemical-safety/contaminants/catalogue/arsenic_en.
 without overpowering As toxicity, according to the spectrometry report submitted by PNRI

it falls less than the recommended amount, the soil will be contaminated with arsenic first

using sodium arsenite to exceed its arsenic content to the set threshold. 1 mg/L adds up to 1

ppm.11 For example, if the soil has only 4 ppm of arsenic content, 14 mg/L of sodium

arsenite will be added to the soil. It will make the soil concentrated enough for the rice to

take up arsenic and for the solution to work with significant effects.

Afterward, the contaminated soil will be placed in a soil box and moistened with

water that is in the same condition as the irrigation water used in the actual rice field. Once

the rice plants are planted, a saturated (flushed) consistency will be applied.

PHASE IV: EX VIVO TESTING

Soil Box Experiment

After preparation and contamination, the soils will be treated with the combination

11 Vancouver Island University, “Units of Concentration - Vancouver Island University,” MORE ABOUT UNITS OF CONCENTRATION (Vancouver
Island University), accessed November 27, 2022, https://web.viu.ca/krogh/chem301/UNITS%20OF%20CONCENTRATION.pdf.
 of oxalic acid and citric acid in different concentrations (0.1 mol/L, 0.5 mol/L, and 1

mol/L), which will then be mixed into the corresponding PVC pots, each with five (5) kg of

soil in varied conditions (three soil settings). 12 After two (2) weeks, the rice plants will be

transplanted into the sample groups. After ten (10) days, 3g of sample from the treated soil

will be collected using a handheld shovel, assuming the chelation reaction has taken place.

At the laboratory, 9 mL of cleaning agent will be infused with the samples while

stirring them at 40 rpm with an overhead shaker (UIC SRC, Davao City). 13
After the

reaction, the suspension will be centrifuged at 6000 rpm for 10 minutes and filtered

through a syringe filter in 0.45 μm (UIC SRC, Davao City). 14 After solid-liquid separation,

arsenic in the solution will be analyzed through a fluorescence spectrometer (PNRI,

Quezon City).

Treatment of Water from Irrigation

The arsenic treatment of water will be done using a reduction reaction. According to

a study, dithionite was used as a reductant to remove As in water and was observed that

arsenic concentrations decreased as dithionite concentration increased.15 Hence, using that

12 Lee, Myeong Eun, Eun-Ki Jeon, Jong-Gook Kim, and Kitae Baek. "Oxalic Acid-based Remediation of Arsenic-contaminated Soil." Journal of Soil
and Groundwater Environment 23, no. 1 (2018): 85-91.
13 Lee et al.,. "Oxalic Acid-based Remediation…”

14 Ibid.
15 Kim, E. J., & Baek, K. Selective recovery of ferrous oxalate and removal of arsenic and other metals from soil-washing wastewater using a
reduction reaction. Journal of Cleaner Production, 221; (2019). 635–643. doi:10.1016/j.jclepro.2019.03.014
 study as a reference, the researchers will utilize oxalic acid and citric acid as reducing

agents to reduce the As content in the water samples significantly.

In the procedure, 30mL of controlled water sample will be treated with oxalic acid

and citric acid with varying amounts of concentration (0.1 mol/L, 0.5 mol/L, and 1 mol/L).

The reduction reaction with oxalic acid and citric acid will be conducted in a mixed batch

reactor system (laboratory to be determined) for 3h at 20°C and 150 rpm. After the

reaction, the water sample will be centrifuged at 8000 rpm for 10 min and filtered with a

0.45-mm pore size membrane filter to separate any solid that may have formed from the

treatment.

After the treatment, the testing of the arsenic content will be done at the Science

Resource Center (UIC, Davao City) to determine if the level of arsenic concentration is

reduced in the water sample. At the same time, it will be placed in its respective

experimental sample group to be absorbed by the rice in flooded conditions for fourteen

(14) days.16 The soil boxes will continue to be irrigated with the treated water solutions in

the same levels of varying concentrations for the next two (2) weeks.

PHASE V: IN VIVO QUANTITATIVE TESTING

16 Ullah, Aman, Yibing Ma, Jumei Li, Nazia Tahir, and Babar Hussain. "Effective amendments on cadmium, arsenic, chromium and lead
contaminated paddy soil for rice safety." Agronomy 10, no. 3 (2020): 359.
 Based on the previous tests, the rice samples from the same source where the soil

and water were collected will be screened through a fluorescence spectrometer (PNRI,

Quezon City) to determine the amount of arsenic before testing. Another set of treated rice

samples (post-experiment) will also be tested.

Before the harvested rice samples are sent to the laboratory for testing, the collected

leaves of the rice samples will be washed thoroughly with deionized water before oven-

drying them at 70 degrees Celsius. After the dehydration process, the leaves will be

grounded in a stainless steel grinder and will be sieved. 17

PHASE VI: STATISTICAL ANALYSIS

All the gathered data will be analyzed using quantitative tools to bring a logical

result to the study. The level of significance for this study is 1% (α = 0.01). With the aid of

the R software (version 4.2.2), Microsoft Excel for the tabular content, and a statistician,

the following statistical tests will be used:

Mean will determine the average arsenic content removed by the two components

(oxalic acid and citric acid) on multiple testing conditions.

Simple linear regression will assess the correlation between the amount of

removed As content (the y variable) and the concentration levels of the mixture of oxalic

acid and citric acid (the x variable). There will be two graphs constructed, one for the As

content in the soil and the other for the As content in water. This test will tell whether there

17 Ullah, Aman, Yibing Ma, Jumei Li, Nazia Tahir, and Babar Hussain. "Effective amendments on cadmium, arsenic, chromium and lead
contaminated paddy soil for rice safety." Agronomy 10, no. 3 (2020): 359.
is an effect on the concentration level of the solution and determine which concentration

level is the most effective.

Two-way ANOVA (Analysis of variance), also called Multivariate ANOVA,

would assess the significant difference in the arsenic content between the soil and water

samples in similar concentration levels by treating them with a combination of oxalic acid

and citric acid.

Tukey’s HSD (Honest Significant Difference) test will assess the differences

among sample means for significance and determine which medium (soil or water) has the

best and most effective concentration level or if both mediums have the same effect with

the treatment method. It comes after the results of the two-way ANOVA have been

analyzed.

T-test will give results to the acid mixture’s efficacy by determining whether there

is a significant difference in the arsenic content in rice treated with oxalic acid and citric

acid in different concentrations, including the one without treatment.

PHASE VII: DISPOSAL, RISK, AND SAFETY

Arsenic

Sodium arsenite will be used for the intended contamination of the soil, which, along

with its toxic and carcinogenic nature, requires strict disposal measures. Arsenic

compounds in occupational settings occur predominantly as As(III) and As(V) compounds

 in mists, fumes, vapors, or dust. Arsenic is listed as a toxic substance under Section 313 of

the Emergency Planning and Community Right to Know Act (EPCRA) under Title III of

the Superfund Amendments and Reauthorization Act (SARA) (EPA 1995c). The disposal

of wastes containing arsenic is controlled by several federal regulations.

In handling arsenic, one should wear protective equipment to prevent skin and eye

exposure and handle it by following good industrial hygiene and safety practices. The

remains or spillage of this element should be collected and shoveled into suitable

containers for disposal. It should not be flushed into surface water or sanitary sewer

systems and should be prevented from entering drains. Chemical waste generators must

determine whether a discarded chemical is classified as hazardous waste. Chemical waste

generators must also consult local, regional, and national hazardous waste regulations to

ensure complete and accurate classification.18

Citric Acid

Waste Citric Acid will be treated as Dangerous Waste if it has a pH of 5 or below or

a concentration of 1% or more. The material should be gathered in an appropriate,

ventilated container with a lid made for storing acids and bases. The container must be kept

away from incompatible substances like metal nitrates, oxidizing agents (such as nitric

acid), reducing agents, sulfuric acid, strong acids, and strong bases.

When waste is first placed in the container, a completed Dangerous Waste label

18 Health, Safety and Environmental Department. Thermo Fisher Scientific Chemicals, Inc. February 14, 2020
 needs to be attached. We will fill out a Chemical Collection Request Form when the

container is full or no longer needed, then give it to the waste accumulation area operator.

The solution may be discharged if its pH is between 5 and 9 and contains less than

1%. The solution cannot be diluted to change its pH or concentration for disposal.19

Oxalic Acid

To dilute the acid for disposal, we will pour it into a sizable bowl filled with icy

water. Splashing and boiling will no longer be risks. The acid will go after the water. In the

diluted Oxalic acid solution, baking soda will be added to dilute the acid. It will begin to

bubble. Baking soda should be added until bubbling ceases.

The recommended action is to keep adding baking soda until the solution's pH is 7.

However, 5.5 pH is also acceptable for drain use. The solution will then be neutralized and

will not provide a toxicity risk. More water will be added when necessary before emptying

it down the drain. It is acceptable to flush about 100 grams or 100 mL down the toilet.20

Oxalic acid, although safe for plants, is considered hazardous for humans, especially

in its powdered form, which is the form that we are going to use for our study. We will

utilize personal protective equipment (PPE) for our experiment with a qualified scientist or

biosystems engineer to do the procedures in the most risk-free way possible. There is no

19 Standard Operating Procedures For Hazardous And Particularly Hazardous Chemicals (2020). Washington State University.

20 (2021). A Comprehensive Guide On Oxalic Acid. https://camachem.com/en/blog/post/oxalic-acid-guide?

fbclid=IwAR3tPkEkLoUUvkv80B2UENTpToeAzrHtZDQiRE4HL54GJqrIFGJgKIGBR3s
 food or water consumption during the experiment hours inside the laboratory.

Other Chemicals

Aqua regia will be used for the sequential extraction process. However, this chemical

imposes several hazards on humans. According to the safety data sheet released by

Columbus Chemical Industries Inc., aqua regia is classified as corrosive, harmful by

ingestion, and lethal on target organs by the Occupational Safety and Health

Administration.21

In disposal, excess and unused amounts of aqua regia will be added to a large

amount of ice. (500 grams of ice per 100 mL of aqua regia). A basic aqueous solution will

help neutralize the combination, such as saturated sodium bicarbonate (NaHCO3) or 1M or

10% sodium hydroxide (NaOH) in water. After that, the neutralized solution can now be

poured down the drain. Heavy metals (such as gold, platinum, lead, or chromium) should

be tagged and removed from the neutralized mixture using the chemical waste management

system.

Aqua regia stoppered bottles should never be stored since they could explode. Every

time aqua regia is used, it should be freshly prepared, and any surplus should be neutralized

immediately.

21 Princeton University, ed., “Aqua Regia,” Princeton University (The Trustees of Princeton University), accessed November 27, 2022,
https://ehs.princeton.edu/laboratory-research/chemical-safety/chemical-specific-protocols/aqua-regia.