10/16/2021Chemical Coagulation of Microplastics in Water Using Tannin Extracted from

Indian Mango (Mangifera indica) Seed Kernels

A Science Investigatory Project Plan

Submitted as Partial Fulfillment

of the Requirements in Research III

Abad, Arianna Janna A.

Cisnero, Angela B.

Danguilan, Karl Vinzent K.

Matubis, Janina D.

Pring, Robin Ace R.

Ragpa, Alexandra Mari H.

Ramos, Kyla Louise M.

Quezon City Science High School

March 2019

Rationale
 Plastic is the most prevalent marine debris that poses a great threat to aquatic ecosystems,

societies, and economic activities such as aquaculture and tourism. Considering the constant

need for plastics in modern life because of its properties, restriction of mass plastic production is

not to be expected anytime soon. Plastics are generally divided into macroplastics which are less

than 2.5 centimeters (cm), and the smaller microplastics which are less than 5 millimeters (mm),

including nano plastics. Sources and pathways of marine litter are diverse and exact quantities

and routes are not fully known.1 Many of these polymers and plasticizers absorb and adsorb

other persistent organic pollutants and metals lost to the environment, resulting in a long list of

toxicants associated with plastic debris.2

The widespread use of plastics facilitates continuous contact of these materials with the

human body and with it daily exposure to ingredients in plastics. Although plastic components

do not have significant bioaccumulation potential (except when accidentally ingested and

becoming entrapped in the gastrointestinal system), biomonitoring studies have demonstrated the

presence of a steady-state concentration of plastics’ components in the human body; thereby,

reflecting the ongoing balance of constant exposure, metabolism, and excretion of these

compounds. This situation implies that in today’s plastics-enabled society there are no control

groups to be found to analyze the effects on human health from low-level, environmental

exposures to plastic constituents.

New solutions have been raised as a replacement for conventional wastewater treatment

with primary and secondary treatment processes efficiently removing microplastics from the

wastewater.3 A study involving the removal of microplastics from effluent in four different
1
Abreo, A. S. (2018). Marine plastics in the Philippines: a call for research. Philippine Science Letters,
11(1), 20-21.
2
GMA News. (2018, May 30). GMA Network. Retrieved from GMA News Online:
https://www.gmanetwork.com/news/lifestyle/healthandwellness/655063/salt-samples-from-phl-tested-for-
microplastics-is-it-already-in-our-food/story/
3
Talvitie, J., Mikola, A., Koistinen, A., & Setälä, O. (2017, October 15). Solutions to microplastic pollution
Municipal Wastewater Treatment Plants (WWTPs) utilizing different advanced final-stage

treatment technologies has been investigated. The study included a bioreactor treating primary

effluent and different tertiary technologies namely disc-filter, rapid sand filtration and dissolved

air filtration. The study showed that advanced wastewater treatment technologies can

substantially reduce microplastic pollution released to both WWTPs and aquatic environments.

WWTPs proved successful in western countries with a success rate of 98.41%. Despite

the large reduction, a study published in May 2016 found that a wastewater treatment plant

claiming the high success rate still was releasing 65 million microplastics into the receiving

water every day.4 This results in microplastics getting discharged with “clean water” with many

of these having hydrophobic surfaces that adhere to common pollutants like polychlorinated

biphenyls (PCBs). WWTPs utilizing sand filter face another problem: sludge production and

removal. Sludge from wastewater treatment plants forms a vital biosolid product that is recycled

to agricultural land. However, increased concentrations of plastics in this product would not just

return plastics back to the environment but put at risk a valuable source of nutrients for the

agricultural sector.5

Indian mango (Mangifera indica) is a native fruit in tropical Asia. It has been developed

in the Indian subcontinent and is currently naturalized in most tropical nations. 6 Mango seed,

which represents between 20-60% of the fruit, has limited food or industrial use in most

– Removal of microplastics from wastewater effluent with advanced wastewater treatment technologies. Water
Research, 123, 401-407.
4
AquaSPE. (2019). Retrieved January, 2019 from The Water Network:
https://thewaternetwork.com/_/water-treatment/article-FfV/removing-microplastics-from-tap-water-starts-at
treatment-plants-LcFIy6NHAtSyp09-Pd3nNg
5
The Editors of Encyclopaedia Britannica. (2016, April 07). Encyclopædia Britannica. Retrieved
December,
2019 from Encyclopædia Britannica: https://www.britannica.com/science/tannin
6
Shah, K., Patel, M., RJ, P., & Parmar, P. (2010). Mangifera Indica (Mango). Pharmacognosy Review, 4(7),
42-48.
producing countries and is therefore wasted. The concentration of tannic acid, a hydrolysable

tannin, (gram/kilogram) in Indian mango seed kernel was found to be 56.5% to 70%.7

Recently, tannin-based coagulants have been utilized in coagulation or flocculation

process for water purification. These coagulants are, in general, used as a point-of-use

technology in less-developed communities since they are relatively cost-effective compared to

chemical coagulants, and can be easily processed and produce less sludge volume and bulk, thus

reducing the amount of materials to manage or dispose of. Coagulants are used in the process of

coagulation. In coagulation, a chemical is added such as tannin. This chemical produces positive

charges and neutralizes the negative charges on the particles. The particles then stick together

and form larger particles that are easier to remove.8

The main objective of this study is to find out if tannin from Indian mango seed kernels

would be effective in coagulating microplastics. Specifically, the student researchers would like

to determine which of the following amounts of tannin among 10 milligrams (mg), 20 mg, and

30 mg will coagulate the greatest amount of microplastics in the river water sample.

This study can benefit the community by presenting an alternative source for inorganic

coagulants that are used as a part of WWTPs, mainly focusing on filtering microplastics and

reducing the discharge of large amounts of microplastics in drinking and tap waters.

Research Question

7
Siaka, D. (2014, May). Potential of Mango (Magnifera Indica L.) seed kernel as a feed ingredient for
poultry: A review. World's Poultry Science Journal, 70(2), 279-288.
8
Frazer, A., Lapina, K., Whaley, A., & Muga, H. (2007). Michigan Environmental Education Curriculum
Drinking Water Treatment. Department of Civil & Environmental Engineering, Michigan Tech.
 1. Which of the following amounts of tannin among 10 milligrams (mg), 20 mg, and 30 mg

extracted from Indian mango (Mangifera indica) seed kernels is the most effective in

coagulating microplastics from water?

Hypotheses

Research Hypothesis

If a higher amount of tannin extracted from Indian mango (Mangifera indica) seed

kernels is applied to the water sample, then more microplastics will be coagulated.

Statistical Hypothesis

Null hypothesis: Different amounts of tannin extracted from Indian mango (Mangifera indica)

seed kernels have no significant difference on the amount of coagulated microplastics in river

water.

Alternative hypothesis: Different amounts of tannin extracted from Indian mango (Mangifera

indica) seed kernels have a significant difference on the amount of coagulated microplastics in

river water.

Expected Outcomes

The student researchers expect that the tannin extracted from Indian mango (Mangifera

indica) seed kernels will be effective in coagulating microplastics. The researchers hypothesized

that the setup with 30 mg of tannin will yield the greatest amount of coagulated microplastics

because it has the highest concentration.

Flowchart

Collection of Indian Mango (Mangifera indica) Seed Kernels

Extraction of Tannin in Indian Mango Seed Kernels

Preparation of Water Sample

Addition of Tannin Extract to Water Containing Microplastics

Filtration of Microplastics in Water Containing Microplastics

Procedure
Determination of the Final Amount of Microplastics in Water
Collection of Indian Mango (Mangifera indica) Seeds

The student researchers will gather 200 grams (g) of Indian mango seeds from Grow

Farm at Bagac, Bataan. The collected seeds will be brought to the University of the Philippines

Diliman - Institute of Biology (UPIB) for authentication.

The conduct of the experiment will also be done in Mach Union Water Laboratory, so

materials such as the 500 milliliters (mL) beaker, bulks, flasks, and vials will be provided by the

laboratory.

Extraction of Tannin in Indian Mango Seed Kernels

Indian mango seeds will be washed and dried in hot air at 60 degrees Celsius (°C) for 3-4

hours. Indian mango kernels will be separated from stone manually using a stainless steel knife.

Kernels will be soaked in distilled water for 6 to 7 hours, blanched for 1 to 2 minutes, chopped,

dried and ground into flour, sieved and stored in an air-tight container. From 100 g of mango

kernel flour, tannin will be extracted with 1000 mL of 80% acetone for 4-5 hours by stirring with

a magnetic stirrer. The slurry will then be strained through a muslin cloth to separate the extract.

The total polyphenols extracted will be subjected to rotary evaporation at 45°C to remove

acetone completely and to obtain a concentrated extract in liquid form. 9 Rotary evaporation is

the process of reducing the volume of a solvent by distributing it as a thin film across the interior

of a vessel at an elevated temperature and reduced pressure. 10 It will be stored in an airtight

container and kept in a cool place.

9
Yatnatti, S., & Vijayalakshmi, D. (2017). . Int.J.Curr.Microbiol.App.Sci. , 6(4), 2303-2314.
10
MindTouch. 2017. Libre Texts. 20 November.
https://chem.libretexts.org/Ancillary_Materials/Demos%2C_Techniques
%2C_and_Experiments/General_Lab_Techniques/Rotary_Evaporation.
Preparation of Water Sample

The student researchers will use 1200 mL of distilled bottled water bought from the

supermarket for the experiment. The water will be divided equally into three parts and each part

will be poured into a 500 mL beaker. The beakers will be named A, B, and C respectively. Each

beaker will receive 100 g of microbeads. The microbeads will have a length of 3 millimeters

(mm) and it will be bought from a trusted seller online.

Addition of Tannin Extract to the River Water Sample

The tannin from Indian mango seed kernels will be added to each setup. Group A will

receive 10 mg of tannin, Group B will receive 20 mg of tannin, and Group C will receive 30 mg

of tannin. Each setup will be mixed using a conventional jar test apparatus provided with a 1-

minute rapid-mixing and a 20-minute slow-mixing at rotational speeds of 200 rotations per

minute (rpm) and 30 rpm respectively. The setups will be left settled for 10 minutes.11

Filtration of Microplastics in Treated River Water Sample

After the coagulation process, the river water sample will be poured into a 500 mL beaker

and filtered using the no.4 mesh sieve previously used in the experiment. The filtered water will

then be collected for the determination of the remaining microplastics in the sample.

Determination of the Final Amount of Microplastics in Treated River Water Sample

11
Kan, C., Chihpin, H., & Pan, J. R. (2002). Time requirement for rapid-mixing in coagulation. Colloids and
Surfaces, 1-9.
 For the determination of the final amount of microplastics, the student researchers will

conduct a final microscope examination, the same process used in the determination of the initial

amount of microplastics in the river water samples. The amount of microplastics left in the

beaker will then be compared to the previous data using percentages to determine the

effectiveness of tannin extracted from Indian mango seed kernels in coagulating microplastics.

Risk and Safety

 A risk that should be considered is the handling of Indian mangoes (Mangifera indica).

Immediate and delayed hypersensitivity reaction can be caused by Indian mangoes. 12 Urushiol, a

substance found in high concentrations in the peel and fruit of mangoes, can induce an allergic

skin response.13 Beakers, bulks, vials and flasks to be used in the laboratory may contain cracks

or damages which can harm the student researcher who will handle them.

Safety measures to be observed are the use of proper laboratory attire and materials in the

experimental process. Guidance of a professional in the field of study will be required and the

researchers must follow the laboratory and professional’s protocols regarding the experiment. All

Personal Protective Equipment (PPE) should be ready and accessible before the start of the

experiment. Inspection of glass materials in the laboratory must be done for weaknesses, cracks,

or damages before and after working.14

Data Analysis

The type of statistical test that this research will be using is one-way ANOVA Test. The

amounts of the tannin from Mango (Mangifera indica) seed kernels is the independent variable,

while the amount of the coagulated microplastics is the dependent variable, with three

independent levels, namely with increasing measurements.

Bibliography

Abreo, Angelo S. 2018. "Marine plastics in the Philippines: a call for research."

12
Shah, K., Patel, M., RJ, P., & Parmar, P. (2010).
13
Sareen, R., & Shah, A. (2011, April). Hypersensitivity manifestations to the fruit mango. International
Journal of Current Microbiology and Applied Sciences, 6(4), 2303-2314.
14
Stony Brook University. (n.d.). Stony Brook University. Retrieved 2019 from
https://ehs.stonybrook.edu//about-us/full-staff-list
Philippine Science Letters 11 (1): 20-21.

AquaSPE. 2019. Accessed January, 2019. https://thewaternetwork.com/_/water-

treatment/article-FfV/removing-microplastics-from-tap-water-starts-at-treatment-plants-
LcFIy6NHAtSyp09-Pd3nNg.

Asrafuzzaman, Md., A.N.M. Fakhruddin, and Md. Alamgir Hossain. 2011. "Reduction of
Turbidity of Water Using Locally Available Natural Coagulants." ISRN Microbiology 1-6.

MindTouch. 2017. Libre Texts. 20 November.

https://chem.libretexts.org/Ancillary_Materials/Demos%2C_Techniques
%2C_and_Experiments/General_Lab_Techniques/Rotary_Evaporation.

Frazer, Aubrey, Kateryna Lapina, Andrew Whaley, and Helen Muga. 2007. "Michigan
Environmental Education Curriculum Drinking Water Treatment." Department of Civil &
Environmental Engineering, Michigan Tech.

Añonuevo-Arcega, and Macy. 2017. Pasig River is world's 2nd biggest source of plastic
waste for its size –study. News Report, GMA News Online.

GMA Network. 2018. Salt samples from PHL tested for microplastics: Is it already in
our food? Lifestyle Report , GMA News Online.

Hidalgo-Ruz, Valeria, Lars Gutow, Richard C. Thompson, and Martin Thiel. 2012. "
Microplastics in the Marine Environment: A Review of the Methods Used for Identification and
Quantification." Environmental Science and Technology 46: 3060-3075.

Kan, Chichuan, Huang Chihpin, and Jill Ruhsing Pan. 2002. "Time requirement for
rapid-mixing in coagulation." Colloids and Surfaces 1-9.

Marine & Environmental Research Institute. 2015. "GUIDE TO MICROPLASTIC

IDENTIFICATION." Marine & Environmental Research Institute.

Sareen, R, and A Shah. 2011. "Hypersensitivity manifestations to the fruit mango."

International Journal of Current Microbiology and Applied Sciences 6 (4): 2303-2314.

Shah, KA, MB Patel, Patel RJ, and PK Parmar. 2010. "Mangifera Indica (Mango)."
Pharmacognosy Review 4 (7): 42-48.

Siaka, D.S. 2014. "Potential of Mango (Magnifera Indica L.) seed kernel as a feed
ingredient for poultry: A review." World's Poultry Science Journal 70 (2): 279-288.

Stony Brook University. n.d. Stony Brook University. Accessed 2019.

https://ehs.stonybrook.edu//about-us/full-staff-list.
 Talvitie, Julia, Anna Mikola, Arto Koistinen, and Outi Setälä. 2017. "Solutions to
microplastic pollution – Removal of microplastics from wastewater effluent with advanced
wastewater treatment technologies." Water Research 123: 401-407.

The Editors of Encyclopaedia Britannica. 2016. Encyclopædia Britannica. 07 April.

Accessed December, 2019. https://www.britannica.com/science/tannin.

Yatnatti, Shilpa, and D. Vijayalakshmi. 2017. "Extraction of Total Polyphenols (TPP)

from Mango Seed Kernels and its Incorporation in Watermelon Squash."
Int.J.Curr.Microbiol.App.Sci. 6 (4): 2303-2314.