Chapter 1

INTRODUCTION

Plastic is the most common littered material both in land and aquatic environment.

Due to its increasing demand from household usage to industrial application, mass

production of plastics has become enormous. In 2009, the production of plastics reached

up to 230 million tons (Nuelle, M.-T., Dekiff, J. H., Remy, D., & Fries, 2014). Eriksen et

al. (2014) estimated plastics afloat at sea to be >5 trillion (over 250, 000 tons).

Plastic litter undergoes weathering processes contributing to its disintegration into

microplastics. In principle, microplastics is defined as tiny pieces <5mm produced from

the fragmentation of large plastics The type and degree of weathering depends on the

time of exposure and the aquatic environmental compartment plastics enter, making its

degradation process extremely slow (Barnes, Galgani, Thompson, & Barlaz, 2009).

Hidalgo-Ruz & Thiel (2012) accounted that high tide beach zone had the greatest micro

litter deposits while the sub litorral zone had the least fragment deposits. These records

only show that microplastics indeed has occupied large portions of the sea body resulting

to prolonged accumulation in rivers, estuaries and oceans.

Such accumulation of microplastics in the marine fauna pose detrimental effects

to the food chain. Thus, microplastics may carry potential harm in terms of food safety,

security and protection to marine life. Mostly reported are the intake of plastic fragments

in shellfish (Naji et al. 2018), molluscs () and other fish species.

In this study, the occurrence of microplastics present in the sand sediments along

Manila Bay Philippines will be investigated. Dated in history, Manila Bay has long

become the harbor of commercial and industrial business. This research will confirm the

presence of microplastics to different sites along the bay

 . Chemical and surface techniques will be utilized using Fourier Transform

Infrared Spectroscopy-Attenuated Total Reflectance (FTIR-ATR) and Secondary

Electron Microscopy-Energy Dispersive X-ray (SEM-EDX). FTIR-ATR, will identify the

type of polymer collected microplastics originate while SEM-EDX will provide elemental

images and elemental information such as metals of the samples. The elemental analysis

of Sem-EDX will not only provide the organic content but also the metals may be present

on the surface.
 Chapter 2

REVIEW OF LITERATURE

Microplastics

The measurement of microplastic commonly found in literature is about < 5mm.

Plastic debris having a size range of 1-6 mm are still recognizable flotsam (Hidalgo-Ruz

& Thiel, 2012). Depending on the type of polymer and additive chemicals incorporated

into their pristine material, microplastics vary in sizes, shapes and densities. Hence these

plastic fragments will have different distributional patterns (Vethaak et al., 2017).

The aquatic ecosystem which has been a large basin of microplastic pollution has

been reported for the accumulation of microplastics in water surface, column and in

beaches. Microplastics which can be found in water, sediment, invertebrate species

(Karlsson et al., 2017) and seabed demonstrate capability of accumulating in the marine

food web, therefore becoming potential hazards on food safety and human health

(Marques et al., 2015).

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Categories of Microplastics

A paper review on different methods for identification of microplastics by

Hidalgo-Ruz & Thiel (2012) summarized the categories of microplastics (Table 1) as

type, shape and erosion collated from the different researches conducted.

Table 1. Categories in Identification of Microplastics

Categories

Type Shape Erosion Color

Plastic Fragment For pellets: Fresh Transparent

Plastic Pellets Cylindrical Unweathered Crystalline

Filaments disks Incipient Alteration White

Plastic Films Flat Conchoidal Fractures Clear-white-cream

Foamed Ovoid Weathered Red

Plastics Granules Spheruloids Grooves Orange

Styrofoam For Fragment: Irregular Surface Blue

Rounded Jagged Fragments Opaque

Subrounded Linear Fractures Black

Subangular Subparallel Ridges Gray

Angular Very Degraded Brown

Green

Pink

Tan

Yellow

Pigmentation

Reference: Hidalgo &Thiel (2012).

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Microplastics originating from different polymers also vary in densities ranging

from 0.9-2.10 gcm-3 (Hidalgo-Ruz & Thiel, 2012). Literatures reported the densities of

polyethylene (PE) and polypropylene (PP) at about 0.917-0.965 gcm-3 and 0.90-0.91 gcm-
3
, respectively.

Sampling

Light-weighted microplastics can deposit on sea surface, water column and in

sediments. Hidalgo-Ruz & Thiel (2012) identified three approaches of microplastic

sampling: selective, bulk and volume-reduced. Selective sampling is applicable for

collection of marine debris which are still visible by the naked eye (e.g. pellets). Bulk

sampling uses an unreduced volume of sample to retrieve plastic fragments no longer

recognizable while volume-reduced sampling chooses a representative portion from the

bulk sample containing the sample of interest for analysis. Nuelle et al. (2014) in their

study randomly collected three sediment samples from a beach of the island Norderney,

located off the German seashore. Each sediment weighed 3 kg from a depth of about

3cm. The sediment was further processed through density separation method.

Sampling-Sediments

Hidalgo-Ruz & Thiel (2012) mentioned that most sedimentary sampling areas for

microplastics are on sandy beaches, between 5-18 beach sites. Most of these studies

focused on the high tide line sampling (Hidalgo-Ruz & Thiel, 2012) and the approach of

sediment sampling were through: (i) linear extension along the strandline, (ii) an aerial

extension using quadrats (0.0079 to 5m2); and (iii) and at different depth strata (usually 0-

32 cm) using sediment coring tools. The quantity of sediment samples ranged from 0.15

kg to 10 kg and a volume from 0.1 L to 8 L (Hidalgo-Ruz & Thiel, 2012).

 6

Sampling-Method

Hidalgo-Ruz & Thiel (2012) reported four main steps in retrieving microplastics

from sea surface, sediments, and water columns:1) through density separation, 2)

filtration 3) sieving and 4) visual sorting. The concept of density separation is to mix the

sediment samples to a solution (saline NaCl with a density of 1. 2 gcm-3) or sodium

polytungstate (with a density of 1.4 gcm-3), shaking it from around 30 s up to 2 h, and

allowing the suspended particles to settle from about 2 min, up to 6 h. The lighter density

microplastics can then be separated from the sediment particles that are settled to the

bottom of the solution.

Filtration works by screening of the supernatant with the aid of a vacuum.

Commonly used pore size ranges from 1-6 µm. In order to distinguish sizes of retrieved

microplastics the supernatant can also be sieved through variable mesh sizes ranging from

0.038 mm to -4.75 mm. Nuelle et al. (2014) employed 0.45 µm nitrocellulose filter upon

vacuum filtration of the saturated salt solution containing microplastics.

Visual sorting is the careful examination of the visible plastic fragments.

Suspected microplastics can be further investigated with the aid of a stereo or compound

microscope. Dai et al. (2018) observed the collected particles from the 120 soil samples

collected from 53 coastal beach sites using magnifying glass and SMZ25

stereomicroscope. Majority of the separated microplastics were composed of < 1mm in

size (Dai et al., 2018).

Density Separation

Density separation utilizes the density gradient between materials of interest and

and other unwanted materials using the buoyant force of a liquid with an intermediate

density to separate the lighter materials from the denser after a series of shaking and

settling of the mixture materials in the liquid (Wenfeng Wang 2018). It a widely used
 7

method to isolate microplastics in water and sediments. The densities of different salt

solutions lie in the range of 1.202 gcm-3 to 1.7 gcm-3 while sand and other deposits have

densities of 2.56 gcm-3 (Wenfeng Wang 2018).

Nuelle et al. (2014) in his study developed a two-step extraction method of

microplastic on sediment sample by mixing the sediments in a lower density saturated

NaCl solution followed by succeeding floatation using a high density NaI salt solution.

Air induced flow method was used during the first step in which pumping of turbulent

gas in the container of the NaCl-sediment solution forced lighter particles to move faster

on the top layer than heavier ones. This method had then easily separated the light-

density microplastics from biogenic and other matter present on the sediments. The

solution containing the lighter particles was then subjected to subsequent shaking,

refilling, sedimentation and decantation with saturated NaI salt solution for further

removal of the lower density microplastics.

In this study NaCl solution (1.202 gcm-3) will be applied for the separation

microplastics due to its wide availability in the market and its affordability. High-density

salts are generally at higher cost and some are harmful in the environment.

Characterization of Microplastics

Several studies conducted utilize microscope to determine the color, shape and

size of the plastic fragments. Secondary Electron Microscope (SEM) Imaging is also a

helpful tool in reporting the morphological structure of the marine debris. SEM equipped

with Energy Dispersive X-ray (SEM-EDS) provides elemental concentration detected on

the microplastics (Kühn, van Oyen, Booth, Meijboom, & van Franeker, 2018) and thus

reveals wide range of shapes and structures. Manifestations on the degree of degradation

such as mechanical weathering can be captured through the SEM images. Thus,
 8

relevance of surface morphology of microplastics can reveal the relationship of their

occurrence and human activities in marine environment (Dai et al., 2018).

The chemical identification of plastic debris is better understood through Fourier

Transform Infrared Spectroscopy (FTIR) or Raman Spectroscopy. Many researchers

maximize the use of FTIR or Raman providing initial screening and confirmatory of the

different types of microplastics. With these techniques, the type of polymer can be

identified, revealing the distinct functional groups of each polymer through the FTIR

spectra obtained. FTIR- Attenuated total Reflectance Accessory is widely used for direct

analysis of the material (Dai et al., 2018) (Kühn et al., 2018).

Chemical additives incorporated during the manufacturing of plastic debris can be

detected using gas chromatography mass spectrometry (GC MS). Nuelle et al. (2014)

used Pyr-GC MS to investigate the thermal degradation products of microplastics.

One research had embarked in exploring the ability of Time of Flight Secondary

Ion Mass Spectrometry (TOF-SIMS) in understanding the degree of oxidation of a

reference PE material and PE plastic debris. ION-TOF (Gmbh) TOF-SIMS IV equipped

with Bi cluster ion source was used to generate positive and negative spectra of PE pellets

collected from the strandline of Maha’ulepu beach at the southeast corner of Hawaii

(Programme, Management, & Asia, n.d.) A comparison of the presence of O- and OH-

ions from generated spectra was determined and Principal Component Analysis (PCA)

further processed the data sets produced. Auger Electron Spectroscopy (AES) was used

for the elemental analysis of the plastic fragments. Both, TOFSIMS and AES can detect

the inorganic ion present in a material.

Another research also investigated the thermal properties of the plastic fragments

using Differential Scanning Calorimetry (DSC).

Differential Scanning Calorimetry (DSC)

 9

DSC is a useful technique for thermal analysis which can analyze various types of

samples such as polymers, organic and inorganic materials. Among the applications of

DSC are the determination of the phase transition temperature-melting and

crystallization, glass transition temperature and heat capacity measurements (Menczel

and BrucePrime.

Fourier Transform Infrared Spectroscopy-Attenuated Total Reflectance (FTIR-

ATR)

Manila Bay

Philippines, comprising of thousand islands, is lavished with aquatic resources as

it is surrounded by bodies of water. Diverse aquatic environment such as beaches, bays

and rivers have been historically used for source of food, route of transportation, trading

and other maritime activities in the country. Manila Bay located in the southwest area of

Luzon has been widely exploited for economic, industrial, and domestic activities (Sta.

Maria, Siringan, Bulos, & Sombrito, 2009). Surrounded by five provinces including

Pampanga and Metropolitan Manila as shown in Figure 1, its shoreline length measures

approximately 220 km from starting point at Mariveles Bataan to Maragondon, Cavite

and a mean depth of 17m (Programme et al., n.d.). This semi-enclosed coastal body has

a surface area of about 1800 km2. The bay has approximately 17000 km2 of watershed

having 26 catchment areas (Programme et al., n.d.). Its two major contributory areas of

freshwater input are Pampanga and Pasig rivers (Sta. Maria et al., 2009).
 10

Figure 1. Map of Manila Bay watershed boundary marked with dark line (Programme et

al., n.d.)

Studies Conducted in Manila Bay

Several studies have conducted in the assessment of marine pollution in Manila

Bay. Commonly studied is the heavy metal determination in surface sand, core

sediments, marine and freshwater species. T. Hosono et al., (2010) in his study

investigated heavy metal pollution of Manila Bay using sediment metal concentration and
210
Pb isotope ratios together with Pb dating of core sediments using inductively coupled

plasma mass spectrometry (ICP-MS; ELAN 6100DRC11). A study was also conducted

in determining the Pb and Cu levels on adult mud crabs from East Bataan coast using

atomic absorption spectrometry (Vince Cruz, Ramos, & Ablan-Lagman, 2015). Another

study focused on trace metal bioaccumulation in mangrove stands in Parañaque Critical

 11

Habitat and Ecotourism Area at the east of Manila Bay (Gabriel & Salmo, 2014). Only

recently that studies in the Philippines are beginning to consider marine plastic debris

determination, specifically microplastics as this becomes a global threat to marine

environment.

The first recorded study on microplastic ingestion of plastics of a recovered dead

body of an adult female green turtle (Chelonia mydas) in Barangay Lapu-Lapu, Agdao

Davao City was investigated by Abreo & Blatchley (2014). During its necropsy, it was

found out that plastic materials had been ingested by the sea turtle. Though the ingested

plastics did not cause internal damage, the study inferred that blockage of the digestive

tract of the recovered turtle could be the reason of its death (Abreo & Blatchley, 2014).

Another assessment of microplastics was done by cultured Asian green mussels

(Perna viridis) in Bacoor Bay Cavite (Argamino, Cristian Ryan and Janairo, 2011). The

mussel tissues were extracted using acid digestion, rinsed with deionized water and

vacuum filtered to remove the accumulated microplastics. Through microscopic analysis

(stereomicroscope SMZ745T), presence of clear and colored microplastics of about <1

mm were reported (Argamino, Cristian Ryan and Janairo, 2011).

Only few studies in the Philippines pursued on microplastics, and mostly focused

on ingestion and accumulation in fish, bivalve, marine invertebrates, etc. So far, one

findings of surface sand microplastic deposits in Macajalar Bay has been

accounted (Galarpe, 2018). No study has yet published in the occurrence of microplastics

sediments in Manila Bay. Thus, exploration on the characterization of microplastics in

surface sand and core sediments and even in water column is a promising research in

providing the extent of marine pollution in the Philippines, particularly in Manila Bay.

By performing surface and analytical techniques on the study of microplastics, the

chemical and mechanical processes that marine debris undergo can be further
 12

comprehended and explained. Thus, embarking on this field of study will be useful in

establishing future microplastic profiling in the Philippines.

Chapter 3

Methodology

Materials

Pellet reference materials with known purity of low density polyethylene (LDPE,

𝜌=0.92g/cm3), polypropylene (PP, 𝜌=0.90g/cm3) and polyethylene terepththalate (PET,

𝜌=0.90g/cm3) will be purchased from Sigma Aldrich and/or from local manufacturers.

Reference materials will be manually grinded with silicon carbide paper and will be
 13

separated by sieving at the following ranges: 256-645um (A) and 645-1000 um (B)

patterned from the study Chialanza et al. (2017).

Sampling

Three (3) stations each with a will be selected from the 7.6 km Roxas Boulevard Manila

Bay, Philippines for the sampling of sediments. An aerial extension zdalong the high

tide line (Hidalgo-Ruz & Thiel, 2012) of Manila Bay through quadrats from 0.0079 to

5m2 at single depth layer within the top 5 cm sediment (Wenfeng Wang, Jun Wang

2018). Sampling will be repeated five (5) times until approximately 8kg of sand

sediments will be collected from the sites. Table 1 shows the collection sites of the

microplastics.

Table 1. Stations for the collection of samples.

Sample Location Classification

Collected bulk samples will be placed in bottles covered with aluminum foil and will be

transported to the laboratory for further processing.

Extraction

In this study, density separation will be used to collect the microplastics from the

sand sediments. The samples from each station will be sieved first using 1mm and then

500 µm sieves. The particles that pass through the sieves will be transferred in a clean

erlenmeyer flask. Saturated NaCl solution (1.85 g/mL) will be added to the flask in a 1:1

ratio. The solution will be mixed together and shaken for about 30s-2h. The particles

will then be allowed to settle for around 2 min- 6hours allowing lighter particles to move
 14

upward while remaining sediments settle to the bottom of the flask. The supernatant will

be separated through decantation and will be filtered in a 1-1.6 µm filters aided by a

vacuum pump. All the collected microplastics will be washed and sonicated with

deionized water. These will be dried in an over and will be stored in a dark and

temperature-controlled environment.

Identification

Optical Microscope

Visualization of microplastics will be through the Axiom stereomicroscope with

magnification from 6.5x to xx. The color, shape and size will be noted per sample. The

extracted microplastics will be classified based on the categories identified by Hidalgo-

Ruz et al.

FTIR

The vibrational spectra of the pure polymers and the microplastics will be obtained using

FTIR Frontier Perkin Elmer 100 Spectrum equipped with Universal Atttenuated Total

Reflectance (U-ATR) Accessory. Analysis will be at 20 scans, 4cm-1 resolution in the

mid-range of 4000-600 cm-1.

DSC

Thermal analysis will be performed using DSC Perkin Elmer STA 4000. Nitrogen gas at

50mL/min will be purged throughout the analysis to prevent the occurrence of oxidation.

About 5-10 mg of the pure polymers will be consumed per run. Standard aluminum pan

with cover will be used. The thermal program will be as follows: i) isothermal at 300C for

5 minutes, ii) first heating from 300C-4550C, iii) isothermal at 4550C for 5 minutes, and

iv) cooling from 4550C -300C. Steps i-iv will be repeated for the second heating and
 15

cooling. Ramp rate will be at 100C /min and nitrogen gas will be applied during the

analysis. Integration of DSC peaks will be processed using Pyris software. The

equipment will be calibrated first with Indium following ASTM method prior to the

actual run of samples.

Characteristic temperatures such as glass transition (Tg), melting onset (T0) and

melting maximum temperature (Tm) will be determined from DSC.

DSC Calibration Curve

Calibration curves of LDPE, PP and PET reference materials will be measured for the

two particle sizes A and B. with masses of 5, 6, 7 and 10 mg. The mass range 5-8 mg

since this is the usual range of mass utilized for DSC analysis. Following equation 1,

finding the calibration constant will aid in the mass determination of microplastics. Table

2 shows the onset temperature determined by the DSC.

Table 2. Onset Temperature (Tx) for each pure polymer and the corresponding calibration

curve with their correlation factor

Polymer Particle size Tx(0C) Slope Intercept R2

LDPE 256-645 (A)

645-1000 (B)

PP 256-645 (A)

645-1000 (B)

PET 256-645 (A)

645-1000 (B)

Analysis of PE and PP Microplastics

The extracted microplastics from the three stations will be analyzed. From the calibration

curve, the PE and PP concentration will be measured.

 16

Figure 3 shows the sampling scheme of the samples.

Bulk Sediment Sample

Rinsed with NaCl solution

Density separation

Sieving through cascade of 1mm and 500 µm

Morphological Analysis (Axiom Microscope)

 17

Thermal Analysis (DSC) and

Spectroscopy (FTIR)

Figure 3. Scheme of sampling, extraction and analyses of microplastic.

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(Chelonia mydas ) in Davao Gulf , a global impact . Philippine Journal of Marine

Science, 145(1), 17–23.

Argamino, Cristian Ryan and Janairo, J. I. B. (2011). Qualitative Assessment and

Management of Microplastics in Asian Green Mussels (Perna viridis) Cultured in

Bacoor Bay, Cavite Philippines. EnvironmentAsia, 4(1), 56–61.

https://doi.org/10.14456/ea.2010.32

Barnes, D. K. A., Galgani, F., Thompson, R. C., & Barlaz, M. (2009). Accumulation and

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Philippines. Bulletin of Environmental Contamination and Toxicology, 93(6), 722–

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 18

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 19

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