UNIVERSITY OF NOVA GORICA

SCHOOL OF ENVIRONMENTAL SCIENCES

ANALYSIS OF POLYCHLORINATED BIPHENYLS

ADSORBED ON PLASTIC PELLETS: COMPARISON OF

EXTRACTION TECHNIQUES

MASTER'S THESIS

Petra Makorič

Mentor: dr. Marilyne Pflieger

Nova Gorica, 2017

II

IZJAVA

Izjavljam, da je magistrsko delo rezultat lastnega raziskovalnega dela. Rezultati, ki so nastali v okviru skupnega raziskovanja z drugimi raziskovalci, ali so jih prispevali drugi raziskovalci (strokovnjaki), so eksplicitno prikazani oziroma navedeni (citirani) v magistrskem delu.

Petra Makorič

III

ACKNOWLEDGMENT

I would like to thank my mentor dr. Marilyne Pflieger for her expert advice and encouragement throughout my research project and study.

I also would like to thank doc. dr. Dorota Korte for her help and advice for statistical evaluation of results.

I am grateful to my employer prof. dr. Mladen Franko for enabling me to combine my work with study obligations.

And also, special thanks to my friends, family and co-workers for their encouragement during the study.

IV

TABLE OF CONTENTS

TABLE OF CONTENTS ............................................................................................... IV

1. INTRODUCTION ................................................................................................... 1

Research goals ............................................................................................... 1

2. THEORETICAL BACKROUND .............................................................................. 2

Plastics and POPs .......................................................................................... 2

2.1.1. Marine plastic debris ................................................................................ 2

2.1.2. Characterization of plastics ...................................................................... 2

2.1.3. Impacts of plastics ................................................................................... 3

Plastic debris and organic pollutants ............................................................... 3

2.2.1. Persistent organic pollutants (POPs) ....................................................... 3

2.2.2. Plastic debris as a source of pollutants .................................................... 3

2.2.3. Plastic debris as a sink and vector of organic pollutants .......................... 4

2.2.4. Impact of organic pollutants adsorbed on plastic fragments ..................... 5

Marine plastic pellets ...................................................................................... 5

Extraction techniques used in extraction of PCBs from plastic matrix ............. 6

2.4.1. Soxhlet extraction .................................................................................... 6

2.4.2. Pressurized fluid extraction ...................................................................... 6

2.4.3. Maceration ............................................................................................... 6

2.4.4. Ultrasonic extraction ................................................................................ 7

3. EXPERIMENTAL WORK ....................................................................................... 7

Presentation of the study ................................................................................ 7

3.1.1. Design of the protocol for determination of PCBs .................................... 7

Equipment and chemicals ............................................................................... 9

Sample preparation ...................................................................................... 11

3.3.1. Stock solution of PCBs mixture .............................................................. 11

3.3.2. Spiking of pellets with standard PCBs mixture solution .......................... 11

3.3.3. Standard calibration solutions of PCB mixtures ..................................... 11

Optimization steps ........................................................................................ 12

3.4.1. Optimization of GC-ECD analysis .......................................................... 12

3.4.2. Optimization of pressurized fluid extractor ............................................. 12

3.4.3. Clean-up step and evaporation .............................................................. 13

............................................................................................................................ 14

Comparison of extraction techniques ............................................................ 15

3.5.1. Soxhlet extraction .................................................................................. 16

3.5.2. Pressurized fluid extraction .................................................................... 16

3.5.3. Ultrasonication ....................................................................................... 17

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3.5.4. Maceration ............................................................................................. 18

3.5.5. Statistical comparison of the results ....................................................... 19

4. RESULTS AND DISSCUSSION .......................................................................... 19

Optimization of experimental protocol for determination of PCBs adsorbed on plastic pellets ........................................................................................................... 19

4.1.1. Optimization of GC-ECD analysis for PCBs ........................................... 19

4.1.2. Optimization step for pressurized fluid extractor .................................... 22

4.1.3. Optimization of the cleaning step ........................................................... 22

4.1.4. Samples of real pellets .......................................................................... 23

Comparison of extraction techniques ............................................................ 25

4.2.1. Adsorption of PCBs on virgin plastic pellets ........................................... 25

4.2.2. Extraction results ................................................................................... 26

4.2.3. Statistical analysis of the extraction techniques ..................................... 31

4.2.4. Comparison of extraction techniques ..................................................... 35

5. CONCLUSIONS .................................................................................................. 37

6. REFERENCES .................................................................................................... 38

APPENDIX 1. Composition of PCB mix 20 and 37 ............................................... 41

APPENDIX 2. Calibration curves of PCBs mix 20 and 37 on GC-ECD ................ 42

APPENDIX 3. Statistical t-test table ..................................................................... 46

VI

LIST OF TABLES

Table 1: Chemicals used in the study ......................................................................... 10 Table 2: Volume, time and temperature for each extraction technique ........................ 15 Table 3: Mass of pellets and final extract volumes for Soxhlet extraction test ............. 16 Table 4: Mass of pellet and final extract volume for PFE test ...................................... 17 Table 5: Mass of pellets and final extract volume for ultrasonic extraction test ............ 18 Table 6: Mass of pellets and final extract volume for maceration test .......................... 18 Table 7: Analytical conditions for PCBs analysis ......................................................... 19 Table 8: Calibration parameters of PCB mix 20 and 37 solution on GC-ECD .............. 21 Table 9: Recovery results (%) from all three clean-up tests ........................................ 23 Table 10: Concentration of PCBs in solution before spiking pellets, concentration of PCBs in recondensed solvent and percentages of PCBs adsorbed on pellets ............ 25 Table 11: Concentration of extracted PCBs form 6 replicates of spiked pellets ........... 26 Table 12: Concentration of extracted PCBs from HDPE pellets with Soxhlet extraction ................................................................................................................................... 27 Table 13: Concentration of extracted PCBs from HDPE pellets with PFE ................... 28 Table 14: Concentration of extracted PCBs from HDPE pellets with maceration technique ................................................................................................................................... 29 Table 15: Concentration of extracted PCBs from HDPE pellets with ultrasonication ... 30 Table 16: Concentration (ng/g) of extracted PCBs after first and second cycle ........... 31 Table 17: Average amounts of extracted PCBs for each extraction technique ............ 32 Table 18: Relative standard deviations for each extraction technique ......................... 34 Table 19: t-test for two independent techniques; 99% confidence interval on the difference between the means (Soxhlet and PFE extractions) .................................... 34 Table 20: t-test for two independent techniques; 99% confidence interval on the difference between the means (maceration and ultrasonication) ................................. 35 Table 21: Composition of PCB mixture 20 .................................................................. 41 Table 22: Composition of PCB mixture 37 .................................................................. 41

VII

LIST OF FIGURES

Figure 1: Molecule of PCB ............................................................................................ 8 Figure 2: Rotational evaporation of solvent during the process of pellet spiking .......... 11 Figure 3: Commercial HDPE pellets ............................................................................ 11 Figure 4: Example of pressure/time diagram of a two-cycle extraction process (BÜCHI Labortechnik AG 2009) ............................................................................................... 13 Figure 5: Solid phase extractor (SPE) ......................................................................... 14 Figure 6: Rotating concentrator miVac ........................................................................ 14 Figure 7: Clean-up device for cleaning 40-mL extracts ............................................... 14 Figure 8: Extraction thimble for Soxhlet ...................................................................... 16 Figure 9: Soxhlet extractor .......................................................................................... 16 Figure 10: Transferring pellets in extraction cell .......................................................... 17 Figure 11: Placing the extraction cell in the PFE ......................................................... 17 Figure 12: Maceration in 20mL amber vials ................................................................ 18 Figure 13: Chromatogram of PCB mix 20 obtained with the final analytical conditions 20 Figure 14: Melted pellets after extraction at 100 °C ..................................................... 22 Figure 15: Pressurized fluid extractor (Büchi) ............................................................. 22 Figure 16: Unsorted pellets sampled on the beach ..................................................... 24 Figure 17: Sorted pellets ............................................................................................. 24 Figure 18: Chromatogram shown the extracted PCBs from Greek pellets ................... 24 Figure 19: Sum of initial adsorbed PCBs and extracted PCBs from spiked pellets for each extraction technique .................................................................................................... 33

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ABBREVIATIONS AND SYMBOLS

ABS acrylonitrile butadiene styrene

DDD Dichlorodiphenyldichloroethane

DDE Dichlorodiphenyldichloroethylene

DDT Dichlorodiphenyltrichloroethane

DEHP diethylhexyl phthalate

DMP dimethyl phthalate

HDPE high density polyethylene

HOC hydrophobic organic compounds

IR Infra-red

OCP organochlorine pesticides

PVA polyvinyl alcohol

PBDES polybrominated diphenyl ethers

PCB polychlorinated biphenyls

PCDD/FS polychlorinated dibenzo-p-dioxins and –furans PE PEF polyethylene

PET polyethylene terephthalate

POP persistent organic pollutant

PP polypropylene

PS polystyrene

PVC polyvinylchloride

SPE solid phase extraction

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NASLOV

Analiza polikloriranih bifenilov adsorbiranih na plastičnih

granulatih: primerjava ekstrakcijskih tehnik

IZVLEČEK

Plastični granulati so ena izmed glavnih sestavin plastičnih delcev v morskem okolju. To

so majhne granule v obliki valja ali diska, s premerom nekaj milimetrov, ki se v industriji uporabljajo kot surovina za proizvodnjo plastičnih predmetov. Plastični granulati lahko

med proizvodnjo in transportom nehote zaidejo v okolje, reke in morja in posledično

zaradi morskih tokov dosežejo oceane. Zaradi njihove obstojnosti v okolju jih najdemo v

oceanih in na obalah. Na plastične granulate se zlahka adsorbirajo toksične snovi

prisotne v okolju, na primer poliklorirani bifenili (PCB).

V okviru magistrskega dela sem izdelala protokol za ekstrakcijo in določanje PCB adsorbiranih na plastičnih granulatih vzorčenih na obmorskih plažah. Na področju mikroplastike visokotlačna tekočinska ekstrakcija (PFE) ni pogosto uporabljena metoda, a je znana kot zelo zanesljiva in učinkovita metoda na drugih raziskovalnih področjih. PFE metodo smo preizkusili za ekstrakcijo PCB-jev iz plastičnih granulatov, kjer so bili ekstrakcijski pogoji 100 bar in 65 °C, topilo uporabljeno pri ekstrakciji pa je bil heksan. Ekstrakt smo skoncentrirali na 1 mL, očistili na SPE Florisil kartuši, ter pred analizo na GC-ECD ponovno skoncentrirali na 1 mL.

V drugem delu magistrskega dela smo primerjali štiri različne ekstrakcijske tehnike za ekstrakcijo PCB-jev iz plastičnih granulatov: Soxhlet ekstrakcijo, visokotlačno tekočinsko ekstrakcijo, ultrazvočno ekstrakcijo in maceracijo. Najboljši izkoristek ekstrakcije in najboljšo ponovljivost smo dosegli z visokotlačno tekočinsko ekstrakcijo. V primeru Soxhlet ekstrakcije rezultati niso bili ponovljivi. V primeru maceracije in ultrazvočne ekstrakcije pa je bil izkoristek ekstrakcije od 40 do 45 % nižji kot v primeru visokotlačne tekočinske ekstrakcije.

KLJUČNE BESEDE

PCB, plastični granulati, ekstracija, visokotlačni tekočinski ekstraktor, Soxhlet, ultrazvočna ekstrakcija, maceracija, mikroplastika

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TITLE

Analysis of polychlorinated biphenyls adsorbed on plastic

pellets: comparison of extraction techniques

ABSTRACT

Plastic resin pellets are one of the main components of plastic fragments in the marine environment. They are small granules, generally cylinder or disk-shaped, with a diameter of a few millimetres used as industrial raw material for the production of plastic manufactured items. Resin pellets can be unintentionally released to the environment during manufacturing and transport, and can reach the ocean through processes such as surface run-off. Because of their environmental persistence, they are widely distributed in the oceans and on the beaches, all over the globe. Plastic pellets are also a sink of toxic compounds such as polychlorinated biphenyls (PCBs) that are present in the environment.

During this thesis, we designed a protocol for extracting and analysing PCBs from marine plastic resin pellets. Although pressurized fluid extraction (PFE) is not commonly used in the field of microplastics, it is a reliable and efficient technique applied in other research areas. Thus, this method was tested in this work. PCBs were extracted from plastic pellets with PFE at 65 ºC and under 100 bar with hexane. Then, the extracts were concentrated to 1 mL, cleaned up on Florisil cartridge through solid-phase extraction. Prior to analysis on GC-ECD, the cleaned extracts were concentrated again.

In the second part of the thesis, the following four extraction techniques of PCBs from plastic resin pellets were compared: Soxhlet extraction, PFE, ultrasonication and maceration. The obtain results have shown that the best efficiency and repeatability of extraction is achieved with PFE. In case of Soxhlet extraction, the results were unrepeatable. Maceration and ultrasonication had 40-45 % lower extraction efficiencies than PFE.

KEY WORDS

PCB, plastic resin pellets, pressurized fluid extraction, Soxhlet, maceration, ultrasonication, microplastics

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1. INTRODUCTION

Marine litter is defined as “any persistent, manufactured or processed solid material discarded, disposed of or abandoned in the marine and costal environment” (UNEP, 2009). These items have been made or used by people and deliberately discarded or unintentionally lost into the sea or on beaches (Galgani et al., 2013). Any material transported into the marine environment from land by rivers, draining or sewage systems, or wind is included in this definition. Marine litter poses a major threat to marine ecosystems in most of the seas worldwide due to its environmental, economic, safety, health and cultural impacts. Marine debris can be organic or inorganic, but a large fraction of debris consists of plastics. Plastics are synthetic organic polymers derived from the polymerisation of monomers extracted from petroleum. Plastic debris is usually persistent and hydrophobic in nature. It can be transported over significant distances (Engler, 2012). Plastic resin pellets are one of the main components of plastic fragments found in the marine environment. Plastic resin pellets are small granules with a diameter of a few millimetres (Mato et al., 2001). During manufacturing and transport, resin pellets can be released to the environment and reach the ocean through streams and rivers and/or surface run-off. Because of their environmental persistence, they are widely distributed in the oceans and on the beaches all over the world (Ogata et al., 2009). Plastic resin pellets can adsorb hydrophobic organic compounds (HOC), including persistent organic pollutants (POPs) (Heskett et al., 2012), such as polychlorinated biphenyls (PCBs) and organochlorine pesticides (e.g. DDT). POPs are present in aquatic systems worldwide (Ogata et al., 2009). POPs are chemical substances that: a) persist in the environment, b) are able to bio-accumulate through the food web, c) and are likely to cause adverse effects to human health and environment (Stockholm convention on persistent organic pollutants as amended in 2009).

Research goals

The overall aim of this study was twofold. The first part was the design of a protocol for the determination of PCBs adsorbed on plastic resin pellets. The optimization of the protocol included: i.) optimization of GC-ECD for analysis of PCBs; ii.) optimization of extraction of PCBs from plastic pellets with PFE; iii.) optimization of clean-up procedure on SPE equipment. The second part of the study focused on the comparison and evaluation of four different extraction techniques: Soxhlet extraction, PFE, ultrasonication and maceration.

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2. THEORETICAL BACKROUND

Plastics and POPs

2.1.1. Marine plastic debris

Marine debris has become a general pollution problem, causing direct injuries in wildlife (Frias et al., 2010). Marine debris is a solid man-produced object or material, that enters the water system (Engler, 2012) by its improper disposal, accidental loss and by natural disasters (Watters et al., 2010). Marine debris can be of organic or inorganic composition (Engler, 2012), but a large fraction (60 to 80 %) consists of plastic material (Derraik, 2002). It is estimated that about 80% of plastic marine litter come from land- based sources (Bakir et al., 2012).

2.1.2. Characterization of plastics

Plastics are ubiquitous in modern life, present, for instance, on a daily basis in the packaging of food and drinks or in household items and shopping bags (Fendall and Sewell, 2009). As a result of relatively low production costs and constant increase in the demand for plastics, the waste items are accumulating in the environment (Bakir et al., 2012).

Plastics are made of polymeric materials including rubbers, technical elastomers, textiles, technical fibres, thermo sets and thermoplastics. There are some 200 plastics families in production including polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinylchloride (PVC), polyethylene terephthalate (PET), nylon, polyvinyl alcohol (PVA) and acrylonitrile butadiene styrene (ABS) (UNESCO 2010).

When plastics are exposed to sunlight, the UV radiation causes chemical damage that results in the eventual physical fragmentation of these polymeric materials into smaller pieces. Even if they break into smaller pieces and their surface is eroded, they are still present as plastic material in the environment (Rios L. 2007). Plastic fragments are classified according to their size as follows:

a) nanoplastics (size < 1µm of diameter) (Elaslie et al., 2011),

b) microplastics (size < 5mm of diameter),

c) macroplastics (size >5 mm of diameter) (Fendall and Sewell, 2009).

Macroplastics do not fully degrade, they break down into microplastics, and subsequently into nanoplastics.

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2.1.3. Impacts of plastics

Plastic marine debris has a number of negative effects on marine wildlife. There can be physical and biological impacts as well as chemical pollution. The importance of plastic debris in the marine environment was first noticed by visual impact of plastics dispersed throughout the oceans and physical direct impacts on marine biota. The two main physical impacts on marine biota are entanglement and ingestion (Ryan and Moore et al., 2009). In addition, there are ecotoxicological effects from ingestion of plastics due to adsorption of persistent, bio-accumulative and toxic pollutants (such as DDT) on their surface. Plastic fragments can also be a vector of microorganisms, including pathogenic species. Finally, they might have various economic and social repercussions (Martins and Sorbal, 2011).

Plastic pellets are floating on the sea surface, so they can easily adsorb contaminants (Ogata et al, 2009). Organic micro-pollutants have been detected in plastic resin pellets stranded on beaches. The hydrophobic organic pollutants can adsorb to pellets from the surrounding seawater with a concentration factor up to 106, because of the hydrophobic nature of most plastic surfaces.

Plastic debris and organic pollutants

2.2.1. Persistent organic pollutants (POPs)

According to the European commission, POPs are chemical substances that persist in the environment, are able to bio-accumulate, and cause a risk of adverse effects to human health and the environment. These pollutants can be found across international boundaries far from their sources, even where they have never been used or produced (Stockholm convention, 2009).

POPs are hydrophobic and lipophilic chemicals. In aquatic systems and soils, they adsorb particularly on organic matter. Among them, there are many families of chlorinated (and brominated) aromatics, including polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins and –furans (PCDD/Fs), polybrominated diphenyl ethers (PBDEs) and different organochlorine pesticides (OCP). Many POPs have been synthesised for industrial (PCB) or agro-chemical (OCP) uses. Some are accidental by-products of combustion or industrial synthesis of other chemicals (Jones and Voogt, 1999).

2.2.2. Plastic debris as a source of pollutants

In the manufacturing of plastics, organic compounds are used as additives in polymers to improve the properties of the resulting products. Release of additives is an unwanted process for the manufacturer, because loss of additives can shorten the polymer lifetime and have negative effects on the environment and living organisms.

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Plasticizers are the largest group of additives in polymers. Their range of molecular weights is approximately 200 to almost 700 g mol-1. The most used plasticizers are phthalates; they can substitute more than 60 % of polymers in PVC. For instance, dimethyl phthalate (DMP) has relatively high water solubility, and can be released from its resin as soon as the DMP containing product is present in the landfill. On the contrary, the higher molecular weight phthalates, such as diethylhexyl phthalate (DEHP), are more resistant to migration. DEHP are released from polymer surface more slowly than DMP because of their hydrophobicity (Teuten et al., 2009).

Plasticizers may have biological effects already at low concentrations in the range of ng L-1 or µg L-1. Because of the low diffusivities of chemicals like bisphenol A (BPA) in plastics, biological effects might be expected (Koelmans et al., 2014). However, it must be kept in mind that plastics ingested by organisms are not the only source of organic pollutants (including additives). In fact, these compounds are also found in other environmental compartments (e.g. air, sea, surface water…) where they can be in direct contact with various organisms.

Plastic resin pellets and fragments are known sources and sinks of xenoestrogens and POPs in marine and aquatic environments. They can be readily ingested by invertebrates at the base of the food web (Frias et al., 2010).

2.2.3. Plastic debris as a sink and vector of organic pollutants

Besides being a source of additives, plastic debris can be a sink of toxic chemicals which adsorb from the environment to the plastic surface. However, these chemicals can also desorb from the plastic debris when the equilibrium changes. POPs (e.g. PCBs and pesticides) have properties that make them priority pollutants. They represent potential risks to human health and ecosystems. These pollutants resist degradation in the environment, so that they can persist for years or even decades. They have very low water solubility, which is why they tend to partition to sediment or concentrate at the sea surface microlayer where floating plastic fragments are present. However, when they come in contact with plastic debris, they tend to adsorb to it.

The amount of substance adsorbed on plastic debris depends on the partition coefficient of the compound (Kd), which is defined as the ratio of the mass of the substance adsorbed on the plastic (µg per kg of debris) to concentration of substance in seawater (µg of substance per L of seawater). This amount also depends on the concentration of the substance in surrounding water.

The sorption of POPs depends on the interactions between a particular pair of POP/plastic. For example, polyethylene sorbs PCB more readily than propylene does. In most cases and especially in floating plastics, the hydrophobicity of POPs and plastics will generally favour sorption. Sorption is also affected by weathering of plastic and by biofouling. Nevertheless, POPs will adsorb to the plastic debris over time. The longer a plastic particle is in the water, the higher concentrations of POPs will adsorb onto it.

For instance, concentrations of PCBs adsorbed on plastics vary with the local concentration of PCBs in the water and the plastic residence time in the water. It may take months for the PCBs to equilibrate between the water and the plastic (Engler, 2012). Floating and stranded plastics have been found with adsorbed PBC concentrations ranging from 10 - 500 ng per g of pellet (Yeo et al., 2015) .

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2.2.4. Impact of organic pollutants adsorbed on plastic fragments

POPs are chemically stable and quite persistent in the environment. They have a strong bioaccumulative nature, are highly toxic and are known to affect endocrine functions. These toxic contaminants can cause physiological dysfunction, which has been suggested in marine mammals. Increased levels of DDT showed reduction of testosterone in blood of marine mammals exposed to organochlorines. The effect of organochlorines has also been observed on steroids and thyroid hormones (Tanabe, 2002).

Organic pollutants adsorbed to plastics can be released to the digestive fluid and can be transferred to the tissues of organisms. A more complex situation is observed for marine organisms due to biomagnification. Hydrophobic and poorly metabolized contaminants are amplified through the food web. Higher trophic level organisms (e.g. seabirds) are exposed to high concentrations of hydrophobic contaminants via their prey (e.g. fish). Plastics as anthropogenic prey can compete with natural prey (Teuten et al., 2009).

Because of their hydrophobic nature and persistence in the environment, PCBs and OCPs can be bioconcentrated and biomagnified through the food chain. There is a possibility of transferring these contaminants from plastics to the tissues of marine organisms that ingested them. This may be a significant pathway for the chemicals present on/in plastics to enter marine organisms. Seabirds are a group of marine animals that often confuse plastics with prey, and ingest it. The same POPs adsorbed on plastics have been found in tissues of seabirds (Colabuono et al., 2010).

Marine plastic pellets

Besides plastic fragments, plastic pellets are also commonly found. They are industrial raw material. Pellets usually enter the marine and coastal environments during their manufacturing and/or transport. In many places, pellets represent the most abundant form of plastic coast litter. Their size is higher than 5 mm (in diameter) and their colour is commonly white, off-white or translucent (Turner, 2011).

The chemical composition of pellets can be determined by infra-red (IR) techniques. It has been shown that they are mainly made of polyethylene and polypropylene (Fotopoulou and Karapanagioti, 2012).

The first significant increase in concentrations of plastic particles, including resin pellets in the sea surface, was observed in the North Pacific from the 1970s to the late 1980s. Mato et al. (2001) were the first researchers, to our knowledge, who detected organic pollutants (including PCBs, DDE, and nonylphenol) in plastic resin pellets on Japanese beaches. They launched the International pellet watch, where pellets from 30 beaches located in 17 different countries were analysed for OCPs and PCBs (Ogata et al., 2009). For example, pellets were sampled on remote islands in the Pacific, Atlantic and Indian Oceans and the Caribbean Sea (Heskett et al., 2012). Only one single study, which was performed in Greece, focuses on the Adriatic region (Karapanagioti et al., 2011).

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Extraction techniques used in extraction of PCBs from plastic matrix

2.4.1. Soxhlet extraction

Soxhlet extraction is one of the main extraction techniques in many scientific fields (environmental science, chemistry, pharmaceutic). Soxhlet is a very useful preparative tool, where the analyte is concentrated or separated from a solid matrix. Soxhlet extraction is a solid-liquid extraction and is one of the oldest methods. Still nowadays Soxhlet extraction is estimated as one of the most relevant techniques in the environmental extraction field (Extraction, 1997).

The Soxhlet technique is also commonly employed for in the extraction of POPs from plastic matrices. In the study of Mato et al. (2001), the contaminants were extracted from PP resin pellets with hexane for 17 h. Soxhlet extraction was also used for extracting POPs from plastic debris (Rios et al., 2010). Rios et al. (2010) applied the Soxhlet technique to extract PCBs, PAHs and DDTs from plastic debris with 150 mL of dichloromethane for 24 h.

Soxhlet extraction was also applied to extract POPs from plastic fragments found in seabirds tissues (Tanaka et al., 2013).

2.4.2. Pressurized fluid extraction

PFE is a newer technique for solid-liquid and semisolid-liquid extractions and is related to Soxhlet extraction (Agency 2007). PFE has the possibility to extract the target sample under high temperature and pressure, which remains constant during the extraction. Compared to Soxhlet, PFE requires less solvent, the technique is automated, and the extraction is more rapid – usually taking less than one hour (Suchan et al., 2004).

To our knowledge, only one study applied the PFE technique to extract POPs from plastic pellets ( Frias et al., 2010). The authors used a mixture of hexane and acetone, and the extraction was done at 100 ºC and 1500 psi (103 bar) for 5 min.

PFE was also applied to extract OCPs and PCBs from fish tissue (Gassel et al., 2013), and the extraction of PCBs from lugworm tissue (L et al., 2013).

2.4.3. Maceration

Ogata et al. (2009) used maceration as a technique to extract PCBs, DDTs and HCHs from plastic resin pellets. In their study, the maceration process is described as follows: 2 to 10 pellets were weighed and put in a 30 mL vial then 15 mL of hexane was added, and the vial was kept in the dark at room temperature for 72h. After 72h the hexane was transferred into a pear-shape flask, and fresh hexane was added to the pellets, and the extraction was repeated.

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The same extraction technique was used by Endo et al. (2005) to extract PCBs from plastic resin pellets.

2.4.4. Ultrasonic extraction

Ultrasonic extraction is a technique for extracting non-volatile and semi volatile organic compounds from solids such as soil, sludge, and waste. It ensures intimate contact of the sample matrix with the extraction solvent (USEPA, 2007).

Ultrasonic extraction was also used for extracting trace metals from plastic resin pellets (Holmes et al., 2012).

Zhang et al. (2015) extracted POPs from plastic resin pellets with ultrasonic extraction. Pellets were put in 10 mL of hexane, spiked with 10 µL of surrogate standard and put in an ultrasonic bath for 30 min. After 30 min the procedure was repeated with fresh solvent and the extracts were combined.

3. EXPERIMENTAL WORK

Presentation of the study

The present study includes two main steps:

- Optimization of the experimental protocol for determination of adsorbed PCBs on plastic pellets

- Comparison of extraction techniques for extraction of PCBs from plastic pellets

3.1.1. Design of the protocol for determination of PCBs

The protocol for the determination of PCBs adsorbed on plastic pellets includes several steps as follows:

- extraction of PCBs from the pellets by means of PFE; - concentration of extract from 40 mL to 1 mL; - clean-up of the extract with solid phase extractor (SPE) in order to remove most

of the unwanted compounds; - concentration of the cleaned extract to get higher concentrations in the solution

that will be analysed; - quantitative analysis on gas chromatography equipped with an electron capture

detector (GC-ECD).

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3.1.1.1. Selected POPs

In previous studies on POPs adsorbed on plastics resin pellets, several pollutants were detected. Ogata et al. (2009) observed high concentrations of PCBs and DDT.

In their study, Ogata et al. (2009) observed the following PCB congeners: no. 66, 101, 110, 105, 118, 128, 149, 153, 183, 170, 180,187, 180 and 206. The most abundant congeners detected were n°52, 101, 118 and 170, the second most abundant were congeners n°105, 138 and 153. Mizukawa et al. (2013) showed in their study that the most abundant PCBs congener were n°66, 101, 105, 110, 118, 128, 138, 149, 153, 170, 180, 187 and 206.

Based on these investigations, the following PCBs congeners were selected: 28, 31, 52, 77, 95, 99, 101, 105, 110, 118, 126, 128, 138, 146, 149, 151, 153, 156, 169, 170, 177, 180, 183, 187. The molecule of PCB is shown in Figure 1.

Figure 1: Molecule of PCB

3.1.1.2. Extraction of PCB from plastic resin pellets

Several solid-liquid extraction techniques can be found in the literature, among which Soxhlet is the most widely used. For instance, Hirai et al. (2011) extracted organic compounds in Soxhlet with dichloromethane at a rate of 3-4 cycles per hour for 18 hours. Ogata et al. (2009) used the maceration method. They mixed the pellets with 15 mL of hexane and kept the solution in the dark at room temperature for 72 h. Then they transferred the hexane solution to a pear-shape flask, added fresh solvent and repeated the extraction. Finally, only Frias et al. performed the extraction with PFE. They used a mixture of hexane:acetone (1:1) at 100 °C, 1500 psi (i.e. 103,4 bar), followed by static extraction for 5 min.

To our knowledge, there is no study focusing on the comparison of the efficiency of these different techniques regarding the extraction PCBs from plastic pellets. Soxhlet is a commonly used method and is known to be comparable to PFE. However, it is time and solvent consuming. Thus, PFE was selected in this investigation. PFE consists in extracting organic compounds from a solid matrix under controlled temperature and high pressure in a small volume of organic solvent.

3.1.1.3. Clean-up of extract on solid phase extractor

Prior to analysing the extract, it is often necessary to clean it in order to remove most of the unwanted compounds. Both Ogata et al. (2009) and Hirai et al. (2011) used an

9

activated silica gel column to clean their extract. Hirai et al. (2011) cleaned five different fractions, for which they changed the ratio between dichloromethane and hexane. Ogata et al. (2009) prepared three fractions with hexane by changing the volume of solvent. Frias et al. (2010) cleaned their extract on Florisil glass column. They eluted the compounds with n-hexane and cleaned up with sulphuric acid. The first fraction contained PCBs and the second fraction contained DDEs, DDDs and DDTs.

In order to be able to clean several samples in parallel, the SPE technique was chosen. Florisil sorbent is commonly used for cleaning up solutions containing PCBs and OCPs. Thus, this type of sorbent was selected.

3.1.1.4. Concentration of extract

The concentrations of OCPs and PCBs on pellets are expected to be low. Heskett et al. (2012) reported that the concentrations of OCPs range from 0.6 to 19 ng per g of pellet, and PCBs range from 0.1 to 10 ng per g of pellet. Ogata et al. (2013) reported concentrations of OCPs and PCBs on pellet ranging from 40 to 400 ng per g and 300 to 600 ng per g, respectively. Thus, a concentration step prior to the analysis is required. For instance, both Ogata et al. (2009) and Hirai et al. (2011) concentrated their extract by rotary evaporator and re-dissolution into the appropriate solvent (e.g. hexane).

3.1.1.5. Analysis of PCBs with GC

In previous studies, PCBs adsorbed on plastic pellets were mostly analysed by gas chromatography equipped with mass spectrometer (GC-MS) and electron capture detector (GC-ECD). Ogata et al., (2009) analysed OCPs on GC-ECD and PCBs on GC-MS. The limits of quantification for the sum of 13 PCB congeners were below 1.0 ng g-1. Colabuono et al. (2010) analysed PCBs on GC-MS, the pollutants were extracted from plastic pellets ingested by animals. Endo et al. (2005) analysed PCBs extracted from PE and PP pellets on GC-ECD, and the concentrations ranged from 28 ng/g to 2300 ng per g of pellet.

Equipment and chemicals

Equipment:

• Gas chromatograph Hawlett Packard HP 6890 Series gas chromatograph with GERSTEL MultiPurpose Sampler MPS 2XL with ECD and FID detector, Agilent technologies, Santa Clara USA;

• Pressurized fluid extractor, Speed Extractor E-916, Buchi, Flawil, Switzerland;

• Solid phase extractor, VACMASTER 20, BIOTAGE, Uppsala, Danska;

• Rotating Evaporating, Laborota 4000, Heidolph;

• Concentrator miVac DUO, Genevac SP Scientific, Suffolk UK;

• Ultrasonic bath, Sonis 4, Iskra Pio d.o.o., Šentjernej, Slovenia,

• GC capillary column Zebron ZB-XLB (30 x 0.25 x 0.25), Phenomenex, USA,

10

• Soxhlet extractor

• Cellulose Soxhlet Extraction thimbles, Whatman, Maidstone, UK,

• Glass fibre filter for extraction cell E-916, Buchi, Germany,

• 20 mL amber vials

• 10 mL, 50 mL and 100 mL volumetric flasks, Brand, Wertheim, Germany,

• 10 µL syringe, Hamilton, Nevada, USA,

• 0.1mL 1 mL, 2 mL, and 5 mL graduated pipettes, Brand, Wertheim, Germany,

• 10 mL, 15 mL and 50 mL volumetric pipettes, Brand, Wertheim, Germany,

• 50 mL tubes

• 10 mL tubes

•

Chemicals used in the study, are shown in Table 1.

Table 1: Chemicals used in the study

Name Formula Molecular weight (g

mol-1)

Purity (%)

Supplier

Hexane C6H14 86,18 HPLC grade

MS Supra Solv, Merck Millipore, Darmstand,

Germany

Acetone C3H6O 58,08 HPLC grade

Chroma Solv Plus, Sigma-Aldrich, St. Louis, USA

PCB mix 20* C12H10−Cl(3 – 7) 257 - 359 97-99,5 Dr. Ehrenstorfer,

Augsburg, Germany

PCB mix 37* C12H10−Cl(3 – 7) 257 - 359 97-99,5 Dr. Ehrenstorfer,

Augsburg, Germany

Product Supplier

SPE Florisil cartridge 1000 mg/6 mL

Strata FL-PR Florisil, Phenomenex, California, USA

Fat free quartz sand 0.3-0.9 mm Buchi, Flawil, Switzerland

Commercial polyethylene pellets PE-HD

Lupolen 5121 B natur, Tera

*The composition of PCB mixture solutions is shown in Appendix 1.

11

Sample preparation

3.3.1. Stock solution of PCBs mixture

The 100 µg L-1 stock solution of PCB mix 37 and PCB mix 20 was prepared by transferring 1 mL of 10 mg L-1 standard solution in 100 mL volumetric flask and diluted in hexane.

The stock solutions were used for preparing PCBs standard solution for spiking plastic pellets and for external calibration of GC-ECD.

3.3.2. Spiking of pellets with standard PCBs mixture solution

25 mL of 50 µg L-1 standard solution of PCBs mix 37 was prepared by diluting 12.5 mL of 100 µg L-1 stock solution in hexane. The solution was transferred to a rotating evaporator flask, and 21.4312 g of high density polyethylene pellets were added. The pellets were left in contact with the standard solution for 24 h at room temperature in the dark. Then the solvent was evaporated on rotating evaporator to complete dryness. In Figure 2 is shown the evaporating process of the solvent during the spiking of commercial pellets, which are shown in Figure 3.

3.3.3. Standard calibration solutions of PCB mixtures

For the optimization of the GC-ECD method, we prepared 20 ppb standard solutions of PCBs mixtures 20 and 37. 2 µL of 100 µg L-1 stock solution were transferred in a 10-mL volumetric flask and diluted in hexane.

From the stock solution, we prepared standard calibration solutions in order to obtain concentrations ranging from 0.5 ppb to 12 ppb in 10-mL volumetric flasks.

Figure 3: Commercial HDPE pellets Figure 2: Rotational evaporation of solvent during the process of pellet spiking

12

Optimization steps

3.4.1. Optimization of GC-ECD analysis

Before analysing PCBs, several parameters had to be adjusted in order to optimize the quantification of the target compounds. The main elements under consideration were the following:

- Injector parameters (e.g. injection mode, purge time, temperature, pressure, total flow)

- Oven temperature program (e.g. temperature, flow) - Detector parameters (e.g. temperature)

The injector temperature is usually set so that all the target compounds are vaporized. The injection mode has a significant impact on the analysis sensitivity. Under a split injection, a liquid sample is introduced into a hot inlet where it quickly vaporizes. Only a very small amount of the vapour reaches the column while the major fraction injected flushes through the purge vent. However, because we expected low concentrations of PCBs, the splitless mode was more appropriate to our study. In this case, the split valve is closed during the injection and for a certain period of time after the injection (e.g. 10 s - 3 min) to allow the sample to be vaporized and transferred to the column. Then, the split valve is opened to clean the liner. During the splitless mode, the initial temperature of the column is kept about 20°C below the boiling point of the solvent so that the solvent is re-condensed and the compounds stay at the head of the column. Usually, the initial temperature of the oven is held constant for the duration of the splitless mode. Once the purge valve is opened, it is possible to raise the temperature of the oven.

In our study, we analysed the selected PCBs on GC-ECD, which is a sensitive technique for chlorinated compounds. The method was developed on a Zebron ZB-XLB column (30 m x 0.25 mm x 0.25 µm). Standard stock solutions were prepared in hexane at a concentration of 100 ppm. The optimization of the GC method was done with standard solutions of 20 ppb in hexane for PCBs.

During the optimization process, the PCBs mixtures were injected to check whether the separation of the peaks and the sensitivity of the method improved. For PCBs, the elution order was provided by the supplier of the commercial mixture.

3.4.2. Optimization of pressurized fluid extractor

The extraction of PCBs from spiked polyethylene pellets and pellets sampled in Greece during a European project (DeFishGear) was performed on PFE. The extraction solvent was hexane.

As previously mentioned, only one study (Frias et al. 2010), to our knowledge, carried out the extraction of plastic pellets by means of PFE. Thus, the extraction parameters were first set according to the investigation of Frias et al. (2010) in combination with the application book provided by the supplier of the instrument (BÜCHI Labortechnik AG, 2009). In Figure 4 the pressure and time diagram of a two-cycle extraction process is

13

shown. As can be seen, first the instrument is heating up to reach the selected temperature. Then, the pressure raises and both parameters are maintained during a certain time. The extract is discharged and a second cycle can start. For the extraction of low concentrations, it is preferable to perform one long cycle than several short ones (i.e. < 2 min).

3.4.3. Clean-up step and evaporation

For the clean-up step, Florisil (magnesium silicate sorbent) cartridges of 6 mL with 1 g of sorbent were used. In the application note of Young (2013), the protocol for cleaning and collecting the PCB fractions is as follows:

- Activation of sorbent with 4 mL of hexane - Loading of 1 mL of sample - Elution with 4 mL of hexane

Different modifications of the clean-up protocol were performed to reach the best recovery of PCBs compounds. The recovery of compounds was assessed after each modification of the protocol.

A 5-ppb stock solution of PCBs mix 20 diluted in hexane was used in these steps. The protocols tested for cleaning-up PCBs on Florisil cartridge were based on the application note of Young (2013) and METHOD 3620 (Tobergte & Curtis 2013). After every modification of the protocol, the extract and the stock solution were injected in the GC-ECD analyser to check the recovery of the compounds. Altogether we performed three different clean-up steps.

First, to avoid concentrating the extract and eventually loosing compounds by evaporation, the 40 mL of PCB mixture solution was directly loaded on SPE. The obtained cleaned extract (i.e. of 4 mL) was injected in GC-ECD. The SPE extractor and the concentrator are shown in Figure 5 and 6, respectively. Figure 7 shows the clean-up device for 40 mL of extract.

In the second test, the SPE cartridge was activated with 4 mL of hexane. Then, 1 mL of sample (PCBs mixed) was loaded. PCBs were eluted with 4 mL of hexane. The 4 mL extracts were injected on GC-ECD.

Figure 4: Example of pressure/time diagram of a two-cycle extraction process (BÜCHI Labortechnik AG 2009)

14

In the third test, we repeated the steps of second test. However, at the end, the obtained 4 mL extracts were concentrated to 1 mL and injected on GC-ECD.

Figure 5: Solid phase extractor (SPE) Figure 6: Rotating concentrator miVac

Figure 7: Clean-up device for cleaning 40-mL extracts

15

Comparison of extraction techniques

Although several types of extraction techniques are used in the literature to extract organic compounds from plastic pellets, their efficiencies have not been compared so far. Thus, we decided to test the four different extraction techniques usually employed.

The extraction techniques that we selected to compare are:

- Soxhlet extraction - Ultrasonic extraction - Maceration - Pressurized fluid extraction

In all experiments, we used 10 polyethylene (PE) pellets spiked with PCBs. The extraction solvent was hexane. For each extraction technique, five extraction replicates and one blank were performed. Cleaned HDPE pellets were used in the blank experiment. The extraction time, volume and temperature were adapted to the different techniques tested. The parameters of each extraction techniques are shown in Table 2.

Table 2: Volume, time and temperature for each extraction technique

Extraction technique Volume Time Temperature

Soxhlet 150 mL 24 h 70 ºC

Maceration 30 mL 144 h Room

Ultrasonic extraction 10 mL 30 min Room

PFE 40 mL 35 min 65 ºC

For each technique, we carried out a second cycle of extraction on the same samples in order to check if some compounds remain unextracted during the first cycle.

The extracts obtained from the first and second cycles were separately concentrated on the rotating concentrator to approximately 1 mL under a controlled temperature of 45 ºC. The 1 mL extracts were then cleaned on solid phase extractor equipped with SPE Florisil cartridge. The cleaning protocol of the extracts was adapted from METHOD 3620 C (Tobergte & Curtis, 2013) (see details in results and discussion, section 4.1.3.). After the clean-up step, the 4-mL extracts were concentrated to approximately 1 mL on rotating concentrator and then injected on GC-ECD analyser to determine the concentration of PCBs extracted from pellets.

In order to obtain the exact concentration of extracted PCBs from pellets, we weighed each set of pellets prior to extraction. After the final concentration step, we measured the exact volume of each extract before injection on GC-ECD.

The PCBs concentration extracted from pellets were expressed as ng of PCBs per g of pellets.

16

3.5.1. Soxhlet extraction

For the Soxhlet extraction, we used a 200 mL distillation flask, a round-bottom flask of 150 mL, cellulose thimble, and condenser. The extraction temperature was set around 70 ºC and 4 to 6 extraction cycles per hour were performed for 24 h. The obtained 150 mL extracts were concentrated to 1 mL and then the previously described protocol was applied. The mass of pellets and the final volume of the first cycle replicates are shown in Table 3. The Soxhlet extractor is shown in Figure 8 and 9.

Table 3: Mass of pellets and final extract volumes for Soxhlet extraction test

Parallel 1

Parallel 2

Parallel 3

Parallel 4

Parallel 5

Blank

Mass of pellets (g) 0.2969 0.3228 0.3618 0.3316 0.3139 0.2786

Volume of extracts (mL)

1.650 1.742 1.7240 1.814 1.722 1.634

3.5.2. Pressurized fluid extraction

The PFE used was equipped with a heating zone for 40-mL stainless steel extraction cells, a gradient HPLC pump for solvents and 250-mL collection vials.

10 HDPE spiked pellets were weighted and transferred in a 40-mL extraction cell. In order to perform efficient extractions, the sample has to be homogenized with fat free

Figure 8: Extraction thimble for Soxhlet

Figure 9: Soxhlet extractor

17

quartz sand 0.3 - 0.9 mm. The bottom and the top of the cell were covered with quartz glass filters. Filled cells were placed in the heated part of the extractor. In this extraction technique, all six replicates including the blank were performed in parallel at once.

The extraction parameters were: heat up time 1 min, hold time 10 min, discharge time 2 min, temperature 65 ºC, pressure 100 bars, one cycle and 40 mL of hexane.

The mass of the ten pellet samples and the final volume of extracts obtained from first cycle are shown in Table 4. Figure 10 depicts how the extraction cell for PTF was filled and Figure 11 shows the placing of extraction cell into PFE.

Table 4: Mass of pellet and final extract volume for PFE test

Parallel 1

Parallel 2

Parallel 3

Parallel 4

Parallel 5

Blank

Mass of pellets (g) 0.3122 0.3120 0.3021 0.3115 0.3068 0.2987

Volume of extracts (mL) 1.663 1.657 1.736 1.630 1.748 1.601

3.5.3. Ultrasonication

Ten HDPE spiked pellets were weighted in 20 mL amber vials, then 10 mL of hexane were added, and vials were placed into an ultrasonic bath for 30 min at room temperature. Five replicates and one blank were performed. After the first cycle, the extracts were removed and replaced with fresh hexane and a second cycle of extraction was repeated.

The mass of the ten pellet samples and the final volume of extracts obtained from the first cycle are shown in Table 5.

Figure 10: Transferring pellets in extraction cell

Figure 11: Placing the extraction cell in the PFE

18

Table 5: Mass of pellets and final extract volume for ultrasonic extraction test

Parallel 1 Parallel 2 Parallel 3 Parallel 4 Parallel 5 Blank

Mass of pellets (g) 0.3092 0.3294 0.3154 0.3029 0.3343 0.3065

Volume of extracts (mL)

0.465 1.578 1.657 1.382 1.624 1.547

3.5.4. Maceration

Ten spiked HDPE pellets were weighed and placed into 20 mL amber vials. 15 mL of hexane were added. The vials were kept at room temperature for 72 h. The extracts were replaced with fresh 15 mL of hexane, and the extraction was repeated again for 72 h. At the end, both extracts were combined (i.e. 30 mL extracts).

The mass of the ten pellet samples and the final volume of extracts obtained from the first cycle are shown in Table 6. The maceration process is shown in Figure 12.

Table 6: Mass of pellets and final extract volume for maceration test

Figure 12: Maceration in 20mL amber vials

Replicate 1

Replicate 2

Replicate 3

Replicate 4

Replicate 5

Blank

Mass of pellets (g) 0.3350 0.3145 0.3415 0.3086 0.3113 0.2986

Volume of extracts (mL)

0.432 0.491 0.406 0.491 0.229 0.399

19

3.5.5. Statistical comparison of the results

The concentrations of PCBs extracted from spiked pellets were calculated in ng of PCBs per gram of pellets. Statistical t-tests were performed to compare the efficiency and repeatability of the different extraction techniques.

The t-test is commonly used to determine whether two methods (sets of data) are significantly different from each other (i.e. whether the mean of a population significantly differs from a specific value or from the mean of another population). It follows a Student’s t-distribution under null hypothesis and a t-value is calculated. When the calculated t-value is higher than observed t-value form table, there is a statistically significant difference between the two compared groups. Tabled t-values can be found in Appendix 3.

The t-test was performed to compare the extraction techniques two by two. It was also applied to compare the efficiency of each extraction technique and to compare their repeatability.

4. RESULTS AND DISSCUSSION

Optimization of experimental protocol for determination of PCBs

adsorbed on plastic pellets

4.1.1. Optimization of GC-ECD analysis for PCBs

PCBs were quantified on GC-ECD. As a first approach, the analytical conditions developed by Ogata et al. (2009), Endo et al. (2005) and Rios et al. (2010) were applied. In total 69 methods were tested on the GC-ECD apparatus. The obtained chromatograms were compared regarding the separation of peaks and the peak areas.

The parameters of the chosen method are shown in Table 7.

Table 7: Analytical conditions for PCBs analysis

Carrier gas Helium Flow in the column 1.0 mL min-1

Total flow 13.6 mL min-1 Detector temperature 300 °C

Injector port 280 °C Column temperature Gradient of

temperature*

Injector mode splitless Pressure 11.82 psi

Purge time 2 min Run time 74.40 min

Injection volume 2 µL

20

* The temperature was held at 50 °C for 1.3 min, then raised at 25°C min-1 to 140 °C, increased again at 1.5 °C min-1 to 240 °C, followed by a ramp of 20 °C min-1 to reach 280 °C, which was held for 10 min.

Commercial PCB mixture (i.e. PCB mix 20), containing 15 PCB congeners, was used in the development of the method.

The separation of compounds for PCB solution mix 20 using the selected method is shown in Figure 13.

Figure 13: Chromatogram of PCB mix 20 obtained with the final analytical conditions

The optimized method allowed a better separation of peaks without loss of sensitivity.

The calibration of PCBs was performed under these final analytical conditions with standard solutions for PCB mix 20 and 37 ranging from 1 to 15 ppb in hexane. The obtained parameters can be found in Table 8. The calibration curves are available in Appendix 2.

21

Table 8: Calibration parameters of PCB mix 20 and 37 solution on GC-ECD

Compound Retention time

(min) LOD (ppb)a LOQ (ppb)b R2c

PCB 31 32.52 1.60 16.02 0.9930

PCB 28 32.89 2.40 23.97 0.9845

PCB 52 35.90 2.28 22.79 0.9860

PCB 95 42.61 0.53 1.61 0.9989

PCB 101 45.40 1.94 19.37 0.9898

PCB 99 46.04 0.64 1.92 0.9985

PCB 110 49.49 0.42 1.26 0.9993

PCB 151 50.48 0.32 0.95 0.9996

PCB 77 50.79 3.50 35.00 0.9676

PCB 118 53.05 1.31 13.07 0.9953

PCB 149 53.17 0.34 1.01 0.9996

PCB 146 54.17 0.39 1.16 0.9994

PCB 153 54.81 2.35 23.53 0.9851

PCB 153 54.91 0.30 0.89 0.9997

PCB 105 55.76 6.89 68.92 0.9932

PCB 138 57.70 2.02 20.22 0.9889

PCB 183 59.10 0.40 1.21 0.9994

PCB 187 59.73 0.40 1.20 0.9994

PCB 126 60.12 1.14 11.40 0.9965

PCB 128 60.65 1.64 16.36 0.9927

PCB 177 62.36 0.44 1.33 0.9993

PCB 156 63.99 1.54 15.44 0.9935

PCB 180 65.02 1.72 17.18 0.9920

PCB 170 67.89 1.83 18.29 0.9909

PCB 169 68.50 1.45 14.52 0.9958 a LOD = limit of detection b LOQ = limit of quantification c R2= coefficient of the linear least square regression

LOD and LOQ were extrapolated from the calibration curves as follows:

- 𝐿𝑂𝐷 = (𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑑𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛 ÷ 𝑠𝑙𝑜𝑝𝑒)×3,33

- 𝐿𝑂𝑄 = (𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑑𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛 ÷ 𝑠𝑙𝑜𝑝𝑒) ×10

where the slope is obtained from the linear regression equation.

22

4.1.2. Optimization step for pressurized fluid extractor

Overall, we tested nine extraction methods. The following parameters were checked: heat up time, hold time, discharge time, number of cycles and volume of the extract. During the optimization step, we observed that high temperature (100 ºC) causes the melting of pellets in the extraction cell (Figure 14), which resulted in whitish extracts that were not analysed. In Figure 15 is presented Pressurized fluid extractor Büchi.

The method with the best extraction efficiency was as follows:

• Heat-up time 1 min and hold time for 10 min

• Discharge time 2 min

• The extraction was done in 1 cycle and performed in 40-mL cells

• Ten spiked virgin HDPE pellets were placed in the cell together with quartz sand

• The extraction temperature was set at 65 °C, and the pressure at 100 bars.

4.1.3. Optimization of the cleaning step

Recovery results for PCBs which were cleaned on SPE with Florisil cartridge following the first protocol described in section 3.3.3. are shown in Table 9 and the recovery results from the third test described.

Figure 14: Melted pellets after extraction at 100 °C

Figure 15: Pressurized fluid extractor (Büchi)

23

Table 9: Recovery results (%) from all three clean-up tests

Compound Recovery (%) from 1st test

Recovery (%) from 2nd test

Recovery (%) from 3rd test

PCB 31 5.79 2.16 61.1

PCB 28 5.44 2.80 93.6

PCB 52 5.32 1.80 162.0

PCB 101 6.12 0.26 65.1

PCB 118 5.36 3.11 105.3

PCB 153 4.95 1.54 80.5

PCB 105 6.28 2.19 64.8

PCB 138 5.65 3.06 104.2

PCB 126 8.05 1.55 105.4

PCB 128 7.32 2.06 106.3

PCB 156 5.19 2.34 102.0

PCB 180 4.84 1.86 100.0

PCB 170 5.59 2.17 82.3

PCB 169 9.45 2.23 105.3

From the results in Table 9, we can see that the recovery results of PCBs compounds after the clean-up step are very low and that a modification of this step is necessary. Similar results were obtained with the second protocol described.

We can also see that the recovery results for PCB compounds are high. The recoveries of PCB congeners range from 60 to 105 % except for PCB 52, which has a recovery of 162.02 %. This might be due to contamination.

Overall, we can say that the last test has the highest recovery of PCBs compounds. It can be used as such. In the third protocol, after extraction of PCBs, the extract is concentrated to 1 mL. The Florisil cartridge is activated with 4 mL of hexane and then 1 mL of sample is loaded. The compounds are finally eluted with 4 mL of hexane. The extract is concentrated to 1 mL before injection on GC-ECD.

4.1.4. Samples of real pellets

Pellets sampled in March 2015 on Corfu, Greece (Issos beach) were extracted and analysed in order to test the experimental protocol on real samples.

The pellets from real samples can be made of different types of plastics (e.g. PE, PP (Fotopoulou and Karapanagioti, 2012)), which is a parameter affecting the adsorption process of PCBs to the pellet surface and that can also impact the efficiency of the analytical protocol. Moreover, the colour of the pellets usually reflects the residence time in the environment. For instance, older pellets are brown/dark yellow. Thus, before weighing, the pellets were visually sorted by colour in order to analyse the aged ones. The unsorted and sorted pellets can be seen in Figure 16 and 17.

24

Ten of the sorted pellets (m = 0.3034 g) were extracted and analysed according to the optimized protocol.

The obtained chromatogram is presented in Figure 18. Two out of the six observed peaks correspond to the retention time of the following PCB congers: PCB 95 and PCB 149. Both signals are in the range of detection corresponding to calculated LOD.

Figure 18: Chromatogram shown the extracted PCBs from Greek pellets

Figure 16: Unsorted pellets sampled on the beach

Figure 17: Sorted pellets

25

Comparison of extraction techniques

4.2.1. Adsorption of PCBs on virgin plastic pellets

For the comparison of the four extraction techniques, virgin plastic pellets were spiked with PCBs. This was done by mixing the pellets in a PCB solution. After 24 h the solvent (hexane) was evaporated to dryness. The recondensed solvent was then concentrated to 1 mL and injected into GC-ECD in order to obtain the concentration of PCBs adsorbed on pellets. The amounts of PCBs in ng/g and the percentages of each PCB adsorbed on pellets are presented in Table 10.

Table 10: Concentration of PCBs in solution before spiking pellets, concentration of PCBs in recondensed solvent and percentages of PCBs adsorbed on pellets

Compound ng of PCB per g of pellet before

spiking

ng of PCBs per g of pellets after spiking

% of adsorbed PCBs on pellets

PCB 28 57.5 5.0 91.4

PCB 52 51.9 4.5 91.3

PCB 95 52.9 3.0 94.3

PCB 101 52.2 2.5 95.2

PCB 99 51.6 1.8 96.6

PCB 110 55.1 1.1 98.1

PCB 151 51.6 1.7 96.7

PCB 118 54.3 1.8 96.7

PCB 149 55.0 1.7 96.9

PCB 146 53.0 1.1 97.9

PCB 153 53.5 5.7 89.4

PCB 105 56.1 1.6 97.2

PCB 138 52.0 1.9 96.3

PCB 183 56.1 1.2 97.9

PCB 187 56.4 2.1 96.4

PCB 177 52.2 1.2 97.8

PCB 180 57.0 1.0 98.3

PCB 170 58.2 1.3 97.7

From the results, we can see that the percentages of PCBs adsorbed on pellets range from 89.4 to 98.3 %, meaning that almost all of the PCBs dissolved in hexane adsorbed on pellets. Spiked pellets were extracted two times on PFE. The results after the first extraction are shown in Table 11. In the second cycle, after extraction we did not detect any compound.

26

Table 11: Concentration of extracted PCBs form 6 replicates of spiked pellets

Replicates 1 2 3 4 5 6 STDV

Compound Conc.

(ng/g of pellet)

Conc. (ng/g of pellet)

Conc. (ng/g of pellet)

Conc. (ng/g

of pellet)

Conc. (ng/g

of pellet)

Conc. (ng/g

of pellet)

PCB 28 54.0 51.2 53.8 46.7 53.3 55.3 3.1

PCB 52 55.9 52.8 46.8 64.9 68.7 56.1 8.0

PCB 95 54.5 54.8 53.9 58.0 63.2 58.5 3.5

PCB 101 51.2 49.6 50.7 56.3 64.0 49.8 5.7

PCB 99 54.3 54.3 51.9 56.8 62.2 57.0 3.5

PCB 110 55.9 56.6 55.9 60.5 64.2 53.8 3.8

PCB 151 50.8 50.3 49.7 58.0 65.7 58.0 6.3

PCB 118 54.8 54.8 51.2 57.3 64.2 47.6 5.6

PCB 149 57.5 55.3 53.8 55.5 61.9 59.8 3.1

PCB 146 51.4 49.3 50.0 50.1 58.9 43.7 4.9

PCB 153 52.4 54.2 50.6 55.6 57. 9 52.6 2.7

PCB 105 57.6 55.1 56.2 64.5 60.5 53.2 4.1

PCB 138 54.6 56.9 55.8 56.3 58.3 56.7 1.2

PCB 183 50.5 52.8 54.3 53.1 62.8 46.4 5.4

PCB 187 53.1 51.5 48.8 51.6 62.7 54.6 4.8

PCB 177 57.8 56.1 58.7 63.2 66.4 54.1 4.6

PCB 180 51.2 48.5 48.7 50.6 60.6 51.6 4.5

PCB 170 54.2 58.8 59.2 57.4 63.6 48.7 5.1

From the results, we can see that the concentrations range from 43.7 to 68.7 ng per g of pellet. Moreover, the standard deviation for all extracted compounds is lower than 7 ng per g. Thus, we can say that we obtained a homogenous adsorption of PCBs on pellets.

4.2.2. Extraction results

For each technique, a blank sample was also extracted and measured. No compounds were detected meaning that no contamination artefact had to be considered. Moreover, a second cycle of extraction (i.e. 3 replicates) was performed after the first one in order to check whether a single extraction step would be sufficient for extracting the possible remaining compounds. No compounds were detected in the second-cycle extracts except with the ultrasonication technique.

Seven replicates were performed for Soxhlet extraction. However, we observed a significant loss of volume (i.e. from 150 to 100 mL) in three experiments probably due to a leakage in the system. Thus, the corresponding results were discarded. The results of Soxhlet extractions are shown in Table 12.

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Table 12: Concentration of extracted PCBs from HDPE pellets with Soxhlet extraction

Replicate 1 Replicate 2 Replicate 3 Replicate 4

Compound Conc. (ng/g) Conc. (ng/g) Conc. (ng/g) Conc. (ng/g)

PCB 28 53.9 57.7 75.1 60.3

PCB 52 44.6 84.7 69.4 49.6

PCB 95 47.9 36.7 62.7 50.8

PCB 101 42.5 34.6 63.2 45.9

PCB 99 40.2 31.2 60.1 43.0

PCB 110 50.9 28.8 62.9 52.3

PCB 151 40.4 29.4 59.5 43.7

PCB 118 47.9 30.9 66.9 50.8

PCB 149 46.2 29.5 67.7 47.8

PCB 146 37.5 24.4 59.0 38.5

PCB 153 47.9 27.7 71.2 50.5

PCB 105 50.5 23.9 69.4 53.4

PCB 138 50.1 24.0 68.9 49.9

PCB 183 39.9 24.1 62.4 42.3

PCB 187 39.2 22.7 60.3 40.9

PCB 177 47.7 23.9 65.7 49.3

PCB 180 43.3 23.6 66.0 43.9

PCB 170 44.1 20.6 63.1 43.4

We can see that the concentrations of PCBs extracted from pellets ranged from 20.6 to 84.7 ng per g of pellet. The second replicate displays the lowest concentration of extracted PCBs whereas high concentrations were obtained in the third replicate. It seems that the extraction was not efficient enough to extract all the PCBs from the pellets in the second replicate. In the third, the high concentration of extracted PCBs may be a consequence of contamination from previous experiments. Nevertheless, the extracted concentrations of PCBs are, on average, in the same range than the estimated initial concentrations (see section 4.3.).

In PFE, we performed six parallel extractions including one blank. The results are shown in Table 13.

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Table 13: Concentration of extracted PCBs from HDPE pellets with PFE

Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5

Compound Conc. (ng/g

of pellet) Conc. (ng/g

of pellet) Conc. (ng/g

of pellet) Conc. (ng/g

of pellet) Conc. (ng/g

of pellet)

PCB 28 46.7 53.3 62.3 42.6 55.3

PCB 52 64.9 68.7 73.0 55.6 56.1

PCB 95 58.0 63.2 62.6 47.5 58.5

PCB 101 56.3 64.0 63.7 47.3 49.8

PCB 99 56.8 62.2 68.1 47.2 57.0

PCB 110 60. 5 64.2 71.7 50.5 53.8

PCB 151 58.0 65.7 65.4 47.1 58.0

PCB 118 57.3 64.2 65.4 46.7 47.6

PCB 149 55.5 61.9 65.7 47.2 59.8

PCB 146 50.1 58.9 60.0 42.5 43.7

PCB 153 55.6 57.9 60.4 42.5 52.6

PCB 105 64.5 60.5 71.1 49.6 53.2

PCB 138 56.3 58.3 64.3 43.1 56.7

PCB 183 53.1 62.8 61.2 43.7 46.4

PCB 187 51.6 62.7 62.1 43.7 54.6

PCB 177 63.2 66.4 73.5 51.5 54.1

PCB 180 50.6 60.6 60.2 42.0 51.6

PCB 170 57.4 63.6 66.7 45.7 48.7

The concentrations of extracted PCBs ranged from 42.03 to 73.49 ng/g of pellets. At first glance, the results of all parallels are comparable. The extracted concentrations of PCBs from pellets are in the same range than estimated initial PCBs concentrations.

Table 14 presents the results obtained with the maceration technique. Five replicates and one blank experiments were performed.

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Table 14: Concentration of extracted PCBs from HDPE pellets with maceration technique

Replicate1 Replicate 2 Replicate 3 Replicate 4 Replicate 5

Compound Conc. (ng/g

of pellet) Conc. (ng/g

of pellet) Conc. (ng/g

of pellet) Conc. (ng/g

of pellet) Conc. (ng/g

of pellet)

PCB 28 33.8 39.7 27.7 37.9 14.3

PCB 52 28.9 33.2 27.2 33.7 12.7

PCB 95 27.7 31.8 23.6 28.8 13.0

PCB 101 27.2 32.2 26.9 33.5 12.7

PCB 99 27.4 33.2 24.8 33.1 12.6

PCB 110 27.6 32.0 27.2 32.5 12.2

PCB 151 26.9 31.5 25.0 31.0 11.6

PCB 118 26.5 31.1 25.7 29.8 11.3

PCB 149 28.3 34.6 26.2 32.8 12.7

PCB 146 26.1 31.2 26.6 30.2 11.4

PCB 153 26.0 31.5 24.2 29.9 11.4

PCB 105 29.1 35.6 29.2 28.2 12.6

PCB 138 26.0 30.3 24.2 29.0 10.8

PCB 183 25.7 29.7 25.3 28.2 10.3

PCB 187 26.5 30.5 24.1 28.8 10.4

PCB 177 25.6 29.7 25.7 27.6 10.1

PCB 180 26.1 30.3 24.3 28.6 10.1

PCB 170 26.1 29.4 26.2 27.8 9.8

The concentrations of PCBs extracted with maceration ranged from 9.76 to 39.69 ng per gram of pellet. We can also see that only the fifth replicate displays lower concentrations of extracted PCBs compared to the other four. The lower concentrations of extracted PCBs may be due to fast passing of sample through the cartridge during the clean-up protocol. In addition, no compounds were detected in the second extraction step. Thus, the maceration technique does not allow the extraction of the most strongly bound compounds and has overall a low extraction efficiency.

In Table 15 the concentrations of PCBs extracted with ultrasonication are shown.

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Table 15: Concentration of extracted PCBs from HDPE pellets with ultrasonication

Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5

Compound Conc.

(ng/g of pellet)

Conc. (ng/g of pellet)

Conc. (ng/g of pellet)

Conc. (ng/g of pellet)

Conc. (ng/g of pellet)

PCB 28 3.7 27.9 32.7 30.7 26.9

PCB 52 2.5 33.2 34.8 28.5 19.6

PCB 95 2.4 35.2 35.5 29.9 16.7

PCB 101 2.2 33.0 34.7 29.0 16.0

PCB 99 2.4 34.1 36.6 28.3 16.6

PCB 110 2.3 32.8 34.6 29.6 14.7

PCB 151 2.0 37.8 36.2 29.7 14.2

PCB 118 1.9 38.4 41.5 30.8 13.1

PCB 149 2.6 33.0 35.0 29.0 17.6

PCB 146 1.9 37.0 32.6 28.0 14.0

PCB 153 1.9 35.6 34.5 27.9 14.4

PCB 105 2.8 32.1 35.1 30.3 19.1

PCB 138 1.9 35.1 38.1 27.9 14.5

PCB 183 1.7 39.7 39.6 28.2 12.7

PCB 187 1.7 38.7 39.3 27.9 13.6

PCB 177 1.7 39.6 35.5 30.8 13.8

PCB 180 1.7 38.2 37.4 27.8 13.3

PCB 170 1.8 37.6 34.6 29.8 14.2

Ultrasonication yielded PCB concentrations between 1.65 and 41.48 ng per g of pellet. We can also see that the first replicate displays the lowest concentrations of PCBs extracted from pellets. This may be due to a low recovery after the SPE clean-up step.

In this case, compounds were detected in the second cycle of extraction. The concentration of PCBs extracted in the second step are shown in Table 16.

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Table 16: Concentration (ng/g) of extracted PCBs after first and second cycle

Replicate 1 Replicate 2 Replicate 3

Compound Cycle 1 Cycle 2 Cycle 1 Cycle 2 Cycle 1 Cycle 2

PCB 28 45.5 27.3 35.2 25.9 37.7 10.6

PCB 52 51.4 31.0 43.9 32.8 43.0 12.0

PCB 95 58.2 35.2 46.5 33.7 46.6 12.9

PCB 101 56.2 33.6 44.6 33.5 44.4 10.4

PCB 99 55.6 33.7 44.3 32.8 43.0 10.4

PCB 110 56.9 33.1 42.2 31,.8 42.2 9.1

PCB 151 63.0 38.2 46.4 35.4 49.1 9.9

PCB 118 63.2 38.1 46.2 35.9 49.1 9.4

PCB 149 52.6 32.3 39.6 32.7 43.1 7.4

PCB 146 50.0 34.5 41.7 33.1 46.0 7.5

PCB 153 47.8 38.4 42.6 33.8 45.0 7.9

PCB 105 39.7 31.8 36.8 30.2 42.5 6.5

PCB 138 57.8 36.1 42.5 33.2 45.4 8.6

PCB 183 70.7 42.4 47.3 36.1 52.9 6.7

PCB 187 68.2 41.5 47.0 35.0 51.1 6.6

PCB 177 69.2 42.3 46.5 33.6 53.1 6.6

PCB 180 71.5 40.6 47.4 35.3 51.1 6.1

PCB 170 67.4 41.5 47.2 35.5 52.2 5.8

The concentrations of extracted PCBs from pellets are still high ranging from 10 to 42 ng per g. This means that two extractions in series are required for achieving an efficient extraction.

4.2.3. Statistical analysis of the extraction techniques

To test which extraction technique showed the best extraction efficiency and repeatability we carried out statistical tests. To this end, we compared the average concentrations and standard deviations of extracted PCBs between techniques. We also applied the t-test to assess the possible statistically significant difference between Soxhlet extraction and PFE extraction, as well as between ultrasonication and maceration.

In Table 17 the average concentrations and the standard deviations of extracted PCBs from pellets are shown for each extraction technique. Figure 19 presents the sum of the initial concentration of PCBs adsorbed on pellets and the extracted concentration of PCBs from pellets for each technique.

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Table 17: Average amounts of extracted PCBs for each extraction technique

Initial conc. Soxhlet PFE Ultrasonication Maceration

Compound Conc.

(ng/g of pellet)

Conc. (ng/g of pellet)

Conc. (ng/g of pellet)

Conc. (ng/g of pellet)

Conc. (ng/g of pellet)

PCB 28 57.4 61.3 ± 9.3 52.0 ± 7.7 24.4 ± 11.7 30.7 ± 10.3

PCB 52 51.9 62.1 ± 18.5 63.7 ± 7.7 23.7 ± 13.2 27.1 ± 8.5

PCB 95 52.8 49.5 ± 10.7 58.0 ± 6.3 23.9 ± 14.3 25.0 ± 8.3

PCB 101 52.2 46.6 ± 12.1 56.2 ± 7.7 23.0 ± 13.7 26.5 ± 8.2

PCB 99 51.6 43.6 ± 12.1 58.3 ± 7.7 23.6 ± 14.6 26.2 ± 8.5

PCB 110 55.1 48.7 ± 14.3 60.1 ± 8.4 22.8 ± 13.9 26.3 ± 8.2

PCB 151 51.6 43.2 ± 12.4 58.8 ± 7.5 24.0 ± 15.5 25.2 ± 8.1

PCB 118 54.3 49.1 ± 14.8 56.3 ± 8.9 25.1 ± 17.0 24.9 ± 7.9

PCB 149 54.9 47.8 ± 15.6 58.0 ± 7.1 23.5 ± 13.5 26.9 ± 8.6

PCB 146 53.0 39.9 ± 14.3 51.1 ± 8.2 22.7 ± 14.5 25.1 ± 8.0

PCB 153 53.4 49.3 ± 17.8 53.8 ± 6.9 22.9 ± 14.5 24.6 ± 7.9

PCB 105 56.1 49.3 ± 18.9 59.8 ± 8.6 23.9 ± 13.3 26.9 ± 8.5

PCB 138 52.0 48.2 ± 18.5 55.7 ± 7.8 23.5 ± 15.1 24.1 ± 7.8

PCB 183 56.1 42.1 ± 15.7 53.4 ± 8.6 24.4 ± 16.8 23.8 ± 7.8

PCB 187 56.4 40.7 ± 15.4 54.9 ± 7.9 24.2 ± 16.4 24.1 ± 8.0

PCB 177 52.2 46.6 ± 17.2 61.7 ± 9.0 24.3 ± 16.0 23.7 ± 7.8

PCB 180 57.0 44.2 ± 17.3 53.0 ± 7.7 23.7 ± 15.9 23.9 ± 8.0

PCB 170 58.2 42.8 ± 17.4 56.4 ± 9.1 23.6 ± 15.2 23.8 ± 8.0

SUM 975.9 855.6 ± 272.1

1021 ± 142.8

427.1 ± 264.6 458.8 ± 147.5

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Figure 19: Sum of initial adsorbed PCBs and extracted PCBs from spiked pellets for each extraction technique

PFE technique is the most efficient (105%) since it enables to extract about 105% on average of the adsorbed compounds when comparing with the estimated initial sum of concentrations. Also, a good efficiency of 88% is achieved by the Soxhlet extraction. Maceration and ultrasonication display an extraction efficiency of 47% and 44%. It is apparent that not all the PCBs are extracted with these two techniques. Contamination occurred during the Soxhlet and ultrasonication extraction, which resulted in recovery of 153% and 140%, respectively.

In the case of ultrasonication, the results of the fractions obtained in both extraction cycles were not taken into account. Combining both cycles, the extraction efficiency would be about 140%.

The obtained results show that the recoveries differ between different extraction techniques.

In Table 18 the relative standard deviations are shown for each technique.

0

200

400

600

800

1000

1200

1400

PCBs on pellets Soxhlet PFE Ultrasonication Maceration

Su

m c

on

ce

ntr

atio

n o

f P

CB

s (

ng

/g)

Type of extraction

Sum of extracted PCBs

34

Table 18: Relative standard deviations for each extraction technique

Soxhlet PFE Ultrasonication Maceration

Compound % % % %

PCB 28 15.0 14.8 48.3 33.4

PCB 52 29.7 12.1 56.0 31.5

PCB 95 21.6 10.9 59.6 92.3

PCB 101 25.9 13.7 59.8 31.2

PCB 99 27.7 13.3 60.0 32.3

PCB 110 29.5 14.0 60.9 31.3

PCB 151 28.8 12.9 64.5 32.2

PCB 118 30.0 15.7 67.7 31.7

PCB 149 32.7 12.2 57.4 32.0

PCB 146 35.8 16.1 63.7 31.8

PCB 153 36.0 12.9 63.2 32.2

PCB 105 38.2 14.4 55.6 31.7

PCB 138 38.3 13.9 64.4 32.3

PCB 183 37.3 16.0 69.1 32.7

PCB 187 37.8 14.4 67.5 33.2

PCB 177 36.9 14.6 65.8 32.8

PCB 180 39.1 14.5 67.2 33.6

PCB 170 40.6 16.2 64.3 33.5

Average RSD

32.3 14.0 61.9 35.7

By comparing relative standard deviations (Table 18), we can see that the highest RSDs are obtained in the case of ultrasionication and the lowest in case of PFE extraction, which means that the repeatability of extraction is lowest with ultrasonication and best with PFE extraction. Overall, the Soxhlet extraction technique comes second and maceration third.

We performed a statistical t-test to compare Soxhlet and PFE extractions, as well as ultrasonication and maceration techniques. The test was performed with 99 % confidence interval, which means that α is 0.01. Table 19 presents the results of the t-test for comparison of Soxhlet and PFE extractions.

Table 19: t-test for two independent techniques; 99% confidence interval on the difference between the means (Soxhlet and PFE extractions)

|t| (Critical value) 5.738

t (Observed value) 5.041

α (confidence level) 0.01

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In Table 19, the statistical results from two tailed t-test are shown. The critical calculated t-value is 5.738, however the observed t value is 5.041. We can see that critical t-value is larger than observed t-value, which shows significant statistical difference between two compared techniques. We therefore have to reject the null hypothesis, and can confirm that there is a statistically significant difference between these two extraction techniques.

Table 20 presents the results of the statistical t-test for comparison of ultrasonication and maceration.

Table 20: t-test for two independent techniques; 99% confidence interval on the difference between the means (maceration and ultrasonication)

|t| (Critical value) 3.169

t (Observed value) 4.587

α 0.01

In table 20 we can observe, that the critical t-value is 3.169 however the observed t-value is 4.587. Since the critical t-value is lower than observed t-value we have to accept the null hypothesis, which means that the test does not show significant statistical difference between these two techniques. With 99% confidence we can say that ultrasonication and maceration are comparable techniques.

From these results we can conclude that all four extraction techniques show statistically significantly difference between concentrations of extracted PCBs.

The best extraction efficiency and repeatability (i.e. regarding the low RSD value) is obtained in case of PFE. Although maceration technique has quite good repeatability, its efficiency is low (<50%). Soxhlet extraction is a less accurate method. In case of ultrasonication, two extraction in series should be performed to obtain satisfying extraction efficiency, but still the repeatability is quite low.

If we compare (Table 18) the RSD of extracted PCBs adsorbed on pellets between four techniques, we can see that the lower RSD value is obtained with PFE (14%) and the highest with ultrasonication (61.9%). In case of the Soxhlet extraction and maceration we obtained similar RSD values: 32.3 and 35.7%. Best repeatability was therefore achieved with PTF extraction, and the worst with ultrasonication.

4.2.4. Comparison of extraction techniques

We saw from the results that the best extraction efficiency is obtained with PFE. However, there are also other parameters to take into account in the selection of an extraction technique, such as the volume of extracting solvent, the extraction time, the costs of equipment and the simplicity of the technique.

The Soxhlet extraction is a solvent and time consuming technique. It is not user-friendly since it requires a long cleaning procedure and possible leakage can occur during the extraction.

36

With PFE, the consumption of solvent is low (around 40 mL), several extractions can be performed in parallel and, the technique is automated. The time of extraction is short (<1h), and the equipment is easy to use. However, we have also to take into account the consumables such as quartz sand used for homogenizing the sample and the consummation of nitrogen gas which is needed for regulating the pressure in the instrument. However, the biggest downside is in the cost of the equipment, which requires an investment in the range of 50.000 EUR and additional possible maintenance costs.

With maceration extraction, we only need amber vials and the consumption of solvent is low. However, this technique is time consuming. Altogether, it takes 6 days to perform the extraction.

Ultrasonic extraction is also performed in amber vials, the consumption of solvent is low, and it is also a fast technique (i.e. 30 min). Furthermore, the costs of an ultrasonic bath are no higher than 1.000 EUR.

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5. CONCLUSIONS

In this study, we developed a method for analysing PCBs on GC-ECD that shows high sensitivity and adequate separation of the chromatographic peaks. In order to validate the identification of each peak, GC-MS could be applied in the future.

After the optimization of the GC-ECD method, the clean-up protocol was designed. The final clean-up method shows good recoveries for all compounds (i.e. higher than 61%), which are comparable to the results obtained by Young (2013). However, one compound (i.e. PCB 52) shows too high recoveries (i.e. 162.02%). It might be the consequence of contamination.

Prior to performing efficiency assessment of the extraction techniques, we showed that it is possible to spike virgin pellets in a homogenous and reproducible way.

References to different extraction techniques can be found in the literature for extraction of organic compounds from plastic pellets and/or microplastics. To our knowledge, no comparison study has been carried out so far to determine the efficiency of the different methods. The four main techniques identified are: maceration, ultrasonication, Soxhlet and PFE.

We showed that the best extraction efficiency and repeatability are obtained with PFE. Moreover, it is fast, it consumes low volume of solvent and it does not require a lot of manually work. The main drawback of this technique is the cost of the equipment and maintenance.

The Soxhlet extraction was the most difficult to handle and probably that is why it led to low efficiency and repeatability.

For ultrasonication extraction we suggest to perform a second cycle of extraction in order to obtain better extraction efficiency. However, the repeatability is insufficient.

The most abundant microplastics found in marine environment are PP and PE. The developed protocol would need to be tested on pellets made of different plastic material and on other type of microplastics of smaller dimensions.

Finally, other compounds (e.g. metals, PAHs, phthalates) can sorb on plastic pellets, as well. In this order in the future work, we would suggest extending our experimental protocol to other organic pollutants such as PAHs and OCPs.

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APPENDIX

APPENDIX 1. Composition of PCB mix 20 and 37

Table 21: Composition of PCB mixture 20

Product Name Cas no.

PCB 28 2,4,4′-Trichlorobiphenyl 7012-37-5

PCB 31 2,4',5-Trichlorobiphenyl 16606-02-3

PCB 52 2,2',5,5'-Tetrachlorobiphenyl 35693-99-3

PCB 101 2,2',4,5,5'-Pentachlorobiphenyl 37680-73-2

PCB 105 2,3,3',4,4'-Pentachlorobiphenyl 32598-14-4

PCB 118 2,3',4,4',5-Pentachlorobiphenyl 31508-00-6

PCB 126 3,3',4,4',5-Pentachlorobiphenyl 57465-28-8

PCB 128 2,2',3,3',4,4'-Hexachlorobiphenyl 38380-07-3

PCB 138 2,2',3,4,4',5'-Hexachlorobiphenyl 35065-28-2

PCB 153 2,2',4,4',5,5'-Hexachlorobiphenyl 35065-27-1

PCB 156 2,3,3',4,4',5-Hexachlorobiphenyl 38380-08-4

PCB 169 3,3',4,4',5,5'-Hexachlorobiphenyl 32774-16-6

PCB 170 2,2',3,3',4,4',5-Heptachlorobiphenyl 35065-30-6

PCB 180 2,2',3,4,4',5,5'-Heptachlorobiphenyl 35065-29-3

Table 22: Composition of PCB mixture 37

Product Name Cas no.

PCB 28 2,4,4′-Trichlorobiphenyl 7012-37-5

PCB 52 2,2',5,5'-Tetrachlorobiphenyl 35693-99-3

PCB 95 2,2',3,5',6-Pentachlorobiphenyl 38379-99-6

PCB 99 2,2',4,4',5-Pentachlorobiphenyl 60233-25-2

PCB 101 2,2',4,5,5'-Pentachlorobiphenyl 37680-73-2

PCB 105 2,3,3',4,4'-Pentachlorobiphenyl 32598-14-4

PCB 110 2,3,3',4',6-Pentachlorobiphenyl 38380-03-9

PCB 118 2,3',4,4',5-Pentachlorobiphenyl 31508-00-6

PCB 138 2,2',3,4,4',5'-Hexachlorobiphenyl 35065-28-2

PCB 146 2,2',3,3',4,4'-Hexachlorobiphenyl 38380-07-3

PCB 149 2,2',3,4',5',6-Hexachlorobiphenyl 38380-04-0

PCB 151 2,2',3,5,5',6-Hexachlorobiphenyl 52663-63-5

PCB 153 2,2',4,4',5,5'-Hexachlorobiphenyl 35065-27-1

PCB 170 2,2',3,3',4,4',5-Heptachlorobiphenyl 35065-30-6

PCB 177 3,3',4,4',5,5'-Hexachlorobiphenyl 32774-16-6

PCB 180 2,2',3,4,4',5,5'-Heptachlorobiphenyl 35065-29-3

PCB 183 2,2',3,4,4',5',6-Heptachlorobiphenyl 52663-69-1

PCB 187 2,2',3,4',5,5',6-Heptachlorobiphenyl 52663-68-0

42

APPENDIX 2. Calibration curves of PCBs mix 20 and 37 on GC-ECD

y = 774,23x + 155,55R² = 0,993

y = 1226,4x + 498,67R² = 0,9845

y = 840,95x + 438,56R² = 0,986

0

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Concentration (ppb)

PCB congeners n° 31, 28 and 52

PCB 31

PCB 28

PCB 52

y = 1253,5x + 650,13R² = 0,9898

y = 676,02x - 123,25R² = 0,9676

y = 1411,8x + 166,51R² = 0,9953

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a

Concentration (ppb)

PCB congeners n° 101, 77 and 118

PCB 101

PCB 77

PCB 118

43

y = 1368,2x + 599,19R² = 0,9851

y = 1867,3x + 281,37R² = 0,9932

y = 1823,7x + 920,87R² = 0,9889

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a

Concentration (ppb)

PCB congeners n° 153, 105 and 138

PCB 153

PCB 105

PCB 138

y = 887,21x - 140,14R² = 0,9965

y = 2332,6x + 918,78R² = 0,9927

y = 2169,3x + 252,24R² = 0,9935

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a

Concentration (ppb)

PCB congeners n° 126, 128 and 156

PCB 126

PCB 128

PCB 156

y = 2081,6x + 798,03R² = 0,992

y = 2239,7x + 1379R² = 0,9909

y = 1123,1x + 393,62R² = 0,9895

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a

Concentration (ppb)

PCB congeners n° 180, 170 and 169

PCB 180

PCB 170

PCB 169

44

y = 929,37x + 10,153R² = 0,9932

y = 737,57x + 144,26R² = 0,999

y = 1031x + 15,756R² = 0,9989

y = 1129,1x + 75,38R² = 0,9992

0

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16000

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a

Concentration (ppb)

PCB congeners n° 28, 52, 95 and 101

PCB 28

PCB 52

PCB 95

PCB 101

y = 1202x + 20,634R² = 0,9985

y = 1509,7x + 48,487R² = 0,9993

y = 1385,6x + 58,145R² = 0,9996

y = 1223,8x + 19,483R² = 0,9993

0

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a

Concentration (ppb)

PCB congeners n° 99, 110, 151 and 118

PCB 99

PCB 110

PCB 151

PCB 118

45

y = 1228,5x + 100,1R² = 0,9996

y = 1409,9x + 55,932R² = 0,9994

y = 1319,3x + 60,029R² = 0,9997

y = 1754,2x - 185,35R² = 0,996

y = 1737,8x - 26,782R² = 0,9989

0

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a

Concentration (ppb)

PCB congeners n° 149, 146, 153, 105 and 138

PCB 149

PCB 146

PCB 153

PCB 105

PCB 138

y = 1548x + 61,703R² = 0,9994

y = 1751,9x + 41,672R² = 0,9994

y = 1608,7x + 40,591R² = 0,9993

y = 1920,8x - 18,105R² = 0,9994

y = 2103,3x - 163,23R² = 0,9988

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a

Concentration (ppb)

PCB congeners n° 183, 187, 177, 180 and 170

PCB 183

PCB 187

PCB 177

PCB 180

PCB 170

46

APPENDIX 3. Statistical t-test table