Cumulative exposure assessment of multiple chemicals in fresh shellfish in South Korea

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CUMULATIVE EXPOSURE

ASSESSMENT OF MULTIPLE

CHEMICALS IN FRESH SHELLFISH IN

SOUTH KOREA

Word count: 17688

Jannes Tondeleir

Student number: 01305928

Promotors: Prof. dr. ir. Liesbeth Jacxsens, Prof. dr. Tanja Cirkovic Velickovic

Master’s Dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Science in Bioscience Engineering Technology: Agriculture and Horticulture - main subject Plant and Animal Production

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This page is not available because it contains personal information.

Ghent University, Library, 2021.

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Preamble concerning COVID-19

The corona situation did not largely effect the work needed to complete this thesis. Once relevant data was transferred or acquired, exposure simulations could be conducted undisturbed.

The 6 week stay at the GUGC in Incheon South-Korea however was impacted but not in a way to undermine the completion of this work. In march 2020, UGent requested all foreign students to return to Belgium due to the growing presence of COVID in Belgium. This premature departure would have led to a lack of reliable data pertaining to microplastics contamination in shellfish. Seeing as Belgium at this point was on the brink of lockdown and reported cases in Korea where already beginning to decrease, ultimately, the wise decision was made to not mandate early return of students.

The last week of experiments was however cut short by two days owing to the large-scale cancellation of international flights. Extra efforts were made to complete all ongoing experiments in time for early departure, which was successful.

This preamble was composed by mutual agreement of the student and the promotor and approved by both.

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Acknowledgements

This thesis was written with the intent of acquiring the degree of Master of Science in Bioscience Engineering Technology: Agriculture and Horticulture - main subject Plant and Animal Production. As you will come to understand, the subject investigated herein does not constitute an integral part of my specialization in agriculture, a deliberate choice.

A wide array of subjects are presented to all students finishing their degrees, why not wonder of the beaten track a bit and open up some new avenues to explore. Seeing as my specialization focusses on primary production of domestic animal and plant based food, I decided to look into subjects a bit more down the line in the “field to fork” pathway. This search is what landed me with this exploration into food safety and risk analysis.

The completion of this thesis would have never been possible without the excellent guidance and support of my promotor Prof. Dr. Ir. Liesbeth Jacxsens and my stay at the Ghent University Global Campus in South Korea made possible by Prof. dr. Tanja Cirkovic Velickovic.

While on the subject of the GUGC, I would like to express my gratitude to everyone there who made my stay enjoyable and possible. In particular Maria Krishna D. de Guzman who allowed me to interject into her research. I wish you the best with the continuation of your investigation into microplastics.

Thank you Marlies for always being there for me and your patience with me whilst you absolutely outperform me in pretty much every aspect in life.

To end I would like to thank my parents who made my education at Ghent University possible. They’ve had to endure a pretty long and mediocre at best studying career from their only son.

Ghent, May 2020 Jannes Tondeleir

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List of abbreviations

ATSDR Agency for Toxic Substances and Disease Registry

BMD Benchmark Dose

BMDL Benchmark Dose Lower confidence limit

BMR Benchmark Response

B.W. Bodyweight

CONTAM Panel on Contaminants in the Food Chain

D.W. Dry Weight

EDI Estimated Daily Intake

EFSA European Food Safety Authority

EPA Environmental Protection Agency

FSA Food Standards Agency

F.W. Fresh Weight

HDPE High Density Polyethylene

IARC International Agency for Research on Cancer

JECFA Joint FAO/WHO Expert Committee on Food Additives KCDC Korea Centers for Disease Control and Prevention

KNHANES Korea National Health and Nutrition Examination Survey

L.B. Lower Bound

LDPE Low Density Polyethylene

LOAEL Lowest Observed Adverse Effect Level

L.O.D. Limit Of Detection

M.B. Medium Bound

MOE Margin Of Exposure

MP-VAT Microplastics Visual Analysis Tool

MRFP Monomeric Red Fluorescent Protein

NOAEL No Observed Adverse Effect Level

NTP National Toxicology Program

PAH Polycyclic Aromatic Hydrocarbon

PET Polyethylene Terephthalate

PP Polypropylene

PS Polystyrene

PTFE Polytetrafluoroethylene

PTWI Provisional Tolerable Weekly Intake

PUR Polyurethane

PVC Polyvinyl Chloride

QRA Quantitative Risk Assessment

SCD Systemic Contact Dermatitis

SCF Scientific Committee on Food

TDI Tolerable Daily Intake

TEF Toxic Equivalency Factor

TWI Tolerable Weekly Intake

U.B. Upper Bound

UL Tolerable Upper Intake Level

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Abstract

The Korean diet includes more shellfish then a typical western diet. Shellfish are bio-accumulators of toxins owing to their filtration based feeding method. The goal of this thesis was to conduct exposure assessments pertaining to the heavy metal and poly-aromatic hydrocarbon content in four widely consumed shellfish species found in Korea. In addition, steps were made into the quantification of microplastics found in shellfish samples. These preliminary findings were also translated into a probabilistic exposure assessment to indicate the levels of microplastics the Korean consumer is exposed to originating from shellfish. The GUGC provided heavy metal and poly-aromatic hydrocarbon contamination data for relevant shellfish samples from which 9 heavy metals and 16 PAH’s of interest were selected. Consumption survey data as well as Korean health statistics and bioaccessibility factors were compiled to construct exposure simulations in @RISK-software. The preliminary exposure assessment pertaining to microplastics was made possible by a four week experiment performed during a six week stay at the GUGC in Incheon, South Korea. It could be concluded that cadmium exposure was found to exceed the toxicological limit for a small percentage of the population making it a potential health issue. In addition to cadmium, arsenic exposure was found to be of concern while its maximum levels in shellfish are not regulated. Exposure to other heavy metals, some of them micronutrients were found not to exceed toxicological limits. The carcinogenic poly-aromatic hydrocarbons are not of health concern with exception to the consumption of the Yesso scallop, a potential health issue is connected to the consumption of this shellfish species in Korea. In regards to microplastics quantification in shellfish it could be stated that more development is needed. As of yet a time-efficient and reliable method has not yet been achieved.

Keywords:

heavy metals, poly-aromatic hydrocarbons, microplastics, probabilistic exposure assessment, shellfish, Korea

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Abstract

Het Koreaanse dieet bevat meer schelpdieren dan een typisch westers dieet. Schelpdieren zijn bio-accumulatoren van toxinen dankzij hun op filtratie gebaseerde voedingswijze. Het doel van dit proefschrift was om blootstellingsbeoordelingen uit te voeren met betrekking tot het gehalte aan zware metalen en poly-aromatische koolwaterstoffen in vier courant geconsumeerde schelpdiersoorten die in Korea voorkomen. Hiernaast zijn stappen ondernomen in de kwantificering van microplastics in schelpdierstalen. Deze provisoire bevindingen werden ook vertaald in een probabilistische blootstellingsbeoordeling om een indicatie te geven aan de hoeveelheden microplastics, afkomstig uit schelpdieren, waaraan de Koreaanse consument wordt blootgesteld. De GUGC leverde concentratiegegevens voor zware metalen en poly-aromatische koolwaterstoffen in relevante schelpdiermonsters waaruit 9 zware metalen en 16 PAK's werden geselecteerd. Zowel consumptiegegevens als Koreaanse gezondheidsstatistieken en “bioaccessibility”-factoren werden gecombineerd om blootstellingssimulaties te construeren in @RISK-software. De preliminaire blootstellingsbeoordeling met betrekking tot microplastics werd mogelijk gemaakt door een vier weken durend experiment dat werd uitgevoerd tijdens een verblijf van zes weken aan de GUGC in Incheon, Zuid-Korea. Er kan worden geconcludeerd dat de blootstelling aan cadmium voor een klein percentage van de bevolking de toxicologische limiet overschrijdt, waardoor het een potentieel gezondheidsprobleem is. Naast cadmium bleek blootstelling aan arseen zorgwekkend te zijn, terwijl de maximale gehaltes hiervan in schelpdieren niet gereguleerd zijn. Blootstelling aan andere zware metalen, waarvan sommige micronutriënten bleken de toxicologische limieten niet te overschrijden. De kankerverwekkende poly-aromatische koolwaterstoffen vormen geen gezondheidsrisico, met uitzondering van de consumptie van de “Yesso scallop”. Een mogelijk gezondheidsrisico is verbonden aan de consumptie van deze schelpdiersoort in Korea. Met betrekking tot de kwantificering van microplastics in schelpdieren kan worden vastgesteld dat er nog veel vooruitgang vereist is. Een betrouwbare en tegelijk tijds-efficiënte methode is tot nu toe niet gerealiseerd.

Sleutelwoorden:

zware metalen, poly-aromatische koolwaterstoffen, microplastics, probabilistische blootstellingsberekening, schelpdieren, Korea

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1 Introduction

Food safety is a fundamental issue in the agrofood chain. The consumption of shellfish is an integral part of Asian cuisine. Due to the comparatively large amount of seafood consumed in Asia we can assume it as a staple food in the Asian culture, which has seen an undeniable “boom” in total population and economic development starting in the early seventies of the previous century. But with rapid economic growth comes growing pains and possible consequences for the ever increasing population.

A fast developing, loosely monitored industry can pose a great risk to the population. Consuming fresh, locally bought seafood was always and is still widely regarded as healthy. In the case of the Korean East coast, seafood caught in the seas between China and Korea, the former the most polluting country in the world. The discharge of industry is bound to creep its way into the food chain. Is to consume food provided by the fishing industry still safe or are pollutants catching up to the average Korean consumers and their diet?

Clams, oysters, cockles, mussels and scallops are some examples of aquatic molluscs the average Korean consumer is familiar with. These species belong to the class of the Bivalvia, of which the majority are so called filter feeders. This method of food acquisition relies on the straining of suspended matter and food particles from water, typically by passing the water over or through a specialized filtering structure. Being mostly immobile they actively pump water and all therein dissolved substances through their body while remaining attached to a substrate or while simply lying on the bottom sediment. It is in this way of feeding the so called bio-accumulation of water soluble toxins is realized.

A wide range of Bivalvian species are consumed. These either originate from the open seas and are therefore wild catch while others are grown and subsequently harvested in controlled environments, so called aquafarms. Different means of collecting different consumed species of filter feeders introduces a wide variety of possible contaminations to which the consumer might be exposed.

The total number of possible chemical hazards that could find their way into the food chain is substantial. Their presence constitutes a potential risk to the health of consumers of which they are mostly unaware. The compilation of dosage, bioaccessibility and the harmfulness of chemicals, to name a few, presents a daunting challenge to anyone in the agro food sector. To tackle food safety we will often regard the biggest risks as the ones to be mitigated first. Every substance has its origin and in an ideal world could thus be traced to its source. To discover it enables us for it to be eliminated from, or controlled in our food.

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2 To protect consumers, risk assessors carry out relevant risk assessments. It is then up to risk managers to inform governing bodies of their findings and aid legislators in drafting propositions and laws which should eventually provide the aforementioned protection to the consumer. Are correct standards in place regarding seafood to protect Koreans if they wish to consume shellfish? The overall objective of this master thesis is to quantify the chemical risk posed to the Korean population when consuming locally caught shellfish.

Specific objectives are :

1. Probabilistic exposure to chemical hazards originating from the consumption of fresh shellfish, how much is the Korean population at risk?

2. Aquatic mollusks are known bio-accumulators of water based toxins, which species pose the greatest risk to the consumers health?

3. Which chemical hazards are most profound and can their origins be traced to certain human activity?

4. How can we quantify microplastics in shellfish?

As stated before, the variety of chemical hazards that could pose a risk to human health is comprehensive. Going forward there will be a distinct focus on hazards of which there is specific data available, in this case originating from the Food Chemistry and Technology Research Center at Incheon Global Campus (South-Korea). At first, it was the objective to come to cumulative exposure calculations, by combining chemical with similar toxicologic end points. However, due to the nature of the chemicals, for which data were available it was not possible to go this far. Therefore, single exposures per type of chemical were conducted.

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Risk assessment:

microplastics

heavy metals

poly-aromatic hydrocarbons

Desk research

Hazard identification and risk characterisation

Shellfish consumption pattern Korean population

Exposure calculations

Explorative lab work

GUGC 6 weeks contaminants in clams

Protocol regarding quanti-fication of microplastics

Data transfer metals and poly-aromatic hydrocarbons

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2 Literature study

The subsequent literature study encompasses five topics related to the exposure assessments carried out in following chapters. Firstly, situating the shellfish species of interest followed by a relevant hazard identification of microplastics, heavy metals and poly-aromatic hydrocarbons. Consumption of shellfish in Korea is touched upon as well as the mitigating factor of bioaccessibility. Closing out the literature study is an exploration of earlier exposure assessments pertaining to shellfish.

2.1 The investigated shellfish species

A central aspect in every exposure assessment is the food matrix which delivers the focused upon contaminants to the consumer. The four investigated shellfish species are elaborated upon in the following table and subsequent images 1 to 4.

Scientific classification

Common name Class Family Genus Species

Bivalvia

Veneridae Ruditapes philippinarum Manila clam Pectinidae Mizuhopecten yessoensis Yesso scallop

Arcidae Tegillarca granosa Blood cockle Arcidae Anadara broughtonii Blood clam Table 1: Scientific and common nomenclature for the four investigated shellfish species (Flanders Marine Institute, 2020)

Images 1-4: Specimens of the four shellfish species. FLTR: Manila clam, Yesso scallop, Blood cockle and Blood clam (Flanders Marine Institute, 2020)

These, in South Korea widely consumed shellfish species, are either sourced naturally or are commercially grown. The latter being an important branch of Korean fishery known as

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5 aquaculture. Regardless of their origin, most Bivalvia share the same feeding method: filter feeding. Through evolution their gills have taken over the role of trapping food particles, such as phytoplankton, as well as respiration. Inherent to this feeding method is the processing of large amounts of inhalant water and subsequent exposure to contaminants via three different routes: contaminants in solution, ingestion of contaminated food or ingestion of particulate matter containing contaminants (Phillips & Segar, 1986). Bivalves occur naturally all around the world and have garnered a reputation as being excellent bio-indicators of different forms of marine pollution including metals, poly-aromatic hydrocarbons and microplastics (Bruner, Fisher, & Landrum, 1994; Januar, Dwiyitno, Hidayah, & Hermana, 2019; Yusof, Yanta, & Wood, 2004).

2.2 Hazard identification of chemical contaminants in seafood

It should be stated that the array of possible contaminants in seafood is substantial. As a result, a selection of contaminants was made based upon the ongoing research at the GUGC food chemistry and technology research center. This selection includes microplastics, 9 heavy metals and 16 poly-aromatic hydrocarbons.

2.2.1 Microplastics

Worldwide annual production of plastics has been steadily increasing since 1950 and was estimated at 359 million tons in 2018. The term plastic envelops more than twenty families of polymers among which 6 are referred to as the “big six”: polyethylene terephthalate (PET), high- and low-density polyethylene (HDPE and LDPE), polypropylene (PP), polystyrene (PS), polyurethane (PUR) and polyvinyl chloride (PVC). They constitute 80% of the plastic production in Europe (Plastics - the Facts, 2019). Very little plastic is recycled and it fragments or degrades at a very slow rate, thus accumulating in all environments. The first observations of microplastic pollution in marine ecosystems were recorded in 1972 (Carpenter, Anderson, Harvey, Miklas, & Bradford, 1972). More recently, it has been estimated that about 10% of plastics produced worldwide eventually end up in oceans. Marine litter is comprised for 60 to 80% out of plastics (Thompson, 2006) (Moore, 2008).

It is widely reported that plastics pose a major threat to the wellbeing of the world’s oceans and seas. An important facet of plastic pollution is the occurrence of microplastics in the aquatic ecosystem (Vandermeersch, et al., 2015). Plastics in oceans are encountered in macro- (>25 mm), meso- (5–25 mm) and microplastic (<5 mm) forms. Primary microplastics are referred to as microparticles produced as such, plastic pellets, exfoliating cosmetics or synthetic clothing fibers, while secondary microplastics are derived from the breakdown of larger plastic debris (Cole, Lindeque, Halsband, & Galloway, 2011).

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6 The lower limit in dimension of what constitutes microplastics is not uniformly agreed upon. Recently however, the scientific community has adopted the term nanoplastic for particles smaller than a couple of micrometers. This differentiation is useful because increasingly smaller particles are harder to isolate from their substrate with simple methods used for microplastics, such as filtration. Additionally, at these small dimensions, there is a potential risk that instead of simply accumulating in the gastro-intestinal system of living organisms, these nanoplastic could penetrate tissues much more easily than larger particles (Nature, 2019). As stated before, nanoplastics are difficult to isolate from their environment with simple methods and as such are not a part of the ongoing research at the GUGC food chemistry and technology research center. Microplastics have manifested themselves in waterbodies across the world and have been encountered in all levels of the marine environment. Microplastic ingestion has been demonstrated in a wide array of marine organisms. They can have chemical, physical and biological impact on organisms that ingest them directly or indirectly through the consumption of contaminated prey (Vandermeersch, et al., 2015).

In table 2 results of previous investigations into microplastic contamination in seafood are summarized.

Origin Matrix Species Levels (n/g w.w.) Size (µm) Reference

South Korea Oyster Mussel Clam Scallop C. gigas M. edulis T. philippinarum P. yessoensis 0,07 ± 0,06 (mean ± st.dev.) 0,12 ± 0,11 ( mean ± st.dev.) 0,34 ± 0,31 (mean ± st.dev.) 0,08 ± 0,08 ( mean ± st.dev.) 100 - 200 100 - 200 100 - 200 100 - 200

(Cho, Shim, Jang, Han, & Hong, 2019)

China Mussel P. viridis 1,52 – 5,36 (range) 250 - 2500 (Qu, Su, Li, Liang, & Shi, 2018) China Mussel M. edulis 2,7 (mean) 5 - 250 (Li, et al., 2016)

China Mussel Ark shell M. galloprovincialis Sc. Subcrenata 2,4 (mean) 10,5 ( mean) 5 - 250 5 - 250

(Li, Yang, Li, Jabeen, & Shi, 2015)

Belgium Mussel M. edulis 0,35 (mean) 1000 - 1500 (De Witte, et al., 2014)

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7 2.2.2 Heavy metals

The concept of heavy metals was defined as constituting all metallic elements that have a comparatively high density to water. They were defined as such in the assumption that their weight and toxicity are inter-related. The assortment of heavy metals includes metalloids, which share some of their properties with metals. Arsenic for example is such a metalloid which is able to cause toxic effects at low levels of exposure (Fergusson, 1990).

Heavy metals found naturally in the environment vary widely in their abundance and toxicity. Certain metals such as copper, iron, manganese and zinc are essential for the human body since they play important roles in various metabolic systems. However, others such as cadmium, lead and mercury can cause harm to the human body, even when one is exposed to trace amounts. In general, heavy metals accumulate in marine organisms, especially in various species of molluscan shellfish to which the Bivalvia belong. The accumulation patterns of heavy metals in aquatic organisms depend on both the specific uptake and elimination rate of said metal in the particular variety. They are absorbed through different organs of the marine organism and accumulated at different levels in various organs of the subject.

To protect public health, the Korean authority has established regulatory limits for three heavy metals being cadmium, lead and mercury in the edible parts of molluscan shellfish. The internal organs of the Bivalvia are officially not considered edible by the Korean health authorities. However, internal organs of these species are consumed in many cuisines including the Korean one. For the consumer the consequences of being exposed to heavy metal pollution can be quite substantial. The regular monitoring of the amount of heavy metals in edible and “non-edible” tissues of molluscan shellfish is necessary to provide accurate information to consumers about the hazard levels (Mok, et al., 2015).

The aforementioned metals and their maximum allowed concentration in the edible parts of shellfish are stated in the table below. Methylmercury is also included to illustrate the lack of a regulatory limit regarding its presence in shellfish whilst metallic mercury is indeed regulated.

Contaminant Cadmium (mg/kg w.w.) Lead (mg/kg w.w.) Mercury (mg/kg w.w.) Methylmercury (mg/kg w.w.) Max. Concentration - Korea < 2,0 < 2,0 < 0,5 Not specified

Max. Level - EU 1,0 1,5 0,50 Not specified

Table 3: Regulatory limits for metals in the edible parts of shellfish expressed in milligrams per kilogram of wet weight (Korean Ministry of Food and Drug Safety, 2019) (European Commision, 2006)

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8 These regulations express the presence of metals on a “wet weight” basis whilst in the remainder of this work the term “fresh weight” is utilized. All these metals will be explored further in the continuation of this chapter.

2.2.2.1 Arsenic (As)

Arsenic is an omnipresent metalloid that is detected in small concentrations in a wide variety of environmental matrices. The most important inorganic forms of this element include the trivalent arsenite (As3+_{) and the pentavalent arsenate (As}5+_{). The organic forms of interest are the so-called}

methylated metabolites: monomethylarsonic acid, dimethylarsinic acid and trimethylarsine oxide (Bentley & Ghasteen, 2002).

Environmental pollution with elemental arsenic or arsenic based compounds occur as a result of certain natural phenomena like volcanic eruptions or the erosion of soil through rainfall or bad agricultural practices. (Agency for Toxic Substances and Disease Registry, 2000). Various arsenic containing compounds are produced industrially for agricultural means. They include insecticides, herbicides, fungicides and rodenticides. They have also been used widely in veterinary medicine for the eradication of tapeworms and roundworms found in pigs, cattle and sheep (Tchounwou, Wilson, & Ishaque, 1999). Arsenic containing compounds have also been used in the medical field for at least a century in the treatment of syphilis and dysentery for example. These drugs are still being used today in the treatment of certain tropical diseases such as African sleeping sickness (Centeno, et al., 2005). Most cases of human toxicity have been associated with exposure to the inorganic forms of arsenic. Trivalent arsenite being two to ten times more toxic in humans than pentavalent arsenate (Goyer, 2001). By binding to thiol or sulfhydryl groups on various proteins, trivalent arsenite can denaturalize over 200 enzymes. This binding is the mechanism responsible for arsenic’s broad array of negative effects on different organs. Pentavalent arsenate, being less toxic, replaces phosphate in certain metabolic processes, disrupting them (Hughes, 2002).

In regards to European legislation for arsenic, the Panel on Contaminants in the Food Chain (CONTAM) concluded in 2015 that the provisional tolerable weekly intake (PTWI) of 15 µg/kg b.w. established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2009 was no longer appropriate. New data had shown that inorganic arsenic causes lung-, urinary bladder- and skin cancer at exposures lower than those previously reviewed by the JECFA. the Panel therefor established a BMDL01 between 0,3 and 8 μg/kg b.w. per day for an increased risk of said

cancers as well as skin lesions (EFSA, 2009). In the following table 4, results of specific studies regarding arsenic contamination in seafood are summarized.

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Origin Matrix Species Levels (µg/g w.w.) Reference

South Korea Freshwater snail Clam Mussel Cipangopaludina japonic Corbicula leana Anodonta woodiana 1,120 ± 1,567 (mean ± st.dev.) 0,997 ± 1,076 (mean ± st.dev.) 3,559 ± 2,961 (mean ± st.dev.) (Habte, et al., 2015)

South Korea Oyster Crassostrea gigas 2,690 ± 0,885 (mean ± st.dev.) (Mok, et al., 2015) South Korea Mussel Mytilus galloprovincialis 2,255 ± 0,902 (mean ± st.dev.) (Mok, et al., 2014)

Peru Octopus Sea snail Scallop O. mimus B. ventricosa Cymatium sp. A. purpuratus 2 - 27 (range) 4 - 101 (range) 2 - 9 (range) 1 - 3 (range)

(Loaiza, De Troch, & De Boeck, 2018)

Sweden Mussel Mytilus edulis 1,94 (mean)

< 2 - 2 (range) (Kollander, et al., 2019) Japan Oyster Clam Mussel Sea snail

Ostrea gigas thunberg Ruditapes philippinarum Meretrix meretrix Linnaeus Scapharca subcrenata Mytilus edulis

Neptunea cumingi crosse

2,01 (mean) 1,38 (mean) 3,18 (mean) 2,76 (mean) 3,28 (mean) 1,41 (mean) 1,17 (mean) 1,30 (mean) 1,17 (mean) 5,12 (mean) 3,36 (mean) (Li, et al., 2019)

Table 4: Literature on arsenic contamination in aquatic organisms

2.2.2.2 Cadmium (Cd)

Cadmium is a heavy metal of substantial environmental and health-related concern. It is found in most soils at an average concentration of 0,1 mg/kg. High levels of cadmium compounds are accumulated in the environment in sedimentary rocks or phosphates, containing about 15 mg /kg (WHO, 1987). Prevalent industrial applications of cadmium include, but are not limited to, the production of metal alloys, pigments, and batteries (Wilson, 1988). The use of cadmium in batteries showed considerable growth at the turn of the millennium but its use has declined steadily following environmental concerns linked to nickel-cadmium battery waste (EPA, 2009). The main pathways of exposure for humans are via smoke inhalation, with cigarette smoking being a key contributor, and ingestion. Exposure to cadmium is possible through a number of several sources, mainly the consumption of contaminated food or employment in the metal industry (IARC, 1993).

The metal is a severe irritant for both the pulmonary and gastrointestinal systems. This can be acutely fatal if inhaled or ingested in a large enough dose. Following ingestion, various symptoms

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10 such as abdominal pain, convulsions, loss of consciousness , muscle cramps, nausea and vomiting could appear within 15 to 30 minutes (Baselt & Cravey, 1995). Chronic exposure to cadmium reduces the body’s produced levels of acetylcholine, norepinephrine and serotonin (Singhal, Merali, & Hrdina, 1976).

In regards to European legislation surrounding cadmium levels in food, the CONTAM Panel analyzed numerous studies which explored the relationship between urinary cadmium levels and a certain protein excreted in the urine called beta-2-microglobulin. This protein acts a biological indicator of kidney function. The Panel established a tolerable weekly intake (TWI) of 2,5 µg/kg b.w. by applying the findings of these studies into a model which translated levels of urinary cadmium into an estimated dietary exposure. The Panel even concluded that the risk of adverse health effects for groups that have exposures at levels above this TWI was very low. This is due to the TWI not being based on actual kidney damage, but on early indications of changes in normal kidney function which would suggest possible kidney damage in the long run (EFSA, 2011). In the following table 5, results of specific studies regarding cadmium contamination in seafood are summarized.

South Korea Freshwater snail Clam Mussel Cipangopaludina japonic Corbicula leana Anodonta woodiana 0,010 ± 0,012 (mean ± st.dev.) 0,256 ± 0,248 (mean ± st.dev.) 0,024 ± 0,020 (mean ± st.dev.) (Habte, et al., 2015)

South Korea Oyster Crassostrea gigas 0,591 ± 0,186 (mean ± st.dev.) (Mok, et al., 2015) South Korea Oyster Crassostrea gigas 0,600 ± 0,156 (mean ± st.dev.) (Mok, et al., 2014) South Korea Mussel Mytilus galloprovincialis 0,197 ± 0.071 (mean ± st.dev.) (Mok, et al., 2014)

Peru Octopus Sea snail Scallop O. mimus B. ventricosa Cymatium sp A. purpuratus 0,1 - 0,2 (range) 0,2 - 5,5 (range) 0,1 - 0,3 (range) 0,2 - 1,1 (range)

(Loaiza, De Troch, & De Boeck, 2018)

Table 5: Literature on cadmium contamination in aquatic organisms

2.2.2.3 Cobalt (Co)

Numerous cobalt compounds are found in nature with an average cobalt soil concentration of around 25 mg/kg. It has some chemical properties that make it highly similar to iron and nickel (Barceloux, 1999). Inorganic cobalt predominantly occurs in two valence states being its cobaltous (Co2+_{) and cobaltic (Co}3+_{) forms. The former being more commercially and}

environmentally available than the latter (Paustenbach, Tvermoes, Unice, Finley, & Kerger, 2013). Cobalt ions are trace elements widely distributed in nature. As trace elements they are essential, in specific quantities, for normal physiological function. Cobalt ions play a role in the normal

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11 functioning of the immune system, regulation of gene expression, the forming of antioxidant defenses and the prevention of chronic diseases. Cobalt has an important biological role as a component of the vitamin B12, also named cyanocobalamin. Other cobalt based compounds have been described as being harmful for the environment and toxic for the human body following excessive exposure (Strachan, 2010). The main route of contamination for the general population is primarily exposure through ingestion of food and drinking water containing Co compounds or inhalation of dust in ambient air (Agency for Toxic Substances and Disease Registry, 2004). In terms of food consumption, the highest cobalt concentrations can be found in chocolate, butter, coffee, fish, fresh grains, leafy vegetables and nuts (Arnich, et al., 2012).

The different mechanisms of action for cobalt ions include generation of reactive oxygen species resulting in lipid peroxidation, the interruption of normal mitochondrial function, alteration of the calcium and iron homeostasis, interruption of thyroid iodine uptake and induction of genotoxic effects. Blood or tissue concentrations dictate which mechanisms are present in the toxic response to cobalt. Chronic exposure manifests itself as a clinical syndrome with a variable presentation of cardiovascular, endocrine and neurological symptoms depending on the systemic Co levels (Paustenbach, Tvermoes, Unice, Finley, & Kerger, 2013).

At this time the European Food Safety Authority (EFSA) has not yet given an opinion on dietary exposure to cobalt and subsequent toxicological values. However the Food Standards Agency (FSA) stated that in rats and mice, cobalt exposure is associated with adverse effects on spermatogenesis. Application of a 23 mg cobalt/kg b.w. per day dosage caused testicular effects, the severity of which increased in a dose related manner. This is the lowest observed adverse effect level (LOAEL) in animals. It is not yet known whether these effects on fertility also occur in humans exposed to cobalt but it would be safe to assume that they indeed do. Application of the following uncertainty factors: 10 for LOAEL to No Observed Adverse Effect Level (NOAEL) extrapolation, 10 for inter species variation and 10 for inter individual variation, a total cobalt intake of 0,023 mg/kg b.w. per day would not be expected to result in any adverse health effects in humans (FSA, 2003). In the following table 6, results of specific studies regarding cobalt contamination in seafood are summarized.

China Clam Mactra veneriformis

0,55 (mean) 0,81 (mean) 0,56 (mean)

(Li, et al., 2015)

India Clam Villorita cyprinoides 0,19 (mean) (Sivaperumal, Sankar, & Viswanathan, 2007) Venezuela Oyster Crassostrea rhizophorae 1,4 (mean) (Alfonso, et al., 2013)

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2.2.2.4 Chromium (Cr)

Chromium is a naturally occurring element which is found in animals, plants, and soil originating from the erosion of chromium containing rocks or volcanic dust and gases. Chromium has oxidation states ranging from Cr2-_{to Cr}6+_{but its elemental forms do not occur naturally in the}

environment. Chromium compounds however are stable with Cr in the trivalent state and it occurs in nature in this state in for example the ore chromite (U.S. Environmental Protection Agency, 1984).

In humans and animals, Cr3+ _{is an essential nutrient that plays its role in the carbohydrate, lipid}

and protein metabolisms by potentiating the action of insulin (Anderson, 1981). The biologically active form of chromium, called chromodulin, is an oligopeptide complex with four chromic ions as integral parts. Humans as well as animals are capable of transforming inactive inorganic chromium to physiologically active structures and although it is classified as an essential nutrient, exposure to high levels through either ingestion, inhalation or dermal contact may cause adverse health effects (Jacquamet, et al., 2003). In regards to legislation the CONTAM panel derived a tolerable daily intake (TDI) of 0,3 mg/kg b.w. per day for chromium based on a U.S. national toxicology program (NTP) chronic oral toxicity study on rats (EFSA, 2014). In the following table 7, results of specific studies regarding chromium contamination in seafood are summarized.

Origin Matrix Species Levels (µg/g w.w.) Reference South Korea Oyster Crassostrea gigas 0,215 ± 0,096 (mean ± st.dev.) (Mok, et al., 2015) South Korea Oyster Crassostrea gigas 0,269 ± 0,096 (mean ± st.dev.) (Mok, et al., 2014) South Korea Mussel Mytilus galloprovincialis 0,234 ± 0,120 (mean ± st.dev.) (Mok, et al., 2014)

1,37 (mean) 0,49 (mean) 1,95 (mean)

(Li, et al., 2015)

Tunisia Ark shell Arca noae

0,27 ± 0,12 (mean± st.dev.) 0,26 ± 0,04 (mean ± st.dev.) 0,24 ± 0,06 (mean ± st.dev.) 0,38 ± 0,16 (mean ± st.dev.)

(Ghribi, et al., 2020)

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13

2.2.2.5 Copper (Cu)

Copper is an omnipresent metal detectable in small concentrations in a wide variety of environmental matrices. A number of human activities release copper into the surroundings with mining operations being the main source of copper discharge to the environment. Release of the metal into water occurs from soil erosion and industrial or sewage treatment plant runoff (International Programme on Chemical Safety, 1998). Copper is found in the environment in three different valence states being Cu0_{, Cu}1+_{and Cu}2+_{. The state of the copper ion affects its solubility,}

therefore the copper forms found in aquatic environments will be different from those found on land (Florence, Morrison, & Stauber, 1992).

Copper is an essential micronutrient and is utilized to maintain cardiovascular integrity and lung elasticity in addition to regulating the iron metabolism and endocrine functions. The EFSA proposes an adequate intake (AI) for this metal of 1,6 mg/day for men and 1,3 mg/day for women (Panel on Dietetic Products, Nutrition and Allergies, 2015). Acute ingestion of excess copper can cause gastrointestinal tract disturbances and chronic ingestion can lead to liver toxicity in sensitive populations (Goyer, 1991). The SCF set a NOAEL of 10 mg/day on the basis of a 12 week supplementation study, which showed the absence of adverse effects on liver function at this dose. Using an uncertainty factor of two, a Tolerable Upper Intake Level (UL) of 5 mg/day was established for adults (SCF, 2003). In the following table 8, results of specific studies regarding copper contamination in seafood are summarized.

Origin Matrix Species Levels (µg/g w.w.) Reference South Korea Oyster Crassostrea gigas 32,48 ± 11,10 (mean ± st.dev.) (Mok, et al., 2015) South Korea Oyster Crassostrea gigas 34,45 ± 12,44 (mean ± st.dev.) (Mok, et al., 2014) South Korea Mussel Mytilus galloprovincialis 1,501 ± 0,722 (mean ± st.dev.) (Mok, et al., 2014)

2,34 (mean) 1,98 (mean) 1,05 (mean)

(Li, et al., 2015)

Tunisia Ark shell Arca noae

4,22 ± 0,77 (mean± st.dev.) 3,78 ± 0,16 (mean ± st.dev.) 5,03 ± 0,72 (mean ± st.dev.) 6,37 ± 0,40 (mean ± st.dev.)

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14

2.2.2.6 Lead (Pb)

Lead is a naturally occurring metal present in small amounts throughout the terrain, in addition various human activities such as fossil fuel usage and mining, contribute to the release of high concentrations. The metal has a wide array of agricultural and industrial applications (USGS, 2011). Since the late seventies, significant efforts have been made to decrease lead exposure. Mainly the elimination of lead as an additive in gasoline, the reduction of lead usage in paints, food cans, and plumbing. Despite these efforts, human exposure to lead remains a serious health problem because of its abundance and toxicity (Pirkle, et al., 1994).

Lead affects multiple systems or organs in the body including the central nervous-, endocrine- and reproductive system as well as the kidneys and liver. Exposure to the metal is of particular concern to pregnant women because it is prone to migrate to the developing fetus. Animal testing in addition to the monitoring of exposed humans link prenatal exposure to lead with various abnormalities in their offspring. Mainly lower birth weights, premature deliveries and neurological anomalies have been observed (Andrews, Savitz, & Hertz-Picciotto, 1994).

Lead has the innate ability to inhibit and even mimic the behavior of calcium in the human body. As a result lead could be incorporated into the bones of the skeletal system in place of calcium. The metal also binds to thiol or other amide groups of various enzymes which diminishes their activities. In addition to this lead competes with essential metallic micronutrients for binding sites. This inhibits normal enzyme activity or alters the transport of essential cations such as copper (Flora, Saxena, Gautam, Kaur, & Gill, 2007).

In regards to European legislation surrounding lead levels in food, following a review of the in 2010 available data the EFSA’s CONTAM panel concluded that the earlier established PTWI of 25 μg/kg b.w. set by the JECFA and endorsed by the SCF was no longer appropriate. Following this statement no new guidance level for the metal was established. The Panel found no clear threshold below which it was confident to state that no adverse health effects would occur (EFSA, 2010). The Panel assessed however that developmental neurotoxicity in young children and cardiovascular effects along with nephrotoxicity in adults were appropriate critical health effects to base a risk assessment upon. The respective Benchmark Dose Lower Confidence Limit (BMDLs) they calculated translated into dietary intake values are as follows:

 developmental neurotoxicity BMDL01, 12 µg/kg b.w. per day

 effects on systolic blood pressure BMDL01, 36 µg/kg b.w. per day

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15 In the following table 9 results of specific studies regarding lead contamination in seafood are summarized.

South Korea Freshwater snail Clam Mussel Cipangopaludina japonic Corbicula leana Anodonta woodiana 0,061 ± 0,062 (mean ± st.dev.) 0,246 ± 0,132 (mean ± st.dev.) 0,195 ± 0,100 (mean ± st.dev.) (Habte, et al., 2015)

South Korea Oyster Crassostrea gigas 0,150 ± 0,062 (mean ± st.dev.) (Mok, et al., 2015) South Korea Oyster Crassostrea gigas 0,117± 0,066 (mean ± st.dev.) (Mok, et al., 2014) South Korea Mussel Mytilus galloprovincialis 0.159 ± 0.122 (mean ± st.dev.) (Mok, et al., 2014)

Table 9: Literature on lead contamination in aquatic organisms

2.2.2.7 Manganese (Mn)

Manganese is an essential micronutrient and is the twelfth most abundant element on earth and the fifth most metal. Manganese is not found in nature in an elemental form but complexed in carbonates, oxides and silicates in abundant minerals. Manganese can mainly be found in its manganese dioxide form. Being an essential ingredient in steel alloys, inorganic manganese is additionally utilized in battery and glass production and is an important component of chemical fertilizers. Organic forms of manganese are used in agriculture as fungicides, gasoline additives and as a medical imaging compound (Agency for Toxic Substances and Disease Registry, 2012). Being a micronutrient, Mn is essential for normal development and body function across the human life span. Manganese binds to and regulates many enzymes throughout the body. For example, it is a required co-factor for arginase, an enzyme responsible for the urea synthesis in the liver (Keen, Ensunsa, & Clegg, 2000). The metal is of toxicological concern because overexposure can lead to a progressive, permanent, neurodegenerative illness with symptoms comparable to those of Parkinson's disease (Levy & Nassetta, 2003).

In general dietary manganese intake has not proven to cause any adverse health effects in the population and thus be a risk at conventional intake levels. The metal intake resulting from the consumption of animal based products is low and not a safety concern for consumers. This is also applicable for sensitive subgroups such as elderly people or infants (EFSA Panel on Additives and Products or Substances used in Animal Feed, 2013). The EFSA proposes an AI of 3 mg/day for adults. For the determination of an UL, the SCF noted that a NOAEL for critical endpoints from animal studies were not available. In addition, data in humans was limited resulting in their conclusion that a reliable UL could not be set. The US Institute of Medicine however established a NOAEL and subsequent UL for manganese of 11 mg/day for adults. This defining was based on a lack of adverse health effect reports from subjects with Western diets ingesting up to 10,9 mg/day

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16 (EFSA, 2013). In the following table 10, results of specific studies regarding manganese contamination in seafood are summarized.

23,95 (mean) 56,12 (mean) 7,99 (mean)

(Li, et al., 2015)

India Clam Villorita cyprinoides 1,07 (mean) (Sivaperumal, Sankar, & Viswanathan, 2007)

Tunisia Ark shell Arca noae

12,11 ± 4,60 (mean ± st.dev.) 11,41 ± 5,34 (mean ± st.dev.) 11,98 ± 3,51 (mean ± st.dev.) 19,60 ± 5,75 (mean ± st.dev.)

Table 10: Literature on manganese contamination in aquatic organisms

2.2.2.8 Mercury (Hg)

Mercury is a peculiar element in the way it occurs in nature in three forms. Elemental-, inorganic- and organic mercury each having their own toxicological profile. At room temperatures elemental mercury appears in a liquid state with a high vapor pressure and releases mercury vapor into the environment. Mercury also exists as the mercurous and mercuric cations, Hg1+_{and Hg}2+

respectively. In addition to this, the most frequently encountered compound of the organic form found in the environment is methylmercury (Clarkson, Magos, & Myers, 2003).

The heavy metal was used in the electrical industry in the production of batteries and thermostats as well as dentistry. Various industrial processes utilize mercury from the production of sodium hydroxide to it being used as a precious metal solvent (Tchounwou, Ayensu, Ninashvilli, & Sutton, 2003). Similar to lead, the demand for mercury peaked in the seventies and began to sharply decline between 1980 and 1995 as the result of legislation. Mercury additives in for example paints and pesticides were banned and the reduction of its use in batteries mandated (Guzzi & La Porta, 2008). The metal is an omnipresent environmental toxicant which instigates severe alterations in different organ systems and thus causes a wide range of adverse health effects (Sarkar, 2005). Limiting exposure is a challenge because of the various chemical forms in which mercury is present in the environment which make it unable to avoid (Holmes, James, & Levy, 2009). With regards to European legislation the CONTAM Panel considered new scientific information regarding the toxicity of these various forms of mercury and evaluated earlier provisional TWIs established in 2003 and 2010 by the JECFA. In its newly constructed opinion, the panel established a TWI for inorganic mercury of 4 µg/kg bodyweight which was in line with the earlier JECFA recommendations (EFSA, 2012).

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17 As stated before methylmercury is by far the most common form of organic mercury found in the food chain. In the aquatic environment, mercury is methylated by microbial and abiotic processes taking place mainly in the sediments of fresh and ocean water. With regards to methylmercury the CONTAM Panel considered several adverse health effects and established a TWI of 1,3 µg/kg b.w. for methylmercury based on the most acute effect, being prenatal neurodevelopmental toxicity (EFSA, 2015). In the following table 11, results of specific studies regarding mercury and methylmercury contamination in seafood are summarized.

Origin Matrix Species Levels Hg (µg/g w.w.) Levels MeHg

(µg/g w.w.) Reference South Korea Freshwater snail Clam Mussel Cipangopaludina japonic Corbicula leana Anodonta woodiana 0,002 ± 0,003 (mean ± st.dev.) 0,005 ± 0,007 (mean ± st.dev.) 0,052 ± 0,006 (mean ± st.dev.) - - - (Habte, et al., 2015) South

Korea Oyster Crassostrea gigas

0,009 ± 0,003 (mean ± st.dev.) - (Mok, et al., 2015) South

Korea Oyster Crassostrea gigas

0,014 ± 0,003 (mean ± st.dev.) - (Mok, et al., 2014) South

Korea Mussel Mytilus galloprovincialis

0,007 ± 0.003 (mean ± st.dev.) - (Mok, et al., 2014) Japan Oyster Clam Mussel Sea snail

Ostrea gigas Thunberg Ruditapes philippinarum Meretrix meretrix Linnaeus Scapharca subcrenata Mytilus edulis

Neptunea cumingi crosse

0,30 (mean) 0,48 (mean) 1,17 (mean) 0,10 (mean) 0,40 (mean) <LOD 0,30 (mean) <LOD <LOD <LOD 1,12 (mean) 2,90 (mean) 0,91 (mean) 4,73 (mean) 0,46 (mean) 2,51 (mean) 1,78 (mean) 1,29 (mean) 6,38 (mean) 0,74 (mean) 2,68 (mean) 4,01 (mean) (Li, et al., 2019)

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18

2.2.2.9 Nickel (Ni)

Nickel is encountered in various mineral forms and can be found throughout the environment. Bodies of fresh- or salt water, soil, air, plants, and animals all contain the metal. When found in nature it is usually combined into oxides or sulfides (Young, 1995). Nickel is released into its surroundings during mining operations and by the metal industry. These industries might also discharge nickel containing waste water into the environment. In addition, the metal is also released into the atmosphere by fossil fuel burning and incineration of waste (Agency for Toxic Substances and Disease Registry, 2005). Oral exposure to certain levels of nickel induce adverse health effect targeting the kidneys. Animal studies have brought to light additional developmental and reproductive toxic effects (Goyer, 2001).

The CONTAM Panel decided on a TDI of 2,8 µg Ni/kg b.w. as derived from a lower 95% confidence limit for a benchmark dose at 10 % extra risk (BMDL10) of 0,28 mg/kg b.w. for fetal loss in rats.

They did remark however that the current dietary exposure to nickel raises concern when considering the chronic exposure levels for all different age groups as some of these groups exceeded this TDI. As a result, systemic contact dermatitis (SCD) was selected as the critical health effect suitable for the assessment of acute effects of nickel. In testing the lowest BMDL10 of 1,1 µg

Ni/kg b.w. was derived for the incidence of SCD following oral exposure to nickel of human volunteers. The Panel applied a margin of exposure (MOE) approach and considered an MOE of 10 to be indicative of a low health concern pertaining to nickel. Overall they concluded that at the current levels of acute dietary exposure to nickel, there is a concern that sensitive individuals may develop these skin reactions (EFSA, 2015). In the following table 12, results of specific studies regarding nickel contamination in seafood are summarized.

Origin Matrix Species Levels (µg/g w.w.) Reference South Korea Oyster Crassostrea gigas 0,153 ± 0,062 (mean ± st.dev.) (Mok, et al., 2015) South Korea Oyster Crassostrea gigas 0,124 ± 0,043 (mean ± st.dev.) (Mok, et al., 2014) South Korea Mussel Mytilus galloprovincialis 0,358 ± 0,201 (mean ± st.dev.) (Mok, et al., 2014)

0,73 (mean) 1,70 (mean) 1,00 (mean)

(Li, et al., 2015)

Tunisia Ark shell Arca noae

0,35 ± 0,03 (mean ± st.dev.) 0,39 ± 0,13 (mean ± st.dev.) 0,49 ± 0,12 (mean ± st.dev.) 0,59 ± 0,12 (mean ± st.dev.)

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19 2.2.3 Polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are hazardous organic compounds consisting of two or more aromatic rings in various arrangements. PAHs, which are also classified as persistent organic pollutants, are well known carcinogens and their risks to human and animal health have been significantly reported over the last few decades. Especially the forms containing four benzene rings, benzo(a)anthracene and chrysene or more than five fused rings, such as benzo(a)pyrene and benzo(b)fluoranthene among others, are generally considered as genotoxic and carcinogenic to humans (Abdel-Shafy & Mansour, 2016).

PAHs are as a result of human activity present in the air, water and soil, and so coming into contact with them unavoidable. Once deposited into our surroundings these compounds can readily enter the agricultural food chain due to their tendencies to accumulate, migrate and transform themselves in the environment. The main sources of these compounds is fossil fuel combustion during various industrial processes. In addition to their presence in raw food, the processing and cooking of foods can also contribute to PAHs occurring. Thermal processes such as grilling, smoking, frying and roasting, are major routes for PAHs formation and accumulation of food. (Jaewook, Jun-Hyun, Shinwoong, & Kwang-Geun, 2018). In the past decade PAHs were evaluated by the International Programme on Chemical Safety, the SCF and by the JECFA.

The SCF concluded that for 15 PAHs, namely:  benz(a)anthracene  benzo(b)fluoranthene  benzo(j)fluoranthene  benzo(k)fluoranthene  benzo(g,h,i)perylene  benzo(a)pyrene  chrysene  cyclopenta(c,d)pyrene  dibenz(a,h)anthracene  dibenzo(a,e)pyrene  dibenzo(a,h)pyrene  dibenzo(a,i)pyrene  dibenzo(a,l)pyrene  Indeno(1,2,3-c,d)pyrene  5-methylchrysene

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20 There is a clear evidence of mutagenicity and or genotoxicity in somatic cells in experimental animals. Accordingly the SCF reasoned that these compounds may as such be regarded as potentially genotoxic and carcinogenic to humans as well and therefore represent a priority group in the assessment of the risk for long term adverse health effects following dietary intake. (SCF, 2002).

The CONTAM Panel also reviewed the available data on occurrence and toxicity of PAHs. They explored whether a so called toxic equivalency factor (TEF) approach to the risk characterization of the PAHs found in food was appropriate. Following their investigation it was deduced that the TEF approach was not scientifically valid in this case. An inherent lack of data from oral carcinogenicity studies on individual PAHs in addition to their different modes of action and some evidence of poor predictivity of the carcinogenic potency of different PAH mixtures led them to this assessment. Taking all of this in account the CONTAM Panel concluded that the risk characterization should be based upon the PAHs for which oral carcinogenicity data was indeed available. These were limited to benzo(a)pyrene and seven other PAHs that were measured in the two coal tar mixtures used in the carcinogenicity studies of (Culp, Gaylor, Sheldon, Goldstein, & Beland, 1998). The CONTAM Panel stated that these eight PAHs (PAH8), either individually or in a combination, are currently the only possible indicators of the carcinogenic potency of PAHs in food. PAH8  Benzo(a)pyrene  Benzo(a)anthracene o (Benzo(b)fluoranthene)  Benzo(k)fluoranthene  Benzo(g,h,i)perylene  Chrysene  Dibenzo(a,h)anthracene  Indeno(1,2,3-c,d)pyrene

For which, the seven highlighted compounds have been measured in the investigated four shellfish species.

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21 Besides the sum of the above mentioned eight PAHs, the sum of:

 Benzo(a)pyrene  Benzo(a)anthracene

o (Benzo(b)fluoranthene)  Chrysene

constitute PAH4 and the sum of:  Benzo(a)pyrene

 Chrysene form PAH2.

The CONTAM Panel concluded that solely benzo(a)pyrene is not a suitable indicator for the occurrence of PAHs in food. Based on currently available data relating to occurrence and toxicity, they stated that PAH4 and PAH8 are the most suitable indicators of PAHs in food, with PAH8 not providing much added value compared to PAH4.

For the evaluation of human and experimental animal data EFSA has proposed to use the Benchmark dose (BMD) methodology to derive a reference point on the dose-response curve. The use of the BMDL calculated for a Benchmark Response (BMR) of the confidence limit 10% (BMDL10) is considered an appropriate reference point for compounds that are both genotoxic

and carcinogenic. The main goal of calculating BMDL’s is to use them to estimate a daily oral exposure level for the human population that are likely to be without adverse health effects during one’s lifetime. Their reported BMDL’s being:

 BMDL10 of 0,07 mg/kg b.w. per day was chosen for benzo(a)pyrene

 BMDL10 of 0,17 mg/kg b.w. per day was chosen for the PAH2

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22 In table 13 results of specific studies regarding PAH contamination in seafood are summarized.

Origin Matrix Species Levels (ng/g w.w.) Reference

BaP PAH2 PAH4 PAH8

South Korea

Clam Venerupis

phlippinarum < 0,03 (mean) 0,31 (mean) 0,43(mean) 0,52 (mean)

(Hwang, Woo, Choi, & Kim, 2012) Oyster Crassostrea

gigas < 0,03 (mean) < 0,04 (mean) < 0,07 (mean) < 0,14 (mean)

Mussel Mytilus

coruscus < 0,03 (mean) < 0,04 (mean) < 0,07 (mean) < 0,14 (mean)

Ark shell

Tegillarca

granosa < 0,03 (mean) < 0,04 (mean) < 0,07 (mean) < 0,14 (mean)

South Korea

Oyster Crassostrea

gigas 0,71 (mean) N.D. N.D. 19,90 (mean) _{(Kim, et al.,}

2010) Clam Venerupis

phlippinarum 0,18 (mean) N.D. N.D. 9,35 (mean)

South Korea

Clam Venerupis

phlippinarum 0,28 (mean) 0,49 (mean) 0,81 (mean) 1,01 (mean)

(Hu, et al., 2005) Oyster Crassostrea

gigas 0,07 (mean) 0,07 (mean) 0,23 (mean) 0,54 (mean)

Ark shell

Tegillarca

granosa 0,02 (mean) 0,02 (mean) 0,23 (mean) 0,31(mean)

Clam N.D. 0,00 (mean) 0,00 (mean) 0,00 (mean) 0,00 (mean) Mussel Mytilus

galloprovincialis 0,00 (mean) 0,00 (mean) 0,40 (mean) 0,45 (mean)

Table 13: Literature on PAH contamination in aquatic organisms expressed in nanogram per gram wet weight, BaP = benzo(a)pyrene

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23

2.3 Bioaccessibility and bioavailability of metals in food

In an exposure assessment of metals, typically concentration data (achieved via analysis of metals in foods) is multiplied with consumption data. However, when doing so an overestimation of exposure is derived due to the fact that the complete metal content of the food matrix is not available for uptake in the human body. The mitigating factors that constitute this effect are called bioavailability and bioaccessibility. To construct an accurate assessment to any given compound, exposure should be calculated based on the amount of the element likely to be released, which is the bioaccessible amount, or absorbed, which signifies the bioavailable amount (Babaahmadifooladi , Jacxsens, Van De Wiele , da Silva, & Du Laing , 2020).

2.3.1 Bioaccessibility

The concept of bioaccessibility can be defined as the quantity of a compound released from a given food matrix into the gastro-intestinal tract during digestion and as such becomes available for absorption. This constitutes numerous digestive transformations of components into material ready to be absorbed by the epithelium cells of the gut (Heany, 2001). This digestive transformation of components is initiated in the mouth upon chewing of the food. The food matrix is predominantly broken down in the small intestines by bile, pancreatic- and other enzymes secreted from the intestinal mucosa (Gropper & Smith, 2009). Bioaccessibility is influenced by numerous factors such as the composition of the digested food matrix (Fernàndez‐Garcìa, Carvajal‐Lérida, & Pérez‐Gàlvez, 2009) but also by physicochemical properties such as pH, temperature and even the food’s texture (Neilson & Ferruzzi, 2011).

In the following tables 14-18, bioaccessibility of different metals reported in studies are summarized.

Arsenic bioaccessibility in shellfish

Matrix Species Bioaccessibility Reference

Clam Mactra quadrangularis 76,1 ± 0,9% (mean ± st.dev.)

(Liao, Wang, Li, & Zhao, 2018)

79,2 ± 2,1% (mean ± st.dev.) Scallop Argopecten irradias 90,4 ± 3,5% (mean ± st.dev.)

83,6 ± 7,1% (mean ± st.dev.) Oyster Ostrea gigas 90,2 ± 3,6% (mean ± st.dev.)

88,4 ± 2,5% (mean ± st.dev.)

Mussel Mytilus galloprovincialis 86 ± 5% (mean ± st.dev.) (Cano-Sancho, et al., 2015)

Clam Mya arenaria

83 ± 32% (mean ± st.dev.)

(Koch, et al., 2007) 36 ± 3% (mean ± st.dev.)

88 ± 7 (mean ± st.dev.) 44 ± 7 (mean ± st.dev.)

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24

Cadmium bioaccessibility in shellfish

Mussel Mytilus galloprovincialis 73,32% (mean) (Gedik, 2018)

Oyster Crassostrea gasar 51 ± 11% (mean ± st.dev.) (Kuranchie-Mensah, et al., 2016)

Oyster Crassostrea hongkongensis

27,1 ± 5,0% (mean ± st.dev.)

(Gao & Wang, 2014) 37,4 ± 6,4% (mean ± st.dev.)

34,9 ± 7,2% (mean ± st.dev.) 32,1 ± 7,8% (mean ± st.dev.) 21,0 ± 6,9% (mean ± st.dev.) Oyster Crassostrea gigas 62 ± 3% (mean ± st.dev.)

(Amiard, et al., 2008) 84 ± 13% (mean ± st.dev.)

Oyster Ostrea edulis 66 ± 14% (mean ± st.dev.)

62 ± 3% (mean ± st.dev.) Oyster Saccostrea cucullata 68 ± 7%(mean ± st.dev.) Mussel Perna viridis 36 ± 7% (mean ± st.dev.) Venus clam Marcia hiantina 48 ± 6%(mean ± st.dev.)

Table 15 : Literature on cadmium bioaccessibility in aquatic organisms expressed as percentage of total amount present

Copper bioaccessibility in shellfish

72,3 ± 3,7% (mean ± st.dev.)

(Amiard, et al., 2008) 97,4 ± 0,8% (mean ± st.dev.)

80 ± 8% (mean ± st.dev.) Oyster Saccostrea cucullata 61 ± 5% (mean ± st.dev.) Mussel Perna viridis 63 ± 20% (mean ± st.dev.) Venus clam Marcia hiantina 68 ± 3% (mean ± st.dev.)

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25

Lead bioaccessibility in shellfish

81,2 ± 9,3% (mean ± st.dev.)

(Amiard, et al., 2008) 34 ± 13% (mean ± st.dev.)

28 ± 2% (mean ± st.dev.) Oyster Saccostrea cucullata 19 ± 7% (mean ± st.dev.) Mussel Perna viridis 44 ± 4% (mean ± st.dev.) Venus clam Marcia hiantina 37 ± 5% (mean ± st.dev.)

Table 17 : Literature on lead bioaccessibility in aquatic organisms expressed as percentage of total amount present

Chromium bioaccessibility in shellfish

Mussel Mytilus galloprovincialis 58,44% (mean) (Gedik, 2018) Cobalt bioaccessibility in shellfish

Oyster Crassostrea gasar 90 ± 3% (mean ± st.dev.) (Kuranchie-Mensah, et al., 2016) Nickel bioaccessibility in shellfish

Mussel Mytilus galloprovincialis 83,11% (mean) (Gedik, 2018) Manganese bioaccessibility in shellfish

Oyster Crassostrea gasar 51 ± 27% (mean ± st.dev.) (Kuranchie-Mensah, et al., 2016)

Table 18 : Literature on chromium, cobalt, nickel and manganese bioaccessibility in aquatic organisms expressed as percentage of total amount present

Arsenic bioaccessibility ranges from 90,4 to 44%, cadmium ranges from 21,0 to 84%, copper from 63 to 97,4%, lead from 19 to 86,7% and manganese from 51 to 69,11%. Furthermore, bioaccessibility for chromium, cobalt and nickel were found to be 58,44%, 90% and 83,11% when consumed through shellfish. It can be concluded that there is wide variability between the different evaluated heavy metals. Furthermore there are substantial differences in accessibility when looked at a single metal’s bioaccessibility between species of shellfish or even different subjects of the same species.

In regards to the bioaccessibility of poly aromatic hydrocarbons from a shellfish matrix, little research has been conducted. The reported results of one study are included in table 19 below.

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26

PAH bioaccessibility in shellfish

Clam Meretrix lyrata 22 to 84% (range) (Fabíola, et al., 2018)

Table 19 : Literature on poly-aromatic hydrocarbon bioaccessibility in aquatic organisms expressed as percentage of total amount present

2.3.2 Bioavailability

Bioavailability is differently defined based on the context in which the term is used. In pharmacology, regarding drugs and other substances that act within the body, it is generally considered to be the quantity of the administered dose of a substance that gets into the circulation and is then not either complexed, excreted or metabolized before it can exert its intended biological effect (Heany, 2001). In nutritional sciences the notion of the bioavailable fraction of a contaminant designates the fraction of its total present concentration in the food matrix that can be absorbed in the body and reaches the systemic circulation (Babaahmadifooladi , Jacxsens, Van De Wiele , da Silva, & Du Laing , 2020).