Paper Kapita Selekta Analytic

Determined PAH (Policyclic Aromatic Hydrokarbon) in Seafood, Mussels by

using HPLC

JURUSAN KIMIAFAKULTAS MATEMATIKA DAN ILMU PENGETAHUAN ALAM

UNIVERSITAS HASANUDDINMAKASSAR

2016CHAPTER I

SENIATI SALAHUDDIN H31112281

RABIATUL ADAWIAH H31112

INTRODUCTION

Environmental monitoring is of great importance to ensure food safety.

That is in particular true when it comes to seafood, as seafood is without doubt an

integral part of any healthy diet. Chemical pollutants have received increased

attention in the last decade in relation to safety of seafood. These chemical

pollutants can either occur naturally or be a result of human activity

(Alfreosdottir, 2014).

Enormous quantities of noxious pollutants have been released into marine

ecosystems over the last few decades. Among these pollutants, heavy metals and

polycyclic aromatic hydrocarbons (PAHs) represent major pollutants of the

marine environment. Marine organisms tend to accumulate different dietary and

waterborne contaminants including heavy metals, PAHs and others from the

environment which they live in (Busaidi et. all, 2013).

Polycyclic aromatic hydrocarbons (PAH) are organic compounds

containing two or more fused aromatic rings made up of carbon and hydrogen

atoms. They belong to a group of ubiquitous environmental contaminants formed

and released during incomplete combustion or by industrial processes. They are

characterized by high mutagenic and carcinogenic potential. PAH can arise both

naturally and as a result of anthropogenic activity. The latter is a much more

important contributor of environmentally hazardous compounds (Węgrzyn et all,

2006).

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental

contaminants that may originate from a variety of incomplete combustion and

pyrolysis processes from anthropogenic and natural sources. A high amount of

PAHs is emitted from processing coal and during the incomplete combustion of

organic matter, such as fuel oils. PAHs and their substituted derivatives are a large

class of organic compounds containing two or more aromatic fused rings. Those

compounds containing five or more aromatic rings are known as heavy PAHs,

whereas those containing fewer than five rings are light PAHs. Many of these

compounds, namely benzo[a]pyrene, benzo[a]anthracene, dibenzo[a,h]anthracene

and chrysene, have been reported to possess carcinogenic and genotoxic

properties. Other PAHs that are not defined as carcinogenic may act as synergists.

It is well documented that causes of PAHs in food sources include contamination

by air pollutants, uptake from the soil and carbonisation of carbohydrates, fats and

proteins during food processing, such as smoking or high-temperature cooking.

PAHs can accumulate on the waxy surfaces of many vegetables and fruits. The

presence of PAHs in uncooked food, such as vegetables, seeds and grains, has

been demonstrated with certainty. Food authorities from different countries

worldwide have recommended different maximum residue limits. Most of these

limits have been related to the sum of the heavy (five- and six-nuclear) PAHs and

benzo[a]anthracene. In Spain, Italy and Canada, a limit of 3–5 ppb has been

recommended. In Germany, the recommended limits are 5 ppb for the sum of

heavy PAHs and 1 ppb for benzo[a]pyrene (BaP) (Yo et.all, 2013).

The analytical methods most frequently used for determination of the

carcinogenic PAH are HPLC with fluorescence detection and GC–MS.

Traditionally, procedures such as Soxhlet, solid-phase, and liquid–liquid

extraction, with previous saponification with KOH–methanol solution, have been

described for sample clean-up. These methods are, however, very difficult and

time and solvent-consuming, and, because they involve long and complex

procedures, are unsuitable for routine analysis. The reason is the complicated

nature of lipophilic matrices, the physicochemical properties of which (solubility,

molecular weight, etc.) are similar to those of PAH. One of the limitations of

single-wave-length UV and fluorescence detection is the lack of peak purity

determination and qualitative analysis other than retention time. A second,

additional, method of analysis, for example GC–MS is, therefore, usually

recommended when the compound cannot be easily identified by HPLC.

Compounds can also be identified by use of a diodearray detector. This detector is

not recommended for PAH determination, however, because of its lack of

selectivity and high detection limit.

According to the contaminant PAH in the enviroment, expecially in

seafoods. It is important to us for learning more about this compound, the damage

in the environtment and analyze the compound by using analytical methods.

1.1 Problem Formulation

1. What is the PAH

2. How to analysis the PAH with HPLC

3. What is the benefit study to analyze PAH

1.2 The Purpose of the paper

1. To learn more about PAH

2. To know analyze the PAH with HPLC

3. To know the benefit to study analyze PAH

CHAPTER II

LITERATURE REVIEW

2.1. Definition

Polycyclic organic matter (POM) defines a broad class of substances

including polyaromatic hydrocarbons (PAHs), also known as polynuclear

aromatics (PNAs). POM compounds are identified as a substance with up to seven

fused rings and theoretically millions of POM compounds can be formed. PAHs

are a group of several hundred organic compounds that are composed of two or

more fused aromatic rings in a linear, angular or clustered arrangement and, as

indicated in their name, they only contain carbon and hydrogen. “By definition all

PAHs compounds can be classified as POM but not all POM compounds can be

classified as PAHs” and today about 660 different PAHs have been. PAHs

normally occur as a complex mixture rather than a single compound. PAHs break

down over a period of days to weeks by reacting to sunlight or other chemicals in

the air. However, most PAHs do not dissolve easily in water where they stick to

solid particles and settle to the bottoms of lakes, oceans and rivers. Polyaromatic

hydrocarbons are included in the US Environmental Protection Agency (EPA,

1987) priority pollution list because PAHs represent the largest group of

compounds that are mutagenic, carcinogenic and teratogenic. According to US-

EPA, the following 16 PAHs are of main concern (see also Figure 1 for their

chemical structure): naphthalene, acenaphthylene, acenaphthene, fluorene,

phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, chrysene,

benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene,

dibenz[a,h]anthracene, benzo[g,i,h]perylene and indeno[1,2,3-cd]pyrene

(Alfreosdottir, 2014).

2.2 Formation

Formation of PAHs occurs largely through the combustion or pyrolysis of organic

matter either naturally or through various human activities. Their production is

favored by oxygen deficient flame in the temperature range of 650-900°C and

fuels that are not highly oxidized (Maliszewaska- PAHs are preferred products of

combustion under this condition because of kinetic pathways and

thermodynamics. However, they are not only formed at high temperature but also

at low temperature (<200°C) combined with high pressure over geological time

scale,e.g. during the generation of coal and mineral oil (Fetzer, 2000).

The mechanism of PAHs formation during combustion is complex and

primarily due to pyrolysis and pyrosynthesis. During heating, organic compounds

are cracked to smaller and unstable fragments (pyrolysis). These fragments are

highly reactive free radicals with a very short lifetime and are converted to more

stable PAHs through pyrosynthesis (Kim, 2008).

2.3 Characteristics

In pure form PAHs are usually colorless, white or pale yellow green at

room temperature as solids and can have a faint pleasant odor. Their physical and

chemical properties vary with their molecular weight and structure (see Table 1).

The general characteristics common to the class are high melting and boiling

points, low vapor pressure, and very low water solubility, which tends to decrease

Figure 1. Structure of the 16 PAHs specified in US EPA

with increasing molecular mass. They are highly lipophilic and easily absorbed in

organic solvents or organic acids International Union of Pure and Applied

Chemistry (IUPAC) has adopted a nomenclature for PAHs that are the accepted

international naming rules (Alfreosdottir, 2014).

2.4 PAHs in Marine Environment

PAHs are some of the most widespread organic pollutants in the marine

environment which enter the sea from offshore activates, operational and

accidental oil spills from shipping, river discharges and the air. therefore it is

possible to determine if the origin is of a pyrogenic or petrogenic source

(Alfreosdottir, 2014).

2.5 PAHs damage human body

Exposure to carcinogens early in life may have greater impact on children

than similar exposure in adults. Animal and human studies indicate that parental

exposure to PAHs can result in serious or irreversible effects in the fetus,

including cancer and low birth weight. A study published in 2012 showed that

children that were exposed to high levels of environmental PAHs in the womb,

show a higher risk of developing anxiety, depression and attention problems

before the age of seven (Perera et al., 2012).

Table 1physical and chemical properties of 16 PAHS

2.6 Mussels as a biomarker

Biomarkers can be useful to provide an early warning signal of harmful

effects on biological systems and for estimating biological effects due to

contaminants. For decades marine bivalves like mussels have been successfully

used as biomarkers to indicate marine pollution due to their ability to

bioaccumulate organic and toxic chemicals such as PAHs from the water,

sediments and their food sources. Mussels are regarded as good candidates

because contaminant levels in their tissue respond to changes in environmental

levels. and they accumulate pollutants with little metabolic transformation

(Alfreosdottir, 2014).

2.7 Sources of PAH in seafood

According to the Thematic Assessment of Hazardous Substances

(HELCOM 2010), the highest levels of PAHs are observed in lagoon areas (e.g.

Szczecin lagoon), in the vicinity of harbours (e.g. port of Copenhagen) or in the

accumulation areas (e.g. Arkona Deep or Gdańsk Deep). Likewise in this core

indicator report, high PAH concentrations were found in similar areas.

Detectable concentrations of anthracene have been found in fish from

Swedish background stations. It has been measured in sediment from the

Stockholm area (with concentrations falling inversely with distance from central

Stockholm) and homogeneous coastal samples, indicating small local impact. It

has also been measured in detectable concentrations in water areas sampled with

the use of passive sampling devices. Fluoranthene is frequently present in fish

from Swedish background stations, and also found in sediment and sludge. It has

been found in all water samples from Sweden taken by means of passive sampling

devices, and it is detectable in groundwater samples (Swedish EPA 2009).

Wergzyn et. all (2006) by using HPLC method with fluorescence detection

has been developed for determination of 8 polycyclic aromatic hydrocarbons

(PAH) with four to six condensed aromatic carbon rings in edible oils and smoked

products. The method employs preparative size-exclusion chromatography for

efficient one-step lipid removal without saponification; benzo[b]chry-sene is used

as internal standard for quantification. Two other methods (liquid–liquid

extraction and solid-phase extraction) were tested for one-step clean-up and

sample enrichment but it was found that one-step procedures did not remove

lipids completely.

Linearity of calibration plots was good for all PAH in the concentration

range from the detection limit (approx. 0.1 ppb) to 100 ppb. The repeatability

(RSD, n = 6) for different PAH ranged from 0.5 to 5%. Analysis of standard

reference materials from the National Institute of Standards and Technology

(mussel tissue, SRM 2978), the Community Bureau of Reference (coconut oil,

CRM 458), and the Central Science Laboratory (olive oils, FAPAS 0615, 0618,

and 0621) resulted in a good agreement between measured and certified

concentrations (Wegrzyn, 2006).

Bejarano and Michel (2008) have been analysis of PAH body burdens in

blue mussels in winter in 2008 by using HPLC. More than 100 kilometers of

coastline on Unalaska Island were oiled by the Selendang Ayu grounding, creating

both short and long term biological consequences, including biochemical evidence

of continued oil exposure through 2008 in harlequin ducks. The objective of this

research is to evaluate potential biological effects to other biota within the spill-

impact zone The Indigenous mussels were collected from intertidal areas within

the Selendang Ayu oil spill area, a reference area, and a human-impacted area –

and analyzed for PAHs. The research were collected mussels in two different

times, in february and in July/August.

Only a handful of studies have reported the relationship between tissue

body burdens and the adverse toxicological effects of total polycyclic aromatic

hydrocarbon (TPAH) in mussels1-4. These studies were used to assess the effect

of TPAH in blue mussels via two effect endpoints: scope for growth and

lysosomal destabilization. Scope for growth (SFG) is a measure of an individual

organism’s available energy for growth, as it balances energy gains and

expenditure losses. SFG has been used as a sensitive indicator of contaminant-

induced stress under field condition scenarios. Lysosomes are intracellular

organelles involved in essential cellular functions (i.e., membrane turnover,

nutrition, and cellular defense), and they can also sequester a variety of

contaminants. Lysosomal destabilization assays have been used to indirectly

quantify the effects of contaminant body burdens on cellular functions, damage,

and apoptosis.

.

The result from analyzed sample was presented on the table, which the

two effect endpoints used in the current analysis suggested little to no adverse

effects of TPAHs in blue mussels. In only one tissue sample (SKN11-C) mild

sublethal effects (SFG reduction > 25%) and increased lysosomal destabilization

(experimental model = 9% and FieldModel = 51%) were found relative to tissue

samples from lightly to moderately oiled sites. However, only one sample

indicating potential adverse effects does not allow generalization of effects to the

entire blue mussel population or to other invertebrates. Duplicate chemical

analysis of this sample suggests potential sample contamination of the chrysene

series.

The other research about the PAH in mussels were analyzed by Busaidi

et.all (2013), using the mussel spesies of Liochoncha ornata from the Omani Sea,

Arabian, East Asian. Mussels samples of Liochoncha ornata were collected for a

period of one year from July 2009 to June 2010 at monthly interval. The soft

tissue of mussel was analyzed to detect some polycyclic aromatic hydrocarbons

(PAHs). The bioaccumulation of PAHs in the mussels appeared to be selective

and ranged from 4.80 to 12.0 ng/g; wet weight. The most carcinogenic PAHs,

such as benzo (a) pyrene and dibenz (a,h) anthracene, were in all cases below the

Table 2 TPAH from the mussels

in winter 2008

^concentration (ng/g, dw)

^Naph=napthalenes,

Fluor=fluorenes

^Dibenz=dibenzothiophenes,

^Phen/Anth=phenanthrenes/anthrac

enes ^Chrys=chrysenes

limits of detection. No distinctive relationship was found between different size

class and contaminant uptake by the mussels.

In the other side of the planet, there was a research by Yoo et.all (2013)

supported by Korea Food Research Institute, they were compare the analytical

methods to analyzed contaminant of PAH in seafood by high-performance liquid

chromatography with fluorescence detection. The seafood samples using in this

research was mussels from the yellow sea, China. The samples were prepared

using two methods: the Quick, Easy, Cheap, Effective, Rugged, Safe

(QuEChERS) method and the alkali digestion method. The QuEChERS method

involved a convenient and effective solid–liquid extraction and a simple

purification. The alkali digestion method was comprised of a liquid–liquid

extraction after saponification with potassium hydroxide followed by purification.

The limits of detection (LODs) and limits of quantification (LOQs) of the

QuEChERS method ranged from 0.05 to 1.60 lg kg-1, and those of the alkali

digestion method ranged from 0.28 to 5.18 lg kg-1. The repeatability for all target

analytes was similar for the two methods, that is, 0.66–4.24% and 0.26–5.75% for

the QuEChERS and alkali digestion methods, respectively. At analyte

concentrations of 2.5–50 lg kg-1, the recovery of the QuEChERS method ranged

from 86.87% to 115.67% and that of the alkali digestion method ranged from

69.22% to 100.21%.

Figure 3Typical high-performance liquid chromatography with fluorescence detection chromatograms of the fourteen polycyclic aromatic hydrocarbons (PAHs): (a) a standard mixture of the selected PAHs prepared by the QuEChERS method, (b) an unspiked sample prepared by the QuEChERS method and (c) a spiked sample containing 2.5–12.5 ng g1- of PAHs prepared by the QuEChERS method.

As a result, the experiments showed that the recovery of fourteen PAHs

from the seafood samples was more than 91%, suggesting that the modified

QuEChERS method is areliable method for the sample preparation of seafoods.

CHAPTER III

METHODS

Analyze the PAHs in seafood (mussels), there are two preparation

methods we can use for analysis of polycyclic aromatic hydrocarbons

(Yoo et.all, 2013):

3.1 Sample preparation for the QuEChERS method

For the sample extraction step, a 2.0 g sample of manila mussels

homogenate was placed into a 50-mL centrifuge tube containing 2.0 g of

anhydrous MgSO4 and 0.5 g of NaCl. Then, 5 mL of acetonitrile was added to the

tube, and the sample was vortexed for 2 min to achieve a homogeneous sample.

After vortexing the samples, the tubes were sonicated for 10 min to enhance the

extraction efficiency and centrifuged for 5 min at 2300 g to produce a clear

supernatant layer. The extraction process was then repeated as described. The

supernatants were combined with those from the first extraction. For the sample

purification step, 1.5 mL of the extract was transferred to a centrifuge tube

containing 50 mg of PSA sorbents and 150 mg of anhydrous MgSO4. The sample

tube was shaken vigorously for 1 min and centrifuged at 2300 g for 2 min. A 1-

mL aliquot of the extract was filtered using a 0.2-lm nonsterile syringe filter, and

then, 500 lL of the cleaned extract was placed in an autosampler vial for the

HPLC/FLD analysis.

3.2 Sample preparation for the alkali digestion method

To analyze the PAHs from seafood using the alkali digestion method, the

shell from manila mussels was removed before being homogenised in a blender.

A 2.0 g manila clams sample was then saponified with 10 mL of 1 M KOH in an

ethanol solution for 30 min at 80 °C. Then, 5 mL of water and 5 mL of n-hexane

were added, and the samples were mixed by a shaker seven times in 5-min

intervals. All of the hexane fractions were collected. The fractions were

transferred into a 50-mL tube containing 1 g of Na2SO4 and 1 g of silica gel to

remove the water and then shaken for 10 min. The extract was filtered through

filter paper and gently concentrated under a nitrogen gas stream to approximately

1 mL. The 1-mL volume of the concentrated extract was filtrated through a 0.20-

lm syringe filter into an Eppendorf tube for the HPLC injection.

CHAPTER IV

DISCUSSION

According to the literature and research about seafood, mussels. We have

found the different consentration and contaminant of PAH. Data from the

literature are obtained may be influenced upon the amount of pollutants that enter

into the marine environment. the content of various PAHs in the marine

environment in knowable because mussels are regarded as good candidates

because contaminant levels in their tissue respond to changes in environmental

levels and they accumulate pollutants with little metabolic transformation.

CHAPTER V

CONCLUSION

PAH (Polycyclic Aromatic Hydrocarbon) study provides important

information about the impact of the body and PAH contamination in the marine

environment in recent years. PAH were danger to all living creature, arms to the

human body, because mutagenic and carcinogenic. There are two efficient, quick

and easy sample preparation procedures for the simultaneous determination of

fourteen PAHs in seafood, expecially in mussels were developed and validated

using HPLC. However, the QuEChERS method showed a higher separation

efficiency than the alkali digestion method for the determination of the fourteen

PAHs in seafood when using the HPLC.

Source References

Bejarano, A.C., Michel, J., 2010, Analysis of PAH in blue mussels in winter 2008.

Busaidi, M.A., Yesudhason, P., Al-Waili, A., Al-Rahbi, Al-Hartry, K., Al-Mazrooei, Al-Habsyi, S., 2013, Accumulation of Some Toxic Metals and Polycyclic Aromatic Hydrocarbons (PAHs) in Marine Clam Liochoncha ornata Collected from the Omani Sea, Academia Journals, 5 (9) : 238-247.

Yoo, M., Lee, S., Kim, S., Seo, H.Y., Shin, D., 2013, A Comparative Study of the Analytical Methods for the Determination of Polycyclic Aromatic Hydrocarbon in Seafood by High-Performanced Liquid Chromatography with Fluorescence Detection, International Journal of Food Science and Technology, 49 : 1480-1489.

Alfreosdottir, B.O., 2014, Polyclyclic Aromatic Hydrocarbon in Mussels from Iceland, Food Science and Nutrition School of Health Science, Iceland.

HELCOM, 2010, Hazardous substances in the Baltic Sea – An integrated thematic assessment of hazardous substances in the Baltic Sea, Sea Environ Proc, No. 120B.

Swedish EPA, 2009, Swedish Pollutant Release and Transfer Register (online).

Wegrzyn, E., Grzeskiewicz, S., Poplawska, W., Glod, B.K., 2006, Modified Analytical Method for Polycyclic Aromatic Hydrocarbons, Using SEC for Samples Preparation and RP-HPLC with Fluorescence Detection Application to Different Food Samples, Acta Cromatographica, 17.

(EPA) Environmental Protection Agency, 1987, Quality criteria for water, EPA 440/5-86-001, United States Environmental Protection Agency, Washington DC.

Fetzer, J.C., 2000, Polycyclic Aromatic Hydrocarbons: Chemistry and Analysis. John Wiley & Sons, Retrieved from http://books.google.com/books?id=X6NpmLR5FnwC&pgis=1

Perera, F. P., Tang, D., Wang, S., Vishnevetsky, J., Zhang, B., Diaz, D., Rauh, V., 2012, Prenatal polycyclic aromatic hydrocarbon (PAH) exposure and child behavior at age 6-7 years, Environmental Health Perspectives, 120(6), 921–6.