Professional Documents
Culture Documents
SENIATI SALAHUDDIN
H31112281
RABIATUL ADAWIAH
H31112
JURUSAN KIMIA
FAKULTAS MATEMATIKA DAN ILMU PENGETAHUAN ALAM
UNIVERSITAS HASANUDDIN
MAKASSAR
2016
CHAPTER I
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 (Wgrzyn 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 35 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 GCMS.
Traditionally, procedures such as Soxhlet, solid-phase, and liquidliquid
extraction, with previous saponification with KOHmethanol 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 GCMS 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
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 USEPA, 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 650-900C 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 (<200C) 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
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).
Table 1physical and chemical properties of 16 PAHS
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 spillimpact 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
organisms available energy for growth, as it balances energy gains and
expenditure losses. SFG has been used as a sensitive indicator of contaminantinduced 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
.
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 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 solidliquid extraction and a simple
purification. The alkali digestion method was comprised of a liquidliquid
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, 0.664.24% and 0.265.75% for
the QuEChERS and alkali digestion methods, respectively. At analyte
concentrations of 2.550 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 3
Typical 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 2.512.5 ng g1- of
PAHs prepared by the QuEChERS
method.
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 0.20lm 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., AlMazrooei, 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),
9216.