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Darren Pouv
Professor Pavia
Honors 222
June 8, 2014

Vertebrate and Invertebrate Metabolism of Polycyclic Aromatic Hydrocarbons

and its Implications on Managing Seafood Safety
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and widespread environmental
chemicals consisting of hydrogens and carbons arranged in a six-membered ring structure also
known as a benzene and or aromatic ring. PAHs consist of two or more fused aromatic rings and
are the main component of crude oil. Due to the aromaticity of its rings, PAHs display a high
melting and boiling point. Moreover, the dominance of carbons and hydrogens in the structure of
PAHs make them extremely hydrophobic and instead lipophilic, rendering them soluble in
organic solvents. Lipophilicity refers to the ability of a chemical compound to dissolve in fats,
lipids, oils, and other non-polar solvents. This property of PAHs poses a great concern because
it allows both vertebrates and invertebrates to bioaccumulate PAHs into the muscles of one
body in concert with PAHs presence, persistence, and disposition in the environment after an oil

Figure 1: The 16 PAHs identified as exhibiting carcinogenic activity.

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spill has taken place. While several PAHs are toxic, a subset of 16 PAHs has been identified by
the EPA as exhibiting carcinogenic activity as can be seen in Figure 1.
The presence of PAHs in sea water especially after an oil spill is of great concern for the
aquatic environment as it has major repercussions for the fishing and seafood industry. Due to
the lipophilic nature of polyclic aromatic hydrocarbons, its bioavailability after ingestion or
inhalation is significant,
especially in muscle tissue where
fat and lipid content is high. Due
to this fact, the main objective of
PAH metabolism in both
vertebrates and invertebrates is to
metabolize the hydrocarbons into
a chemical compound that is
polar. This would thereby allow
the chemical compound to be
soluble in an aqueous solvent
where it can then be excreted from the body in the feces or urine. The main enzyme system used
to metabolize PAHs is the mixed function oxidase system, also known as the mixed-function
oxygenase system, the aryl hydrocarbon hydroxylase system, the mixed-function oxidase, or the
cytochrome p-450-dependent mixed-function oxidase. The term refers to the fact that the enzyme
oxidizes two different substrates simultaneously using the two oxygen atoms of O2. The PAHs
are first expoxidated, then conjugated with glutathione, a step mediated by the enzyme
glutathione transferase. Epoxides not conjugated with glutathione are converted to phenols and

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diols where they proceed through a number of successive reactions with glucuronic and sulfuric
acid. These hydrolated PAH derivatives continue on through more successive reactions to
become a phenol epoxide, diphenol, triols, diol-epoxide, etc. The complexity of the number of
different metabolites and reactions involved in metabolizing PAHs can be illustrated in the
diagram above provided by IARC using benzo(a)pyrene as an example.
The metabolites and intermediates involved in this metabolic process pose a large concern
due to their carcinogenic effects. The epoxides and dihydrodiols produced as intermediates
during metabolism are capable of binding and reacting with the proteins and DNA in ones body.
As a result of this carcinogenic effect, developmental malfunctions may take place as well as the
growth of malignant and benign tumors. In terms of humans, the effects vary depending on the
length of exposure, the concentration of PAH, the length of PAH exposure, and also the innate
toxicity of the particular PAH. Benzo(a)pyrene for instance is the most common PAH to initiate
cancer in animals. Naphthalene has been observed to cause the breakdown of red blood cells, as
well as cause irritation, nausea, vomiting, cataracts, kidney damage, liver damage, and breathing
problems.
Both vertebrate and invertebrates combat the acute and chronic effects of PAHs in ones body
by utilizing the mixed function oxygenase enzyme systems to metabolize PAHs. However, what
strikes the difference between the metabolic capacities of vertebrates versus marine invertebrates
is that marine invertebrates are simply slower and less efficient in their ability to metabolize
PAHs. While invertebrates do have the necessary enzymes to metabolize PAHs through the
mixed function oxygenase enzyme system, they invertebrates lack the quantity of enzymes to
efficiently and quickly metabolize PAHs. As a result, this slow metabolic process allows for the

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high accumulation of toxic PAHs in the muscles and tissues of invertebrates considering PAHs
are highly available to seafood once the oil particles are dispersed into the water column.
Taking this into consideration, seafood safety is one area of concern particularly after any oil
spill takes place. Since humans usually consume seafood whole, one is essentially consuming all
the PAHs and metabolites present in body. Thus, a strategic and efficient method for monitoring
PAH levels in seafood with respect to human health is integral. While the slow and inefficient
metabolism of invertebrates is a factor responsible towards high PAH accumulation, feeding
strategies also affect invertebrates exposure to PAH. While fish absorb water-soluble PAHs
through gills, filter feeders such as oysters which possess feeding bivalves ingest oil droplets
mixed into the water column. Not only that, the lipid content of an organism can also affect the
levels of PAH accumulated in the body. For example, Heras and colleagues found that salmon,
who have a muscle lipid content of about 4.0%, have higher concentrations of PAHs than cod,
who only have about 0.75% muscle lipid content. These patterns can also be attributed to
invertebrates as well. Bender and colleagues observed that clams and oysters sampled during
spawning cycles in the fall displayed higher PAH levels than what was measured in the spring.
During the fall, lipid contents are higher because the production and deposit of eggs requires a
great investment in not only energy but also fat to ensure a successful spawning season. This
higher lipid/fat content inside the body correlates to increased PAH accumulation. Intertwining
this information as one, the concentration of PAHs in seafood can be contributed to a number of
physiological and behavioral factors. Moreover, measurement of PAH concentrations in seafood
is one that requires accuracy and precision to ensure the safety of human health in the aftermath
of an oil spill.

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The process of accessing safety levels in seafood is a two-step process that depends on the
results of both a sensory and chemical test. After the Exxon Valdez oil spill of 1989, the FDA
has developed an approach in developing a risk assessment on seafood; many state and local
government authorities also possess their own protocol for measuring seafood safety. According
to the FDA, there are two ways in which seafood is rendered inappropriate to be sold on the
market due to the effects of an oil spill. One of these is the presence of dangerous levels of PAHs
in seafood, which can possibly lead to carcinogenic metabolites that can affect DNA and protein
function. The second is a sensory test, also known as the sensory analysis. Using a persons
sense of smell, if a particular seafood smells or tastes like petroleum, it is rendered as tainted
and cannot be sold as food. While this does not necessarily mean that the particular food source
is of harm to human health, the fact that it is labeled as tainted due to the peculiar smell or
taste prevents it from being sold for human consumption.
Determining the safety of seafood with regard to human health after an oil spill is two-fold
process that first begins with the sensory analysis where trained staff members smell raw and
cooked seafood to determine whether there may be the presence of PAHs. According to NOAAs
website, Using your sense of smell is one of the best methods for determining the safety and
acceptability of seafood, moreover, NOAA continues to back up this procedure by stating that
sensory analysis is a commonly used tool in seafood safety and quality inspections. A NOAA
seafood inspector attempts to smell the distinct scent of oil or chemicals that may originate from
dispersants that would differ from the normal odor of regular fish. NOAA attempts to train their
inspectors to fine tune their sense of smell. Given the subjective nature of smelling, what may
appear to be of odor to one person could be interpreted as a normal scent for another. Thus,
NOAA attempts to train prospective seafood inspectors to not only repeat their sensory ability,

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but also standardize how they describe what they smell. The sensory analysis is the first step
performed to determine the risk of seafood before any chemical testing is done. A range of 6 to
11 samples of raw fish randomly chosen to be representative of a particular area out in the ocean
effected by the oil spill are brought to be analyzed by 10 seafood inspectors. Once the seafood
inspectors have smelled each sample of raw fish and recorded what he/she smells, the seafood is
then cooked, and the process repeats once again. At this point in time the seafood inspectors may
even try to sample the cooked seafood to determine if there are any irregular or foul tastes
present in the seafood. The cooking step is done in the hopes of releasing polycyclic aromatic
hydrocarbons that may be less detectible in its raw form. Furthermore, seafood inspectors may
also find themselves more sensitive to cooked seafood than they are to raw seafood.
Sensory analysis is our first line of defense in assessing seafood safety levels regarding
human health. If the seafood inspectors do not detect any presence of foul or chemical odors
permeating from the seafood due to the bioaccumulation of PAHs, a 200 gram mixture of all the
fish sampled in that trial is sent to the NOAA Northwests Fisheries Science Center in Seattle,
Washington for chemical testing. However if seafood inspectors detect a chemical odor
indicative of the presence of PAHs, then the area of which the samples were collected will
remain closed until subsequent sensory and chemical tests later on determine that the seafood
from that particular area does not harbor any unhealthy PAH levels.
After passing the initial sensory test, the main chemical tests performed to determine the
levels of PAHs in seafood is traditionally a gas chromatography and mass spectrometry
(GC/MS). Gas chromatography is a type of chromatography used for analyzing and separating
compounds that can be vaporized using an inert gas. Consisting of a moving phase which is our
inert gas and a stationary phase, our gaseous compounds from our mixture interact with walls in

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the column coated with the stationary phase. As a result, each fragmented compound elutes at a
different time, causing different compounds to have different retention times. This distribution of
retention times is what allows us to analyze the composition of PAHs in our sample. In fact, new
technology in chromatography has allowed for more effective ways of separating the different
fragments. New Liquid Chromatography (LC) with Fluorescense Detection (FLD) and tandem
mass spectrometry allows for more efficient separation of 26 PAHs, 6 alkylated derivative
metabolites, as well as 11 photo-oxidated byproducts in a single liquid chromatography run.
Mass spectrometry is a technique that produces fragments of atoms that comprise to form our
original sample being analyzed. A sample is first ionized or shot with electrons, causing the
original sample to break into an array of charged fragments. By accelerating these charged
fragments into a magnetic field, we are then able to separate these fragments by their mass-tocharge ratio. With this distribution, mass spectrometry allows us to piece out the mass and
structure of a particular molecule. In concert with gas chromatography, both of these chemical
tests allow scientists to identify individual PAH compounds, the different types of PAHs present,
and above all whether detected levels of PAH pose a threat to humans or not.
The importance of data reporting and interpretation should not be underestimated while
performing a GS/MS on a seafood sample. As we had discussed in class, the notion of calibration
and standardizing our observations is one that transcends into this topic of chemical testing.
Chemists need to ensure that when these tests are performed, the units that of which the results
are reported in need to be the same (whether the sample was dry or wet). Moreover, chemists
need to ensure that minimal detection limits of PAHs are the same among different tests as well
as that they are lipid-normalized. Since PAHs are highly lipophilic, it is natural that the
concentration of PAHs is higher in areas containing more lipids such as muscle. Thus, lipid-

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normalization must be a factor in interpreting and reporting these figures since we dont want to
attribute high PAH concentration simply because of a higher lipid concentration.
Once these chemical tests have been performed the only task left is to interpret these results
and determine whether the PAH levels present in the seafood pose a risk to human risk. A basic
equation summarizing a multitude of risk factors is used to assess cancer risks for PAHs in
seafood. The equation, using the polycyclic aromatic hydrocarbon benzo(a)pyrene as an
example:

RL stands for acceptable Risk Level. The risk level accepted by risk managers is 1 x 10-6
This number means that at a defined consumption rate over a defined exposure period would
yield a lifetime cancer risk of no greater than 1 in 1,000,000.
BW stands for Body Weight. This number is intended to represent the average body weight
of a consumer, and any number between 132-154 pounds is typically used.
AT stands for Averaging Time represents the average length of a human lifetime, which is
about 70 years. This time period of 70 years is of interest for examining whether cancer will act
as the endpoint for ones lifetime.
ED stands for Exposure Duration, which correlates nicely with AT, thus the default for this
factor is 70 years.
CR stands for seafood consumption rate. This number can vary depending whether we are
calculating this figure for a regular consumer or an upper-end consumer. Moreover, serving sizes
and the frequency of seafood meals one consumes is another critical juncture for uncertainty.
Using information from the Continuing Survey of Food Intake by Individuals (CSFII), we can

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approximate that 7.5 grams of seafood is consumed per person per day. Synthesizing these
factors into a relatively functional equation allows seafood risk assessments to be conducted in a
standardized fashion that also accommodates to the fluctuating factors affecting seafood risks
relative to human consumption.
Adherence to the guidelines set forth in performing seafood risk assessments after an oil spill
would assume equal adherence to the level of uncertainty the assessment brings forth. Upon
inhalation or ingestion of PAHs, vertebrates and invertebrates begin to metabolize these
chemical compounds through the mixed-function oxidase system, resulting in carcinogenic
metabolites that alter with protein function and DNA replication. It comes to no surprise that the
deficiency of enzymes present in invertebrates slows down this metabolic process and
subsequently leads to the bioaccumulation of PAHs, metabolites, and photo-oxidated products
that can be transferred to humans through consumption. The sensory tests, chemical tests, and
advisory/action level equations are prime lines of defense to prevent the adverse effects of PAHs
towards human health yet each tier of testing brings a certain level of uncertainty. Seafood
sensory inspectors need to be specially trained to sense chemical odors, chemists need to
calibrate their measurements to ensure accuracy when comparing different sets of data, and those
involved in assessing the overall health risk of the PAHs present need to make decisions such as
what an acceptable risk level is or what the seafood consumption of a particular demographic is.
The very nature of seafood risk assessment is founded on assumptions and uncertainty. Without
a doubt, seafood risk assessment has triumphed in the efforts to protect the safety and well-being
of humans. From a different point of view, the level of uncertainty underlying risk assessments
attributed to the complexity of PAH metabolism is problematic and misplaced.

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Eisler, R., Patuxent Wildlife Research Center., U.S. Fish and Wildlife Service., U.S. Fish and
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Seafood Safety After an Oil Spill | response.restoration.noaa.gov. (n.d.). Seafood Safety After an
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