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CHAPTER 2
LITERATURE REVIEWS
1. Polycyclic Aromatic Hydrocarbons, PAHs Generally, PAHs are
hydrocarbon compounds which can be found in coal
and all oil types, such as crude oil, petroleum, benzene, and
diesel. The PAHs structure
consists carbon and hydrogen atoms in the form of two or more
fused aromatic
(benzene) rings. A benzene ring shares a pair of carbon atoms
with another ring (Gary
& Petrocelli, 1985). In the purest form, PAHs are flat,
solid, and range in appearance
from colorless to white or pale yellow-green. In general, PAHs
in the environment are
produced from incomplete combustion of substances with carbon
molecules such as
oil, wood, or coal. Other anthropogenic sources include motor
vehicles, cooking
ovens, and cigarettes. Furthermore, PAHs can come from natural
sources which are
forest fires and volcanic eruptions. Additional, PAHs are also
found naturally in the
environment especially in some plant species.
The basic structure of PAHs includes carbon and hydrogen atoms
which are
arranged in the form of two or more fused aromatic rings. Two
aromatic rings are
fused together by sharing a pair of carbon atoms. Their
structures are in a single plane.
Generally, their molecular weight is in the range of 166 to 328.
The molecular weight
of each molecule depends on its number and position of fused
rings and other
components. PAHs are crystalline solid with high melting and
boiling points but with
low vapor pressure and water solubility. Furthermore, they have
a high affinity for
solid particles, especially with high organic content. Thus,
when they reach to the
aquatic environment, they tend to rapidly adsorb on solid
surfaces such as suspended
particles and sediments. Generally, PAHs in linear form are less
soluble than angular
or condensed form (NRCC, 1983). Some PAHs structures are shown
in Fig 2-1.
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5
Naphthalene (Naph) Fluorene (Fl) Anthracene (Ant)
Pyrene (Pyr) Chrysene (Chr) Benzo[a]pyrene (BaP)
Figure 2-1 The structure of some PAHs (Witt, 1995)
PAHs are classified as highly toxic pollutants; they can cause
both acute and
chronic effects in the organisms (Narro, Cerniglia, Balen, &
Gibson, 1992). PAHs can
cause mutation and cancer and also affect endocrine systems. Low
molecular weight
PAHs tend cause more acute effects, while the high molecular
weight PAHs tend to
have chronic effects (Ashok & Saxena, 1995). The
characteristics and toxicities of
some PAHs are shown in table 2-1.
Table 2-1 The characteristics and toxicities of some PAHs
(Adapted from Ashok &
Saxena, 1995; Chen et al., 2004)
PAHs Formula Sw, mg/L
(25 °C)
Kow Koc WDL SDL Acute
Toxicity
Fluorene C13H10 1.9 1.5x104 3.9x103 42.25 4.507 +
Phenanthrene C14H10 1.0-1.3 2.9x104 1.4x104 17.14 1.829 +
Anthracence C14H10 0.05-0.07 2.8x104 1.4x104 11.65 1.243 ?
Fluoranthrene C16H10 0.26 3.4x105 3.8x104 22.39 2.388 ?
Pyrene C16H10 0.14 2.x105 3.8x104 24.29 2.591 +
Benzo(a)anthracene C18H22 0.01 4x105 2x105 15.46 1.649 ?
Chrysene C18H22 0.002 4x105 2x105 10.66 1.137 ?
Benzo(e)pyrene C20H12 0.0038 106 - 76.05 8.112 ?
Benzo(k)fluoranthene C20H12 - 7x106 5.5x105 26.43 2.819 ?
Benzo(a)pyrene C20H12 0.0038 106 5.5x106 33.52 3.575 ++
(WDL: Determination limits in water, ng/L; SDL: Determination
limits in solid, ng/g)
+ mean Relative Degree of effect on Organism, - mean No effect,
?means Unknown
effect
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2. Sources of PAHs in the environment Sources of PAHs in the
environment can be classified to 2 sources.
2.1 Pyrogenic sources
Pyrogenic or Anthropogenic PAHs occur as effects of many
human
activities. These PAHs arise during incomplete combustion of
organic compounds or
fossil fuel, fuel combustion in vehicle engines (diesel and
gasoline) and in industry,
and other combustion of organic materials such as papers,
rubber, and organic waste.
2.2 Petrogenic source
Petrogenic or Natural PAHs are PAHs arising from natural sources
such as
forest fires, and volcanic eruptions, and also from some
plants.
3. The accumulation of PAHs in the aquatic environment PAHs can
reach to the aquatic environment by several routes such as
leakage
during transportation and illegal disposal. When oil reaches the
aquatic environment, it
will cover and spread over the water surface and then cover the
water as a gas. As a
consequence, the photolysis rate of algae is reduced by about
50%. Moreover, the cell
membrane of the algae is destroyed resulting in a loss of
potassium and manganese
(Rungreunsin, 1980). Almost PAHs will be adsorbed on suspended
particles and then
settling onto the bottom (IPCS, 1998).
Chen, Zhu, and Zhou (2006) studied on PAHs accumulation in water
and
sediments from Qiantang River which flow to Yanzi River China.
This river flows
through industrial, agricultural, and urban areas. They found
that most PAHs in the
water had 2-3 aromatic rings such as naphthalene, acenaphthene,
fluorene, and
phenanthrene. PAHs in sediments were slightly larger with 3-4
rings, such as pyrene,
fluoranthrene, and benzo(a)anthracene. However, the amount of
PAHs in sediments
from urban areas was higher than in remote areas.
Pattarasiriwong and Bunyoy (2002) studied on the distribution of
PAHs;
benzo(a)pyrene, benzo(k)fluoranthrene, and
benzo(g,h,i)perlyrene, in water sources in
the Bangkok, Nontaburi, Patumthani, and Samutpragran provinces.
They found only
benzo(k)fluoranthrene in water, while in sediments there was
benzo(a)pyrene,
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7
benzo(k)fluoranthrene, and benzo(g,h,i)perlyrene. They concluded
that the main
source of PAHs is transportation.
Table 2-2 PAHs contamination in sediments and water in several
countries (Chen,
Zhu, & Zhou, 2006)
Total PAHs concentration Site River / Country
Mean Range Water Quiantang River, China
Gao-ping River, China Tianjing River, China Minjiang River,
China Seine River, France Yellow River, China
283.3 ng/L 430 ng/L 174 ng/L
72400 ng/L 20 ng/L
70.3-1644.4 ng/L 10-940 ng/L
45.8-1272 ng/L 9900-474000 ng/L
4-36 ng/L 185-2182 ng/L
Sediments
Quiantang River, China Athabasca River, Canada Kishon River,
Israel Yellow River, China
313.3 ng/g
153.2 ng/g 76.8 ng/g
91.3-614.4 ng/g 10-34700 ng/g
59.5-298.9 ng/g 31-133 ng/g
Minjiang River, China River in Thailand Pear River, China
Tianjing River, China
433 ng/g 263±174 ng/g
4892 ng/g 10980 ng/g
122-877 ng/g
1434-10811 ng/g 787-1943000 ng/g
4. Effects of PAHs on aquatic organisms PAHs can exert effects
on aquatic organisms, especially filter feeder
organisms, such as mussel. Filter feeders filtrate water and
suspended particles into
their body, thus they also ingest PAHs which are adsorbed on
particles. Moreover, the
organisms which live on the bottom can absorb PAHs through their
skin because of
the hydrophobic characteristic of PAHs.
Bunyatumanon and Srilachai (2003) studied PAHs accumulation
around the
Gulf of Thailand. The amounts of low molecular weight PAHs was
higher than that of
high molecular weight, and PAHs were highly accumulated in green
mussels. Both
pyrogenic and petrogenic PAHs were found.
Since considerable amounts of fish are consumed by us, any
accumulation of
PAHs in the fish is of great concern. Following entry into the
fish, PAHs become
concentrated in the liver and then excreted in the bile.
4.1 Gall bladder
Toxic agents in the body are detoxified in the liver by coupling
to various
compounds, and the resulting molecules are secreted into the
bile ducts. The bile then
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8
flows into the gall bladder, where it is concentrated, and it is
then emptied into the
duodenum. The bile, together with the secretion from the
pancreas and the intestinal
epithelium, is responsible for the digestion of lipids,
carbohydrates and proteins in our
food.
4.2 Liver
The liver has several functions which are demonstrated
below.
4.2.1 Metabolizes nutrients such as carbohydrate, protein, and
lipid.
4.2.2 Produces plasma proteins
4.2.3 Eliminates xenobiotics
4.2.4 Produces bile
When toxicants reach the liver, they will be bound to proteins
and
transformed to inactive forms. However, some transformed
toxicants can not be
excreted from the body and, moreover, the may become more toxic
(Torsakulkaew,
Klinsukon, & Temjaleun, 1996).
5. PAHs toxicity testing A study of the dose-response
relationship of a toxicant is performed by
exposing the animal to increasing doses the toxic compound,
usually by injection or by
inhalation. The mean lethal dose (LD50), the most commonly used
term, is the dose
causing death in 50% of the animals. In the case of aquatic
animals, the exposure
duration must be determined because the effect of each toxicant
in the environment is
not equal. In dose-response relationship testing, there should
be at least 10
individuals/group, and the toxicant should be used at at least 3
concentrations.
Figure 2-2 Dose response relationship testing (Bunsaneor,
2002)
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9
No observed effect level (NOEL) is the level of toxicant which
does not exert
any observable effect on the animal (Torsakulkaew, 1996).
Lowest observed effect level (LOEL) is the lowest level of
toxicant which
causes an observable effect in the animal (Bunsaneor, 2002).
In dose-response relationship testing, there are several factors
which affect
the testing, such as water conditions, tested organ, age and sex
of the animal, route of
exposure, and animal species.
Montizaan (1989) studied the acute toxicity (LC50 in 24 h) of
benzo(a)pyrene
in a fish, Poeciliopsis lucida. The result revealed that LC50 in
24 h was 1.2-3.7 mg/l.
6. Biochemical alterations of PAHs in the organism Most of
toxicants which are absorbed into the organisms are in non
ionic
forms, especially PAHs. PAHs are highly soluble in lipid, and
thus they are rapidly
absorbed into the body. In the body, they accumulate and are
mainly metabolized in
the lung, kidney, and liver.
Figure 2-3 Biotransformation and excretion of PAHs
(Torsakulkaew, 1986)
Phase 1 Metabolism
Phase 2 Conjugation
More toxicity Conjugation with endogenous substances
Toxicant
inactive
More soluble in lipid More soluble in water
Absorption Toxicant metabolism Excretion
Conjugation with endogenous substances
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10
The metabolic process of PAHs (Timbrell, 1999)
Xenobiotic Metabolite Conjugate Increasing Polarity
The enzyme which is responsible for detoxification of PAHs is
Aromatic
Hydrocarbon Hydroxylase (AHH) which is a Cytochrome P-450
Microsomal Enzyme.
The first reaction in the metabolism of PAHs is an oxidation by
AHH
enzyme; PAHs are transformed to epoxide by several ways.
1. Epoxide is transformed to dihydrodial by the epoxide
hydrolase enzyme.
Then, dihydrodial is oxidized to dihydrodial epoxide by AHH.
Next, dihydrodial
epoxide can react further in two ways: 1). Transformation to
tetraols and 2). Reaction
with glutathione (GSH) and transformation to become a
glutathione conjugate by the
enzyme glutathione-s-transferase. Finally, the glutathione
conjugate is transformed in
several steps to mercapturic acid and then excreted out of the
body.
2. Epoxide directly reacts with glutathione-s-transferase enzyme
and is then
excreted out of the body in mercapturic acid form.
3. Epoxide is transformed to phenol, quinine, and diols. These 3
metabolites
can react with glucuronic and sulfuric acid and transform to
glucuronide and sulfate
conjugates which are excreted out of the body (U.S. DHHS,
1995).
Endogeneous Compound
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Figure 2-4 Metabolisms of PAHs in mammals (Adapted from U.S.
DHHS, 1995)
For cancer induction, PAHs in the body are metabolized to
epoxide
intermediates. Some forms of epoxide may be degraded to phenolic
derivatives which
can bind with glucuronic acid in the liver and transform into
glucuronidesulfate which
has a high water solubility. This compound can be excreted in
urine.
After metabolism, some PAHs metabolites are excreted, while
other
metabolites increase their toxicity and accumulate in lipid
tissues (Torsakulkaew,
1996). Moreover, some PAHs metabolites cause both acute and
chronic effects (Narro,
Cerniglia, Balen, & Gibson, 1992) such as mutations and
cancer related to their effects
on endrocrine systems (figure 2-5).
PAH-epoxide
PAH-dihydrodiols
PAH-epoxide
PAH-phenol diols
PAH-quinones
Reaction glucuronic acid
and sulfuric acid
Glucuronide Sulfate conjugates
PAH-epoxides
PAH-tetraols
glutathione
Premercapturic acid
mercapturic acid
Method 1 Epoxide hydrolase
Method 2 Glutathione (GSH)
GSH-S-trasferase
+
Aromatic hydrocarbon hydroylase:AHH Method 3
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Figure 2-5 Transformation of toxicants (Torsakulkaew, 1996)
7. Cytochrome P450 1A Cytochrome P450-dependent mono-oxygenases
(CYP450) are membrane-
bound enzymes which metabolize a range of exogenous (organic
contaminants) and
endogenous (steroids, fatty acids) substrates. They are
ubiquitous throughout the
animal kingdom, but in fish are often present at higher
concentrations in tissues such
as liver. CYP450s are Phase I enzymes which catalyse the
insertion of an oxygen atom
into xenobiotic molecules to facilitate the conjugation to an
endogenous substrate. The
terminal oxygenase of this enzyme system is an iron-containing
haemoprotein,
namely, cytochrome-P450. The system is called P450 because of
the wavelength
absorption maximum, after reduction with carbon monoxide, is at
450 nm. The
conjugation reactions are carried out by a second group of
enzymes (Phase II) such as
uridinediphosphate (UDP)-glucuronyl transferase (UDP-GT) and
glutathione S-
transferase (GST). These combined reactions have the effect of
making neutral, lipid-
soluble xenobiotic compounds more water soluble, thus
facilitating their elimination
from the body. Although this sequence of events is generally a
detoxification
mechanism, it can result in the activation of relatively inert
chemicals to highly
reactive and damaging intermediates.
In fish, one form of CYP450 termed CYP450 1A is induced by
environmental exposure to a diverse range of planar molecules,
including polycyclic
aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs),
dioxins and
pesticides. The enzymes responsible for this activity belong to
the 3-
Initial Chemical Interaction with DNA
Repair of DNA
Original state
Repair of DNA Carcinogenicity
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13
methylcholanthrene inducible family of cytochrome P450 and in
fish there appears to
be only a single gene (CYP450 1A1) in this family (Nebert &
Gonzalez, 1987; Nelson
et al., 1993).
The basis for the use of CYP450 1A in monitoring is the
phenomenon of
induction, whereby levels of the enzyme are increased by the
synthesis of new protein
following exposure and the levels of enzyme activity are
measurably increased. The
induction of CYP450 1A can therefore be used to monitor exposure
to bioavailable
planar contaminants (Stagg & Mcintosh, 1998).
7.1 Induction of CYP450 1A in Fish
If the mechanisms of induction in different groups of animals
are similar, it
may lead to a predictive capability regarding the nature of
inducing compounds.
Understanding those mechanisms, therefore, could be important in
interpreting results
of biomarker analysis. Representatives of teleost CYP450 1A are
induced by many of
the same compounds that elicit induction of CYP1A1 proteins in
mammalian species,
where mechanisms are better known. The results suggest that the
induction of CYP450
1A in teleost and mammalian species occurs by similar
mechanisms. Thus, the
analysis of mRNA has demonstrated that fish treated with
compounds such as ß-
naphthoflavone show increased amounts of mRNA for CYP450 1A
prior to increases
in the CYP450 1A protein itself (Stegeman et al., 1992).
The response involves a soluble protein present in cells at low
concentration
and known as the Ah (aryl hydrocarbon) receptor. The Ah receptor
binds strongly to
incoming inducing agents (figure 2-6). The receptor-inducer
complex apparently binds
to another protein (a translocating factor), which allows the
complex to enter the
nucleus. Once inside the nucleus, the complex attaches to
specific sites on DNA,
distorting the DNA chain and resulting in transcription of mRNA
that codes for
CYP450 1A. The mRNA is subsequently translated into new CYP450
1A, which is
inserted into the endoplasmic reticulum and supplied with a
molecule of heme. Heme
required at the active site of P450 is synthesized in the
mitochondria
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Figure 2-6 Induction of cytochrome CYP450 1A (Di Guilio, Benson,
Sanders, & Van
Veld, 1995).
7.2 Reaction Mechanism
The CYP450 reaction is initiated by substrate (RH) binding to
the oxidized
CYP (Fe3+). This binding induces conformational changes around
the heme, increasing
the heme iron redox potential, by which CYP450 becomes reduced.
Electrons are
transferred to the substrate-CYP complex from specific electron
carrier proteins by
reductase enzymes. The reduced state CYP (RH-Fe2+) has high
affinity for oxygen,
and one electron at the heme iron is donated to the bound oxygen
molecule (RH-Fe3+-
O2-). A second electron stabilizes the (RH-Fe2+-O2-)-form, one
oxygen reacts with
hydrogen ions and water is released. The other oxygen is
incorporated in the substrate,
forming a hydroxyl group (ROH). The substrate is released and
CYP450 is oxidized
(Fe3+) again (figure 2-7) (Omure, Ishimura, &
Fujii-Kuriyama, 1993).
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The general reaction catalyzed by a CYP enzyme can be expressed
as:
RH + NADPH + H+ + O2 ROH + NADP+ + H2O
Figure 2-7 The CYP450 catalytic cycle. (Stegeman & Hahn,
1994).
CYP450 1A mono-oxygenase activity is most conveniently measured
as 7-
ethoxyresorufin O-deethylase (EROD) activity and the method
described here is that
originally developed by Burke and Mayer (1974).
7-Ethoxyresorufin is an artificial
substrate, but is used because the assay is simple, sensitive,
highly specific for
CYP450 1A and also presents a low hazard to the operator. Aryl
hydrocarbon
hydroxylase (AHH) was used in many earlier studies, but it is
catalysed by a broader
spectrum of P450s. A previous TIMES leaflet (Galgani &
Payne, 1991) gives details
of a microplate method using a fluorescent plate reader.
Although advantageous from
the point of view of being able to process many samples very
quickly, it suffers from
high levels of variability and the lack of internal
standardization.
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Microsomal mono-oxygenase activity is the overall activity of a
multi-
enzyme system residing in the sacroplasmic recticulum which
comprises an NADPH
reductase and multiple CYP450s. Other components involved in the
reaction are
membrane lipids, cytochrome B5 reductase and cytochrome b5.
NADPH reductase is a
flavoprotein which accepts electrons from NADPH and passes them
on to CYP450.
The CYP450 component provides the system with its substrate
specificity, which in
the case of CYP450 1A is selective for planar aromatic
molecules. 7-Ethoxyresorufin
O-deethylase (EROD) reacts exclusively with CYP450 1A and is
converted from 7-
ethoxyresorufin to resorufin by deethylation of the hydroxyl
group on the para
position (figure 2-8).
Figure 2-8 The microsomal 7-ethoxyresorufin O-deethylase (EROD)
reaction
(Stegeman et al., 1992).
8. EROD activity To measure enzyme activity, the liver is
dissected from fish, a sub-sample is
homogenized in an appropriate buffer and then centrifuged
usually to yield a 10,000 x
g supernatant (the S9 fraction). Many investigators prefer to
work with a more refined
sample in which the S9 fraction is further centrifuged to yield
a 100,000 x g pellet
which is resuspended in buffer (microsomal preparation).
However, preparation if this
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refined sample would exclude measurements on research vessels
and restrict the
application of the technique in monitoring the marine
environment. Therefore, only the
use of S9 fractions will be described here.
EROD is measured in the S9 fraction in the presence of NADPH
using 7-
ethoxyresorufin (ethoxyphenoxazone) as substrate; the product,
resorufin, is
determined fluorometrically (Burke & Mayer, 1974). The
reaction can be followed in
the reaction cuvette because the characteristic emission spectra
of 7-ethoxyresorufin
(λEx 470 nm; λEm 560 nm) and resorufin (λEx 535 nm; λEm 585 nm)
show not only
good separation of the maxima, but also the relative
fluorescence intensity for
resorufin compared to that of 7-ethoxyresorufin is at least one
order of magnitude
greater (Prough, Burke, & Mayer, 1978). An important source
of variability in EROD
measurements is the purity of resorufin used to calibrate the
assay. Therefore,
resorufin standards should be made up taking into account the
purity of the resorufin
stocks used. This is most conveniently done by reference to the
extinction coefficient
of resorufin (Klotz, Stegeman, & Walsh, 1983) and adjusting
the weight of the
standard used to account for the purity measured.
The resorufin formed is derived from a resorufin standard curve
using the
following equation:
y = mx + b
where, y = samples fluorescence in fluorescence units
(subtracted with the
blanks)
m = slope of the resorufin standard curve (fluorescent units
nM-1)
x = resorufin concentration in the sample wells in nM
The resorufin concentration in the well in nM is then converted
in pmol. And
then the specific activity is calculated (Trudeau, 1995).
Resorufin (EROD activity) = [resorufin] nM x [200 x 10-6 L] x
1000 pmole.nmol-1
(pmoles/min/mg protein) 8 min x Total protein (mg)
where, 200 x 10-6 = Total volume (L)
8 min = Reaction time
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9. Fluorescence technique Fluorescence technique can be used for
measuring bile PAH metabolites and
EROD activity in liver because this technique is simple, fast,
and cheap (Aas et al.,
1998). The principle is to measure the luminescence of the
metabolite. When PAH
metabolites are excited by a beam with a certain wavelength,
they will emit a beam
with another wavelength . Fluorescence techniques can be
classified into 2 techniques.
9.1 Fixed wavelength fluorescence (FF)
For this technique, the wavelength of both excitation and
emission for each
metabolite are fixed. The metabolites are excited and emit at
the specific wavelength;
then the intensity of the beam (exciting and emitted) will be
used for calculating the
amount of PAH metabolites. Thus, the Em/Ex for naphthalene
metabolite is 290/335
nm, for pyrene metabolite 341/383 nm, and for benzo(a)pyrene
380/430 nm (Lin,
Cormier, & Torsella, 1996).
9.2 Synchronous fluorescence spectrophotometry (SFS)
The range of wavelength which is used determined, but the
specific
wavelength for each metabolite is not fixed. The result of this
technique is shown in
the area under curve. The standard range of wavelength for
measuring is about 200-
600 nm.
The luminescence level is measured by using Fluorometric or
Fluorescence
spectrophotometers which can be classified to Cuvette and Plate
reader (48 or 96-
well). Cuvette which is widely used can measure one sample in a
time. This technique
is appropriate for measuring fish bile PAH metabolites by fixed
wavelength technique.
The plate reader which can measure many samples in a time, is
appropriate for
measuring EROD activity in fish liver.
Jonsson, Sundt, Aas, and Beyer (2004) used Fluorescence
technique for
screening the specific wavelength of chrysene metabolite
luminescence in bile of
Atlantic Cod (Gadusmorhua L.) which had been exposed to
chrysene. They found that
the Ex/Em specific wavelength for chrysene is 272/374 nm.
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10. Respiration and oxygen consumption The respiration can be
divided to three phrases.
1. The external respiration: This is the mechanism which
exchanges carbon
dioxide in the body with oxygen from the external
environment.
2. The gas transport mechanisms: The oxygen obtained from
external by
respiratory organs is distributed to all cells in the body.
Moreover at the same time,
carbon dioxide is excreted.
3. The internal respiration: The oxygen is consumed within the
cells by
oxidation-reduction reactions producing carbon dioxide and
energy.
10.1 Respiratory and Circulatory system
Most fish exchange gases using gills on either side of the
pharynx. Gills
consist of threadlike structures called filaments. Each filament
contains a capillary
network that provides a large surface area for exchanging oxygen
and carbon dioxide.
Fish exchange gases by pulling oxygen-rich water through their
mouths and pumping
it over their gills. In some fishes, capillary blood flows in
the opposite direction to the
water, causing counter current exchange. The gills push the
oxygen – poor water out
through opening in the sides of the pharynx. However, most
fishes have a single gill
opening on each side. This opening is hidden beneath a
protective bony cover called
an operculum.
Many fish can breathe air via a variety of mechanisms. Lungfish
and bichris
have paired lungs similar to those of tetrapods and must surface
to gulp fresh air
through the mouth and pass spent air out through the gills.
Fish have a closed-loop circulatory system. The heart pumps the
blood in a
single loop throughout the body. In most fish, the heart
consists of four parts,
including two chambers and an entrance and exit. The first part
is the sinus venosus, a
thin walled sac that collects blood from the fish’s veins before
allowing it to flow to
the second part, the atrium, which is a large muscular chamber.
The atrium serves as a
one-way antechamber, sends blood to the third part, ventricle.
The ventricle is another
thick-walled, muscular chamber and it pumps the blood, first to
the fourth part,
bulbous arteriosus, a large tube, and then out of the heart. The
bulbus arteriosus
connects to the aorta, through which blood flows to the gills
for oxygenation.
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10.2 Metabolic rate
The metabolism is adjusted to the availability of oxygen
(Boutilier, 1990)
and is often expressed in terms of oxygen consumption.
The metabolic rate is the energy metabolism per unit time. It
can be
determined in three different ways.
The first is by calculating the difference between the energy
value of all food
taken in and the energy value of all excrete (primarily feces
and urine). This method
assumes that there is no change in the composition of the
organism. It therefore cannot
be used for growing organisms or in organisms that have an
increase or a decrease in
storage of fat or other material. The method is technically
cumbersome and is accurate
only if carried out over a sufficiently long period of
observation to assure that the
organism has not undergo significant changes in size and
composition.
The second method of determining metabolic rate is from the
total heat
production of the organism. This method should give information
about all fuel used,
and in principal it is the most accurate method. In practice,
determinations are made
with the organism inside a calorimeter. This can yield very
accurate results, but
technically it is a complex procedure. Such items as heating of
ingested food and
vaporization of water must be entered into the total heat
account.
The third measure that can be used to determine the metabolic
rate is the
amount of oxygen used in oxidation processes, provided
information is available about
which substances have been oxidized (and there is no anaerobic
metabolism). The
determination of oxygen consumption is technically easy and is
commonly used for
estimation of metabolic rates.
10.3 Oxygen consumption
The oxygen requirement of aquatic organisms influences on growth
rate,
activities, and survival rate. In general, the minimum oxygen
for living of the aquatic
organisms is 5 mg/l while for sea water fish is 3 mg/l. For
shrimp, crab, and shell, the
minimum oxygen requirement is 4 mg/l (Poxton & Allouse,
1982). The utilization of
oxygen at rest varies in different animal (Wickins, 1981) and
increases with varying
degrees with activity (Wilson, 1972).
In low oxygen contents, the response of animals to decreasing of
oxygen
content is higher than the response to increase oxygen content
demand. Adjustment to
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21
the availability of oxygen deficiency is carried out by
adjustment of blood circulatory
system and gill ventilation.
Factors influencing oxygen consumption are described below.
1. Sex: Oxygen consumption rate in male is higher than in
female, and
oxygen demand of seasonal maturation is higher than normal.
2. Size: Smaller animals have lower oxygen consumption than
larger ones
(Wilson, 1972).
3. Activity: High activities of animals result in an increased
oxygen
consumption. When an animal is stimulated or excited,
respiration and metabolism
increases markedly.
4. Stage of life cycle: During growth, the organisms use more
energy than
during normal conditions (Gardiner, 1972).
The oxygen consumption measures the amount of oxygen which the
animals
consumes per time unit. The oxygen consumption can be calculated
from the equation
is shown below (Napeetaput & Kabilrom, 1996).
Mo2 = (ΔPo2 sample – ΔPo2 blank) x ax V x 60/t/W (μmol. G-1.
h-1) ΔPo2 sample = Po2 (final) – Po2 (start) of water
ΔPo2 blank = Po2 (final) – Po2 (start) of water (without
fish)
a = water solubility of oxygen at that temperature (μmoll-1
torr-1)
V = water volume in chamber (l)
t = experimental time period (hr)
W = Weight of fish (g)
11. Osmoregulation Osmoregulation is the ability to regulate
water and solute concentrations
(Kay, 1998). It is possible to classify the osmotic response of
animals into two
categories; osmoconformer and osmoregulator. The vast majority
of marine crustacean
are osmoconformers, i.e., the osmotic concentration of their
body fluids is the same as
that of the seawater they live in. This means that they are in
osmotic equilibrium (there
is no net gain or loss of water).
The body and cell fluids of most marine organism are osmotically
similar to
seawater with salinity close to 33-35 ppt. When exposed to a
hypotonic environment
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22
(water with lower salinity than that of their internal fluids)
organisms tend to take in
excessive fluid. In hypertonic situations (where the ambient
fluid has higher salinity)
they lose fluids to the environment. Either situation can be
fatal.
All animals can survive in media of different salinities. They
may be good or
poor osmoregulators depending on the salinity range which they
can withstand. The
good osmoregulators will rely on very efficient mechanisms of
osmoregulation while
the poor will take advantage of several peculiarities which
enable them to escape too
large modifications in the salinity of the external medium for a
more or less long
period of time.
Osmoregulation of aquatic organisms can be obtained in two
different ways.
The first consists of trying to keep the osmoconcentration of
the extracellular fluids
constant whatever the salinity of the external medium may be.
The second is an
attempt to maintain the intracellular fluid isosmotic to the
extracellular fluid. Both
methods tend towards the same goal which is to avoid water
movement (loss or gain)
at the cellular level.
There are only a few things an osmoregulator can do to prevent
or
compensate for the loss or gain of water due to osmosis or the
loss or gain of ions by
diffusion.
1. The osmoregulator can actively transport ions in the
direction opposite
from that of diffusion. The mechanism for such active transport
is generally an ionic
pump located in the plasma membranes of certain cells. Such
pumps enable animals
living in a hyperosmotic environment to pump out excess ions to
compensate for
diffusion and intake with food. Ionic pumps can also be used to
set up a concentration
difference that will transport water osmotically. Using this
arrangement, an animal in a
hyperosmotic environment can pump in ions to bring in water,
then pump the ions
back out. Conversely, animals living in hypo-osmotic
environments can pump ions in
to compensate for loss, or use ion pumps to eliminate excess
water.
2. The osmoregulator can vary its permeability to water or ions.
This
approach allows for controlling the amount of water and ions
entering or leaving the
body.
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23
12. Physiological change response to toxicant As toxicants are
assimilated into the organism’s body or some surrounding
factor is changed, they can affect and induce physiological
adaptation in the body.
There are several studies conducted about this effect. Claireaux
and Fariba (2010)
found that after Sole (Solea solea) exposed to spilled oil and
then challenged with an
acute rise in temperature (from 15 to 30 °C at 1.5 °C h−1),
cardio-respiratory responses
were impaired and unable to meet the temperature-driven increase
in tissues oxygen
demand. Ansari et al. (2009) studied the effect of naphthalene
on growth, metabolic
index, and biochemical constituents in juvenile Metapenaeus
affinis. They found that
growth rate in term of weight gain was lower than that in the
control, metabolic rate
assessed in the term of oxygen consumption rate was reduced,
activity increased
initially but reduced subsequently as well. Protein content and
organic carbon in the
body was decreased while lipid content was increased. This
showed that its physiology
was changed after exposure to naphthalene and then recovered
during detoxification.
Lemarie et al. (2003) studied the effect of chronic ammonia
exposure on growth of
European seabass (Dicentrarchus labrax) juveniles. They found
that it caused a
decrease in weight gain compared to the control until after 13
days the fish could adapt
to increased ammonia concentration. However after 55 days,
plasma ammonia levels
were positively related to aqueous ammonia concentrations, and
oxygen consumption
recorded in fasting fish was significantly dependent on ammonia
concentrations.
Couture and Puja (2003) found that heavy metal (Cu and Cd)
exposure caused an
upregulation of liver protein metabolism, presumably at least in
part for the purpose of
metal detoxification. By contrast, tissue biosynthetic capacity
decreased with
increasing liver Cd concentration. They indicated that heavy
metal exposure impaired
aerobic capacities in the muscle of contaminated fish. They also
suggested that
mitochondria may be the primary targets for inhibition by Cu,
while Cd may reduce
gill respiratory capacity. Muscle aerobic and anaerobic
capacities were inversely
related.
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24
13. Nile tilapia Phylum Chordata Class Actinopterygii Order
Perciformes Family Cichildae Genus Oreochromis Species Oreochromis
niloticus
Figure 2-9 Nile tilapia (Oreochromis niloticus)
Nile tilapia is a fresh water fish. Its origin is in Africa.
Tilapia are shaped
much like sunfish or crappie but can be easily identified by an
interrupted lateral line
characteristic of the Cichlid family of fishes. They are
laterally compressed and deep-
bodied with long dorsal fins. The forward portion of the dorsal
fin is heavily spined.
Spines are also found in the pelvis and anal fins. There are
usually wide vertical bars
down the sides of fry, fingerlings, and sometimes adults.
Moreover, the adult which
can be identified sex is about 10 cm in size. They are found
widely in rivers, ponds,
and lakes. They can live in both fresh water and brackish water.
They endure and
adjust properly to varied environment. They are easy to nourish,
good taste, and high
growth rate, thus they are economical fish in many country.
Nile tilapia is omnivore; they can eat phytoplankton, juvenile
of insects,
copepods, mosses, and degraded organic matter on the bottom. For
adult, they also eat
algae and duckweed; it is a way to control the growing of weed
flora. In aquatic farms,
they are fed by the food which consists of rice bran chaff, dry
ground-up fish, broken-
milled rice, dried weed flora, and food particles.
Nile tilapia is mouth brooders; eggs are fertilized in the nest
but parents
immediately pick up the eggs in their mouths and hold them
through incubation (about
3-5 days) and for several days (about 10-14 days) after hatching
for protecting their
fingerlings.
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25
14. Relevant study Lin, Cormier, and Torsella (1996) compared
fixed wavelength fluorescence
(FF) technique and High-performance liquid chromatography –
fluorescence (HPLC-
F) technique for measuring benzo(a)pyrene metabolites and
naphthalene metabolites in
bile of brown bullhead, white sucker, and common carp. The
statistic analysis revealed
that benzo(a)pyrene metabolites and naphthalene metabolites
which are measured by
FF technique and HPLC-F technique have correlation (r2) as 0.89
and 1.00
respectively. Thus, both of FF technique and HPLC-F technique
can be used for
evaluating PAH contamination in fish.
The Marine Research Institute in Norway (1996) studied the
effects of oil
production in Statfjord, Oseberg, and Brent (North Sea) by using
Egersundbanken area
as reference site. The effects were studied in three fish
species, Atlantic cod (Gadus
morhua), Haddock (Melanogrammus aeglefinus), and Long rough
dad
(Hippoglossoides platessoides). Their bile was diluted in ratio
of 1:1,600 by 48%
ethanol and then subjected to measurements of the luminescence
of PAH metabolites
by FF technique: naphthalene (ex290/em335 nm), pyrene
(ex341/em383 nm), and
benzo(a)pyrene (ex380/em430 nm). It was found that the measured
luminescence level
of PAH metabolites in fish from the area which was close to oil
production sites was
higher than that in the reference site.
Aas, Beyer, and Goksoyr (1998) studied on the effects of oil
production in
the same area (1996). They used turbot and cod fish from the
area close to oil
production sites. The luminescence of PAHs metabolites in the
bile from these fishes
was measured using SFS technique (∆λ42nm). It was found that the
luminescence
level of PAH metabolites in fish from the area close to the oil
production (Statfjord
region) site was higher than that from at a distance.
Aas, Baussant, Balk, and Liewenborg (2000) studied bile PAH
metabolites
and EROD activity in liver of fish (juvenile cod) which were
exposed to crude oil at
different concentrations; 1, 0.25, and 0.06 ppm, and recorded
the result at 12 h, 24 h,
3 d, 16 d, and 30 d. They measured the luminescence of PAHs
metabolites by FF
technique, naphthalene (ex290/em335nm), pyrene (ex341/em383nm),
and
benzo(a)pyrene (ex380/em430nm) and measured EROD activity by
plate reader at
ex544/em584nm. They found that the fish which were exposed to
crude oil of high
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26
concentration had higher luminescence levels of PAHs metabolites
and EROD activity
than those exposed to oil at lower concentrations.
Figure 2-10 Bile PAHs metabolites and EROD activity in
liver(Aas, Baussant, Balk, &
Liewenborg, 2000)
Escartin and Porte (1999) evaluated PAH contamination in coastal
areas of
the Mediterranean Sea. They measured the luminescence level of
fluorescence
metabolites in bile of two fish species, the red mullet (Mullus
barbatus) and the sea
comber (Serranus cabrilla) by Liquid Chromatography Fluorescence
(LC-F)
technique and by Gas Chromatography-mass spectrometry (GC-MS).
They found that
the results from both techniques gave the same trend, which
indicated that the
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27
luminescence level in each samples was different depending upon
the sampling site.
The samples from urban areas had higher luminescence levels than
those from remote
or agricultural areas. However, the LC-F technique was simple
and fast, so it was
appropriate for monitoring and evaluating PAH contamination in
coastal areas.
Yang and Baumann (2005) studied PAHcontamination in benthic fish
and
brown bulhhaed (Ameiurus nebulosus) collected from areas near
industries (Detroit,
Ottawa, Black, and Cuyahoga), from watershed areas (Ashtabula,
Buffalo, and
Niagara), and from pristine areas (Old Woman Creek in Erie
tributaries lake). They
found that the luminescence level of bile PAHs metabolites
increased with PAH
concentration in sediments. Thus, the luminescence of PAHs
metabolites could be
used as an indicator of PAHs contamination.
Dissanayake and Galloway (2004) investigated PAH contamination
in shore
crabs fish (Carcinus maenas), which were collected from a nearby
oil refinery plant,
by measuring the luminescence of bile PAHs metabolites (by FF
technique and
synchronous fluorescence spectrophotometry, SFS). They concluded
that both
techniques could be used for monitoring and effect evaluation of
PAHs contamination
in the water source.
Camus, Aas, and Borseth (1998) studied the long-time effects on
juvenile
turbot fish (Scophthalmus maximus) which had been exposed to
leaked oil at sublethal
concentrations (0.125, 0.5, and 2 mg/l) by measuring cytochrome
P 450 from EROD
activity level in liver and the luminescence level of bile PAHs
metabolites (FF). The
luminescence could be measured after 24 hours – 4 days, and then
it was slightly
decreased. In contrast, cytochrome P 450 from EROD activity was
slightly increased
with time. The results showed that the detoxification occurred
mainly in bile sac;
however toxicants could also accumulate in the liver. They also
concluded that EROD
activity measurement was appropriate for the study of long-time
effects of oil
contamination.
Gorbi and Regoli (2004) determined PAH contamination in an
aquatic
environment during different seasons by measuring EROD activity
in liver. They
collected European eel (Anguilla anguilla) in January, April,
August, and October and
found that EROD activity was very high in August and October
(rainy season). They
also found that the temperature influenced EROD activity. EROD
activity level in
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28
summer was higher than that in winter because metabolic
processes and other
activities in fish body were influenced by temperature. Thus,
the detoxification rate in
summer was higher than that in winter. Moreover in winter, the
toxicants tended to be
accumulated in fish body especially in liver.
Jonsson, Sundt, Aas, and Beyer (2004) measured bile chrysene
metabolites in
four fish species: Atlantic cod (Gadus morhua L.), Flounder
(Platichthys flesus),
Plaice (Pleuronectes platessa) and Sheepshead minnow (Cypriodon
variegates), which
were exposed to 1 mg/kg of diluted chrysene in acetonitrile. The
bile, diluted with
50% of methanol in a ratio of 1:100 displayed luminescence of
chrysene metabolites
by the SFS and HPLC-F methods. The results from both methods
showed the same
trend. The luminescence levels of metabolites were increased
with PAH concentration.
Barra, Sanchez-Hernandez, Orrego, Parra, and Gavilan (2001)
studied PAH
accumulation in the aqautic organisms in Biobio River, Chile.
They compared
benzo(a)pyrene accumulation in rainbow trout fish (Oncorhynchus
mykiss) from two
sources, field (cage in La Mochita area) and laboratory, by
measuring the level of
monooxygenase (MFO) activity in liver and Total Biliary
Fluorescence (TBF365/520) in
bile using FF techniques. They found that the luminescence level
of metabolites was
increased with time (both methods reported the same trend).
However, the
luminescence level in fish collected from the field was higher
than that from the
laboratory, because in the field study PAH exposure of fish was
affected by many
factors, such as temperature, pH, salinity, and water flow
rates.
Fuentes-Rios, Orrego, Rudolph, Mendoza, Gavilan, and Barra
(2005)
investigated the relationship between EROD activity level in
liver and the
luminescence level of bile metabolites for the detection of PAH
contamination in
coastal areas in Chile. They found that EROD activity in fish
from each site was
dependent upon the level of toxicants. Moreover, EROD activity
level in liver related
with luminescence levels of metabolites in bile and varied with
toxicant concentration
in the area.
Inzunza, Orrego, Penalosa, Gavilan, and Barra (2006) studied
PAH
contamination in sediments from Biobio River, in central Chile
by using GC-FID.
They found that range of PAH concentration in sediments from the
sampling sites with
potential sources was about 2,000-7,000 ng/g dry weight of
sediments. The results also
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29
revealed that PAH concentration in sediments increased with the
amount of organic
matter. Then they placed these sediments in aquatic tanks (in
the laboratory) with 60 L
clean water and rainbow trout (Oncorhynchus mykiss) and tested
CYP1A1 induction at
0 and 21 days. CYP1A1 induction could be found after 21 days.
They concluded that
the aquatic organisms could be use as bioindicators for PAHs
contamination in an
aquatic environment.
Vuorinen et al. (2006) studied bile PAH metabolites as
bioindicators of PAH
contamination in the Baltic Sea. They used flounder fish
(Platichthys flesus) as
representative for the Lithuanian coast, Wismar Bay, and the
Gulf of Gdansk, perch
fish (Perca Fluviatilis) for Kvadöfjärden, and eelpout fish
(Zoarces viviparus) for
Kvadöfjarden, Wismar Bay, and the Gulf of Gdansk. They measured
1-OH pyrene by
HPLC täechniques and Total Biliary Fluorescence (TBF365/520) by
FF techniques. They
found that Total Biliary Fluorescence (TBF365/520) in the male
was higher than that in
the female. They concluded that both techniques could be used
for measuring the
luminescence of bile metabolites, but the results varied because
of sex influence.
Trun-Duy et al. (2008, A) studied on the effects of oxygen
concentration and
body weight on maximum feed intake, growth and hematological
parameters of Nile
tilapia (Oreochromis niloticus). They compared the feed intake,
growth and
hematological parameters of small fish with that of big fish at
different DO levels.
They found that feed intake and growth of the fish at a high DO
level were
significantly higher than that at a low DO level. Moreover,
these two parameters in
small fish were significantly higher than that in big fish. Fish
at low DO levels made
no hematological adjustments. The differences of these
parameters were influenced by
the limitation of gill surface in the small fish and the
relationship between the gill
surface and body weight.
Sparks et al. (2003) studied effects of environmental salinity
and 17α-
methyltestosterone (MT) on growth and oxygen consumption in the
tilapia,
Oreochromis mossambicus. They found that in the same age,
seawater-reared tilapia
had a significantly lower routine metabolic rate than the fresh
water-reared fish.
Moreover, treatment with MT in fresh water caused a significant
increase in oxygen
consumption, whereas MT treatment was without effect on routine
metabolic rate in
seawater-reared fish. These results indicated that
seawater-rearing and MT treatment
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30
accelerated growth of tilapia. It was also likely that the
increased routine metabolic
rate and growth in MT-treated tilapia in fresh water may be due
to the metabolic
actions of MT.
Allen et al. (2006) studied effects of temperature and salinity
on the standard
metabolic rate (SMR) of the caridean shrimp, Palaemon
peringueyi. They found that
at a constant salinity of 35 ‰, the respiration rate increased
with an increase in
temperature. At a constant temperature of 15°C the respiration
rate also increased with
an increase in salinity. The results suggested that the shrimp
could adapt to variations
intemperature and salinity which most likely occur in estuarine
conditions.
Gracia-Lopez et al. (2006) studied effects of salinity on
physiological
conditions in the juvenile common snook, Centroponus
undecimalis. They found that
the changing of salinity had a direct effect on the physiology,
inducing changes in the
oxygen consumption, nitrogen excretion, the energetic substrate
and plasma osmotic
pressure.
Santos et al. (2006) studied effects of naphthalene on metabolic
rate and
ammonia excretion of juvenile Florida pompano, Trachinotus
carolinus. They
investigated the effects of different concentrations of
naphthalene, acute (50-min and
24-h) and chronic (12-day) exposure, on the physiological
parameters of the species.
After acute exposure there was a tendency to increase specific
oxygen consumption.
After chronic exposure, a decrease was observed at the highest
concentration,
indicating a narcotic effect of naphthalene. Moreover, ammonia
excretion was
reduced significantly in all the exposed fishes. The effects
caused by naphthalene were
time and dose dependent for this fish.
Das et al. (2005) studied effects of temperature on oxygen
consumption of
Labeo rohita. The experiments were performed at varied
temperature of 26, 31, 33,
and 36°C. They found that oxygen consumption rate increased
significantly with
increasing temperature. Preferred temperature decided from
relationship between
acclimation temperature and Q10 values were between 33 and 36°C,
which gives a
better understanding of optimum temperature for their
growth.