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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.
Transcript
  • 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.

  • 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

  • 6

    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,

  • 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

  • 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)

  • 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

  • 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

  • 11

    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

  • 12

    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

  • 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

  • 14

    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).

  • 15

    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.

  • 16

    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

  • 17

    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

  • 18

    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.

  • 19

    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.

  • 20

    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

  • 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

  • 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.

  • 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.

  • 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.

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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.


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