CHAPTER 6

Oxidative Stress
François Gagné

Chapter Outline
6.1 Antioxidant Enzymes 104
6.1.1 SOD 104
6.1.1.1 Reagents 105
6.1.1.2 Procedure 105
6.1.1.3 Data Calculation and Normalization 106
6.1.2 Peroxidase 106
6.1.2.1 Reagents 106
6.1.2.2 Procedure 107
6.1.2.3 Data Calculation and Normalization 107
6.1.3 Catalase 107
6.1.3.1 Reagents 108
6.1.3.2 Procedure 108
6.1.3.3 Data Calculation 108
6.1.4 Measurement of Antioxidant Capacity 109
6.1.4.1 Reagents and Tissue Preparation 109
6.1.4.2 Procedure 109
6.1.4.3 Data Calculation, Normalization, and Comments 110
6.1.5 Lipid Peroxidation (Oxidative Damage) 110
6.1.5.1 Reagents 110
6.1.5.2 Procedure 111
6.1.5.3 Data Calculation, Normalization, and Comments 111
6.1.6 Measurement of Age-Related Pigments and Lipofuscins 111
6.1.6.1 Reagents and Solutions 112
6.1.6.2 Procedure 112
6.1.6.3 Data Calculation 113
6.2 Applications 113
References 115

Oxidative stress represents a common if not universal denominator of toxicity

that could lead to tissue injury and threaten organism health. During respira-
tion, the release of uncoupled high-energy electrons by mitochondria and
chloroplasts represents one of the major sources of reactive oxygen species in

Biochemical Ecotoxicology r 2014 Elsevier Inc.

DOI: http://dx.doi.org/10.1016/B978-0-12-411604-7.00006-4 All rights reserved. 103
104 Biochemical Ecotoxicology

cells. Many xenobiotics have the potential to uncouple electron transport in

mitochondria, which could lead to important leakage of electrons in the intra-
cellular environment and produce oxidative damage. The production of reac-
tive oxygen species could also result from inflammation reactions in which
nitric oxide and hydrogen peroxide are produced and during oxidative metabo-
lism of xenobiotics. We invite the reader to consult recent reviews on oxidative
stress in the environment for a more detailed account on the role of oxidative
stress during pathogenesis [1].
Oxidative stress occurs when excess oxygen radicals are produced in cells,
which could overwhelm the normal antioxidant capacity. When the concen-
tration of reactive species is not controlled by internal defense mechanisms
such as antioxidants (tocopherols, ascorbic acid, and glutathione) or enzymes
involved in oxygen radical scavenging (catalase, peroxidase, and superoxide
dismutase, SOD), oxidative damage occurs to proteins, lipids, and DNA,
which could lead to cytotoxicity, genotoxicity, and even carcinogenesis when
damaged (mutated) cells can proliferate. Oxidative stress could results from
the following: (1) the presence of xenobiotics, (2) the activation of the
immune system in response to invading microorganisms (inflammation), and
(3) radiation, which makes oxidative stress a common denominator of toxic-
ity or stress. In this chapter, the reader will find a variety of assays to measure
oxidative stress and damage.

6.1 ANTIOXIDANT ENZYMES

6.1.1 SOD
SODs are found in both the mitochondria and cytoplasm in cells where the
latter is dependent of Cu and Zn. SOD catalyzes the dismutation of oxygen
radicals to produce hydrogen peroxide, which in turn is transformed into water
and oxygen by catalase:
Superoxide anion-H2 O2 ðSODÞ
H2 O2 -O2 1 H2 O ðcatalaseÞ

In the present assay, a nonenzymatic means of superoxide generation is pre-

sented in which the generated radicals are detected by the reduction of the col-
orimetric dye p-iodonitrotetrazolium (p-INT) [2]. This method could be run
on 96-well microplates for high-throughput analysis.
 Oxidative Stress 105

6.1.1.1 Reagents
Homogenization buffer: 50-140 mM NaCl containing 10 mM HEPES-NaOH,
pH 7.4, containing a protease inhibitor (phenylmethylsulfonylfluoride 0.01%
or apoprotinin 1 μg/mL) and a reducing agent (5 mM β-mercaptoethanol or
5 mM dithiothreitol).
Assay buffer: 50 mM KH2PO4 at pH 7.5 with NaOH 1 M: add 0.68 g
KH2PO4 in 90 mL of SQ water, adjust pH to 7.5, and complete to 100 mL
SQ water. Keep at 4 C.
Stock EDTA (10 3 ): 9 mg of EDTA disodium salt in 10 mL water.
NADH stock: Resuspend 9.5 mg of reduced NADH in 1.4 mL of water.
Prepare daily.
p-INT stock (5 mM): Dissolve 25 mg of p-INT in 10 mL of SQ water.
Keep in the dark at 4 C, stable for one week.
Phenazine methosulfate (PMS) stock solution: Dissolve 6 mg in 20 mL of SQ
water. Keep at 220 C. If the clear liquid becomes brown then discard.
SOD enzyme concentrate: Dissolve commercial SOD (4000 U/mg solid) in
10 mL of assay to obtain 1000 U/mL. Conserve 100 μL aliquot at 220 C
for up to 2 years. Dilute again 1/10 in the assay buffer on the day of
the assays.
Reagent 1: Prepare at the day of the assay the following: 165 μL of PMS and
250 μL of EDTA stock and complete to 5 mL with assay buffer.
Reagent 2: 1 mL of EDTA, 3.2 mL of p-INT stock, and 1.96 mL of NADH
and complete to 10 mL with the assay buffer.

6.1.1.2 Procedure
The tissues are weighed and minced with a scalpel on ice. The minced tissues
are homogenized by using a Teflon pestle tissue grinder and the homogenate
centrifuged at 15,000 3 g for 30 min at 24 C. The supernatant (S15) is kept
aside for the assay.
In a clear polystyrene 96-well microplate, add 50100 μL of S15, blank, or
SOD sample to an equal volume of Reagent 2. Start the reaction by adding
25 μL of Reagent 1. Determine the absorbance at 560 nm at time 0, 2, 4, 8,
and 16 min. The SOD sample contains 10 U/mL and is prepared as follows:
22 μL of SOD stock solution and 88 μL of assay buffer (100 μL). Continue
as with the S15 sample by adding 1 volume of Reagent 2 and 25 μL of
Reagent 1. The blank solution contains only the assay buffer.
106 Biochemical Ecotoxicology

6.1.1.3 Data Calculation and Normalization

The presence of SOD will block the reduction of the dye by the electron gen-
eration system. The decrease in absorbance is determined over time and the
slope is calculated to have Δ absorbance/min. The percentage of inhibition by
SOD is first determined as follows:
½ðΔA560 nm=minBlank 2 ðΔA560 nm=minSOD Þ 3 100 5 %inhibition;
ðΔA560 nm=minBlank Þ

and the percentage of inhibition of the S15 sample determined:

½ðΔA560 nm=minBlank 2 ðΔA560 nm=minS15 Þ 3 100 5 %inhibition
ðΔA560 nm=minBlank Þ
The percentage of inhibition in the S15 fraction is converted into SOD
equivalents by the following:
SOD ðU=mLÞ 5 ðx% inhibition S15=y% inhibition of SODÞ 3 10 U=mL
The SOD activity is then normalized by the amount of proteins in the S15
fraction or by the tissue weight to give SOD U/mg proteins or g tissue weight.

6.1.2 Peroxidase
Peroxidase is a hemoprotein catalyzing the oxidation of a number of substrates
by hydrogen peroxide:
H2 O2 1 substrate 2 H2-substrate 1 2 H2 O

Endogenous substrates are, for example, ascorbate, ferrocyanide,

31
Fe -cytochrome c, and reduced glutathione (2 GSH). The enzyme requires a
substrate susceptible to oxidation and represents a fundamental pathway of
oxidation of many xenobiotics. In the present assay, a highly sensitive fluores-
cence measurement of fluorescein is proposed as a convenient procedure. For
more sensitivity, luminol (3-aminophthalhydrazide) could be also used as lumi-
nescent assay if a luminescence reading instrument is available.

6.1.2.1 Reagents
Assay buffer: 50 mM KH2PO4 at pH 7.5 with NaOH 1 M: add 0.68 g
KH2PO4 in 90 mL of SQ water, adjust pH to 7.2, and complete to 100 mL
SQ water. Keep at 4 C.
 Oxidative Stress 107

Dichlorofluorescein diacetate 10 mM (DCF): Dissolve 48.5 mg DCF in 10 mL

100% dimethylsulfoxide.
Horseradish peroxidase (HRP): 1 mg/mL in 0.1 M KH2PO4 buffer, pH 6.
The buffer is prepared by dissolving 1.36 g of monobasic potassium phos-
phate in 90 mL water, pH adjusted to 6 with HCl, and complete to 100 mL
with SQ water. A volume of 10 μL of the stock is added to 100 mL of assay
buffer to produce 0.1 μg/mL.
Reagent solution: Mix 100 μL of DCF and 100 μL of H2O2 at 1 mM con-
centration and complete to 10 mL with assay buffer. Prepare just before the
assays and keep on ice in the dark.
Fluorescein standard solution (100 µg/mL): Dissolve 1 mg fluorescein in 10 mL
of assay buffer.
6.1.2.2 Procedure
In a dark 96-well microplate, add 20 μL of S15, standard, or blank to
160 μL reagent solution and 20 μL of water. Incubate at 2530 C and mea-
sure fluorescence at 485 nm excitation and 520 nm emission wavelengths at
time 0, 5, 10, 15, and 20 min. The blank consists of the assay buffer only.
A positive internal control using 10 ng/mL peroxidase is added to the S15
to express the data as peroxidase equivalents (standard addition method). A
volume 20 μL of the diluted stock solution is added to 180 μL reagent solu-
tion with S15 (final peroxidase concentration 10 ng/mL). The activity could
also be expressed as nmol fluorescein formed by preparing an external 1
μg/mL fluorescein standard: 2 μL of fluorescein standard solution to 198 μL
of assay buffer.
6.1.2.3 Data Calculation and Normalization

Peroxidase activity ðng=mLÞ 5 ½RFU ðsample 2 blankÞ=min=½RFU

ðspiked sample 2 blankÞ=min 3 added peroxidase ðng=mLÞ 3 dilution factor
The equivalent amount of peroxidase is then normalized against total pro-
tein content in the S15 fraction or the tissue weight (g/mL homogenization
buffer) to give ng peroxidase/mg proteins.

6.1.3 Catalase
Catalase is the enzyme responsible for the breakdown of H2O2 into water and
oxygen. It is the major antioxidant defense enzyme system. Catalase is inhibited
by aminotriazole at concentrations between 1 2 10 mM.
108 Biochemical Ecotoxicology

2 H2 O2 -2 H2 O 1 O2

The assay is based on the time-dependent elimination of H2O2, which is

determined by the DCF substrate described earlier.

6.1.3.1 Reagents
Assay buffer: 100 mM KH2PO4, pH 7.0. Dissolve 1.36 g KH2PO4 in 90 mL
bidistilled water, adjust pH to 7 with NaOH 1 M, and complete to 100 mL
with bidistilled water.
Hydrogen peroxide reagent: Prepare a 100 mM (or 0.2%) solution in bidistilled
water. Hydrogen peroxide is usually sold at B30% concentrate. Prepare
daily and keep at 4 C.
Peroxidase reagent: Dissolve 10 mg HRP and 1 mg of DCF in 100 mL of
assay buffer. Dilute 1/10 in the assay buffer.
Fluorescein standard (10 µM): Dissolve 3 mg in 10 mL of bidistilled water.
Dilute 1/100 in assay buffer to obtain the final concentration 3 μg/mL.
6.1.3.2 Procedure
Catalase is a cytosolic enzyme and the assay is commonly practiced on the
post-mitochondrial fraction ( . 10,000 3 g supernatant of tissue homogenates).
Mix 1050 μL of 10 to 15 000 3 g supernatant, blank (homogenization
buffer), or hydrogen peroxide standard (110 mM H2O2) and complete to
180 μL with the assay buffer. Start the reaction by adding 20 μL of hydrogen
peroxide reagent and incubate for 0, 10, 20, 30, and 40 min at 2022 C.
At each incubation time, pipette 20 μL of reaction mixture and mix with
80 μL of peroxidase reagent and incubate for 15 min and measure fluorescence
at 485 nm excitation and 520 nm emission. Blanks consist of 20 μL of bidis-
tilled water and a fluorescein standard of 1 μM could be used for calibration
(10 μL of fluorescein standard in 80 μL of the assay buffer). The amount of
H2O2 will decrease over time by catalase.
6.1.3.3 Data Calculation
Catalase activity will eliminate hydrogen peroxide, which in turn will reduce
the amount of fluorescein (less oxidation of the DCF substrate). It is calculated
as follows:
½ðFluorescein formed=15 minÞ at 0 min2½ðFluorescein formed=15 minÞ at 20 min
5loss of hydrogen peroxide=min=mL
 Oxidative Stress 109

This activity is then normalized against total protein content (mg/mL) or

tissue weight (g/mL) to give loss of hydrogen peroxide/min/mg proteins or g
tissue weight.

6.1.4 Measurement of Antioxidant Capacity

During oxidative stress, reactive oxygen species reach levels that could over-
whelm antioxidant defense in cells; a decrease in the levels of antioxidants such
as ascorbic acid or reduced glutathione levels leads to general reduction of the
antioxidant capacity of the cytoplasm. An extremely cheap and rapid method
to determine the antioxidant capacity in tissue extracts is presented therein.
The assay consists of the reduction of the phosphomolybdate complex by the
presence of antioxidants in the biological sample [3,4]. This complex forms a
dark blue chromophore when reduced that is measured between 660 and
850 nm with a maximum at 810 nm. This assay requires only a standard spec-
trophotometer for tubes or microplates. These data are usually expressed as
ascorbic acid equivalents, which are used as positive controls.

6.1.4.1 Reagents and Tissue Preparation

Tissues are usually homogenized in a buffer that is devoid of any antioxidant
and the buffer should be used in the blank. Trichloroacetic acid (TCA) is
added to the homogenate sample to give a final concentration of 5% (w/v) and
placed in ice. The mixture is then centrifuged at 10,000 3 g for 5 min at 4 C
and the supernatant collected.
Phosphomolybdate reagent: Dissolve 10 mg phosphomolybdate [H3(P(Mo3)
O10)4] in 1 mL of 20% ethanol (10 mg/mL) in a dark container and store at
4 C. Dilute at 50 μg/mL in 0.5% TCA before the assays.
Ascorbic acid reagent: Dissolve 10 mg of ascorbic acid in 1 mL of SQ water.
Prepare fresh (daily) and place on ice in the dark to prevent oxidation (max-
imum concentration).

6.1.4.2 Procedure
To 50 μL of blank, ascorbic acid standards, and tissue homogenate acid extract
mix 150 μL of phosphomolybdate reagent and wait for 15 min at room tem-
perature. Read absorbance at 810 nm. The blank is 5% TCA in the homogeni-
zation buffer.
110 Biochemical Ecotoxicology

6.1.4.3 Data Calculation, Normalization, and Comments

½A810 sample 2 A810 blank 3 Standard concentration of ascorbic acid=

½A810 standard 2 A810 blank 3 ðsample volume μL=total assay volume μLÞ
5 Y mg=mL
This concentration is then normalized against total proteins (mg/mL) or
homogenate tissue weight in g/mL:
Y μg=mL=g tissue weight=mL or mg=mL protein 5 Z μg=g or Z μg=mg proteinsÞ:

The concentration of the standard solution should be close to the signal

value of the sample, i.e., within 6 0.2 absorbance unit. A standard curve could
also be constructed to convert the increase in absorbance at 810 nm as function
of ascorbic acid concentration. The concentration of ascorbic acid is usually
between 1 and 10 mg/mL. If the test sample significantly absorbs at 810 nm
then an operational correction blank could be used, which consists of 50 μL of
the sample with 150 μL of 0.5% TCA only. These data are expressed as mg
ascorbic acid equivalents/mg proteins.

6.1.5 Lipid Peroxidation (Oxidative Damage)

Excess reactive oxygen radicals that are not removed by antioxidants or catalase
could lead to deleterious oxidative reactions in cells. The carbon double bonds
in polyunsaturated lipids could react with hydrogen peroxides to give the
breakdown product malonaldehyde (MDA). The thiobarbituric test represents a
very convenient and simple assay for the measurement of lipid peroxidation
[5]. MDA is detected by the thiobarbituric acid, which forms a chromophore
detectable by either spectrophotometry or fluorometry. Therefore this assay
represents the extent of breakdown products resulting from the oxidation of
polyunsaturated lipids and hence a biomarker of oxidative damage. The assay
is usually practiced on lipid-rich samples such as tissue homogenates and
mitochondrial and microsomal fractions.

6.1.5.1 Reagents
Reagent A: Add 10 mL of trichloroacetic acid (6.1 M) to 70 mL of water,
dissolve 0.028 g of FeSO4, and complete to 100 mL with water;
Reagent B: Add 0.67 g of thiobarbituric acid to 100 mL of SQ water and
heat at 5060 C to assist dissolution;
 Oxidative Stress 111

Tetramethoxypropane (TMP) standard: TMP is the stabilized formulation of

MDA. A standard stock solution is prepared by adding 10 μL of TMP in
10 mL of 0.1 M HCl. This stock solution is stable for one week at 4 C in
the dark. The working solution 10 μg/mL is freshly prepared before the
assay by mixing 10 μL of stock solution in 1 mL SQ water.

6.1.5.2 Procedure
In a dark 96-well microplate, add 150 μL of Reagent A and 75 μL of Reagent
B to 50 μL of blank, standard, or sample. Mix, heat in water bath at 7075 C
for 10 min, and cool at room temperature. The microplate could be centri-
fuged briefly if there is precipitation (protein denaturation) at 3000 3 g for
5 min. Read fluorescence at 535 nm excitation and 635 nm emission or read
the absorbance at 540 nm.

6.1.5.3 Data Calculation, Normalization, and Comments

½A540 sample 2 A540 blank 3 Standard concentration=

½A540 standard 2 A540 blank 3 ðsample volume μL=total assay volume μLÞ
5 Y μg=mL

This concentration is then normalized against total proteins (mg/mL) or

homogenate tissue weight in g/mL:
Y μg=mL=g tissue weight=mL or mg=mL protein 5 Z μg=g or Z μg=mg proteinsÞ:

The concentration of the standard solution should be close to the signal

value of the sample, i.e., within 6 0.2 absorbance units or 6 20% of the fluo-
rescence signal. A standard curve could also be constructed to convert the
absorbance values into MDA equivalents. Since other aldehydes could react
with the reagent, these data are usually expressed as μg thiobarbituric acid
reactants (TBARS).

6.1.6 Measurement of Age-Related Pigments and Lipofuscins

Age-related pigments such as lipofuscins represent a simple way to determine
the rate of living or the physiological age of organisms living in polluted envir-
onments [6]. Age-related pigments are formed by the chronic oxidation of
proteins, lipids, and carbohydrates in cells. These pigments form insoluble
112 Biochemical Ecotoxicology

inclusion bodies and are associated with neutral lipids and lipofuscins during
histocytochemical observations [7]. These inclusions are fluorescent and accu-
mulate over time and persist in cells (no turnover) that can be used to assess the
“physiological” age of organisms provided these inclusions are related with
chronological age during normal conditions. This needs to be checked when
working with novel tissues as in reference [6] where a correlation between age-
related pigments and chronological age was found at reference sites.

6.1.6.1 Reagents and Solutions

Homogenization buffer: 25 mM HEPES-NaOH buffer, pH 7.4, containing
125 mM NaCl, 10 μg/mL apoprotinin, and 1 mM dithiothreitol.
Ethanol: Analytical grade absolute ethanol (99100%), 100 mL.
Extraction buffer: Prepare 10 mM sodium phosphate buffer, pH 7.4, contain-
ing 125 mM NaCl and 0.5% Tween 20.
Quinine sulfate: Prepare a stock solution of quinine sulfate at 1 mg/mL in
100% ethanol. Dilute at 0.1 μg/mL in 0.05 N H2SO4.

6.1.6.2 Procedure
The levels of age-related pigments in the foot of the mussel were measured
according to the fluorescence methodology [8]. Age-related pigments are usu-
ally performed on tissues with low turnover such as the skin, mantle, or foot in
mussels or clams. Tissues are homogenized at a 1:5 ratio in the homogenization
buffer using a Polytron or Teflon pestle tissue grinder at 24 C. The homoge-
nate was decanted to remove tissue clumps and 2 volumes of absolute ethanol
were mixed to 1 volume of homogenate for 10 min at room temperature. The
mixture was then centrifuged at 10,000 3 g for 5 min to precipitate denatured
proteins. The supernatant is referred to as lipid-like and the pellet as protein-
associated age-related pigments, respectively. The protein pellet was mixed
with 23 volumes of the extraction buffer for 15 min and recentrifuged at
10,000 3 g for 5 min at room temperature. The supernatants were diluted 1:5
in either absolute ethanol or the extraction buffer. Fluorescence was deter-
mined at 350 and 460 nm excitation and emission, respectively. Quinine sulfate
at the concentration 100 ng/mL was used as an internal standard for normaliza-
tion of the lamp excitation energy, which can change in time and between
 Oxidative Stress 113

instruments. Total proteins are also determined using the method of Bradford
as described in Chapter 8.

6.1.6.3 Data Calculation

The same concentration of quinine should give the same fluorescence units
within 6 20% of the measured value. If the value differs from this then a cor-
rection factor should be applied to sample batch showing higher/lower values
in fluorescence units. For example, if the quinine fluorescence values for
batches 1 and 2 are 100 and 85 fluorescence units then no correction is
applied, because they are within 20% tolerance interval. If a third batch of sam-
ple gives a fluorescence value of 135 then the fluorescence values should be
corrected against the mean fluorescence value of batches 1 and 2: The mean
value of batches 1 and 2 is 92.5 fluorescence units and a correction factor is
derived, 135/92.5 5 1.459. The fluorescence units of the samples in batch 3
should be divided by 1.459 before comparing with the values obtained for
batches 1 and 2. Some investigators just apply the correction factor regardless
of the 6 20% rule between sample batches.
The data are expressed as corrected relative fluorescence units/
mL 3 dilution factor [5] and normalized against total proteins (mg/mL) or g
tissue wet or dry weight.

6.2 APPLICATIONS
The occurrence of pharmaceutical compounds in treated municipal waste-
waters has raised concerns about their potential for harming nontarget aquatic
organisms. Indeed, pharmaceuticals such as carbamazepine are designed to have
specific therapeutic effects and to be eliminated either directly or indirectly by
mammalian-based biotransformation activity. However, aquatic organisms such
as the hydra may not have the biotransformation capacity to biotransform them
for elimination (see Figure 6.1). In the example provided below, exposure of
the Hydra to increasing concentrations of carbamazepine for 48 hr at room
temperature. In Figure 6.1A, heme oxidase activity (a generic assay of cyto-
chrome P450 activity) was significantly increased at 6 μM concentration. In
Figure 6.1B, lipid peroxidation was significantly induced at 600 μM which
indicates oxidative stress-induced cell damage.
114 Biochemical Ecotoxicology

(A) 0,22
0,20 *
Mean Stand. error
0,18
0,16

Heme oxidase activity

0,14
0,12
0,10
0,08
0,06 *
* *
0,04
0,02
0,00
–0,02
0 6 60 600 6000
CBZ (uM)

(B) 7
*
Mean Stand. error

6
(ug TBARS/mg proteins)

5
Lipid peroxidation

*
3

1
0 6 60 600 6000
CBZ (uM)
Figure 6.1 Increase in heme oxidase and lipid peroxidation in hydra exposed to carbamazepine.
The activity of heme oxidase is involved in xenobiotic metabolism (A) and lipid peroxidation is a
biomarker of oxidative damage to polyunsaturated lipids (B).
 Oxidative Stress 115

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generator. Anal Biochem 1995;232:2438.
[3] Verlecar XN, Jena KB, Chainy GBN. Seasonal variation of oxidative biomarkers in gills and digestive
gland of green-lipped mussel Perna viridis from Arabian Sea Estuarine. Coastal Shelf Sci
2008;76:74552.
[4] Mitusi A, Ohata T. Photooxidative consumption and photoreductive formation of ascorbic acid in
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[5] Wilbur KM, Bernheim F, Shapiro OW. The thiobarbituric acid reagent as a test for the oxidation of
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Mya arenaria populations: the effects of clam bed distance from the shore. STOTEN
2009;407:584454.
[7] Sheehy MR, Greenwood JG, Fielder DR. Lipofuscin as a record of “rate of living” in an aquatic
poikilotherm. J Gerontol A Biol Sci Med Sci 1995;50:32736.
[8] Hammer C, Braun E. Quantification of age pigments (lipofuscin). Comp Biochem Physiol
1988;90B:717.