Abstract
In order to measure organic pollutants and heavy metals in coastal waters.
previous studies have directly assayed for these compounds in tissues of bioaccumulators
such as Mytilus galloprovincialis. In contrast, this study measured the activity of the
detoxification enzyme glutathione S-transferase (GST) in the digestive gland of M.
galloprovincialis as a bioindicator of the state of organic pollution at three sites in the
San Francisco and Monterey Bays with varying pollutant levels. Glutathione S¬
transferase activity was measured through the conjugation of 1-chloro-2,4-dinitrobenzene
(CDNB) to reduced glutathione (GSH) to form CDNB-SH, at a wavelength of 340 nm.
The activity data were collected in milliabsorbance units/min and were normalized by
dividing by total tissue wet weight. Activity was highest in M. galloprovincialis from the
most polluted site, Moss Landing (0.0573 + 0.0226 mAbs/min/g tissue), intermediate in
specimens from the Palo Alto site (0.0327 + 0.0140 mAbs/min/g tissue), and lowest in
samples from the least polluted site, Monterey Marina (0.0228 + 0.0068 mAbs/min/g
tissue, ANÖVA p = 3.8 x 10°). Organisms from Moss Landing also have greater
digestive gland masses per total tissue mass and have higher protein concentrations than
organisms from less polluted sites. Mytilus galloprovincialis was transplanted from
polluted water at Moss Landing into pristine seawater and GST activity was measured
over 30 days. There was a significant decrease in GST activity with reduced exposure to
organic pollutants.
Introduction
Anthropogenic marine pollution is a hazard for marine invertebrates inhabiting
coastal waters, especially when they live in close proximity to areas of agricultural run¬
off, or inhabit bays where such pollution may collect. In recent decades, the application
of pesticides and other organic chemicals to farmlands in California’s agricultural valleys
has increasingly affected the coastal oceanic and bay environment. Toxins such as DDT
and its breakdown products, polyaromatic hydrocarbons (PAH’s), and polychlorinated
biphenyls (PCB's) have been found in the Monterey and San Francisco Bays (SWRCB
1996). Although concentrations of these pollutants in seawater can be measured directly.
many of these lipophilic compounds are not very soluble in water, so data analysis can be
difficult. For example, contamination can be an issue when concentrations are low, and
the methods involved in direct water sampling can be complicated and time-consuming.
An alternative method is to examine animal biomarkers. Biomarkers are defined
as organisms that tolerate and accumulate into tissues toxins from the environment,
There are many characteristics that define good biomarkers: 1) They must amplify as
well as accumulate lipophilic compounds. Organic pollutants are often hydrophobic and
they enter the food web through phytoplankton, which store energy in lipid droplets. The
lipid droplets draw lipophilic pesticides from the surrounding water. Thus, any organism,
including marine filter feeders, that gets most of its energy from phytoplankton quickly
accumulates pesticides in its tissues if these compounds are present in the water (J.
Watanabe, pers. comm.). 2) They are sessile rather than motile, ensuring that they are
exposed to contaminants in a limited area. 3) They are transplantable for the purpose of
comparison studies. 4) They are naturally abundant and occur over a wide geographical
range as well as over a wide range of pollutant concentrations.
In the past, water quality monitoring programs have used assay techniques on the
tissues of biomarker organisms to determine directly the amount of specific pollutants at
a monitoring site. For example, the State of California Mussel Watch program and the
San Francisco Estuary Institute use tissues of native or transplanted bivalves to assess the
abundance and distribution of several heavy metals and organic toxins along the
California coast (SWRCB 1996; SFEI 1999).
Another way to use biomarkers to determine relative contaminant levels in the
marine environment is to examine an organism’s biochemical response to the toxicity of
the contaminant. By observing the activity of enzymes involved in detoxification, a more
integrated measure of toxin exposure can be obtained than by assaying directly for
individual chemicals. Specific chemical tests may not find all of the toxins present in
seawater or biomarker tissue. Thus, by studying a detoxification pathway in a biomarker
organism, one can evaluate the effect of all pollutants of a particular type (e.g. organics)
on an organism.
There are a variety of detoxification pathways that biomarker organisms may use
to metabolize the organic compounds common in agricultural run-off. These include the
mixed-function oxidase system, which includes the cytochrome p450 group (Livingstone
et al. 1989); flavin-containing monooxygenase (Schlenk and Buhrer 1989).
multixenobiotic transport proteins (Cornwall et al. 1995), and glutathione S-transferase
(Fitzpatrick et al. 1995).
The activity of glutathione S-transferase (GST) is an ideal marker for the presence
of organic pollutants. Glutathione S-transferase detoxifies xenobiotic compounds by
catalyzing the conjugation reaction between reduced glutathione (GSH) and any
electrophile. Consequently, GST will link any compound with an electrophilic center to
GSH, and the resulting product, a glutathione S-conjugate, will be less reactive and more
polar than the original substrate. The glutathione S-conjugate can then be
compartmentalized and exported from the cell (Karam 1998). Many organic toxins such
as DDT and its derivatives are electrophilic and act as substrates for GST. Thus,
examination of GST activity provides an integrated assessment of the exposure of
organisms to organic compounds in the environment.
Other contaminant types that are currently measured through water sampling or in
biomarker tissues may be detected by increased activity from other detoxification
pathways. For example, the presence of heavy metals, which do not induce a GST
response (Regoli and Principato 1995), may instead increase levels of metallothionein
(Bolognesi et al. 1999)
An ideal organism for study of GST activity is the bay mussel, Mytilus
galloprovincialis. In addition to being a filter-feeding accumulator, M. galloprovincialis
occurs over a wide geographical range and can tolerate high concentrations of organic
pollutants. The mussel’s digestive gland is responsible for most of the organism’s
detoxification of pollutants. Thus, the highest level of GST activity in Mytilus is found in
the digestive gland (Regoli 1998; Kaaya et al. 1999).
This study compares GST activity in M. galloprovincialis from three sites varying
in pollutant levels. The sites studied were chosen based on water quality data from the
California Mussel Watch Program (SWRCB 1996). Moss Landing, at the mouth of the
Salinas River, is subject to toxin-rich agricultural run-off from the entire Salinas Valley.
Mussel Watch has found high levels of many anthropogenic pollutants such as DDT and
chlordane in the water at Moss Landing Harbor. Monterey Marina does not receive or
collect contaminated run-off. It is also likely to experience more ocean flushing with
clean water than Moss Landing Harbor. Bixbee Park, near Palo Alto in South San
Francisco Bay is not as polluted as Moss Landing (SWRCB 1995; SFEI 1999). Figure 1
shows the locations of these sites, and Figure 2 provides data on representative pollutant
concentrations at each site.
Given these toxin concentrations, I hypothesized that M. galloprovincialis from
Moss Landing would show more GST activity than mussels from Palo Alto, and that
GST activities would be lowest in Monterey Marina. I also expected that mussels
transplanted from Moss Landing into less contaminated water in the laboratory would
show a decrease in GST activity with time.
Materials and methods
Collection of specimens
Mytilus galloprovincialis were collected from floating docks in three different locations.
chosen for their relative concentrations of organic toxins: Monterey Harbor and Moss
Landing Harbor in Monterey Bay, and Bixbee Park in the South San Francisco Bay,
Mussels were between 4 cm and 6 cm in length. During transport, individuals from each
blotted with a kimwipe, and the tissue (not including gonad or byssal threads) was
weighed.
Preparation of samples
Each digestive gland was homogenized in 4 volumes O.05M potassium phosphate buffer,
pH 7.4 (Ig tissue 2 1mL), for 15-20 seconds, using an IKA Ultra-Turrax model T
tissue homogenizer. One mL of the resulting homogenate was spun in a refrigerated
microcentrifuge for 20 min at 4° C and 10,000 g. The supernatant was divided into two
parts, one for measurement of protein concentration, which was kept on ice for up to one
week, and one for the GST activity assay, which was used immediately,
Measurement of GST activity
The homogenate sample, to which 0.2mM dithiothreitol and 0.25 ug/mL Pefabloc (a
protease inhibitor, Boehringer Mannheim) were added, was kept on ice. The conversion
of 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GSH) to form oxidized
glutathione was measured at 340 nm on a Shimadzu UV-160 1 spectrophotometer,
following the method of Lima and Storey (1993). The spectrophotometer was connected
to a PC and data were collected using the Kinetics software provided by the
manufacturer. To begin, 3 mL cuvettes were filled with 2.0 mL of assay buffer (0.05 M
potassium phosphate (pH 7.4), 2mM GSH and 2mM CDNB). A 60-second time course
was run at 20 °C to determine the background activity in the absence of GST. To initiate
the assay, 50 uL of the homogenate was added to the cuvette and activity was measured
for another 60 seconds. GST activity was determined after subtracting the background
activity from that of the homogenate. Activity was expressed in milliabsorbance
units/minute/g total tissue and was normalized in three ways: with respect to total protein
concentration; the whole digestive gland; and total tissue mass of the organism (see
results below).
Total protein concentration
Total protein in the homogenate sample was assayed using the Coomassie Plus Protein
Assay (Pierce, Rockford, IL). The homogenates were diluted 20x with distilled water to
adjust protein concentration to within the working range of the system. The standard
used was bovine serum albumin.
Measurement of GST activity: Time Course
To test how GST activity levels change as mussels experience a decrease in concentration
of organic toxins, fifty individuals were transplanted from Moss Landing Harbor into the
flow-through tanks at Hopkins Marine Station. These mussels were acclimated to the
local water as described above. A group of individuals was removed from the tanks and
GST activity was measured at t = 0 days, 4, 10, 15, and 30 days.
Results
Estimation of total tissue wet weight
The correlation between tissue wet weight and shell length is shown in Figure 3a.
The r’ value (0.71) was low, so I included other morphological parameters to create a size
index more representative of the volume of a mussel shell, and thus to characterize more
accurately the tissue mass. The size index was the product of the three dimensions of
each shell, length * width * depth. Using the same group of individuals as before, the
relationship between measured total tissue wet weight and size index (Figure 3b) shows a
strong correlation (r* = 0.96), thus the equation for the regression line can be used to
estimate tissue mass from shell dimensions in cm:
Mass (g) = 0.10791*(1*w*d) - 0.32598.
This estimation of mass was used to normalize GST activity to total tissue wet weight
(see below).
Measurement of GST activity: Location
Figure 4 shows GST activity normalized to total protein concentration. Activity
from mussels at Moss Landing was 0.359 + 0.0923 mAbs/min/mg protein (average +
standard deviation; n = 14 from each site); that from Monterey specimens was 0.398 +
0.0848 mAbs/min/mg protein; and that of Palo Alto specimens was the highest, 0.488 +
0.0153 mAbs/min/mg protein. According to single factor ANOVA, there was a
statistically significant effect of site (p = 0.0147), and a Tukey-Kramer test indicated the
Moss Landing and Palo Alto groups were statistically different (p « 0.05).
The data normalized in this way do not support the hypothesis that organisms
from Moss Landing, the most polluted site, have higher levels of GST activity. This
normalization was based on the convention of expressing enzyme activity in units per mg
protein (Regoli and Principato, 1995), which requires the assumption that total protein
concentrations in M. galloprovincialis digestive glands vary randomly among individuals
regardless of location. Since the normalized GST activity data do not support my
hypothesis, 1 decided to examine the assumptions underlying the use of total protein
concentration as the normalization factor. If, for example, protein concentration were not
independent of site or pollutant concentration, then normalization by total protein
concentration would obscure any effect of site on GST activity.
To test this possibility, I correlated protein concentration per gram digestive gland
with mass of digestive gland for all three sites. The protein concentration is higher in M.
galloprovincialis digestive glands from Moss Landing than in those from Monterey and
Palo Alto (Figure 5), contradicting the assumption that protein concentration varies
independently of location. Because protein concentration appears to be correlated with
site and pollution level, it is inappropriate to normalize GST activity with this factor. This
figure also suggests that the mass of the digestive gland varies with location. To confirm
this observation, I compared the mass of M. galloprovincialis digestive glands to total
tissue wet weights. These data are shown in Figure 6, and indicate that for their total
tissue wet weight, mussels from Moss Landing have larger digestive glands than mussels
from Monterey or Palo Alto. Thus, the data in Figures 5 and 6 illustrate both that the
digestive gland comprises a greater percentage of total tissue, and that the total protein
concentration in the digestive gland is higher in M. galloprovincialis from a polluted site.
To analyze the GST activity in a more meaningful and appropriate way, activity
data in mAbs/min/gram digestive gland (from each aliquot tested in the
spectrophotometric assay) was multiplied by the mass of the digestive gland of each
individual (Figure 7). This manipulation gives activity for each organism—that is, the
total amount of GST detoxifying power each individual mussel has in its digestive gland.
Glutathione S-transferase activity at Moss Landing (0.170 + 0.101 mAbs/min) was
statistically different (by Tukey-Kramer, p « 0.05) from activity levels at Palo Alto
(0.072 + 0.034 mAbs/min) and Monterey (0.063 + 0.021 mAbs/min) (ANOVA p = 6.66 x
103
However, Figure 7 does not take into account the effect differing body size on
GST activity. To correct for this, the data were normalized to total tissue wet weight, as
shown in figure 8. Total tissue wet weight was extrapolated in each case from valve
dimensions using the equation noted above. The activity at Moss Landing (0.0573 +
0.0226 mAbs/min/g tissue) was significantly higher than the activities at both Palo Alto
(0.0327 + 0.0140 mAbs/min/g tissue) and Monterey (0.0228 + 0.0068 mAbs/min/g
tissue) (ANÖVA p = 3.8 x 10“, Tukey-Kramer p 50.05). This analysis, like that shown in
Figure 7, supports the hypothesis that GST activity is higher in organisms from a polluted
site.
Measurement of GST activity: Time Course
Figure 9 shows the GST activity data from the 30-day transplantation time-course,
normalized to total tissue mass. At t = 0 days, GST activity is 0.0439 + 0.0152
mAbs/min/g tissue and at t = 30 days, GST activity is 0.0315 + 0.0155 mAbs/min/g
tissue. There is a significant decrease in GST activity over time (two-sample t-test p =
0.0283), supporting the hypothesis that mussels that experience a decrease in pollutant
concentration will show a decrease in GST activity.
Discussion
The influx of anthropogenic pollutants from industry, agriculture, and other
human activities into coastal water has a direct effect on marine life. In this study. I
chose to examine an aspect of that effect—one of many biochemical pathways that
metabolize those chemicals—as an analytical tool to determine the level of pollution in
coastal marine habitats. Currently, testing for both organic chemicals and heavy metals is
conducted in two ways: direct water sampling, and measurement of pollutant
concentration in bioaccumulator tissue.
Most organic pesticides are lipophilic and sparingly soluble in water, thus the
direct sampling method sometimes used by regional monitoring programs (SFEI, 1999) is
complicated and costly due to the low concentrations of pollutants in the water.
Lipophilic compounds accumulate in filter-feeding organisms, so higher concentrations
may be found in the tissues of filter-feeders like mussels and clams, and an alternative
testing method for organic compounds uses the tissues of these filter-feeders. This
method of testing provides specific information on concentrations of individual pollutants
accumulated by the organism, but because pollutants may accumulate at different rates
(Bjork and Gilek 1997), the relative concentrations of accumulated toxins in tissues may
not represent the actual concentrations of those tissues in the surrounding environment.
An alternative to these direct methods is to study enzymatic detoxification
pathways to gauge organic stress in marine bioaccumulators. There are a variety of
enzymes involved in detoxification. In addition to GST, some of these enzymatic
pathways, such as cytochrome p450 and multixenobiotic transfer proteins have been
considered as biomarkers to study toxic stress on an organism. Most studies on
cytochrome p450 have been performed on fish or other vertebrates, although Livingstone
et al. (1989) have assayed for cytochrome p450 activity using a fluorescence assay based
on ethoxy-o-deethylase.
The multixenobiotic transport system has been studied in M. californianus.
Cornwall et al. (1995) determined that moderately hydrophobic pesticides such as dacthal
and chlorbenside are substrates for the multixenobiotic transport protein, but extremely
hydrophobic pesticides such as DDT and its derivatives are not. Thus, studying GST--
which does detoxify these hydrophobic pesticides-in conjunction with multixenobiotic
transport protein activity, would provide a more complete illustration of the concentration
of most types of toxic pesticides in coastal waters. Furthermore, examining a variety of
detoxification pathways in several aquatic organisms linked in the food web would show
the course of toxins from phytoplankton into higher trophic levels.
The results from three different sites show a significant correlation between GST
activity in the digestive gland of M. galloprovincialis and organic pollutant levels. The
results from the time-course transplantation experiment indicate that GST activity is
dependent on the ambient concentration of organic pollutants.
This study uncovered unexpected correlations between the pollutant level of the
three sites and both mass of the digestive gland and total protein concentration in the
gland. That mussels from Moss Landing, the most polluted site, have larger digestive
glands per total tissue wet weight and more total protein per gram digestive gland
suggests that these two factors are themselves responsive to pollutant levels. The
digestive gland is the primary detoxifying organ in M. galloprovincialis, and it may
contain several detoxification pathways that are induced by high pollutant concentrations.
Thus, the total protein concentration in the digestive gland would increase with more
pollutants in the water, and the digestive gland may itself respond by increasing its size to
increase its detoxification power. Thus, a very simple estimate of the pollution in a
habitat may be made by examining the relative mass of the digestive gland compared to
those of individuals from pristine sites.
There are many directions to take in future studies on GST activity in M.
galloprovincialis as an indicator of marine pollution. Though this paper suggests a strong
correlation between GST activity and marine pollution, more studies would provide
evidence for whether organic pollutants are the cause of elevated detoxification enzyme
activity. Including other enzyme pathways, as well as expanding the time course
experiment as mentioned above would supply more conclusive data for the dependence
of enzymatic detoxification on pollutants in the water.
Future studies: possible confounding factors
There are many factors other than pollutant concentration that may vary among
sites, but which this study did not consider. What are the effects of seasonal changes,
including climate and run-off, on the mussels in each site? Do GST activity levels vary
with the reproductive cycle of each individual? How does food availability vary among
the sites?
In future studies, I would perform variations of the time course with more
controls. In addition to moving M. galloprovincialis from a polluted site into a clean site,
1 would transplant individuals from a clean site to a site higher in pollutant concentration.
1 would also control for seasonal and shorter-term variations by taking parallel
measurements of individuals that had not been transplanted. Dosing organisms from a
clean site with known concentrations of organic chemicals in the laboratory would show
the organism’s detoxification response to only one pollutant at a time.
Future studies: other enzyme systems
Unlike the other detoxification enzymes mentioned, metallothionein is not
induced by organic compounds, but instead is induced by heavy metal pollutants. For a
complete look at pollution in the coastal marine environment, metallothionein must be
included along with enzymes that catalyze the breakdown of organic pollutants. For
example, M. galloprovincialis from Monterey Marina, the location with the lowest
concentration of organic pollutants, has the lowest level of GST activity, indicating that
these mussels experience relatively low levels of organic pollutants. However, it is
thought that this population of mussels is experiencing toxic stress due to copper and
tributyltin pollution from anti-fouling paints and other byproducts of heavy boat use in
the marina. Mussels from the Monterey Marina exhibit shell morphologies (large shell
width to length ratio) that suggest they are not healthy (Mark Stephenson, pers. comm.).
and assays of other enzyme systems, which detoxify compounds other than organic
pollutants, may be useful in determining the source of this stress.
Conclusions
GST activity is higher in the digestive glands of M. galloprovincialis from water with
higher concentrations of anthropogenic organic pollutants. GST activity does decrease
when organisms are transplanted from a site with high concentrations of organic
pollutants to a less contaminated site. From these two conclusions, I believe that activity
levels of GST and other detoxification enzymes in marine organisms are useful indicators
of the general level of marine pollution. In combination with direct sampling methods,
this biochemical assay system can provide a comprehensive measure of the
concentrations of organic pollutants in certain environments, as well as the ecological
implications of extended exposure to these pollutants in the biomarker species, M.
galloprovincialis.
Acknowledgements
1 would like to thank George Somero, Peter Fields, and Nancy Eufemia for their guidance
and ideas. Thank you to Mark Stephenson at Moss Landing Marine Labs. Thank you
Caren Braby, Kevin Brown, Gina Kang, Chris Patton, Eric Sanford, the Somero
Laboratory, and the Spring 2000 students at HMS.
Literature Cited
Bayne, B.L. 1978. Mussel watching. Nature 275:87-88.
Bjork, M. and M. Gilek. 1997. Bioaccumulation kinetics of PCB 31, 49 and 153 in the
blue mussel, Mytilus edulis L as a function of algal food concentration. Aquat. Toxicol.
38:101-123.
Bolognesi C., E. Landini, P. Roggieri, R. Fabbri, and A. Viarengo. 1999. Genotoxicity
biomarkers in the assessment of heavy metal effects in mussels: experimental studies.
Environ. Mol. Mutagenesis 33:287-292.
Cornwall, R., B.H. Toomey, S. Bard, C. Bacon, W. Jarman, and D. Epel. 1995.
Characterization of multixenobiotic/multidrug transport in the gills of the mussel Mytilus
californianus and identification of environmental substrates. Aquat. Toxicol. 31:277-296.
Fitzpatrick, P.J., D. Sheehan, and D.R. Livingstone. 1995. Studies on isoenzymes of
glutathione S-transferase in the digestive gland of Mytilus galloprovincialis with
exposure to pollution. Mar. Environ. Res. 39:241-244
Grelle, C. and M. Descamps. 1998. Heavy metal accumulation by Eisenia fetida and its
effects on glutathione-S-transferase activity. Pedobiologia 42:289-297.
Habig, W. H. and W. B. Jakoby. 1981. Assays for differentiation of Glutathione S-
Transferases. Meth. Enzymol. 77:398-405.
Habig, W. H. and W. B. Jakoby. 1981. Glutathione S-Transferases (Rat and Human).
77.218-231.
Meth. Enzymol. 11.2
Hermes-Lima, M. and K. B. Storey. 1993. Antioxidant defenses in the tolerance of
freezing and anoxia in garter snakes. Am. J. Physiol. 265:R646-R652.
Kaaya, A., S. Najimi, D. Ribera, J. F. Narbonne, and A. Moukrim. 1999. Characterization
of glutathione S-transferases (GST) activities in Perna perna and Mytilus
galloprovincialis used as a biomarker of pollution in the Agadir Marine bay (south of
Morocco). Bull. Environ. Contam. Toxicol. 62:623-629.
Karam, D. 1998. Glutathione S-transferase: an enzyme for chemical defense in plants.
http:/www.colostate.edu/Depts/Entomology/courses/en570/papers 1998/karam.htm
Livingstone, D. 1989. Cytochrome P-450 and oxidative metabolism in invertebrates.
Biochem. Soc. Trans. 18:15-19.
Livingstone, D. 1991. Organic xenobiotic metabolism in invertebrates. Adv. Comp.
Environ. Physiol. 7:45-185
Livingstone, D., M. A. Kirchin, and A. Wiseman. 1989. Cytochrome P-450 and oxidative
metabolism in molluscs. Xenobiotica 19:1041-1062.
Pederson, K. 2000. Characterizing watersheds in mussel hybrid zone: central California
Coast. Unpublished work. California State University Monterey Bay. Geographical
Information Systems Class, Spring 2000.
Regoli, F. 1998. Trace metals and antioxidant enzymes in gills and digestive gland of the
mediterranean mussel Mytilus galloprovincialis. Arch. Environ. Contam. Toxicol. 34:48¬
63.
Regoli, F. and G. Principato. 1995. Glutathione, glutathione-dependent and antioxidant
enzymes in mussel, Mytilus galloprovincialis, exposed to metals under field and
laboratory conditions: implications for the use of biochemical biomarkers. Aquat.
Toxicol. 31:143-164.
SFEI. 1999. 1997 Annual Report: San Francisco Estuary Regional Monitoring Program
for Trace Substances. San Francisco Estuary Institute, Richmond, CA.
SWRCB (State Water Resources Control Board). 1996. State Mussel Watch Program.
1993-1995 Data Report. http:/ww.swrcb. ca. gov/programs/smw/index.html
Schlenk, D. and D. Buhler. 1989. Xenobiotic biotransformation in the pacific oyster
(Crassostrea gigas). Comp. Biochem. Physiol. 94C:469-475.
Figure Legend
Figure 1. Map of study sites. (Pederson 2000)
Figure 2a. Anthropogenic organic pollutants in Mytilus tissue. Locations are PA: Palo
Alto, ML: Moss Landing, and MM: Monterey Marina. The y-axis shows concentrations
of accumulated organic chemicals in ppb, or ug/L, from O to 2000 (SWRCB 1996; SFEI
1999)
Figure 2b. Anthropogenic organic pollutants in Mytlius tissue. Same locations and data
as Figure 2a. The scale on the y-axis is increased by a factor of 10 (SWRCB 1996; SFEI
1999)
Figure 3a. Total tissue wet weight (g) vs. shell length(mm).
Figure 3b. Total tissue wet weight (g) vs. size index (cm’) (see text).
Figure 4. GST activity normalized to total protein concentration. Each vertical bar is the
average of the activities of 14 individuals from each site. Sites are MM: Monterey
Marina, ML: Moss Landing, and PA: Palo Alto. Error bars are standard deviations from
the mean. Shared letters (A, B, AB) indicate groups whose means are not significantly
different (Tukey-Kramer p 2 0.05).
Figure 5. Total protein concentration (mg/mL) vs. mass digestive gland (g).
Figure 6. Mass digestive gland (g) vs. total tissue wet weight (g).
Figure 7. GST activity normalized to whole digestive gland. Each vertical bar is the
average of the activities of 14 individuals from each site. Sites are as above. Error bars
are standard deviations from the mean. Shared letters (A, B) indicate groups whose
means are not significantly different (Tukey-Kramer p 2 0.05).
Figure 8. Activity normalized to total tissue mass. Each vertical bar is the average of the
activities of 14 individuals from each site. Sites are as above. Error bars are standard
deviations from the mean. Shared letters (A, B) indicate groups whose means are not
significantly different (Tukey-Kramer p2 0.05).
Figure 9. GST Activity: Time Course. GST activity is normalized to total tissue wet
weight.
Figure 1: Study Sites
(Pederson 2000)
Concentration (ppb, dry weight)
o8888888.
EDEE

OZO
9
8 0
0
a
Concentration (ppb, dry weight)
o88888888
EDEE
93
O
8
8
Total Tissue Wet Weight (no gonad) (g)
—

R
O
8
Total Tissue Wet Weight (no gonad) (g)

O
O
c
Figure 4
GST Activity Normalized to Total Protein Concentration
MM
ML
PA
AB
B
A
Site
Total Protein Concentration (mg/mL)
NOONO
2
PE
9:

+ 5
85
9
+
%
8
+2
O
Mass digestive gland (g)
oaoo
8
3 5
0


X5
8
0.4
0.3
0.2
0.1
0.0 -
Figure 7
GST Activity Normalized to Whole Digestive Gland


PA
MM
ML
B
Site
0.10
0.08
0.06
0.04
0.02
0.00 +
Figure 8
Activity Normalized to Total Tissue Mass

.
PA
ML
MM
Site
O
8
8
GST Activity (mAbs/min/g)
N0
S

990
90
0
009
00°0
9
o°% * 9
•