Interseasonal variability in biomarkers of exposure in fish inhabiting a southwestern Australian...

Interseasonal Variability in Biomarkers ofExposure in Fish Inhabiting a SouthwesternAustralian Estuary

Diane Webb,1 Marthe Monique Gagnon,1 Tom Rose2

1Department of Environmental Biology, Curtin University of Technology, Bentley Campus,Perth, Western Australia 6845, Australia

2Department of Environment, Hyatt Centre, East Perth, Western Australia 6892, Australia

Received 14 February 2005; revised 13 May 2005; accepted 13 May 2005

ABSTRACT: The Swan–Canning Estuary, in southwestern Australia, undergoes distinct seasonalchanges, with freshwater discharge predominant in the winter (wet) season and low flow with high salinitypredominant in the dry summer season. To investigate seasonal variability in biomarkers of exposure infish, black bream (Acanthopagrus butcheri) were collected from seven sites in the Swan–Canning Estuaryin winter 2000 and in summer 2001. No interseasonal or intersite differences in serum sorbitol dehydro-genase concentration were found, indicating the measured mixed-function oxygenase (MFO) enzymeswere not influenced by liver damage. The ethoxyresorufin-O-deethlyase (EROD) activity of the postspawn-ing females was higher in summer than in winter but was significantly lower than that in males in both sea-sons, suggesting estradiol suppression in females. Sexual differences in ethoxycoumarin-O-deethylase(ECOD) activity were not evident in either season. Both EROD and ECOD activities and polycyclic aro-matic hydrocarbons (PAH) biliary metabolites had significantly different patterns of induction betweenseasons. The ratio of naphthalene-type to benzo(a)pyrene-type biliary metabolites was significantly higherin summer, indicating the sources of petroleum hydrocarbons were petrogenic compared to in winter,when the source was a mixture of pyrogenic and petrogenic PAHs. There was no upstream or down-stream gradient of response in any biomarker in either season, demonstrating that there were multiplesources of contaminant input into the estuary. Although winter biomarker levels were triggered by the dis-charge runoff from major roads and drains, summer biomarker levels appear to have been related to rec-reational boating use on the estuary. ' 2005 Wiley Periodicals, Inc. Environ Toxicol 20: 522–532, 2005.

Keywords: biomarker; biomonitoring; black bream; ECOD; EROD; PAH bile metabolites; Swan–CanningEstuary

INTRODUCTION

The Swan–Canning Estuary, in the southwestern part of

Australia, is the focal point of the city of Perth, whose pop-

ulation is approximately 1.5 million people. The estuary

has a long narrow entrance channel (ca. 5 km, the lower

estuary), two large basins (middle estuary), and long saline

reaches of two tributary rivers, the Swan and Canning riv-

ers (the upper estuary, > 20 km; Kanandjembo et al., 2001;

Fig. 1). Southwestern Australia has a Mediterranean-type

climate characterized by hot, dry summers and cool, wet

winters. The estuary experiences highly seasonal condi-

tions, with freshwater discharge dominating tidal influences

during the wet winter months, whereas during the dry

summer, there is cessation of freshwater flow with marine

�C 2005 Wiley Periodicals, Inc.

522

Correspondence to: D. Webb; e-mail: [email protected]

Contract grant sponsor: Curtin University.

Contract grant sponsor: ARC SPIRT (to M.M.G.).

Contract grant sponsor: Water & Rivers Commission, Perth, Western

Australia.Published online in Wiley InterScience (www.interscience.wiley.com).

DOI 10.1002/tox.20141

conditions prevailing throughout the estuary (Riggert,

1978; Twomey and John, 2001). As seasonal winter rainfall

declines, there is a gradual increase in salinity, creating a

salt wedge that advances upstream over the course of the

spring and summer until, by autumn, the middle estuary

becomes saline to hypersaline and the upper estuary brack-

ish to saline. Estuarine conditions can extend inland up to

60 km from the mouth of the Swan River and up to the Kent

Street Weir on the Canning River (Stephens and Imberger,

1996; Twomey and John, 2001; Fig. 1).

The Swan–Canning Estuary is an important historical,

recreational, and economic focus for the city of Perth

(Swan River Trust, 1999a). During the summer the estuar-

ine waters are used extensively for recreational activities

such as fishing, yachting, and jet- and water-skiing, as well

as by ferries for tourism purposes. This summer increase in

recreational and commercial vessels could amplify the level

of burned and unburned fuels from outboard motors input

into the estuary. Although the input of freshwater via road

runoff from winter rainfall ceases over the dry summer

months, the potential remains for inputs from wash downs

of commercial premises, the illegal dumping of waste, and

accidental spills. Over the dry summer months exhaust and

oil from cars, as well as other debris, accumulate on road

surfaces and are flushed into the estuary at the onset of the

wet season. The impact of these inputs of nonnutrient con-

taminants on the biota inhabiting the estuary from these

sources is little understood (Swan River Trust, 1999b).

In winter 2000, while the river was flowing, a prelimi-

nary study was undertaken to investigate the response of

black bream (Acanthopagrus butcheri) to contamination

under field conditions. Biomarkers of exposure measured

were: activity of the mixed-function oxygenase (MFO)

enzyme ethoxyresorufin-O-deethylase (EROD) and levels

of naphthalene-, pyrene-, and benzo(a)pyrene-type metabo-

lites of polycyclic aromatic hydrocarbons (PAHs) in bile

(Webb and Gagnon, 2002). It was concluded that black

bream was a suitable bioindicator species for field studies

in the Swan–Canning Estuary, although EROD activity

suppression in prespawning female fish suggested that PAH

biliary metabolites were a more suitable biomarker of expo-

sure to PAH compounds in the estuary than was EROD

activity. The study also found no downstream enrichment

or accumulation in larger PAH compounds in the estuary

following heavy winter rainfall (Webb and Gagnon, 2002).

The objective of this subsequent study was to evaluate

the interseasonal variability of biochemical markers of

exposure in the black bream. In addition, as an alternative

to using EROD activity as a biomarker, the MFO enzyme

ethoxycoumarin-O-deethylase (ECOD) was evaluated as a

biomarker of exposure of the black bream to urban contam-

inants. This study was part of ongoing research into the via-

bility of using a suite of biomarkers in native fish from the

Swan–Canning Estuary to incorporate into a routine moni-

toring program.

MATERIALS AND METHODS

Chemicals

All chemicals were purchased from Sigma Aldrich Pty Ltd.

(Castle Hill, NSW, Australia) unless otherwise indicated.

Sampling Stations

Black bream were collected by a commercial fisherman at

the end of the winter of 2000 from six sites in the Swan

River and from one site in the Canning River. Fish also

were collected in late summer 2001 from five sites in the

Swan River and two sites in the Canning River. As all areas

in the Swan–Canning Estuary have been affected by human

activity (Swan River Trust, 1999a), none of the sampling

sites could be considered a reference site. Water quality

was measured for temperature, pH, and salinity 1.5–2.0 m

below the surface of the water, using a TPS WP-81 Con-

ductivity-Salinity-pH-Temperature Meter at each site dur-

ing fish collection. Dissolved oxygen measurements were

taken with an Oxi 320 oximeter.

The following sites were sampled (Fig. 1):

� Helena: This site is approximately 40 km upstream from

the Swan River Estuary mouth, downstream from the

major tributaries of Bennett Brook and the Helena River.

The area is surrounded by developed land with 415%

native vegetation. The site receives drainage from roads,

bridges, residential properties, hobby farms, light indus-

try, and parks.

� Ascot: The Ascot station is about 6 km downstream from

the Helena site. Bordered by a racecourse and a major

Fig. 1. Field collection sites in the Swan–Canning Estuary(adapted from Swan River Trust, 1999).

523INTERSEASONAL VARIABILITY IN FISH BIOMARKERS OF SW AUSTRALIA

residential development, it receives drainage from the

international and domestic airports, to the south, and to

the north, a major drain (Bayswater Main Drain) with a

catchment area of 26.2 km2 brings storm-water and road

drainage from high-density residential, commercial, and

light- to medium-industrial areas (DoE, 2003). An im-

portant arterial road crosses the river at this point.

� Belmont: An additional 6 km downstream from the Ascot

site, this station has the Belmont Park Racetrack on it

southern banks, the site of a major pesticide spill in 1997

(Swan River Trust, 1998). A recently constructed major

arterial road crosses the river near the site. The site

receives drainage from high-density residential and com-

mercial properties.

� Barrack Street: This site is 20 km from the estuarine

mouth near the northern bank of a section of the estuary

known as Perth Water. It has the Perth Central Business

District (CBD) on its northern shore and significant resi-

dential and commercial development along the southern

banks. Perth Water has undergone many changes since

European settlement, with river reclamations, channel

deepening, and riverbank modifications. It is regularly

dredged for navigational purposes. The banks are bound

by major roads and a freeway interchange carrying heavy

vehicular traffic. The site has jetties from which river fer-

ries operate, moor, and conduct maintenance. Barrack

Street receives storm-water drainage from the CBD

including air conditioning discharges, road run off from

its immediate surrounds and the Spring Street Drain,

which collects road and storm-water drainage from high-

density urban and commercial development up to 8 km

north of the estuary. The Swan River Trust annually

reports spillages occurring near the Barrack Street Jetty

(e.g., 600 L of diesel in January 2002 and 30 kL of sew-

age in February 2002; Swan River Trust, 2002).

� Crawley: Approximately 3 km downstream from Barrack

Street, black bream were captured near the northern bank

of Matilda Bay in part of the estuary known as Melville

Water. The estuarine substrate is sandy in the shallows

and fine gray mud in the deeper waters. A yacht club sits

in Matilda Bay, and the site receives drainage from a busy

arterial road and Kings Park and Botanic Gardens (172 ha

of natural bushland set aside as public open space).

� Freshwater Bay: The most downstream site on the Swan

River, 6 km from the estuarine mouth, Freshwater Bay

has two large yacht clubs with slipping facilities servic-

ing both sail and motorized watercraft. The banks are

bound by long-established suburban residential develop-

ment containing roads, parks, and gardens that drain

directly into the estuary.

� Salter Point: This site is in the Canning River about 6 km

upstream from its confluence with the Swan River. Some

restoration work to replace fringing vegetation is being

undertaken. However, storm-water drainage directly enters

the estuary from medium-density residential development

as well as from supporting roads, parks, and gardens.

� Riverton: About 3.5 km upstream from Salter Point in

the Canning River is the Riverton site. This site has sedi-

ment composed of sulfurous black mud often mixed with

old bivalve shells, grit, and small pebbles. Slightly down-

stream from where the black bream were captured are

two bridges that carry road traffic across the river. The

site receives drainage directly from surrounding urban

roads and gardens as well as from the Mills Street Drain,

which brings runoff from the surrounding medium- to

high-density residential and intensive industrial areas.

Fish and Sample Collection

One hundred and six black bream were collected during

winter 2000, and 137 were captured in summer 2001. Tis-

sue collection was undertaken using the same techniques

described in Webb and Gagnon (2002). The biopsies col-

lected were analyzed for the following biomarkers: ECOD

and EROD activity, serum sorbitol dehydrogenase (s-SDH)

activity, and PAH biliary metabolites of the naphthalene-,

pyrene-, and benzo(a)pyrene types. These biomarkers are

known to be responsive to the contaminants in the estuary

(Webb and Gagnon, 2002). In addition, they are more sen-

sitive than is chemical analysis for petroleum hydrocarbon

detection and indicate bioavailability and uptake by fish.

EROD activity has been shown to be influenced by the sex

of the fish (Hodson et al., 1991; Goksoyr and Forlin, 1992;

Webb and Gagnon, 2002). As a consequence, ECOD activ-

ity was assessed as a possible alternative to EROD activity,

as other studies have indicated that ECOD is not affected

by sex in some species of fish (Holdway et al., 1994;

Machala et al., 1997).

The weight of each liver and gonad was recorded. Phys-

iological indices of condition factor (CF), liver somatic

index (LSI), and gonadosomatic index (GSI) were calcu-

lated using equations (1), CF ¼ [(BW � GW)/TL3] � 100,

(2), LSI ¼ (LW/CW) � 100, and (3), GSI ¼ (GW/CW) �100, where BW is total body weight, GW is gonad weight,

TL is total length, LW is liver weight, and CW is carcass

weight. The condition factor is based on gonad-free weight

to avoid any bias from variation in sexual maturation, and

the LSI and GSI are based on carcass weight to avoid bias

from variability in the fat content of the gonads and intes-

tines and in gonad weight (Hodson et al., 1991).

Postmitochondrial Supernatant (PMS)Preparation

Individual liver samples were thawed on ice, then homo-

genized in HEPES (pH 7.5) using a Heidolph DIAX 900

524 WEBB, GAGNON, AND ROSE

homogenizer. The homogenate was centrifuged (Jouan

CR3i centrifuge) at 9800 rpm for 20 min at 48C, and the S9

postmitochondrial supernatant (PMS) was collected for

immediate use. The protein content of the PMS was deter-

mined according to Lowry et al. (1951).

EROD Assay

EROD activity was measured using the method of Hodson

et al. (1991) optimized for black bream, as detailed in

Webb and Gagnon (2002). EROD activity is expressed as

picomoles of resorufin produced per milligram of total pro-

tein per minute (pmol R mg Pr�1 min�1).

ECOD Assay

ECOD activity was assessed using a modified method

based on that of Holdway et al. (1998). The reaction mix-

ture, containing 0.1M Tris (hydroxymethyl aminomethane)

buffer (pH 7.4), KCl, MgCl2, 0.125 mM NADPH solution,

and PMS, was incubated for 2 min in a water bath at 308C.The reaction was initiated by adding 2 mM ethoxycoumarin

and incubated for an additional 10 min at 308C and then

terminated by the addition of 5% ZnSO4 and saturated

Ba(OH)2. Umbelliferone (C9H6O3, 7-hydroxycoumarin)

standards (0.000–0.093 nM) and samples were centrifuged

to precipitate proteins, and 1 mL of the supernatant was

transferred to a test tube. To each tube was added 0.5 M

glycine-NaOH buffer (pH 10.4), and the fluorescence of the

buffered supernatant was read on a Perkin-Elmer LS-45

luminescence spectrometer at excitation and emission

wavelengths of 380 and 452 nm, respectively (slit 10 nm

ex/10 nm em). ECOD activity is expressed as picomoles of

7-hydroxycoumarin produced per milligram of total protein

per minute (pmol H mg Pr�1 min�1).

Serum Sorbitol Dehydrogenase (s-SDH) Assay

Serum SDH activity was analyzed using a Sigma Diagnos-

tic Kit (procedure 50-UV). The change in absorbance was

read on a Pharmacia UV-visible spectrophotometer at

340 nm and expressed as milli–international units (mU) in

the serum of black bream.

Determination of Bile Metabolites

Three biliary metabolite types—naphthalene, pyrene, and

benzo(a)pyrene [B(a)P]—were measured by fixed wave-

length fluorescence. The term metabolite type was used as

the method of detecting groups of compounds that fluoresce

at specific wavelengths. For example, nearly all naphtha-

lene-type metabolites fluoresce at ex290/em335 nm. This

method offers the advantage of enabling particularly sensi-

tive detection of a group of metabolites originating from a

common parent compound (Lin et al., 1996). Consequently,

the results are expressed by metabolite group, with naph-

thalene-type metabolites as one group and pyrene- and

B(a)P-type metabolites the other two groups. The concen-

trations of PAH bile metabolites are reported as the metab-

olite equivalents of their respective standards, the amount

of a metabolite that would be present if a group of metabo-

lites originated from the same parent compound (Krahn

et al., 1986).

Biliary metabolites were measured by fixed wavelength

fluorescence (FF) using the methods of Krahn et al. (1986)

for pyrene-type metabolites and of Lin et al. (1996) for both

naphthalene-type and B(a)P-type metabolites.

The protein content of the bile was determined using the

method of Lowry et al. (1951) in order to account for

changes in PAH levels from differences in the feeding status

of fish (Collier and Varanasi, 1991). The protein content of

the bile reflects the amount of water in the bile, or how

diluted it is, when collected from the gall bladder. Biliary

PAHs were standardized to biliary protein (metabolite mg

protein�1).

Statistical Analysis

For each biomarker, the data were tested for normality and

homoscedasticity and log-transformed to achieve normality.

Statistical analysis was undertaken using the SPSS 10 for

Macintosh statistical package. Student t tests were carried

out to identify differences between sexes, and where differ-

ences were identified (p < 0.05), data for each sex were

treated separately. For each biomarker, a two-way analysis

of variance (ANOVA) was run initially to investigate

whether the data were affected by site/season interaction. As

no such interactions were found, sites and seasons were com-

pared using one-way ANOVA. Where significant differences

between sites were found (p < 0.05), Tukey’s W test was

used to identify differences between the means. Data are pre-

sented as mean6 standard error (SEM).

RESULTS

The measured water-quality parameters were within previ-

ously reported ranges in the estuary for both collection peri-

ods (Table I). Surface estuarine water was well oxygenated

(70%–98%), except for that at Salter Point, which showed a

lower level of dissolved oxygen (46%).

During both seasons a larger number of female than male

black bream were collected (winter: male 34, female 72;

summer: male 49, female 88). On the basis of data on fish

length (Table II), all fish were estimated to be > 3 years of

age (Sarre and Potter, 1999). With the exception of a few

individuals, the male black bream in winter 2000 were in

stage 5 (milting) of gonad development and the females in


stage 4 (according to Nikolskii, 1969), whereas in summer

2001 the majority of both sexes were in early stage 2.

Physiological Indices

No site differences were detected in winter 2000 for CF

(male: p ¼ 0.06; female: p ¼ 0.25). Intersite differences,

however, did appear in the summer sampling. The CF in

both males and females was significantly higher at the Hel-

ena site than at the others (p < 0.001). Seasonal differences

were detected in the male CF at the Helena site only, which

was higher in summer 2001 than in winter 2000 (p ¼ 0.01);

however, female black bream showed seasonal differences

at Helena, Ascot, and Belmont (p < 0.05), with the summer

CF higher than the winter CF at Helena, but lower at both

Ascot and Belmont (Table III).

Overall, the LSI of females was higher than that of males

in both seasons at all sites (p < 0.001). No site differences

were recorded in male LSI during winter (p ¼ 0.06), whereas

female LSI in winter was significantly higher at Freshwater

Bay than at all other sites except Belmont (p < 0.001) and

was lower at Crawley than at all other sites. The summer LSI

of male black bream was significantly lower at Freshwater

Bay than at Ascot, Belmont, and Riverton but not Helena,

Barrack Street, or Salter Point (p ¼ 0.005). The summer LSI

of female black bream was significantly lower at Freshwater

Bay than at all other sites (p < 0.001), whereas no statistical

differences between the remaining sites were identified. For

both sexes, the LSI was generally lower in summer 2001

than in winter 2000 (p < 0.001). This seasonal difference

was only significant at Salter Point for male black bream,

whereas the seasonal difference between sites for female fish

was significant at all sites except Barrack Street (Table III).

Pooled data for each sampling period revealed signifi-

cant sex-related differences in GSI in black bream. The

male GSI was higher relative to the female GSI during the

winter (p < 0.001); however, this was reversed in the

summer, with the female GSI higher than that of the males

(p < 0.001). In winter 2000 no site differences in males

were detected (p ¼ 0.05), whereas female GSI was higher

at Freshwater Bay relative to Belmont, Barrack Street, and

Crawley and at Salter Point relative to Crawley (p <0.001). Site differences in male GSI were identified in

summer 2001, with Ascot higher than Belmont, Barrack

Street, Freshwater Bay, and Salter Point (p ¼ 0.001). No

significant difference was detected in female GSI between

sites in summer 2001 (p ¼ 0.29). The GSI of both male and

female black bream was significantly lower in summer

2001 than in winter 2000 (p < 0.001) at all sites, except for

that of male GSI at Ascot (p ¼ 0.11; Table III).

EROD Activity

The EROD activity in male black bream was significantly

higher than that in the female black bream (p < 0.001) in

both seasons. Both sexes showed some differences in

EROD activity between sites, as detailed below.

Winter 2000: The EROD activity of male black bream

was higher at Barrack Street than at any other site; how-

ever, this difference was statistically significant only

between the Barrack Street and Freshwater Bay sites [p ¼0.01; Fig. 2(a)]. Female EROD activity was 1.8 times

higher at Ascot than at Belmont and 1.7 times higher at

Ascot than at Barrack Street [p < 0.001; Fig. 2(b)].

Summer 2001: The EROD activity of male black bream

was 2.3 times higher at Freshwater Bay relative to Belmont

[p ¼ 0.03; Fig. 2(a)], and female EROD activity was 1.9

times higher at Ascot than at Riverton [p ¼ 0.04; Fig. 2(b)].

A seasonal difference in the EROD activity of male

black bream was detected at Freshwater Bay (p < 0.001)

but not at any other site [p > 0.05; Fig. 2(a)], whereas for

female black bream, seasonal differences in EROD activity

occurred at all sites [p < 0.05; Fig. 2(b)].

ECOD Activity

No significant differences between male and female black

bream in ECOD activity were found in either sampling

period, so results for each sex were pooled for each season

(winter: p ¼ 0.11; summer: p ¼ 0.42).

Winter 2000: No significant differences were detected

between sites in the ECOD activity of black bream col-

lected in the Swan–Canning Estuary (p ¼ 0.49; Fig. 3).

Summer 2001: ECOD activity was higher at Ascot and

Salter Point than at any other sites; however, only Ascot

and Salter Point differed significantly with Belmont, Barrack

TABLE I. Water quality parameters of theSwan–Canning Estuary at end of winter 2000 andend of summer 2001

Site Temp (8C)Salinity

(ppt) pH

Dissolved

Oxygen (%)

Winter 2000

Helena 14 2.6 7.3 85

Ascot 15 2.7 7.3 83

Belmont 14 2.1 7.3 72

Barrack Street 16 4.6 7.5 70

Crawley 16 4.6 7.4 81

Freshwater Bay 15 9.2 8.4 98

Salter Point 17 1.2 7.6 76

Summer 2001

Helena 17 26.4 8.0 80

Ascot 18 29.8 7.6 71

Belmont 20 33.1 7.8 74

Barrack Street 20 37.1 7.9 88

Freshwater Bay 17 38.2 8.1 91

Salter Point 19.5 37.0 7.9 46

Riverton 17.5 35.3 7.8 87

Note: Measurements taken 1.5–2.0 m below surface of water.


Street, Freshwater Bay, and Riverton, but not with Helena

(p < 0.001; Fig. 3).

Significant seasonal differences in ECOD activity only

occurred at Helena (p ¼ 0.01), Belmont (p < 0.001), and

Freshwater Bay (p < 0.001), with activity higher in winter

than in summer (Fig. 3). Similar (but not significant) patterns

also were observed at Ascot and Barrack Street. This trend

was reversed at Salter Point, which showed that ECOD

activity was higher in summer than in winter, although this

difference was not statistically significant (p ¼ 0.06).

Serum Sorbitol Dehydrogenase (s-SDH)Activity

s-SDH activity did not differ significantly by sex (winter:

p ¼ 0.96; summer: p ¼ 0.59) or by site (winter: p ¼ 0.28;

summer: p ¼ 0.46). No seasonal differences were detected

at any site (p > 0.05; Table IV).

Bile Metabolites

No significant differences between male and female black

bream in any of the three biliary metabolites were identi-

fied, so the results for male and female fish were pooled for

each season (p > 0.05).

Winter 2000: Significant differences were found for each

of the biliary metabolites. Naphthalene-type biliary metab-

olites were 4.8 times higher at Barrack Street than at Fresh-

water Bay and 2 times higher at Ascot than at Freshwater

Bay (p < 0.001). The trend was for naphthalene-type bili-

ary metabolites at the remaining sites also to be higher than

at Freshwater Bay; however, the difference was not signifi-

cant [p > 0.05; Fig. 4(a)]. The level of pyrene-type biliary

metabolites at Barrack Street was 7.6 times higher relative

to that at Freshwater Bay. The remaining sites also had

higher levels (3–4.6 times) of pyrene-type biliary metabolites

compared to that at Freshwater Bay [p < 0.001; Fig. 4(b)].

TABLE II. Body length, weight, and reproductive status of black bream captured in the Swan–Canning Estuary inwinter 2000 and summer 2001

Site

Date

Sampled Sex NLength

6 SEM (cm)

Total Weight

6 SEM (g)

Carcass Weight1

6 SEM (g)

Reproductive

Stage2

Winter 2000

Helena Aug. 28 Male 8 30.66 0.5 5506 47 488 6 44 4–5

Female 7 30.36 0.5 5246 35 467 6 31 3–4

Ascot Aug. 30 Male 2 31.96 0.0 6476 8 565 6 13 4–5

Female 17 31.36 0.6 6226 40 543 6 35 3–4

Belmont Sept. 4 Male 8 31.66 0.8 6566 76 565 6 64 5

Female 9 31.96 0.9 6596 80 579 6 67 4

Barrack Street Aug. 15 Male 5 29.86 0.7 4856 58 433 6 51 4–5

Female 4 26.86 0.9 3616 32 321 6 35 3–4

Crawley Aug. 15 Male 2 29.66 1.2 4766 89 421 6 74 4

Female 4 29.16 1.3 4536 84 404 6 75 4

Freshwater Bay Sept. 21 Male 5 30.36 0.7 5096 23 439 6 16 5

Female 15 31.36 0.6 6296 50 541 6 42 3–4

Salter Point Sept. 11 Male 4 32.86 0.6 6616 63 565 6 51 5

Female 16 36.46 1.1 9676 77 841 6 67 4

Summer 2001

Helena May 8 Male 11 30.96 0.5 6066 31 556 6 28 2, 3

Female 10 30.86 0.5 5846 32 532 6 28 2, 3

Ascot April 9 Male 5 32.96 1.2 6706 75 606 6 71 2, 3, 5

Female 15 32.36 0.4 6066 26 550 6 23 2

Belmont April 3 Male 7 31.96 0.6 6046 42 541 6 36 2, 3

Female 13 32.96 0.6 6326 29 573 6 27 2

Barrack Street April 1 Male 8 36.96 1.3 9086 101 833 6 101 2

Female 12 36.26 0.8 8856 51 805 6 44 2, 3

Freshwater Bay April 29 Male 6 35.56 1.6 8056 101 749 6 93 2

Female 14 34.96 1.1 8026 99 750 6 94 2

Salter Point April 10 Male 4 34.36 2.3 7396 178 675 6 164 2

Female 16 33.26 2.1 6876 61 620 6 55 2, 3

Riverton April 30 Male 8 32.16 0.6 5706 31 514 6 30 1, 2, 6

Female 8 32.96 0.7 6176 27 552 6 25 2, 6

1Carcass weight ¼ total weight less viscera.2According to Nikolskii (1969).


The B(a)P-type bile metabolite level was 4.6 times higher at

Barrack Street than at Freshwater Bay [p ¼ 0.02; Fig. 4(c)].

Summer 2001: Significant differences between sites

were not found in either the naphthalene-type (p ¼ 0.28) or

the B(a)p-type (p ¼ 0.29) metabolites in the summer of

2001 [Fig. 4(a,c)]. However, site differences for pyrene-

type biliary metabolites were found to be significant, with

the level 2.2 times higher at the Barrack Street site than at

the Salter Point site and 2 times higher at the Belmont site

and 1.9 times higher at the Helena site than at the Salter

Point site [p < 0.001; Fig. 4(b)].

Naphthalene-type biliary metabolite levels showed sea-

sonal variation at all sites (p < 0.05), with summer levels

higher than those in winter, exception for Barrack Street

(p ¼ 0.59), which showed higher levels in winter than in

summer, although this trend was not statistically significant

[Fig. 4(a)]. Levels of pyrene-type metabolites were higher

in winter compared to summer at all sites [p < 0.001;

Fig. 4(b)]. B(a)P-type metabolite levels also were higher in

winter relative to summer at Ascot (p ¼ 0.01), Barrack

Street (p < 0.001), and Salter Point (p ¼ 0.03). There also

were higher levels at Helena, Belmont, and Freshwater

Bay, but this was not significant [p 5 0.05; Fig. 4(c)].

DISCUSSION

During both seasons the recorded physicochemical meas-

urements of salinity, temperature, pH, and dissolved oxy-

gen were all within previously recorded ranges for the

Swan–Canning Estuary for those seasons (Stephens and

Imberger, 1996; Kanandjembo et al., 2001; Twomey and

John, 2001). Black bream collected in both seasons were of

a similar size and age. The sexual bias toward female black

bream may have been the result of mesh-size selection,

resulting in smaller black bream being able to escape the

net. The black bream is a member of the Sparid family,

known to be hermaphroditic—beginning life as male, then

changing to female at a certain size. However, there is cir-

cumstantial evidence that the hermaphroditism found in

black bream populations in western Australia is only rudi-

mentary and that sex change does not occur (Sarre and Pot-

ter, 1999). In addition, the data on length of the black

bream captured for this study do not indicate any bias in

size between the sexes.

Three of the 16 black bream collected from Riverton in

the summer of 2001 had scarring from a prior bacterial

‘‘red spot’’ infection (i.e., epizootic ulcerating syndrome).

Anecdotal evidence suggests that this condition occurs reg-

ularly in black bream captured in the Swan–Canning Estu-

ary, with up to 10% of fish in the Swan River and 50% in

the Canning River suffering from this condition over some

years (K. Littleton, commercial fisherman, personal com-

munication). These black bream appeared to be in good

physical condition despite the evidence of past bacterial

infection, and no evidence suggested that the past infection

had any influence on biomarker responses in the black

bream in this study.

TABLE III. Mean (6 SEM) physiological indices of for black bream collected from the Swan–Canning Estuary in winter2000 and summer 2001

Site

Condition Factor1 Liver Somatic Index2 Gonadosomatic Index3

Male Female Male Female Male Female

Winter 2000

Helena 1.81a 6 0.04* 1.80a 6 0.06* 1.26a 6 0.08 1.67b 6 0.11* 5.00a 6 0.48* 4.01bcd 6 0.57*

Ascot 1.90 1.91a 6 0.03* 1.31 1.91b 6 0.08* 5.72 4.03bcd 6 0.23*

Belmont 1.90a 6 0.06 1.91a 6 0.05* 1.27a 6 0.08 1.95bc 6 0.10* 7.59a 6 0.50* 4.00cd 6 0.56*

Barrack Street 1.72a 6 0.06 1.82a 6 0.03 1.47a 6 0.27 1.51bd 6 0.09 4.09a 6 0.58* 3.16cd 6 0.71*

Crawley 1.75 1.73a 6 0.04 .94 1.16d 6 0.08 4.69 2.55d 6 0.51

Freshwater Bay 1.69a 6 0.04 1.88a 6 0.05 .94a 6 0.07 2.38c 6 0.13* 8.61a 6 1.79* 6.64b 6 0.57*

Salter Point 1.73a 6 0.06 1.86a 6 0.03 1.21a 6 0.07* 1.90b 6 0.06* 8.11a 6 2.28* 5.51bc 6 0.68*

Summer 2001

Helena 2.02b 6 0.04* 1.96b 6 0.04* 1.10ab 6 0.07 1.24a 6 0.07* 1.05ab 6 0.24* 1.46a 6 0.19*

Ascot 1.82a 6 0.05 1.76a 6 0.04* 1.23a 6 0.08 1.46a 6 0.08* 2.32b 6 0.77 1.52a 6 0.22*

Belmont 1.83a 6 0.04 1.75a 6 0.03* 1.20a 6 0.10 1.43a 6 0.11* 0.78a 6 0.25* 1.06a 6 0.03*

Barrack Street 1.75a 6 0.06 1.82a 6 0.04 1.01ab 6 0.08 1.31a 6 0.06 0.36a 6 0.03* 1.39a 6 0.22*

Freshwater Bay 1.74a 6 0.03 1.76a 6 0.03 0.77b 6 0.05 0.93b 6 0.06* 0.67a 6 0.11* 1.19a 6 0.04*

Salter Point 1.73a 6 0.05 1.83a 6 0.03 1.00ab 6 0.25* 1.29a 6 0.07* 0.73a 6 0.26* 1.51a 6 0.30*

Riverton 1.706 0.01 1.726 0.05 1.19a 6 0.08 1.43a 6 0.07 1.02ab 6 0.27 1.20a 6 0.05

Note: Within columns, values with the same superscript letter have no significant site differences (p � 0.05) based on Tukey’s W test, whereas sites

marked with an asterisk have significant seasonal differences. The N is as indicated in Table II.1Condition factor ¼ [(total body weight – gonad weight)/total length3] � 100.2Liver somatic index ¼ (Liver weight/carcass weight) � 100.3Gonadosomatic index ¼ (gonad weight/carcass weight) � 100.


The lack of difference in the CF between postspawning

male and female fish suggests that the differences between the

sexes in prespawning black bream detected in winter 2000

related to the different energy demands and reproductive

investment by male and female fish at this time of the year.

The higher CF of the black bream at Helena relative to other

sites in summer 2001 may have been a result of food avail-

ability in the upper reaches of the estuary compared to farther

downstream; however, this could not be discerned from the

gut contents, which in all fish predominantly consisted of

mussel shells and undigested bryophytes and macrophytes.

In fish vitellogenin is synthesized in the liver and trans-

ferred to the oocyte in the ovary via plasma (Arcand-Hoy

and Benson, 2001), resulting in gonadal enlargement. During

vitellogenesis dramatic structural changes occur in a female

fish’s liver, resulting in an increase in rough endoplasmic

reticulum and enlargement of the Golgi bodies (Bun Ng and

Idler, 1983). It therefore was not surprising to observe cova-

riation in the LSI and GSI of the prespawning female fish in

winter, with a correlation of r ¼ 0.90 during this winter sam-

pling season. No such significant correlation was observed

between these two parameters in the postspawning black

bream in the summer months. In addition, no significant rela-

tionship was found between the measured LSI and GSI in

the pre- and postspawning male black bream.

The GSI in male and female black bream indicated that

gonad development and spawning activity followed the same

pattern as that observed by Sarre and Potter (1999), who estab-

lished that gonad development in both sexes of black bream

from the Swan–Canning Estuary steadily increased from

February to July, rose sharply from July to August, peaked in

October, and then fell progressively until February of the

following year. In summer the GSI was smaller in males than

in females, with the majority of fish at a similar stage of gona-

dal development (stage 2 to early stage 3). This differs from

prespawning male black bream in winter 2000, which showed

more advanced gonadal development (late stage 4 to stage 5)

than did the females (stage 3 to late stage 4).

Although EROD activity in females was 1.5 to 2 times

higher in summer 2001 than in winter 2000, EROD activity

in female black bream was likely to have been inhibited by

estradiol competition (Lee, 1988; Waxman, 1988) in both

sampling seasons compared to male EROD activity. Of the

seven sites investigated, both the lowest level of EROD

activity of male black bream, which occurred in winter, and

the highest, which occurred in summer, were recorded at

Freshwater Bay. In fact, the EROD activity of male black

bream from Freshwater Bay was 4 times higher in summer

than in winter. This finding is consistent with the concept

that tidal flushing dilutes EROD-inducing contaminants in

winter (Webb and Gagnon, 2002), whereas the nearby mar-

inas in Freshwater Bay contribute to localized contamina-

tion that is not flushed during summer because of a lack of

river flow. Similarly, the EROD activity of female black

bream was higher in summer at Freshwater Bay than in

Fig. 3. ECOD activity (mean 6 SEM) in black bream col-lected from the Swan–Canning Estuary. Bars with the sameletter have no significant site differences. Sites with a sea-sonal difference are denoted in the table by an asterisk.Numbers in the bars represent the number of fish.

Fig. 2. EROD activity (mean 6 SEM) in black bream col-lected throughout the Swan–Canning Estuary: (A) male, (B)female. Bars with the same letter have no significant site dif-ferences. Sites with a seasonal difference are denoted in thetable by an asterisk. Numbers in the bars represent the num-ber of fish.


winter 2000, although the difference was not as great. None

of the other sites were subject to major tidal flushing in

winter, and it is possible that the higher EROD activity in

summer compared to winter at Ascot and Salter Point was a

result of increased use of these areas by two-stroke carbu-

retor recreational watercraft. The lower summer EROD

activity at Barrack Street reflects the reduced storm-water

and road runoff from the Perth Central Business District

and the Spring Street Drain.

Arukwe and Goksoyr (1997) and Lange et al. (1999)

reported differences by sex in ECOD activity in turbot

(Scophthalmus maximus) and dab (Limanda limanda),respectively. However, there was no sexual difference in

the ECOD activity of black bream in either season during

this study, in agreement with the findings of Holdway et al.

(1994) in the sand flathead (Platycephalus bassensis) andMachala et al. (1997) in the carp (Cyprinus carpio).

There were no intersite differences in the ECOD activity

of black bream among the winter 2000 samples, although

the high variability in measured activity at the sites may

have masked detection of site-related differences. This vari-

ability, most noticeable in the samples from the Barrack

Street and Crawley sites, was influenced by the small num-

ber of fish collected from these sites. High variability in

ECOD activity also was recorded at the Barrack Street site

(Fig. 3) in summer 2001. High variability also was evident

in both EROD activity and bile metabolite level at several

sites in both seasons. Published field studies typically report

Fig. 4. Biliary metabolites (mean 6 SEM) in black bream collected throughout the Swan–Canning Estuary: (A) naphthalene type, (B) pyrene type, (C) B(a)p type. For each metabolitetype, bars with the same letter have no significant site differences. Sites with a seasonaldifference are denoted in the table by an asterisk. Numbers in the bars on graph (A) repre-sent the number of fish for each metabolite.

TABLE IV. Mean (n, SEM) serum sorbitol dehydrogenase(s-SDH) of black bream collected from theSwan–Canning Estuary in winter 2000 and summer 2001

Site Winter 2000 (mU) Summer 2001 (mU)

Helena 54.4 (15, 6.2) 55.5 (21, 2.9)

Ascot 53.2 (19, 5.8) 58.8 (20, 3.5)

Belmont 42.8 (17, 6.2) 55.8 (20, 3.0)

Barrack Street 57.8 (9, 6.3) 57.1 (20, 3.6)

Crawley 64.8 (6, 10.0) N/A

Freshwater Bay 48.1 (20, 4.2) 49.8 (20, 3.3)

Salter Point 59.3 (20, 6.2) 55.7 (20, 2.7)

Riverton N/A 51.2 (16, 3.5)

Site significance (p) 0.28 0.46

Note: N/A—not available.


variability. Krahn et al. (1986) stated that variability is a

result of the different rates at which individual fish take up

and metabolize aromatic compounds.

Site differences in ECOD activity occurred during the

summer of 2001, with Salter Point and Ascot having the

highest differences. Unlike EROD activity, Freshwater Bay

ECOD activity was lower in summer 2001 than in winter

2000. There was no correlation between EROD and ECOD

activities in either season, a finding supports the hypothesis

that ECOD activity represents a cytochrome P450 pattern

that is different from that for EROD activity (Machala et al.,

1997; Stegeman et al., 1997). Lange et al. (1999) demon-

strated that each activity has species- and xenobiotic-specific

differences, indicating that EROD and ECOD activity are

catalyzed by more than one protein. In addition, it has been

established that several chemicals can differentially induce

EROD and ECOD activity (Goksoyr and Forlin, 1992).

The seasonal variability in both EROD and ECOD activ-

ity in black bream found in the present study is consistent

with the variability found in a study using pre- and post-

spawning dab (Lange et al., 1999). There was no gradient

of response in the black bream in either season for either

EROD or ECOD activity, indicating there had been no

upstream or downstream enrichment of MFO-inducing con-

taminants in the Swan–Canning Estuary.

Normally, the concentration of SDH is negligible in the

blood of fish, so its presence in serum indicates that hepato-

cellular injury has occurred (Ozretic and Kranovic-Ozretic,

1993). Fish livers with hepatocellular injuries are less capa-

ble of MFO induction than are uninjured livers (Holdway

et al., 1994). The lack of both site and seasonal differences

in s-SDH suggests that there was no bias from hepatic tis-

sue damage in the MFO activity measured (Dixon et al.,

1987; Holdway et al., 1994).

Fixed wavelength fluorescence (FF) analysis of bile meas-

ures groups of PAH metabolites, with the naphthalene type

mainly comprised of two- and three-ring structures, the pyrene

type mainly four-ring structures, and the B(a)P type mainly

five-ring structures (Aas et al., 2000). PAH contamination

originating from a petrogenic source showed a dominance of

two- and three-ring compounds, whereas pyrogenic PAHs

were dominated by four- and five-ring compounds (Neff,

1990; Aas et al., 2000). Therefore, ratios of naphthalene-, pyr-

ene-, and B(a)P-type metabolites can be used as an indicator

of the source of PAH contamination (Aas et al., 2000). Higher

ratios indicate exposure to naphthalene-rich petroleum prod-

ucts such as unburned fuel and crude oil, whereas lower ratios

indicate exposure to petroleum compounds of pyrolytic origin

such as combusted petroleum hydrocarbons.

The naphthalene/B(a)P ratios in winter in black bream

from the Swan–Canning Estuary ranged from 246 to 396

and were proportionally similar throughout the estuary,

indicating the dominance of PAHs originating from burned

fuel (e.g., from motor vehicles) from winter runoff into the

estuary. Higher ratios were detected in the summer (1300–

2204), indicating that naphthalene-type compounds from

petrogenic sources such as unburned fuels (outboard

motors, fuel spills or leakage from industry) were more

dominant in the estuary than they were in the winter of

2000. Low-molecular-weight PAHs (e.g., naphthalene) tend

to dissipate rapidly in ecosystems, whereas larger PAHs

show less dispersion (Neff, 1990). The measured biliary

metabolites indicated that downstream enrichment of large

PAH compounds in the river did not occur in either summer

or winter. This suggests that these compounds have multi-

ple sources of input along the estuary.

The results show high interseasonal variability in bio-

marker response in black bream from the Swan–Canning

Estuary, with no seasonal upstream/downstream trends.

The large number of female black bream captured at most

sites and the suppression of their EROD activity, although

to a lesser degree in summer, make interpretation of this

biomarker difficult. Although suppression of ECOD activ-

ity in female black bream did not occur, its lack of correla-

tion with EROD activity suggests that ECOD activity is not

a suitable substitute for EROD activity. However, ECOD

activity does have the potential to be a suitable biomarker

for exposure to a mixture of contaminants other than PAHs.

Biliary metabolites appear to be the most suitable bio-

marker of exposure of black bream collected from the

Swan–Canning Estuary to PAH compounds.

Winter biomarker responses in the Swan–Canning Estu-

ary were influenced by the proximity of major roads and

drainage discharge to the collection sites. Storm-water

drainage did not have the same effect on biomarker

responses in black bream in summer as in winter because of

the notably lower rainfall during the summer months,

resulting in minimal runoff into the estuary. Summer bio-

marker levels reflected exposure to motorized recreational

craft (particularly inefficient two-stroke outboard motors),

contaminated bilge water, and refueling spillages (Swan

River Trust, 2001), in addition to localized runoff from

poorly irrigated suburban parks and gardens.

The authors extend special thanks to Mr. Kerry Littleton, com-

mercial fisherman, for his assistance in fish collection. The treat-

ment of animals was in accordance with Curtin University Animal

Experimentation Ethics N-25-00.

REFERENCES

Aas E, Beyer J, Goksoyr A. 2000. Fixed wavelength fluorescence

(FF) of bile as a monitoring tool for polyaromatic hydrocarbon

exposure in fish: an evaluation of compound specificity, inner

filter effect and signal interpretation. Biomarkers 5(1):9–23.

Arcand-Hoy LD, Benson WH. 2001. Toxic responses of the repro-

ductive system. In: Schlenk D, Benson WH, editors. Target

organ toxicity in marine and freshwater teleosts. Volume 2—

Systems. London: Taylor & Francis. p 175–202.


Arukwe A, Goksoyr A. 1997. Changes in three hepatic cytochrome

P450 subfamilies during the reproductive cycle in turbot (Scoph-thalmus maximus L.). J Exper Zool 277:313–325.

Bun Ng T, Idler DR. 1983. Yolk formation and differentiation in

teleost fishes. In: Horn WS, Randall DJ, Donaldson E, editors.

Fish physiology. Volume IX, Reproduction Part A, endocrine

tissues and hormones. London: Academic Press. p 483.

Collier TK, Varanasi U. 1991. Hepatic activities of xenobiotic

metabolizing enzymes and biliary levels of xenobiotics in Eng-

lish sole (Parophrys vetulus) exposed to environmental contam-

inants. Arch Environ Contam Toxicol 20:462–463.

Dixon DG, Hodson PV, Kaiser KLE. 1987. Serum sorbitol dehy-

drogenase activity as an indicator of chemically induced liver

damage in rainbow trout. Environ Toxicol Chem 6:685–696.

[DoE] Department of Environment. 2003. Assessment of water,

sediment and fish quality in the Bayswater drains and adjacent

Swan River, April/May 2003. Perth, Western Australia: Depart-

ment of Environment. 56 p.

Goksoyr A, Forlin L. 1992. The cytochrome P-450 system in fish,

aquatic toxicology and environmental monitoring. Aquat Toxi-

col 22:287–312.

Hodson PV, Klopper-Sams PJ, Munkittrick KR, Lockhart WL,

Metner DA, Luxon PI, Smith IR, Gagnon MM, Servos M,

Payne JF. 1991. Protocols for measuring mixed function oxy-

genases of fish liver. Canadian Technical Report of Fisheries

and Aquatic Sciences 1829, Department of Fisheries and

Oceans, Quebec. 51 p.

Holdway DA, Brennan SE, Ahokas JT. 1994. Use of hepatic MFO

and blood enzyme biomarkers in sand flathead (Platycephalusbassensis) as indicators of pollution in Port Phillip Bay, Aus-

tralia. Mar Pollut Bull 26:683–695.

Holdway DA, Brennan SE, Haritos VC, Brumley CM, Ahokas JT.

1998. Development and evaluation of standardised methods for using

liver MFO enzymes in two Australian marine fish as biomarkers of

xenobiotic exposure. Melbourne: RMIT. Report no. 24. 34 p.

Kanandjembo AN, Potter IC, Platell ME. 2001. Abrupt shifts in

fish community on the hydrologically variable upper estuary on

the Swan River. Hydrolog Process 15:2503–2517.

Krahn MM, Rhodes LD, Myers MS, Macleod WD, Jr, Malins DC.

1986. Association between metabolites of aromatic compounds

in bile and the occurrence of hepatic lesions in English sole

(Parophrys vetulus) from Puget Sound, Washington. Arch

Environ Contam Toxicol 15:61–67.

Lange U, Goksoyr A, Siebers D, Karbe L. 1999. Cytochrome

P450 1A-dependent enzyme activities in the liver of dab

(Limanda limanda); kinetics, seasonal changes and detection

limits. Comp Biochem Physiol B Biochem Mol Biol 123:361–

371.

Lee RF. 1988. Possible linkages between mixed function oxygen-

ase systems, steroid metabolism, reproduction, molting, and

pollution in aquatic animals. In: Evans MS, editor. Toxic con-

taminants and ecosystem health: a Great Lakes focus. New

York: John Wiley and Sons. p 201–213.

Lin ELC, Cormier SM, Torsella JA. 1996. Fish biliary polycyclic

aromatic hydrocarbon metabolites estimated by fixed-wave-

length fluorescence: comparison with HPLC-fluorescent detec-

tion. Ecotoxicol Environ Saf 35:16–23.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein

measurement with the Folin phenol reagent. J Biol Chem 193:

265–275.

Machala M, Nezvada K, Petrivalsky M, Jarosova A, Piacka V, Svo-bodova Z. 1997. Monooxygenase activities in carp as biochemicalmarkers of pollution by polycyclic and polyhalogenated aromatichydrocarbons: choice of substrates and effects of temperature,gender and capture stress. Aquat Toxicol 37:113–123.

Neff JM. 1990. Composition and fate of petroleum and spill-treat-

ing agents in the marine environment. In: Geraci JR, St Aubin

DJ, editors. Sea mammals and oil: confronting the risks. Lon-

don: Academic Press. p 1–33.

Nikolskii GV. 1969. Theory of fish population dynamics as thebiological background for rational exploitation and manage-ment of fishery resources. Bradley JES, translator. Edinburgh:Oliver & Boyd. 323 p.

Ozretic B, Kranovic-Ozretic M. 1993. Plasma sorbitol dehydro-

genase, glutamate dehydrogenase, and alkaline phosphatase as

potential indicators of liver intoxication in grey mullet (Mugil aur-atus Risso). Bull Environ Contam Toxicol 50:586–592.

Riggert TL, editor. 1978. The Swan River Estuary development,

management and preservation. Perth Australia: Swan River

Conservation Board. 137 p.

Sarre GA, Potter IC. 1999. Comparisons between the reproductive

biology of black bream Acanthopagrus butcheri (Teleostei:

Sparidae) in four estuaries with widely differing characteristics.

Int J Salt Lake Res 8:179–210.

Stegeman JJ, Woodin BR, Singh H, Oleksiak MF, Celander M.

1997. Cytochromes P450 (CYP) in tropical fishes: catalytic

activities, expression on multiple CYP proteins and high levels

of microsomal P450 in liver of fishes from Bermuda. Comp

Biochem Physiol C Toxicol Pharmacol 116(1):61–75.

Stephens R, Imberger J. 1996. Dynamics of the Swan River Estu-

ary: the seasonal variability. Mar Freshw Res 47:517–529.

Swan River Trust. 1998. Annual Report 1997–98. Perth, Australia:

Swan River Trust. 40 p.

Swan River Trust. 1999a. Swan–Canning cleanup program: an

action plan to clean up the Swan–Canning rivers and estuary.

Available at: http://www.wrc.wa.gov.au/srt/sccp/index.html.

Accessed 2004 Nov 9.

Swan River Trust. 1999b. Swan–Canning Ccleanup program: con-taminants in the Swan–Canning rivers and estuary: a supportingdocument to the Swan–Canning Cleanup Program Action Plan.Report no. 15. Perth, Western Australia: SCCP. 31 p.

Swan River Trust. 2001. Annual report 2000–2001. Perth, West-

ern Australia: Swan River Trust. 108 p.

Swan River Trust. 2002. Annual report 2001–2002. Perth, West-

ern Australia: Swan River Trust. 102 p.

Twomey L, John J. 2001. Effects of rainfall and salt-wedge move-

ment on phytoplankton succession in the Swan–Canning

Estuary, Western Australia. Hydrolog Process 15:2655–2669.

Waxman DJ. 1988. Interactions of hepatic cytochromes P-450with steroid hormones: regioselectivity and stereospecificity ofsteroid metabolism and hormonal regulation of rat P-450enzyme expression. Biochem Pharmacol 37:71–84.

Webb D, Gagnon MM. 2002. Biomarkers of exposure in fish

inhabiting the Swan–Canning Estuary, Western Australia—a

preliminary study. J Aquat Ecosys Stress Rec 9:259–269.


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