P Krikowa

B Honkoop

Bayne

A Burt

W Maher

A Roach

F Krikowa

P Honkoop

B Bayne

The Accumulation of Zn, Se, Cd, and Pb and Physiological Condition of Anadara trapezia Transplanted to a Contamination Gradient in Lake Macquarie, New South Wales, Australia

come     The Accumulation of Zn, Se, Cd, and Pb and Physiological Condition of Transplanted to a Contamination Gradient in Lake Macquarie, New South Wales, Australia

Introduction

Industrial development around Lake Macquarie, NSW, Australia (Figure 1) is extensive and consists of a Pb-Zn smelter, a fertiliser plant, steel foundry, collieries, sewage treatment works and two coal-fired power stations. The lake supports a large recreational fishery and provides breeding and nursery grounds for many commercial fish species.

In comparison to other NSW estuaries, Lake Macquarie has significantly higher concentrations of trace metals 1 in its sediment, especially in the northern reaches [START_REF] Batley | Current Trace Metal Status of Lake Macquarie[END_REF]Kirby et al., 200a, b;Peters et al., 1999a, b;[START_REF] Roy | Trace Metals in a Contaminated Australian Estuary-Dispersion and Accumulation Trend[END_REF].

A Pb-Zn smelter to the North of the lake has been in operation since 1897, and is a known source of Zn, Se, Cd, and Pb (SPCC, 1983, AWACS, 1995). Metal concentrations above background levels in the southern reaches and elevated metal concentrations near power stations indicate that the coal-fired power stations are also contributing trace metals to the lake (Kirby et al., 2001a, b;[START_REF] Roach | Assessment of metals in sediments from Lake Macquarie, New South Wales, Australia, using normalisation models and sediment quality guidelines[END_REF].

To evaluate if trace metals in Lake Macquarie sediments are bioavailable and likely to cause harm to benthic aquatic organisms we undertook a study to establish the exposure-dose-response relationships [START_REF] Widdows | Mussels and Environmental Contaminants: Bioaccumulation and Physiological Aspects. The Mussel Mytilus: Ecology, Physiology, Genetics and Culture[END_REF] of the sedimentdwelling cockle Anadara trapezia (which is indigenous to Lake Macquarie) transplanted along a suspected sediment trace metal contamination gradient in Lake Macquarie. Environmental trace metal concentrations were established in the sediments of Lake Macquarie and related to the uptake and accumulation of trace metals in A. trapezia. Finally, to indicate the biological response of the transplanted cockles the physiological measurements, clearance rate, absorption efficiency, oxygen consumption were made and the stress index Scope for Growth calculated [START_REF] Bayne | The effects of stress and pollution on marine animals[END_REF][START_REF] Widdows | Practical Procedures for the Measurment of Scope for Growth[END_REF].

Experimental

Study area and locations

Lake Macquarie is a large estuarine barrier lake about 100km north of Sydney on the southern outskirts of Newcastle, NSW (Figure 1). The lake extends 22km in a northsouth direction, has a maximum width of about 10 km and a surface area of approximately 109 km 2 . It is the largest coastal lake in eastern Australia and has a catchment area of approximately 622 km 2 (AWACS, 1995). The average depth of the lake is 6.7 m with a maximum depth of 11 m near Pulbah Island [START_REF] Batley | Trace Metal Speciation in Waters, Sediments and Biota from Lake Maquarie, New South Wales[END_REF]. A narrow connection to the ocean at Swansea reduces the tidal range to between 0.1 -0.2 m, resulting in poor tidal flushing of the lake (AWACS, 1995). Also there are only small salinity fluctuations due to its relatively small catchment and associated freshwater inflows.

Five locations (Figure 1) were selected to examine how trace metal concentrations diminish with distance southward from the mouth of Cockle Creek. Locations were selected to follow the dispersion of sediments from Cockle Creek, the major freshwater input into Lake Macquarie and were of similar depths (~ 1.5 m). In addition a location with no industrial or sewage inputs was chosen at Bagnalls Beach, Port Stephens as a relatively pristine external control from which the cockle A. trapezia would be collected for transplant studies.

Study design and Statistical Analyses and sampling

Sediments

The sediment study was designed for a three-factor nested analysis of variance (Time, Location and Site). There were four sampling times and six locations (including control), with two sites nested in each location. Tukey's HSD test was performed on random times and raw data to determine where the significant differences occurred.

Samples were collected by pushing an acid-washed 50-mL urine vial vertically down into the top 5 cm of the sediment layer. Vials were then withdrawn and frozen for transportation back to the laboratory.

Transplantation experiments

This study was designed for a four-factor nested analysis of variance (time, location, site and cage). Locations were the general area of sampling (Figure 1) while sites were specific areas at least 100 m apart, randomly chosen with locations for sampling.

There were three sampling times (30, 60 and 90 days) and six locations (including control), with two sites nested in each location and two cages nested in each site.

Tukey's HSD test was performed on cockles sampled after 60 days (time for cockles to reach a steady state with their environment) to determine where the significant differences occurred.

Transplantation experiments

A.trapezia Collection

The Sydney cockle, A. trapezia, was chosen to assess bioaccumulation and effects as it is native to Lake Macquarie. It is a sediment-dwelling organism and been shown to accumulate trace metals in areas where sediments are contaminated with trace metals (Bately, 1987;[START_REF] Furner | Lead, Cadmium, Zinc and Copper in Organisms of Lake Macquarie[END_REF][START_REF] Kirby | Selenium, cadmium, copper, and zinc concentrations in mullet (Mugil cephalus) from the southern basin of Lake Macquarie, NSW, Australia[END_REF]Peters et al., 1999a, b, Rai, 1998).

Insufficient cockles were present at locations to use native cockles. Collection occurred over a three-day period in April 2000, shortly after the active spawning season, that occurs during the summer months [START_REF] Rai | The use of Anadara trapezia as a bioindicator of cadmium, copper and zinc pollution in estuarine environments[END_REF]. The collection of 450 cockles was from a native population at Bagnalls Beach, Port Stephens, located North of the study area. The area was chosen for its abundance of A.trapezia, close proximity to the study area and the relatively uncontaminated state of its environment (NSW Fisheries Research Institute Staff, Port Stephens pers comm.). Collection from a single population was favoured to decrease the genetic variability of the sample population, and to ensure that organisms had a similar exposure history to contaminants. To confirm that the collection site and cockles were relatively free from contaminants, a sub-sample of 22 cockles was used to determine baseline trace metal concentrations.

Transplantation of A.trapezia

In total, sixty plastic cages 35cm length x 30cm width x 10cm height were deployed on 10th April 2000, to randomly selected sites at the study locations. Divers, using SCUBA, embedded the cages into the benthic sediment of the lake. Cages were placed subtidally in 1-1.5 m of water. Cockles were unable to be caged at the 'source' location, as Bagnalls Beach proved to be a busy public beach, with a large tidal fluctuation, making the placement of control cages at this site prone to theft and impractical. Previous studies [START_REF] Cain | Copper and silver accumulation in transplanted and resident clams (Macoma balthica) in South San Francisco Bay[END_REF]Couillard et al., 1994a, b;[START_REF] Dewitt | Do amphipods have the same concentration-response to contaminated sediment in situ as in vitro?[END_REF]Martincic et al., 1992) have concluded that cages per se have no treatment effects so this became an assumption for this study. Three days later on the13th of April 2000, after sediment was allowed to equilibrate in the cages, SCUBA divers, deployed seven cockles into each cage. Cages were then secured to avoid predation and escape of the cockles. Three retrieval trips occurred, once a month, until July 2000. On each occasion two randomly chosen cages were retrieved from each site, nested within the locations, and five cockles randomly selected from each cage for metal analyses. For the same purpose ten cockles were collected from Bagnalls Beach each month. Cockles were depurated for 72 hours in fresh aerated seawater from the location they were collected, then frozen at -20 °C until dissection.

Examinations of cockle digestive systems showed no evidence of the presence of sediment.

Water samples were also collected at each location on each sampling occasion using a 1L acid-washed low density polyethylene container. A 50-ml subsample was filtered through a 0.45 µm filter (Millipore HA), frozen on ice in the field and on return to the laboratory frozen at -20 °C until analysed.

Trace metal analysis

Sediments

Sediments were oven dried at 60°C for 72 hours and hand sieved through a nylon mesh (63µm) and a 0.2 gram sub-sample weighed into a 50-ml polypropylene centrifuge tube. Five mL of nitric acid (Aristar Grade) was added, the tube was then capped and a fiberoptic probe placed in one of the sample tubes, and used as a temperature control for all vessels. Samples were digested in a CEM 2000 microwave oven, for 60 minutes at 60% power, which produced a stable temperature of 115°C.

Samples were allowed to cool, diluted to a final volume of 50mL with deionised water, centrifuged and the supernatant removed for analysis.

Cockles

Thawed cockles were thoroughly washed of any detrital and epiphytic matter to reduce the chance of contamination. Tissues were frozen at -20 °C prior to freezedrying at -83 °C (LABCONCO, Freezone Plus 6) for 24 hours and homogenised samples digested using a low-volume microwave digestion technique [START_REF] Baldwin | Measurement of trace elements in marine biological tissues using microwave digestion in low volume pressurised vessels[END_REF]. Digested samples were allowed to cool down to room temperature, transferred to 10-mL polyethylene centrifuge tubes and diluted to 10 mL with deionised water.

Trace Metal Analysis

Extracts were analysed for Zn, Se, Cd and Pb using a Perkin-Elmer Elan 6000 Inductively Coupled Plasma -Mass Spectrometer (ICP -MS) with a Scott double pass spray chamber and cross-flow nebuliser [START_REF] Maher | Measurement of trace metals and phosphorus in marine animal and plant tissues by low volume microwave digestion and ICPMS[END_REF] 1) Water samples were analysed after on-line preconcentration with iminodiacetate resin and removal of salts [START_REF] Warken | Performance optimization of a commercially available iminodiacetate resin for thedetermination of Mn, Ni, Cu. Cd and Pb by on-line preconcentration inductively coupled plasma-mass spectrometry[END_REF][START_REF] Willie | Determination of Cu, Ni, Zn, Mn, Co, Pb, Cd and V in seawater using flow injection ICP-MS[END_REF]. The accuracy of water analyses was confirmed by the analysis of two water CRMs, NASS-5 and SLEW-3 (Table 1)

Scope for growth measurements

Physiological response measurements (clearance rate, food absorption efficiency, respiration rate and ammonia excretion) were measured for a sub-sample of predeployment cockles (n = 40), Bagnall Beach cockles and all cockles collected 90 days after transplantation using published procedures [START_REF] Bayne | Physiological Integrations. Marine mussels -their ecology and physiology[END_REF][START_REF] Bayne | Physiological ecology of two populations of Mytilus edulis L[END_REF]. Trace metal concentrations in the algae feed to cockles, Isochrysis galbana (Tahitian strain), Chaetoceros gracillis/muellrri and Tetraselmis species (Reed Mariculture Inc., USA. were measured to detect any additional trace metal exposure that may have occurred through food intake during scope for growth trials.

Trace metal concentrations in food were low (Zn: 12-72 µg g -1 ; Se: < 0.02 µg g -1 ; Cd: < 0.02 µg g -1 ; Pb: < 0.02.-2.3 µg g -1 dry mass), and can be disregarded as a possible influence on cockles..

Other analyses

For particle size analysis a portion of the dried sediment was wet sieved through 63µm mesh, oven dried and re-weighed to obtain the proportions of the silt/clay and sand fractions in the sediment samples

Results

Spatial and Temporal Variability in Sediment Trace Metal Concentrations

Initially a range of trace metals were measured in sediments (Ag, As, Cd, Co, Cu, Fe, Ni, Mn, Mo, Pb, Se, Sn and Zn), but only Zn, Se, Cd and Pb had elevated concentrations with respect to background concentrations. Bagnalls Beach had low Zn, Se, Cd and Pb concentrations relative to the locations in Lake Macquarie (Figure 2). This confirms that Bagnalls Beach, Port Stephens, was an appropriate choice to use as a less contaminated control and as a collection site for A. trapezia.

Zn, Cd and Pb

Zinc, Cd and Pb concentrations in surficial sediments differed significantly between the five locations (Table 2). The concentrations decreased in the order of locations, Cockle Bay > Warners Bay ≥ Koorooa Bay > Eraring Bay > Whiteheads Lagoon (Figure 2). The order of concentrations from highest to lowest at locations did not change with time other than for Zn and Pb at Koorooa Bay where considerable variation between sites occurred in July. Whole sediment Zn concentrations were near ANZECC/ARMCANZ (2000) sediment quality guidelines levels of concern (low: 200 µg g -1 , high: 410 µg g -1 ) at Cockle Creek (183 µg g -1 ). Whole sediment Cd concentrations were above or near ANZECC/ARMCANZ (2000) sediment quality guidelines levels of concern (low: 1.5 µg g -1 , high: 10 µg g -1 ) at Cockle Bay (9.6 µg g - 1 ), Warners Bay (2.8 µgg -1 ) and Koorooa Bay (1.54 µg g -1 ). Whole sediment Pb concentrations were also above or near ANZECC/ARMCANZ (2000) sediment quality guidelines levels of concern (low: 50 µg g -1 , high: 220 µg g -1 ) at Cockle Bay (95 µg g -1 ) and Warners Bay (49 µg g -1 ).

Zinc, Cd and Pb concentrations in sediments were significantly correlated and showed a strong relationship (Table 3) indicating they share a common source of contamination. Zinc, Cd and Pb concentrations in sediments were significantly negatively correlated with Fe concentrations (Table 3). When Whiteheads Lagoon data are removed from the correlations there are no longer any significant correlations of any kind. The similarity of grain size at Cockle Bay ( < 63 um, 28 ±5 %), Warners Bay ( < 63 um, 27 ± 7%) and Koorooa Bay ( < 63 um, 29 ± 5 %) indicates that the significant differences in Zn, Cd and Pb concentrations at these locations cannot be attributed to location grain-size effects. Whiteheads Lagoon had significantly different grain size ( < 63 um, 59 ± 13 %) Se Se concentrations in sediments differed significantly between the five locations in Lake Macquarie (Table 2). Se concentrations were similar for Cockle Bay, Warners Bay, Koorooa Bay, and Eraring Bay, but significantly higher at Whiteheads Lagoon (Figure 2b). The order of concentrations from highest to lowest at sites and locations did not change with time. At Koorooa Bay considerable variation of Se concentrations between sites occurred in July. At Whiteheads Lagoon, Se concentrations varied each month. There are no ANZECC/ARMCANZ (2000) sediment quality guidelines levels of concern for Se.

Zn -Se, Cd-Se and Pb-Se correlations were negative and statistically significant (Table 3). A significant positive relationship was found between Se and Fe concentrations and Se concentration and grain size (Table 3), suggesting that higher Se concentrations at Whiteheads Lagoon may partially be explained by enriched Fe concentrations and grain size.

Spatial and temporal variability of trace metal concentrations in A. trapezia transplanted to Lake Macquarie

Zn, Cd and Pb Zinc, Cd and Pb concentrations in whole tissues were significantly different among locations after 60 days (Table 4). Mean concentrations decreased in the order Cockle Bay > Warners Bay > Koorooa Bay > Eraring Bay > Whiteheads Lagoon and were significantly higher than Bagnalls Beach (Figure 3a). Concentrations reached a maximum after 60-days, with the exception of Zn and Cd in cockles from Warners Bay which had higher concentrations after 90-days (Figure 3a and3c). Zn-Cd (r 2 = 0.537, p = 0.0001), Zn-Pb (r 2 = 0.780, p = 0.0001) and Cd-Pb (r 2 = 0.612, p = 0.0001) concentrations were strongly correlated suggested that co-accumulation of trace metals is occurring.

Se

Se concentrations in whole tissues were significantly different between locations after 60 days (Table 4). Mean concentrations decreased in the order Cockle Bay = Koorooa Bay = Whiteheads Lagoon > Warners Bay = Eraring Bay and were significantly higher than Bagnalls Beach (Figure 3). Se concentrations increased significantly with time (Table 4), reached a maximum after 60-days and slightly decreased after this time (Figures 3b).

Spatial variation of Physiological Responses in Lake Macquarie

Physiological data for cockles is given in Table 5. Clearance rates (F = 7.21; d.f. = 4,80; p < 0.0001), absorption efficiency (F = 17.10; d.f. = 4,80; p < 0.0001), respiration rates (F = 13.41; d.f. = 4,80; p < 0.0001) and Scope for Growth (F = 5.65; d.f. = 4,80; p < 0.0001) were significantly different among locations after 90 days.

Clearance rate was reduced to < 0.4 L g -1 h -1 at Cockle Bay, then increased southward along the contamination gradient. Food absorption efficiencies were similar throughout Lake Macquarie. Of the transplanted cockles, Cockle Bay cockles had the highest respiration rates (>23 µmol O 2 g -1 h -1 ).

For Scope for Growth (Table 5) there was a general trend of low values in the north (< 8 J g -1 h -1 ) with values increasing southward (>70 J g -1 h -1 at Eraring Bay). Partially lower values were evident at Whiteheads Lagoon (52 J g -1 h -1 ). Mean values of Scope for Growth of cockles from Bagnall Beach varied from 36 -58 J g -1 h -1 over the study period (Table 5) and considered indicative of "healthy" cockles.

Exposure-dose-response relationships

Exposure-dose

Trace metal concentrations measured 60 days after transplantation were used for these analyses, as this was the approximate timing that A. trapezia trace metal concentrations equilibrated with environmental contamination (Figure 2). Zinc and Pb concentrations in cockle tissues were strongly positively correlated with concentrations of these trace metals in sediments (Figures 4a and7a). Strong correlations between Zn concentrations in A. trapezia and Cd and Pb concentrations in sediments (r 2 = 0.873, p = 0.0532; r 2 = 0.953, p = 0.0122 respectively) and whole Pb in A. trapezia and Cd in surficial sediments (r 2 = 0.984, p = 0.0024) were also found. These relationships probably are probably coincidental as Zn, Cd and Pb concentrations increase together in the sediments (Figure 2).

Dose-Response Relationships

Neither significant relationships nor trends were found between Se and Cd concentrations in the tissues of A. trapezia, their physiological responses and Scope for Growth (Table 6, Figures 5c and6c).

There was a significant relationship between tissue Zn concentration and absorption efficiency (Table 5).

Clearance rate and Scope for Growth were significantly inversely correlated with Pb concentrations in A. trapezia (Table 6, Figure 7c) Absorption efficiencies were significantly positively correlated with Pb concentrations (Table 6).

Exposure-Response Relationships

No significant relationships were evident between sediment Zn and Se concentrations and the physiological responses of A. trapezia (Table 6). Significant inverse relationships were found between Cd and Pb sediment concentrations and clearance rates and Scope for Growth (Table 6; Figures 6b and7b).

Exposure-Dose -Response Relationships

Significant Zn exposure-dose relationship (Fig 4a ) indicates that Zn bioaccumulation is occurring in response to sediment contamination but no significant impairment of physiological responses was detected. The significant Cd exposure-response relationship (Figure 6b) indicates that Cd may be indirectly influencing the health of cockles. The significant Pb exposure-dose, exposure-response and dose-response relationships (Figures 7a,b,c) indicate that Pb is probably affecting the health of cockles in Lake Macquarie.

Discussion

Spatial and Temporal Variability in Sediment Trace Metal Concentrations

Zinc, Cd and Pb concentrations decreased in a southward direction in the order Cockle Bay > Warners Bay > Koorooa Bay > Eraring Bay > Whiteheads Lagoon (Figure 2), indicating Cockle Creek as the source of contamination. Correlation analysis (Table 3) showed a common source of Zn, Cd and Pb further supporting Cockle Creek as the contamination source. This was expected as the smelter and other industries located on Cockle Creek release Zn, Cd and Pb into Lake Macquarie (AWACS, 1995). Smelter waste that has accumulated in Cockle Creek in the past is most likely the main source of trace metals, especially during flood periods when highly contaminated sediments may be scoured and transported into the lake. The absence of efficient tidal flushing in Lake Macquarie ensures that bottom sediments receive and retain virtually all of the trace metals introduced to the lake.

Sediments of Lake Macquarie are enriched in trace metals. Trace metal concentration ratios relative to background concentrations are Zn: 1.5-10, Se: 2-27, Cd: 40-700, Pb: 3.5-58 with Zn, Cd and Pb concentrations at Cockle Creek and Warners Bay (Figure 2) near or above those considered to be detrimental to sediment dwelling organisms.

Selenium concentrations in sediments were low throughout the northern section of the lake (1-2 µg g -1 ), while elevated concentrations (4-9 µg g -1 ) were found in Whiteheads Lagoon (Figure 2b). Atmospheric deposits from power station stacks and the smelter at Cockle Creek are the likely sources of Se to northern sediments. Selenium most likely enters Whiteheads lagoon from the overflow channel from the power station's ash-fly dam. High concentrations of Se in the sediments of Lake Macquarie have been previously associated with power generation stations [START_REF] Kirby | Selenium, cadmium, copper, and zinc concentrations in mullet (Mugil cephalus) from the southern basin of Lake Macquarie, NSW, Australia[END_REF]Peters et al., 1999a, b) and smelting operations (AWACS, 1995). One such study (Peters et al., 1999) found a range of 5-12 µg g -1 (mean 8.3 µg g -1 ) of Se in surficial sediments located in Mannering Bay, west of Vales Point power station. This study found no evidence of elevated Se concentrations at the northern study locations situated near the lead-zinc smelter.

The elevated Se concentrations at Whiteheads Lagoon can partially be accounted for due to a high percentage of fine grain particles (59 % relative to 28 % at most of the other sites) and the enrichment of Fe concentrations in surficial sediments (30,000 ± 9000 µg/g relative to 13,000 ± 5000 µg/g at other sites). The amount of fine sediments in the lagoon is evidence of an extremely low energy depositional environment, where minimum transportation velocities are virtually non-existent, resulting in the precipitation and accumulation of fine particles. Trace metals, are known to be associated with fine grain particles [START_REF] Luoma | Temporally intensive study of trace metals in sediments and bivalves from a large river-estuarine system: Suisun Bay/Delta in San Francisco Bay[END_REF]. A strong Fe-Se relationship (Table 3) indicates that large amounts of Se may be adsorbed to the Fe particulates. Therefore, the flocculation of fine Fe-enriched particles in Whiteheads Lagoon probably accounts for the elevated concentrations of Se in these sediments.

Generally, sediment trace metal concentrations did not vary over time during the study (Table 2; Figure 2). Small variations at Cockle Bay, Warners Bay and Eraring Bay measured over the temporal scale of this study are probably due to heterogeneity of sediments rather than temporal variations. Temporal variability in concentrations of trace metals results from the deposition of new sediments to the lake that have lower or higher concentrations of trace metals. This temporal variation would occur over larger time scales, than used in this study, as new sediments are deposited at <1 mm per year to Lake Macquarie (Peters et al., 1999a).

Zinc and Pb concentrations at Koorooa Bay varied significantly at one site in July compared to other months (Figures 2a and2d) and Se concentrations at Whiteheads Lagoon varied each month (Figure 2b). There was evidence of land-derived sediments entering Koorooa Bay near the anomalous site during the study i.e. sediment plumes and local observations indicate that mine spoil was being used for foreshore reclamation work. These factors may explain, the variability found in sediment trace metal concentrations of the anomalous site in Koorooa Bay, especially during July 2000. Temporal variation of Se concentrations at Whiteheads Lagoon, is likely to be related to the variability of discharges from the Eraring power station fly-ash dam overflow channel.

Spatial and temporal variations in transplanted Anadara trapezia trace metal concentrations

Baseline concentrations of trace metals in A. trapezia collected from Bagnalls Beach were lower than trace metal concentrations measured in A. trapezia after transplantation to Lake Macquarie (Figure 3). This justifies the initial decision of collecting A. trapezia from Bagnalls Beach to represent a lesser-contaminated location and establish baseline concentrations of trace metals. Small fluctuations in trace metal concentrations of Bagnall Beach cockles occurred over the 4-month study (data not presented) but were all below concentrations accumulated by transplanted cockles.

Zinc, Cd and Pb concentrations in transplanted A. trapezia, also decreased in a southward direction, in the order Cockle Bay > Warners Bay > Koorooa Bay > Eraring Bay > Whiteheads Lagoon (Figure 3). Zinc and Pb concentrations in A.

trapezia were significantly correlated with concentrations of these trace metals in the surficial sediments of the lake (Figures 4a and7a) indicating significant exposuredose relationships. These relationships were strong, and 98% of Zn and 96% of Pb concentration variation in A. trapezia transplanted to Lake Macquarie was explained by the concentration of these trace metals in the surficial sediments that form their habitat. Overlying dissolved water concentrations of trace metals were <2-5 µg/l, < 0.05 ug/l and < 0.03 µg/l for Zn, Cd and Pb respectively over the period of this study.

These findings are supported by previous findings of Bately, (1987) who found that Zn and Pb are in highly bioavailable forms in the sediments of Lake Macquarie and support the conclusions of Bately (1987) and [START_REF] Furner | Lead, Cadmium, Zinc and Copper in Organisms of Lake Macquarie[END_REF], that bivalves in Lake Macquarie primarily accumulate Zn and Pb from contaminated surficial sediments. However, it is not known whether cockles accumulate trace metals from resuspended sediment particles or contaminated food derived from the sediment. Selenium concentrations in A. trapezia were similar across all locations within Lake Macquarie, but concentrations were significantly greater than Se concentrations in A. trapezia from Bagnalls Beach (Figure 3). Selenium enrichment in the surficial sediments at Whiteheads Lagoon was not reflected in the tissues of A.. trapezia (Figure 5a) and not in agreement with the previous measurements made on native A. trapezia and other biota from the southern section of Lake Macquarie [START_REF] Jolley | Selenium accumulation in the cockle Anadara trapezia[END_REF][START_REF] Kirby | Selenium, cadmium, copper, and zinc concentrations in mullet (Mugil cephalus) from the southern basin of Lake Macquarie, NSW, Australia[END_REF]; Peters, et al.,1999a). These studies concluded that Se concentrations in biota in close proximity to power stations were elevated. This study indicates that Se in Whiteheads Lagoon sediments is not in a bioavailable form to A. trapezia. Se in Whiteheads Lagoon is either associated with the high amounts of Fe found in the sediments or present as reduced species such as Se o and selenium sulphides due to the reducing conditions of the anoxic sediments. These forms of Se may be less available for biological uptake, furthermore, the lack of bioturbative activity in Whiteheads Lagoon, due to a paucity of organisms previously found at his location, may break an important link between abiotic (i.e. surficial sediments) and biotic (A. trapezia) trace metal uptake at this site. The resuspension of sediments by biota (i.e. bioturbation) is a potentially important mechanism by which sediment particulate material and their adsorbed contaminants are reintroduced to the water column, making contaminants available for consumption by higher organisms [START_REF] Wainwright | Sediment-to-water fluxes of particulate material and microbes by resuspension and their contribution to the planktonic food web[END_REF]. Experimental evidence (Peters et al. 1999a) has indicated that the burrowing and feeding activities of benthic organisms in Lake Macquarie can increase Se flux to interstitial waters, resulting in higher concentrations of Se becoming available for other organisms in the ecosystem, including the benthic bivalve A. trapezia.

The uptake and retention of Zn, Cd and Pb in A. trapezia reached a maximum approximately 60 days after transplantation to Lake Macquarie (Figure 3). Zinc, Cd and Pb concentrations varied little over the next month, with the exception of cockles at Cockle Bay, where Cd and Pb concentrations slightly decreased and at Warners Bay in July where Cd and Pb concentrations increased. The increase at Warners Bay was for only one site that was visible affected by land derived sediment inputs (Figures 2a and2d). The increased variability in trace metal concentrations after 60 days also suggests that other biological and/or environmental processes could be affecting the accumulation of these trace metals.

Other studies [START_REF] Andres | Field transplantation of the freshwater bivlave Corbicula fluminea along a polymetallic contaimination gradient (River Lot, France): I. Geochemical characteristics of the sampling sites and cadmium and zinc bioaccumulation kinetics[END_REF][START_REF] Elliott | The influence of cyclic exposure on the accumulation of heavy metals by Mytilus edulis planulatus (Lamarck)[END_REF][START_REF] Hickey | Metal concentrations of resident and transplanted freshwater mussels Hyridella menziesi (Unionacea: Hyriidae) and sediments in the Waikato River, New Zealand[END_REF][START_REF] Klumpp | Investigations of the Potential of Bivalve Molluscs as Indicators of heavy metal levels in tropical marine waters[END_REF]Martincic et al., 1992;[START_REF] Romeril | The uptake and distribution of 65Zn in oysters[END_REF][START_REF] Simpson | Uptake and loss of zinc and lead by mussels (Mytilus edulis) and realtionships with body weight and reproductive cycle[END_REF][START_REF] Wallner-Kersanach | Accumulation and Elimination of trace metals in a transplantation experiment with Crassostrea rhizophorae[END_REF], have found that equilibration of trace metals in bivalves with their environment can range between 30 days to 77 days (11 weeks) and longer for some species of clams. Such a wide range in equilibration times among species can be associated with previous exposure history, life stage of bivalve, associated metabolic activities associated with adjustment to a new environment, temperature changes, and food availability. Our results showed that A.trapezia reached "steady state" with its environment and doesn't continue to accumulate trace metals as other bivalves such as Mytilis edulis do [START_REF] Langston | Metal handling strategies in molluscs[END_REF]. Thus, A.trapezia is a good biomonitor as it reflects bioavailable trace metals in its environment.

Strong relationships between tissue Zn -Cd, Zn -Pb, Cd -Pb concentrations were also found, suggesting that the accumulation of these metals may be dependent on one another or have a common source. Sediment data in this study indicates a common source of Zn, Cd and Pb. Molluscs readily take up Cd, most likely via the same pathways as Zn and Cu [START_REF] Simkiss | Transport of Metals Across Membranes[END_REF]. [START_REF] Elliott | The influence of cyclic exposure on the accumulation of heavy metals by Mytilus edulis planulatus (Lamarck)[END_REF] concluded that for the bivalve Mytilus edulis, the accumulation of Cd was greatly reduced in the presence of Zn, Pb and Cu; and, that in the presence of Cd and Pb, Zn accumulation was not directly proportional to exposure time. Laboratory uptake studies of trace metals will need to be conducted to determine the interaction of trace metals during uptake.

Spatial variation of Physiological Responses in Lake Macquarie

There was a decline in the physiological condition of A. trapezia transplanted to Lake Macquarie after a 90-day period (Table 5). By July, there was a marked difference in clearance rates at Cockle Bay and Warners Bay, absorption efficiencies at all locations, and respiration rates at Warners Bay, Koorooa Bay, Eraring Bay and Whiteheads Lagoon (Table 5). Mean Scope for Growth values at Cockle Bay were markedly lower than other locations in Lake Macquarie (Table 5).

The ability of A. trapezia to filter particulates (i.e. clearance rates) improved southward in the order Cockle Bay < Warners Bay ≤ Koorooa Bay ≤ Whiteheads Lagoon < Eraring Bay (Table 5). The decline in the condition of A. trapezia at Cockle Bay can be associated with the low food clearance rates (mean 0.31 L g -1 h -1 ) and the increase in oxygen consumption (mean 23.13 µmol O 2 g -1 h -1 ) associated with an animal under stress. As a result of enhanced oxygen consumption a marked reduction in the Scope for Growth or growth potential was recorded in cockles from the Cockle Bay mean compared to other locations (Table 5). Scopes for Growth values at all locations, except Cockle Creek, were similar to those measured in cockles from Bagnalls Beach (36-58 J g -1 h -1 ) and consistent with values reported for 'healthy' cockles [START_REF] Din | Changes in the Scope for Growth of Blood Cockles (Anadara granosa) Exposed to Industrial Discharge[END_REF]. This indicates that organisms in the southern section of Lake Macquarie, other than at Cockle creek, are not under physiological stress. However, direct comparisons should be made with caution as the water temperature was markedly higher at Bagnalls Beach (Table 5).

Absorption efficiencies were statistically similar throughout the lake; however, there was trends of decreasing efficiencies in a southward direction as feeding rates improve (Table 5). Decreases in the food absorption efficiencies of bivalves are known to be a function of high feeding rates. Similar results were found in a study on Mytilus edulis transplanted along a pollution gradient in Narragansett Bay, USA (Widdows et al., 1980-81). Several bivalve species have also demonstrated that food absorption is independent of seston concentration, however, it is dependent on the seston quality, or percentage of organics (MacDonald et al., 1998) i.e. absorption efficiencies increase with the quality of the food.

The observed decline in Scope for Growth of A. trapezia transplanted to Lake Macquarie was significantly correlated with the increasing concentrations of Cd in the surficial sediments (Figure 7b) and increasing concentrations of Pb in the surficial sediments (Figure 4d) and tissues of A. trapezia (Figure 7c). The significant Cd exposure-response relationship implies that Cd may be influencing the health of A. trapezia, probably through indirect effects on the cockle's health such as a decrease in the quality of food, rather than because of the bioaccumulation of Cd. The significant Pb exposure-dose, dose-response exposure-response relationships indicate that Pb concentrations are elevated sufficiently in Lake Macquarie surficial sediments to cause adverse effects on biota. Pb and Cd concentrations exceeded the ANZECC/ARMCANZ (2000) sediment quality guidelines levels of concern at several sites.

Trends were also evident between Scope for Growth and concentrations of Zn in A. trapezia (Figure 4c), however, caution must be exercised with interpreting this correlation as A. trapezia are known regulators of Zn [START_REF] Rai | The use of Anadara trapezia as a bioindicator of cadmium, copper and zinc pollution in estuarine environments[END_REF] and it is unlikely that elevated Zn concentrations are causing the decline in the physiological condition of this organism. No significant exposure-dose-response relationships are evident for Se (Figure 2), indicating that the accumulation of Se in the tissues are not high enough to induce stress in the organism and limit its ability for growth and reproduction.

. Natural stressors, such as temperature and salinity fluctuations, have been suggested as possible influences on the Scope for Growth. Bayne (1975, op. cit. Martin et al., 1984) and [START_REF] Bayne | Physiological Integrations. Marine mussels -their ecology and physiology[END_REF] found Scope for Growth in mussels remained relatively independent of temperature from 10 -20°C, and that the mussels suffer transient stress from reduced salinities, but recover within 2 -3 days. In the present study the natural stressors that are most likely to affect physiological responses, remained relatively constant along the length of Lake Macquarie (Table 5), furthermore, due to the nature of Lake Macquarie, in general, these environmental factors are known to vary little throughout the year (AWACS, 1995). Thus the reduction in the condition of A. trapezia transplanted to the northern section on lake can be assumed to be independent of temperature and salinity, and caused by anthropogenic stressors rather than natural environmental variables. Other contaminants such as organochlorine pesticides and ammonia in pore waters are low (NSW DEC, unpublished data), therefore are not stressors in this environment.

Risk assessment of contaminated sediments

Many papers have been published that relate exposure-dose-response for transplanted bivalves in contaminated waters (e.g. [START_REF] Gagne | Health status of Mya arenaria bivalves collected from contaminated sites in Canada (Saguenay Fjord) and Denmark (Odense Fjord) during their reproductive period[END_REF][START_REF] Zorita | Biomarkers in mussels from a copper site gradient (Visnes, Norway): An integrated biochemical, histochemical and histological study[END_REF] but none to our knowledge have applied this framework to bivalves transplanted to contaminated sediments. Zinc, Cd and Pb concentrations at Cockle Creek, Warners Bay and Koorooa Bay are above or near those considered to be detrimental to sediment dwelling organisms, based on ANZECC/ARMCANZ (2000) sediment quality guidelines. Clearly based on the criteria that exposure-dose and dose-response should be significant, Pb concentrations are likely causing adverse effects to the transplanted cockles and by inference on other sediment dwelling biota. The effects of Zn and Cd accumulation in sediments and biota are unclear. For Zn, exposure-dose is significant but dose response is not. For Cd, although the sediment quality guidelines are exceeded, exposure-dose and dose-response relationships are not significant. Zinc and Cd in sediment appear not to be directly affecting the health of transplanted cockles. These relationships, although only correlative, form the basis of a weight of evidence approach. Thus, on balance all three metals are probably contributing to the impaired physiological condition of the transplanted cockles. We are at present developing other measures of health based on the production of oxygen radicals in cells and cell damage [START_REF] Ercal | Toxic metals and Oxidative Stress Part 1: Mechanisms involved in metal oxidative damage[END_REF] as additional lines of evidence to increase the confidence in making predictions about the effect of contaminated sediments on benthic organisms. )

Sediment Zn (µg g-1) Scope for growth (J g -1 h -1

)

Sediment Zn (µg g-1)

Scope for growth (J g -1 h -1 )

A. trapezia Zn (µg g-1)

Fig 6

A. trapezia Cd (µg g -1

)

Sediment Cd (µg g-1) Scope for growth (J g -1 h -1

)

Sediment Cd (µg g-1)

Scope for growth (J g -1 h -1 )

A. trapezia Cd (µg g-1)

Fig 7

A. trapezia Pb (µg g -1

)

Sediment Pb (µg g-1) Scope for growth (J g -1 h -1

)

Sediment Pb (µg g-1)

Scope for growth (J g -1 h -1 )

A. trapezia Pb (µg g-1)

Figure 1 .Figure 2 Figure 3

 123 Figure 1. Sampling locations in Lake Macquarie, NSW, Australia

Figure 4 Figure 5 Figure 6 Figure 7

 4567 Figure 4 Exposure-dose-response for Zn. *Indicates significance (p < 0.05)

Table 1

 1 Recoveries of Zn, Se, Cd, and Pb from certified standard reference material. Values are means ± SE

			Zinc		Selenium	Cadmium	Lead
						µg g -1 dry mass or µg l -1
	CRM	Supplier	Certified	Found	Certified	Found	Certified	Found	Certified	Found
	Oyster Tissue	NIST	830 ± 57	769 ± 25	2.21 ± 0.24	2.1 ± 0.2	4.15 ± 0.38	4.7 ± 0.7	0.37 ± 0.01	0.4 ± 0.3
	SRM 1566a			(93)		(97)		(113)		(102)
	Lobster Hepatopancreas	NRCC	177 ± 10	194 ± 98	6.88 ± 0.47	5.6 ± 0.2	26.3 ± 2.1	23.9 ± 0.1	10.4 ± 2	11 ± 1
	TORT -1			(110)		(81)		(91)		(104)
	Sargasso	NIES	15.6 ± 1.2	13.7 ± 0.4	0.05 1	B.D.L.	0.15 ± 0.02	0.17 ± 0.09	1.35 ± 0.05	1.12 ± 0.05
	CRM No. 9			(88)				(113)		(83)
	Chlorella	NIES	20.5 ± 1	20 ± 1	N.A.	0.06 ± 0.01	0.026 b	B. D.L.	0.6 a	0.4 0± 0.02
	CRM No. 3			(95)						(66)
	Estuarine Sediment	NIST	138 ± 6	100 ± 3	0.6	0.5 ± 0.11	0.39 ± 0.7	0.35 ± 0.8	28.2 ± 1.8	20.7 ± 1.1
	SRM 1646			(73)		(84)		(90)		(73)
	Marine Sediment	NRCC	119 ± 12	92 ± 4	0.43 ± 0.06	0.35 ± 0.13	0.25 ± 0.94	0.23 ± 0.1	22.7 ± 3.4	19.8 ± 0.6
	BCSS-1			(77)		(81)		(92)		(87)
	Open Ocean Seawater	NRCC	0.102 ± 0.039 0.11 ± 0.04	0.018 1	B.D.L.	0.023 ± 0.003 0.022 ± 0.008	0.008 ± 0.005	0.090 ± 0.007
	NASS-5			(108)				(96)		(113)
	Estuarine water	NRCC	0.201 ± 0.037 0.18 ± 0.03	N.A.		0.048 ± 0.004 0.043 ± 0.008	0.0090 ± 0.0014 0.008 ±0.001
	SLEW-3			(90)				(90)		(89)
	1 Reference values not certified
	B.D.L. = Below Detection Limit < 0.02 µg g -1 dry mass
	N. A. = Not available
	( ) = % Recovery

Table 2

 2 Nested ANOVA results for spatial and temporal differences of Zn, Se, Cd and Pb concentrations in Lake Macquarie and Bagnalls Beach surficial sediments (n = 48). Tukey's results are presented in decreasing order (L -R) and locations underlined are not significantly different (p>0.05).

	Metal	Source	Df	F value	p > F
	Zinc	Location	5	25.79	0.0001
		Site(Location)	6	2.83	0.0417
		Time	3	1.00	NS
		Time*Location	15	1.83	NS
		Time*Site(Location)	18	19.40	0.0001
	Selenium	Location	5	24.64	0.0001
		Site(Location)	6	2.19	NS
		Time	3	2.17	NS
		Time*Location	15	9.91	0.0001
		Time*Site(Location)	18	1.57	NS
	Cadmium	Location	5	302.48	0.0001
		Site(Location)	6	6.20	NS
		Time	3	0.578	NS
		Time*Location	15	7.45	0.0001
		Time*Site(Location)	18	1.17	NS
	Lead	Location	5	95.51	0.0001
		Site(Location)	6	2.50	NS
		Time	3	1.18	NS
		Time*Location	15	2.53	0.0317
		Time*Site(Location)	18	2.97	0.0006
	Zn
	Cockle Bay Koorooa Bay Warners Bay Eraring Bay Whiteheads Lagoon Bagnalls Beach
	Se
	Whiteheads Lagoon Koorooa Bay Cockle Bay Eraring Bay Warners Bay Bagnalls Beach
	Cd
	Cockle Bay Warners Bay Koorooa Bay Eraring Bay Whiteheads Lagoon Bagnalls Beach
	Pb
	Cockle Bay Warners Bay Koorooa Bay Eraring Bay Whiteheads Lagoon Bagnalls Beach

Table 3

 3 Pearson's Correlation Coefficients (P values in italics) for trace metal, iron and silt concentrations in Lake Macquarie surficial sediments. Significant relationships are in bold where p<0.05.

	n		Zn	Se	Cd	Pb	Fe	%<63µm
	120	Zn	1
		Se	-0.3711	1
			0.0001
		Cd	0.5443	-0.3752	1
			0.0001	0.0001
		Pb	0.7747	-0.4924	0.8762	1
			0.0001	0.0001	0.0001
		Fe	-0.3943	0.8269	-0.4859	-0.5577	1
			0.0001	0.0001	0.0001	0.0001
	10	%<63µm	-0.2344	0.8032	-0.1005	-0.2091	0.7837	1
		inc. W.L	0.5145	0.0051	0.7824	0.5620	0.0073
	8	%<63µm	0.8348	0.2859	0.5936	0.7907	0.0482	1
		excl. W.L	0.0099	0.4923	0.1208	0.0195	0.9098
	W.L = Whiteheads Lagoon.

Table 4

 4 Nested ANOVA results for spatial and temporal differences of Zn, Se, Cd and Pb concentrations in transplanted Anadara trapezia tissues (n = 244) after 60-days. Tukey's results are presented in decreasing order (L -R) and locations underlined are not significantly different (p>0.05).

	Source	df Zn		Se		Cd		Pb
			F value	p>F	F value	p>F	F value p>F	F value p>F
	Location	5	9.28	0.0015	56.85	0.0001	5.64	0.0190	64.59 0.0001
	Site(Location)	5	0.66	NS	0.14	NS	0.77	NS	1.34	NS
	Cage (Site (Location))	10 0.39	NS	0.37	NS	0.40	NS	0.52	NS
	Time	2	2.44	NS	9.99	0.0046	0.80	NS	5.82	0.0205
	Time*Location	10 8.69	0.0031	0.71	NS	3.91	0.0148	2.86	0.0424
	Time*Site(Location)	9	0.34	NS	2.31	NS	0.51	NS	0.42	NS
	Time*Cage(Site(Location)) 14 1.26	NS	1.46	NS	2.27	0.0085	2.34	0.0064
	NS = not significant p>0.05
	Zn: Cockle Bay Warners Bay Koorooa Bay Eraring Bay Whiteheads Lagoon
	Se: Cockle Bay Whiteheads Lagoon Koorooa Bay Eraring Bay Warners Bay
	Cd: Cockle Bay Warners Bay Eraring Bay Whiteheads Lagoon Koorooa Bay
	Pb: Cockle Bay Warners Bay Koorooa Bay Eraring Bay Whiteheads Lagoon

Table 5

 5 Summary of physiological responses of A. trapezia from Bagnalls Beach and cockles transplanted to Lake Macquarie after 90 days July 2000 (mean ± SE) Cockle Bay Koorooa Bay Eraring Bay Warners Bay Whiteheads LagoonScope for GrowthCockle Bay Warners Bay Koorooa Bay Whiteheads Lagoon Eraring Bay

		Bagnalls			Transplant location
		Beach
			Cockle Bay	Warners Bay Koorooa Bay	Eraring Bay	Whiteheads
		n = 22	n = 20		n = 20	n = 7	Lagoon
							n = 16
	Clearance rate (Lg -1 h -1 )	1.65 ± 0.17	0.31 ± 0.35	0.84 ± 0.37	1.03 ± 0.35	1.78 ± 0.59	1.18 ± 0.51
	Absorption efficiency (%)	39	85	86	78	70	69
	Oxygen consumption	24.48 ± 1.21	23.13 ± 2.10	14.37 ± 2.21	18.91 ± 2.10	15.17 ± 3.55	13.10 ± 3.00
	(µmol O 2 g -1 h -1 )
	C = Joules consumed	119.45	23.68	51.40	65.97	133.31	110.34
	A = Joules absorbed	50.84	20.32	44.87	52.64	96.13	72.88
	R = Joules respired	11.16	10.75	6.56	8.62	6.91	6.23
	U = Joules excreted	0.46	1.43	0.50	1.02	16.18	1.67
	Scope for growth (Jg -1 h -1 )	35.95 ± 4.79	7.96 ± 19.11	37.09 ± 20.27	41.63 ± 19.11	70.35 ± 32.30	52.51 ± 27.36
	Environmental Factors
	Water Temperature ( 0 C)	22.3	13.8	14.4	14.7	15.3	15.2
	Salinity (‰)	33	34	35	35	35	35

Table 6

 6 Pearson's Correlation Coefficients (P values in italics) for A. trapezia trace metal concentrations and physiological responses and sediment trace metal concentrations. Location means were used for analysis. Significant relationships are bold where p<0.05. (n=5)

			Clearance rate	Absorption	Respiration rates	Scope for Growth
				efficiency
	A. trapezia	Zn	-0.823	0.992	0.405	-0.806
			0.0873	0.0009	0.4985	0.0999
		Se	-0.706	0.337	0.600	-0.687
			0.1827	0.5789	0.2843	0.2004
		Cd	-0.179	0.467	-0.473	-0.089
			0.7742	0.4279	0.4209	0.8857
		Pb	-0.915	0.939	0.582	-0.915
			0.0293	0.0177	0.3029	0.0295
	Sediment	Zn	-0.504	0.475	0.637	-0.532
			0.3867	0.4183	0.2480	0.3557
		Se	0.365	-0.668	-0.408	0.452
			0.5462	0.2177	0.4954	0.4446
		Cd	-0.881	0.747	0.835	-0.927
			0.0483	0.1469	0.0783	0.0233
		Pb	-0.895	0.858	0.854	-0.932
			0.0403	0.0626	0.0657	0.0209

)

Sediment Se (µg g-1) Scope for growth

Sediment Se (µg g-1)

Scope for growth (J g -1 h -1 )

A. trapezia Se (µg g-1)


Ka-Man Lee

Emma L Johnston

Low levels of copper reduce the reproductive success of a mobile invertebrate predator

Keywords: Copper, Reproductive success, Flatworms, Barnacles, Stylochus pygmaeus

Marine organisms that occur in urbanised bays can be exposed to low-level chronic pollution that results in sublethal changes to behavior or reproduction. The effects of low levels of copper on the reproductive success of a mobile invertebrate was assessed.

Free living flatworms are common predators of bivalves and barnacles. Flatworms (Stylochus pygmaeus) were exposed to low levels of copper ranging from 0 to 25 µg L -1 in the presence and absence of their barnacle prey (Balanus variegatus). Flatworms laid fewer egg batches when exposed to copper and the hatching success of the eggs was also reduced. Exposure to 25 µg L -1 copper for 10 d reduced the reproductive success of flatworms by up to 80%. Results were consistent regardless of the presence or absence of prey (barnacles). Barnacles were only moderately affected by copper but exhibited major avoidance behavior (feeding inhibition) in the presence of flatworm predators. This is the first ecotoxicological study on marine flatworms. Experiments are required to quantify the effects of flatworm predator populations on sessile invertebrate community structure in the field.

Introduction

Many near-shore marine ecosystems are strongly influenced by heavy metals discharged in domestic sewage, mining and industrial effluents [START_REF] Mance | Pollution threat of heavy metals in aquatic environments[END_REF]. Often the contaminants may not result in obvious or instant mortality, however they may disrupt the physiology or behaviour of exposed organisms [START_REF] Matthiessen | Contaminants and their effects on estuarine and coastal organisms in the United Kingdom in the late twentieth century[END_REF].

Behavioural responses can be an early and sensitive indicator of toxic stress [START_REF] Lam | Effects of cadmium on the development and swimming behvaior of barnacle larvae Balanus amphitrite Darwin[END_REF] that may lead to deterioration in the health of the organisms and the affected ecosystems [START_REF] Mccahon | Episodic pollution: causes, toxicological effects and ecological significance[END_REF][START_REF] Deangelis | Indirect effects: concepts and approaches from ecological theory[END_REF]. Behavioural changes in controlled laboratory exposures may provide a better understanding of the possible environmental consequences of toxicants especially if behavior is disrupted at environmentally realistic toxicant concentrations [START_REF] Faimali | Swimming speed alteration of larvae of Balanus amphitrite as a behavioural end-point for laboratory toxicological bioassays[END_REF]. The sublethal effects of heavy metal pollution include changes in feeding and reproductive behavior which may affect growth and reproductive success (e.g. [START_REF] Conradi | Population responses of the marine amphipod Corophium volutator (Pallas, 1766) to copper[END_REF][START_REF] Cebrian | Sublethal effects of contamination on the Mediterranean sponge Crambe crambe: metal accumulation and biological responses[END_REF].

Copper is an essential trace metal to all marine invertebrates but it is toxic at higher concentrations [START_REF] Lewis | The biological importance of copper in oceans and estuaries[END_REF]. It is a common pollutant in ports and estuaries and the harmful effects of copper on individual marine invertebrates has been widely studied and well documented [START_REF] Hall | Ecological risk assessment of copper and cadmium in surface waters of Chesapeake Bay watershed[END_REF]. However, there are fewer studies of the effects of copper on multi-species test systems [START_REF] Balczon | A comparison of the responses of two microcosm designs to a toxic input of copper[END_REF][START_REF] Johnston | Competition modifies the response of organisms to toxic disturbance[END_REF].

Flatworms from the family Stylochidae are common pests of commercial bivalves and can cause huge economic losses (e.g. [START_REF] Newman | Stylochus (Imogene) matatasi n. sp. (Platyhelminthes, Polycladida): pest of cultured giant clams and pearl oysters from Solomon Island[END_REF][START_REF] O' Connor | Halotolerance of the oyster predator, Imogine mcgrathi, a stylochid flatworm from Port Stephens, New South Wales, Australia[END_REF]. They can also reduce their prey populations alter the community structure [START_REF] Skerman | Note on Stylochus zanzibaricus Laidlaw, (Turbellaria, Polycladida), a suspected predator of barnacles in the Port of Auckland, New Zealand[END_REF]. Few studies have examined the reproductive and predatory behavior of marine flatworms.

Stylochids flatworms are also major predators of barnacles [START_REF] Skerman | Note on Stylochus zanzibaricus Laidlaw, (Turbellaria, Polycladida), a suspected predator of barnacles in the Port of Auckland, New Zealand[END_REF] and the abundant local flatworm Stylochus pygmaeus preys extensively on Balanus variegatus , the most common barnacle in the study area. The empty barnacle shells are used as a shelter and cradle for flatworm eggs and young (personal observation but see also [START_REF] Merory | A new stylochid flatworm (Platyhelminthes, Polycladida) from Victoria, Australia and observations on its biology[END_REF]. In our laboratory exposure study the following hypotheses were tested:

(1) exposure to sublethal concentrations of copper does not affect the reproductive success of S. pygmaeus (tested in the absence of prey in Experiment 1, and the presence of prey in Experiment 2)

(2) The feeding rate of the barnacle prey (B. variegatus) does not change with exposure to copper and/or flatworm predators (Experiment 2).

Materials and methods

Sample collection

Assemblages of sessile marine invertebrates were first allowed to develop on artificial substrata at Kurnell Pier, Botany Bay, New South Wales, Australia (33"59.92 S,151"12.62' E). The site is well flushed with oceanic water and ambient copper concentrations are not elevated [START_REF] Piola | Differential resistance to extended copper exposure in four introduced bryozoans[END_REF] with less than 5 µg L -1 copper. Settlement plates consisting of 6 x 6 cm black Perspex tiles attached to the underside of a 60 x 60 cm PVC backing plate. Backing plates were suspended horizontally from the pier at a depth of 3 m below the low water for 5-7 months.

Approximately 50 settlement plates were retrieved for each experiment and any flatworms found between the settlement and backing plates, and inside empty barnacle shells were collected immediately using a small paintbrush. Settlement plates were retained and brought back to the laboratory. For transport to the laboratory, each settlement plate was placed in a well-aerated transparent plastic container (15 x 8 x 8 cm) with 1 L of filtered field seawater in a dark constant-temperature room (23.5 0.5 o C). After 24 h, any flatworms emerging from the assemblages were collected using a paintbrush. Sessile organisms other than Balanus variegatus were then removed from the settlement plates. Flatworms and barnacles were observed for 2 d prior to exposure to allow them to recover from collection and transport stress they were not fed during this period [START_REF] Clesceri | Standard methods for the examination of water and wastewater[END_REF]. Abundances of flatworms are seasonal (appearing in greater numbers in warmer months) and during this study, we recorded approximately two flatworms and 14 barnacles per 100 cm 2 settlement panel.

Copper treatments

Analytical grade copper II chloride hydrous (CuCl 2 .2H 2 O) (Sigma-Aldrich USA) was used as the reference toxicant in all experiments. A 1000 mg L -1 primary copper stock solution was prepared by dissolving 2.6828g of CuCl 2 .2H 2 O in 1000 ml of Milli-Q ® filtered water. Stock solution was stored in the refrigerator at 4 o C to prevent the reduction of copper ions in the solution. A 1000 µg L -1 copper solution was prepared from this stock solution every day and diluted to obtain the sublethal experimental treatment solutions of 10 and 25 µg L -1 copper. Earlier pilot studies indicated 100% flatworm mortality occurred following 24 h exposure to approximately 100 µg L -1 copper. Seawater collected from Kurnell Pier used as a dilution medium was filtered through a 0.2 µm filter to minimise the reaction of copper ions with organic particles which could reduce the amount of free copper ions in the treatment solutions. The added copper chloride would therefore readily dissociate to release copper ions (Cu 2+ ) that cause toxicity. Treatment solutions were replaced every 24 h. The equipment used in all experiments was acid washed in 10% nitric acid for 24 h and rinsed in Milli-Q ® filtered water before use.

In preparation for copper concentration analysis, 50 ml sub-samples of the experimental copper treatments were taken at the commencement of and 24 h after the experiments commenced and were immediately acidified with analytical grade concentrated nitric acid (1.5 µl of acid per 1 ml of sample) and stored in a refrigerator at 4 o C according to [START_REF] Clesceri | Standard methods for the examination of water and wastewater[END_REF]. The concentrations of copper in the stock and experimental solutions were tested independently at the NATA accredited Australian Government National Measurement Institute using an inductively coupled plasma source mass spectrometer (ICP-MS) with a detection limit of 5 µg L -1 .

Experimental design

In all experiments, transparent plastic containers (15 x 8 x 8 cm) were used as the experimental containers. Each container was pre-soaked in the appropriate copper solution for 24 h before the start of the experiments to ensure minimal chelation of copper ions during the exposure period. Each container was filled with 1 L of treatment solution and was continuously aerated. Exposure to treatments was maintained for 10 d. In all experiments, the length and width of flatworms were measured when they were in quiescent and relaxed state. The size of opercular opening of the barnacles was measured using a pair of callipers prior to exposure to ensure that the size range of organisms in all treatments was consistent, and any changes in the response of the organisms during the experiment were not related to size differences.

Experiment 1: Reproductive success in the absence of food

Brooding behavior of adult flatworms and the subsequent hatching success of their eggs were observed with exposure to copper. The nominal copper concentrations used in this experiment were 0, 10 and 25 µg L -1 with seven replicates for each treatment. A pair of similar size flatworms were placed in each treatment container. Flatworm larvae are planktonic immediately post-hatching and barnacles, as filter feeders are potential predators of flatworm larvae. In order to collect the flatworm larvae and calculate the hatching success of the flatworm eggs it was important not to have live barnacles inside the experimental containers. In pilot studies flatworms were not observed to consume dead barnacles nor any similar crustacean food so flatworms were not provided with a food-source in this experiment.

Digital photographs of the flatworms and location of the egg batches in each container were taken every 24 h using Olympus compound microscope (SZX-ILLK200) connected to Pixelink (PL-A 642) with a magnification of 40 x. The number of eggs laid from each pair of flatworms was counted from the photographs using Image-Pro Express 4.0.1 software. Flatworm larvae were collected by pouring all the one-day old solutions from each treatment container into a 1.25 L sample jar and preserved at 7% formalin. New copper solutions were then poured to each treatment container. All the preserved solutions from the same treatment were combined and poured through a 10 µm sieve, rinsed with Milli-Q ® filtered water and made up to a 500 ml sample solution. The number of egg batches laid and the time taken for the flatworms to lay the first egg batch was recorded. Flatworm activity was recorded every four to six hours from when the first egg batch was observed until larvae were observed hatching from the eggs. Flatworms were considered to be actively "brooding" if their body covered any part of an egg batch.

Experiment 2: Reproductive success with food provided

In order to ensure that the response of flatworms and the timing of egg laying were not influenced by the absence of food, barnacles were placed in each treatment container as a food source in this experiment. A settlement plate with 3-5 barnacles (4 ± 0.3 SE) and a pair of similar size flatworms were placed in each treatment container.

Containers holding a settlement plate with barnacles and no flatworms were used as barnacle mortality controls. The nominal copper concentrations used in this experiment were 0, 10 and 25 µg L -1 , with four replicates for each treatment and three replicates for each barnacle mortality control at each copper concentration. During the 10 d exposure period treatment solutions were replaced daily and barnacles were fed three times with 5 ml of hatched artemia cysts to ensure that any mortality of barnacles was not a result of starvation. The total number of egg batches laid and the time taken for the flatworms to lay the first egg batch was recorded. Flatworm activity was recorded every four to six hours from when the first egg batch was observed until larvae were observed hatching from the eggs. Flatworms were considered to be actively "brooding" if their body covered any part of an egg batch. The feeding rate and mortality of barnacles in all treatments was recorded every four to six hours for the entire 10 d observation period.

Feeding rate of barnacles was recorded as the number of beats of cirri in one minute.

Data analysis

All statistical analysis was completed in Statistical Package for Social Sciences (SPSS version 11.5) with all data assessed for homogeneity of variance and normality using plots of residuals versus means and descriptive statistics. In each experiment, barnacle size was tested using a one-factor analysis of variance (ANOVA) with the presence of flatworms as a fixed factor. Flatworm length was tested using a one-factor ANOVA with copper treatments as the categorical factor. The number of egg batches laid, time spent brooding (averaged the observation period) and the hatching success of flatworm eggs in all treatments were tested separately using a one-factor ANOVA with copper treatment as the categorical factor. The feeding rate of barnacles (cirri beats per minute averaged over the 10 d period) in Experiment 2 was analyzed using a two-factor ANOVA with copper treatments and presence of flatworms as fixed factors. Planned comparison tests were conducted on all significant results to determine the differences between controls and individual copper treatments. Planned comparisons used the error term from the main test of copper treatment [START_REF] Quinn | Experimental design and data analysis for biologists[END_REF]. Planned comparisons are conducted when specific post-hoc tests are required. They are usually more powerful than procedures that test every treatment combination (e.g. Tukeys). In ecotoxicology studies such as this, the tests of interest are usually a comparison of the control treatment against individual toxicant concentrations.

Results

Reproductive success of flatworms

Copper reduced the reproductive success of flatworms; approximately 40% fewer egg batches were laid by exposed flatworms (ANOVA P = 0.006; Fig. 1a). Planned comparisons showed that this effect was evident at 25 µg L -1 Cu (P = 0.007) but not at 10 µg L -1 Cu (P = 0.763). Hatching success was highly consistent among replicates. In control treatments, about 92 0.4% of eggs hatched (Fig. 1b). Hatching success of flatworm eggs was reduced by 10% at 10 µg L -1 Cu and 40% at 25 µg L -1 Cu compared to that in the controls (ANOVA and Planned comparisons, P < 0.001; Fig. 1b).

Flatworms were able to endure protracted fasting and fasting did not appear to affect Stylochus pygmaeus reproductive behavior. In Experiments 1 (flatworms unfed) and 2 (flatworms fed), copper exposure caused no differences in the time taken for the flatworms to lay the first batch of eggs (five days) (Experiment 1: ANOVA, P = 0.303; Experiment 2: ANOVA; P = 0.142). Copper caused no difference in the time spent brooding egg batches (Experiment 1: ANOVA, P = 0.296; Experiment 2: ANOVA; P = 0.698). Flatworms were observed brooding eggs for more than 90% of the time.

Predation rate of Stylochus pygmaeus

No barnacles were observed to die in the mortality controls in Experiment 2 so any mortality of barnacle was therefore due to the presence of the flatworm predators.

Flatworms consumed an average of one barnacle over the experimental period (0.5 barnacles per flatworm 10 d -1 ) and this was not affected by exposure to 10 or 25 µg L -1 Cu in (Kruskal-Wallis, P = 0.735; Fig. 2).

Feeding rate of Balanus variegatus

Feeding rate of barnacles was reduced by both the presence of flatworms and exposure to copper. However, the presence of flatworm predators had a much larger effect than copper exposure (Fig. 3). Barnacle feeding rate was reduced by < 10% when exposed to copper, but it was reduced by > 30% by the presence of flatworm predators.

In the presence of flatworms, barnacle feeding rate was significantly reduced in the 10 and 25 µg L -1 Cu treatments when compared to that in the control (Planned comparison P < 0.001). However, in the absence of flatworm predators, this effect was only observed at 25 µg L -1 Cu (Planned comparison P < 0.001).

Size of barnacles and flatworms

Length of flatworm was used as an indicator of overall flatworm size. The average length of flatworms collected and tested in Experiment 1 was 12.7 0.14 mm; Experiment 2 was 11.6 0.46 mm and Experiment 3 was 16.4 0.63 mm. Flatworm length was similar across treatments within each experiment: Experiment 1 (ANOVA, P = 0.27) and Experiment 2 (ANOVA, P = 0.06).

was consistent between treatments (ANOVA, P = 0.31). There were 4 0.3 barnacles (6.8 0.2 mm) in each treatment and 4 0.17 barnacles (6.5 0.25 mm) in each barnacle mortality control.

Chemical analysis

The nominal and measured copper concentrations taken at the commencement of and 24 hr after the experiment are shown in Table 1. All filtered field seawater samples contained < 5 -1 copper. Since all treatment solutions were prepared and changed every 24 h from a 1000 mg L -1 stock solution and the measured copper concentrations of the treatment solutions from the experiment are close to their nominal values, we have confidence in the accuracy of our treatment solutions.

Discussion

Low levels of metal pollution had a major impact on the reproductive success of the mobile flatworm, Stylochus pygmaeus. The barnacle prey, Balanus variegates, reduced its feeding rate in the presence of a predator but was only slightly affected by exposure to the pollutant. Copper concentrations used in this study fall into the lowest range of those typically used in laboratory toxicity tests and they reflect realistic concentrations experienced by marine assemblages in polluted harbours [START_REF] Stauber | Copper bioavailability and amelioration of toxicity in Macquarie Harbour, Tasmania, Australia[END_REF] or close to vessels coated with copper antifouling paints . The differential sensitivity of a predator and prey observed in this study warrants field experiments investigating the likely structural changes to the marine invertebrate community in areas of low level heavy metal pollution.

Reproductive success of Stylochus pygmaeus

Copper reduced the reproductive success of flatworms. There were fewer eggs laid when flatworms were exposed to low levels of copper, and the likelihood that any one of those eggs would hatch into a young larva was lowered. Copper was clearly reducing the capacity of flatworms to produce eggs. However, the mechanism by which the hatching success was affected is not clear from this study. The early life stages of many organisms are more sensitive to pollutants and are often used in toxicity tests [START_REF] Mora | Heavy metals and metalloids in egg contents and eggshells of passerine birds from Arizona[END_REF]. The toxic effects of copper on adult health may have resulted in the laying of poor quality eggs, but eggs may have also been directly affected by copper exposure, and the newly hatched larvae may also have been directly affected. This situation mimics toxicant exposure in the field where adults, eggs and larvae are likely to be simultaneously exposed.

Exposure to copper negatively affects the reproductive success of other marine organisms, such as sponges [START_REF] Cebrian | Sublethal effects of contamination on the Mediterranean sponge Crambe crambe: metal accumulation and biological responses[END_REF] amphipods [START_REF] Conradi | Population responses of the marine amphipod Corophium volutator (Pallas, 1766) to copper[END_REF] and polychaetes [START_REF] Xie | Responses of polychaete Hydroides elegans life stages to copper stress[END_REF]. However the flatworms used in this study appear to be more sensitive than other marine invertebrates. Toxic effects of copper on the survival, growth and development of barnacles have been widely studied (e.g. [START_REF] Pyefinch | The sensitivity of barnacles and their larvae to copper and mercury[END_REF][START_REF] Koryakova | Using barnacle larvae for evaluation of the toxicity of antifouling paint compounds[END_REF]. It is apparent that barnacles are capable of tolerating quite high copper concentrations (e.g. 24 h LC 50 for copper for adult Balanus balanoides and B. crenatus was 320 and 1350 µg L -1 respectively, [START_REF] Pyefinch | The sensitivity of barnacles and their larvae to copper and mercury[END_REF]. Flatworms have no shell to cover their fragile body, and they rely heavily on diffusion directly across the epidermis for respiration and excretion of waste products. Hence, they may be generally more susceptible to changes in water quality than their barnacle prey, which are protected by both a calcium carbonate external skeleton and a thick integument.

The response of flatworms to copper exposure was the same in the presence and absence of food (barnacles). The predation rate of Stylochus pygmaeus in this study (0.5 barnacle consumed per worm 10 d -1 ) was relatively low when compared to the field observations of other stylochids. For example, Stylochus tauricus consumed five to ten to the relatively small size of S. pygmaeus compared to other stylochids [START_REF] Merory | A new stylochid flatworm (Platyhelminthes, Polycladida) from Victoria, Australia and observations on its biology[END_REF] or a result of reduced energy expenditure of flatworms in well-controlled laboratory conditions. S. pygmaeus also spent more than 90% of its time "brooding" egg batches. [START_REF] Chintala | Reproduction of Stylochus ellipticus (Platyhelminthes: Polycladida) in response to temperature, food, and presence or absence of a partner[END_REF] found that flatworms are able to maintain a high reproductive effort by using stored energy to produce eggs despite reductions in available food. It may be that S. pygmaeus invests time in mating and brooding rather than foraging, when a mate is available.

Predator Avoidance

Predator-avoidance behaviour has been widely studied in terrestrial and aquatic ecosystems (e.g. [START_REF] Nelson | Predators reduce prey population growth by inducing changes in prey behaviour[END_REF][START_REF] Peacor | Positive effect of predators on prey growth rate through induced modifications of prey behaviour[END_REF]. Such behaviour has both direct and indirect effects upon the reacting prey such as reduction of survival and growth .

Predator-avoidance behaviour (feeding inhibition) was clearly shown by Balanus variegatus in response to the presence of flatworms in this study. The presence of flatworms reduced the feeding rate of barnacles to a much greater extent than exposure to low levels of copper. B. variegatus reduced its encounter rate with its flatworm predator by closing opercular valves. Such a defense mechanism was noted in [START_REF] Hurley | The polyclad flatworm Stylochus tripartitus Hyman as a barnacle predator[END_REF] which examined the establishment of B. pacificus in the presence of its flatworm predator, Stylochus tripartitus. The mechanism by which B. variegatus identified the presence of S. pygmaeus is not known. However, chemical cues may be involved [START_REF] Hagen | Alarm responses of the green sea urchin, Strongylocentrotus droebachiensis, induced by chemically labelled durophagous predators and simulated acts of predation[END_REF]. This behavioral response is presumably performed to reduce the predation risk but it comes at the cost of reduced foraging time and hence potentially reduced growth, reproduction and development of the prey species [START_REF] Lefcort | Ramifications of predator avoidance: predator and heavy metal-mediated competition between tadpoles and snails[END_REF].

Conclusion

This study provides new information on the impacts of low concentrations of copper in sea water on the reproductive success of a mobile invertebrate predator.

Copper had obvious detrimental effects on flatworms, reducing their reproductive success by up to 80%. It has been suggested that long-term exposure of organisms to elevated copper concentrations might have serious consequences (such as local extinction) through effects on reproduction and growth [START_REF] Conradi | Population responses of the marine amphipod Corophium volutator (Pallas, 1766) to copper[END_REF].

If concentrations used in this study were to occur in coastal waters in a similarly uncomplexed chemical form, decreased reproductive success in Stylochus pygmaeus would ultimately lead to a decline in flatworm population size in the area exposed.

Within a couple of generations this may result in complete recruitment failure as the density of mature flatworms drops below a critical encounter threshold [START_REF] Russell | Failure of adult recruitment in Quercus buckleyi populations on the eastern Edwards Plateau, Texas[END_REF]. Patterns of population change in barnacles would need to be examined over a longer period and under field conditions in order to determine how the barnacle

Table captions

Table 1. Nominal and measured total copper concentrations (µg L -1 ) at the commencement of and 24 h after the experiments began (±1SE, n = 2). Lowest detection limit for analysis was 5 µg L -1 . Letters indicate significant difference at = 0.050.

Figure captions

  Solutions were replaced once every 24 h and were prepared from the stock solution immediately before use. All experiments were conducted in a constant-temperature room (23 o C 0.5 o C) subjected to 11:13 light: dark cycle. Salinity (33.7-34.5 ppt), pH (7.93-7.98), temperature (23-23.5 o C) and dissolved oxygen (5.56-5.74 mg L -1 ) were monitored daily using and YSI 556 MPS (Yellow Springs, OH, USA) water quality meter and remained constant throughout the experiments. Following 10 d exposure to copper solutions organisms were placed in clean filtered field seawater for 2 d to assess latent mortality.

  Flatworm larvae in each sample solution were counted by sub-sampling 20 ml of the sample solution five times. The number of flatworm larvae present in each treatment was estimated by multiplying the average number of larvae present in the 20 ml sub-sample by twenty five. Hatching success of flatworm eggs was calculated as: (Number of larvae/ Number of eggs) * 100%.

Figure 1 .

 1 Figure 1. Effects of 0, 10 and 25 µg L -1 Cu treatment on (a) the number of flatworm egg

Figure 2 .

 2 Figure 2. Effects of 0, 25 and 50 µg L -1 Cu treatments on the number of barnacles eaten

Figure 3 .

 3 Figure 3. Effects of 0, 10 and 25 µg L -1 Cu treatments on the feeding rate of barnacles in

Table 1 .

 1

	Nominal concentration	Measured concentration 0 hr 24 hr
	0	< 5	< 5
	10	13 ± 1	13 ± 0
	25	23 ± 3	24 ± 2

Fig. 1.

Fig. 2.

Acknowledgements

We would like to thank N. Hill and R. Piola, and two anonymous reviewers for useful comments on earlier drafts of the manuscript. We thank R. Piola for assistance in the field and experimental setup and T. Kwok and C. Wong for collection of seawater. This research was supported by a Faculty Research Grant awarded to Dr. E. L.

Johnston.