Histological and feeding response of Sydney rock oysters, Saccostrea glomerata, to acid sulfate soil outflows.

ABSTRACT Estuarine acidification, caused by disturbance of acid sulfate soils (ASS) is a problem that affects many estuaries in eastern Australia. ASS outflows have low pH and elevated concentrations of metals, principally iron and aluminum. Most production of Sydney rock oysters Saccostrea glomerata occurs in estuaries along the Australian east coast and estuarine acidification has been implicated in localized declines in oyster production. Estuarine areas recurrently impacted by estuarine acidification have higher levels of oyster mortality and reduced oyster growth compared with sites that are not acidified. Two laboratory experiments were conducted to investigate reasons for poor oyster production at sites exposed to ASS-affected waters. Behavioral response, soft tissue lesions and filtration rates of S. glomerata when exposed to ASS-affected waters were examined. It was found that ASS-affected water altered oysters' valve movements and significantly reduced filtration rates at pH 5.5. Acidic treatments (pH 5.1) containing 7.64 mg [L.sup.-1] of aluminum or ASS-affected water caused changes in the mantle and gill soft tissues after short-term (6 h) exposure. Degenerative effects were also caused by iron contained in ASS-affected water. Iron precipitates accumulated on the gills and mantle and were observed in the stomach, intestine, digestive tubules and rectum. Results from this study highlight the rapid deleterious effects of reduced pHs to oysters and the impacts of iron and aluminum contained in ASS-affected waters.

KEY WORDS: estuarine acidification, Saccostrea glomerata, acid sulfate soil, filtration rate, histopathology, iron accumulation

INTRODUCTION

Commercial production of Sydney rock oysters, Saccostrea glomerata (Gould 1850) occurs in eastern Australia. The state of New South Wales is the largest producer with an industry worth approximately US $26 million annually (NSW Department of Primary Industries 2007). Diseases, competition from other species and declining water quality of estuaries have reduced production of S. glomerata to approximately half that of the levels attained in the mid 1970s (Nell 1993).

Localized oyster production declines in some estuaries have also been attributed to estuarine acidification, caused by acid sulfate soils (ASS) (Dove & Sammut in press). ASS contain the mineral iron pyrite, which is benign when left undisturbed in waterlogged soils of coastal lowlands (Dent 1986). However, when iron pyrite is exposed to the atmosphere, through land drainage or excavation, it oxidizes and produces sulfuric acid, which then dissolves metals, such as iron, aluminum and manganese, from the soil matrix (Dent 1986).

Artificially constructed floodplain drains that dissect ASS and are connected to the estuary are a common feature of many floodplains on the east coast of Australia. After heavy rain, large volumes of acidic water laden with toxic concentrations of metals severely acidify tributaries and the main channel of estuaries (Sammut et al. 1995). ASS discharges are capable of acidifying estuarine water to around pH 3 for weeks at a time (Sammut et al. 1996). Estuarine acidification associated with ASS is also known to occur throughout Asia, in Africa and parts of Europe and North America (Klepper et al. 1992, Galle & Montoroi 1993, Astrom & Bjorkland 1995, Soukup & Portnoy 1986).

Estuarine acidification has degraded aquatic ecosystems on the east coast of Australia (Sammut et al. 1996) and threatens the biodiversity, amenity, fisheries production, and overall value of an estuary (Sammut et al. 1995). Hydrogen ions present in ASS-affected waters rapidly damage the gills and skin of fish causing massive fish kills and fish disease (Callinan et al. 2005, Callinan et al. 1993, Sammut et al. 1996, Sammut 1998). ASS leachate causes significant increases in abnormalities in developing S. glomerata embryos at low concentrations (3.3%) in seawater (Wilson & Hyne 1997). Field investigation of the impacts of estuarine acidification to S. glomerata revealed that oysters at sites exposed to ASS-affected waters had significantly higher levels of mortality and significantly reduced growth (Dove & Sammut in press). Additionally, mortalities in small oysters (shell height = 34 mm) were significantly higher than the mortalities in large oysters (shell height = 61 mm) (Dove & Sammut in press). American studies on the effects of acidification on bivalves demonstrated, using laboratory experiments, that exposures to pH values <7 reduces feeding activity (Bamber 1987, 1990) and pumping rates (Loosanoff & Tommers 1947).

Two laboratory experiments were conducted to expose S. glomerata to artificially and naturally acidified treatments in this study. The first experiment investigated oysters' behavioral and soft tissue response to acidified treatments and the second experiment examined feeding behavior of oysters in the presence of ASS-affected water at three pH levels. Both experiments were designed to resemble actual environmental conditions and utilized water quality information from Dove (2003). A combination of artificially acidified treatments and naturally acidified treatments were used for Experiment 1 to focus on the effects of specific elements of acidified water. Naturally acidified water needed for experiments was collected from an ASS drain outflow site. The treatments in this study had similar pH, electrical conductivity (EC) levels and aluminum and iron concentrations that were measured in estuaries impacted by ASS outflows (Dove 2003). Histopathology was used to assess the impacts of acidified water on the gill and mantle soft tissues, examine iron accumulation and provide further information relating to changes in filtration rates caused by ASS-affected waters.

MATERIALS AND METHODS

General Methods

Experimental oysters were supplied from the Manning River estuary (NSW) by a commercial oyster grower and were collected from, and held at, areas that were not impacted by ASS-affected waters (Dove & Sammut in press) before transfer to the laboratory. A Greenspan Smart Sonde (Model SD300) submersible data logger was installed at the site that was used to hold oysters to record pH, EC and temperature to ensure oysters were not exposed to reduced pHs before the laboratory experiments. The mean ([+ or -] SD) shell height and whole weight of oysters used for these experiments was 58.2 [+ or -] 6.0 mm and 19.41 [+ or -] 4.32 g, respectively.

A flow-through system based on Widdows' (1985) apparatus for measurement of clearance rate was used for Experiment 1 and 2. This system comprised of a 60 L header tank, which supplied water to eight 2.9 L trays (120 mm x 300 mm x 80 mm) each containing a baffle to reduce turbulence. The trays overflowed into a 200 L reservoir where pH, EC, dissolved oxygen (DO) and temperature were monitored using a Yeo-Kal 611 Intelligent Water Quality Analyser (Yeo-Kal Electronics Pry Ltd, Sydney, Australia) before the water was pumped back to the header tank. All components were made from food-grade or stabilized plastic to prevent any reaction with the acidic test water. A flow rate of 0.5 L [min.sup.-1] was maintained in each tray so that biodeposits were not resuspended and to minimize sedimentation.

Seawater for experiments was collected near Port Macquarie, NSW (31[degrees]25'30"S, 152[degrees]55'20"E) and was filtered to 1 [micro]m (nominal). Water temperature during experiments was maintained at 25 [+ or -] 1[degrees]C. The ASS-affected water used to acidify seawater in Experiments 1 and 2 was collected from Fernbank Creek, an acidified tributary of the Hastings River. NSW, immediately before the start of both experiments. The pH was stabilized in all acidified treatments using 0.1 M Analar hydrochloric acid (HCl). AR grade aluminum chloride (AJAX Chemicals Ltd.) was added to Treatment 3 (Experiment 1) and AR grade iron chloride (AJAX Chemicals Ltd.) was added to Treatment 4 (Experiment 1). Inductively Coupled Plasma Atomic Excitation Spectroscopy (ICPAES, Model Perkin Elmer Optima 3000 DV) was used to determine the concentration of selected dissolved ions of water samples and total metal concentrations were determined using the Nitric Acid Digestion method detailed in APHA (2005).

Experiment 1: Behavioral and Soft Tissue Response to Acidified Water

Information regarding the test water for the five treatments used for Experiment I is listed in Table 1. Valve activity of exposed oysters was closely observed and the following valve responses were noted during laboratory exposures: excessive gaping (valve separation beyond the range of normal feeding): repetitive shell adductions; no activity (valves remain closed and inactive); and open valves (normal valve separation for feeding) (Bamber 1987, 1990). Twenty-four oysters were observed for behavioral responses during each treatment. The duration of exposure was six-hours (approximate duration of acid exposure in one tidal cycle), commencing from the time each oyster opened its valves. Bamber (1987, 1990) found that oysters were slow to respond to stimuli in acidified treatments. Oysters were prodded every hour to assess their response to a tactile stimulus.

All 24 oysters were removed from each treatment of Experiment 1, six hours after opening their valves. Twelve of these oysters were returned to the estuary to measure post experiment survival. The remaining 12 oysters were opened from the hinge and examined for any gross changes in appearance before the soft tissue was placed into Formalin (10% sea water) for histopathology (Howard & Smith 1983). Formalin (10% sea water) was used in preference to Davidson's fixative because acid fixatives can interfere with iron (Howard & Smith 1983).

Two transverse tissue cross sections were taken, the first through the intestine, digestive diverticula, stomach, and labial palps and the second through the adductor muscle, kidney, and gills. Paraffin embedded sections were cut at 5-7 [micro]m, mounted on acid washed glass slides and stained with hematoxylin and rosin (H&E). Oysters from Treatments 1, 4, and 5 were also stained with Perls' Prussian Blue (PPB) to investigate iron accumulation on the oysters' soft tissues.

Experiment 2: Effect of Acidified Water on Filtration Rate

Experiment 2 measured oyster feeding rates at three pH levels. Treatment 1 was a mixture of seawater and deionized water and had a pH of 7.96: Treatment 2 used ASS-affected waters to acidify the test water to pH 6.5; and, Treatment 3 also used ASS-affected waters to acidify the test water to pH 5.5 (Table 2). These three treatments represented pH conditions often measured in the Manning River estuary (Dove 2003). A water sample was collected before the start of each treatment to determine the concentration of aluminum, iron, manganese, zinc, and silicon.

Natural silt was used as the diet for oysters in all treatments of Experiment 2 and was collected from intertidal mud flats, close to the location that oysters were held in the estuary. Silt was scraped from surface sediments to a depth of 2 3 mm, sieved through 140 [micro]m and 11 [micro]m nylon mesh, and stored at 4[degrees]C prior to each treatment Bayne et al. (1999a).

Six trays were used to measure oyster true feces and pseudofeces and the two empty trays were used as controls. Water samples were collected from the control trays to measure the concentration of suspended particles. One oyster was placed into each of the six trays and left undisturbed for a period of 2-3 h before a measurement of true feces and pseudofeces was performed.

Quantitative measurement of seston concentration (total particulate matter, TPM) using the gravimetric method (Iglesias et al. 1998) was done on samples collected from the control trays. Water samples were filtered onto preashed and preweighed glass microfiber filters (Whatman GFC, Catalogue Number 1822 047). Filters were ashed at 450[degrees]C for 4-6 h, weighed and placed in a desiccator before use. A one-liter aliquot of the treatment water was filtered before the filter was rinsed with 10 mL of 0.9% ammonium formate (Bayne et al. 1999b). Filters were oven dried for 12 h at 80[degrees]C, then ashed at 560[degrees]C for 4-6 h before filter weight was recorded.

A wide-mouth pipette was used to sample the true feces and pseudofeces from the trays. All of the pseudofeces and true feces produced in a one hour time period were collected from the trays and filtered onto preashed and preweighed Whatman GFC filters. A second measurement of pseudofeces and true feces was performed immediately after the first measurement for the same period of time to obtain an average value for the weight of biodeposits produced. This entire procedure was repeated three times so that 18 oysters were exposed to each treatment. The filters containing the oyster true feces and pseudofeces were analyzed using the same methodology as described for suspended particles.

After each treatment oysters were weighed and the soft tissue was removed from the valves. The soft tissue was dried at 80[degrees]C for 12 h and then weighed to determine soft tissue dry weight. Feeding rates were corrected to a standard body size using the allometric equation (Bayne & Newell 1983):

Y = [aX.sup.b]

where Y = measured feeding variable, X = dry body mass in grams, and a is the intercept. The slope, b is the allometric exponent in the equation, which describes the physiological rate as a function of body size (Bayne et al. 1999a). Mean dry body mass ([+ or -] SD, n - 54) of the experimental oysters was 0.77 [+ or -] 0.28 g. This mean body mass was used as the standard body mass in place of a standard 1 g animal and the corrections for weight differences were calculated (Widdows 1985). The weight-exponent was taken from Bayne et al. (1999b) that measured clearance rate in Sydney rock oysters and estimated this to be 0.641.

Single factor ANOVA was used to test for differences between treatments for weight-corrected filtration rate data. Post hoc pairwise comparisons of the results were made using the Least Significant Difference test. SPSS Version 11.0.0 (SPSS Inc.) statistical software package was used for these analyses.

RESULTS

Behavioral Response to Acidified Water

The pH, EC, temperature and Fe and A1 concentration for each of the five treatments used to investigate behavioral response are listed in Table 1. The water used for Treatments 4 and 5 appeared orange and suspended iron floccules were clearly visible in the water column. There was 7.71 mg [L.sup.-1] of suspended iron in Treatment 4 and 0.201 mg [L.sup.-1] and 13.25 mg [L.sup.-1] of dissolved and suspended iron, respectively, in Treatment 5.

The observed behavioral traits displayed by oysters in the five treatments were: open valves; excessive gaping; repetitive shell adductions; and, no activity. Five oysters in Treatment 5 and two oysters in Treatment 4 were inactive for the entire exposure period. In Treatments 1, 2, and 3, all oysters opened their valves and produced true feces and pseudofeces. Excessive gaping was only observed in the acidified test waters and occurred after 4 h of exposure. Clomping occurred in Treatment 5 and was attributed to the high concentrations of suspended particles in the treatment water.

Oysters in Treatments 2-5 were slower to react after prodding, particularly after 4 h of exposure. Oysters actively fed at pH 5.1 and produced true feces and pseudofeces. No postexperiment mortality occurred in oysters that were returned to the estuary and inspected 4 wk later.

Histopathology

Treatment 1 (pH 8.0, No Added Iron and Aluminum)

There were no significant aggregations of hemocytes in the gills or mantle soft tissues of oysters from this treatment. However, there was focal necrosis of the frontal and lateral cells of the ordinary filaments in two oysters. Two oysters had very mild, focal accumulations of hemocytes located in the gills. This response was not typical of the other oysters from Treatment 1.

Treatment 2 (pH 5.1, No Added Iron and Aluminum)

Oysters from Treatment 2 typically had increased hemocyte activity in the gills when compared with oyster sections from Treatment 1. There were mild, focal aggregations of hemocytes located in the interlamellar junctions and the hemolymph sinuses of plicae and ordinary filaments of the gill of particular oysters (Fig. 1, A). Frontal and lateral cell necrosis was observed to a greater extent in Treatment 2 oysters than was observed in Treatment 1. No significant findings were observed in the mantle soft tissue of the 12 oysters from this treatment.

Treatment 3 (pH 5.1, 7.6 mg [L.sup.1] of Aluminum)

Oysters from Treatment 3 had extensive hemocyte activity throughout the gills. Large accumulations of hemocytes were observed in the interlamellar junctions and hemolymph sinuses of plicae and ordinary filaments of the gill. There were gill lesions present in all oysters from this treatment. The most common lesion was in the hemolymph sinuses of plicae. Rupturing of this sinus caused infiltrations of hemocytes into the adjacent water tube (this occurred in l I oysters) (Fig. 1, C). There were infiltrations of hemocytes into the pallial cavity through necrotic frontal and lateral cells of ordinary filaments (Fig. 1, B). This response was observed in six oysters. Hemocytes were commonly observed in the junctions between adjacent filaments, congesting the gills. There was also necrosis and sloughing of mantle epithelial cells predominately on the pallial surface in oysters from Treatment 3.

Treatment 4 (pH 5.1, 7.7 mg [L.sup.1] of Iron)

There were mild to moderate, focal aggregations of hemocytes located in the interlamellar junctions and the hemolymph sinuses of plicae and ordinary filaments of oysters from Treatment 4. There was necrosis and sloughing of the mantle epithelial cells on the pallial surface in two oysters. Corresponding thin sections stained with PPB showed iron at the sites where mantle necrosis and sloughing occurred. The degree of hemocyte activity throughout the gills in this treatment was comparable to that observed in Treatment 2.

Treatment 5 (ASS-Affected Waters Adjusted to pH 5.1)

Treatment 5 contained 13.5 mg [L.sup.-1] of dissolved and suspended iron and 6.2 mg [L.sup.-1] of dissolved and suspended aluminum, which was from the added ASS-affected water. Oysters from Treatment 5 had mild to moderate hemocyte activity throughout the gills. Moderate aggregations of hemocytes were observed in the interlamellar junctions and hemolymph sinuses of plicae and ordinary filaments. Focal necrosis and sloughing of the mantle epithelial cells on the pallial surface was observed in the thin sections as well (Fig. 1, D). There was necrosis of the frontal cells and lateral cells of particular gill filaments. Hemocytes in the sinuses of the ordinary filaments were escaping into the pallial cavity through necrotic frontal cells and lateral cells of these filaments. This was observed in 5 oysters from this treatment. Corresponding thin sections stained with PPB showed iron had accumulated at sites of necrosis and sloughing of mantle epithelial cells. The soft tissue response in Treatment 5 was not as severe as was observed in oysters from Treatment 3 even though the aluminum levels were similar.

Iron Precipitate

All oysters removed from Treatments 4 and 5 displayed gross signs of iron floccules in the shell fluid and on the gill surface. No iron was observed in any of the thin sections from oysters removed from Treatment 1. Table 3 lists the presence and extent of iron accumulation on and in the soft tissues of oysters from Treatments 4 and 5.

Iron precipitates were observed: on the gill epithelium; on the mantle epithelium; in the stomach; in the intestine; and, in the rectum of oysters removed from Treatment 4 (Table 3). Similarly, iron precipitates were observed: on the gill epithelium (Fig. 2, A); on the mantle epithelium and in the pallial cavity (Fig. 2, B and C); in the stomach (Fig. 2, D); in the intestine (Fig. 3, A); in the digestive gland ducts (Fig. 3, B); in the digestive tubules (Fig. 3, C); and, in the rectum (Fig. 3, D) of oysters removed from Treatment 5.

Filtration Rate

Exposure to acidified water caused a significant reduction in filtration rate of oysters (F = 7.11; df = 2/53; P < 0.001). Filtration rate at pH 5.5 was significantly lower than the filtration rate at pH 6.5 and 7.96 (Fig. 4). At pH 5.5, the TPM to particulate inorganic matter (PIM) ratio was lower compared with pH 7.96 and pH 6.5 (Table 2). The Treatment 3 test water was orange, suggesting that iron was precipitating out of solution: and an elevated concentration of dissolved aluminum (0.11 mg [L.sup.-1]) was also measured in this treatment compared with Treatments 1 and 2 (Table 2).

The mean TPM concentration for pH 7.96 (Treatment 1) and pH 6.5 (Treatment 2) were similar, however the organic content is slightly greater at pH 6.5 (Table 2). At pH 5.5 there was an elevated TPM concentration compared with the other two treatments (Table 2). This was attributed to ASS oxidation products (iron and aluminum) in the treatment water being in a suspended state and is reflected by the high PIM value (Table 2). Attempts were made to remove iron precipitates from the treatment water by filtration (1 [micro]m nominal) and allowing time for suspended particles to settle before the ASS-affected water was added to Treatments 2 and 3 to achieve similar compositions of suspended particles for all treatments. However, this process did not remove the dissolved iron from the treatment water, which appears to have precipitated out of solution during the experiment (Table 2).

DISCUSSION

Behavioral and Soft Tissue Response

Exposure to acidified water (pH 5.1) containing aluminum or ASS-affected waters caused abnormal valve movements in some experimental oysters. Oysters opened their valves in all treatments, which directly exposed their soft tissue to acidic conditions and all of the associated contaminants in ASS-affected waters, particularly iron and aluminum. This finding meant that filtration rates could be quantified for S. glomerata as they produced true feces and pseudofeces at reduced pHs.

[FIGURE 1 OMITTED]

Changes were observed in the gill and mantle soft tissues resulting from exposure to acidic waters. Changes were most noticeable in acidic treatments containing added iron, aluminum or ASS-affected waters. The extensive inflammatory response and gill lesions observed in Treatment 3 suggests that the presence of aluminum in combination with low pH causes a more intense response in the gill and mantle soft tissues than water of a low pH with no added aluminum. It is likely that only the initial stages of changes in the soft tissue were observed, because exposure time was only for a short duration (6 h). Sammut (1998) showed that gill lesions in sublethally exposed fish can repair within 24-48 h of exposure but in moribund fish, lesions became more severe before death occurred in the recovery waters.

Comparison of corresponding thin sections stained with H&E and PPB revealed aggregations of inflammatory cells were not only associated with iron accumulation. Further research is required to examine the effects of suspended iron precipitates at neutral and alkaline pH levels. Winter (1972) found that exposure of the mussel Mytilus edulis to iron did not result in acute toxicity but caused high mortality and decreases in body weight. The mobilization of suspended iron precipitates can be several kilometers from the ASS outflow location (Sammut et al. 1996, Dove 2003). Iron floccules were observed grossly in oysters removed from field sites exposed to ASS-affected waters (Dove & Sammut in press). Findings from the present study suggest that the high concentrations of iron at acidified field sites were contributing to the high mortality rates and slow growth measured by Dove and Sammut (in press).

Other studies have established that iron is not toxic to bivalves at neutral pH levels. Sunila (1988) found that ferric iron did not cause a toxic reaction in the gills of M. edulis. It has also been established that not all of the iron that enters the gut of M. edulis is absorbed. George et al. (1976) estimated that 30% of the iron presented to the gut is not absorbed and is passed by way of the feces in this mussel species. Iron chloride added to Treatment 4 of Experiment 1 was not observed in digestive tubules. However, iron in the treatment containing ASS-affected water (Treatment 5) was observed in the secretory-absorptive cells of digestive tubules. Iron transformations in ASS-affected waters are likely to be different than in the artificial test waters because of the reaction of iron with other pyrite oxidation products and other elements present in natural waters (Sammut 1998). The resulting iron chemical species are therefore, likely to be different to those in the artificial test waters. It is also likely that the species of aluminum contained in Treatment 3 was different from the aluminum in Treatment 5 for the same reasons. This would account for the more intense response in the soft tissues of oysters in Treatment 3.

Soft tissue responses observed in oysters from Treatment 4 were likely to be induced by the combination of acidity and iron as opposed to the iron alone. Although there is no evidence of direct iron toxicity in S. glomerata, it is very probable that iron impairs gill function by congesting the ciliary junctions thereby affecting feeding processes and gas exchange. Furthermore, iron was being ingested by oysters, which would have implications for long-term oyster health.

Elevated iron concentrations are known to harm fish (Cruz 1969). Cruz (1969) investigated the pathological action of iron by injecting iron salt (at 200 mg of FeS[O.sub.4] by kg of body weight) into the digestive tract of fish. The survival time of the inoculated fish ranged from 2.5 h to 90 h. Cruz (1969) found internal lesions in various organs when iron was absorbed in large quantities by the digestive tract. Common responses of the fish were: destructive gill lesions; congestion of the gills, liver and kidney; and, liver necrosis. This is a relevant finding because intracellular iron was commonly observed in the epithelium of digestive tubules in oysters exposed to ASS-affected waters.

[FIGURE 2 OMITTED]

Sunila (1986) investigated the changes in the soft tissue in M. edulis caused by the discharge waters of a titanium dioxide plant located near Pori, Finland. Discharge waters were very acidic (pH 1) and contained sulphuric acid, ferro-sulphate, titanium dioxide, aluminum, manganese, vanadium, zinc, chrome, nickel, and cobalt. This polluted an area of 8 [km.sup.2] and ferric hydroxide flakes were spread over a region of approximately 120 [km.sup.2]. Histopathology detected changes in the structure of the mature ova of M. edulis as a response to low pH, however, no changes in the gills were observed (Sunila 1986). The long-term implications of iron accumulation in the digestive tract of S. glomerata are unknown. Winter (1972) demonstrated that, after adding 4 mg [L.sup.-1] of iron as ferric hydroxide, 94% of the iron was expelled as pseudofeces. However, this was at neutral pHs and low concentrations of iron can be accumulated, over time, to high concentrations in the soft tissues of bivalves (George et al. 1976).

Filtration Rate

As the concentration of ASS-affected water was increased, which also increases the acidity, oyster filtration rates were reduced. Filtration rate was significantly reduced at pH 5.5 compared with pH 6.5. Loosanoff and Tommers (1947) recorded increased pumping rates at pH values between 7.0 and 6.75, but when the pH dropped below 6.5 pumping rates dramatically decreased in adult O. virginica. Loosanoff and Tommers (1947) also observed abnormal shell movements when pH was less than 6.5. Bamber (1987) measured feeding inhibition and a significant reduction in tissue and shell growth for the species Venerupis decussata at or below pH 7.0. Bamber (1990) investigated the effects of acidic conditions on feeding activity in Crassostrea gigas, M. edulis and Ostrea edulis. For C. gigas, suppression of feeding activity occurred below pH 7.0 and behavioral inhibition was observed below pH 6.5. Feeding activity was reduced at or below pH 7.2 for O. edulis and M. edulis.

[FIGURE 3 OMITTED]

In the studies done by Loosanoff and Tommers (1947) and Bamber (1987, 1990), acidification was not caused by ASS, but was the consequence of inflows of large quantities of slightly acidic fresh water or industrial pollution. Also, these studies only used artificially acidified test waters and not naturally acidified test waters.

At pH 5.5 there was a higher concentration of TPM and PIM compared with pH 6.5 and 7.96. The presence of oxidation products contained in ASS-affected waters, namely iron and aluminum, could be responsible for this. However, it could not be established whether the significant reduction in filtration at pH 5.5 was because of a reduction in pH alone or from the influence of the oxidation products contained in ASS-affected waters. The results obtained from the other two treatments (pH 7.96 and 6.5) indicate that the reduction in filtration is likely to be predominantly influenced by pH as the experimental conditions were similar in all other respects apart from pH. Data from American studies (Bamber 1987, 1990, Loosanoff & Tommers 1947) strongly suggests that the reduction in pH caused by the addition of ASS-affected water was the main factor inhibiting oyster feeding.

[FIGURE 4 OMITTED]

The increased concentrations of TPM and PIM measured in Treatment 8 have implications for the oyster diet. The experimental conditions used in Experiment 2 were frequently measured in the estuary after high rainfall at oyster production sites in the Hastings and Manning Rivers (Dove 2003, Dove & Sammut in press). Dove (2003) measured TPM and PIM of 28 and 21.5 mg [L.sup.-l], respectively, at an oyster lease impacted by ASS-affected waters. ASS-affected water in this pH range (5.5-6.5) commonly contains high concentrations of ASS oxidation products, such as iron floccules (Sammut et al. 1996).

These results highlight the change in the dietary abundance of food available to oysters in ASS-affected waters. High concentrations of colloidal iron and aluminum alter the ratio between the inorganic component and the organic component of ASS-affected waters. This difference can be seen in Table 2 where PIM greatly exceeds particulate organic matter (POM) at pH 5.5. This equates to a low organic content, or a small proportion of food, and a large proportion of non-utilizable matter within the available seston (Hawkins et al. 1996). Therefore, the nutritional quality of ASS-affected waters is low when quality is expressed as organic content per unit volume of diet.

ASS outflows dramatically alter the biochemical composition of suspended particles in the estuarine waters that it affects. ASS-affected waters bind phosphorus (Simpson & Pedini 1985) and increase the concentrations of iron and aluminum (Sammut et al. 1996). The chemical and physical nature of suspended particles in areas of the estuary impacted by ASS-affected waters is different to the properties of suspended particles that are present under normal estuarine conditions. Exposure of S. glomerata to ASS-affected waters alters their valve movements, significantly reduces filtration rate and causes a degenerative soft tissue response in the gills and, to a lesser extent, the mantle of S. glomerata after only a short period of exposure. Furthermore, iron contained in ASS-affected water is extensively accumulated on the gill and mantle and in the intestine, stomach digestive tubules and rectum of oysters exposed to ASS-affected waters. Findings from this laboratory investigation help to explain high mortalities and reduced growth at sites recurrently exposed to ASS-affected waters measured by Dove and Sammut (in press) and reported by oyster growers working in impacted estuaries.

ACKNOWLEDGMENTS

The authors thank Ian and Rose Crisp for supply of oysters and use of their facilities for this research. They also thank Dr Richard Callinan who provided assistance with the histopathology for this study. Dorothy Yu is thanked for processing ICPAES samples and laboratory support. Allison Dove and Elissa Bishop are thanked for laboratory assistance and thanks are also due to Drs G. Allan and W. O'Connor for valuable editorial comments during the preparation of this manuscript. This study was supported by a Fisheries Research and Development Corporation grant 96/205.

LITERATURE CITED

APHA. 2005. Standard methods for the examination of water and wastewater centennial edition. New York: American Public Health Association. 1368 pp.

Astrom, M. & A. Bjorkland. 1995. Impact of acid sulfate soils on stream water geochemistry in western Finland. Journal of Geochemical Exploration 55:163-170.

Bamber, R. N. 1987. The effects of acidic sea water on young carpetshell clams Venerupis decussata (L.) (Mollusca: Veneracea). J. Exp. Mar. Biol. Ecol. 108:241-260.

Bamber, R. N. 1990. The effects of acidic seawater on three species of lamellibranch mollusc. J. Exp. Mar. Biol. Ecol. 143:181-191.

Bayne, B. L. & R. C. Newell. 1983. Physiological energetics of marine molluscs. In: K. M. Wilbur & A. S. Saleuddin, editors. The Mollusca, Volume 4. New York: Academic Press. pp. 407-515.

Bayne, B. L., D. Hedgecock, D. McGoldrick & R. Rees. 1999a. Feeding behaviour and metabolic efficiency contribute to heterosis in Pacific oysters. [Crassostrea gigas (Thunberg)]. J. Exp. Mar. Biol. Ecol. 233:115-130.

Bayne, B. L., S. Svensson & J. A. Nell. 1999b. The physiological basis for faster growth in the Sydney rock oyster, Saccostrea commercialis. Biol. Bull. 197:377-387.

Callinan, R. B., G. C. Fraser & M. D. Melville. 1993. Seasonally recurrent fish mortalities and ulcerative disease outbreaks associated with acid sulphate soils in Australian estuaries. In: D. Dent & M. E. F. van Mensvoort, editors. Selected Papers of the Ho Chi Minh City Symposium on Acid Sulphate Soils. Wageningen, The Netherlands: International Institute for Land Reclamation and Improvement. pp. 403-410.

Callinan, R. B., J. Sammut & G. C. Fraser. 2005. Dermatitis, branchitis and mortality in empire gudgeon Hypseleotris compressa exposed naturally to runoff from acid sulfate soils. Dis. Aquat. Organ. 63:247-253.

Cruz, M. 1969. About the possibility of iron absorption by fishes and its pathological action. Annales De Limnologie 2:187-193.

Dent, D. 1986. Acid sulphate soils: a baseline for research and development. Wageningen, The Netherlands: International Institute for Land Reclamation and Improvement. pp. 183-184.

Dove, M. C. & J. Sammut. Impacts ofestuarine acidification on survival and growth of Sydney rock oysters Saccostrea glomerata (Gould 1850). J. Shellfish Res. (In press).

Dove, M. C. 2003. Effects of estuarine acidification on survival and growth of the Sydney rock oyster Saccostrea glomerata. PhD. thesis, The University of New South Wales, Sydney, Australia. 241 pp.

Galle, G. & J. P. Montoroi. 1993. Ecological consequence of high aluminium content in acidified estuarian (sic) waters: the case of tilapia fishes in lower Casamance (Senegal). Acta Ecologica 14:87-100.

George, S. G., B. J. S. Pirie & T. L. Coombs. 1976. The kinetics of accumulation and excretion of ferric hydroxide in Mytilus edulis (L.) and its distribution in the tissues. J. Exp. Mar. Biol. Ecol. 23:71-84.

Hawkins, A. J. S., R. F. M. Smith, B. L. Bayne & M. Heral. 1996. Novel observations underlying the fast growth of suspension-feeding shellfish in turbid environments: Mytilus edulis. Mar. Ecol. Prog. Ser. 131:179-190.

Howard, D. W. & C. S. Smith. 1983. Histological techniques for marine bivalve mollusks. Massachusetts: NOAA Technical Memorandum NMFS-F/NEC-25.97 pp.

Iglesias, J. I. P., M. B. Urrutia, E. Navarro & I. Ibarrola. 1998. Measuring feeding and absorption in suspension-feeding bivalves: an appraisal of the biodeposition method. J. Exp. Mar. Biol. Ecol. 219:71-86.

Klepper, O., G. T. Chairuddin, Iriansyah & H. D. Rijksen. 1992. Water quality and distribution of some fishes in an area of acid sulphate soils, Kalimantan, Indonesia. Hydrobiology Bulletin 23:217-224.

Loosanoff, V. L. & F. D. Tommers. 1947. Effect of low pH upon rate of water pumping of oysters, Ostrea virginica. Anat. Rec. 99:668-669.

Nell, J. A. 1993. Farming the Sydney rock oyster (Saccostrea commercialis) in Australia. Rev. Fish. Sci. 1:97-120.

NSW Department of Primary Industries. 2007. Aquaculture production report 2004/2005. Taylors Beach, Australia: NSW Department of Primary Industries. 20 pp.

Sammut, J. 1998. Associations between acid sulfate soils, estuarine acidification, and gill and skin lesions in estuarine and freshwater fish. PhD. thesis, The University of New South Wales, Sydney, Australia 254 pp.

Sammut, J., I. White & M. D. Melville. 1996. Acidification of an estuarine tributary in eastern Australia due to drainage of acid sulfate soils. Mar. Freshw. Res. 47:669-684.

Sammut, J., M. D. Melville, R. B. Callinan & G. C. Fraser. 1995. Estuarine acidification: impacts on aquatic biota of draining acid sulfate soils. Aust. Geogr. Stud. 33:89-100.

Simpson, H. J. & M. Pedini. 1985. Brackishwater aquaculture in the tropics: the problem of acid sulphate soils. Rome, Italy: Food and Agriculture Organisation of the United Nations. 32 pp.

Soukup, M. A. & J. W. Portnoy. 1986. Impacts from mosquito control-induced sulphur mobilisation in a Cape Cod estuary. Environ. Conserv. 13:47-50.

Sunila, I. 1986. Histopathological changes in the mussel Mytilus edulis L. at the outlet from a titanium dioxide plant in Northern Baltic. Annals of Zoology Fennici 23:61-70.

Sunila, I. 1988. Acute histological responses in the gill of the mussel, Mytilus edulis, to exposure by environmental pollutants. J. Invertebr. Pathol. 52:137-141.

Widdows, J. 1985. Physiological procedures. In: B. L. Bayne, D. A. Brown, K. Burns, D. R. Dixon, A. Ivanovici, D. R. Livingstone, D. M. Moore, A. R. D. Stebbing & J. Widdows, editors. New York, NY: The effects of stress and pollution on marine animals: Praeger Scientific. pp. 161-178.

Wilson, S. P. & R. V. Hyne. 1997. Toxicity of acid-sulfate soil leachate and aluminitun to embryos of the Sydney rock oyster. Ecotoxicol. Environ. Saf. 37:30-36.

Winter, J. E. 1972. Long-term laboratory experiments on the influence of ferric hydroxide flakes on the filter-feeding behaviour, growth, iron content and mortality in Mytilus edulis L. In: M. Ruvio, editor. London, England: Marine pollution and sea life. pp. 392-396.

MICHAEL C. DOVE (1) * AND JESMOND SAMMUT (2)

(1) NSW Department of Primary Industries, Port Stephens Fisheries Centre, Taylors Beach, NSW 2316, Australia; (2) School of Biological, Earth & Environmental Sciences, The University of New South Wales, Sydney NSW 2052, Australia

* Corresponding author. E-mail: [e-mail]

TABLE 1.
Mean ([+ or -] 95% CI) pH, EC and temperature values and
concentrations of dissolved and suspended iron and aluminium
measured in Experiment 1 (Treatments 1-5).

Treatment         Treatment                 Mean               pH
   No.              Water                    pH              Range

    1       seawater + deionized     8.02 [+ or -] 0.009   7.99-8.12
              water
    2       seawater + deionized     5.11 [+ or -] 0.005   5.05-5.18
              water + HCl
    3       seawater + deionized     5.12 [+ or -] 0.007   5.04-5.18
              water + Al + HCl
    4       seawater + deionized     5.08 [+ or -] 0.007   5.01-5.16
              water + Fe + HCl
    5       seawater + ASS-          5.09 [+ or -] 0.008   5.01-5.18
              affected water + HCl

Treatment         Treatment                Mean EC
   No.              Water              (dS [m.sup.-1])

    1       seawater + deionized     29.3 [+ or -] 0.01
              water
    2       seawater + deionized     29.3 [+ or -] 0.02
              water + HCl
    3       seawater + deionized     29.3 [+ or -] 0.02
              water + Al + HCl
    4       seawater + deionized     30.8 [+ or -] 0.01
              water + Fe + HCl
    5       seawater + ASS-            28.9 (no shift)
              affected water + HCl

Treatment         Treatment           Dissolved Fe      Dissolved Al
   No.              Water            (mg [L.sup.-1])   (mg [L.sup.-1])

    1       seawater + deionized           ND                ND
              water
    2       seawater + deionized           ND                ND
              water + HCl
    3       seawater + deionized           ND                1.4
              water + Al + HCl
    4       seawater + deionized           ND                ND
              water + Fe + HCl
    5       seawater + ASS-               0.201              0.292
              affected water + HCl

Treatment         Treatment           Suspended Fe      Suspended Al
   No.              Water            (mg [L.sup.-1])   (mg [L.sup.-1])

    1       seawater + deionized           ND                ND
              water
    2       seawater + deionized           ND                ND
              water + HCl
    3       seawater + deionized           ND               6.24
              water + Al + HCl
    4       seawater + deionized           7.71              ND
              water + Fe + HCl
    5       seawater + ASS-               13.25             5.86
              affected water + HCl

TABLE 2.
Mean ([+ or -] 95% CI) pH, EC, DO and temperature and concentrations
of dissolved Al, Fe, Mn, Zn and Si measured in Experiment 2. Mean
([+ or -] 95% CI) total particulate matter (TPM), particulate organic
matter (POM) and particulate inorganic matter (PIM) measured in each
treatment of Experiment 2 are also listed.

Treatment          Mean                 Mean EC
 Number             pH              (dS [m.sup.-1])

    1       7.96 [+ or -] 0.017   29.2 [+ or -] 0.02
    2       6.50 [+ or -] 0.002   29.3 [+ or -] 0.02
    3       5.50 [+ or -] 0.003   29.3 [+ or -] 0.02

Treatment         Mean DO               Al                 Fe
 Number          (% Sat.)         (mg [L.sup.-1])    (mg [L.sup.-1])

    1       88.7 [+ or -] 0.14          ND                 ND
    2       88.1 [+ or -] 0.67          ND                0.01
    3       85.6 [+ or -] 1.51         0.11               0.03

Treatment           Mn                  Zn                 Si
 Number       (mg [L.sup.-1])     (mg [L.sup.-1])    (mg [L.sup.-1])

    1              0.02                0.02               0.22
    2              0.20                0.05               3.20
    3              0.15                0.03               3.92

                   Mean                Mean               Mean
Treatment           TPM                 POM                PIM
 Number       (mg [L.sup.-1])     (mg [L.sup.-1])    (mg [L.sup.-1])

    1        3.8 [+ or -] 0.4    1.2 [+ or -] 0.4   2.6 [+ or -] 0.5
    2        4.0 [+ or -] 0.0    1.8 [+ or -] 0.3   2.2 [+ or -] 0.3
    3        7.5 [+ or -] 0.4    1.1 [+ or -] 0.5   6.4 [+ or -] 0.4

ND = Not detectable.

TABLE 3.
List of iron accumulation in Experiment 2 (Treatments 4 and 5) on the
gills, on the mantle, in the stomach, in the digestive gland tubules
and in the rectum of S. glomerata.

                               Iron Accumulation

Treatment   Oyster
   No.       No.         Gills       Intestine   Stomach

    4         1            D            --          B
              2            D             A          B
              3            D            --          A
              4            D             B          A
              5            D             C          A
              6            D             D          --
              7            D            --          --
              8            D            --          --
              9            D             B          A
              10           C            --          A
              11           D             B          B
              12           D             C          B
    5         1            D            --          A
              2            D             D          C
              3            D            --          B
              4            D            --          --
              5            C             C          --
              6            C             D          B
              7            D             D          C
              8            D             B          B
              9            D            --          B
              10           D            --          A
              11           C             B          A
              12           C             A          A

                         Iron Accumulation

Treatment   Oyster     Digestive
   No.       No.     Gland Tubules    Rectum      Mantle

    4         1           --             A          C
              2           --             A          C
              3           --            --          B
              4           --             A          C
              5           --             A          D
              6           --             A          C
              7           --            --          C
              8           --            --          C
              9           --            --          C
              10          --            --          C
              11          --            --          C
              12          --             A          C
    5         1           --            --          A
              2            C            --          B
              3            B            --          A
              4            A            --          C
              5           --             C          C
              6            C            --          A
              7           --            --          B
              8           --            --          B
              9           --            --          C
              10          --             A          C
              11          --            --          C
              12          --             A          B

A = very minor accumulation, B = minor accumulation, C = moderate
accumulation, D = extensive accumulation.