Estrogen mediated effects in the Sydney rock oyster, Saccostrea glomerata, following field exposures to sewage effluent containing estrogenic compounds and activity.

The Sydney rock oyster, Saccostrea glomerata, has been demonstrated as a useful biomonitor of estrogenic compounds following laboratory exposures, yet its utility in the assessment of estrogenic exposure and effects under field conditions requires investigation. To achieve this aim, S. glomerata were deployed in Newcastle, Australia in the effluent receiving marine waters of Burwood Beach WWTP (Burwood Beach "near", <50 m from outfall and Burwood Beach "far", 100-150 m from outfall) and reference locations (Redhead, Fingal Island 1 and Fingal Island 2) at depths of 4, 8 and 12 m for six weeks. Effluent receiving waters of Burwood Beach WWTP were found to be a suitable impact location, demonstrated via measurement of estrogenic compounds and activity throughout the deployment. Estrogenic compounds were detected (average of combined solids and liquid fractions) at average concentrations of: 1.42 ng/L for estrone, 0.69 ng/L for 17ß estradiol, 3.83 ng/L for estriol (E3), 0.56 ng/L for 17α-ethynylestradiol, 64.2 ng/L for bisphenol A, 7.51 ng/L for 4-nonylphenol and 5.93 ng/L for 4-tert-octylphenol. Total estrogenic activity was estimated at 4.48 ng/L EEQ via the Yeast Estrogen Screen (YES(®)) assay (average of combined solid and liquid fractions). Female vitellogenin gene expression was highest at Burwood Beach locations, yet no significant differences were detected among locations for either sex. Vitellogenin protein was significantly higher (p<0.05) in S. glomerata at Burwood Beach Near compared to reference locations for the 4 and 12 m depths. Increased proportions of females were found at Burwood Beach Near, at 4m depth (p<0.05). Both Burwood Beach locations had higher proportions of mature female gonadal development stages compared to reference locations (p<0.05). Oocyte area was highest at both Burwood Beach locations, but no significant differences were detected among locations. Findings provided further evidence that female S. glomerata may be a suitable candidate species for assessment of effects of estrogenic compounds in Australian waters.

Exposure to 17α-ethynylestradiol causes dose and temporally dependent changes in intersex, females and vitellogenin production in the Sydney rock oyster.

Although mounting evidence suggests exposure to estrogenic contaminants increases vitellogenin production in molluscs, demonstration of dose-response relationships and knowledge of the temporal nature of the vitellogenin response with continual exposure is currently lacking for biomarker utility. To address this knowledge gap, adult Sydney rock oysters, Saccostrea glomerata, were exposed to a range of environmentally relevant concentrations of 17α-ethynylestradiol (EE2) (0, 6.25, 12.5, 25 or 50 ng/l) in seawater under laboratory conditions. Vitellogenin induction and gonadal development was assessed following 4, 21 and 49 days exposure to EE2. Vitellogenin was found to increase in a dose dependent manner with EE2 exposure for females (4 and 49 days) and males (4 and 21 days). Histological examination of gonads revealed a number of individuals exhibited intersex (ovotestis) in 50 ng/l EE2 (after 21 days) and in 6.25 and 12.5 ng/l EE2 (after 49 days). Furthermore, a significant shift towards females was observed following 49 days exposure at 50 ng/l EE2 suggesting estrogenic exposure is capable of facilitating a progression for protandric males from male-intersex-female gametal status. Increases in female vitellogenin (4 days) were predictive of later increases in female developmental stages at 21 days and increases in oocyte area following 49 days. Male vitellogenin (4 days) was predictive of decreased male percentages and lower male developmental stages at 49 days. Vitellogenin in S. glomerata is a predictive biomarker of estrogenic exposure and effect if sampled soon after exposure and at the commencement of a gonadal development cycle.

Effects of 4-nonylphenol and 17alpha-ethynylestradiol exposure in the Sydney rock oyster, Saccostrea glomerata: Vitellogenin induction and gonadal development.

Adult Saccostrea glomerata were exposed to environmentally relevant concentrations of 4-nonylphenol (1microg/L and 100microg/L) and 17alpha-ethynylestradiol (5ng/L and 50ng/L) in seawater over 8 weeks. Exposures were performed to assess effects on vitellogenin induction and gonadal development during reproductive conditioning. Chronic direct estrogenicity within gonadal tissue was assessed via an estrogen receptor-mediated, chemical-activated luciferase reporter gene-expression assay (ER-CALUX). Estradiol equivalents (EEQ) were greatest in the 100microg/L 4-nonylphenol exposure (28.7+/-2.3ng/g tissue EEQ) while 17alpha-ethynylestradiol at concentrations of 50ng/L were 2.2+/-1.5ng/g tissue EEQ. Results suggest 4-nonylphenol may be accumulated in tissue and is partly resistant to biotransformation; maintaining its potential for chronic estrogenic action, while 17alpha-ethynylestradiol, although exhibiting greater estrogenic potency on biological endpoints possibly exerts its estrogenic action before being rapidly metabolised and/or excreted. A novel methodology was developed to assess vitellogenin using high-performance liquid chromatography (HPLC). Exposure to both 17alpha-ethynylestradiol (50ng/L) and 4-nonylphenol (100microg/L) produced increases in vitellogenin for females, whereas males exhibited increases in vitellogenin when exposed to 50ng/L 17alpha-ethynylestradiol only. Females exhibited greater vitellogenin responses than males at 50ng/L 17alpha-ethynylestradiol only. Histological examination of gonads revealed a number of individuals exhibiting intersex (ovotestis) in 50ng/L 17alpha-ethynylestradiol exposures. Male individuals in 1microg/L and 100microg/L 4-nonylphenol exposures and 5ng/L 17alpha-ethynylestradiol were at earlier stages of spermatogenic development than corresponding controls.

Arsenic and heavy metal accumulation by Pteris vittata L. and P. umbrosa R. Br.

This study compared the accumulation of arsenic, copper and chromium by Pteris vittata and Pteris umbrosa grown in a glasshouse in soil from a timber treatment facility. Soil was collected from three locations. Accumulation (as percentage removed) varied between these soils but was not related to soil concentration. P. vittata was more efficient than P. umbrosa, both in accumulating As and metals in the below-ground plant parts and in translocating As to the fronds. Under the experimental conditions, only P. vittata could be effectively used in soil from one location for phytoremediation purposes.

Biomarkers of heavy metal contamination in the red fingered marsh crab, Parasesarma erythodactyla.

Variation in glutathione antioxidant biochemistry in response to metal contamination and accumulation under field conditions was examined in the brachyurid grapsid, Parasesarma erythodactyla. Significant relationships suggesting accumulation were found between sediment metals and metals in crab tissue for Pb, Cu, Cr, Zn, and Se in males and Cd, Pb, Cr Zn, As, and Se in females. Higher pH and lower organic content were associated with greater uptake of selected metals in males and females. Higher salinity was related to increased metal uptake for Cu and Zn in males and lower salinities to increased Se uptake for males and females. When examining metals, which were elevated in crabs, patterns of site discrimination were similar to sediment metal site discrimination for both males and females. In terms of biochemical responses, glutathione levels remained constant while glutathione peroxidase activity was elevated in individuals where metals were elevated. Only females with the highest levels of accumulated metals exhibited increases in lipid peroxidation products. Glutathione peroxidase activity may be a sensitive biomarker of metal exposure and biological effect and lipid peroxides as a secondary marker when accumulated metals are high.

The Akoya pearl oyster shell as an archival monitor of lead exposure.

The Akoya pearl oyster (Pinctada imbricata) was experimentally exposed to (a) constant levels of lead (Pb) at 180 microg L(-1) for nine weeks, or (b) two short term (pulse) exposures of Pb at 180 microg L(-1) (three weeks each) with an intervening depuration period (three weeks), to assess its utility as an (i) accumulative monitor of Pb contamination and an (ii) archival monitor for discriminating constant versus pulsed Pb exposure events. P. imbricata showed similar reductions in growth (based on shell morphology and wet weight) and Pb accumulation patterns for whole tissue and shell in response to both Pb exposure regimes. Thus the whole oyster was deemed an inappropriate accumulative monitor for assessing short-term temporal variation of Pb exposure and effect. However, using secondary ion mass spectrometry, Pb was shown to accumulate in the successively deposited nacreous layers of the shell of P. imbricata, documenting the exposure history of constant versus pulsed Pb events. Patterns of Pb deposition not only reflected the frequency of Pb exposure events but also their relative durations. Thus, the shell of P. imbricata may be employed as a suitable biological archive of Pb exposure.

Quantification of in situ nutrient and heavy metal remediation by a small pearl oyster (Pinctada imbricata) farm at Port Stephens, Australia.

The use of pearl oysters has recently been proposed as an environmental remediation tool in coastal ecosystems. This study quantified the nitrogen, phosphorus and heavy metal content of the tissue and shell of pearl oysters harvested from a small pearl oyster farm at Port Stephens, Australia. Each tonne of pearl oyster material harvested resulted in approximately 703 g metals, 7452 g nitrogen, and 545 g phosphorus being removed from the waters of Port Stephens. Increasing current farm production of 9.8 tyr(-1) to 499 tyr(-1) would balance current nitrogen loads entering Port Stephens from a small Sewage Treatment Plant (STP) located on its southern shores. Furthermore, manipulation of harvest dates to coincide with oyster condition would likely remove substantially greater quantities of nutrients. This study demonstrates that pearl aquaculture may be used to assist in the removal of pollutants from coastal waters while producing a commercially profitable commodity.

Potential biomarkers of crude oil exposure in the gastropod mollusc, Austrocochlea porcata: laboratory and manipulative field studies.

Surveys conducted after a crude oil spill indicated that the intertidal gastropod mollusc Austrocochlea porcata may be highly sensitive to the pollutant, and therefore also valuable as a biomonitoring organism. Toxicity tests conducted in the laboratory and field established cause-effect for A. porcata mortalities on exposure to environmentally relevant concentrations of crude oil constituents. Glutathione antioxidant system components (glutathione and glutathione peroxidase, GPx) and oxidative damage (lipid peroxidation) in A. porcata were measured to determine whether any of these biochemical parameters showed potential as biomarkers of sublethal oil exposure. GPx was the most promising candidate for field-based biomarker studies after showing a dose-dependent induction to a crude oil water accommodated fraction (WAF) in laboratory assays. However, subsequent manipulative field experimentation indicated that the GPx response was not sufficiently sensitive and not necessarily predictive of population level effects when measured in situ.

Accumulation and distribution of heavy metals in the grey mangrove, Avicennia marina (Forsk)Vierh: biological indication potential.

The accumulative partitioning of the heavy metals Cu, Pb and Zn in the grey mangrove, Avicennia marina, were studied under field conditions. Copper and Pb were accumulated in root tissue to levels higher than surrounding sediment levels. Zinc was accumulated to levels reflecting sediment concentrations. Strong linear relationships existed for all metals in sediments with metals in root tissue. Accumulation of Cu in leaf tissue followed a linear relationship at lower sediment concentrations, with an exclusion or saturation mechanism at higher sediment concentrations. Lead showed little mobility to leaf tissue. Zn showed restricted accumulation in leaf tissue, which correlated with sediment concentrations. Decreases in sediment pH were found to increase Zn accumulation to root tissue. Increasing concentrations of Pb and Zn in sediments resulted in a greater accumulation of Pb to both root and leaf tissue. A. marina roots may be employed as a biological indicator of environmental exposure of Cu, Pb and Zn and leaves for Zn, with temporal monitoring.

Toxicity, growth and accumulation relationships of copper, lead and zinc in the grey mangrove Avicennia marina (Forsk.) Vierh.

The effects of three heavy metals Copper (Cu), lead (Pb) and zinc (Zn), and the interaction of an essential (Zn) and non-essential (Pb) metal on germination, growth, and accumulation of metals in the grey mangrove, Avicennia marina (Forsk.) Vierh var. australasica (Walp.) Moldenke, were studied under laboratory conditions. Avicennia marina was found to be highly tolerant to the metals applied. Copper was accumulated in root tissue in a linear relationship at lower sediment concentrations, but at concentrations of 200 microg/g and higher, no further increases in root Cu levels occurred. Translocation of Cu from the root to leaf tissue was low, yet revealed similar accumulation patterns as root tissue. Significant reductions in seedling height leaf number and area were found with significant increases in Cu concentrations in tissues at 100 microg/g sediment Cu. At Cu sediment levels of 400 microg/g, a decrease in total biomass and root growth inhibition was observed. Emergence was retarded with increasing copper concentration, with 800 microg/g sediment Cu resulting in a total inhibition of emergence. The LC50 for emergence and EC50 for biomass was 566 and 380 microg/g Cu respectively. Lead accumulation in root tissue was lower that other metals, yet increased in a dose dependant fashion across the sediment Pb concentration range examined. Lead was excluded from leaf tissue at Pb sediment concentrations up to 400 microg/g, above which limited transport of Pb occurred. Little negative effects on growth were observed due to the low accumulation of Pb. Zinc uptake was high, and was accumulated in a linear fashion in root tissue across the sediment Zn concentration range applied. Zinc translocation to leaf tissue exhibited a dose dependant relationship with both root and sediment Zn levels. Emergence decreased with increasing sediment Zn concentrations, with 1000 microg/g sediment Zn showing 100% mortality. Significant reductions in seedling height, leaf number, area, biomass and root growth inhibition were found at concentrations of 500 microg/g sediment Zn. The LC50 for emergence and EC50 for biomass was 580 and 392 microg/g Zn respectively. Lead and Zn in combination resulted in an increased accumulation of both metals in leaf tissue and increased toxicity than individual metals alone, and is the first noted occurrence of a Pb and Zn additive response in angiosperms. Possible mechanisms of accumulation and toxicity are discussed.

Leaf biochemical parameters in Avicennia marina (Forsk.) Vierh as potential biomarkers of heavy metal stress in estuarine ecosystems.

Sediment loadings and leaf accumulation of the heavy metals copper (Cu), lead (Pb) and zinc (Zn) with accompanying changes in leaf chlorophylls' (a + b), carotenoids and the antioxidant enzyme peroxidase were examined in the grey mangrove Avicennia marina (Forsk.) Vierh, in order to determine the applicability of these parameters as biomarkers of heavy metal stress under field conditions. Copper was found to show limited accumulation to leaf tissue, following a linear relationship at lower sediment concentrations, with saturation at higher sediment Cu concentrations. Copper accumulation relationships to leaf tissue were maintained temporally, and increases in sediment Cu, salinity, and decreases in sediment pH and Zn contributed to the accumulation of Cu to leaf tissue. Lead showed a significant relationship between sediment and leaf Pb levels, but accumulation was minimal. Accumulation relationships for Pb were not maintained temporally, and high sediment Pb, low pH and organic content increased bioavailability and accumulation of Pb. Zinc was the most mobile of all metals and was accumulated to the greatest quantities in leaf tissue in a dose-dependant relationship. Some temporal variation in Zn accumulation occurred, and higher sediment pH, organic content Zn and Pb promoted leaf Zn accumulation. Leaf Cu and Zn showed the strongest relationship with peroxidase activity and to a lesser degree Pb. Zinc was the only accumulated metal to show relationship maintenance with peroxidase activity over time. It was found that peroxidase activity best reflects the total phytotoxic effect from the combined metal stress of all three accumulated leaf metals. The only significant photopigment relationship evidenced was that of leaf Zn with the chlorophyll a/b ratio, but was not maintained temporally. Peroxidase activity may be an appropriate biomarker for Zn or total metal accumulation in leaf tissue, and the chlorophyll a/b ratio a suitable biomarker of Zn accumulation though requires temporal monitoring under field conditions.

Estuarine macrobenthic community structure in the Hawkesbury River, Australia: relationships with sediment physicochemical and anthropogenic parameters.

Macrobenthic community assemblage diversity and abundance were monitored on both Cowan and Berowra Creeks in the estuarine reaches of the Hawkesbury River, Sydney Australia during 1997-1998. Natural sediment physicochemical differences were assessed, along with low-level anthropogenic contaminants including copper, lead, zinc, phosphorus and nitrogen. Contaminant levels at all sites were below sediment guideline values for biological effects. Natural physicochemical sediment differences were the main determinants in species assemblage patterns among sites. Three groupings of sites with similar assemblages were observed during February 1998. Berowra Creek sites, which were higher in organic content and silt/clay (and thus metals and nutrients), higher in pH and lower in salinity, were similar in terms of contributions by the polychaetes Ceratoneresis aequisetis, Scoloplos normalis, the isopod Cyathura hakea and the bivalve mollusc Soletellina alba. Sites on upper Cowan Creek, higher in organic content, silt/clay (nutrients and metals) and lower in salinity, were similar and separated from other sites in terms of the polychaete Carazziella victoriensis and the gastropod mollusc Nassarius jonasii. Sites lower in Cowan Creek, tended to be higher in sand content, more saline and lower in organic content and thus nutrient and metal concentrations. These sites were similar in terms of contributions of the polychaete Sigalion bandaensis and the bivalve molluscs, Mysella vitrea and Tellina deltoidalis. The biotic assemblage patterns were not maintained temporally, suggesting the importance of monitoring over time to assess possible future impacts. Aggregating species data to the family level resulted in similar site discrimination. Site differences were less distinct at higher taxonomic levels and suggests future monitoring at the family level is sufficient to detect assemblage differences among sites. The results obtained represent the difficulty in detecting responses to low-level contamination at the community level, and provide a sound anticipatory baseline for the assessment of future possible anthropogenic disturbance in the Hawkesbury River.

Photosynthetic pigments and peroxidase activity as indicators of heavy metal stress in the Grey mangrove, Avicennia marina (Forsk.) Vierh.

Mangroves have been observed to possess a tolerance to high levels of heavy metals, yet accumulated metals may induce subcellular biochemical changes, which can impact on processes at the organism level. Six month-old seedlings of the grey mangrove, Avicennia marina (Forsk.) Vierh, were exposed to a range of Cu (0-800 micrograms/g), Pb (0-800 micrograms/g) and Zn (0-1000 micrograms/g) concentrations in sediments under laboratory conditions, to determine leaf tissue metal accumulation patterns, effects on photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids), and the activity of the antioxidant enzyme peroxidase. Limited Cu uptake to leaves was observed at low sediment Cu levels, with saturation and visible toxicity to Cu at sediment levels greater than 400 micrograms/g. Leaf Pb concentrations remained low over a range of Pb sediment concentrations, up to 400 micrograms/g Pb, above which it appeared that unrestricted transport of Pb occurred, although no visible signs of Pb toxicity were observed. Zn was accumulated linearly with sediment zinc concentration, and visible toxicity occurring at the highest concentration, 1000 micrograms/g Zn. Significant increases in peroxidase activity and decreases in photopigments were found with Cu and Zn at concentrations lower than those inducing visible toxicity. Significant increases in peroxidase activity only, were found when plants were exposed to Pb. Positive linear relationships between peroxidase activity and leaf tissue metal concentrations were found for all metals. Significant linear decreases in photosynthetic pigments with increasing leaf tissue metal concentrations were observed with Cu and Zn only. Photosynthetic pigments and peroxidase activity may be applicable as sensitive biological indicators of Cu and Zn stress, and peroxidase activity for Pb stress in A. marina.