Geoff R. MacFarlane

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.

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.

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.

Zinc distribution and excretion in the leaves of the grey mangrove, Avicennia marina (Forsk.) Vierh

Abstract Mangroves are important as primary producers in estuarine food chains. Zinc is often a major anthropogenic contaminant in estuarine ecosystems and has potential ecotoxicological consequences for mangrove communities. Accumulation, distribution and excretion of zinc in the leaf tissue of the grey mangrove, Avicennia marina was studied using SEM X-ray microanalysis and Atomic Absorption Spectroscopy. The first leaves of A. marina grown in 500 μg Zn as ZnCl 2 per g of dry soil were found to accumulate 106.3±18.5 μg Zn per g dry tissue, significantly higher than control plants, after a 7-month period. Washings from first leaves contained significantly higher amounts of zinc (0.30±0.14 μg/cm 2 Zn) than control plants after 1 month, suggesting excretion of zinc from glandular trichomes. SEM X-ray microanalysis revealed salt crystals exuded from glandular tissue on the adaxial surface of first leaves to be composed of alkaline metals and zinc in zinc treated plants. SEM X-ray microanalysis of seedlings dosed with 4 g/l Zn as Zn Cl 2 revealed a decreasing Zn gradient from xylem tissue, through photosynthetic mesophyll, to hypodermal (water) tissue. A subsequent increase in Zn concentration was observed in glandular tissue. Cell wall Zn concentrations were consistently higher than intracellular Zn concentrations.

The Semaphore crab, Heloecius cordiformis: bio-indication potential for heavy metals in estuarine systems

Although alterations at the organism level in decapod crustaceans on exposure to heavy metals have been evidenced in the laboratory, little examination of metal effects on morphology and population parameters have been explored in a field-based situation. Relationships between morphological parameters, population demography and heavy metal sediment loadings were examined in conjunction with the accumulation of metals in the Semaphore crab, Heloecius cordiformis, in the Port Jackson and Hawkesbury River estuaries, Sydney, Australia. H. cordiformis exhibited sexual dimorphism, with males having larger carapace width, carapace length, chelae length and total mass than females. Sexes were subsequently treated separately to assess morphological differences among locations. Locations that had greater proportions of females with purple chelae and less females in the population tended to have higher sediment metal levels. These relationships were maintained over time, and could be employed as population-level biological indicators of heavy metal stress. Copper and zinc were regulated in the hepatopancreas of H. cordiformis. Lead was accumulated in the hepatopancreas of H. cordiformis in proportion to sediment lead levels, suggesting the species is both an appropriate candidate for bio-indication of lead pollution, and Pb is the main metal linked with population level differences. Accumulation of lead varied between sexes, indicating that sexes must be monitored separately. Smaller males accumulated more lead than larger males, suggesting size is an important consideration for lead accumulation.

Effects of 4-nonylphenol and 17α-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.

Effects of Salinity and Temperature on the Germination of Phragmites australis, Juncus kraussii, and Juncus acutus: Implications for Estuarine Restoration Initiatives

We describe effects of salinity and temperature on germination characteristics of three dominant macrophytes, Phragmites australis, Juncus acutus, and J. kraussii, located in wetlands along the Hunter River, New South Wales, Australia. These wetlands were altered, from estuarine to freshwater habitats, by flood mitigation activities initiated during the 1970s. Tidal restoration to approximately 300 hectares of the marsh is planned to occur by 2008, with the goal to reduce freshwater vegetation in favor of salt marsh species. We determined if timing restoration projects to coincide with natural germination cycles or seasonal conditions of high salinity would be disadvantageous to P. australis or J. acutus germination. Germination trials lasted 25 days under two temperature range treatments (10–25 and 15– 30°C) and a salinity gradient (0–30 ppt). Many P. australis seeds commenced decomposition after three days (up to 58%). Increased salinity lowered germination in all species; however, only P. australis was influenced by temperature. Phragmites australis germinated in all conditions, although germination rate was low (2% ± 1.7) in the highest salinity treatment at high temperature regime. Both Juncus species obtained 100% germination in freshwater, failed to germinate in the highest salinity, and seed viability was not affected by 25 days emersion in high salinity. For areas dominated by P. australis in eastern Australia, we suggest that tidal reinstatement should be initiated in late autumn when P. australis seed banks are low. Additionally, periods of heavy rainfall, which reduce soil salinity, may help other species colonize the area. Further studies are required to determine characteristics of J. acutus that can be used to repress the species spread along the eastern coast of Australia. Currently, active measures involving chemical and physical weed suppression, litter removal, and mass planting of native species, are likely to be required to achieve management goals.

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.

Mammalian hair as an accumulative bioindicator of metal bioavailability in Australian terrestrial environments

The current study represents the first investigation of the suitability of marsupial and eutherian mammalian hair as indicator tissue for metal exposure and accumulation within contaminated Australian terrestrial ecosystems. A soil metal contamination gradient was established across 22 sites at increasing distances from a decommissioned Lead/Zinc smelter in NSW, Australia. Within each site, soil and small mammal populations were sampled. An Australian native marsupial, the insectivorous Brown Antechinus, Antechinus stuartii: Dasyuridae, and introduced rodents, the omnivorous Brown or Norway Rat, Rattus norvegicus: Muridae and the Black Rat, Rattus rattus: Muridae were assessed for hair concentrations of Cadmium (Cd), Copper (Cu), Lead (Pb) and Zinc (Zn). Metals in soil were most elevated at sites within close proximity to the smelter, with soil metal concentrations decreasing with distance from the smelter. The non-essential metals Pb and Cd were accumulated in hair, both metals exhibiting positive linear relationships with environmental exposure (soil metal concentrations). When the variables of weight and snout-vent length were considered, no further contribution in terms of explaining the variability in hair Cd or Pb was observed for all species examined. The essential metals Cu and Zn were regulated in hair, remaining similar across the metal contamination gradient. A significant negative correlation between snout-vent length and hair Cu concentration was found for the Brown Rat; greater hair Cu concentrations were found in smaller individuals of this species. Accumulation of Pb to hair was similar among species while concentrations of Cd in Brown Rat hair were higher than both Black Rat and Brown Antechinus hair. As each of the three aforementioned species exhibit similar bioaccumulation relationships for Pb, we suggest that sampling hair from introduced rodents (pest species) may provide a suitable proxy for the assessment of Pb bioavailability for a range of small mammals within Australian urban remnants.

The glutathione antioxidant system as a biomarker suite for the assessment of heavy metal exposure and effect in the grey mangrove, Avicennia marina (Forsk.) Vierh

Alterations in the glutathione antioxidant system and lipid peroxidation in Avicennia marina were studied under laboratory and field conditions. The activity of glutathione peroxidase (GPx) was found to respond to Zn exposure, and a significant positive relationship between leaf Zn concentration and GPx activity was observed after 96 h and 8 weeks. Lipid hydroperoxides increased proportionally with increasing leaf Zn concentration after 2 and 8 weeks, while no changes in total glutathione were observed. Induction of GPx at 96 h predicted effects at the individual level at a later time interval (reduced biomass at 8 weeks). Results from the field revealed that increasing leaf metal concentration (Zn, Cu or Pb) produced a proportional increase in GPx activity whereas lipid hydroperoxides and total glutathione were not affected. The utility of GPx as an early warning biomarker is suggested, since GPx activity increases in a dose-dependant fashion in response to accumulated leaf metals, and is predictive of later effects on growth.