Philip T. Orr

Release and degradation of microcystin following algicide treatment of a Microcystis aeruginosa bloom in a recreational lake, as determined by HPLC and protein phosphatase inhibition assay

Abstract Algicide treatment of an hepatotoxic Microcystis aeruginosa Kuetzing emend. Elenkin bloom on Lake Centenary caused cell lysis and the release of microcystin into the surrounding water. Where M. aeruginosa was confined to accumulations along the leeward shore of the lake, dissolved microcystin was detected for only 24 hours after spraying. It was presumed that the toxin was rapidly diluted by uncontaminated water from the main body of the lake. At an enclosed site in the south-west corner of the lake, microcystin persisted at high levels (1300–1800 μg l−1) for 9 days before degradation commenced. Microcystin degradation kinetics following this 9 day lag phase were bi-phasic with a rapid phase lasting 3 days (90–95% loss), and a slower phase which continued until a flash flood on day 21. HPLC analysis and protein phosphatase assay revealed the same overall trend of microcystin release, persistence and then degradation. However, there was a rapid increase in protein phosphatase inhibition from day 5 to day 9 before degradation commenced. These results suggest that the initial bacterial transformation of microcystin resulted in a product more inhibitory to protein phosphatase than the parent toxin.

Cellular microcystin content in N-limited Microcystis aeruginosa can be predicted from growth rate.

ABSTRACT Cell quotas of microcystin (QMCYST; femtomoles of MCYST per cell), protein, and chlorophyll a(Chl a), cell dry weight, and cell volume were measured over a range of growth rates in N-limited chemostat cultures of the toxic cyanobacterium Microcystis aeruginosa MASH 01-A19. There was a positive linear relationship betweenQMCYST and specific growth rate (μ), from which we propose a generalized model that enablesQMCYST at any nutrient-limited growth rate to be predicted based on a single batch culture experiment. The model predicts QMCYST from μ, μmax(maximum specific growth rate), QMCYSTmax(maximum cell quota), and QMCYSTmin (minimum cell quota). Under the conditions examined in this study, we predict aQMCYSTmax of 0.129 fmol cell−1 at μmax and a QMCYSTmin of 0.050 fmol cell−1 at μ = 0. Net MCYST production rate (RMCYST) asymptotes to zero at μ = 0 and reaches a maximum of 0.155 fmol cell−1 day−1at μmax. MCYST/dry weight ratio (milligrams per gram [dry weight]) increased linearly with μ, whereas the MCYST/protein ratio reached a maximum at intermediate μ. In contrast, the MCYST/Chla ratio remained constant. Cell volume correlated negatively with μ, leading to an increase in intracellular MCYST concentration at high μ. Taken together, our results show that fast-growing cells of N-limited M. aeruginosa are smaller, are of lower mass, and have a higher intracellular MCYST quota and concentration than slow-growing cells. The data also highlight the importance of determining cell MCYST quotas, as potentially confusing interpretations can arise from determining MCYST content as a ratio to other cell components.

GENETIC, MORPHOLOGICAL, AND TOXICOLOGICAL VARIATION AMONG GLOBALLY DISTRIBUTED STRAINS OF NODULARIA (CYANOBACTERIA)

Morphological, toxicological, and genetic variation was examined among 19 strains of Nodularia. The strains examined could be morphologically discriminated into four groups corresponding to N. spumigena Mertens, N. sphaerocarpa Bornet et Flahault, and two strains that did not clearly correspond to currently accepted Nodularia species. Genetic variation was examined using nucleotide sequencing of the phycocyanin intergenic spacer region (cpcBA‐IGS) and RAPD‐PCR. The PCR‐RFLP of the cpcBA‐IGS differentiated four genotypes corresponding to the four morphological groups. However, nucleotide sequencing of 598 bp of the 690‐bp fragment showed that one of the three strains corresponding to N. sphaerocarpa (PCC 7804) was genetically divergent from the other two, suggesting that it constitutes a distinct species. Nucleotide variation within the morphospecies groups was limited (<1%), and all 14 Australian strains of N. spumigena possessed identical cpcBA‐IGS sequences. The RAPD‐PCR differentiated the same groups as the cpcBA sequencing and discriminated each of the seven different Australian populations of N. spumigena. Strains from within a bloom appeared genetically identical; however, strains isolated from different blooms could be separated into either a western or a southeastern Australian cluster, with one strain from western Australia showing considerable genetic divergence. The pattern of variation suggests that individual blooms of N. spumigena are clonal but also that Australian N. spumigena populations are genetically distinct from each other. Examination of genetic distance within and between blooms and within and between morphological groups showed clear genetic dicontinuities that, in combination with the cpcBA‐IGS data, suggest that Nodularia contains genetically distinct morphospecies rather than a continuous cline of genetic variation. Furthermore, these morphospecies are genetically variable, exhibiting hierarchical patterns of genetic variation on regional and global scales. Production of the hepatotoxin nodularin was not restricted to one genetic lineage but was distributed across three of the five genotypic groups. A strain of N. spumigena from a nontoxic Australian population was found to fall within the range of genetic variation for other toxic Australian strains and appears to be a unique nontoxic strain that might have arisen by loss of toxin production capacity.

Response of cultured Microcystis aeruginosa from the Swan River, Australia, to elevated salt concentration and consequences for bloom and toxin management in estuaries

A mixed bloom of Microcystis aeruginosa forma aeruginosa and forma flos-aquae from the Swan River, Western Australia, was confirmed toxic by HPLC analysis. At least four, and possibly 11, microcystins were detected in cell-free extracts. Live bloom material was cultured at salt concentrations up to 21.2 g L–1 (total salts). The cultures were salt tolerant up to 9.8 g L–1. Reduction in the total cell concentration in the first 23 h was only observed in the highest salt treatment and first-order rate constants for cell lysis were higher than the rates for reduction of the intracellular microcystin pool size for that treatment. This suggests preferential lysis of genotypes with lower salinity tolerance and toxigenicity. This increased the toxicity of the mixed bloom population and the apparent microcystin cell quota without any change to the intracellular microcystin pool size. Therefore, the toxicity of bloom material may change through preferential lysis of cells with lower tolerances to changing environmental conditions, including salinity. Managers should be aware that the World Health Organization alert levels of 105 cells mL–1 for human contact exposure to cyanobacteria may not be a suitable prima facie test during these periods.

Exposure of beef cattle to sub-clinical doses of Microcystis aeruginosa: toxin bioaccumulation, physiological effects and human health risk assessment.

Yearling beef cattle were fed 1 x 10(5) cells ml(-1) of toxic Microcystis aeruginosa in their drinking water for a period of 28 d. Feed and water intakes of four control and four treated animals remained unchanged following introduction of M. aeruginosa into the drinking water of the treatment animals, and there were no significant differences in feed and water intakes between control and treated animals. We tested the blood plasma of both control and treated animals twice each week for elevated concentrations of the liver enzymes gamma-glutamyl transferase (GGT), glyceraldehyde dehydrogenase (GADH), amino aspartate transferase (AST) and bilirubin. All tests were negative indicating no measurable liver dysfunction resulting from microcystin intoxication. We also tested for the presence of free microcystin in the liver and blood plasma by HPLC and ELISA and for total microcystin (free+bound) in the liver by GC-MS. If all the ingested microcystin was bioaccumulated within the liver, the concentration would have exceeded 3 microg MCYST-LR g(-1) fresh weight. HPLC and GC-MS analysis of the liver tissue and blood plasma from treated animals failed to detect any microcystins. ELISA analysis of liver tissue extracts from the treated animals indicated microcystin concentrations as high as 0.92 microg MCYST-LR equivalents g(-1) fresh weight although none was indicated in the blood plasma. The microcystin concentrations measured by ELISA in livers of treated animals were more than 1000 times higher than the limit of quantification by HPLC and GC-MS indicating the ELISA results were almost certainly due to cross reactivity with something other than intact MCYST-LR. Based on the detection limits of the HPLC and the per capita daily consumption of beef in Australia, it appears that consumption by beef cattle of water containing M. aeruginosa cell concentrations up to 1 x 10(5)cells ml(-1) for 4 weeks would not produce concentrations of microcystin within the liver or blood plasma that would present an unacceptable risk to human health based on World Health Organization protocols for determining such risks.

Ingestion of toxic Microcystis aeruginosa by dairy cattle and the implications for microcystin contamination of milk.

Microcystin (MCYST) toxins can be produced by the bloom-forming cyanobacterium Microcystis aeruginosa. They are chemically stable compounds and have both acute and chronic effects on the health of mammals, including cattle and humans. Cattle will drink water containing lethal cell concentrations of M. aeruginosa. When cattle consume sub-lethal doses of microcystins, the fate of those toxins is unknown. We provided drinking water containing 1 x 10(5) cells ml(-1) M. aeruginosa (strain MASH01-A19) to four lactating Holstein-Friesian dairy cattle for 21 days to determine if MCYST-LR produced by the cyanobacteria, could be detected in milk produced by the cattle. Cattle consumed up to 15 mg MCYST-LR at an ingestion rate of 1.21 microg kg (live weight) d(-1). Analysis by HPLC and ELISA indicated that no detectable amounts of microcystin from the cyanobacteria were present in the milk obtained from the treated animals. Based on the level of quantitation of the ELISA analyses, the maximum possible concentration in the milk was less than 2 ng l(-1). This is more than three orders of magnitude less that the concentration that could be considered problematic for milk of 0.86 microg l(-1) which we calculated using the World Health Organization derived tolerable daily intake for MCYST-LR and the per capita daily consumption of milk in Australia.

Photosynthetic response of Myriophyllum salsugineum A.E. orchard to photon irradiance, temperature and external free CO2

Abstract The photosynthetic capacity of Myriophyllum salsugineum A.E. Orchard was measured, using plants collected from Lake Wendouree, Ballarat, Victoria and grown subsequently in a glasshouse pond at Griffith, New South Wales. At pH 7.00, under conditions of constant total alkalinity of 1.0 meq dm−3 and saturating photon irradiance, the temperature optimum was found to be 30–35°C with rates of 140 μmol mg−1 chlorophyll a h−1 for oxygen production and 149 μmol mg−1 chlorophyll a h−1 for consumption of CO2. These rates are generally higher than those measured by other workers for the noxious Eurasian water milfoil, Myriophyllum spicatum L., of which Myriophyllum salsugineum is a close relative. The light-compensation point and the photon irradiance required to saturate photosynthetic oxygen production were exponentially dependent on water temperature. Over the temperature range 15–35°C the light-compensation point increased from 2.4 to 16.9 μmol (PAR) m−2 s−1 for oxygen production while saturation photon irradiance increased from 41.5 to 138 μmol (PAR) m−2 s−1 for oxygen production and from 42.0 to 174 μmol (PAR) m−2 s−1 for CO2 consumption. Respiration rates increased from 27.1 to 112.3 μmol (oxygen consumed) g−1 dry weight h−1 as temperature was increased from 15 to 35°C. The optimum temperature for productivity is 30°C.

Gas exchange of Typha orientalis presl. communities in artificial ponds

Abstract Large field chambers were used for measuring the net carbon dioxide exchange of vigorously growing communities of Typha orientalis Presl. Uptake was greatest at the beginning of summer, when that of a community with leaf area index 6.8 reached almost 6 g CO 2 m −2 (water surface area) h −1 . In a second community with LAI 4.8 maximum uptake was 3.8 g CO 2 m −2 h −1 . In both communities there was a marked decline in uptake over the middle part of each day: uptake also declined quite rapidly as the communities aged. Maximum dry weight increases of 30 and 24 g m −2 24 h −1 for the 2 communities, respectively, were calculated from day-time net assimilation and night-time respiration. Measurements of maximum water loss from the first community showed a water-use efficiency of 7.86, which declined as the summer progressed. The photosynthetic performance of the Typha communities was similar to that of some well-irrigated C 3 crop plants grown in a similar climate.

Growth responses of Typha orientalis presl to controlled temperatures and photoperiods

Abstract Experiments are described in which seedlings of Typha orientalis Presls were grown for up to 6 months under precise conditions of temperature and photoperiod; photosynthesis was by natural daylight and did not vary between treatments. Variable treatments were imposed either from the seedling stage or on large plants raised under constant conditions. In general, total dry matter production increased as photoperiod increased from 8 to 16 h and also as day or night temperature increased, maximum production occurring when there was a warm day (30 or 27°C) and a small temperature drop (to 22°C) at night. The distribution of dry matter was also markedly affected by the imposed variables, leaf growth being favoured by high temperatures (to 30°C) and long photoperiods, and production of roots and rhizomes by low temperatures (to 10°C) and short photoperiods. None of the treatments resulted in floral initiation. The results are considered in relation to growth in the natural habitat.