Exposure of Microcystis aeruginosa to Hydrogen Peroxide under Light: Kinetic Modeling of Cell Rupture and Simultaneous Microcystin Degradation.

The effect of hydrogen peroxide on the cell integrity of a cyanobacterium, Microcystis aeruginosa, and on the release and degradation of microcystins (MCs) under simulated sunlight was investigated. The cyanobacterium was exposed to H2O2 in the range of 0-60 mg·L(-1) for 3.5 h. Production of OH radical in the solution was estimated by a chemical probe method. More than 99% (2 log) of the M. aeruginosa cells were ruptured or damaged by 3 h for all the treatments. Loss of cell integrity over time revealed two distinct phases. Cells retained their integrity during the initial lag phase and rapidly ruptured following first-order reaction afterward. A linear relationship was found between the duration of the lag phase and the steady-state concentration of OH radical. Release of MCs was closely correlated with the loss of cell integrity. Sequential reaction models were developed to simulate the release and degradation of MCs. These models were able to quantitatively describe the kinetics of all reactions under different H2O2 doses and extended exposure time. In particular, the models successfully predicted the concentration change of MCs using independently measured parameters. These models provide a simple and quantitative means to estimate the interaction of oxidants and cells and the consequent release of metabolites during oxidation treatment of cyanobacterium-laden waters.

Earthy odor compounds production and loss in three cyanobacterial cultures.

Geosmin and 2-methylisoborneol (MIB) related odor events caused by cyanobacteria have been a very common problem to water supply. This paper investigated the effects of temperature (18 and 25 °C) and light intensity (10 and 100 µmol photons m(-2) s(-1)) on the production behaviors of earthy odor compounds by three odorous cyanobacteria, i.e., the geosmin-producing planktonic Anabaena circinalis (Ana 318), geosmin-producing benthic Phormidium amoenum (Pho 012) and MIB-producing benthic Phormidium sp. (Pho 689). At the same time, the effects of biodegradation and volatilization on the fates of the released odor compounds in water were also evaluated. The combination of high temperature (25 °C) and light intensity (100 µmol photons m(-2) s(-1)) favored the growth of the three cyanobacteria and the production of chl-a and odor compounds. However, higher chl-a and odor yields (average odor compounds per cell) were achieved for the two benthic cyanobacteria at the temperature of 18 °C. Most of geosmin was included within the cells for Ana 318 (95-99%) and Pho 012 (85-60%), while only 20-40% MIB was bound to the cells for Pho 689. The half-life times of MIB and geosmin due to volatilization varied between 18.8 and 35.4 days, while 8 out of 10 samples exhibited a half-life time (t(1/2)) for geosmin biodegradation shorter than 1 day (0.38-15.0 h), showing that biodegradation could affect the fate of geosmin significantly in aquatic environments. In comparison, biodegradation of MIB was much slower (t(1/2): 122-2166 h). Denaturing gradient gel electrophoresis (DGGE) analysis showed that Pseudomonas- and Sphingomonas-like bacteria coexisted with cyanobacteria in the cultures, and may have played an important role in geosmin/MIB biodegradation. The result of this study will be helpful for better understanding and managing the earthy odor problems caused by cyanobacteria in water supply.

In situ measurement of odor compound production by benthic cyanobacteria.

A simple technique was developed to make in situ measurements of emission rates of two common odorants, 2-MIB and geosmin, and was validated with different natural communities of benthic cyanobacterial mats in Hope Valley Reservoir (HVR), South Australia, and Kin-Men Water Treatment Plant (TLR-WTP), Taiwan. A pair of parallel columns was used to differentiate between emission and loss rates caused by biodegradation, volatilization, and other mechanisms. Experimental results indicated that the loss rates followed a first-order relationship for all cases tested, with biodegradation and volatilization being the key mechanisms. The loss rates were comparable to those reported in the literature for biodegradation and those calculated from two-film theory for volatilization. After accounting for the loss rates, the net emission of geosmin and 2-MIB was estimated from experimental data. Odorant emission rates on the basis of column surface area, cyanobacterial cell number, and chlorophyll a (chl-a) were 4.2-4.4 ng h(-1) cm(-2), 1.0-5.5 x 10(-6) ng h(-1) cell(-1), and 3.2-3.5 ng h(-1)microg-chl(-1), respectively for 2-MIB released from benthic mats in TLR-WTP, and, 18-190 ng h(-1) cm(-2), 0.053-1.8 x 10(-3) ng h(-1) cell(-1), and 48-435 ng h(-1)microg-chl(-1) respectively for geosmin from benthic mats in HVR. The method developed provides a simple means to estimate the emission rates of odorants and possibly other algal metabolites from benthic cyanobacterial mats.

Effective doses, guidelines & regulations.

A number of countries have developed regulations or guidelines for cyanotoxins and cyanobacteria in drinking water, and in some cases in water used for recreational activity and agriculture. The main focus internationally has been upon microcystin toxins, produced predominantly by Microcystis aeruginosa. This is because microcystins are widely regarded as the most significant potential source of human injury from cyanobacteria on a world-wide scale. Many international guidelines have taken their lead from the World Health Organization's (WHO) provisional guideline of 1 microg L(-1) for microcystin-LR in drinking-water released in 1998 (WHO 2004). The WHO guideline value is stated as being 'provisional', because it covers only microcystin-LR, for reasons that the toxicology is limited and new data for toxicity of cyanobacterial toxins are being generated. The derivation of this guideline is based upon data that there is reported human injury related to consumption of drinking water containing cyanobacteria, or from limited work with experimental animals. It was also recognised that at present the human evidence for microcystin tumor promotion is inadequate and animal evidence is limited. As a result the guideline is based upon the model of deriving a Tolerable Daily intake (TDI) from an animal study No Observed Adverse Effects Level (NOAEL), with the application of appropriate safety or uncertainty factors. The resultant WHO guideline by definition is the concentration of a toxin that does not result in any significant risk to health of the consumer over a lifetime of consumption. Following the release of this WHO provisional guideline many countries have either adopted it directly (e.g., Czech Republic, France, Japan, Korea, New Zealand, Norway, Poland, Brazil and Spain), or have adopted the same animal studies, TDI and derivation convention to arrive at slight variants based upon local requirements (e.g., Australia, Canada). Brazil currently has the most comprehensive federal legislation which includes a mandatory standard of 1 microg L-(1) for microcystins, and also recommendations for saxitoxins (3 microg L(-1)) and for cylindrospermopsin (15 microg L(-1)). Although guidelines for cyanotoxins and cyanobacterial cell numbers for recreational waters are in place in a number of countries, it is consid ered that there is currently insufficient information to derive sound guidelines for the use of water contaminated by cyanobacteria or toxins for agricultural production, fisheries and ecosystem protection. In relation to the need for specific regulations for toxins for the US, the surveys that have been carried out to date would indicate that the priority compounds for regulation, based upon their incidence and distribution, are microcystins, cylindrospermopsin and Anatoxin-a. Additional research is required to support guideline development, including whole-of-life animal studies with each of the known cyanotoxins. In view of the animal studies that indicate that microcystins may act as tumor promoters, and also some evidence of genotoxicity and carcinogenicity for cylindrospermopsin, it may be appropriate to carry out whole-of-life animal studies with both toxicity and carcinogenicity as end-points. In relation to microcystins, it is known that there a large number of congeners, and the toxico-dynamics and kinetics of these variants are not well understood. Further research is needed to consider the approach to take in formulating health advisories or regulations for toxin mixtures, i.e. multiple microcystins, or mixtures of toxin types. An important requirement for regulation is the availability of robust monitoring and analytical protocols for toxins. Currently rapid and economical screening or quantitative analytical methods are not available to the water industry or natural resource managers, and this is a priority before the release of guidelines and regulations. There is insufficient information available in a range of the categories usually required to satisfy comprehensive risk assessment process for the major toxins to currently adopt any of the international guidelines as regulations in the US. The major limitations that need to be overcome include: the capacity to deal with multiple toxin congeners, the absence of robust analytical methods for compliance monitoring, and the absence of certified toxin standards to support analyses. However, the current WHO provisional guideline for microcystin-LR, or the other national guideline variants that are based upon it, (e.g., Canadian, Australian) may be appropriate to adopt as a health advisory in the short-term, while regulations are developed. The bathing and recreationa water guidelines developed in other countries could also be translated fo use as recreational water guidelines situation in the US.

Relative value of surrogate indicators for detecting pathogens in lakes and reservoirs.

This study investigated the relative behavior of pathogens, fecal indicator organisms, and particles of varying size during transport through a reservoir following a storm event inflow in Myponga Reservoir, South Australia. During the inflow, samples were collected from the river and at various locations within the reservoir to determine the fate and transport of microroganisms as they progressed through the water body. Microbiological analysis included the indicator organisms Escherichia coli, enterococci, Clostridium perfringens, aerobic spores, and somatic coliphages, the protozoan pathogens Cryptosporidium spp. and Giardia spp., and the potential physical surrogates of pathogen contamination including particle size and turbidity. Of the microbial indicator groups, C. perfringens spores were the most highly correlated with Cryptosporidium spp. concentrations (Spearman Rho = 0.58), closely followed by enterococci (Spearman Rho = 0.57). Cryptosporidium spp. oocysts were predominantly associated with small sized particles (range of 14.3-27.7 microm). All of the microbial indicator groups tested were associated with larger sized particle ranges (> 63.3 microm) except C. perfringens spores which were associated with particles in the size range of 45.5-63.3 microm. Although indicators may rank correlate with Cryptosporidium spp., the variation in settling rates of different microorganisms has significant implications for the use of surrogates to estimate pathogen attenuation within reservoirs. For example, concentrations of Cryptosporidium spp. oocysts were reduced by a factor of 3 on reaching the dam wall, whereas enterococci were reduced by a factor of 10.

Fate and transport of pathogens in lakes and reservoirs.

Outbreaks of water-borne disease via public water supplies continue to be reported in developed countries even though there is increased awareness of, and treatment for, pathogen contamination. Pathogen episodes in lakes and reservoirs are often associated with rain events, and the riverine inflow is considered to be major source of pathogens. Consequently, the behaviour of these inflows is of particular importance in determining pathogen transport and distribution. Inflows are controlled by their density relative to that of the lake, such that warm inflows will flow over the surface of the lake as a buoyant surface flow and cold, dense inflows will sink beneath the lake water where they will flow along the bathymetry towards the deepest point. The fate of pathogens is determined by loss processes including settling and inactivation by temperature, UV and grazing. The general trend is for the insertion timescale to be shortest, followed by sedimentation losses and temperature inactivity. The fate of Cryptosporidium due to UV light inactivation can occur at opposite ends of the scale, depending on the location of the oocysts in the water column and the extinction coefficient for UV light. For this reason, the extinction coefficient for UV light appears to be a vitally important parameter for determining the risk of Cryptosporidium contamination. For risk assessment of pathogens in supply reservoirs, it is important to understand the role of hydrodynamics in determining the timescale of transport to the off-take relative to the timescale of inactivation. The characteristics of the riverine intrusion must also be considered when designing a sampling program for pathogens. A risk management framework is presented that accounts for pathogen fate and transport for reservoirs.

Acute skin irritant effects of cyanobacteria (blue-green algae) in healthy volunteers.

OBJECTIVE: To assess the skin irritant potential of a range of laboratory grown cyanobacterial species using skin-patch testing on human volunteers. METHODS: Cell suspensions and extracts of cyanobacterial cultures of Microcystis aeruginosa (non-toxic strain), Anabaena circinalis and Nodularia spumigena were applied to 64 volunteers in one trial, and Microcystis aeruginosa (toxic strain), Apanocapsa incerta and Cylindrospermopsis raciborskii were applied to 50 volunteers in a second trial. Six cell concentrations of each organism in the range from less than 5000 to greater than 200,000 cells/mL were applied in random order using adhesive skin patches (Finn Chambers). In addition, the applications included two treatments of each cyanobacterial species, involving whole and lysed cells, and positive (sodium lauryl sulphate) and negative (culture media) controls. Patches were removed after 24 hours and assessment of erythema was made by a dermatologist blinded to the species, cell type and concentration. RESULTS: On average, between 20% and 24% of individuals with 95% confidence interval +/-8% reacted across the concentration range tested for these cyanobacterial species. The reaction rates were lower (11% to 15%) among the subset of subjects not reacting to negative controls. The reaction was mostly mild, and in all cases was resolved without treatment. This was the case for both whole and lysed cells with little difference in reaction rates between these two treatments. There was also no dose-response across the concentration range for any of the cyanobacterial species tested. CONCLUSION: A small proportion of healthy people (around 20%) may develop a skin reaction to cyanobacteria in the course of normal water recreation, but the reaction is mild and resolved without treatment.