Program 2B: Reservoir Management
CURRENT PROJECTS
2.3.3.1 IDENTIFICATION AND ENUMERATION OF CYANOBACTERIA
Project Leader Paul Monis Research Staff Catherine Bernard Organisations Involved AWQC
Budget $378,948
Duration Mar 2001 – Mar 2005 Background
Excessive growth of cyanobacteria is of concern due to their production of toxins or metabolites with off-flavours and tastes.
Semi-automated protocols for the analysis of cyanobacteria should provide rapid more reliable quantitative data on algal samples (including biomass estimates), while releasing expert biologists for other tasks such as data assessment and providing advice.
Principal Aims
To semi-automate the tasks of:
• counting cyanobacteria to estimate population density.
• estimating cell size and biomass.
• acquiring morphometric data to apply to taxon identification and recognition.
Research Plan
1. Preliminary development and sampling issues: A preliminary protocol will be developed for producing binary images of phototroph autofluorescence, which will then be used to examine some problems anticipated in sample preparation and presentation. This phase will address:
• selection of appropriate microscope magnification for analysis.
• system calibration using fluorescent beads of specific sizes.
• comparison of bright-field and autofluorescence images for specificity, resolution and signal/noise ratio.
• assessment of natural variability in morphology within and between species.
2. Optimisation of image segmentation: Accurate image discrimination (or segmentation) is the key to collecting images that can be analysed in a meaningful way. The important steps in this process will be:
• Selection and validation of suitable thresholding routines.
Validation will include use of fluorescent beads of specific sizes.
• programming of thresholding algorithms in a compiled language (contribution of Lyn Jarvis).
• testing of operations for image processing
• integration of sample preparation, digitisation and image processing operations into a cohesive image acquisition procedure
Program 2B: Reservoir Management
3. Development and validation of counting and measurement protocols: The image acquisition procedure will be made semiautomated for counting and measurement by serially linking operations from the Video Pro command menu.
Testing will include:
• design of suitable spreadsheet files for collection and manipulation of data.
• validation of counting for dispersed cells.
• determination of the errors in size measurements and their relationship to microscope magnification.
• examination and testing of alternative procedures for estimating cell numbers in filamentous and colonial organisms.
4. Preliminary development and validation of identification criteria:
• create a database of measurements of specific organisms which can be used to develop criteria for species identification.
• testing identification criteria using model assemblages of organisms.
• integration of criteria with counting protocols and validation for mixed assemblages of varying complexity.
• preliminary consideration of fluorescence in situ
hybridisation for highly specific identification and counting.
5. Development and validation of biovolume/biomass computations:
• development of biovolume calculations that take account of likely 3-dimensional shape, from linear or area measurements.
• testing of correlations of calculated biomass and
measurements made using traditional biomass measures (eg extracted chlorophyll) and with data from flow cytometry.
6. Integration of protocols and reporting of project
• integration of image analysis and data management protocols, including an alert process for detection of putative problem organisms.
• publication of the integrated image analysis process in an appropriate forum.
Milestones Achieved
• Protocols to count and size single-celled Microcystis and trichomes of Cylindrospermopsis developed.
• Semi-automation of the system achieved.
• Assessment of the efficacy of disaggregation techniques of colonial Microcystis was made using the image analysis system and a paper has been submitted for internal review.
• Protocols have been developed to estimate biomass from Microcystis and trichomes of Cylindrospermopsis.
Milestones Planned for Next Year
• AWQC staff training in the basic use of the IA system.
• Validation of counting and/or measuring (biomass) cultures of Microcystis, trichomes of Cylindrospermopsis and other similar shapes.
• Development of methods for measuring coiled shapes.
• Validation of methods for counting and measuring (biomass) cells from blooms.
• Develop methods for screening cells in field samples by fluorescence.
2.2.0.1 HYDRODYNAMIC DISTRIBUTION OF PATHOGENS IN LAKES AND RESERVOIRS Project Leader
Mike Burch Research Staff
Justin Brookes, Peter Hobson, Paul Monis, Chris Saint, Daniel Deere, Christobel Ferguson, Nick Ashbolt, Melita Stevens, Shane Haydon, Peter Nadebaum
Organisations Involved
AWQC, SCA, UNSW, Melbourne Water, GDH Collaborators
AwwaRF, USEPA, UWA Budget $2,158,000
Duration Mar 2002 – Apr 2004 Background
Pathogens present a challenge to drinking water quality worldwide, as demonstrated by the Sydney water crisis in 1999. One of the major issues involved is being able to predict potential problems at the water supply offtake to a filtration plant.
Pathogens usually enter reservoirs from the catchment following heavy rainfall, often as patchy “flood fronts”. Since it is often not known how fast they move through the reservoir from inlet to outlet, they are hard to sample and so potential problems are hard to predict. Monitoring is also currently very expensive, which increases the difficulty in determining how much effort to put in to assess risk.
This project will track and model these flood and transport processes in a number of different reservoirs, including Burragorang (Sydney), Myponga (Adelaide) and Sugarloaf (Melbourne) Reservoirs. The outcome of this project, for utilities both in Australia and in the US, will be to determine the most cost-effective monitoring approach for these pathogens in reservoirs and therefore help minimise the risk they pose.
The project is a collaborative venture between the CRC for Water Quality and Treatment, the Centre for Water Research (University of Western Australia), AwwaRF and the USEPA. It also builds upon a significant existing project being undertaken by the SCA to model reservoir processes in Burragorang Reservoir (NSW).
Principal Aims
• The project will develop, test and verify optimum and cost-effective sampling strategies for detecting pathogens in reservoirs. This will include development of a model for pathogen movement and fate in reservoirs (pathogen module) that can be run within a reservoir 3-D hydrodynamic model. The combined models can be used to simulate pathogen transport and therefore to design a sampling scheme to detect or “capture” them with a sampling program in a flood event.
Program 2B: Reservoir Management
Research Plan
The project will develop, test and verify optimum and cost-effective sampling strategies for detecting pathogens in reservoirs. This will include development of a model for pathogen movement and fate in reservoirs (pathogen module) that can be run within a reservoir 3-D hydrodynamic model. The combined models can be used to simulate pathogen transport and therefore to design a sampling scheme to detect or
“capture” them with a sampling program in a flood event. The 3-D hydrodynamic model will be validated for three different reservoirs in Australia, and will be used to design an actual sampling program, which will then be applied in an inflow event.
These events will be extensively sampled to allow for verification of the optimum sampling scheme suggested by the modelling work. The findings will allow for the refinement and development of a validated robust sampling/detection strategy for pathogen detection in reservoirs. The optimum sampling strategy or “best-practice” scheme will be presented so that it can be utilised by utilities, with or without the operation of the coupled physical-hydrodynamic models.
The project is able to incorporate separate significant large-scale 3-D reservoir modelling work currently being undertaken by a research partner within another project. The Centre for Water Research is contracted under a separate project, for the Sydney Catchment Authority (SCA), to investigate the hydrodynamics of a flood inflow event at Lake Burragorang (Sydney, New South Wales) during 2002. The current project (AwwaRF project 2752) will build upon that research and incorporate investigations on pathogen distribution, which are not currently scheduled within the SCA project. The hydrodynamic model will, in the first instance, be developed for Lake Burragorang This is largely an inkind contribution to project 2752. The information collected for Lake Burragorang will be incorporated into the Centre for Water Research hydrodynamic-physical models (ELCOM-CAEDYM).
The coupled models will be applied to Lake Burragorang to firstly simulate pathogen distribution during an inflow event, and secondly design an optimum sampling strategy for testing and validation in July - December, 2002. The validation would include monitoring at the sites where the model predicts the pathogen occurrence and at other control sites. Subsequent additional model validation will then occur in the winter 2003 (May-September) at Myponga Reservoir (South Australia) and Sugarloaf Reservoir (Victoria).
Three periods that will be investigated with respect to pathogen distribution at each study site: during a flood event, post-flood and during a period when there is no significant input. Intensive field work will be programmed to investigate features of the hydrodynamics and pathogen distribution during each of these periods.
The sampling programs for model validation will focus specifically on Cryptosporidium parvum. Although the pathogen module to model the transport and fate of pathogens will include characteristics for Giardia cysts, Cryptosporidium oocysts, E. coli, and coliphages, the field sampling and validation experiments in the three reservoirs will monitor for Cryptosporidium only.
The modelling scenarios and validation experiments will include tracking a range of particle sizes, which will represent free pathogens and pathogens attached to other material. Modelling will also incorporate inactivation characteristics for Giardia cysts, Cryptosporidium oocysts, E. coli, and coliphages. The coupling of the hydrodynamic model with the pathogen fate module will enable assessment of the risk associated with pathogens
reaching the off-take before inactivation of the representative group.
Milestones Achieved
Milestones Planned for Next Year
• A model for pathogen movement and fate in reservoirs (pathogen module) that can be run within a reservoir 3-D hydrodynamic model will be developed.
• The 3-D hydrodynamic model will be validated for the three different reservoirs in Australia, and will be used to design an actual sampling program, which will then be applied in an inflow event.
• These events will be extensively sampled to allow for verification of the optimum sampling scheme suggested by the modelling work.
2.2.0.6 DETERMINATION AND SIGNIFICANCE OF EMERGING ALGAL TOXINS
(CYANOBACTERIA). AWWARF PROJECT 2789
Project Leader/Principal Investigator Brenton Nicholson
Co-Principal Investigators
John Papageogiou, Glen Shaw, Wayne Carmichael Research Staff
Chris Saint, Andrew Humpage, Brett Neilan, Tom Linke Organisations Involved
AWQC, NRCET, UNSW, Wright State University Budget $1,630,000
Duration Jan 2002 – Dec 2004 Background
Algal toxins have been identified as an emerging issue in the USA. The Centre has signed an agreement with AwwaRF to jointly fund research in the USA and Australia to determine the significance of algal toxins in water supplies and refine the methods for their detection.
Principal Aims
• Develop methods for detection of cyanotoxins.
• Assessment of the significance of these toxins in water supplies in Australia and the USA.
Research Plan
The analytical method development will include the following toxins:
(A) Microcystins
• The colorimetric phosphatase inhibition assay as available in kit form will be compared with existing ELISA methods and validation against HPLC-DAD and HPLC-MS/MS. The timing of this work will depend on the availability of the phosphatase inhibition kit.
Program 2B: Reservoir Management
(B) Lipopolysaccharides
• Extraction and purification.
(C) Saxitoxins
• Neuroblastoma assays.
• HPLC-MS/MS method using hydrophilic interaction liquid chromatography.
• ELISA (existing kits).
(D) Cylindrospermopsin and anatoxin-a
• Combine anatoxin-a with cylindrospermopsin method (LC/
MS/MS) to determine both toxins in the same run.
• The feasibility of including saxitoxin analysis in the cylindrospermopsin/anatoxin (a) method will also be assessed.
(F) Monitoring US waters used as source waters for drinking Once the analytical methods have been developed they will be utilised in surveys of selected water supplies in the USA.
(G) Genetic methods for identifying toxic species The existing DNA based methods for microcystin and cylindrospermopsin production and for saxitoxin producing species of Anabaena will be applied to field samples that potentially contain these toxins.
Milestones Achieved
• The individual project agreements have been finalised and staff have been appointed at AWQC, NRCET and Wright State University.
• Wayne Carmichael of WSU has negotiated the involvement of ten utilities in the USA and one from Canada.
• Australian utilities involved are SA Water, SEQW, Sydney Water and Melbourne Water.
• Progress reports have been provided to AwwaRF in November 2002 and May 2003.
Milestones Planned for Next Year
• Assessment of the significance of LPS toxins.
• Progress reports on combined method forcylindrospermopsin, anatoxin-a and saxitoxin.
• Progress report on the evaluation of ELIA kits.
• Progress report on the analysis of samples from Australia and USA.
• Draft final report.
2.2.0.2 INVESTIGATION OF SURVIVAL OF CRYPTOSPORIDIUM IN ENVIRONMENTAL WATERS
Project Leader Paul Monis Research Staff
Alex Keegan, Chris Saint, Peter Cox, Monica Logan Organisations Involved
AWQC, Sydney Water, WSAA Budget $890,666
Duration Jan 2002 – Jan 2005
Principal Aims
• Stage 1 Laboratory experiments to:
• Identify a surrogate species of Cryptosporidium for stage 2 and 3.
• Develop container for suspending oocysts in environmental waters.
• Stage 2 Pilot study to assess rates of inactivation of oocysts in natural waters.
• Stage 3 Assessment of inactivation rates of oocysts in a range of environmental conditions.Determine the effects of light and temperature on the survival of fresh oocysts.
Research Plan
The primary method used in this project will be cell culture infectivity, with the same assay to be used for all stages of the project. This assay is based on the method of Hijjawi et al [8], with the exception that a molecular technique (real-time PCR) will be used instead of microscopy to quantify the level of infection. Cell culture has been established at the AWQC since the start of 2001 and this has been linked to a real-time PCR assay for Cryptosporidium that has been developed at the AWQC. These methods have been successfully used in combination to examine the effectiveness of various disinfection methods against live oocysts as part of CRC project 3.1.4 (Novel methods of pathogen destruction). The real-time PCR assay has a sensitivity level of a single oocyst and the cell culture infectivity assay can detect one in ten infectious oocysts. In addition to the cell culture assay, fluorescence microscopy and flow cytometry will be used in the preparation of the oocyst seeds that will be used in experiments. All survival experiments will be conducted to allow for the detection of three logs of inactivation. Facilities for both of these are available at the AWQC and Sydney Water laboratories. Standard methods used at the AWQC will be employed to gather data on the physical characteristics of the raw waters used in experiments (eg pH, turbidity, alkalinity, BOD, total plate counts, etc). For stages 2 and 3, existing online sensor systems will be used to measure water quality and environmental parameters at the test sites. The models developed in AWWA / EPA RFP 2752 - Hydrodynamic Distribution of Pathogens in Lakes and Reservoirs (which should be available by 2003) will be used to select test sites within reservoirs. The experimental system of Robertson et al, which describes semi-permeable containers that can be used to contact oocysts with defined environments, will be assessed for suitability for stages 2 and 3. A surrogate (C. andersoni) will be investigated for use in any field studies involving on-line reservoirs. If a surrogate is not available, sealed containers will be used to suspend C. parvum oocysts in reservoirs.
Milestones Achieved
• Preliminary laboratory-scale assessment of the survival of Cryptosporidium oocysts in reagent water at temperatures ranging from 4°C - 25°C completed.
Milestones Planned for Next Year
• Completion of lab-scale experiments for raw and reagent water.
• Evaluation of specifically designed containers for use in field experiments
• Technology transfer to collaborating lab (Sydney Water).
Program 2B: Reservoir Management
• Selection of field sites for years 2 and 3.
• Completion of Milestone report for year 1.
2.2.1.2 IMPACTS OF DESTRATIFICATION ON RESERVOIR NOM AND ITS REMOVAL BY WATER TREATMENT
Project Leader John van Leeuwen Research Staff
Justin Brookes, Mike Burch, K Grice, N Jayasuriya, Alan Wade, Leon Linden,
Organisations Involved
University of Adelaide, AWQC, Actew Budget $702,630
Duration Jul 2002 – Dec 2005 Principal Aims
To determine the changes in NOM during water storage in reservoirs and the implication of those changes on removal of NOM by conventional water treatment processes with particular reference to:
(i) Impacts of water storage in reservoirs on the character of NOM
(ii) Impacts of de-stratification of Myponga Reservoir on the character of NOM
(iii) The significance of the changes in NOM found in (i) and (ii) above, to the capacity of conventional water treatment (using the coagulant alum) to remove NOM from raw water.
Research Plan
• This project utilises the models developed in other projects, ie. “Hydrodynamic distribution of pathogens in lakes and reservoirs (Project 2.2.0.1) and with “Modelling coagulation to maximise removal of organic matter” CRC Project 3.2.8.
• To assess the input of allochthonous NOM into the reservoir, inputs from major sources (stream flows of the Myponga catchment) will be investigated. Modelling of the Myponga catchment hydrology will be investigated through RMIT University.
• Reservoir hydrology modelling will be investigated through project 2.2.0.1.
• Characterisation of the natural organic matter based on assimilable organic carbon content and microbial activities will be investigated using extracelluar enzyme assays and assays incorporating the use of Pseudomonas florescens P17. A range of chemical-structural characterisation techniques will also be incorporated including pyrolysis-gas chromatography-mass spectrometry and C13 CPMAS NMR.
• NOM in water samples obtained following in situ and/or laboratory simulated de-stratified and stratified conditions will be characterised using the above methods and concurrently assessed for treatability with alum. The alum treatment conditions will be determined using predictions from models developed through CRC project 3.2.8.
Milestones Achieved
• Assessment of several assays for measurement of extracellular enzyme (including _-glucosidase, leucine aminopeptidase and chitinase) activities in source and reservoir waters.
• Applied jar tests on source and Myponga Reservoir waters using alum as coagulant to determine the treatability of organics.
• Characterised NOM in source and reservoir waters using UV-vis spectrometry.
• Tested the treatability of an associated Canberra drinking water source (Bendora Reservoir) using alum.
• Implemented a flow-weighted monitoring program to determine the influence of flow on water quality parameters.
• The PhD study literature review has been completed.
Milestones Planned for Next Year
• Establish laboratory-based experiments that simulate de-stratification.
• Apply jar tests under standard conditions to compare removals of NOM by alum from Myponga (South Australia) and Googong (ACT) reservoir waters following stratified, de-stratified or simulated conditions.
• Isolate key NOM samples and characterise these using methods that give chemical-structural information.
• Evaluate the potential for modelling the Myponga catchment hydrology to provide information on the transport of NOM into the reservoir.