Fish, frogs, and other aquatic animals sometimes show signs of ‘endocrine disruption’; aberrant changes to their hormone or reproductive systems that are thought to be caused by chemicals in the water they inhabit. Very few of these chemicals have been identified, and this prevents the use of classical chemistry-based analytical methods. The other problem is that the levels of hormone-like chemicals which have endocrine-disrupting biological effects tend to be so low that standard chemical methods cannot detect them. This research developed a suite of biological tests sensitive enough to detect very low levels of chemicals associated with certain types of endocrine disruption. These tests were used to examine wastewater, surface water and drinking water collected from Australia, South Africa and four European countries. The water samples were also subjected to standard chemical analysis, and the datasets compared. It was concluded that some wastewater and surface water samples contained compounds that interacted with components of the estrogen, progesterone, androgen and mineralocorticoid hormone systems, but none of the biological tests detected endocrine disrupting activity in drinking water.

Wastewater must be treated to remove harmful pathogens and chemicals before it can be released to the environment, but the cost of proving that all pollutants have been removed is prohibitive because potentially thousands of separate chemicals would have to be measured. Another problem is that classical chemistry measurement tests are sometimes not sensitive enough to be able to detect the very low levels of chemicals which still harm animals and plants. This research developed a suite of extremely sensitive in vitro cell culture tests and an in-situ laboratory test in which mosquitofish were observed when swimming in recycled treated water. These bioassays measured the effects of mixtures of contaminants and were compared with traditional chemical measurements of separate contaminants. The in vitro cell culture, in situ mosquitofish and classical chemical analyses of selected contaminants generated equivalent results and led to the conclusion that combining multiple lines of evidence into a toolbox approach for the assessment of water quality provides data which is more informative and relevant when assessing potential impacts on the environment than traditional chemical measurements alone.

Wastewater (WW) contains harmful chemicals, including pesticides, that can disrupt normal gene function or hormone activity. The cost of measuring each separate contaminant at the frequency needed to demonstrate the safety of recycled WW is prohibitive. This research reviewed the risk assessment and regulation of chemicals in Australian water, with a focus on ‘thresholds of toxicological concern’. Laboratory techniques were developed to extract and concentrate WW contaminants into solutions suitable for analysis using both new in vitro cell culture assays and analysis in expensive, established chemical tests. WW and treated samples were collected from nine Australian water reclamation plants. The total effect of each sample (which contained a mixture of contaminants) on cell death, gene integrity and aspects of liver, hormone, nerve and immune system activity, was determined using in vitro cell culture bioassays, and compared with the classical chemical measurement of each separate contaminant. The cheaper cell-culture tests correlated well to the levels of groups of chemicals and could be used to find thresholds of toxicological concern. Both testing regimens also demonstrated that reverse osmosis is a highly effective method that removes harmful chemicals to levels much lower than those designated safe by regulatory authorities.

Before treated wastewater can be discharged to the environment, utilities are required to perform a direct toxicity assessment (DTA), which usually requires that live aquatic animals, such as fish, swim in the clean discharge water for 4-10 days while their growth and activity are observed and measured. This preparatory research investigated the potential of in vitro cell culture assays for replacing whole animal tests. It was concluded that there are no legal or regulatory barriers, that in vitro cell culture has higher social and ethical acceptance than in vivo animal tests, and that three organisms (bacteria, algae and fish) could provide the cells needed to conduct seven toxicity tests that are good candidates for future development as alternatives to existing DTA methods.

Cryptosporidium, a microscopic pathogen, forms infectious oocysts which are removed by specific and targeted water treatments. Oocysts can only be seen by using a microscope but finding an infectious dose of 10 oocysts in a litre of water is like finding a needle in a haystack. Usually a larger volume of water, 1000mL, is filtered to recover all the oocysts into a small volume of 0.1 to 1 mL, because this is small enough to be examined under a microscope. It is scientific practice to add some dead, colour-dyed oocysts to the large volume of water. If all the coloured oocysts are counted on the filter, the recovery is 100%. From this it became clear that there was a problem with oocyst recovery. This research found that different elements reduced recovery from different types of water, for example, iron and silica reduced oocyst recovery from river or groundwaters. The approved method for quantifying the environmental occurrence of oocysts can now be modified to increase recovery, and this change improves analysis and consequent management which further reduces risks to public health.

Source waters are disinfected to remove harmful pathogens, but chlorine reacts with organic matter and bromides to form disinfection by-products (DBPs) which can affect health. Water treatment reduces DBPs to safe levels by using alum to remove organic matter before disinfection but some water sources, particularly those with high bromine levels, are still difficult to treat. This research aimed to compile the best protocols for alum coagulation and disinfection when source waters contain different levels of organic matter and bromides, and to relate these to health risks. When organic matter was removed with 125 mg/L alum, and this treated source water was disinfected twice, and the first dose was calculated to generate chlorine levels of 0.5 mg / L for two days before administering the second dose, then DBP levels in drinking water were minimised. Neither alum-treated nor disinfected water caused toxicity in two laboratory tests used to examine risks to health.

It is prohibitively expensive and time-consuming to monitor drinking water by individually quantifying every possible polluting contaminant. Instead, living cells can be cultured in samples of the test water and if one or more toxic contaminants are present the cells die. Less toxic, more subtle effects can also be measured, for example, a toxicity test that uses cells collected (decades ago) from one monkey kidney, quantifies cell death but also measures the amount of protein made by each live monkey kidney cell. The problem with this, and other toxicity tests, is that chlorine disinfectants and harmless low levels of other substances, such as aluminium or copper, which occur naturally in water, can sometimes have inhibitory effects on the ‘bare’ cells that are often more sensitive when cultured inside laboratory culture vessels than when they were in a normal situation within a body. This research identified commonly used toxicity tests that are not affected by disinfectants or naturally occurring harmless substances, and also worked out some solutions that quench disinfectants and allow cost-effective and useful cell-based toxicity tests to be used to monitor the safety and quality of drinking water.

This paper discusses various water quality risk management techniques and proposes a step-by-step catchment risk assessment methodology that is compatible with the Australian Drinking Water Guidelines.

Cryptosporidium is a waterborne microscopic parasite with different forms at various stages of its lifecycle. One form, the spherical oocyst, is excreted by infected people and transported in rivers and surface waters. The problem is that it is not known if oocysts found in water are dead or are alive and infectious. This leads to an overestimation of the public health risk posed by oocysts found in source waters.

This research developed an in vitro cell culture test to differentiate between dead and infectious oocysts. The immortalised HCT-8 cell line was derived from cancerous human intestinal cells. When the cells are grown in a culture vessel, they display many characteristics of normal human gut cells. It was discovered that treating oocysts with acid to mimic stomach pH and using centrifugal force to ensure oocysts contact the HCT-8 cells, live (but not dead) oocysts react as though they are infecting a human host. The oocysts ‘hatch’ the next sporozoite form of the lifecycle, and these can be counted using a microscope. This ‘infectivity assay’ gives an improved, more accurate method for quantifying risks to human health presented by unidentified cryptosporidium oocysts in water.