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.

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.