The Northern Australian Community Groundwater Security Project

• Most groundwater sampled across northern Australia was fresh and suitable for drinking and agriculture
• Every bore had a distinctive microbial community
• More than 1,000 different types of microbes (bacteria and other microorganisms) were detected
• Treated drinking water had much lower microbial diversity
• eDNA proved to be an effective tool for monitoring groundwater health
• The project established the first groundwater microbial baseline for northern Australia

Our main aim was to find out more about the quality of the groundwater in northern Australia. We wanted to know how fresh or salty, and acidic or alkaline, the groundwater is, because this indicates how suitable water is for drinking, and supporting cattle, crops, industry and biodiversity. We also wanted to know more about groundwater microorganisms because they control the processes that determine groundwater chemistry.

• Developed a standardised groundwater quality sampling protocol that includes an app providing step-wise sampling instructions, a website for collecting and disseminating results, and a report card.
• More than 200 sampling kits were distributed. Around one-third were returned, and almost every returned sample produced usable DNA.
• The sampling locations spanned a 1,000 km tropical to arid zone climatic gradient from Warruwi on South Goulburn Island (-11.616669, 133.4305254) to Watarrka National Park (-24.291412, 131.549616) in central Australia.
• To examine changes across the north (tropical) to south (arid) climatic gradient we statistically analysed 41 bores assigned to six regions based on the criteria that four or more bores were sampled in that region, and their locations were representative of the region’s land use.
• We have also analysed treated drinking water sampled from four NT towns (Darwin, Katherine, Mataranka and Alice Springs) to as reference samples.

• The lowest EC, 20.4 μS/cm, was recorded from a Darwin rural area bore, whilst the highest, 2,810 μS/cm, was recorded from an arid zone (Western Davenport) bore. Both the lowest pH, 6.17 (Watarrka National Park) and the highest pH, 7.98 (Western Davenport) were recorded from arid zone bores. Almost all the groundwater samples were fresh and close to neutral (pH~7 ) and were within the Australian Drinking Water Guidelines: EC <1,090 μS/cm, 6.6 < pH < 8.5. This result is not surprising because almost all the bores sampled were providing water for drinking, cattle or crops.
• Bacteria dominated in all samples, both in numbers and variety, with over 1000 types of bacteria and archaea (similar to bacteria but with a different evolutionary history) detected by eDNA analysis.
• Less common, but also important, groundwater biota detected included over 190 types of fungi, 90 amoebae and 65 ciliates.
• Treated water supplies contained only a tiny fraction of the microbial variety found in raw groundwater, and almost no amoebae or ciliates. These results are consistent with water treatment effectively removing most microorganisms from drinking water. It also gave us a useful benchmark. It provided a clear contrast between “biologically stripped-back” treated water that is safe to drink and the rich communities of natural groundwater.
• One of the most important findings was that every bore was different. While tropical and arid regions showed some broad differences, there was also enormous variation between individual bores. This means no single bore can represent an entire region. Effective groundwater monitoring requires multiple sampling locations. The loss of the diversity described here will be indicative of impacts.

The use of eDNA to detect groundwater microorganisms is a relatively new, but very powerful way of monitoring and reporting groundwater health. Microbes respond quickly to changes in their environment. This includes pollution, shifts in nutrients, and changes in water chemistry (salinity and pH).

Microbes can often act as faster indicators of change than chemical tests alone can reveal. Because microorganisms perform the processes, known as ecosystem services, that maintain good water quality, tracking how they change over time can act as an early-warning system for the health of an aquifer.

The baselines established in this project can now serve as a reference point. If future monitoring shows a loss of variety, a shift in the balance of organisms, or the rise of pollution-associated microbes, those changes can be measured against what has been recorded here.

No study answers every question, and this project is no exception.

• We were not yet able to reliably detect stygofauna (small animals that live underground in groundwater systems). Unfortunately, the DNA reference libraries needed to identify them barely exist for the Northern Territory, only two local species are currently catalogued. Building these libraries is one of the most valuable next steps and would make eDNA a far more powerful tool for tracking this poorly known part of our biodiversity.
• Sampling captured moments in time, so we have a spatial snapshot, but not a timeseries. Groundwater microbiota can change with the seasons, and from year to year, especially after rainfall recharge, and so understanding temporal changes is an important future task.
• Citizen science proved highly effective. Of the 200 kits we distributed, about a third were returned, but of those returned, 97% yielded usable DNA. That high success rate shows the sampling method is robust and reliable in the hands of scientists and non-scientists alike.
• More information is needed about groundwater microbes. Bacteria and archaea, collectively known as prokaryotes, are the most common and abundant groundwater organisms. These small, single-celled microbes control the chemistry of groundwater. They cycle nutrients, break down contaminants, and help keep water clean. Fungi are also present and they play an important role in the decomposition of organic material. Amoebae and ciliates are small single-celled predators which graze on bacteria much like grazing animals crop grass on the surface. Together, these organisms form an underground food web that helps maintain the quality of the water we drink. Despite their importance, these groundwater communities are almost completely undescribed in northern Australia, however, this project has made a major contribution addressing this knowledge gap.

Detecting and identifying groundwater microorganisms is tricky. Most of them can't be seen, grown in a lab, or identified by eye. However, eDNA (shorthand for environmental DNA) is a relatively new method that makes recording the presence of groundwater microbiota possible.

Every living organism sheds traces of DNA into its surroundings. By collecting a litre or so of bore water, and recording the DNA it contains, we can build a list of the organisms living in that water, without ever needing to catch or see them.

In the past the detection of eDNA has needed the involvement of a specialised research laboratory. However, the advent of commercial eDNA analysis services, similar to commercial chemical water testing services, means that a standard analytical service for eDNA detection was available to us.

It also means that standard methods can be applied for environmental impact assessments and monitoring programs where future land use changes and resource extraction activities are occurring.

The large size and remoteness of the NT meant that we couldn't sample every bore ourselves. However, the participation of citizen scientists meant that many more bores could be sampled within the time available for our study.

The purpose-built sampling kit and a free smartphone app, Groundwater North, were developed to guide people step by step with the collection of a groundwater sample. The app automatically records the bore's location, date, and time, and uploads the information to our project website once the phone reconnects to the internet.

The development of a standard sampling protocol meant that community members, landholders, water managers, and scientists could all take part in this project.

• The maps showing the EC, pH and total types of microorganisms recorded in the bores sampled.
• Examples of report cards for bores in the Darwin rural district.
• The scientific paper reporting the project results.