Subject - Faculty of Science, Technology and Engineering

Pettina Love commenced her Phd in 2006. Pettina is currently "Investigating the source of Arsenic contamination in the Bogong Moth and the impacts on the ecology of the Australian Alps".

AIMS AND BACKGROUND

The aim of this research is to identify the source of the Arsenic which is contaminating the adult aestivating Bogong Moth, Agrotis infusa (Lepidoptera: Noctuidae) .

(1) identify the original Arsenic based compounds, transformation products and companion chemicals which are present in potential sources of Arsenic within the different habitats during different stages of the Bogong Moth's lifecycle.

(2) identify the transformation products and companion chemicals which are present within the different tissues of the Bogong Moth as it matures from larvae to migrating moth to aestivating adult, and

(3) link the types of Arsenic sources which occur in different Bogong Moth habitats, to the transformation products identified within the tissues of the contaminated Bogong Moth.

In 2001 Arsenic was discovered in the adult aestivating Bogong Moth (Green, Broome et al. 2001) . The Bogong Moth migrates to the alpine regions of Australia annually, and is a fundamental food source for many of the endemic alpine species. The discovery that the adult aestivating Bogong Moth was contaminated with Arsenic was followed by an analysis of the scats of its main predators. The presence of Arsenic in the scats of the Mountain Pygmy Possum, Burramys parvus (Green, Broome et al. 2001) confirms its introduction into this endangered and threatened species. Arsenic has also been identified in the scats of the Dusky Antechinus, Antechinus swainsonii and the Bush Rat, Rattus fuscipes (Green, Broome et al. 2001) . Other species which are known to feed on Bogong Moths are birds such as the Australian Kestrel ( Falco cenchroides) , the Little Raven ( Corvus mellori) , the Pied Currawong ( Strepera graculina) , Richard's Pipits ( Anthus novaeseelandiae) , and the Smoky Mouse ( Pseudomys fumeus) and it is reasonable to assume that any species which preys on insects in the alpine region (Green and Osbourne 1994) could also have consumed Bogong Moths and hence, be contaminated with Arsenic.

As it is at the bottom of the food web, the contamination of the Bogong Moth with Arsenic puts numerous endemic species at risk. Sub-lethal quantities of Arsenic can cause cancer, mutation and birth defects. Due to its cumulative nature, continued exposure to small sub-lethal amounts can ultimately cause death (Shirley and Rachel Carson Council 1992) .

The initial exposure could have been through ingestion, inhalation or contact (CHEC 2001-2002) . Exposure could have occurred during the larval stage of its lifecycle, or as a migrating moth or as an adult aestivating moth. Each life stage also has different habitats; therefore, contamination could have occurred as larvae in the soils of Queensland, N.S.W. or Victoria, or during migration in the skies of Queensland, N.S.W. or Victoria, or as an adult aestivating moth in the alpine regions of N.S.W. or Victoria (Common 1954) .

There are many products which are a direct anthropogenic source of Arsenic, such as, treated wood, insecticides, herbicides, fungicides, semiconductors, and some veterinary medications. Indirect anthropogenic sources are industrial based practices, which release Arsenic into either the air or water. These include mines and smelters, cotton gins, glass manufacturing operations, coal burning facilities, municipal incinerators, as well as leaching of Arsenic from landfills that contain Arsenic containing ash produced by coal burning power plants and treated wood (Childrens Health Environmental Coalition 2001-2002) .

Arsenic can also occur naturally in the environment. Natural sources of Arsenic includes groundwater (Smith, Jankowski et al. 2003) and the minerals arsenopyrite (FeAsS), realgar (As 2S 2), orpiment (As 2S 3), and arsenolite (As 2O 3) (Costa 2000) .

When the Bogong Moth was exposed to an Arsenic source, it would have been exposed to Arsenic based compounds and other contaminants (companion chemicals). The Arsenic based compounds would have begun to break down into its constituent parts. The original compound would have been altered as it was metabolized by the moth and these different chemicals (transformation products) incorporated into different tissue types. Furthermore the original compound may have already been in an altered condition due to contact with light, water, other chemicals in the soil or plants (Shirley and Rachel Carson Council 1992) . This process would result in the presence of Arsenic based transformation products and companion chemicals in the tissues of Bogong Moth.

Recent Progress in the field

To date there has only been one published theory regarding the source of Arsenic contamination in adult aestivating Bogong Moth (Green, Broome et al. 2001) . Current theory is that the Bogong Moth ingests Arsenic during the larval stage of its lifecycle, after feeding on plants which have been exposed to Arsenic based agricultural products. This initial exposure as larvae results in Arsenic being stored within the moth throughout its lifecycle. The contaminated moth then migrates to the alpine region, transporting Arsenic to the alpine region where it aestivates.

Bogong Moth larvae (also called the Bogong Moth cutworm) inhabit the soil of the inland plains of eastern Australia from autumn (March) to spring (September). The larval grounds extend from the Darling Downs in Queensland , south to the north-western plains of Victoria . These areas contain agricultural and grazing land. The Bogong Moth larvae feed on annuals and have been considered an agricultural pest. Over summer adult Bogong Moths migrate to the Australian alpine regions. It is here that they aestivate in rock crevices and caves. During aestivation they are believed to fast (Common 1954) . This has led to the conclusion that Arsenic contamination is most likely to take place during the larval stage of the Bogong Moth lifecycle (Green, Broome et al. 2001) when feeding takes place.

Of the three broad possible sources of Arsenic, industry, agricultural or natural, it has been suggested that the source of the Arsenic contamination is agricultural (Green, Broome et al. 2001) . The possibility of a industry based source of Arsenic contamination has been dismissed as the larval grounds are located upwind of the eastern seaboard of Australia where Arsenic producing industry is concentrated (Green, Broome et al. 2001) . An agricultural source of Arsenic contamination is considered to be a more likely than a natural source of Arsenic contamination (Green, Broome et al. 2001) , because of the extensive use of Arsenic in both present and past agricultural products within the larvae habitat.

Adult aestivating Bogong Moths have been found to contain Arsenic for four sequential years, leading to the possibility that Arsenic is being transported to the alpine region annually (Green, Broome et al. 2001) . If this is the case then the uptake of Arsenic must be a regular event. Given that Bogong Moth larvae are known to feed on annuals it is probable that the Arsenic is ingested when feeding (Green, Broome et al. 2001) . Two possible explanations for the contamination of these food plants with Arsenic are 1) the present practice of directly applying herbicides which contain monosodium methylarsonate (MSMA) on agricultural crops which are food plants for larvae and 2) the uptake of Arsenic from the soil by the plants. The historic extensive use of Arsenic based products in agriculture from the 1860's to the 1960's (particularly insecticides including Lead Arsenate and DDT), has lead to broad-acre contamination of intensively farmed soil.

The Arsenic is stored in the tissues of the Bogong Moth larvae and subsequently, in the tissues of the adult migrating moths. The amount of Arsenic stored in each moth is of a sub lethal quantity (Green, Broome et al. 2001) and as such, the contaminated moth is still able to migrate to the alpine region to aestivate.

It is the belief that, the migrating moth is acting as a vehicle for the long-distance transport of Arsenic, from Australia's lowlands to its alpine regions (Green, Broome et al. 2001) . The presence of Arsenic in the adult aestivating Bogong Moth which inhabit the alpine regions is believed to have its origins in the lowland agricultural region. Initial exposure is through ingestion of contaminated plants by the Bogong Moth larvae. The Arsenic is incorporated into the tissues of the larvae and is still present within its tissues as an adult aestivating moth.

Relationship of this project to work in the field generally

It is important to note that the current theory is based on only one study and provides a useful starting point for this research. The compound containing the Arsenic has not been identified (Green, Broome et al. 2001) and the possibility that the contamination of adult aestivating Bogong Moth with Arsenic takes place during the larval stage and is agricultural is origin, either in location or product, has simply been stated as one of the more likely scenarios. At this stage the only fact that we know is that Arsenic is present in the adult aestivating Bogong Moth in the alpine regions.

Much more research is required to clarify how, when and where the adult aestivating Bogong Moth is contaminated with Arsenic (Heinze, Broome et al. 2004) .

SIGNIFICANCE AND INNOVATION

This research addresses an important problem

The presence of Arsenic in the Australian alpine environment is a problem. It is a problem because we know that its presence could threaten these ecosystems, and the consumption of Bogong Moths by insectivorous mammals, including the rare and endangered Mountain Pygmy Possum, may result in the accumulation of Arsenic in the alpine food web and may threaten these populations. But, another reason the presence of Arsenic in the Australian alpine environment is a problem is because it raises more questions than it answers. Are Bogong moth populations declining? What species are showing signs of decline due to Arsenic contamination? At what Arsenic concentration does the alpine skink reach its lethal limit? How should Arsenic contamination be managed?

This research will answer one question about the presence of Arsenic in the alpine environment: What is the source of the Arsenic which contaminates the adult aestivating Bogong Moth?

Why the research is significant

This research is significant in two ways. Firstly, it is significant in terms of the practical application of its findings: to assist in the continued management of the Mountain Pygmy Possum. Secondly, it is significant in terms of acknowledging the interest that Indigenous people have in the conservation of the Bogong Moth: to assist in the conservation of an Indigenous Icon.

It has been recommended that identifying the source of the Arsenic contaminating the adult aestivating Bogong Moth could aid in the management of the Mountain Pygmy Possum (Heinze, Broome et al. 2004) . The Mountain Pygmy Possum is an endangered (Environment Protection and Biodiversity Act 1999) and threatened species (Victorian Flora and Fauna Guarantee Act 1988 and the New South Wales Threatened Species Conservation Act 1995).The conservation of the Mountain Pygmy Possum has been actively managed through retention of habitat, construction of a 'possum' corridor to link fragmented habitats, implementation of cat and fox control programs, and captive breeding (Heinze, Broome et al. 2004) . The Mountain Pygmy Possum includes Bogong Moths as a major part of its diet (Heinze, Broome et al. 2004) . It is the female Mountain Pygmy Possum and its young which are particularly reliant upon the adult aestivating Bogong Moths over spring and summer. The abundance of Bogong Moths allows the female Mountain Pygmy Possum to raise up to four young to independence and, furthermore, is believed to allow both adults and young to accumulate essential pre-hibernation body fat stores (Heinze, Broome et al. 2004) . If Arsenic contamination is affecting the survivability of the Mountain Pygmy Possum, identifying the source of the adult aestivating Bogong Moth contamination will enable strategies for its management.

Any research involving Bogong Moth would be of interest to local indigenous people. This project has included visits to Indigenous communities for the purpose of informing people about the project, its progress and the final results. Often researchers involve Indigenous people in research because they believe they are researching a problem which directly impacts on Indigenous people (e.g. education, welfare, social justice) or because Indigenous people are the subject of the research (e.g. anthropology, artistic expression). This research is innovative because Indigenous people will be involved simply because this is a project that Indigenous people would be interested in.

Historic records recorded the importance of Bogong Moths as a food source for indigenous peoples who visited the alpine regions (Flood 1980) . These visits also allowed different tribal groups and families to meet for corroborees, story telling, marriage arrangements, trade and settling disputes (Bennett 1834; Howitt 1904; Tindale 1974) . A physical reminder of the continued importance of the Bogong Moth and the traditions surrounding its annual migration into the alpine region is evidenced through the annual Ngan Girra Festival which is held at Mungabareena Reserve in Albury , New South Wales and was originally called the 'Bogong Moth Festival'. It also features in the logo of Mungabareena Aboriginal Corporation in Wodonga , Victoria and is seen as a cultural icon representing unity. It would also be reasonable to assume that the consumption of the aestivating adult Bogong Moth still occurs. A perusal of bushtucker books in any bookstore will provide details of how to collect and cook Bogong Moths (Isaacs 1996; Cherikoff 1997) , including recipes such as Bogong Moth with Popcorn (Pop-Moth), Moth Damper and Bogong Au Naturel (Cherikoff 1997) . The participation of Indigenous people in this research will ensure that Indigenous communities are kept involved and informed, will assist in dissemination of the final results and, will ultimately result in the protection of an Indigenous Icon.

APPROACH AND METHODOLOGY

Outline conceptual framework

This research is based on the concept that it may be possible to use the transformation products stored within the tissues of the Bogong Moth to identify the source of the Arsenic contamination.

There are three major modes of Arsenic biological transformations that can take place in the environment. These are the redox transformation between arsenate and arsenite (AsO 2), the reduction and methylation of Arsenic, and the biosynthesis of organoarsenic compounds such as Arsenobetaine (Waring, Maher et al. 2005) . It is these biotransformations that cycle Arsenic through the environment and into the food web. The most important inorganic, organic and biological forms of Arsenic in the environment (O'Day 2006) are inorganic Arsenic (Arsine, Arsenious Acid or Arsenite and Arsenic Acid or Arsenate), methylated Arsenic compounds (Methylarsine, Dimethylarsine, Trimethylarsine, Monomethylarsonous Acid, Dimethylarsinous Acid, Monomethylarsonic Acid, Tromethylarsine Oxide, Dimethylarsinic Acid, Tetramethylarsonium ion), organoarsenic compounds (Arsenocholine, Arsenobetaine, Roxarsone) and organoarsenic lipids. Transformation products that will be identified in this research will include these more important species of Arsenic. Other transformation products that may provide evidence of the origin of the Arsenic contamination include companion chemicals, or other contaminants present in the tissues of the Bogong Moth which may have been present with the contaminating Arsenic source.

Designs and methods

Sampling sites are located throughout Qld, NSW and Victoria. The distribution of the larvae is largely unknown, and as such, we will need to sample widely, to establish the present distribution. 30 lowland sites will be sampled for larvae and, later in its lifecycle, these same sites will be used to sample migrating moths. 30 highland sites will sampled for aestivating moths.

Sampling will be repeated once for each lifecycle to determine if the Arsenic is being excreted, stored and/or consistently transferred from one life stage to another.

Ten individuals will be collected from each site during each sampling trip. Samples of larval Bogong Moths will be collected by hand. Samples of migrating and aestivating Bogong Moths will be collected either by hand or using ultra violet moth lights (Common 1954; Green, Broome et al. 2001) and nets.

Samples will be stored in cryo-tubes and will be stored in liquid Nitrogen. Upon completion of a sample trip samples will then be stored at -80 oC in the La Trobe University laboratory.

Analysis of samples will follow the following order. Three samples from each lifecycle stage and each site will be analysed for total Arsenic. If no Arsenic is present then no further testing will be conducted. If Arsenic is present then a further three samples from the same lifecycle stage and site will be dissected into 5 body tissues (Fatty tissue, digestive tissue, respiratory tissues and exterior; skin or 'fur', wings). Each part will then be analysed for the presence of individual Arsenic species and companion chemicals.

Total Arsenic analysis and detection of companion chemicals will be conducted using an Ion Chromatograph available at La Trobe University, Wodonga.

Arsenic speciation can be conducted using a combination of synchrotron analysis (through x-ray absorption spectroscopy/x-ray fluorescence), and surface analysis (through x-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectrometry) available at La Trobe University, Bundoora Campus (pers. comm. Associate Professor Paul Pigram). If required Arsenic speciation is also available at the University of Canberra using inductively coupled plasma mass spectrometry (ICPMS) but only in the event that there are not too many samples as they are now operating close to full capacity (pers. comm. Professor William Maher).

Summary of relevant work

Over the last three years research has been conducted on Bogong Moths which concentrated on using genetic material from aestivating adult Bogong Moths and Bogong Moth larvae from larval grounds. The aim of the project was to identify a genetic match between aestivating adult Bogong Moths and Bogong Moth larvae to demonstrate if the Bogong Moth did selectively migrate to different aestivating sites. If successful this approach could also be used to refine the search for the source of the Arsenic contamination in Bogong Moths (Neil Murray pers.comm.)

E8-REFERENCES

Cherikoff, V. (1997). The Bushfood Handbook . Boronia Park, Australia, Bush Tucker Supply Australia Pty Ltd.

Common, I. F. B. (1954). "A study of the ecology of the adult Bogong Moth, Agrotis infusa (Boisd.) (Lepidoptera: Noctuidae), with special reference to its behaviour during migration and aestivation. ." Australian Journal of Zoology 2 : 223-263.

Costa, M. (2000). Trace Elements: Aluminium, Arsenic, Cadmium and Nickel. Environmental Toxicants: Human exposures and their health effects . M. Lippman. New York, John Wiley & Sons : 817-824.

Flood, J. M. (1980). The Moth Hunters . Canberra, Australian Institute of Aboriginal Studies.

Green, K., L. Broome, et al. (2001). "Long Distance Transport of Arsenic by Migrating Bogong Moths from Agricultural Lowlands to Mountain Ecosystems." The Victorian Naturalist 118 (4): 112-116.

Green, K. and W. Osbourne (1994). Wildlife of the Australian Snow-Country . Chatswood, Australia, Reed Books.

Heinze, D., L. Broome, et al. (2004). A review of the ecology and conservation of the mountain pygmy-possum Burramys parvus . The Biology of Australian Possums and Gliding Possums . R. Goldingay and S. Jackson, Surrey Beatty and Sons : 254-67.

Howitt, A. W. (1904). The native tribes of south-east Australia . London, Macmillan and Co.

Isaacs, J. (1996). A Companion Guide To Bush Food . Sydney, Australia, Lansdowne Publishing.

O'Day, P. A. (2006). "Chemistry and Mineralogy of Arsenic." Elements 2 (2): 77-83.

Shirley, A. B. and Rachel Carson Council (1992). Basic guide to pesticides: their characteristics and hazards . Washington, Taylor and Francis.

Smith, J., V,S, J. Jankowski, et al. (2003). "Vertical distribution of As(III) and As(V) in a coastal sandy aquifer: factors controlling the concentration and speciation of arsenic in the Stuarts Point groundwater system, northern New South Wales, Australia." Applied Geochemistry (18): 1479-1496.

Tindale, N. B. (1974). Aboriginal tribes of Australia . Los Angeles, University of California Press.

Waring, J., W. Maher, et al. (2005). "Occurence and Speciation of Arsenic in Common Australian Coastal Polychaete Species." Environmental Chemistry 2 (2): 108-118.

Current Doctor of Philosophy - Investigating the source of Arsenic contamination in the Bogong Moth and the impacts on the ecology of the Australian Alps. La Trobe University , Wodonga Campus

2004 Research Honours in Applied Science - The decomposition of fire-affected Eucalyptus viminalis leaves in a freshwater ecosystem. University of Canberra , A.C.T.

2001 Advanced Certificate - Conflict Management Training. Australian Institute of Public Safety

1997-1999 Bachelor of Science - Environmental Management and Ecology. La Trobe University , Wodonga Campus

1995 Advanced Certificate in Koori Community Management and Development . Wodonga College of T.A.F.E

1992 Certificate of Occupational Studies - Horticulture. Wodonga College of T.A.F.E.

Lawler, S., Brown, S., Edney, G., Howlett, S., and Love, P. (1998). Buffalo Sallee at the Back Wall: An Alpine Species Adapted to Fire and Drought. The Victorian Naturalist 115 (5), 201-205.

1995-1996 Action Researcher - Teenage Pregnancy in a Rural Area, an Action Research Project

'A' Grade, Full-bore competition rifle shooter with the Albury Rifle Club. Compete in local club competitions and travel to state Queen's competitions.

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Last Updated: October 20, 2011

Science, Technology and Engineering

School of Life Sciences

Department of Environmental Management & Ecology

Ms Pettina Love