Submitted Manuscript: Confidential 1 Title: Environmental DNA is one tool among many in the biosecurity toolbox Authors: C. L. Hewitt 1* , M.L. Campbell 2 , K Dafforn 3 , J Davis 2 , M. R. Deveney 4 , J. I. McDonald 5 , A. D. Miller 6 , C. D. H. Sherman 6 Affiliations: 5 1 School of Science, University of Waikato, Hamilton, New Zealand. 2 Harry Butler Institute, Murdoch University, Western Australia, Australia. 3 Macquarie University, New South Wales, Australia 4 South Australian Research and Development Institute, Aquatic Sciences, West Beach, South Australia, Australia. 10 5 Department of Primary Industries and Regional Development, Fisheries Division, Government of Western Australia, North Beach, Western Australia, Australia. 6 Deakin University, Geelong, School of Life and Environmental Sciences, Centre for Integrative Ecology, Victoria, Australia *Correspondence to: chad.hewitt@waikato.ac.nz. 15 Abstract: Pikitch (15 June, p. 1180) extolled the virtues of environmental DNA (eDNA) in her recent Perspective “A tool for finding rare marine species: Environmental DNA analysis shows promise for studying rare and elusive marine species.” We agree that eDNA can be transformative with significant benefits, however the challenge for this novel methodology is highly contextual particularly for regulatory contexts such as marine biosecurity. Unfortunately, 20 the simplistic presumption that eDNA provides more robust outcomes and can always be used as a substitute for physical sampling and taxonomic analyses is flawed for many systems, and could result in a perverse loss of capability and capacity where needed most. To paraphrase Pikitch, “eDNA should only complement, rather than replace, other research approaches.” One Sentence Summary: eDNA offers transformative opportunity for marine monitoring and 25 biodiversity discovery, however its application for marine biosecurity requires a complimentary approach with traditional tools. Main Text: In the Perspective “A tool for finding rare marine species: Environmental DNA analysis shows promise for studying rare and elusive marine species” Pikitch (15 June, p. 1180) extolled the 30 virtues of environmental DNA (eDNA), providing a compelling argument for its increasing application (1). We agree that eDNA can be transformative with significant benefits, particularly high sensitivity for detecting rare species (2) however the challenge for this novel methodology is highly contextual. Marine biosecurity applications of eDNA methods for early detection of new pest 35 incursion are increasing (2-3). However, pin-pointing exact locations and characterising spatial extent can be difficult in a marine context (4), and the detection of DNA from a “pest” does not indicate introduction or establishment. eDNA can persist in the environment for days and even