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In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. rsc.li/process-impacts Page 1 of 24 Environmental Science: Processes & Impacts 1 2 3 Environmental Impact Statement 4 5 6 7 To derive remediation targets and environmental quality guidelines, Species Sensitivity Distribution 8 (SSD) models require toxicity data from a minimum of eight species from at least four taxonomic 9 10 group. Prior to this study there was no toxicity data available for sensitive early life stages of native 11 subantarctic plants exposed to total petroleum hydrocarbons (TPH) from diesel fuel. The TPH 12 concentrations of contaminated soils required to inhibit germination and root and shoot growth in 13 early life stages was high, but due to the climate of subantarctic regions, the hydrocarbon Manuscript 14 15 concentrations at spill sites may persist over time, so these high concentrations remain 16 environmentally relevant. Therefore the data obtained here makes a significant contribution to the 17 SSD model currently being developed to guide remediation activities at fuel contaminated sites at 18 Macquarie Island and in subantarctic regions more generally. 19 20 21 Accepted 22 23 24 25 26 27 Impacts 28 29 & 30 31 32 33 34 35 36 37 38 Processes 39 40 41 42 43 44 45 Science: 46 47 48 49 50 51 52 53 54 55 56 57 Environmental 58 59 60 Environmental Science: Processes & Impacts Page 2 of 24 1 2 3 4 Impact of hydrocarbons from a diesel fuel on the germination and early 5 6 7 growth of subantarctic plants 8 9 10 11 12 13 † ‡ ‡ § 14 Gabriella K Macoustra , Catherine K King , Jane Wasley , Sharon A. Robinson and Manuscript 15 16 †* 17 Dianne F. Jolley 18 19 20 21 22 Accepted 23 24 † School of Chemistry, University of Wollongong, NSW 2522, Australia 25 26 27 ‡ Australian Antarcc Division, Antarcc Conservaon and Management Program, Kingston, 28 29 Tasmania, Australia Impacts 30 31 & φ 32 School of Biological Sciences, University of Wollongong, NSW 2522, Australia 33 34 35 36 37 38 39 Corresponding author email: [email protected], tel: +61 2 4221 3516 40 Processes 41 42 43 44 45 46 47 48 Science: 49 50 51 52 53 54 55 56 57 58 59 60 Environmental 1 Page 3 of 24 Environmental Science: Processes & Impacts 1 2 3 4 ABSTRACT 5 6 Special Antarctic Blend (SAB) is a diesel fuel dominated by aliphatic hydrocarbons 7 8 that is commonly used in Antarctic and subantarctic regions. The past and present use of 9 10 SAB fuel at Australia’s scientific research stations has resulted in multiple spills, 11 contaminating soils in these pristine areas. Despite this, no soil quality guidelines or 12 13 remediation targets have been developed for the region, primarily due to the lack of 14 Manuscript 15 established indigenous test species and subsequent biological effects data. In this study, 16 17 twelve plant species native to subantarctic regions were collected from Macquarie Island and 18 evaluated to determine their suitably for use in laboratory-based toxicity testing, using 19 20 germination success and seedling growth (shoot and root length) as end points. Two soil 21 22 types (low and high organic carbon (OC)) were investigated to reflect the variable OC Accepted 23 24 content found in soils on Macquarie Island. These soils were spiked with SAB fuel and aged 25 for 14 d to generate a concentration series of SAB-contaminated soils. Exposure doses were 26 27 quantified as the concentration of total petroleum hydrocarbons (TPH, nC9-nC18) on a soil 28 29 dry mass basis. Seven species successfully germinated on control soils under laboratory Impacts 30 31 conditions, and four of these species (Colobanthus muscoides Hook.f., Deschampsia & 32 33 chapmanii, Epilobium pendunculare A.Cunn. and Luzula crinita Hook.f.) showed a dose- 34 dependent inhibition of germination when exposed to SAB contaminated soils. 35 36 Contaminated soils with low OC were generally more toxic to plants than high organic 37 38 carbon soils. Increasing soil-TPH concentrations significantly inhibited shoot and root 39 growth, and root length was identified as the most sensitive endpoint. Although the test 40 Processes 41 species were tolerant to SAB contaminated soils in germination assays, development of early 42 43 life stages (up to 28 d) were generally more sensitive indicator of exposure effects, and may 44 45 be more useful endpoints for future testing. 46 47 48 Science: 49 50 51 52 Keywords: total petroleum hydrocarbons, total organic carbon, monocots, dicots, 53 54 germination, root and shoot growth. 55 56 57 58 59 60 Environmental 2 Environmental Science: Processes & Impacts Page 4 of 24 1 2 3 4 INTRODUCTION 5 One of the greatest challenges in protecting Antarctic and subantarctic regions is the 6 7 effective management of the widespread and damaging effects of petroleum hydrocarbons 8 9 arising from fuel spills [1-3]. Investigations of hydrocarbon contamination and degradation 10 11 under polar conditions commenced more than 30 years ago, and have mostly been limited to 12 the Arctic region [4, 5]. However, Snape and colleagues [6] estimated up to 10 million cubic 13 14 meters of hydrocarbon contaminated soils in the Antarctic, with concentrations up to 59,000 Manuscript 15 16 mg/kg hydrocarbons reported in soils at Casey’s Station [7]. In contrast to the relatively 17 18 rapid recovery of oil spills in tropical climates [8, 9], cold climates have a number of 19 20 characteristics that reduce the natural attenuation of petroleum hydrocarbons to negligible 21 rates. Low temperatures, intrinsic nutrient limitations, and the anaerobic nature of soils 22 Accepted 23 caused by water saturation, combine to significantly inhibit the metabolic activity of 24 25 hydrocarbon-degrading microbes; low temperatures will also reduce rates of hydrocarbon 26 27 volatilisation and evaporation [1, 3, 10]. 28 While numerous fuel spills have occurred in Antarctic and subantarctic regions there 29 Impacts 30 have been relatively few attempts to remediate contaminated sites [2]. Remediation to 31 & 32 pristine levels in Antarctica and subantarctic regions is prohibitively difficult and costly. The 33 34 negative environmental impacts and high costs associated with excavating and transporting 35 contaminated soils for off-site processing and treatment have led the Australian Antarctic 36 37 Division (AAD) to abandon dig-and-haul methods in favour of implementing alternative on- 38 39 site in situ remediation techniques [2, 3]. The in situ techniques utilised on subantarctic 40 Processes 41 Macquarie Island include injection of nutrients and aeration into the soils, both of which 42 43 promote natural attenuation by encouraging hydrocarbon-degrading microbes [3, 10-14]. 44 In order to set practical remediation targets for fuels, a comprehensive understanding 45 46 of the availability of petroleum hydrocarbons in fuel-contaminated soils is required. 47 48 Contaminant availability may be evaluated by surrogate chemical approaches, however, Science: 49 50 biological assays more clearly represent the potential risk contaminants pose to their local 51 environment. Plant-based bioassays are well established, with standard protocols developed 52 53 for temperate climates and plant species by the Organisation for Economic Co-operation and 54 55 Development [15] and International Organization for Standardization [16, 17]. These 56 57 protocols have subsequently been adapted for various Arctic and cold-climate species [18], 58 59 however, there is still limited information available on the effects of hydrocarbons on cold- 60 climate plants, and no established methods to assess hydrocarbon effects on native Environmental 3 Page 5 of 24 Environmental Science: Processes & Impacts 1 2 3 subantarctic plants. Consequently, there is insufficient evidence to establish robust 4 5 remediation guidelines, and site-specific testing is required for an accurate assessment of the 6 7 biological effects of fuel-contaminated soils in Antarctica and subantarctic regions. 8 9 For comprehensive site remediation guidelines to be established, contemporary best 10 11 practices recommend that toxicity data be obtained for a minimum of eight species across at 12 least four taxonomic groups that reflect the range of those present in the natural community 13 14 [19, 20]. This study is focused on subantarctic Macquarie Island, for which there is data for Manuscript 15 16 biological responses to total petroleum hydrocarbons (TPH) exposure for earthworms [21], 17 18 soil microbes [13, 14] and the grass Poa foliosa [22]. This is insufficient information to 19 enable guideline development to assess the risk of fuel-contaminated soils on native 20 21 subantarctic species. This study evaluated seeds from twelve native plant species for 22 Accepted 23 germination success on control soils under laboratory conditions. Germinating species were 24 25 then assessed in a 28-d bioassay using a concentration series of soils contaminated with 26 27 Special Antarctic Blend (SAB) fuel.