Environmental Science Processes & Impacts Accepted Manuscript

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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

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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

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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

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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. To account for the variable organic carbon (OC) content 28 in subantarctic soils, parallel bioassays were performed using both low and high OC soils. 29 Impacts 30 Effects were measured using germination rates, and seedling growth (root and shoot length),

31 & 32 reported against mean soil TPH concentration. 33 34 35 METHODS 36 37 General procedures. Glassware used for bioassays and hydrocarbon extraction and analyses 38 39 were new or cleaned before use by washing with phosphate free detergent and rinsing with

40 Processes 41 acetone, dichloromethane (DCM; Suprasolv, Merck) and Milli-Q deionised water (18 42 43 M/cm; Merck Millipore). 44 45 46 Soil preparation and characterisation. Artificial soils were used to provide a standardised 47 48 soil matrix. Two soil matrices were prepared: the low OC soil consisted of (w/w) 80% Science: 49 50 propagating sand (sieved to <1 mm) and 20% kaolin clay (<40 µm); and the high OC soil had 51 52 (w/w) 70% propagating sand, 20% kaolin clay, and 10% Sphagnum moss (sieved to <2 mm). 53 Test soils were characterised for physical and chemical properties (Table 1). Soil pH 54 55 was measured using a combined pH-mV-temperature meter (TPG Pty Ltd) calibrated as per 56 57 manufacturer’s instructions. Soil moisture was determined after heating to 110°C for 24 h, 58 59 and dry bulk density and porosity determined after drying for 48 h at 60°C [23]. Particle size 60 distribution was determined using a Mastersizer 2000 particle size analyser (Malvern Environmental

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1 2 3 Instruments Ltd, Worcestershire, United Kingdom), calculated using Mie and Fraunhofer 4 5 scattering. Total organic carbon (TOC) was determined using the loss on ignition (LOI) 6 7 method [24], where soils (<2 g) were heated (105°C, 24 h), combined with HCl, heated 8 9 (450°C, 24 h), and mass loss determined. Total Kjeldahl nitrogen (TKN) was determined 10 11 using macro-Kjeldahl digestion (4500-Norg B.) and titrimetric (4500-NH3 C.) methods 12 adapted from Eaton et al. [25]. Total available phosphorous was determined following 13

14 persulphate digestion (4500-P B.5) and ascorbic colorimetric detection (4500-P E.) as per Manuscript 15 16 Eaton et al. [25]. 17 18 Soils were spiked with fuel at nominal concentrations of 20,000 and 40,000 19 mg SAB/kg soil (dry mass) in amber Winchester bottles. Bottles were immediately capped 20 21 and homogenised (20±1°C, 24 h, dark) using a mechanical tumbler (Environmental 22 Accepted 23 Express®). Spiked and control soils were aged in uncapped bottles (15±1°C, 14 d, ventilated, 24 25 dark) to allow the most volatile hydrocarbons to evaporate, reflecting a more ecologically 26 27 relevant soil contamination composition. Aged soils were diluted with control soils to 28 produce a concentration series between 1,250 and 40,000 mg SAB/kg soil, and toxicity 29 Impacts 30 testing was commenced within 48 h. Soil subsamples for TPH analysis were collected on Day

31 & 32 0 and Day 28 (test commencement and termination) and stored at -20°C (dark) until analysed. 33 34 35 Total petroleum hydrocarbon (TPH) analysis. Subsamples of soils (12 g) were extracted with 36 37 1:1 Milli-Q:hexane for TPH analysis (n-C9 to n-C18), measured by gas chromatography with 38 39 flame ionisation detection (GC-FID, Agilent GC 6890N, SGE BP1 column (35 m x 0.22 mm

40 Processes 41 ID, 0.25 m film thickness)). Carrier gas (helium) velocity at the injector was 23.9 mL/min, 42 43 and on column was 1.3 mL/min for 17 min increased to 3.0 mL/min for 7 min. Samples (3 44 L) were injected with a split ratio of 15:1 (30 psi) at 310°C. The samples were cross 45 46 calibrated with an in-house SAB fuel internal standard composed of 250, 50, 50 and 250 47 48 g/mL of bromoeicosane, p-terphenyl, tetracosane and cyclooctane, respectively. Oven Science: 49 50 temperature was maintained at 50°C for several minutes and then ramped to 320°C at 51 52 18°C/min, with the FID heated to 330°C. Combined areas under resolved peaks and the 53 unresolved complex mixture (UCM) were integrated relative to internal standards. For 54 55 quality assurance/quality control (QA/QC) purposes, method blanks, duplicates and spike 56 57 recoveries were performed on at least 10% of samples. All blanks were below the limit of 58 59 detection (≤ 20 mg/kg). Spiked recoveries (equivalent to 5,000 mg SAB /kg for 10 g soil,

60 Environmental extracted into 10 mL hexane) were between 95-113% (mean ± SD, 103 ± 6%) and the mean

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1 2 3 Table 1. Physico-chemical properties of test soils in comparison to reported values for 4 5 standardised organic carbon soils [18] (mean  SE, n≥3) 6 7 Soil properties Low organic High organic Standardised 8 carbon soil carbon soil organic carbon 9 soil [18] 10 11 Particle size distribution (%) 12 Sand, 63 - 1000 µm 85.1±0.7 66±3 77.3 13 Silt, 4.0 - < 63 µm 7.5±0.3 3.3±0.4 7.8

14 Clay, < 4 µm 7.4±0.4 15±2 14.9 Manuscript 15 16 17 Added Organic matter Not added 15.6±0.7 9 18 (Sphagnum moss) 1- 2 mm 19 20 Moisture - storage (%) 6.0±0.1 18.6±0.5 ~20 21 Moisture - testing (%) ~70-80 ~70-80 ~70-80 22 3 Accepted 23 Wet bulk density (g/cm ) 1.23±0.03 0.24±0.02 0.98 24 Water-holding capacity (%) 35±11 171±14 71.5 25 Porosity (%) 54±1 90.8±0.6 - 26 27 pH 6.5±0.5 6.5±0.5 6 28 Total organic carbon (%) 2.3±0.6 6.5±0.6 4.46 29 Impacts 30 Phosphorous (mg/kg) 9.1±0.9 30±5 23

31 Kjeldahl Nitrogen (mg/kg) 54.2 345 500 & 32 33 34 35 36 relative standard deviations (RSDs) of site and method duplicates were typically within 20- 37 30%. The certified reference material analysed (CRM-560, Diesel Soil 4, 448-522 mg/kg 38 39 nC12-nC28) were within the predicted intervals for the nC9-nC18 component. Analysis of

40 Processes 41 control samples detected no TPH compounds within the nC9-nC18 and associated UCM 42 43 hydrocarbon range, therefore no interference from the components of the soil matrix was 44 observed in the spiked samples. 45 46 47 48 Test species, bioassay conditions, and seed viability bioassay. Seeds from twelve terrestrial Science: 49 50 plant species (detailed in Table 2) were collected from Macquarie Island in January-February 51 52 2013. Most of the twelve species occur throughout the subantarctic region, including New 53 Zealand’s subantarctic islands, with two of the species also occurring on Australia’s Heard 54 55 and McDonald Islands. A description and history of the seed collection site are provided in 56 57 the supplementary information. Seeds from each species were stored separately in paper bags 58 59 under ambient laboratory conditions until returned to Australia where they were kept at

60 2±2°C in sealed plastic bags containing silica beads (Silica Gel Australia) to reduce moisture Environmental

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1 2 3 and prevent fungal infestation. Mature, healthy seeds were selected for testing based on seed 4 5 size and colour. To assess seed viability and suitability for laboratory-based bioassays, 20 6 7 seeds of each of the twelve species were planted on unspiked (control) high organic soils 8 9 (n=3) and were observed daily using microscopy (Dino-Lite digital microscope and 10 11 accompanying DinoXcope software) for up to 28 d. Germination was defined as the rupture 12 of the testa (seed coat) by the epicotyl. 13

14 All seed germination bioassays were conducted in Normax glass petri dishes Manuscript 15 16 (diameter 90 mm). Each dish contained one soil type at a single test concentration and was 17 18 divided into quadrants with twenty seeds of a single species evenly planted in each quadrant. 19 Petri dishes spiked with the same SAB concentration were sealed in plastic containers 20 21 (Sistema©, 5 L, 21.0 x 24.2 x 10.5 cm). A systemic fungicide (Garden King Fongarid®) was 22 Accepted 23 used as a preventative and treatment for fungal infections. Seeds were pre-soaked in 24 25 fungicide for 20–30 min and if fungus appeared during testing, the test units were sprayed 26 27 with fungicide. Seeds were incubated in plastic containers at 15±1ºC which is the maximum 28 average daily temperature in surface soils at Macquarie Island on sunny summer days, 29 Impacts 30 reflecting field-realistic and optimal temperatures for minimising germination time and

31 & 32 maximising germination success. Maximum light intensity inside the plastic containers was 33 2 34 55 µmol/m /s. Observations of seeds throughout bioassays were performed using a Dino-Lite 35 digital microscope and accompanying DinoXcope software. 36 37 38 39 Exposure bioassays. To determine a suitable dose–response concentration range of SAB for

40 Processes 41 each species, a 28-d rangefinder bioassay was performed. The seven species that germinated 42 on at least one of the soil types in the viability bioassay underwent further testing on SAB 43 44 contaminated soils. Twenty seeds from each species were tested on a soil concentration 45 46 series (1,250 to 40,000 mg SAB/kg) on both the low and high OC soils. 47 48 The four species that successfully germinated during the rangefinder assay were Science: 49 50 subsequently tested in a definitive toxicity test. In definitive bioassays, twenty seeds were 51 used per test, and tests were performed in triplicate over 28 d on the low and high OC soils. 52 53 Germination was recorded daily, and at test completion, early plant growth (root and shoot 54 55 lengths) was measured using the program ImageJ (public domain Java image processing). 56 57 58 59

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1 2 3 Table 2. Native subantarctic plant species investigated to determine the sensitivity of seeds to 4 5 SAB contaminated soils. Description is adapted from George et al. [27]. 6 7 Species Family Description Distribution 8 9 Monocots 10 11 Agrostis magellanica Poaceae Tufted grass Circumpolar. Wide range of 12 Lam. subantarctic islands, New Zealand 13 and South America

14 Manuscript 15 Deschampsia Poaceae Tufted grass Macquarie Is., New Zealand’s 16 chapmanii Petrie subantarctic islands, plus New 17 Zealand 18 19 Festuca contracta Poaceae Tufted grass Wide range of subantarctic islands 20 Kirk and South America 21 Luzula crinita Juncaceae Tufted grass-like Macquarie Is. and New Zealand’s 22 Hook.f. herb subantarctic islands Accepted 23 24 Dicots 25 26 Acaena minor Rosaceae Prostrate herb Macquarie Is. and New Zealand’s 27 (Hook.f.) Allan subantarctic islands 28 29 Impacts 30 Cardamine Brassicaceae Low herb Macquarie Is. and New Zealand’s 31 corymbosa Hook.f. subantarctic islands & 32 33 Colobanthus Caryophyllaceae Cushion-forming Macquarie Is., plus New Zealand’s 34 muscoides Hook.f.* herb subantarctic and Chatham islands 35 Colobanthus sp. Caryophyllaceae Cushion-forming Macquarie Is. and New Zealand. 36 (C. affinis (Hook.) herb 37 C. affinis: also Australia Hook.f. or C. 38 apetalus var. alpinus C. apetalus var. alpinus: also New 39 (Kirk) L.B.Moore Zealand’s subantarctic islands

40 Processes 41 Epilobium Onagraceae Mat-forming herb Macquarie Is., New Zealand’s 42 brunnescens subantarctic islands and New

43 (Cockayne) Zealand 44 P.H.Raven & 45 Engelhorn subsp. 46 brunnescens 47 48 Epilobium Onagraceae Mat-forming herb Macquarie Is., New Zealand Science: 49 pendunculare subantarctic islands and New 50 A.Cunn. Zealand 51 Montia fontana L. Portulacaceae Mat-forming, herb Almost cosmopolitan in cool 52 subsp. fontana * regions. Macquarie Is., Heard Is., 53 New Zealand’s subantarctic islands, 54 55 New Zealand, and Australia 56 (Tasmania) 57 Pleurophyllum Asteraceae Large rosette- Macquarie Is. and New Zealand’s 58 hookeri Buchan. forming herb subantarctic islands 59 * These species were observed growing in areas of known fuel spills at time of seed collection 60 Environmental

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1 2 3 Data analysis. All data represents the actual soil-TPH concentrations that were not 4 5 normalised to soil-TOC. Two-tailed t-tests were used to identify differences in soil TPH over 6 7 time, at 0.05 significance levels. Dose-response curves were constructed and point estimates 8 9 calculated by linear interpolation using ToxCalc for Microsoft Excel (Version 5.0.23, 10 11 TidePool Scientific Software, California, 1994). Data was tested for normality using the 12 Shapiro-Wilk test, and for homogeneity of variance using Bartlett’s Test. Point estimates 13

14 including the concentrations at which 50% of the population response was inhibited (IC50) Manuscript 15 16 were determined using Dunnett’s Multiple Comparison Test for parametric data or Wilcoxon 17 18 Rank Sum test with Bonferroni correction for non-parametric data. In cases where the IC50 19 was calculated to be greater than the highest treatment concentration, confidence intervals 20 21 were not calculated. No observed effective concentrations (NOEC) and lowest observable 22 Accepted 23 effective concentrations (LOEC) values were determined using Steel’s Many-One Rank test. 24 25 Equality of variance and normality were tested at 0.01 and hypothesis tests and control 26 27 comparisons were tested at 0.05 significance levels. 28 29 Impacts 30 RESULTS

31 & 32 Soil characteristics. The low OC soil was marginally sandier than the high OC soil (62.5 µm 33 34 - 1 mm at 85.1±0.7 and 66±2%, respectively), and the high OC soil contained added organic 35 matter (15.6±0.7% in 1-2 mm particle range). Therefore the high OC soil had almost three 36 37 times more TOC than the low OC soil (6.5±0.6 and 2.3±0.6%, respectively), which increased 38 39 the soil’s water-holding capacity by almost five times (171±14 and 35±11%, respectively).

40 Processes 41 The high OC content also produced a higher porosity, a lower wet-bulk density, and total 42 43 phosphorus and total Kjeldahl nitrogen concentrations that were over three and four times 44 higher, respectively, than in the low OC soil. Therefore the Sphagnum moss contributed 45 46 substantial nutrients to the high OC soil (Table 1). 47 48 Science: 49 50 Total petroleum hydrocarbons. Both the low and high OC control soils contained <20 mg 51 TPH/kg (n ≥ 8), and thus had a minimal contribution of hydrocarbons to the total TPH 52 53 measured in test soils. For all test soils, there was a significant decrease (p < 0.05) in TPH 54 55 concentrations over the 28-d test period (Table 3). Greater losses occurred in the high OC 56 57 soils, with average 28-d losses of 70 and 38% for TPH from the high and low OC soils, 58 59 respectively. Soils dosed with lower fuel concentrations also lost a larger percentage of their

60 initial TPH over time than did soils at higher concentrations. For example, initial TPH Environmental

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1 2 3 concentrations decreased in high OC soils by 91% for treatments starting with ≤ 2,500 ± 600 4 5 mg TPH/kg and by 48% for soils ≥ 3,000 ± 750 mg TPH/kg. Time-averaged mean exposure 6 7 concentrations for each treatment were used in all statistical analyses and data interpretations. 8 9 10 11 Germination success on control soils (seed viability). Seven of the twelve species examined 12 were able to germinate on control soils and were identified as potentially suitable for 13

14 laboratory-based bioassays. These were C. corymbosa, C. muscoides, Colobanthus sp., D. Manuscript 15 16 chapmanii, E. pendunculare, L. crinita, and M. fontana (Figure 1). Germination on the high 17 18 OC soil was generally more successful than on low OC soil, particularly for C. muscoides in 19 which 80% of the seeds germinated within 21 d on high OC soil, compared to 5% of seeds on 20 21 low OC soil. In the low OC soil, only four species germinated (Figure 1); L crinita and E. 22 Accepted 23 pendunculare were most successful, with 55% and 40% of the seeds germinating, 24 25 respectively. Other species were less successful with between 10-30% of the seeds 26 27 germinating on either soil. 28 29 Impacts 30

31 & 32 Table 3. Nominal-SAB exposure doses and measured total petroleum hydrocarbon (TPH) 33 concentrations in aged soils at the start and termination of the subantarctic plant germination 34 bioassays (Days 0 and 28), and as the mean time-averaged exposure concentration (mean ± 35 SD, n=3). Percentage TPH losses from soils over exposure period are also presented. 36 37 Nominal-SAB TPH in soil (mg/kg) § % Loss of . 38 fuel spike Day 0 Day 28 Time averaged Total Loss TPH over 39 (mg/kg) exposure (D0-D28) 28 d

40 Processes 41 High organic carbon soil 42 1,250 190 ± 50 0 ± 25 100 ± 20 190 ± 50 100 ± 25 43 2,500 1,100 ± 260 230 ± 60 640 ± 160 800 ± 200 78 ± 20 44 5,000 2,500 ± 600 100 ± 20 1,300 ± 300 2,400 ± 600 96 ± 24 45 46 10,000 3,000 ± 750 1,300 ± 300 2,200 ± 540 1,700 ± 400 56 ± 14 47 20,000 7,100 ± 1,800 3,800 ± 900 5,400 ± 1,400 3,300 ± 800 47 ± 12 48 40,000 13,000 ± 3,000 7,600 ± 1,900 10,000 ± 2,600 5,600 ± 1,400 42 ± 11 Science: 49 Low organic carbon soil 50 1,250 400 ± 100 80 ± 20 240 ± 60 320 ± 80 80 ± 20 51 52 2,500 1,200 ± 300 1,200 ± 300 1,200 ± 300 * * 53 5,000 2,600 ± 650 2,200 ± 550 2,400 ± 600 400 ± 100 16 ± 4 54 10,000 8,500 ± 2,100 6,000 ± 1,500 7,300 ± 1,800 2,500 ± 600 30 ± 8 55 20,000 16,000 ± 4,000 11,000 ± 2,700 13,000 ± 3,300 5,300 ± 1,300 33 ± 8 56 40,000 35,000 ± 9,000 25,000 ± 6,000 30,000 ± 7,400 10,500 ± 2,600 30 ± 8 57 58 * Measurement error occurred 59 § High and low organic carbon control soils had <20 mg TPH/kg

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1 2 3 Germination response to contaminated soils. Rangefinder bioassays showed that SAB- 4 5 contaminated soils reduced germination success, and the degree of inhibition varied across 6 7 species and soil types. Four species (C. muscoides, D. chapmanii, E. pendunculare, and L. 8 9 crinita) showed dose-dependent responses to fuel-contaminated soils at concentrations up to 10 11 10,000 and 30,000 mg SAB/kg for high and low OC soils, respectively, and as such these 12 concentrations and species were used in the definitive bioassay. Again there was greater 13

14 germination success on the high OC fuel contaminated soils, which is consistent with the Manuscript 15 16 trend observed in seed viability assays. However, a clearer and less variable dose-dependent 17 18 response was observed on the low OC soils (Supplementary Information Figure S3). Three 19 species showed no or insignificant germination in SAB contaminated soils across the entire 20 21 concentration series investigated; C. corymbosa, Colobanthus sp. and M. fontana. 22 Accepted 23 24 25 Definitive toxicity tests. Definitive bioassays were performed in triplicate for 28 d in a 26 27 concentration series of soils ranging from 0-10,000 and 0-30,000 mg SAB/kg in high and low 28 OC soils, respectively. In these bioassays the suitability of germination and early-life growth 29 Impacts 30 (root and shoot length) as toxicity endpoints were evaluated. The contaminated soils had a

31 & 32 pronounced inhibitory effect on the germination and subsequent growth of early-life stages, 33 34 with the degree of inhibition varying with species, endpoints and soil types (Figure 2, Table 35 4). Germination success was the least variable endpoint, with most species having the same 36 37 or similar IC50 values (~5,000 and >10,400 mg TPH/kg for low and high OC soils, 38 39 respectively). The exception to this was E. pendunculare, which was particularly tolerant,

40 Processes 41 with an IC50 of 28,000 mg TPH/kg on low OC soil. Germination was generally less sensitive 42 to SAB fuel exposure on the high OC soils, again with the exception of E. pendunculare. 43 44 For the early life stages, the response to contaminated soils in terms of root growth 45 46 was similar between species. Root growth was inhibited by 50% in all species at 47 48 concentrations >2,300 mg TPH/kg, except for L. crinita on low OC soils and E. Science: 49 50 pendunculare on high OC soils (both with estimated IC50 of 190 mg TPH/kg, which was 51 below the lowest tested exposure concentration of 240 mg TPH/kg). The effect of soil OC 52 53 content on the IC50 root growth in contaminated soils was not consistent across the species, 54 55 with the high OC soils resulting in less tolerance to fuel in E. pendunculare (190 mg 56 57 TPH/kg), but more tolerance in C. muscoides and L. crinita (5,400 and 4,100 mg TPH/kg, 58 respectively). Soil type did not affect the toxicity of SAB contaminated soils on root growth 59

60 Environmental of D. chapmanii (IC50 of ~2,300 mg TPH/kg).

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1 2 3 4 5 6 7 8 9 10 11 12 13

14 Manuscript 15 16 17 18 19 20 21 22 Accepted 23 Figure 1. Germination success of native subantarctic plants seeds in low and high organic 24 carbon control soils after a minimum of 21 d (mean ± SD, n=3; 20 seeds per replicate). Data 25 26 not presented for non-germinating plant species (A. magellanica, A. minor, E. brunnescens, 27 F. contracta and P. hookeri). 28 29

Impacts 30 a 31 & 32

33 34 35 36 37 38 39 a

40 Processes 41 a a a a 42 43 a 44 45 a a a b 46 b 47 48 Science: 49 50 51 52 53 54 55 56 Figure 2. TPH-IC50 values generated in toxicity tests with subantarctic plants. Tests were 57 performed using two soil types (high and low organic carbon soils) for germination success 58 and subsequent early-life stage growth (root and shoot length). Mean ± standard deviation, n 59 ≥ 3. Where a indicates that the IC estimate is greater than highest test concentration, and 60 50 Environmental b that the IC50 estimate is less than the lowest test concentration.

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1 2 3 Shoot length was the most variable endpoint with the four test species dividing into 4 5 two groups: D. chapmanii and L. crinita were significantly more sensitive to SAB 6 7 contaminated soils than C. muscoides and E. pendunculare. The level of total OC in soils did 8 9 not affect IC50 values for D. chapmanii, L. crinita, and C. muscoides with both low and high 10 11 OC soil matrices having similar responses (570-1,200 and 1,300-2,300 mg TPH/kg, 12 respectively, Figure 2, Table 4). Interestingly for shoot length the high OC content in soils 13

14 enhanced the effect of SAB contaminated soils, with all species having lower IC50 values in Manuscript 15 16 the high OC assays. This interaction was most pronounced with E. pendunculare, as it was 17 18 more sensitive to all three endpoints on the high OC soils (germination success, root length 19 and shoot length, Figure 2). 20 21 22 Accepted 23 Discussion 24 25 Soil characteristics. Organic carbon content in the soil had a significant impact on 26 27 the toxicity of the SAB contaminated soil to the plant species tested, with clear differences 28 between the low and high OC soil types across all three endpoints (Figure 2). Arguably two 29 Impacts 30 of the most important factors in determining the toxicity of petroleum hydrocarbons in soil

31 & 32 are the level of organic matter in the soil and the duration or age of contamination. The 33 34 ameliorating effect of OC on hydrocarbon bioavailability is well established, with 35 hydrocarbon content generally normalised to the OC concentration in sediment toxicity 36 37 assessments [28]. Lowered hydrocarbon bioavailability is attributed to increased 38 39 hydrocarbon sequestration onto organic matter and therefore, decreased bioavailability,

40 Processes 41 which is consistent with equilibrium partitioning for neutral organics based on sorption 42 coefficients [29-32]. 43 44 A significant effect of higher OC content was the greater losses of TPH from soils 45 46 that occurred during the bioassay, which is consistent with previous studies [12, 22, 33]. 47 48 Hydrocarbons are volatile compounds that chemically transform and degrade at different Science: 49 50 rates depending on the physicochemical characteristics of the soil and environmental factors 51 such as light intensity, temperature, oxygen availability and microbial activity. The oil spill 52 53 itself can increase soil temperatures by up to 10°C due to decreased soil surface albedo [49], 54 55 which in turn increases rates of hydrocarbon volatilisation. In addition, petroleum 56 57 hydrocarbons undergo complex physical and chemical interactions with soils, strongly 58 sorbing to OC, making it less available for biological uptake [4]. Organic matter provides 59

60 more nutrients in soils (Table 1) that will facilitate not only seedling growth, but also higher Environmental microbial activity, and this microbial activity will contribute to the higher TPH losses in high 13

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1 2 3 Table 4. 28-d IC values for germination success, root and shoot length of seedlings of 4 50 5 subantarctic plants exposed to Special Antarctic Blend fuel. Fuel concentration measured as 6 total petroleum hydrocarbon (TPH, mg/kg). *Where standard deviation was not calculated by 7 ToxCalc. ^Concentrations lower than lowest test concentration. 8 9 Endpoint Species Organic IC50 (mg TOC normalised 10 carbon soil TPH/kg) IC (mg 11 50 12 type TPH/kg) 13 Germination Deschampsia chapmanii High >10,400 >1,600

14 (%) Low 4,800±800 2,090±350 Manuscript 15 Luzula crinita High >10,400 >1,600 16 17 Low 4,700±700 2,040±300 18 Colobanthus muscoides High >10,400 >1,600 19 Low 5,400±1,400 2,350±610 20 Epilobium pendunculare High >10,400 >1,600 21 Low* 28,000 12,200 22 Accepted 23 Root length Deschampsia chapmanii High 2,300±960 350±150 24 (mm) Low >2,400 >1,050 25 Luzula crinita High 4,100±970 630±160 26 Low^ 190±10 80±4 27 28 Colobanthus muscoides High >5,400 >830 29 Low >2,400 >1,040 Impacts 30 Epilobium pendunculare High 190±40 80±20

31 Low >2,400 >1,040 & 32 33 Shoot Deschampsia chapmanii High 1,230±130 190±20 34 length (mm) Low^ 2,300±780 1,000±340 35 Luzula crinita High 570±140 88±20 36 Low 1,800±1,400 780±600 37 Colobanthus muscoides High 7900±50 1,600±10 38 39 Low >13,300 >5,800

40 Epilobium pendunculare High* 9,060±60 1,390±10 Processes 41 Low >30,000 >13,000 42 43 44 45 46 47 48 Science: 49 50 51 52 53 54 55 56 57 58 59

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1 2 3 organic carbon soils. A dose-dependent increase in microbial respiration (IC of 350 mg 4 20 5 fuel/kg) has been reported in fuel contaminated soils up to 50,000 mg SAB/kg in the presence 6 7 of sufficient nitrogen [14], which illustrates that the additional nutrients from the organic 8 9 matter (particularly TKN) in the high OC soil may be important in facilitating the microbial 10 11 activity and degradation of the fuel. 12 During hydrocarbon exposures, acute toxicity is likely caused by lighter, more 13

14 volatile hydrocarbons with a lower equivalent carbon number; heavy fractions with a high Manuscript 15 16 equivalent carbon number are more likely to cause chronic toxic effects [34-37]. Toxicity of 17 18 petroleum hydrocarbons is strongly correlated with hydrocarbon fractions that have lower 19 boiling points and octanol-water coefficients [log(K )], especially those within the nC10– 20 ow 21 nC19 range [12, 37-38]. Differences in toxicity between hydrocarbon fractions has been 22 Accepted 23 addressed by Environment Canada, and with some exceptions, hydrocarbons have been split 24 25 into four specific fractions (F1-F4) as the basis in their tiered risk assessment framework (F1 26 27 nC6–nC10; F2 nC11–nC16; F3 nC17–nC34; F4; ≥nC35) [39]. In terms of their toxicity, the 28 fractions may be ranked in the following order F1>F2>F3>F4 which is consistent with the 29 Impacts 30 hypothesis that toxicity decreases with increasing equivalent carbon number. Due to the rapid

31 & 32 volatilisation of nC6–nC10 hydrocarbons (F1) [35], hydrocarbons with an equivalent carbon 33 34 number between nC11–nC16 (F2) represent the fraction with the greatest toxicological effect 35 [12]. This is significant in the context of SAB fuel as it is primarily composed of nC9–nC14 36 37 hydrocarbons, a range that encompasses some of the most toxic components [2]. 38 39

40 Processes 41 The effect of SAB on seed germination and seedling growth in subantarctic plants. Plant 42 responses to contaminated soils are influenced by both the physiological characteristics of the 43 44 plant species and the physical and chemical parameters of the soil [4]. Thus assessing the 45 46 toxicity of fuel-contaminated soils to plants requires multiple plant species, different 47 48 biological endpoints and soils that reflect site conditions and the range of soil properties at Science: 49 50 that site. 51 In this study, each test species differed in its response to SAB-contaminated soils 52 53 across three endpoints (germination, root and shoot growth; Figure 2). Initial tests with 54 55 freshly harvested seeds revealed that not all species were able to germinate under controlled 56 57 laboratory conditions. This may be due to unsuitable conditions for plant germination in the 58 laboratory setting, or alternatively to seed maturity or dormancy mechanisms. Dormancy is 59

60 an innate “whole-seed” characteristic that defines the environmental conditions that must be Environmental satisfied to initiate germination. Dormancy will prevent germination until specific 15

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1 2 3 environmental conditions trigger testa rupture, and these conditions are typically species- 4 5 specific [40-41]. In seed viability bioassays, seeds of seven of the twelve plant species were 6 7 able to germinate on high organic control soils, whereas only four species germinated on the 8 9 low OC soil. The effect of OC content on the species germination success suggests that seed 10 11 dormancy was not the only mechanism influencing germination. 12 When soils were spiked with SAB, germination was completely inhibited in three 13

14 species, all of which were dicot herbs (M. fontana, C. corymbosa and Colobanthus sp.). This Manuscript 15 16 suggests that these species are particularly vulnerable to SAB-contaminated soils, and risk 17 18 being lost from the impacted region, potentially decreasing species diversity at contaminated 19 sites. However, M. fontana (a perennial, mat-forming, herb) was found growing at the fuel- 20 21 spill sites during seed collection (Table 2). This suggests that either M. fontana seed 22 Accepted 23 production is more tolerant to soil-SAB concentrations than germination, that the SAB- 24 25 concentration in soils at the seed collection site was lower than bioassay concentrations, 26 27 or/and that other factors are influencing the field-based germination. 28 The fact that the three species with 100% germination inhibition were dicot herbs is 29 Impacts 30 likely to be coincidental, as two of the species that did germinate were monocot and dicot

31 & 32 herbs. Robson et al. [42] also noted high variability in test species tolerance to hydrocarbon- 33 34 contaminated soil, with drastic dose-dependent reductions in biomass and relative growth rate 35 (RGR) in some species, while others were not impacted. This variation was related to the 36 37 RGR, with plants with low RGR showing the least impact from fuel contamination [42], 38 39 which is consistent with our findings, where higher tolerance to fuel contaminated soil

40 Processes 41 occurred in slower growing species. Interestingly species with low RGR are also more 42 successful on nutrient-deficient soils [43]. 43 44 The seed coat also plays a major role in species-specific variability in germination 45 46 success. The coat protects the seed prior to germination by preventing mechanical or 47 48 chemical damage to the embryo. To assess the protective nature of the seed coat from fuel Science: 49 50 contamination, Amakiri and Onofeghara [44] soaked seeds of Capsicum frutescensin crude 51 oil from between 1 h to > 32 weeks prior to planting. Whilst C. frutescens maintained 100% 52 53 germination regardless of the period of pre-exposure of seeds to oil, the time to achieve 54 55 germination increased significantly in seeds pre-exposed for longer periods, delaying seed 56 57 emergence [44]. This implies that germination was physically inhibited by the oil coating on 58 the seeds, preventing imbibition and likely increasing microbial respiration around the seeds, 59

60 thereby depleting available oxygen. This physical inhibition can be overcome in time as the Environmental hydrocarbons degrade, enabling water and oxygen to penetrate the seed. Thus, if inhibition of 16

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1 2 3 germination is overcome as a result of a non-renewed coating of oil, it is possible that the 4 5 three herbs that germinated on control soils but not on SAB-contaminated soils in the current 6 7 study, may have germinated had the test duration been increased beyond 28 d. The four 8 9 species that did successfully germinate on both the low and high OC soils (C. muscoides, D. 10 11 chapmanii, E. pendunculare, and L. crinita) had comparable sensitivities to SAB 12 contaminated soils, with similar 28-d IC550 values based on germination inhibition (except E. 13

14 pendunculare on sandy soil, Figure 2). Manuscript 15 16 Germination was more tolerant than root growth on SAB-contaminated soils for all of 17 18 the native subantarctic species, and was generally more tolerant when the soils contained 19 higher OC. Once germination had occurred, the level of soil OC had no significant effect on 20 21 SAB toxicity for root growth (p > 0.05), however, higher OC enhanced the SAB toxicity on 22 Accepted 23 shoots, with a greater inhibition of the shoot growth. Thus, the added organic matter reduced 24 25 the toxicity of SAB contaminated soils in germination tests, but increased toxicity in terms of 26 27 above ground shoot growth. This may be related to nutrient cycling within contaminated 28 soils, as microbial-driven nitrification was inhibited within SAB-contaminated fuels in 29 Impacts 30 subantarctic regions (IC20 at 660 mg SAB/kg soil over 21 days [14]), most likely due to the

31 & 32 enzymatic inhibition of ammonia monooxygenase (AMO), however, denitrification process 33 34 were very tolerant. 35 The greater sensitivity of root growth than germination to SAB contaminated soils 36 37 (Figure 2) is most likely because the roots are in direct contact with the soil. Roots are highly 38 39 permeable tissues of the early seedlings that are no longer protected by the hard, impermeable

40 Processes 41 seed coat. Because early roots have yet to develop protective layers, such as a thickened 42 epidermis and cuticle, the lipid bilayer may be more vulnerable to the presence of 43 44 hydrocarbons. The lipid bilayer structure of the plasma membrane is, in essence, a colloidal 45 46 micelle and as such, will follow the same principles of other colloidal micelles [45]. This 47 48 includes the solubilisation when a foreign molecule is incorporated into the colloid. As Science: 49 50 hydrocarbons are readily solubilised, they are able to move into the plasma membrane and 51 displace fatty acids, thereby reducing the integrity of the membrane. This in turn causes the 52 53 cell to become increasingly permeable, eventually leading to collapse of the cell [45]. In 54 55 addition, the high tolerance of the shoots with the dicots C. muscoides and E. pendunculare 56 57 to SAB contaminated soils may be related to the epigeal germination. Monocots undergo 58 hypogeal germination in which the cotyledons remain below ground where they are more 59

60 exposed to the surrounding soil contamination (such as D. chapmanii and L. crinita), whereas Environmental

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1 2 3 dicots perform epigeal germination pushing the cotyledons above ground upon emergence 4 5 from the seed, away from the contaminated soils. 6 7 In this study, three biological endpoints were used to evaluate the effect of SAB 8 9 contaminated soils on germination, and shoot and root growth on subantarctic plant species. 10 11 Given the mode of action of hydrocarbon toxicity discussed above and in past research [12, 12 38, 46-48], root growth was expected to be the most sensitive endpoint as roots are in direct 13

14 contact with contaminated soil. In contrast, inhibition of shoot growth is thought to arise, not Manuscript 15 16 only from direct contact with hydrocarbons, but also as a result of systemic shock of 17 18 translocation of hydrocarbons to stems [46]. Unexpectedly shoot growth was less adversely 19 affected on the low than on the high OC soil. This could be due to the plant favouring shoot 20 21 growth over root growth given the less hospitable nature of the low organic carbon soil for 22 Accepted 23 root growth (which has higher wet bulk density and lower water holding capacity and 24 25 porosity, Table 1). 26 27 Using the IC50 data, the sensitivity of each endpoint was ranked using “1” as most 28 sensitive to “3” as least sensitive (Table 5). The ranking is presented for both the high and 29 Impacts 30 low OC soils following 28-d exposure to concentration series of SAB-contaminated soils.

31 & 32 Overall the least sensitive endpoint was germination, and the most sensitive endpoint was 33 34 root growth, but this varied between root and shoot growth depending on the species and the 35 soil type tested (Figure 2, Table 5). Other studies have also shown that germination success is 36 37 a less sensitive endpoint than root and shoot growth, resulting in an underestimation of the 38 39 toxicity of fuel-contaminated soils [36, 40]. Interestingly for D. chapmanii and E.

40 Processes 41 pendunculare, the order of sensitivity of endpoints was conserved between soil types, but this 42 order differed between soil types for L. crinita and C. muscoides (Table 4). These species 43 44 sensitivities to SAB-contaminated soils are far greater than that of the tolerant subantarctic 45 46 grass P. foliosa following an 8 month exposure (no impact on biomass production, plant 47 48 morphology and photosynthetic efficiency), which demonstrated ideal characteristics of a Science: 49 50 plant with phytoremediation properties [22] rather than a toxicity test species. 51 This study contributes to the growing evidence of species-specific responses of plants 52 53 to petroleum hydrocarbon contamination, as well as differences in sensitivity of test end 54 55 points. This study therefore highlights the need for toxicity tests to include multiple species 56 57 from different phylogenetic groups, a range of end points, and multiple soil types that are 58 representative of the impacted environment, in order to have a comprehensive and accurate 59

60 site-specific risk-assessment. Environmental

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1 2 3 Table 5. Comparison of endpoint sensitivity (based on IC values) ranked for each 4 50 5 subantarctic plant species from the most sensitive (1) to the least sensitive (3). The ranking is 6 presented for both the high and low organic carbon soils following 28-d exposure to SAB- 7 contaminated soils. Where the same number appears, there was no significant difference in 8 the sensitivity of endpoints. 9 10 Species High Organic Carbon Soil Low Organic Carbon Soil 11 12 Deschampsia chapmanii Shoot 1 Shoot 1 13 Root 1 Root 1

14 Germination 2 Germination 2 Manuscript 15 Luzula crinita Shoot 1 Root 1 16 Root 2 Shoot 2 17 Germination 3 Germination 3 18 Colobanthus muscoides Root 1 Root 1 19 20 Shoot 2 Germination 2 21 Germination 2 Shoot 3 22 Epilobium pendunculare Root 1 Root 1 Accepted 23 Shoot 2 Germination 2 24 Germination 2 Shoot 2 25 26 27 28 Conclusions 29 Impacts 30 In this study, site-specific remediation targets for subantarctic plants on fuel contaminated

31 & 32 soils utilised multiple species and early life cycle endpoints (germination, root length and 33 34 shoot length) to fully assess toxicity endpoints. Variable germination rates between species 35 under control conditions highlights that not all native subantarctic plants were suitable for use 36 37 in laboratory toxicity testing or for year round testing. Of the species that were suitable for 38 39 laboratory bioassays, germination, root and shoot growth varied with exposure to fuel

40 Processes 41 contaminated soil, with root and shoot growth exhibiting greater sensitivity than germination. 42 The TPH concentrations of contaminated soils required to induce growth inhibition was very 43 44 high, and only likely to be experienced directly at a spill site, but due to the climate of 45 46 subantarctic regions, the high concentrations may persist over time, so these high 47 48 concentrations are environmentally relevant. Future research is required to optimise test Science: 49 50 procedures for the species identified as suitable test species in this study, primarily to identify 51 the factors around the seed viability, and the effect of seed dormancy on subantarctic seed 52 53 germination during laboratory bioassays. 54 55 In order to derive remediation targets and environmental quality guidelines, Species 56 57 Sensitivity Distribution (SSD) models require toxicity data from a minimum of eight species 58 from at least four taxonomic groups. Prior to this study there was no toxicity data available 59

60 for sensitive early life stages of native subantarctic plants exposed to TPH. Therefore the data Environmental obtained here makes a significant contribution to the SSD model currently being developed to

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1 2 3 guide remediation activities at fuel contaminated sites at Macquarie Island and in subantarctic 4 5 regions more generally. 6 7 8 9 ACKNOWLEDGMENTS 10 11 We thank Corrine de Mestre for collecting plant seeds from Macquarie Island and TOC Art 12 13 photo, Anna Nydahl for technical assistance during soil preparation, and Lauren Wise for

14 assistance with chemical analysis. This study was funded by the Australian Government Manuscript 15 16 through an Australian Antarctic Science Grant (AAS 4100) awarded to C. King. 17 18 19 20 21 22 Accepted 23 References 24 25 1. Aislabie JM, Balks MR, Foght JM, and Waterhouse EJ. 2004. Hydrocarbon spills on 26 Antarctic soils: Effects and management. Environmental Science & Technology 38(5): 27 1265-1274. 28 29 2. Snape I, Harvey PM, Ferguson SH, Rayner JL, and Revill AT. 2005. Investigation of Impacts 30 evaporation and biodegradation of fuel spills in Antarctica I. A chemical approach 31 using GC-FID. Chemosphere 61: 1485-1494. & 32 33 3. Rayner JL, Snape I, Walworth JL, Harvey PM, and Ferguson SH. 2007. Petroleum- 34 hydrocarbon contamination and remediation by microbioventing at sub-Antarctic 35 Macquarie Island. Cold Regions Science and Technology 48(2): 139-153. 36 37 4. Atlas RM. 1981. Microbial-Degradation of Petroleum-Hydrocarbons - an 38 Environmental Perspective. Microbiological Reviews 45(1): 180-209. 39

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40 Processes 41 hydrocarbon spills on the temperature and moisture regimes of Cryosols in the Ross sea 42 region. Antarctic Science. 14: 319-326 43 44 45 46 47 48 Science: 49 50 51 52 53 54 55 56 57 58 59

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