Accepted Manuscript

Title: Assessing germination characteristics of Australian native plant species in metal/metalloid solution

Authors: J. Guterres, L. Rossato, D. Doley, A. Pudmenzky, C. Bee, V. Cobena

PII: S0304-3894(18)30919-1 DOI: https://doi.org/10.1016/j.jhazmat.2018.10.019 Reference: HAZMAT 19839

To appear in: Journal of Hazardous Materials

Received date: 5-6-2018 Revised date: 21-9-2018 Accepted date: 8-10-2018

Please cite this article as: Guterres J, Rossato L, Doley D, Pudmenzky A, Bee C, Cobena V, Assessing germination characteristics of Australian native plant species in metal/metalloid solution, Journal of Hazardous Materials (2018), https://doi.org/10.1016/j.jhazmat.2018.10.019

This is a PDF ﬁle of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its ﬁnal form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Assessing germination characteristics of Australian native plant species in metal/metalloid solution

J. Guterresa, L. Rossatoa,*, D. Doleyb, A. Pudmenzkyc, C. Beed, V. Cobenae aThe University of Queensland, School of Pharmacy, Woolloongabba, Queensland 4102, Australia bThe University of Queensland, School of Agriculture and Food Sciences, St Lucia, Queensland 4072, Australia cThe University of Queensland, School of Information Technology and Electrical Engineering, St Lucia, Queensland 4072, Australia dThe University of Queensland, St Lucia, Queensland 4072, Australia eKawsay Consulting, Lima, Peru

*Corresponding author. E-mail addresses: [email protected] and [email protected]

Word count excluding references: 4,802 words

Highlights:

 Radicle tolerance index (RTI) was the most sensitive indicator for plant metal tolerance during germination  Selected native plant species were highly tolerant to high concentrations of metal/loids tested  Species showed different metal toxicity thresholds and levels of tolerance based on RTI as tolerance indicator  Metal/loids tolerance thresholds were established for each native species  Species could all be classified as metallophytes, suitable for use for mine site rehabilitation ACCEPTED MANUSCRIPT Abstract

This study investigated tolerance of Australian native grass species Astrebla lappacea, Themeda australis, and Austrostipa scabra and a tree species Acacia harpophylla to different concentrations of arsenic As(V) (13.34 - 667.36 μM), Cu2+ (0.5 - 200 μM), Zn2+ (9 - 500 μM), Mn2+ (8 - 10240 μM) and Pb2+ (240 - 9600 μM) in single solutions in germination experiments. Metal/loid tolerance indicators used were maximum germination percentage

(Gmax), mean germination time (MGT), radicle and shoot tolerance indexes (RTI & STI). Radicle tolerance index was the most sensitive indicator of metal tolerance in germinating seeds. All native species were highly tolerant to the metal/loids tested, however, they showed different metal toxicity thresholds and levels of tolerance based on RTI as a metal tolerance indicator during germination. Overall, all four species could be classified as metallophytes, confirming their current suitability for and established use in mine site rehabilitation. This work may also serve as a basis for future studies on metal/loid tolerance of other plant species during germination.

Keywords: germination, Australian native plant species, metal/loid phytotoxicity threshold, tolerance indicator, radicle tolerance index

1. Introduction

Metal accumulation in the environment has become a widespread problem as toxic soluble metals have been released into the biosphere through both natural processes and human activities, such as mining and agriculture [1-3]. Although low concentrations of metals such as manganese (Mn), copper (Cu), zinc (Zn), molybdenum (Mo) and nickel (Ni), are essential to microorganisms, plants, and animals [4,5], at high concentrations, they have strong toxic effects and are environmental threats [6-8].

A common objective of dealing with waste metals in mining environments is to revegetate waste storage facilities that may contain toxic concentrations of these metals [9]. Where metal properties are highly problematic, they may be covered with impermeable barriers and layers of ACCEPTEDbenign material deep and stable enough toMANUSCRIPT be secure almost indefinitely [10]. While the design of these facilities may aim to intercept and evaporate all of the incident rainfall and

prevent the release of soluble forms of these metals [11,12], this is rarely achieved in Australian coal and metalliferous mines [13,14]. As a result, various remediation procedures are applied to the surfaces of tailings dams and waste heaps in order to establish vegetation [15-17]. However, where sufficient cover materials of suitable quality are not available, plant establishment may be compromised by toxic concentrations of metals in the surface material unless economical soil amendment can be achieved. The application of polymer particles to surface soils is one such amendment that has enabled the germination and establishment of plant species on otherwise toxic substrates [18,19]. However, it is difficult to totally eliminate soluble metals from these environments, and it is important to understand the tolerance to elevated metal concentrations of plant species that may be selected for revegetation work.

Trees and grasses are commonly used for revegetation of degraded and/or metal contaminated land [15,20,21]. In Australia, many native plant species have been used in mine site rehabilitation as they are tolerant to drought [17,22,23], low soil fertility [15,24], salinity [15] and poor soil structure [14,24,25]. Native plants are also preferred over exotic (i.e. non- native) species [17,19,24] as they do not present the risk of becoming invasive and reducing local plant biodiversity [26,27].

Metal tolerance has been reported widely for plants native to temperate regions on the basis of field observations [28,29] and for crop and pasture species on the basis of experimental solution culture studies [30,31]. There have been comprehensive listings for mine sites in different areas of Australia of native species generally [17,32,33] and grasses in particular [23] that can be recommended for revegetation work. These valuable studies of the outcomes of plant adaptation did not always describe quantitative responses of plant growth to plant- available metal concentrations in the soils.

Experimental studies of the effects of plant available metal concentrations on the growth of seedlings in soil or culture solution have been conducted by Reichmann [34] and Reichmann et al. [35]. Germination is often the stage of plant development that is most sensitive to enviroACCEPTEDnmental stress [36] so it is important to MANUSCRIPTunderstand its response to plant-available metal concentrations in the soil solution. This aspect of mined land restoration has been considered

for some situations in southern Australia [22,24], but not in detail for tropical environments or for mining situations in which site amelioration by techniques such as polymer particle application may be advantageous [19].

The present study was undertaken to establish the limits within which selected semi-arid Australian native plant species including grasses (Astrebla lappacea, Austrostipa scabra and Themeda australis) and a tree (Acacia harpophylla) might be able to germinate on different plant-available concentrations of As(V), Cu2+, Zn2+, Mn2+ and Pb2+ commonly found in mine wastes.

2. Materials and Methods

2.1. Plant materials

Grass and tree species native to Australia were chosen for the study. Seeds of Astrebla lappacea var. yanda and Themeda australis were obtained from AustraHort Pty Ltd (Queensland, Australia) and from Native Seeds Pty Ltd (Cheltenham, Victoria, Australia), respectively. Seeds of A. lappacea were hand collected between March and April 2006 from Central Queensland (Australia), and in March 2007 from Walgett (NSW, Australia), and stored for the 4-6 months required for after-ripening [37]. Seeds of T. australis were collected between March and April 2006 from Penrith (NSW, Australia), and in December 2007 from Coonabarabran (NSW, Australia). T. australis seeds collected from this area of south-east Australia will generally only have a small percentage of seeds that are dormant [37]. Seeds of Acacia harpophylla were obtained from AustraHort Pty Ltd (Queensland, Australia), and seeds of Austrostipa scabra from Native Seeds Pty Ltd (Cheltenham, Victoria, Australia). Seeds of A. harpophylla were collected from Roma (Queensland, Australia) in January 2006 and did not require any pre-treatment to germinate [38]. Seeds of A. scabra were collected in January 2008 from Ardlethan (NSW, Australia), easily accommodating the storage period of four months recommended by Ralph [37] to overcomeACCEPTED dormancy. Given that A. lappacea MANUSCRIPT and A. scabra seeds were stored for longer than the recommended time required to overcome dormancy, and T. australis and A.

harpophylla seeds were not expected to display dormancy, no pre-treatment was applied to seeds. All seeds collected were stored air-dry at 15˚C before the start of germination experiments.

2.2. Chemicals

Water soluble metal and metalloid compounds were used throughout. As(V) . 2+ . (Na2HAsO4 7H2O) was purchased from Sigma-Aldrich (Germany), Cu (CuCl 2H2O) and 2+ 2+ . Zn (ZnCl2) from Chem-Supply Pty Ltd (South Australia, Australia), Mn (MnCl2 4H2O) 2+ from Asia Pacific Specialty Chemicals Ltd (NSW, Australia) and Pb (PbCl2) from Lomb Scientific (Aust.) Pty Ltd (Coopers Plains, Queensland, Australia).

2.3. Germination experiments

Effects of various concentrations of As(V), Cu2+, Zn2+, Mn2+ and Pb2+ on the germination of the selected native species were evaluated using single metal solutions freshly prepared in sterilized deionized water (SDIW). Two sets of experiments were conducted and metal/metalloid concentrations tested are presented in Table 1.

Prior to germination experiments, seeds were surface-sterilised in 20% sodium hypochlorite for 10 min and then rinsed three times in SDIW for one min. All seed manipulations and examinations were then conducted in a laminar flow cabinet (AES Environmental, CLYDE- APAC & Contamination Control Laboratories, Australia) to minimise fungal and bacterial contamination throughout the experiment. Disposable Petri dishes (9 cm) each containing two 84 mm filter papers discs (Advantec) autoclaved for 15 min at 121˚C prior to experiments (Tomy ES-315 Autoclave, Tokyo, Japan) were used for germination. For all species, 25 seeds wereACCEPTED placed in each Petri dish. Five (A. lappacea MANUSCRIPT and A. harpophylla), eight (A. scabra), or either five, eight or ten (T. australis) replicate dishes were used for each species depending on

their viability. Ten mL of metal solution was added to each treatment Petri dish and 10 mL of SDIW was used for the control.

The Petri dishes were closed, sealed with Parafilm and placed in a transparent plastic zip resealable bag to reduce water evaporation. They were then incubated in a controlled temperature cabinet at optimum conditions of day and night temperatures of 30.2 ± 0.1 °C and 25.2 ± 0.1 °C [39-41] with 12:12 hours white light and dark conditions, and with moisture non-limiting. The bags and dishes were opened daily for measurements and to maintain oxygenation.

Newly germinated seeds were counted and removed daily for grasses, and either daily (experiment 1) or at 3.08, 6.25, 9.25, 12.25, 15.41, 18.91, 21.91, 24.91, 48.91, 72.91, 103.41, 120.41 and 151.91 hours (experiment 2) for A. harpophylla, until the maximum germination percentage was reached i.e. after 14 days for A. lappacea and A. scabra, either after 14 or 16 days for T. australis, and after 7 days i.e. 168 hours (experiment 1) or 151.91 hours (experiment 2) for A. harpophylla.

Seeds were considered germinated when 0.5 mm (A. lappacea, A. scabra and T. australis) and 1 mm of radicle (A. harpophylla) had emerged, or alternatively, if no radicle had emerged, when 2 mm of shoot had emerged. In cases where a single propagule contained more than one germinated caryopsis, the length of only the longest radicle or shoot was recorded and a single germinated diaspore counted.

2.4. Determination of metal tolerance indicators

Four indicators were used to assess the tolerance of the plant species to the selected metals during germination, namely maximum germination percentage (Gmax), mean germination time (MGT),ACCEPTED radicle tolerance index (RTI) and shoot MANUSCRIPT tolerance index (STI). For grasses, indicators used to determine species’ tolerance to metal/loids were Gmax, MGT, RTI and STI (As(V) and

Pb treatments only). For A. harpophylla, in experiment 1, only Gmax and MGT were

calculated and experiments were monitored daily while in experiment 2, monitoring was done hourly and included Gmax, MGT and RTI.

2.4.1. Maximum germination percentage (Gmax)

Gmax was calculated as the mean of the maximum cumulative germination percentage observed in each replicate Petri dish for each treatment. In some experiments, the plumule emerged from some seeds without the prior or contemporaneous appearance of the radicle

(germination was described as “with shoot only” or Gmax S), while in others, radicle emergence was the first response (germination was described as “with radicle” or Gmax R).

2.4.2. Mean Germination Time (MGT)

MGT was calculated using the following formula of Ellis and Roberts [42]:

Dn MGT(hours / days)   (1) n

Where: D = number of days (grasses) or hours (tree) since the beginning of the experiment, n = number of germinated diaspores/seeds on day/hour D

As for Gmax, mean germination time of seed germinated “with shoot only” was defined as

MGT S while germinated seed “with radicle” was defined as MGT R.

2.4.3. Radicle (RTI) and Shoot (STI) Tolerance Indices

Radicle length of germinated seeds was measured from the radicle-shoot junction to the tip of the longest radicles after 3 days for the grass species and after 9 hours for the tree species A. harpophyllaACCEPTED and expressed as RTI using the followingMANUSCRIPT formula [18]:

Length of the longest radicle in metal treatment (2) RTI (%)  100 Length of the longest radicle in SDIW control

Shoot length was measured from the radicle-shoot junction to the tip of the longest shoot after 3 days and expressed as shoot tolerance index (STI):

Length of the longest shoot in metal treatment STI (%)  100 (3) Length of the longest shoot in SDIW control

2.5. Statistical Analysis

Germination data were presented as the mean and standard error of five (A. lappacea and A. harpophylla), eight (A. scabra), or either five, eight or ten (T. australis) replicates. Data were tested for significant differences between treatments using one-way analysis of variance (ANOVA), p<0.05) and mean separation performed using the Tukey’s multiple comparison test and 95% confidence interval (p<0.05) (Matlab® Statistics Toolbox, version 2007b, The Mathworks, USA) [18].

3. Results

3.1. Effect of metal/loids on metal tolerance indicators during germination

Species’ tolerance thresholds based on maximum germination percentage (Gmax), mean germination time (MGT), radicle and shoot tolerance indexes (RTI & STI) as tolerance indicators during germination on As(V), Cu2+, Zn2+, Mn2+ and Pb2+ are presented in Table 2.

Gmax, MGT and STI were not reliable indicators of metal/loids toxicity to germination in A. lappacea, T. australis, A. scabra and A. harpophylla (detailed data in Supplementary Material)ACCEPTED. RTI was the most sensitive indicator MANUSCRIPT (section 3.2) of species’ metal/loid tolerance during germination.

3.2. Effect of metal/loids on radicle tolerance index (RTI)

3.2.1. As(V)

There was no significant difference between RTI of A. lappacea in As(V) concentration compared to control until the concentration reached 533.88 µM where the RTI began to decrease significantly (P<0.05) and reached 9.4% of the control value at 667.36 µM As(V) (Table 3 and Fig. 1). The results show that the RTI toxicity threshold was between 400.41 and 533.88 µM As(V). Consistent toxic effects of higher concentration of As(V) suggests that RTI was responsive to the range of concentrations tested.

Themeda australis’ RTI was not affected by As(V) up to 66.73 µM but it was significantly (P<0.05) reduced at 133.47 µM As(V) and progressively reduced further at higher concentrations compared to the control treatment (Table 3). This indicates that T. australis’ RTI toxicity threshold was between 66.73 and 133.47 µM As(V).

The RTI of A. scabra was considerably higher than the control at 13.34 µM As(V) and was not significantly different up at any of the other As(V) concentration tested, suggesting a RTI toxicity threshold >667.36 µM (Table 3).

For A. harpophylla, there was no significant (P>0.05) difference between RTI in any As(V) treated seeds compared to the control, also suggesting a RTI toxicity threshold >667.36 µM (Table 3).

3.3.2. Cu

Copper at all concentrations tested did not affect significantly (P>0.05) the RTI of A. lappacea and T. australis compared to the control (Table 3), suggesting that the species’ RTI tolerance threshold to Cu was >200 µM.

3.3.3.ACCEPTED Zn MANUSCRIPT

As with Cu, there were no significant (P>0.05) differences in the RTI of A. lappacea and T. australis in any treatment of Zn compared to the control (Table 3). This indicates that the species’ RTI toxicity thresholds were >500 µM.

3.3.4. Mn

Manganese did not affect (P>0.05) RTI of A. lappacea, T. australis and A. harpophylla at any concentration tested compared to the control (Table 3), indicating a RTI toxicity threshold >10240 µM for these species.

3.3.5. Pb

There was no significant difference between RTI of A. lappacea in Pb treatments compared to the control up to 4800 µM, where it was significantly reduced to 14.4% of the control value and further reduced to 0% in 9600 µM Pb (Table 3). At 9600 µM Pb, all the germinated seeds were without a radicle (shoot only) (Table 3 and Fig. 2). RTI appeared to be a good indicator of Pb toxicity to A. lappacea during germination and the Pb effect was consistent across the treatments. The species RTI toxicity threshold to Pb was between 3840 and 4800 µM Pb.

Themeda australis’ RTI was significantly (P<0.05) reduced compared to the control at Pb concentrations of 240 µM and higher (Table 3). This suggests that the species’ RTI toxicity threshold was <240 µM.

The RTI of A. scabra was considerably higher at 240 µM Pb compared to the control but remained not significantly (P>0.05) different from the control at all other Pb concentrations tested (Table 3), indicating a RTI toxicity threshold >9600 µM for this species.

4. DiscussionACCEPTED MANUSCRIPT

Our study suggests that radicle tolerance index (RTI) was the most sensitive indicator to reflect native plant species tolerance to metal/loids during seed germination. The sensitivity order of tolerance indicators during species germination was RTI > maximum germination percentage (Gmax) > shoot tolerance index (STI) > mean germination time (MGT). The finding in this study is in agreement with previous studies demonstrating high sensitivity of root length in response to toxicity of various metals (As, Cd, Cr, Pb) compared to other germination indicators such as germination rate, shoot length, maximum germination percentage and shoot biomass in both crop plants [43-47] and recognized metal-tolerant species [18,48]. In addition, Hou et al. [49] studied toxicity effects of chromium (Cr) on various plant species including Brassica oleracea, Cucumis sativus, Lactuca sativa, Triticum aestivum and Zea mays and found that root elongation was one of the most sensitive indicators for phytotoxicity of Cr in soil. Mishra and Choudhuri [50] reported that shoot and root tolerance indexes were the most sensitive indicators for biomonitoring of phytotoxic effects of Pb and mercury (Hg) on rice cultivars. Roots are often used to measure heavy metal tolerance of plants because they are more responsive to metal toxicity in the environment [44]. This might be because roots function as the initial absorptive organs and are more directly exposed to metals compared to the other plant tissues [44,49]. Therefore, the presence of metals may reduce the ability of roots to absorb water and nutrients from soils and may inhibit cell division in roots [49,51]; consequently, germinating seeds are not able to develop and grow. This is confirmed by the present study where substantial proportions of germinated seeds of A. lappacea were observed without a radicle in higher concentrations of As(V) and Pb treatments.

Overall, all native species were highly tolerant to the metal/loids tested during germination. However, species showed different metal/loid toxicity thresholds and levels of tolerance to As(V), Cu, Zn Mn and Pb using RTI as metal tolerance indicator. Acacia harpophylla and A. scabra were extremely tolerant to increasing concentrations of As(V), with no effects on the species RTI at the highest concentration tested (667.36 µM), while A. lappacea was tolerant to slightly lower As(V) concentrations (between 400.41 and 533.88 µM). In contrast, T. australisACCEPTED was least tolerant to As(V), with itsMANUSCRIPT RTI being affected at relatively much lower As(V) concentrations (between 66.73 and 133.47 µM) than the other species. Overall, the

As(V) tolerance rank during germination of the species was A. harpophylla = A. scabra > A. lappacea > T. australis. Although these species have been frequently used for rehabilitation of mine sites [52], their tolerance thresholds to As(V) had never been studied. This study was the first to establish the species tolerance threshold during germination in As(V) solutions. The four native species tested in this study were more tolerant or showed similar tolerance levels to As(V) during germination compared to previously studied plant species such as Phaseolus aureus, Helianthus annuus L., Hordeum vulgare L. and Triticum aestivum L.. For example, radicle length of H. vulgare L. was reduced at 4 mg As (arsenate)/L (equivalent to 53.38 µM As) [53], P. aureus at 10 µM As (arsenate) [54], H. annuus L. at 6-10 mg As (sodium arsenate) (equivalent to 19.22 – 32.04 µM) [55], early root development of T. aestivum L. was reduced at 4 - 16 mg As (arsenite)/L (equivalent to 53.38 – 213.55 µM As) [56], and there was a gradual reduction of germination percentage, shoot and root length elongation of T. aestivum L. at 5 - 30 mg As (arsenate)/L (equivalent to 66.73 – 400.41 µM As) [57].

The present study found that A. lappacea and T. australis were tolerant to high concentrations of Cu and both showed a similar level of tolerance to Cu, with RTI not affected by Cu at the highest concentration tested (200 µM). This suggests that both A. lappacea and T. australis have a greater tolerance to Cu during germination (> 200 µM) than Alyssum montanum and Thlaspi ochroleucum where germination percentage and growth rate of radicle were reduced at 80 and 160 µM Cu [58]. Germination percentage of Zea mays was not affected at 12 ppm (equivalent to 188.82 µM Cu) but root and shoot length of the seedlings were greatly reduced at the same concentration of Cu [59]. Root length of 6-day-old seedlings of Triticum aestivum

L. was reduced at 50 ppm Cu (CuSO4.5H2O) (equivalent to 200.64 µM Cu) [60] and root and shoot length of Agropyron elongatum seedlings was reduced at 30 mg/L Cu (equivalent to 472.06 µM Cu) [61]. Other species showed higher tolerance to Cu, such as Pisum sativum, where seed germination percentage was reduced at 80 ppm Cu (equivalent to 1258.85 µM Cu) [62], maximum inhibition of seed germination percentage and decrease of shoot and root length of Cassia angustifolia Vahl occurred at 200 mg/L Cu (3147.12 µM Cu) [63] and reductionsACCEPTED of germination rate of Oryza sativa MANUSCRIPT L. cv. Hwayeong occured at 1 mM, shoot length at 0.2 mM and root formation at 250 µM [81] [64].

Our study showed that both A. lappacea and T. australis showed the same level of tolerance to Zn as RTIs of both species were not significantly affected by Zn and RTI toxicity threshold was > 500 µM. Rossato et al. [18] reported RTI of A. lappacea was significantly reduced at 10 mM Zn in solution during germination. The tolerance of T. australis to toxic levels of Zn during germination was not previously known. Tolerance of other Australian native species to Zn has been studied by Reichman et al. [65] using much lower concentrations (up to 100 µM Zn) than those tested in the study (up to 500 µM), but during the seedling growth stage. Kunjam et al. [62] reported Zn at 80 ppm (equivalent to 1223.61 µM Zn) did not affect root growth of P. sativum seedlings during germination and growth. Zinc at 50 mg/L (equivalent to 764.75 µM Zn) also markedly reduced root and shoot lengths of 28-day-old Cassia angustifolia seedlings [63], and root growth of Salvia coccinea was inhibited at 100 mg/L (equivalent to 1529.51 µM Zn) [66]. Dorycnium pentaphyllum germination was slightly delayed and final germination percentage was greatly reduced at 10000 µM Zn [51].

Astrebla lappacea, T. australis and A. harpophylla were equally tolerant to Mn at all concentrations tested with a RTI toxicity threshold > 10240 µM. The species tolerance rank to toxic levels of Mn during germination was A. lappacea = A. harpophylla = T. australis. Manganese toxicity thresholds for these species have not been studied previously. Other species, such as Nicotiana tabacum have been found to tolerate 2 mM Mn during tissue regeneration as callus induction, callus growth and shoot regeneration were not affected at 2 mM Mn [67]. In Arabidopsis, primary root growth was only affected by 2 mM Mn [68]. In addition, germination of Xanthium sibiricum was only affected by Mn concentration above 5000 μM where germination potential, germination index, vigor index, root length, fresh weight, dry weight and root-shoot ratio were greatly reduced [69].

Austrostipa scabra, A. lappacea and T. australis were tolerant to high levels of Pb. Based on RTI as a tolerance indicator, the order of species tolerance to Pb was A. scabra (> 9600 µM) > A. lappacea (between 3840 and 4800 µM) > T. australis (< 240 µM). Toxicity of Pb in solutions on germination of A. lappacea was studied previously by Rossato et al. [18], who reportedACCEPTED a significant decrease in RTI (0% ofMANUSCRIPT the control value), at 9600 µM Pb. In contrast, tolerances of germinating A. scabra and T. australis’ seeds to Pb have never been tested. In

comparison, previous studies reported that germination percentage of Matricaria chamomilla was not affected by Pb at 60 µM but root and shoot lengths and weight of seedlings of the species were reduced at 30 µM Pb [70]. Germination rate of Triticum aestivum was reduced at 0.3 mM Pb but root and leaf elongations of germinated seeds were reduced at 1.5 mM Pb [71], and in Festuca arundinacea, inhibitory effects of Pb on relative germination rate and germination index were apparent at 1000 mg Pb/L (equivalent to 4826.25 µM) [72].

5. Conclusion

Radicle tolerance index was found to be the most sensitive indicator of As(V), Cu, Zn, Mn and Pb tolerance in A. lappacea, T. australis, A. scabra and A. harpophylla during germination. All native species were highly tolerant to the metal/loids tested, however, they showed different metal toxicity thresholds and levels of tolerance based on RTI as a metal tolerance indicator during germination. Our study was only able to identify toxic concentration ranges for some metal/loids and for some of the species, and further work will be needed to identify more precise RTI toxicity thresholds (actual concentrations or at least limited concentration ranges as opposed to the ranges identified here). All four species could be classified as metallophytes, confirming their current suitability and use for mine site rehabilitation. This work may also serve as a basis for future studies on metal/loid tolerance of other plant species during germination. The resulting RTI toxicity thresholds will only apply to germinating seeds and additional studies will be required to confirm whether these thresholds can also be applied to species during plant establishment.

Acknowledgements

This work has been funded by a competitive University of Queensland (Brisbane, Australia)

Early Career Researcher grant awarded to Dr Laurence Rossato. ACCEPTED MANUSCRIPT

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Figure captions

Figure 1 Radicle elongation of Astrebla lappacea, Themeda australis and Austrostipa scabra after 3 days on various concentrations of As(V) solutions. Arrows indicate emergent radicle.

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Figure 2 Radicle elongation of Astrebla lappacea, Themeda australis and Austrostipa scabra after 3 days on various concentrations of Pb solutions. Arrows indicate emergent radicle.

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Table 1 Metal/loid concentrations tested on seed germination of native plant species Astrebla lappacea, Themeda australis, Austrostipa scabra and Acacia harpophylla.

Table 2 Metal/loid tolerance levels of Astrebla lappacea, Themeda australis, Austrostipa scabra and Acacia harpophylla during germination on As(V) (13.34 - 667.36 μM), Cu2+(0.5 - 200 μM), Zn2+(9 - 500 μM), Mn2+(8 - 10240 μM) and Pb2+ (240 - 9600 μM), based on maximum germination percentage (Gmax), mean germination time (MGT), radicle and shoot tolerance indexes (RTI & STI) as tolerance indicators.

Table 3 Radicle tolerance index (RTI) of Astrebla lappacea, Themeda australis and Austrostipa scabra after 3 days or Acacia harpophylla after 9 hours on various concentrations of As(V), Cu, Zn, Mn and Pb solutions.

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Table 1 Metal/loid concentrations tested on seed germination of native plant species Astrebla lappacea, Themeda australis, Austrostipa scabra and Acacia harpophylla.

Metal/loid concentration (μM) Plant species As(V) Cu Zn Mn Pb A. lappacea 13.34 0.5 9 8 240 T. australis 66.73 10 25 128 480 A. scabra 133.47 40 100 2048 960 266.94 80 200 4096 1920 400.41 200 500 10240 3840 533.88 - - - 4800 667.36 - - - 9600 A. harpophylla 1st experiment 13.34 0.5 9 8 - 66.73 10 25 128 - 133.47 40 100 2048 - 2nd experiment 133.47 - - 2048 - 266.94 - - 4096 - 667.36 - - 10240 -

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Germination Species' metal/loid toxicity threshold (μM) Metal/loid indicators A. lappacea T. australis A. scabra A. harpophylla

As Gmax R <13.34 >667.36 133.47 - 266.94 >667.36 MGT R >667.36 >667.36 >667.36 >667.36 RTI 400.41 - 533.88 66.73 - 133.47 >667.36 >667.36 STI >667.36 13.34 - 66.73 >667.36 nt

Cu Gmax R >200 >200 nt >40 MGT R >200 >200 nt >40 RTI >200 >200 nt nt STI nt nt nt nt

Zn Gmax R >500 >500 nt >100 MGT R >500 >500 nt >100 RTI >500 >500 nt nt STI nt nt nt nt

Mn Gmax R >10240 >10240 nt >10240 MGT R >10240 >10240 nt >10240 RTI >10240 >10240 nt >10240 STI nt nt nt nt

Pb Gmax R 1920 - 3840 >9600 4800 - 9600 nt MGT R 4800 - 9600 Inconclusive 3840 - 4800 nt RTI 3840 - 4800 <240 >9600 nt STI 4800 - 9600 240 - 480 >9600 nt 'G max R' stands for 'maximum germination percentage of germinated seeds with radicle'; 'MGT R' stands for 'mean germination time of germinated seeds with radicle'; 'nt' stands for 'not tested’.

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RTI (%)* Metal or metalloid A. lappacea T. australis A. scabra A. harpophylla

nd 2 exp. Control (SDIW) 100±0a 100±0a 100±0a 100±0a As(V) (13.34 μM) 123.1±48.3a 72±37.2a 623.2±264.2b nt As(V) (66.73 μM) 104.5±23.1a 51.0±38.7a 261.9±139.5a nt As(V) (133.47 μM) 119.6±29.0a 30.4±22.2b 94.6±37.5a 86.7±6a As(V) (266.94 μM) 61.5±10.1a 21.5±17.5b 65.5±16.3a 66.7±6.2a As(V) (400.41 μM) 39.6±18.2a 6.5±4.2b 76.8±26.4a nt As(V) (533.88 μM) 22.6±14.8b 11.1±8.3b 62.5±28.1a nt As(V) (667.36 μM) 9.4±9.4b 1.4±1.4b 63.7±27.7a 66.7±13.6a Control (SDIW) 100±0a 100±0a nt nt Cu (0.5 μM) 106.7±6.7a 66.7±33.3a nt nt Cu (10 μM) 65.3±16.9a 104.4±54a nt nt Cu (40 μM) 96.7±13.3a 61.1±2a nt nt Control (SDIW) 100±0a 100±0a nt nt Cu (40 μM) 130±8.6a 36.5±16.2a nt nt Cu (80 μM) 111±17.3a 146.1±64.1a nt nt Cu (200 μM) 64±16.1a 65.8±29.9a nt nt Control (SDIW) 100±0a 100±0a nt nt Zn (9 μM) 73.3±11.3a 58.3±14.4a nt nt Zn (25 μM) 78.7±21.7a 116.7±35.4a nt nt Zn (100 μM) 96.7±15.5a 46.1±28.2a nt nt Control (SDIW) 100±0a 100±0a nt nt Zn (100 μM) 98±14.5a 87.2±33.5a nt nt Zn (200 μM) 122.7±8.2a 64.6±25.6a nt nt Zn (500 μM) 100±13.9a 54.18±14.8a nt nt Control (SDIW) 100±0a 100±0a nt nt Mn (8 μM) 101.7±9.3a 44.4±5.6a nt nt Mn (128 μM) 91.7±13.9a 64.6±45.8a nt nt Mn (2048 μM) 93.8±6.3a 109.2±34a nt nt 2nd exp. Control (SDIW) 100±0a 100±0a nt 100±0a a a a Mn (2048 μM) 101.7±8.4 157.7±93 nt 66.7±6.2 Mn (4096 μM) 117.7±10.3a 189.6±104.8a nt 66.7±6.2a Mn (10240 μM) 101±7.1a 89.6.4±28.4a nt 86.7±13.6a Control (SDIW) 100±0a 100±0a 100±0a nt Pb (240 μM) 137.6±27.4a 35.9±22.6b 554.3±317.4b nt Pb (480 μM) 139.6±46.8a 10.9±6.8b 258.4±163.6a nt Pb (960 μM) 152.4±38.9a 0±0b 144.0±70.4a nt Pb (1920 μM) 92.8±24.2a 49.5±34b 32.9±10.9a nt ACCEPTEDa b MANUSCRIPTa Pb (3840 μM) 48.8±25.5 0±0 64±35.3 nt Pb (4800 μM) 14.4±9.0b 6.3±6.3b 36.5±24a nt Pb (9600 μM) 0±0b 15.6±11.8b 35.2±14.2a nt

*Results are given as the mean and standard error of five (A. lappacea and A. harpophylla) or eight (A. scabra) or either five or eight or ten (T. australis) replicates. Treatments with the same letter do not differ significantly (P>0.05) from the control. ‘SDIW’ stands for ‘sterilized deionized water’. ‘nt’ stands for ‘not tested’. ‘2nd exp.’ refers to ‘second experiment’.

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