Toxic Effects of Rare Earth Elements (Cerium, Europium, and Neodymium) on Radish, Tomato, and Durum Wheat

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Toxic Effects of Rare Earth Elements (Cerium, Europium, and Neodymium) on Radish, Tomato, and Durum wheat

Amanda Pellegrino

A Thesis presented to The University of Guelph

In partial fulfilment of requirements for the degree of Master of Science in Environmental Science, Toxicology

Guelph, Ontario, Canada

ABSTRACT

TOXIC EFFECTS OF RARE EARTH ELEMENTS (CERIUM, EUROPIUM, AND NEODYMIUM) ON RADISH, TOMATO AND DURUM WHEAT

Amanda Pellegrino Advisor: University of Guelph, 2018 Dr. Beverley A. Hale

Rare earth elements (REEs), or rare earth metals, are a group of 17 elements including the lanthanide series, yttrium and scandium. They occur naturally together in mineral deposits, have similar properties, and are being used at increasing rates in new technology. At low REE concentrations in soils, plant growth stimulation has been identified; however, at higher concentrations, toxicity has been determined for some REEs. Toxic effect concentrations (shoot/root length, and shoot biomass) for radish, tomato and durum wheat were determined for three REEs: cerium, neodymium, and europium. A hormetic response was not characterized by any of the data, however many endpoints displayed thresholds. Overall, effect concentrations

(EC25) ranged from 900-7600 mg/kg; these were higher than those reported in previous literature, likely due to low bioaccessibility of REEs in the highly organic soil. Internal tissue concentrations might therefore be a better predictor of toxicity.

Dedication

To my Opa, for being a constant source of encouragement in my pursuit of education.

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Acknowledgements

Above all, I would like to thank my advisor Dr. Beverley Hale for her continuous support and expert advice throughout the process of working on this research, and without whom I would not have had many incredible opportunities. Thank-you Bev, for making it possible for me to attend many conferences throughout these two years where I was able to present this work and meet people in my field. I would also like to acknowledge the work of Bianca Pereira, who contributed significantly to this project, not only due to the link between our experiments but also through her friendship and support. I’d like to thank Luba Vasiluk for her assistance with many aspects of my project including experimental setup and laboratory training, and Peter Smith for his assistance with analytical procedures. I would like to thank all of the Hale lab members for their help with plant harvests, many hands make light work. In particular, I would like to thank Elizabeth Jones for her help with sample analysis by sharing in the workload of processing and analysing thousands of plant and soil samples. I would like to thank everyone, not only for helping to keep me on track through this process and helping out in the lab, but also for providing hours of friendship, fun, and lab lunches. The support of my family and friends throughout my MSc has been invaluable. Thank- you to my parents and siblings and to Jeffrey for listening to not only the fun stories of this adventure but also to my grumblings when things didn’t go as planned. I’d like to thank Dr. Gladys Stephenson and Dr. Paul Sibley for being on my advisory committee and providing me with guidance on my project. Finally, I would like to acknowledge the support of Environment and Climate Change Canada for the funding provided for this project.

Table of Contents

ABSTRACT ...... ii Acknowledgements ...... iv Table of Contents ...... v List of Tables...... vii List of Figures ...... ix List of symbols and abbreviations ...... xii 1.0 Introduction ...... 1 2.0 Literature review ...... 2 2.1 Rare Earth Elements ...... 2 2.2 Hormesis ...... 4 2.3 Bioaccessibility ...... 6 2.4 Hypotheses ...... 6 3.0 Materials and Methodology ...... 7 3.1 Range-finding studies ...... 7 3.1.1 Soil preparation, analysis and amendment ...... 7 3.1.2 Plant assay ...... 10 3.2 Definitive studies ...... 11 3.2.1 Soil Preparation, analysis and amendment ...... 12 3.2.2 Plant assay ...... 14 3.3 Positive control assessment ...... 14 3.3.1 Soil amendment ...... 15 3.3.2 Plant Assay ...... 15 3.4 Leaching experiment ...... 15 3.4.1 Soil preparation...... 15 3.4.2 Plant assay ...... 16 3.5 OECD Soil comparison ...... 17 3.5.1 Soil preparation...... 17 3.5.2 Bioaccessibility measurement ...... 18 3.6 Statistical analyses ...... 18

3.6.1 Range-finding studies ...... 18 3.6.2 Definitive studies...... 19 4.0 Results and Discussion ...... 20 4.1 Soil analysis ...... 20 4.1.1 Range-finding studies ...... 20 4.1.2 Definitive studies ...... 23 4.2 Toxic effect concentrations related to total soil concentration ...... 27 4.2.1 Range-finding study ...... 27 4.2.2 Definitive studies ...... 29 4.3 Tissue concentration ...... 39 4.4 Leaching experiment ...... 47 4.4.1 Soil analysis ...... 47 4.4.2 Plant growth inhibition ...... 48 4.5 OECD soil comparison ...... 50 4.5.1 Soil analysis ...... 51 5.0 Conclusions ...... 57 6.0 Bibliography ...... 60 7.0 Appendix – Supplementary Materials ...... 63

List of Tables

Table 1: Nominal concentrations of REE-amended soils for the range-finding test (mg/kg dry soil) .... 9

Table 2: Nominal concentrations of REE-amended soil for the definitive test (mg/kg dry soil) ...... 13

Table 3: Total and bioaccessible (mg/kg dry soil) plant nutrients in black garden soil prior to use for range-finding study (NM: not measured) ...... 20

Table 4: Mean (± SD) total and bioaccessible REE concentrations (mg/kg) of each amended dry soil at the end of the 14-day test ...... 21

Table 5: Total and bioaccessible plant nutrients (mg/kg) in black garden soil prior to use for definitive studies (NM: not measured) ...... 24

Table 6: Mean (± SD) total REE concentrations (mg/kg) in soil from each treatment at the end of the 14-day definitive tests with wheat and tomato ...... 24

Table 7: Mean (± SD) bioaccessible REE concentrations (mg/kg) of Ce and Nd at the end of the 14- day definitive tests with wheat ...... 24

Table 8: Effect concentrations (EC10, EC20, EC25, EC50) and 95% confidence intervals for radish shoot length exposed to REEs (mg/kg dry soil) ...... 29

Table 9: Effect concentrations (EC10, EC20, EC25) and 95% CI for tomato and durum wheat shoot and root lengths exposed to REEs in soil for 14 days (mg/kg dry soil)...... 39

Table 10: Internal effect concentrations (EC10, EC20, EC25) of REEs derived for tomato and durum wheat shoot biomass (mg/kg dry biomass). 95% CI excluded due to lack of significance at measurable precision level...... 46

Table 11: Internal (tissue) effect concentrations (mg/kg dry tissue) for tomato and durum wheat shoot and root lengths exposed to REEs (EC10, EC20, EC25, EC50) ...... 47

Table 12: Total Ce concentrations after leaching, and total and bioaccessible Ce concentrations of leached soil following plant growth (mg/kg dry soil)...... 48

Table 13: Physicochemical properties of garden soil and artificial soil ...... 50

Table 14: Mean (± SD) total and bioaccessible concentrations (mg/kg) of Ce in CeCl and Ce NO3 amended artificial soil...... 51

Table 15: Mean (± SD) total and bioaccessible concentrations (mg/kg) of Eu in EuCl3 and Eu NO3 amended artificial soil...... 52

Table 16: Mean (± SD) total and bioaccessible concentrations (mg/kg) of Nd in NdCl3 and Nd NO3 amended artificial soil...... 52

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Table 17: Comparison of EC25 values between this research and published literature for radish and tomato (mg/kg dry soil) ...... 56

Table 18: Percent bioaccessibility of REE-NO3 in garden soil after 2 week aging and REE-Cl3 in artificial soil after 24 hours of aging used to normalize effect concentration values ...... 56

Table 19: Effect concentrations (EC25) for radish and tomato from this research and the literature that have been normalized for bioaccessibility (mg/kg dry soil) ...... 57

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List of Figures

Figure 1: Hypothetical exposure concentration-response curves depicting (A) threshold, (B) linear non-threshold, (C) sigmoidal, and (D) J-shaped hormetic models...... 4

Figure 2: Relationship between total and bioaccessible Ce in soil for the range-finding study. Confidence bands are set at 2.5% and 97.5%...... 22

Figure 3: Relationship between total and bioaccessible Eu in soil for the range-finding study. Confidence bands are set at 2.5% and 97.5%...... 22

Figure 4: Relationship between total and bioaccessible Nd in soil for the range-finding study. Confidence bands are set at 2.5% and 97.5%...... 23

Figure 5: Relationship between total and bioaccessible Ce in soil for the 14-day definitive test with durum wheat. Confidence bands are set at 2.5% and 97.5%...... 25

- Figure 6: Nitrification test NO3 concentration (mg/L) over time for 28 days in definitive test soil. Confidence bands are set at 2.5% and 97.5%...... 26

Figure 7: Concentration-response curve for shoot length of radish when exposed to Ce amended soil for 14 days. The approximate EC50 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%...... 28

Figure 8: Concentration-response curve for shoot length of radish when exposed to Eu amended soil with approximated EC50 value. The approximate EC50 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%...... 28

Figure 9: Concentration-response curve for shoot length of radish when exposed to Nd amended soil with approximated EC50 value. No EC50 could be approximated. Confidence bands are set at 2.5% and 97.5%...... 29

Figure 10: Mean (±SD) shoot and root lengths of radish, tomato and durum wheat grown in positive and negative control soils with and without KNO3 respectively...... 32

Figure 11: Root length of tomato exposed to Ce amended soil for 14 days. Solid line at 80% root length shows that the EC20 was not bounded. Confidence bands are set at 2.5% and 97.5%. N.S. denotes relationship is not significant...... 33

Figure 12: Shoot length of tomato exposed to Ce amended soil for 14 days. Approximated EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%...... 33

Figure 13: Root length of tomato exposed to Eu amended soil for 14 days. Approximated EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%. N.S. denotes relationship is not significant...... 34

Figure 14: Shoot length of tomato exposed to Eu amended soil for 14 days. Solid line at 80% shoot length shows that the EC20 was not bounded. Confidence bands are set at 2.5% and 97.5%...... 34

Figure 15: Root length of tomato exposed to Nd amended soil for 14 days. Approximated EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%. N.S. denotes relationship is not significant...... 35

Figure 16: Shoot length of tomato exposed to Nd amended soil for 14 days. Approximated EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%...... 35

Figure 17: Root length of durum wheat exposed to Ce amended soil for 14 days. Approximated EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%...... 36

Figure 18: Shoot length of durum wheat exposed to Ce amended soil for 14 days. Solid line at 80% root length shows that the EC20 was not bounded. Confidence bands are set at 2.5% and 97.5%...... 36

Figure 19: Root length of durum wheat exposed to Eu amended soil for 14 days. Approximated EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%. N.S. denotes relationship is not significant...... 37

Figure 20: Shoot length of durum wheat exposed to Eu amended soil for 14 days. Solid line at 80% root length shows that the EC20 was not bounded. Confidence bands are set at 2.5% and 97.5%...... 37

Figure 21: Root length of durum wheat exposed to Nd amended soil for 14 days. Approximated EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%. N.S. denotes relationship is not significant...... 38

Figure 22: Shoot length of durum wheat exposed to Nd amended soil for 14 days. Approximated EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%...... 38

Figure 23: Radish root accumulation of REEs in the range-finding study after growing in REE amended soils for 14 days...... 41

Figure 24: Radish shoot accumulation of REEs in the range-finding study after growing in REE amended soils for 14 days...... 42

Figure 25: Tomato root accumulation of REEs in the definitive study after 14 days of growth in REE amended soils...... 44

Figure 26: Tomato shoot accumulation of REEs in the definitive study after 14 days of growth in REE amended soils...... 45

Figure 27: Durum wheat root accumulation of REEs in the definitive study after 14 days of growth in REE amended soils...... 45

Figure 28: Durum wheat shoot accumulation of REEs in the definitive study after 14 days of growth in REE amended soils...... 46

Figure 29: Shoot length of radish exposed for 14 days to Ce in soil following leaching. Confidence bands are set at 2.5% and 97.5%. N.S. denotes relationship is not significant...... 49

Figure 30: Hypocotyl length of radish exposed for 14 days to Ce in soil following leaching. The approximated EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%...... 49

Figure 31: Mean (± SD) percent hypocotyl development by treatment for radish exposed to Ce in soil following leaching. Error bars show standard deviation...... 50

Figure 32: Bioaccessible Ce in CeCl3 amended artificial soil after 24 hour and 2 week aging periods in OECD comparison study ...... 53

Figure 33: Bioaccessible Ce in CeNO3 amended artificial soil after 24 hour and 2 week aging periods in OECD comparison study ...... 53

Figure 34: Bioaccessible Eu in EuCl3 amended artificial soil after 24 hour and 2 week aging periods in OECD comparison study ...... 54

Figure 35: Bioaccessible Eu in EuNO3 amended artificial soil after 24 hour and 2 week aging periods in OECD comparison study ...... 54

Figure 36: Bioaccessible Nd in NdCl3 amended artificial soil after 24 hour and 2 week aging periods in OECD comparison study ...... 55

Figure 37: Bioaccessible Nd in NdNO3 amended artificial soil after 24 hour and 2 week aging periods in OECD comparison study ...... 55

List of symbols and abbreviations

CCME – Canadian Council of Ministers of the Environment

EC – Electrical conductivity

ICP-MS – Inductively coupled plasma - mass spectrometry

ICP-OES – Inductively coupled plasma - optical emission spectrometry

LOD – Limit of detection

NOEC – No observed effect concentration

NNR – Net nitrification rate

OECD – Organisation for Economic Co-operation and Development

REE – Rare earth element

SOM – Soil organic matter

SQG – Soil quality guideline

TOC – Total organic carbon

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1.0 Introduction

Toxicity testing of trace elements and its application to the regulation of environmental concentrations has been well studied. The literature on these subjects includes methods of testing for different end points such as growth inhibition and various other detrimental factors to all types of biota. With such a large breadth in the toxicity information available, protocols for integrating this information into regulation have been derived.

In the current Canadian federal regulatory framework, elements are assessed through the creation of species sensitivity distributions (SSDs) from toxicity data for several species of plants, animals and microbes (CCME, 2006). Low effect concentration data are preferred, including the NOEC (no observed effect concentration) and EC10 (concentration that affects 10% of the population) endpoints, which are considered to be protective. In order to establish a guideline, the minimum dataset must include at least 10 endpoints from at least three studies, from at least two invertebrate and two plant species. For regulatory purposes, the level which is hazardous to 5% of the species on the SSD (i.e. the median of the 5th percentile of the

NOEC/EC10 values in the SSD, known as the HC5) is identified and compared to the current concentrations of the substance in question within the environment. The level which is considered to be safe is then used to establish a soil quality guideline (SQG) (CCME, 2006).

Currently, there are insufficient toxicity data for REEs to develop an SSD.

To help address these data gaps, the major objective of this thesis was to determine toxic effect concentrations for REEs, in the context of soil chemistry, to aid in risk assessment. To achieve this, the toxicity of cerium (Ce), europium (Eu) and neodymium (Nd) to three plant species was determined for three end points: lengths of roots and shoots, and shoot dry biomass.

Hormesis of the REEs was quantified, as well as the bioaccessibility of REEs in soil. Tissue concentrations of the REEs were also quantified to help understand the relationship between total and bioaccessible soil REE concentration and toxicity.

2.0 Literature review

2.1 Rare Earth Elements

Rare earth elements (REEs), sometimes called rare earth metals, are a group of 17 elements including scandium (Sc), yttrium (Y), and the 15 lanthanides (“REE Handbook: The ultimate guide to Rare Earth Elements,” 2013). Though their name implies rarity, their crustal abundances range from cerium (Ce), at 66 ppm, the 25th most abundant element, to lutetium

(Lu), the least abundant REE, at 0.8 ppm (Tyler, 2004). The REEs are split into two groups,

“light” REEs (LREEs) and “heavy” REEs (HREEs), according to the pairing of electrons in their valence shells. LREEs do not have paired valence electrons and include elements with atomic numbers from 57 through 64; HREEs have at least one set of paired valence electrons and include elements 65 through 71 (“REE Handbook: The ultimate guide to Rare Earth Elements,”

2013). In Canadian soils, LREEs are more common, and it is most likely that they would become elevated in soils due to mining (Avalon Advanced Metals Inc., 2016). REEs exist as mixtures within deposits in the earth’s crust and as such, natural soils elevated in REEs also contain mixtures. The ratios between elements in mixtures of REEs are generally similar amongst soils

(Anawar et al., 2010).

REEs have become an essential component of modern lifestyle. Their use in hand-held electronics, as well as green technologies such as wind turbine batteries, has led to an increase in their extraction (mining) from 50 kt/year in 1990 to 150 kt/year in 2010 (Chen, 2011). All REEs

can be found in a divalent state, which allows them to be taken up into plants by non-specific divalent cation transporters; they are thought to be translocated within the plants as trivalent ions, though the mechanism is unknown (Babula et al., 2008). Exposure to low concentrations (10-200 ppm) of some REEs, in particular Ce, Nd, lanthanum (La), and samarium (Sm), is known to enhance crop production. They can increase photosynthesis, uptake and utilization of nutrients and water, and enhance respiration and stress tolerance (Emmanuel et al., 2010). Many of these effects are proposed to be due to REEs interfering with plant uptake of Ca(II) (Diatloff et al.,

2008, Zeng et al., 2000) or Mg(II) (Liu et al., 2009). Diatloff et al. (2008) found that exposure to

Ce and La had no effect on shoot length in corn, but concentrations of micro-nutrients in corn shoots were generally decreased, especially Ca; Mg and P concentrations were found to have increased. However, the findings of Shtangeeva, in 2014 showed that uptake of Eu and Ce had no impact on the concentrations of micro-nutrients within tissues of rye and wheat seedlings grown in soils with 10-50 mg/kg of REEs.

At concentrations of 1.20 - 2.40 mM, Hu et al. (2016) found that La caused rupturing of chloroplast ultrastructure, as well as decreased concentrations of mineral elements within the chloroplasts of rice. At much higher concentrations (400-1500 ppm), REEs have been reported to reduce plant biomass (Carpenter et al., 2015; Thomas et al., 2014). Thomas et al. (2014) determined toxic effect concentrations (EC25) for Y, La, and Ce, using REE chloride salts, for three crop plant species and three plant species native to Canada, in (OECD) artificial soil. The

EC25 values for Ce, one of the elements examined in this research, ranged from 55 to 239 mg/kg of dry soil (Thomas et al., 2014). Toxic effect concentrations (EC25, EC50) were determined for the same six plant species, but for REEs praseodymium (Pr), terbium (Tb), dysprosium (Dy), erbium (Er), Nd, and Sm, following the same procedure as the previous study (Carpenter et al.,

2015). The EC25 and EC50 values for Nd, another of the elements examined in this research, ranged from 216 to 1025 mg/kg of dry soil and 455 to 1482 mg/kg of dry soil, respectively

(Carpenter et al., 2015). All of these effect concentrations are expressed as total metal in the soil.

2.2 Hormesis

Hormesis is a bi-phasic stress 120 response. This means that, 100 A depending on the concentration of

80 B the stressor, the organism exhibits 60 D both stimulation and inhibition of % Response % 40 the measured endpoint. Thus, the C 20 exposure concentration-response curve is either a “β-curve” or “j- 0 0 5 10 15 curve” (Figure 1D), instead of the Concentration Figure 1: Hypothetical exposure concentration- more common linear (Figure 1A), response curves depicting (A) threshold, (B) linear non-threshold, (C) sigmoidal, and (D) J-shaped threshold-linear (Figure 1B), or hormetic models. sigmoidal curves (Figure 1C) (Hanekamp,(Hanekamp, 2008). Hormesis 2008) is hypothesized to be due to an organism’s overcompensation in response to low levels of stress, leading to increased fitness (Calabrese and Baldwin, 2003; Hanekamp, 2008; Kefford et al., 2008). This can result in stimulation of the parameter measured by up to 30-60% over the control. Hormesis has been observed in plants in response to ozone (Finnan et al., 1996), nanoparticles (Nascarella and

Calabrese, 2012), and herbicides (Cedergreen et al., 2005). For metal stress, the biochemical response is typically stimulation of antioxidant systems or detoxification molecules (DalCorso et al., 2013). Plants that tolerate or thrive in a metal rich environment, known as metallophytes,

exposed to As, Cd, Cu, Ni, Pb and/or Zn, expressed more proteins linked to energy and carbohydrate metabolism, cellular metabolism, and stress and antioxidant mechanisms, but did not show increased expression of proteins for metal chelation or transportation (Visioli and

Marmiroli, 2013). These effects could be linked to hormesis, as these elements are not essential at high concentrations. Babula et al. (2008) proposed that REEs can induce hormetic effects in plants. This is supported by showing growth promotion at low concentrations and toxicity at high concentrations as mentioned previously (Carpenter et al., 2015; Liu et al., 2009; Thomas et al.,

2014). Experiments conducted on rice with Ce (Xia et al., 2013) and La (Hu et al., 2016) found hormetic responses in mitochondrial metabolism and chloroplast ATPase transcription, respectively. Research conducted by He and Loh (2000) which showed increased shoot length of

Arabidopsis thaliana at concentrations of Ce and La up to 2.5 µM, and decreased shoot length at

10-50 µM, further supports this hypothesis.

Though hormesis is rarely included in the derivation of toxic effect concentrations, the response is widespread across toxic agents, endpoints, and species. Whether or not hormesis is included in the derivation of the effect concentration can misidentification, which can translate further up the regulatory chain to the establishment of inaccurate SQGs. If the EC used for determination of the SQG is higher due to inclusion of hormesis, it might lead to a less environmentally protective guideline than intended (Kefford et al., 2008). Since a controlled range in concentrations of REEs in soils will be used for the present study, it is likely that hormesis will be identified.

2.3 Bioaccessibility

Metal in soil is partitioned between exchangeable metal and solid phase metal.

Exchangeable metal is either bound to readily exchangeable sites on soil solids or dissolved in the soil solution. The ratio of solid-liquid partitioning is expressed as Kd (Degryse et al., 2009), the larger the value of Kd, the greater the distribution to the soil. Thus, bioaccessible metal is considered to be the metal within the exchangeable or liquid portion and accessible by soil organisms. Metal bioaccessibility is dependent on physical and chemical soil characteristics including effective cation exchange capacity (eCEC), soil pH, % organic carbon, and % clay, which all contribute to variation in Kd (Degryse et al., 2009). Bioaccessibility can be estimated by single extraction, such as with CaCl2 (Brand, 2009), or through biogeochemical modelling

(Degryse et al., 2009).

2.4 Hypotheses

To address the overall objective to determine toxic effect concentrations for REEs, in the context of soil chemistry, the following three hypotheses were tested:

1) Toxic effect concentrations for Ce, Eu and Nd are similar among radish, tomato and

durum wheat;

2) The major variation in lanthanide toxicity among the three plant species can be attributed

to differential tissue concentration;

3) Toxic effect concentrations are more strongly correlated with bioaccessible REE

concentrations than by the total REE concentrations in the soil.

3.0 Materials and Methodology

3.1 Range-finding studies

3.1.1 Soil preparation, analysis and amendment

Black garden soil with no known history of contamination was purchased from

Greenhorizons, Cambridge, ON in 2016. The soil was air-dried and sieved to less than two millimeters prior to analysis and use. Soil texture and content of inorganic and organic carbon were determined by University of Guelph Laboratory Services. Soil organic matter (SOM) was calculated from the total organic carbon (TOC) and the van Bemmelen factor (1.724) following

Equation 1 (Pribyl, 2010). Soil pH was determined by the H2O slurry method (Carter, 1993), whereby 2 g of oven-dried soil was mixed with 20 mL of water, stirred for 30 minutes, allowed to sit for 1 hour and measured by a pH meter (Mettler Toledo SevenExcellenceTM Benchtop pH/Conductivity Meter). Electrical conductivity (EC) was also measured on samples following preparation for pH measurement by use of a conductivity probe attached to the pH meter.

푆푂푀 = 푇푂퐶 ∗ 1.724 (Equ. 1)

Pseudo-total (hereafter referred to as “total”) concentrations of background Ce, Eu and

Nd were determined by following a version of U.S. EPA Method 3051a modified by using an oven (110oC) instead of microwave heating (U.S. EPA, 2007). In this procedure, 0.5 g of oven- dried soil was placed into a Teflon digestion vessel with three mL of HCl and nine mL of HNO3

(reagent grade, Sigma Aldrich Canada Co., Oakville, ON). The vessel was then sealed and placed into an oven at 110oC for 14 hours. Following digestion, samples were filtered using

Whatman 42 acid washed filter paper, diluted to 25 mL with ultrapure water, and analysed by

ICP-OES (Varian Vista Pro ICP-OES with axial viewed plasma, LOD = 0.1 ppm). Hydrochloric acid was used in this procedure rather than the more dangerous hydrofluoric acid, and as such the measurement was of “pseudo-total” rather than “total” metal concentration. This method was also followed for analysis of soil nutrients (Al, Fe, K, Mg, Mn, P, S, Zn) before experimental use. All samples were analysed in triplicate alongside a blank and two standard reference materials, NIST SRM 2711a (Montana II Soil, National Institute of Standards Technology) and

CRM SU-1b (Nickel-Copper-Cobalt Ore, Natural Resources Canada).

Extraction with a 0.01 M CaCl2 solution identified the concentrations of plant-available background and experimental REEs and nutrients: this is the “bioaccessible” fraction of the element. To prepare the 0.01 M CaCl2 solution, 1.47 g of CaCl2 dehydrate was added to 1 L of ultrapure water. A 1:10 ratio of oven-dried soil to CaCl2 solution was placed onto a rotary shaker for 2 hours, then filtered through Whatman 42 filter paper (Gupta and Sinha, 2007; Houba et al.,

2000). The resulting solution was then analysed by ICP-MS (Laboratory Services at the

University of Guelph, Bruker 820-MS with CETAC ASX-520 autosampler, LOD = 1 ppb). This extraction method was chosen because it is considered one of the best weak extraction methods, and due to its’ previous use in the lab.

This soil was then divided for amendment with REEs. The experimental design was a two factor factorial: four REE concentrations, and three REEs. A single reference treatment was used concurrently with the 12 treatments. Three replicates of each treatment were used for a total of 39 concurrent experimental units. Within each experimental unit there were five plants which were treated as subsamples and averaged.

Nitrate salts of Ce (Ce(NO3)3·6H2O), Eu (Eu(NO3)3·5H2O), Nd (Nd(NO3)3·6H2O)

(obtained from Sigma Aldrich Canada Co., Oakville, ON) were selected with the expectation that this form would minimize the impact of the non-REE component on plant health (Smart

Fertilizer Inc., 2016); and because nitrate salts are highly soluble in water. Exposure- concentration ranges were chosen based on review of the literature as well as crustal concentrations of these elements. These ranges (Table 1) were chosen to encompass previously reported EC25s, be large enough to determine EC50s, and allow for potential reductions in bioaccessibility because of soil properties. Four concentrations of each element were chosen for range-testing, to ensure that the range of concentrations included toxic effect concentrations.

Table 1: Nominal concentrations of REE-amended soils for the range-finding test (mg/kg dry soil)

Element Ref 1Trt 1 Trt 2 Trt 3 Trt 4 Cerium 0 160 2500 5000 7400 Europium 0 150 1100 3600 5000 Neodymium 0 110 1000 2500 5000 1Trt = treatment

To amend the soil, 1.1 kg of air-dried soil was added to a large zipper-seal bag. The necessary mass of REE(NO3)3 to achieve each treatment concentration was dissolved in 1.5 L of ultrapure water, which was then added to the bag and mixed by hand into the soil (Environment

Canada, 2007). At this time, 1.5 L of water was also added to the reference/control soil. The soil was aged in open bags at room temperature for 14 days while being mixed by hand for approximately five minutes each day to ensure even distribution of the REE, as well as to allow solid-liquid partitioning of the REEs to stabilize, as metal bioaccessibility in amended soils tends to be higher than in field contaminated soils (Smolders et al., 2009). During the aging period, soil

was allowed to air dry, as the slurry formed was not suitable for plant growth. After aging, the soil was reanalysed for the same parameters as before spiking to quantify the effects of amendment. Soil was then partitioned equally among the three replicate pots for each treatment each of 300 g dry soil, and closed with lids; the pots were 1 L clear polypropylene containers with lids, 10 cm ø x 20 cm h.

3.1.2 Plant assay

Radish (Raphanus sativus, var. ‘Cherry Belle’) was chosen as the test species for the range-finding experiment, as described in the Environment Canada Biological Test Method: Test for Measuring Emergence and Growth of Terrestrial Plants Exposed to Contaminants in Soil. It was chosen due to its ability to grow well in various soil types, has a known sensitivity to metals, and is recommended as a test species for toxicity testing with established validity criteria

(Environment Canada, 2007).

Radish seeds were germinated before planting by enclosing seeds in a petri dish with a damp paper towel for 2 days prior to planting. On the day of planting, five seedlings were placed approximately 0.5 cm below the soil surface, covered over with soil, watered to approximately

75% of the soil water holding capacity, and covered with lids according to Environment Canada protocol (Environment Canada, 2007).

All pots were then transferred to a controlled environment chamber with a photoperiod of

18:6 hour light:dark, with lighting set to 300 µmol/m2·s at the soil surface, humidity at 60%, day time temperature at 24 ± 3 oC, and night time temperature at 15 ± 3 oC, for the duration of the 14 day test (Environment Canada, 2007). During the 14 day growth period, ultrapure water was

sprayed onto the soil surface every 1-2 days to achieve approximately 75-80% soil water holding capacity (by weight). Once plants began to touch the lids of the pots, lids were removed.

On the 14th day of growth, plants in each pot were photographed and then processed.

Plants were carefully removed from the soil so as not to break roots and then rinsed thoroughly with ultrapure water to remove all soil particles. Shoot length and hypocotyl length of each plant were recorded, as well as the longest root length in each pot. Lengths were measured by laying the plants out flat against a ruler and recording length to the nearest 0.5 cm. Plants were then cut at the joint between root and shoot (the hypocotyl included with the roots), placed into separate paper bags, and oven dried at 65oC for one week.

Following plant harvest, samples of soil, as well as root and shoot tissue, were analysed for total REE content, according to the method previously described. Modifications to the method were made to keep the same acid volume to sample mass ratio, as some treatments had low plant mass. Due to low total REE concentrations in plant tissues, ICP-MS was used, because it has lower detection limits than ICP-OES. Bioaccessible REE concentrations in the soil extracted by CaCl2, and pH by the H2O slurry method, were also determined as described previously.

3.2 Definitive studies

Based on the range-finding test carried out with radish in soils with four REE concentrations, the concentration range for further experimentation was determined. Variations in methods from the range-finding experiment are discussed in section 3.2.1.

3.2.1 Soil Preparation, analysis and amendment

More soil was purchased from Greenhorizons in 2017 for the definitive studies.

Differences in background REE content, fertility, texture and pH are noted below. A 1:1 mix of this soil with the soil left from the range-finding study (unamended) was used for the following experiments.

The net nitrification rate (NNR) was measured before amendment of the soil with REEs on the assumption that amendment would not change it. The NNR is a measurement of soil health, and indicates the nitrification capacity of the soil microorganisms; it is the rate at which ammonium in the soil is converted to nitrate by soil bacteria, and is dependent on the level of

+ NH4 in the soil as well as environmental factors. It was measured over time by adding 100 mL of 2 M KCl to one 20 g sample of soil; this was the initial, or day 0, time point. Six other 20 g samples were brought to field capacity with ultrapure water and set aside. The day 0 sample with

KCl solution was shaken on a rotary shaker for 1 hour before being filtered through a Whatman

42 filter paper. On days 1, 3, 7, 14, 21, and 28 the extraction process was repeated with one of

+ the six samples set aside initially (at field capacity). All filtrates were analysed for NH4 and

- - NO3 and the net nitrification rate calculated as the change in NO3 concentration over time

(Carter and Gregorich, 2008). Ammonium and nitrate were both measured using a SEAL AAIII auto analyzer, following method G-102-93 Rev.7 and G-200-97 Rev.6, respectively.

This soil was then divided for amendment with REEs. Separate experiments were carried out for each REE. For each REE experiment, the experimental design was a two factor factorial: nine REE concentrations including the reference, and two plant species. There were five

replicates of each treatment for a total of 90 concurrent experimental units. Within each experimental unit there were five plants which were treated as subsamples and averaged.

Soil was amended with REEs as previously described, with the following modifications.

REE concentrations were chosen to expand the range, with the intent to achieve a greater than

50% decrease in plant endpoints. In addition to using a positive control, eight concentrations were chosen in even increments of 1000 mg/kg for Ce and Nd, from 1000-8000 mg/kg dry soil, and for Eu from 750-6000 mg/kg dry soil, in increments of 750 mg/kg (Table 2).

Table 2: Nominal concentrations of REE-amended soil for the definitive test (mg/kg dry soil)

Element Ref Trt 1 Trt 2 Trt 3 Trt 4 Trt 5 Trt 6 Trt 7 Trt 8 Cerium 0 1000 2000 3000 4000 5000 6000 7000 8000 Europium 0 750 1500 2250 3000 3750 4500 5250 6000 Neodymium 0 1000 2000 3000 4000 5000 6000 7000 8000

The increasing amount of nitrate across treatments that was added to soil as the counter- ion in REE-nitrate was identified as a potentially confounding factor. To address this, KNO3 was added to all treatments at a concentration that, when added to the NO3 from the REE-nitrate treatment, would make the NO3 in all treatments equal. This was also applied to the control treatment, resulting in the positive control.

Five replicate pots were used, and as such, soil was amended in larger batches of 4.5 kg, in large heavy duty garbage bags, to prevent soil from leaking out. In order to hydrate the soil evenly, 4 L of water were added to each treatment, of which 1 L was used to dissolve the REE- nitrate, another 1 L to dissolve the KNO3, and 2 L used to rinse the containers to ensure no residue was left in the glassware.

3.2.2 Plant assay

As radish had been studied previously, two other plant species were chosen; one dicotyledonous species and one monocotyledonous species. The dicotyledonous species was tomato (Solanum lycopersicum, var. ‘Bonnys best’), due to its ability to grow well in a variety of soils, its demonstration of clear dose-response relationships, and its relevance to Canadian horticulture (Environment Canada, 2007). The monocotyledonous species chosen was durum wheat (Triticum durum, var. ‘Kyle’), for many of the same reasons as tomato, as well as its quick time to germination and emergence. Both species are recommended test species from the

Environment Canada protocol that was followed (Environment Canada, 2007). The varieties of both species had been used previously in this laboratory. Tomato took 4 days to germinate; in comparison, durum wheat and radish took only 2 days.

In addition to the overall longest root length for each pot, the five plants were separated from each other and the lengths were measured separately, as sub-samples. The total dry mass of plant tissue in each pot was also recorded, following one week of drying in an oven at 65oC.

Bioaccessible REE concentration of soil was only assessed for Ce, in order to confirm that it followed the same trend as previously noted.

3.3 Positive control assessment

In the previous study, plants grown in positive control (KNO3 amended) soil did not grow as well as expected, especially when compared to the negative control (unamended soil) plants from the range-finding experiment. To assess the effect of KNO3, a comparison study was undertaken, where all three species (radish, tomato, durum wheat) were grown under both negative and positive control conditions.

3.3.1 Soil amendment

The 1:1 soil mix described above was used for this experiment. The positive control was amended with only KNO3, to achieve the nitrate concentration used in the definitive study experiment. The negative control was hydrated with water only and kept free of KNO3.

3.3.2 Plant Assay

Seeds for all species were pre-germinated so all planting occurred on the same date; plants grew for 14 days, and were then harvested. Due to the large mass of the roots for the negative control plants, only the longest root length per pot, and the overall wet and dry mass per pot, were measured.

3.4 Leaching experiment

An experiment was undertaken to assess the degree to which nitrate in the soil that was amended with the REE-nitrates could be leached, in an attempt to understand this potentially confounding variable.

3.4.1 Soil preparation

Soil from the initial range-finding experiment was used for this study, as it had been amended with REEs and had nitrate concentrations that were confounded with the REE concentration effects. This soil did not have KNO3 added to it, so only the nitrate in the REE salt needed to be leached to achieve equal nitrate levels across the treatments. The soil amended with the Ce nitrate salt was selected, because it had the best defined exposure concentration-response in the range-finding test.

To remove nitrate from the soils, the soils were leached with artificial rainwater (Li et al.,

2010). Soil was placed into plastic containers from which most of the bottom had been replaced with a fine mesh screen. Artificial rainwater was placed into larger plastic containers, and the containers with the soil in them were placed into these larger containers to allow the soil to draw the rainwater up. Once the rainwater had reached the surface of the soil, one pore space equivalent of rainwater was added to the top of the soil, below the rim of the container, and allowed to sit overnight. Pore space equivalents were calculated as the amount of rainwater taken up into the soil, by subtracting the amount left in the large container from the amount added to it.

The next morning the containers with the soils in them were lifted out of the rainwater and allowed to drain for 24 hours, when samples of the leachate water and soil were collected for measurement of EC and pH. This procedure was repeated until the EC of the soil and leachate solution became equal across all treatments and remained stable between leaching events. The soil was then removed from the leaching apparatus, oven-dried at 105 oC, and the total Ce concentration measured.

Because this soil had been used in a previous experiment, there was some loss of soil from each treatment, so dried soil was divided evenly to create treatments with three replicate pots of slightly less than 300 g each. The day before planting, water was added to the pots of soil to approximately 60% of the water holding capacity.

3.4.2 Plant assay

For better comparison to previous experimental results from this soil, radish was chosen as the test species and planted as before with five seedlings per pot. Shoot length and hypocotyl

lengths of all plants were measured, as well total shoot dry mass per pot. The development of the hypocotyl was also visually assessed based on colour and shape for each plant.

Bioaccessibility of Ce in this soil was measured in order to determine the effect of leaching.

3.5 OECD Soil comparison

Previous research done with OECD artificial soil and chloride forms of REEs indicated that toxicity thresholds were much lower than what was identified in the current experiments. As such, it was undertaken to measure the bioaccessibility of REE-chlorides in OECD soil as well as the difference in bioaccessibility due to different soil properties.

3.5.1 Soil preparation

Artificial soil was prepared by mixing 10% peat, 20% Kaolin clay and 70% silica sand according to EC protocol (SOP 15.09/1.3/S). Water was added to the soil to bring it to an initial moisture of approximately 20%. To compensate for the acidity of the peat, calcium carbonate was added to the soil to achieve a soil pH of approximately 5.8. Soil was placed in a zipper seal bag and allowed to sit for at least 2 days before further use.

Chloride forms of Ce (CeCl3·7H2O), Eu (EuCl3·6H2O), and Nd (NdCl3·6H2O) were purchased from Sigma Aldrich, Canada. In order to test the full range of experimental concentrations, four concentrations were chosen; two lower concentrations within the range of previous work and two higher concentrations to cover the full range of the definitive studies. Soil amendment was carried out as before, however less water (100 mL) was used to dissolve the

REE-chloride due to the smaller mass of soil (200 g). For better comparison, artificial soil was

also amended with REE-nitrates at the same rates as the REE-chlorides. Soils were amended in zipper seal bags to allow for easy mixing and storage during 14 days of aging.

3.5.2 Bioaccessibility measurement

After 24 hours, and again after 14 days of aging, samples from each REE treated artificial soil were oven dried and extracted with 0.01 M CaCl2 solution according to the procedure described previously. At 14 days, soils were also analysed for pseudo-total REE content, as previously described.

3.6 Statistical analyses

3.6.1 Range-finding studies

Radish shoot length data were normalized to the negative control, by transformation to percent decrease in response where the negative control was 100%. In order to assess the datasets for hormesis, data for each element were analysed separately using hormetic and non-linear models in the drc package (Ritz et al., 2015) in R (version 3.4.3, R Core Team, 2017) and SAS

(version 9.4, Statistical Analysis System, SAS Institute Inc., Cary, NC, USA) statistical software.

Data were also graphed and analysed through fitting of polynomial curves using SigmaPlot, and the highest order polynomial model where each parameter was significantly different from zero was selected as the model. From this model, EC10, EC20, EC25 and EC50 effect concentration values were determined. The data were tested for normality using the Shapiro-Wilk test, and the

Levene’s test for constant variance. In all cases where data failed one or both of these tests, other model parameters were also not significant and the model was considered not to fit the data. For all statistical tests a significance level of α = 0.05 was used.

3.6.2 Definitive studies

The statistical analyses applied to definitive study data were the same as those used for the range-finding studies; the increased number of treatments was expected to increase the likelihood of identifying a significant model to fit the data, and identify hormesis, if present. The endpoints measured included shoot and root length, as well as shoot dry biomass. These were regressed against both the measured total soil concentrations of REEs and the internal tissue concentrations. To assess datasets for hormesis, Weibull models with a separate hormetic shape parameter and non-linear models were applied to the data. Following those, simpler polynomial models were fitted, and finally piece-wise regression was used in order to capture shape. This shape was either an initial decrease followed by a plateau, or initial plateau followed by a decrease, that was not captured effectively by linear models, which would have an impact on determination of effect concentrations. The most appropriate model for each dataset was determined by statistical significance of the parameters as well as use of the R2 values. Once significant concentration-response curves were plotted, and the equations determined, the effect concentrations (same as previous) were calculated. Intervals containing 95% of the observations were calculated for each effect concentration. The data were tested for normality using the

Shapiro-Wilk test, and the Levene’s test for constant variance. In all cases where data failed one or both of these tests, other model parameters were also not significant and the model was considered not to fit the data. For all statistical tests a significance level of α = 0.05 was used.

4.0 Results and Discussion

4.1 Soil analysis

4.1.1 Range-finding studies

Soil used for the range-finding study was obtained from Greenhorizons, and had no known history of contamination. The soil was a very dark clay loam with a high soil organic matter (49.5%) and neutral pH (6.97). Total concentrations of required plant nutrients were adequate to high (Table 3); bioaccessible concentrations were determined, though adequate bioaccessible concentrations are not well identified in the literature (Table 3). Background REE levels were close to crustal concentrations, and considered unlikely to cause an effect (Ce 20 mg/kg, Eu undetectable, Nd 93 mg/kg). Recovery of REEs following soil amendment was found to be consistent with dosing (Table 4).

Table 3: Total and bioaccessible (mg/kg dry soil) plant nutrients in black garden soil prior to use for range-finding study (NM: not measured)

Al Fe K Mg Mn P S Zn Total 10615 9700 2850 7340 270 1200 4530 54 Bioaccessible < 0.01 3.6 540 600 2.5 3.6 NM < 0.01

Table 4: Mean (± SD) total and bioaccessible REE concentrations (mg/kg) of each amended dry soil at the end of the 14-day test

REE Ref Trt 1 Trt 2 Trt 3 Trt 4 19 158 2526 4999 7397 Total (± 0.6) (± 6.1) (± 63) (± 69) (± 131) Ce 0.01 0.13 0.67 1.65 2.62 Bioaccessible (± 0.002) (± 0.02) (0.01) (± 0.08) (± 0.51) 229 787 2180 4302 Total < 5 (± 14) (± 18) (± 106) (± 93) Eu 0.12 0.42 0.75 1.29 Bioaccessible < 0.01 (± 0.02) (± 0.08) (± 0.10) (± 0.21) 76 154 1291 3570 5036 Total (± 1.5) (±8.1) (± 131) (± 308) (± 26) Nd 0.01 0.10 0.50 1.04 1.92 Bioaccessible ( ± 0.0004) (± 0.01) (± 0.11) (± 0.16) (± 0.18)

There was a linear relationship between total and bioaccessible [REE] in the soil following plant growth, where the bioaccessible portion was 0.03% (i.e. the slope was 0.0003) of the total for Ce and Eu (Figures 2, 3) and 0.04% for Nd (Figure 4). The low bioaccessibility is speculated to be due to the physicochemical characteristics of this soil, namely the high organic matter content. Soil organic matter provides many opportunities for REE binding and complexation, reducing the amount that is in the soil solution (Basta et al., 2005).

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

-0.5 Confidence Band R2 = 0.95 Bioaccessible Ce concentration (mg/kg) concentration Ce Bioaccessible -1.0 0 1000 2000 3000 4000 5000 6000 7000 8000 Total Ce concentration (mg/kg)

Figure 2: Relationship between total and bioaccessible Ce in soil for the range-finding study. Confidence bands are set at 2.5% and 97.5%.

2.0

1.5

1.0

0.5

0.0 Confidence Band Bioaccessible Eu concentration (mg/kg) concentration Eu Bioaccessible R2 = 0.95 -0.5 0 1000 2000 3000 4000 Total Eu concentration (mg/kg) Figure 3: Relationship between total and bioaccessible Eu in soil for the range-finding study. Confidence bands are set at 2.5% and 97.5%.

2.5

2.0

1.5

1.0

0.5

0.0 Confidence Band 2 Bioaccessible Nd concentration (mg/kg) Nd concentration Bioaccessible R = 0.96 -0.5 0 1000 2000 3000 4000 5000

Total Nd concentration (mg/kg)

Figure 4: Relationship between total and bioaccessible Nd in soil for the range-finding study. Confidence bands are set at 2.5% and 97.5%.

4.1.2 Definitive studies

Soil used for the definitive studies was the 1:1 mixture of soil used for the range-finding study and black garden soil purchased the following year, which had the same texture and colour as the soil used for the range-finding study. Fertility testing showed that total concentrations of required plant nutrients were adequate to high (Table 5); bioaccessible nutrient concentrations were determined (Table 5), though adequate bioaccessible concentrations are not well identified in the literature. Background REE levels were again close to crustal concentrations, and considered unlikely to cause an effect (Ce 10 mg/kg, Eu undetectable, Nd 78 mg/kg). REE concentrations following amendment and plant growth are shown in Table 6. Bioaccessible REE concentrations are shown in Table 7.

Table 5: Total and bioaccessible plant nutrients (mg/kg) in black garden soil prior to use for definitive studies (NM: not measured)

Al Fe K Mg Mn P S Zn Total 7765 10352 2812 8328 NM 893 8087 39 Bioaccessible 5.3 6.3 883 670 NM 20 1170 NM

Table 6: Mean (± SD) total REE concentrations (mg/kg) in soil from each treatment at the end of the 14-day definitive tests with wheat and tomato

Plant REE Ref Trt 1 Trt 2 Trt 3 Trt 4 Trt 5 Trt 6 Trt 7 Trt 8 species 18 800 1589 2822 3646 4442 5344 6862 7824 Wheat (± 1.4) (± 16) (± 49) (± 149) (± 221) (± 125) (± 336) (± 277) (± 190) Ce 20 773 1525 2426 3362 4372 5050 5869 6739 Tomato (± 0.79) (± 28) (± 41) (± 61) (± 186) (± 196) (± 263) (± 94) (± 127) 613 1144 1688 2568 3238 3899 4604 5270 Wheat < 5 (± 16) (± 62) (± 70) (± 142) (± 56) (± 275) (± 306) (± 148) Eu 570 1123 1651 2480 3108 3838 4049 4897 Tomato < 5 (± 21) (± 42) (± 69) (± 54) (± 51) (± 157) (± 229) (± 125) 91 818 1556 2586 3383 4332 4972 6011 6689 Wheat (± 0.71) (± 33) (± 47) (± 35) (± 225) (± 136) (± 250) (± 80) (± 265) Nd 98 883 1658 2605 3505 4574 5446 5885 6917 Tomato (± 2.1) (± 19) (± 45) (± 95) (± 118) (± 109) (± 232) (± 371) (±305)

Table 7: Mean (± SD) bioaccessible REE concentrations (mg/kg) of Ce and Nd at the end of the 14-day definitive tests with wheat

Plant REE Ref Trt 1 Trt 2 Trt 3 Trt 4 Trt 5 Trt 6 Trt 7 Trt 8 species 0.01 0.13 0.31 0.46 1.09 0.83 1.03 1.09 1.61 Ce Wheat (± 0.01) (± 0.03) (± 0.09) (± 0.10) (± 0.64) (± 0.13) (± 0.29) (± 0.26) (± 0.30) 0.13 0.46 0.45 0.69 0.99 0.99 0.93 1.71 Nd Wheat < 0.01 (± 0.04) (± 0.08) (± 0.19) (± 0.27) (± 0.13) (± 0.51) (± 0.45) (± 0.62)

Based on the Ce treatments, the same linear relationship between total and bioaccessible concentrations observed in the range-finding study was observed with the new soil and fuller

range of concentrations (Figure 5). However, greater variation in the data was observed, which might be partially due to the larger number of samples. The range of bioaccessibility was also somewhat depressed when compared to the range-finding test; the highest total Ce concentration corresponded to a lower bioaccessible Ce concentration than observed for the range-finding test

(definitive study slope of 0.0002, range-finding slope of 0.0003). This might be due to the

+ - increased concentrations of K and NO3 in the solution due to amendment with KNO3 causing interference with both extraction of Ce from the soil, as well as quantification of the Ce in solution.

2.5

2.0

1.5

1.0

0.5

0.0

-0.5 Confidence Band Bioaccessible Ce concentration (mg/kg) Ce concentration Bioaccessible R2 = 0.73 -1.0 0 1000 2000 3000 4000 5000 6000 7000 8000 Total Ce concentration (mg/kg) Figure 5: Relationship between total and bioaccessible Ce in soil for the 14-day definitive test with durum wheat. Confidence bands are set at 2.5% and 97.5%.

- In the NNR test, the concentration of NO3 increased until day 14 then plateaued (Figure

- 6). When tested statistically, the slope of the apparent drop in NO3 concentration was not different from zero. The initial rate was 3.69 mg/L·day-1, and the slope of days 14 to 28 was not

+ different from 0. The concentration of NH4 however, remained low over the course of the 28 days (data not shown).

The positive NNR during the first 14 days shows that the soil contains healthy nitrifying bacteria that are able to convert ammonium, which has low plant availability, to nitrate, which is generally the dominant form of plant-available nitrogen (International Plant Nutrition Institute,

2018). The plateau after 14 days is likely due to nitrification reaching equilibrium where the bacteria are converting ammonium to nitrate at the same rate that nitrate is converting to other forms of nitrogen.

160

140

120

100

concentration (mg/L) concentration

80 Confidence Band

Soil NO

60 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

Day - Figure 6: Nitrification test NO3 concentration (mg/L) over time for 28 days in definitive test soil. Confidence bands are set at 2.5% and 97.5%.

4.2 Toxic effect concentrations related to total soil concentration

4.2.1 Range-finding study

All three REEs caused concentration-dependent decreases in plant size, to which quadratic curves could be fit (Figures 7, 8, 9). Shoot length data had less variability within treatments than root length data, likely due to breakage of roots which occurred during removal from pots and soil. This breakage would bias toward underestimating root length. Due to this inconsistency, threshold concentrations were not determined for radish roots. When compared to the control, Ce, Eu and Nd all caused a significant reduction in shoot length. EC10, EC20 and

EC25 values were within the bounds of experimental concentrations for Ce, Eu and Nd; however

EC50 values were bounded only for Ce and Eu (Table 8). Sensitivity of radish shoot length to

REEs increased from Nd (least sensitive), to Eu (most sensitive).

130 120 Confidence Band 110 y= 104 - 0.0005x - 1.3E-6x2 100 R2 = 0.97 90 80 70 60 50

Shoot length (%) Shoot length 40 30 20 10 0 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 Cerium concentration (mg/kg) Figure 7: Concentration-response curve for shoot length of radish when exposed to Ce amended soil for 14 days. The approximate EC50 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%.

130 120 Confidence Band 110 y = 103.4 - 0.013x 100 R2 = 0.83 90 80 70 60 50

Shoot length (%) Shoot length 40 30 20 10 0 0 1000 2000 3000 4000 5000 Soil Eu concentration (mg/kg) Figure 8: Concentration-response curve for shoot length of radish when exposed to Eu amended soil with approximated EC50 value. The approximate EC50 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%.

140 130 120 110 100 90 80 70 60 50

Shoot length (%) Shoot length 40 30 Confidence Band 2 20 y = 99.2 + 0.02x - 5.4E-6x 2 10 R = 0.94 0 0 1000 2000 3000 4000 5000

Soil Nd concentration (mg/kg) Figure 9: Concentration-response curve for shoot length of radish when exposed to Nd amended soil with approximated EC50 value. No EC50 could be approximated. Confidence bands are set at 2.5% and 97.5%.

Table 8: Effect concentrations (EC10, EC20, EC25, EC50) and 95% confidence intervals for radish shoot length exposed to REEs (mg/kg dry soil). 95% CI excluded due to lack of significance at measurable precision level.

REE EC10 EC20 EC25 EC50 Ce 3095 4109 4535 6256 Eu 1038 1813 2201 4139 Nd 4065.6 4444.7 4614.9 >5000

4.2.2 Definitive studies

Growth inhibition of tomato and durum wheat was observed for shoots as well as roots.

While some breakage of roots is inevitable during the soil removal process, for these plant species it was deemed to be negligible and the root data were statistically analysed, as were shoot

- data. The positive control (highest level of NO3 , added as KNO3) plants overall were found to be approximately 30-50% of the size of negative control plants (Figure 10). This discrepancy between the negative and positive controls can be attributed to the nitrate added to the positive control, the same nitrate concentration as in the highest REE concentration treatment. While nitrate is a plant nutrient, at high levels (greater than 10 mM, d’Aquino et al., 2009) it can be toxic, and it contributes to soil salinity. Dissolved salts, including nitrate, contribute to the EC of the soil (Grisso et al., 2009), which in this experiment was found to be consistently high (in the range of 3500-5000 µS/cm). In Alberta, an EC of 2000 µS/cm is recommended for salt remediation of agricultural land, soil with an EC of 4000-8000 µS/cm is considered poor, and for crop production EC of >8000 µS/cm is unsuitable (Alberta Environment, 2001). The high EC in the positive control likely explains the discordance with the negative control. However, because the nitrate levels were kept constant across the treatments through application of KNO3, the growth response measured in the definitive studies is most likely caused by the REEs.

The concentration-responses for tomato roots and shoots are shown in Figures 11-16, and Figures 17-22 show the concentration-responses of durum wheat roots and shoots to Ce, Eu and Nd. Root and shoot lengths are normalized to the positive control. Depicted on each graph is also the estimated EC20 value for the response endpoint. A linear model, either continuous or segmented, was fitted to each dataset. Several types of hormetic models were assessed, including piecewise regression for positive slope in the first segment; however, none of the exposure concentration-responses demonstrated hormesis, which was unexpected considering previous research that suggests plant responses to REEs are often hormetic (Babula et al., 2008; d’Aquino et al., 2009; He and Loh, 2000; Hu et al., 2016; Xia et al., 2013). This may be due to the concentrations assessed in the present study being much higher than those which have been

shown to cause beneficial effects, therefore causing hormesis to be hidden between lower concentration data points. For all three REEs, there was a threshold in tomato root length response: for Ce and Nd the decrease in length occurred after a threshold concentration, whereas for Eu the roots decreased at the lower concentrations and then reached a threshold where concentration no longer had an effect. Tomato shoot responses were negative and linear for all three REEs. Durum wheat was different: for Ce, both root and shoot responses were negative and linear, for Eu the shoot response was negative and linear, but the root response had a threshold, and for Nd the root and shoot responses both had thresholds. Effect concentrations (EC10, EC20,

EC25) were calculated from each model (Table 9). Many of the higher effect concentrations, including all EC50s, were greater than the concentrations tested in this experiment. For Ce and

Eu, effect concentrations for tomato were lower than for durum wheat; the opposite is the case for Nd.

Toxic effect concentrations for Ce and Nd identified by Carpenter et al., (2015) and

Thomas et al., (2014) were much lower than the effect concentrations identified in this experiment. Those effect concentrations (IC25) were determined for plant shoot biomass (which is highly correlated with shoot length) for tomato exposed to Ce and Nd, which were 315 mg/kg and 1025 mg/kg, respectively. This contrasts with the EC25s from this experiment for Ce and Nd of 6130 mg/kg and 6350 mg/kg, respectively. The discordance between these values might be due to the differences in the soil used for that study, namely OECD artificial soil, whereas commercial garden soil was used in the present study. These two soils have very different characteristics (Table 13); in particular, garden soil has a much higher SOM (50% as opposed to

8% in artificial soil), and higher clay content, which might contribute to low REE bioaccessibility (Basta et al., 2005), and thus higher soil effect concentrations.

40 Positive control shoot length 35 Negative control shoot length Positive control root length Negative control root length 30

Length (cm) Length 15

0 Radish Tomato Durum wheat Plant species Figure 10: Mean (±SD) shoot and root lengths of radish, tomato and durum wheat grown in positive and negative control soils with and without KNO3 respectively.

130 Confidence Band 120 110 100 90 80 70 60 y = -0.0006x + 101.43 N.S. y = -0.0066x + 127.65 2 50 R2 = 0.01 R = 0.29 40 30 20

Root length (% control) ofRoot positive length 10 0 0 1000 2000 3000 4000 5000 6000 7000 Soil Ce concentration (mg/kg) Figure 11: Root length of tomato exposed to Ce amended soil for 14 days. Solid line at 80% root length shows that the EC20 was not bounded. Confidence bands are set at 2.5% and 97.5%. N.S. denotes that the relationship is not significant.

140 130 Confidence Band 120 y = 100.74 - 0.0042x 2 110 R = 0.50 100 90 80 70 60 50 40 30 20

Shoot length (%control) of Shootpositive length 10 0 0 1000 2000 3000 4000 5000 6000 7000 Soil Ce concentration (mg/kg)

Figure 12: Shoot length of tomato exposed to Ce amended soil for 14 days. Approximated EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%.

140 y = -0.027x + 98.99 y = -0.0023x + 67.50 N.S. 130 R2 = 0.49 R2 = 0.06 120 Confidence Band 110 100 90 80 70 60 50 40 30 20

Root length (%control) ofRoot positive length 10 0 0 1000 2000 3000 4000 5000 Soil Eu concentration (mg/kg)

Figure 13: Root length of tomato exposed to Eu amended soil for 14 days. Approximated EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%. N.S. denotes that the relationship is not significant.

130 120 110 100 90 80 70 60 50 Confidence Band 40 y = 100.39 - 0.0035x 30 R2 = 0.22 20

Shoot length (%control) of Shootpositive length 10 0 0 1000 2000 3000 4000 5000 Soil Eu concentration (mg/kg) Figure 14: Shoot length of tomato exposed to Eu amended soil for 14 days. Solid line at 80% shoot length shows that the EC20 was not bounded. Confidence bands are set at 2.5% and 97.5%.

130 Confidence Band 120 110 100 90 80 70 60 50 y = 0.0006x + 97.40 N.S. y = -0.0072x + 129.61 R2 = 0.0041 R2 = 0.30 40 30 20

Root length (%control) ofRoot positive length 10 0 0 1000 2000 3000 4000 5000 6000 7000 Soil Nd concentration (mg/kg)

Figure 15: Root length of tomato exposed to Nd amended soil for 14 days. Approximated EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%. N.S. denotes that the relationship is not significant.

120 Confidence Band 110 y = 99.77 - 0.0039x 2 100 R = 0.59 90 80 70 60 50 40 30 20 Shoot length (%control) of Shootpositive length 10 0 0 1000 2000 3000 4000 5000 6000 7000 Soil Nd concentration (mg/kg)

Figure 16: Shoot length of tomato exposed to Nd amended soil for 14 days. Approximated EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%.

140 y = -0.004x + 107.06 130 R2 = 0.4463 120 Confidence Band 110 100 90 80 70 60 50 40 30 20

Root length (%control) ofRoot positive length 10 0 0 1000 2000 3000 4000 5000 6000 7000 8000 Soil Ce concentration (mg/kg) Figure 17: Root length of durum wheat exposed to Ce amended soil for 14 days. Approximated

EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%.

170 160 Confidence Band y = 114.81 - 0.0029x 150 2 140 R = 0.13 130 120 110 100 90 80 70 60 50 40 30

Shoot length (%control) of Shootpositive length 20 10 0 0 1000 2000 3000 4000 5000 6000 7000 8000 Soil Ce concentration (mg/kg) Figure 18: Shoot length of durum wheat exposed to Ce amended soil for 14 days. Solid line at

80% root length shows that the EC20 was not bounded. Confidence bands are set at 2.5% and 97.5%.

140 y = 0.0043x + 96.52 y = -0.0070x + 116.41 2 130 R = 0.065 R2 = 0.45 120 Confidence Band 110 100 90 80 70 60 50 40 30 20

Root length (%control) ofRoot positive length 10 0 0 1000 2000 3000 4000 5000 Soil Eu Concentration (mg/kg) Figure 19: Root length of durum wheat exposed to Eu amended soil for 14 days. Approximated

EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%. N.S. denotes that the relationship is not significant.

140 130 120 110 100 90 80 70 60 50 40 30 Confidence Band 20 y = 102.87 - 0.0027x Shoot length (%control) of Shootpositive length 2 10 R = 0.13 0 0 1000 2000 3000 4000 5000 Soil Eu concentration (mg/kg) Figure 20: Shoot length of durum wheat exposed to Eu amended soil for 14 days. Solid line at

80% root length shows that the EC20 was not bounded. Confidence bands are set at 2.5% and 97.5%.

130 120 Confidence Band 110 100 90 80 70 60 50 40 y = -0.0068x + 101.85 y = -0.0016x + 82.14 N.S. 2 2 30 R = 0.47 R = 0.029 20

Root length (%control) ofRoot positive length 10 0 0 1000 2000 3000 4000 5000 6000 7000 Soil Nd concentration (mg/kg) Figure 21: Root length of durum wheat exposed to Nd amended soil for 14 days. Approximated

EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%. N.S. denotes that the relationship is not significant.

150 y = -0.0080x + 107.17 y = 0.0028x + 62.00 N.S. 140 2 R = 0.34 R2 = 0.028 130 Confidence Band 120 110 100 90 80 70 60 50 40 30 20

Shoot length (%control) of Shootpositive length 10 0 0 1000 2000 3000 4000 5000 6000 7000

Soil Nd concentration (mg/kg) Figure 22: Shoot length of durum wheat exposed to Nd amended soil for 14 days. Approximated

EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%.

Table 9: Effect concentrations (EC10, EC20, EC25) and 95% CI for tomato and durum wheat shoot and root lengths exposed to REEs in soil for 14 days (mg/kg dry soil). 95% CI excluded due to lack of significance at measurable precision level.

REE Plant Endpoint EC10 EC20 EC25 Root 5704 >7000 >7000 Tomato Shoot 2561 4942 6132 Ce Root 4266 6766 >8000 Durum wheat Shoot >8000 >8000 >8000 Root 337 711 898 Tomato Shoot 2968 >5000 >5000 Eu Root 3772 5201 >5300 Durum wheat Shoot 4517 >5300 >5300 Root 5502 6891 >7500 Tomato Shoot 2505 5069 6351 Nd Root 1742 3213 >3500 Durum wheat Shoot 2146 3396 4021

4.3 Tissue concentration

4.3.1 REE accumulation in radish tissues

Shoot concentrations of the REEs, generally increased with concentration (Figure 23).

Due to variability in the data, it is unclear whether the trend for Ce is linear or reaches a maximum at high concentrations, whereas Eu and Nd might accumulate either linearly or increase exponentially. Root accumulation of Ce increased exponentially with increasing exposure concentrations; however, root accumulation of Eu and Nd increased linearly.

This variation in the exposure concentration-tissue concentration relationship suggests different mechanisms of uptake and accumulation in the roots and translocation to the shoots, or similar mechanisms with different affinities for uptake channels or transporters. It is to be noted that the hypocotyl of the radishes, due to being part of the below ground biomass, was included with roots, though it is a modified shoot. Accumulation of Ce in the roots appears to be linear below 5000 mg/kg, but at high soil concentrations increases greatly. The initial uptake might be due to passive diffusion into the root tissue; however, the change at high concentrations might be due to active transport into the root once a threshold in soil concentration is reached. Another explanation might be that the control mechanism functions well at low concentrations, but the system becomes overwhelmed above 5000 mg/kg and tissue concentration increases greatly. In the shoots, however, the tissue concentration increases initially, then levels off. This could be due to initial diffusive translocation through the plant, but with a control mechanism in place which limits the amount of Ce reaching the shoot tissue.

For Nd a somewhat different trend appears, although soil concentrations covered a smaller range of concentrations than for Ce. Root accumulation of Nd was linear without a threshold. Accumulation in the shoot tissue increased exponentially, which might mean that some barrier was overcome, allowing more Nd into the shoot as soil and root concentration increase.

The accumulation of Eu in radish tissue differed from that of Nd and Ce. For radish, Eu concentrations in root tissue increased linearly but remained below 30 mg/kg; shoot Eu concentration was greater than that for root. In contrast, the tissue accumulation data for Ce and

Nd, indicated that concentrations in root tissue were greater than those in shoots. Due to low plant mass at the highest Eu treatment, shoot tissues for all replicates were combined for

analysis; therefore, variability and significance were not quantified. Shoot accumulation displays a linear trend only to the second highest soil concentration. When the highest concentration is considered, there is uncertainty if the linear trend exists because an exponential increase similar to that shown by Nd, occurred with Eu. The ratio of shoot:root concentration was greater than one, which suggests that the Eu is being readily translocated to the shoots at lower root concentrations than the other REEs (Figures 23, 24). The increased translocation of Eu as compared to Ce has also been reported to occur in rye and wheat (Shtangeeva, 2014) and has been hypothesized to be related to fractionation during uptake by plants, which was previously thought not to occur (Shtangeeva, 2014).

1600 Cerium 1400 Neodymium Europium 1200 Cerium Europium 1000 Neodymium

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Root tissue REE concentration (mg/kg) Root tissue REE concentration 0

0 1000 2000 3000 4000 5000 6000 7000 8000 Soil REE concentration (mg/kg) Figure 23: Radish root accumulation of REEs in the range-finding study after growing in REE amended soils for 14 days.

300 275 Cerium Europium 250 Neodymium 225 Cerium Europium 200 Neodymium 175 150 125 100 75 50

Shoot REE concentration (mg/kg) Shoot REE concentration 25 0 0 1000 2000 3000 4000 5000 6000 7000 8000 Soil REE concentration (mg/kg)

Figure 24: Radish shoot accumulation of REEs in the range-finding study after growing in REE amended soils for 14 days.

4.3.2 Tomato and durum wheat internal dose-response

Accumulation of REEs in tomato roots and shoots (Figures 25 and 26) durum wheat roots and shoots (Figures 27 and 28) are summarized graphically. Tomato shoot uptake of Ce increased to a maximum where it plateaued, Nd was linear, and Eu accumulation was more variable and linearity uncertain. Tomato root accumulation of all three REEs was linear and overall the slopes were similar. Generally, accumulation of Eu in tomato shoot and root was the highest, followed by Ce, and then Nd. Durum wheat roots (Figure 27) and shoots (Figure 28) showed trends in accumulation similar to those for tomato. For durum wheat roots accumulation of the three REEs was linear. In the shoots Ce and Nd accumulation was linear; however, accumulation of Nd appeared to reach a threshold at higher concentrations, although it was not quantifiable. Eu accumulation in durum wheat shoots did not display linearity. Overall, REE

concentrations in durum wheat tissues for the three REEs were similar; they could be ranked

(from high to low) as Ce, Nd, and Eu.

Europium concentrations in the shoots of both species were the highest of the three REEs and it was also the highest in the roots of tomato. Shtangeeva (2014) also found higher plant accumulation of Eu, as compared to Ce. Though the REEs have similar chemistry and were assumed not to partition into different fractions during plant uptake, it has been suggested that there is some fractionation causing changes in speciation, where Ce(III) could oxidize to Ce(IV) in the rhizosphere and become less soluble, accounting for comparatively lower uptake by plants.

Tomato REE accumulation in roots and shoots was very similar to that of radish. Durum wheat had much higher accumulation in the roots, but much lower accumulation in shoot tissues.

This difference in shoot accumulation may be attributed to the differences in water use efficiency between monocotyledonous (wheat) and dicotyledonous (tomato and radish) species. Although the effect of transpiration rate on translocation of elements by plants is element specific, the higher transpiration rate of dicotyledons, due to their larger leaf size, might explain the differences exhibited between these species (Marschner, 2012), though transpiration rates were not measured in this experiment.

Table 10 summarizes internal shoot effect concentrations (EC10, EC20, EC25) for shoot mass. Due to the high variability in root masses and root accumulation, effects were not quantified for this endpoint. The effect of internal concentration on length of shoots was highly similar to the effect on biomass (Table 11). Tomato reaches higher ECx values at lower soil concentrations than durum wheat, which is consistent with its higher transpiration rate. Tissue concentrations which cause the same effect are consistently higher in tomato shoots than durum

wheat shoots, suggesting tomato has a higher tolerance (lower sensitivity) to REEs as internal

(tissue) concentrations. This contrasts with the external (soil) concentrations, where tomato shows higher sensitivity to REEs than durum wheat to Ce and Eu, though for Nd they show the same level of sensitivity. This demonstrates that true bioavailability is difficult to determine without measuring it directly in plant tissue, due to variability among plant species.

1000 Cerium 900 Europium Neodymium 800 Cerium Europium 700 Neodymium 600

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Tomato root REE concentration (mg/kg) REE root concentration Tomato 0 0 1000 2000 3000 4000 5000 6000 7000 8000 Soil REE concentration (mg/kg) Figure 25: Tomato root accumulation of REEs in the definitive study after 14 days of growth in REE amended soils.

200 Cerium 175 Neodymium Europium Cerium 150 Europium Neodymium 125

100

Tomato shoot REE concentration (mg/kg) shoot REE concentration Tomato 0 0 1000 2000 3000 4000 5000 6000 7000 8000 Soil REE concentration (mg/kg) Figure 26: Tomato shoot accumulation of REEs in the definitive study after 14 days of growth in REE amended soils.

2000 Cerium 1750 Europium Neodymium Cerium 1500 Europium Neodymium 1250

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Durum wheat root REE concentration (mg/kg) root REE concentration wheat Durum 0 0 1000 2000 3000 4000 5000 6000 7000 8000 Soil REE concentration (mg/kg) Figure 27: Durum wheat root accumulation of REEs in the definitive study after 14 days of growth in REE amended soils.

20 Cerium 18 Europium Neodymium 16 Cerium 14 Europium Neodymium 12

Durum wheat shoot REE concentration (mg/kg) shoot REE concentration wheat Durum 0 1000 2000 3000 4000 5000 6000 7000 8000 Soil REE concentration (mg/kg) Figure 28: Durum wheat shoot accumulation of REEs in the definitive study after 14 days of growth in REE amended soils.

REE Plant EC10 EC20 EC25 Tomato 15 32 41 Ce Durum wheat 8 10 11 Tomato 33 60 >70 Eu Durum wheat 5 9 10 Tomato 4 28 40 Nd Durum wheat 2 3 4

Table 11: Internal (tissue) effect concentrations (mg/kg dry tissue) for tomato and durum wheat shoot and root lengths exposed to REEs (EC10, EC20, EC25, EC50).

REE Plant Endpoint EC10 EC20 EC25 EC50 442 772 Root >800 >800 441-443 771-773 Tomato 28 50 Shoot >50 >50 Ce 18-37 40-60 Root 687* 1104* >1250 >1250 Durum wheat 12 17 Shoot >18 >18 -1.0-29 4-37 31 Root >70 >70 >70 24-38 Tomato 49 Shoot >90 >90 >90 Eu 44-54 Root 602* >1083 >1083 >1083 Durum wheat 7 13 16 Shoot >17 -3-17 3-24 6-28 Root 518* >872 >872 >872 Tomato 12 40 53 Shoot >64 6-19 33-46 46-60 Nd 74 Root >130 >130 >130 69-78 Durum wheat 3 5 6 Shoot >8 -3-9 -0.6-12 0.6-13 *95% CI excluded due to lack of significance at measurable precision level.

4.4 Leaching experiment

4.4.1 Soil analysis

After leaching and prior to use for the plant growth test, soils had the same total concentrations of Ce as before leaching (Table 4). Total and bioaccessible concentrations of Ce after radish growth are summarized in Table 12.

Table 12: Total Ce concentrations after leaching, and total and bioaccessible Ce concentrations of leached soil following plant growth (mg/kg dry soil).

Reference Trt 1 Trt 2 Trt 3 Trt 4 Total after leaching at start 24 182 2345 5301 7787 of growth test

Total at end of growth test 26 189 2166 4721 7202

Bioaccessible at end of < 0.01 0.11 0.59 2 3 growth test

4.4.2 Plant growth inhibition

Following leaching of soil, there was no exposure-response of radish shoot length to Ce

(Figure 29), although total soil Ce concentrations remained high and bioaccessible concentrations were the same as in the unleached study, as measured by CaCl2. Since bioaccessibility remained the same before and after leaching, the growth dose-response seen previously must not have been due to the Ce within the plant tissue, and are more likely due to the NO3 counter-ion. This suggests that the accumulation was only indicative of exposure to Ce, not of plant growth effects. Radish hypocotyls were shorter as concentration increased (Figure

30), and hypocotyl development decreased in response to Ce after leaching (Figure 31).

Therefore, the radish shoot responses noted previously, were due either to the increasing concentrations of NO3 present across treatments and/or to the increased soil salinity. It is possible that the toxicity was caused by NO3 itself (d’Aquino et al., 2009), or could be due to the poor salt tolerance of radish (Environment Canada, 2007).

130 120 110 100 90 80 70 60 50 40 30 Confidence Band Shoot length (% of Shootcontrol) length y = 91.15 + 0.001x N.S. 20 R2 = 0.078 10 0 0 1000 2000 3000 4000 5000 6000 7000

Cerium concentration (mg/kg) Figure 29: Shoot length of radish exposed for 14 days to Ce in soil following leaching. Confidence bands are set at 2.5% and 97.5%. N.S. denotes that the relationship is not significant.

130 Confidence Band 120 y = 88.40 - 0.0041x 110 R2 = 0.50 100 90 80 70 60 50 40 30 20

Radish hypocotyl length (% of control) Radish hypocotyl length 10 0 0 1000 2000 3000 4000 5000 6000 7000

Soil Ce concentration (mg/kg) Figure 30: Hypocotyl length of radish exposed for 14 days to Ce in soil following leaching. The approximated EC20 is shown by the arrow. Confidence bands are set at 2.5% and 97.5%.

110 Hypocotyl development % 100 90 80 70 60 50 40 30 20

% Radish hypocotyl development 10 0 0 1000 2000 3000 4000 5000 6000 7000 8000 Soil Ce concentration (mg/kg) Figure 31: Mean (± SD) percent hypocotyl development by treatment for radish exposed to Ce in soil following leaching. Error bars show standard deviation.

4.5 OECD soil comparison

To better compare the results of this thesis to those in the literature, this study was undertaken to determine how using a different soil influenced toxic effect concentrations. The soil characteristics of the garden soil and the artificial soil that was used for previous research are summarized in Table 13.

Table 13: Physicochemical properties of garden soil and artificial soil

Soil TOC % Sand % Silt % Clay % Texture pH (H2O slurry) Garden soil 28.7 28.9 36.1 35.1 Clay loam 7.0 Artificial soil 4.7 75.4 16.6 8.04 Artificial soil 6.0

4.5.1 Soil analysis

Following aging periods of 24 hours and 14 days, bioaccessible REE concentrations were measured by CaCl2 extraction (Tables 14-16). Overall, bioaccessibility of each REE was very similar between Cl and NO3 forms for each aging time period; and the bioaccessibility of all

REEs in the artificial soil decreased when allowed to age for 14 days instead of 24 hours

(Figures 32-37). Linear models were the best fit for the data; however, they do not capture the potential nonlinearity at higher concentrations. The percent bioaccessibility reported on the graphs was calculated based on the slope of the linear model and overestimates the bioaccessibility at soil concentrations in the middle of the range.

Table 14: Mean (± SD) total and bioaccessible concentrations (mg/kg) of Ce in CeCl and Ce

NO3 amended artificial soil. Reference Trt 1 Trt 2 Trt 3 Trt 4

Bioaccessible CeCl3 after 24 hours < 0.01 1.08 10.4 76.7 1040

Bioaccessible CeNO3 after 24 hours < 0.01 1.09 8.37 59.9 649

Bioaccessible CeCl3 after 14 days < 0.01 0.46 4.23 35.91 687

Bioaccessible CeNO3 after 14 days < 0.01 0.40 2.60 28.7 410

Total CeCl3 after 14 days 10.2 190 1160 4420 7210

Total CeNO3 after 14 days 10.2 182 987 3680 6830

Table 15: Mean (± SD) total and bioaccessible concentrations (mg/kg) of Eu in EuCl3 and Eu

NO3 amended artificial soil. Reference Trt 1 Trt 2 Trt 3 Trt 4

Bioaccessible EuCl3 after 24 hours < 0.01 0.55 4.48 13.1 82.0

Bioaccessible EuNO3 after 24 hours < 0.01 0.59 4.20 13.6 98.1

Bioaccessible EuCl3 after 14 days < 0.01 0.71 4.87 17.8 73.6

Bioaccessible EuNO3 after 14 days < 0.01 0.74 4.89 17.7 91.1

Total EuCl3 after 14 days < 5 121 857 2760 5380

Total EuNO3 after 14 days < 5 131 849 2810 5580

Table 16: Mean (± SD) total and bioaccessible concentrations (mg/kg) of Nd in NdCl3 and Nd

NO3 amended artificial soil. Reference Trt 1 Trt 2 Trt 3 Trt 4

Bioaccessible NdCl3 after 24 hours < 0.01 0.95 6.95 32.5 417

Bioaccessible NdNO3 after 24 hours < 0.01 1.04 6.37 29.8 416

Bioaccessible NdCl3 after 14 days < 0.01 0.85 4.69 27.2 319

Bioaccessible NdNO3 after 14 days < 0.01 0.83 4.64 25.4 307

Total NdCl3 after 14 days < 5 186 1010 3570 6920

Total NdNO3 after 14 days < 5 177 955 3420 6630

1100 CeCl 1000 24 hour 12.5% 3 900 2 week 8.3% 800 700 600 500 400 300 200 100 0

Bioaccessible Ce(mg/kg) concentration Bioaccessible -100

0 1000 2000 3000 4000 5000 6000 7000

Soil Ce concentration (mg/kg)

Figure 32: Bioaccessible Ce in CeCl3 amended artificial soil after 24 hour and 2 week aging periods in OECD comparison study.

700 24 hour 8.8% CeNO3 600 2 week 5.5%

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Bioaccessible Ce concentration (mg/kg) Ce concentration Bioaccessible

0 1000 2000 3000 4000 5000 6000 7000

Soil Ce concentration (mg/kg)

Figure 33: Bioaccessible Ce in CeNO3 amended artificial soil after 24 hour and 2 week aging periods in OECD comparison study.

90 24 hour 1.4% EuCl3 80 2 week 1.3% 70

Bioaccessible Eu concentration (mg/kg) concentration Eu Bioaccessible -10 0 1000 2000 3000 4000 5000

Soil Eu concentration (mg/kg)

Figure 34: Bioaccessible Eu in EuCl3 amended artificial soil after 24 hour and 2 week aging periods in OECD comparison study.

110 EuNO 100 24 hour 1.7% 3 2 week 1.6% 90 80 70 60 50 40 30 20 10

Bioaccessible Eu concentration (mg/kg) concentration Eu Bioaccessible 0 -10 0 1000 2000 3000 4000 5000

Soil Eu concentration (mg/kg)

Figure 35: Bioaccessible Eu in EuNO3 amended artificial soil after 24 hour and 2 week aging periods in OECD comparison study.

450 24 hour 5.6% NdCl3 400 2 week 4.3%

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Bioaccessible Nd concentration (mg/kg) Nd concentration Bioaccessible -50 0 1000 2000 3000 4000 5000 6000 7000

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Figure 36: Bioaccessible Nd in NdCl3 amended artificial soil after 24 hour and 2 week aging periods in OECD comparison study.

450 24 hour 5.9% NdNO3 400 2 week 4.3% 350

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Bioaccessible Nd concentration (mg/kg) Nd concentration Bioaccessible -50 0 1000 2000 3000 4000 5000 6000

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Figure 37: Bioaccessible Nd in NdNO3 amended artificial soil after 24 hour and 2 week aging periods in OECD comparison study.

For simpler comparison between this research and published studies, the effect concentrations are summarized in Table 17. Effect concentrations were normalized to bioaccessibility by multiplying the percent bioaccessibility (Table 18) of the appropriate REE- salt in each soil by the reported effect concentration value. The percent bioaccessibility used for normalization was calculated from the data points at that total soil concentration, rather than from the linear model, due to the poor fit at concentrations in the middle of the range.

Effect concentrations normalized to bioaccessibility (Table 19) were much closer between the current research and the published studies, particularly for Ce, which were almost the same. This further suggests that the differing bioaccessibility, likely due to differences in soil type and aging duration accounts for the major variability between this research and the work done previously.

Table 17: Comparison of EC25 values between this research and published literature for radish and tomato (mg/kg dry soil).

Effect Ce Nd

Current radish EC25 4535 4615

Literature radish EC25 314 452

Current tomato EC25 6132 6351

Literature tomato EC25 315 1024

Table 18: Percent bioaccessibility of REE-NO3 in garden soil after 2 week aging and REE-Cl3 in artificial soil after 24 hours of aging used to normalize effect concentration values.

REE Garden soil – 2 week aging NO3 Artificial soil – 24 hour aging Cl Ce 0.03 0.57 Eu 0.03 0.45 Nd 0.04 0.51

Table 19: Effect concentrations (EC25) for radish and tomato from this research and the literature that have been normalized for bioaccessibility (mg/kg dry soil).

Effect Ce Nd

Current radish EC25 136 185

Literature radish EC25 179 230

Current tomato EC25 184 254

Literature tomato EC25 180 522

5.0 Conclusions

The major objective of this thesis was to contribute new knowledge describing the toxicity of three REEs to aid in their risk assessment. Toxic effect threshold concentrations were identified for three plant species which will make a significant contribution to the eventual formation of an SSD for risk assessment purposes. For completion of this objective, three hypotheses were addressed:

1) Toxic effect concentrations for Ce, Eu and Nd are similar among radish, tomato and

durum wheat;

2) The major variation in lanthanide toxicity among species can be attributed to differential

tissue concentration;

3) Toxic effect concentrations are more closely represented by bioaccessible REE

concentrations than by the total REE concentrations in the soil.

Results from both the range-finding and definitive studies were able to be used to determine toxic effect concentrations for radish, tomato and durum wheat to the three REEs studied. The models that best describe the datasets however; did not include models with

hormetic parameters. Because of this, hormesis was not able to be quantified within the scope of this research; likely due to the design of the experiment leading to concentrations that would cause hormetic effects not being included.

According to results of the range-finding study, and definitive study results, toxic effect concentrations were determined for Ce, Eu and Nd. Bioaccessibility of all three REEs was assessed and found to be linearly correlated to total soil concentration, and generally approximately 0.03% of the total concentration in soil was bioaccessible. This low degree of bioaccessibility might help explain the magnitude of the ECx values determined as compared with other studies of these trace elements; however, due to the linearity of the association, bioaccessible concentrations do not explain toxicity any better than total soil concentrations.

When effect concentrations are expressed as tissue concentrations, as opposed to external concentrations, a trend forms when tomato and durum wheat are compared across the metals.

Effect concentrations for tomato are consistently higher than those for durum wheat, which suggests that the tomato is more tolerant of internal doses of REEs. This was not the case for external concentrations; durum wheat effect concentrations were consistently somewhat higher than those for tomato.

When comparing internal tissue effect concentrations among the metals, Ce and Nd are very similar in tomato, but not in durum wheat, where Ce and Eu concentrations are much more similar. This is in contrast with external effect concentrations, where effect concentrations for all three REEs are fairly similar for tomato, and none of the three are similar for durum wheat.

Overall, there are still questions left unanswered, including how soil properties affect bioaccessibility of these elements, and what differences there are between the elements and the

mechanisms within the plants that contribute to the differences in the comparative toxicities among elements and among species. One way to expand this study would be through further measurement of bioaccessibility of REEs in different soil types, after different periods of aging and after leaching. Expansion of research into the mechanism of REE accumulation and movement through plants is also necessary to better understand the mechanisms driving REE toxicity.

6.0 Bibliography

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7.0 Appendix – Supplementary Materials

Appendix 1: Summary of models for external concentration vs plant length Linear Piecewise Metal Plant Endpoint Model chosen R2 Equation R2 Equation of significant linear portion Root 0.2818 Y=-0.0025x+104.17 0.5450 Y=-0.0066x+127.65 Piecewise Tomato Shoot 0.5028 Y=-0.0042x+100.76 0.3295 Linear Ce Durum Root 0.4463 Y=-0.004x+107.06 0.4724 Linear wheat Shoot 0.1289 Y=-0.0029x+114.81 0.1289 Linear Root 0.0724 Y=-0.0023x+83.88 0.3610 Y=-0.0267x+98.99 Piecewise Tomato Shoot 0.2207 Y=-0.0035x+100.39 0.2659 Linear Eu Durum Root 0.3310 Y=-0.0037x+102.87 0.4197 Y=-0.007x+116.41 Piecewise wheat Shoot 0.1344 Y=-0.0027x+102.20 0.1688 Linear Root 0.1841 Y=-0.0023x+102.12 0.2876 Y=-0.0072x+129.61 Piecewise Tomato Shoot 0.5882 Y=-0.0039x+99.77 0.5938 Linear Nd Durum Root 0.5443 Y=-0.0046x+98.61 0.5759 Y=-0.0068x+101.85 Piecewise wheat Shoot 0.2712 Y=-0.0042x+101.31 0.3339 Y=-0.008x+107.17 Piecewise

Appendix 2: Summary of models for internal shoot concentration vs shoot mass Linear Piecewise Metal Plant Model chosen R2 Equation R2 Equation of significant linear portion Tomato 0.3132 Y=-0.0005x+0.16 0.4632 Y=-0.0010x+0.17 Piecewise Ce Durum wheat 0.1176 Y=-0.0062x+0.14 0.1586 Y=-0.0055x+0.15 Piecewise Tomato 0.1011 Y=-0.0001x+0.14 0.2675 Y=-0.0005x+0.14 Piecewise Eu Durum wheat 0.2377 Y=-0.0064x+0.24 0.2567 Linear Tomato 0.1227 Y=-0.0004x+0.09 0.1401 Linear Nd Durum wheat 0.369 Y=-0.0129x+0.19 0.4038 Y=-0.0182x+0.20 Piecewise

Appendix 3: Summary of models for internal root vs shoot concentrations Linear Piecewise Metal Plant Model chosen R2 Equation R2 Equation of significant linear portion Tomato 0.4677 Y=0.0791x+16.50 0.5449 Y=0.3317x-3.94 Piecewise Ce Durum wheat 0.5486 Y=0.0066x+0.99 0.5523 Linear Tomato 0.4266 Y=0.1226x+13.71 0.4633 Y=0.1736x+4.85 Piecewise Eu Durum wheat 0.5891 Y=0.0125x+0.03 0.5891 Linear Tomato 0.5822 Y=0.0495x+6.92 0.6095 Y=0.0668x+3.97 Piecewise Nd Durum wheat 0.4441 Y=0.0067x+1.35 0.505 Y=0.0099x+0.85 Piecewise

Appendix 4: Summary of models for internal concentration vs plant length

Linear Piecewise Model Metal Plant Endpoint Equation of significant linear R2 Equation R2 chosen portion Root 0.1636 Y=-0.0165x+101.31 0.2004 Y=-0.0303x+103.3915 Piecewise Tomato Shoot 0.3719 Y=-0.2705x+98.46 0.4506 Y=-0.4493x+102.4838 Piecewise Ce Durum Root 0.3207 Y=-0.0173x+103.76 0.6516 Y=-0.0240x+106.4857 Piecewise wheat Shoot 0.2429 Y=-2.1863x+116.72 0.2958 Linear Root 0.0144 Y=-0.0053x+79.91 0.3493 Y=-0.2397x+97.4324 Piecewise Tomato Shoot 0.1034 Y=-0.0739x+96.37 0.2279 Y=-0.2209x+100.8988 Piecewise Eu Durum Root 0.0761 Y=-0.0115x+96.93 0.0781 Linear wheat Shoot 0.3516 Y=-1.7322x+101.99 NA* Linear Root 0.1298 Y=-0.0189x+99.79 0.1304 Linear Tomato Shoot 0.2438 Y=-0.3698x+94.62 0.2682 Linear Nd Durum Root 0.3185 Y=-0.0363x+93.98 0.4586 Y=-0.1763x+102.9783 Piecewise wheat Shoot 0.3839 Y=-4.7381x+104.42 0.3521 Linear *Not able to fit piecewise model

Appendix 5: 95% Confidence intervals for EC values of plant length vs external (soil) concentration (mg/kg dry soil)

EC10 EC20 EC25 Metal Plant Endpoint Lower Upper Lower Upper Lower Upper Root 5704.107 5704.185 Tomato Shoot 2560.891 2560.916 4941.845 4941.872 6132.314 6132.350 Ce Durum Root 4266.315 4266.370 6766.309 6766.348 wheat Shoot 8555.514 8555.587 Root 336.383 336.792 710.878 711.273 898.069 898.542 Tomato Shoot 2968.273 2968.300 Eu Durum Root 2772.238 2772.277 5200.798 5200.856 wheat Shoot 4517.135 4517.162 Root 5501.958 5502.024 6890.83 6890.932 Tomato Shoot 2505.043 2505.062 5069.144 5069.165 6351.192 6351.219 Nd Durum Root 1742.495 1742.540 3213.072 3213.144 wheat Shoot 2145.912 2146.010 3395.893 3396.030 4020.876 4021.047