**2. Methods**

## *2.1. Soil Sampling*

Metals in roadside soils have been deposited from vehicle emissions over varying time periods of up to multiple decades depending on the age of the roads. Thus, roadside soils provide an opportunity to study bioavailability and metal aging processes in long-term in situ metal-contaminated soils. Soil samples were collected in December 2012 from roadside soils formed on chromosol [22] of newer volcanic basaltic geology [23] in the west of Melbourne, Australia. The selection of roads from the same geology ensured that the soil derived from similar parent material reduced variations due to natural soil derivatives. All chosen roads were constructed from the same surface material, i.e., asphalt. Samples were collected adjacent to roads carrying ≥ 1500 vehicles/day and represented three different road ages, i.e., new (N = 1.5–5 years), medium (M = 5–10 years) and old (O ≥ 15 years). Three soil

replicates for each road type were collected. Three sites in parklands at least 1 km away from roads and industry were included as control sites. Approximately 500 g of topsoil samples from 0–10 cm were collected using a clean stainless steel spatula and placed in zip-lock polythene bags. The immediate road edge up to 1 m was avoided to minimise the risk of sampling refilled or recently disturbed soil from road constructions and upgrades [24]. Soil samples were air-dried (23 ± 2 ◦C), sieved (≤2 mm), sealed in zip lock polythene bags and stored at ambient laboratory conditions (20 ± 2 ◦C) in the dark until further analysis [20].

## *2.2. Wheat Assay*

Soil aliquots (75 g) were placed into 24 polystyrene plastic pots (180 mL). The pots were moistened to 50% of the maximum water holding capacity (MWHC) with ultrapure water (18 MΩ.cm) and left to equilibrate for 24 h at 21 ◦C in the dark. *Triticum aestivum* cv. AXE (bread wheat) seeds (Department of Primary Industries, New South Wales, Australia) were surface sterilised with 0.3% NaOCl solution and rinsed with deionised water [25]. Twenty seeds were sown into each pot and the pots placed in a growth chamber at 21 ◦C/15 ◦C on a 14 h/10 h light/dark cycle for 28 days. On the 5th day after sowing, the seedlings were thinned to 10 plants per pot. Aliquots (5 mL) of Ruakara nutrient solution [6] were added to the pots on days 5, 10, 15, 20 and 25 prior to watering. Throughout the experiment, pots were regularly watered with ultrapure water to maintain 50% MWHC.

On day 28, seedlings were harvested by cutting shoots 1 cm above the soil surface. Shoots were washed thoroughly with ultrapure water, dried for 24 h at 45 ◦C, and the shoot dry weight was recorded for each pot. Dried shoot samples were finely ground and stored in polypropylene plastic storage vials at ambient laboratory conditions (20 ± 2 ◦C) in the dark until further processing. Nitric acid (HNO3, 5 mL, 70%, Sigma Aldrich analytical grade), together with hydrogen peroxide (H2O2, 1 mL, 30% *w*/*w*, Sigma Aldrich analytical grade), was added to aliquots of the dried plant samples (0.2 g) and digested at 115 ◦C using a microwave digester (Milestone, Ethos d' model). After digestion, samples were filtered (pore size 0.45 μm) and diluted with ultrapure water before being analysed for total metals using inductively coupled plasma-mass spectrometry (ICP-MS, Agilent Technologies 7700x element analyser). The isotopes chosen for this investigation were Cd (111), Cu (63), Ni (60), Mn (55) and Zn (66). The accuracy of the method was verified by analysing reagent blanks, which represented 10% of the total digested sample population and replicates of certified reference citrus plant material [NCS ZC73018 (GSB-11)]. The recovery of the certified reference material was >90% for all certified metals and no signs of contamination were found.
