Experimental setup

The experiment was conducted in two parts, referred to as Part 1 and Part 2. Part 1 was begun on April 10th, 2007 and completed on May 24th, while part 2 was begun on September 5th, 2007 and completed on October 19th, 2007.

2.10.1 Treatments

Growth media treatments included: a triple acid washed inert sand, five residue sand treatments containing varying concentrations of salts and 15 residue fines addition treatments (Table 2.6). Three pretreatments of residue fines were used: seawater- washed, carbonated and unaltered fines. Residue fines were added on a w/w basis. Table 2.6. Bauxite residue treatments assessed for germination and emergence of

Acacia saligna. Treatment Fines Addition (%) Gypsum Addition (%) Leaching reduction of EC Initial EC(1:5) (dS m-1) Study Part

Inert Sand 0 0 0 0.02 1 and 2

Residue Sand 0 0 0 1.15 2

Residue Sand 0 1 0 1.64 1 and 2

Residue Sand 0 1 4/5 original 1.53 2

Residue Sand 0 1 3/5 original 1.18 2

Residue Sand 0 1 2/5 original 0.67 2

Seawater fines 3 1 0 3.20 2 Seawater fines 5 1 0 2.67 1 Seawater fines 8 1 0 4.39 2 Seawater fines 10 1 0 4.36 1 Seawater fines 20 1 0 8.49 1 Carbonated fines 3 1 0 2.18 2 Carbonated fines 5 1 0 2.90 1 Carbonated fines 8 1 0 2.54 2 Carbonated fines 10 1 0 2.68 1 Carbonated fines 20 1 0 3.23 1 Unaltered fines 3 1 0 2.12 2 Unaltered fines 5 1 0 3.22 1 Unaltered fines 8 1 0 2.51 2 Unaltered fines 10 1 0 3.17 1 Unaltered fines 20 1 0 3.94 1

For explanations to pretreatments (i.e. seawater-washing and carbonation) of residue fines refer to Section 2.4.1.

2.10.2 Propagation trays

In each part, the setup was similar with each replicate having three propagation trays with a total of 512 cells; each cell was approximately 3 cm3 in volume (Premium Plastic, Wangara, WA). Each tray was divided into four - 100 cell sections, and each cell was filled with approximately 4.5 grams of the appropriate treatment growth media. This allowed for up to twelve treatments of 100 seeds per replicate. Two replicates were completed simultaneously during each part. This resulted in a total of two

replicates using six propagation trays and 200 seeds per treatment being evaluated, with a total of 2200 or 2400 seeds, for part 1 and part 2 respectively. In both parts, the treatments inert sand and residue sand were repeated for comparison, and thus they were evaluated for 400 seeds each.

2.10.3 Seed handling

Seeds for part 1 and 2 were collected at the same time from seed stocks of Alcoa World Alumina Australia. Seeds were kept cool and dry in a dark storage until planted. Acacia saligna seeds were pretreated by boiling in distilled water for 30 seconds (Bell 1999a) in two allotments. Seeds from each allotment were immediately removed and placed onto and covered by damp paper towels and kept moist until all seeds were planted into growth media over roughly 12 hours. Both allotments of A. saligna seeds were planted within 48 hours of each other. Seeds of A. saligna were planted into all treatment mixtures to a depth of approximately one centimetre.

2.10.4 Watering and emergence counts

Planted seeds were allowed 45 days to germinate and emerge in a constant temperature room set at 25 °C on a 12 hour light schedule. All treatments were hand watered daily to keep the small portions of growth media and seeds moist. This

seedlings were counted each day, but for part 2, it was considered sufficient to count emerged seedlings every two days. A seedling was determined “emerged” as soon as any visible green material was found to be protruding from the growth media surface in the individual cell.

2.10.5 Categorizing non-emerged seeds

After 45 days, the non-emerged seeds were excavated and each seed was categorized into: germinated (but not emerged), imbibed, rotten, or non-active but viable. These categorizes were defined as follows:

 Germinated – obvious penetration of the seed coat by the cotyledon with green healthy plant material visible.

 Imbibed - Swollen seed with softened seed coat, when pressed between finger and thumb green solid material will protrude.

 Rotten – Soft seed coat with either nothing in it, or when pressed between finger and thumb soft unconsolidated material of brown or grey color is produced.

 Non-active, but viable – Intact seed coat with no visible signs of swelling or damage and the seed is firm when pressed between finger and thumb.

2.10.6 Part 1

In the initial emergence and germination study, treatments were inert sand, residue sand (with 2 % w/w phosphogypsum), and fines additions of 5, 10, 20 % (w/w) for each fines treatment. This allowed for emergence in a control (inert sand) to be established, along with the current rehabilitation practice (residue sand with

2.10.7 Part 2

In the second portion, the experiment was expanded to include: residue sand with no phosphogypsum addition, three leached residue sands, 3 and 8 % fines

additions. To ensure conditions were not significantly different between the two parts, an inert sand and residue sand (with phosphogypsum) were repeated in part 2.

Leaching of residue sand to 4/5, 3/5, 2/5 of initial EC was done in PVC columns 25 cm high and 10 cm in diameter. Two kg of residue sand material was placed in each leaching column and distilled water was added in estimated quantities necessary to achieve desired EC levels. Materials were subsequently removed and oven dried at 60 °C for > 24 hour. Materials were thoroughly mixed and EC measurements were taken on five sub-samples of the materials (Rayment and Higginson 1992) to ensure

appropriate EC levels.

2.10.8 Extractable bases (Ca, Mg, K and Na), pH and EC

Subsamples of the growth media (n = 3 per treatment) were collected for analysis prior to seeds being sown (initial) and after the completion of the 45 day experiment (final). Initial and final samplings were analyzed for extractable bases extracted with alcoholic 1M ammonium chloride at pH 8.5 (Section 2.13.2.2 for details of extraction), without pre-washing for soluble salts (Rayment and Higginson 1992). Leaching and extraction was done in an especially designed leaching rack (see Section 2.13.2.1) and analysis of extractant for Ca, Mg, K and Na was completed by the Marine and Freshwater Research Laboratory (MAFRL) (Determination of elements in waters and other appropriate solutions by ICP-AES, MAFRL Method: ICP 001,Varian (Vista AX) ICP-AES CCD Simultaneous, ISO 15587-1:2002 (MAFRL 2009a)) at Murdoch University. Electrical conductivity and pH (1:5) were also completed (Rayment and Higginson 1992).

2.11 Experiment 4. Mobility of cations (Ca, Mg, K, Na) in altered bauxite residue