Methods and materials

4.2.1 Hydroponics glasshouse experiment

The study contains two experiments. The first experiment measures the P use efficiency of SSP and LWSSP, which differ in their ratios of MCP, DCP and TCP, and MCP, DCP and TCP in their pure forms for pasture growth in hydroponic conditions. The second experiment measures the solubility of SSP, LWSSP, MCP, DCP and TCP in leaching columns with simulated rainfall and no plants.

The P forms and predicted solubility based on their chemistry as the ratio of calcium to P increases, the solubility decreases as shown in Table 4.2. The solubility of the two P fertilisers (SSP and LWSSP) is a function of their ratio of each phosphate form.

Table 4.2 Phosphorus fertilisers with their composition percentage and phosphorus analysis.

The phosphorus fertilisers used in these experiments with the amount of phosphorus present within each of them and the percentage of the total phosphorus of each phosphate form (within brackets). Single superphosphate (SSP), low water- soluble superphosphate (LWSSP), monobasic calcium phosphate (MCP), dibasic calcium phosphate (DCP), tribasic calcium phosphate (TCP).

SSP LWSSP MCP DCP TCP

Total P 9.1 8.3 26.2 18.1 15.9

Water-soluble Pᴬ 7.8 (86) 2.9 (35) 26.2 (100) 0 (0) 0 (0) Citrate-soluble Pᴬ 0.8 (9) 3.7 (45) 0 (0) 18.1 (100) 0 (0) Citrate-insoluble Pᴬ 0.5 (5) 1.7 (20) 0 (0) 0 (0) 15.9 (100) ᴬ Measured by standard (AOAC 1975).

The design comprised three replicates of 12 treatments; six fertiliser treatments – SSP, LWSSP, MCP, DCP, TCP and a nil-P on two common pasture species used on the SCP, Dalkeith subterranean clover (Trifolium subterranean L.) and Wimmera annual ryegrass (Lolium rigidum). The experiment comprised a total of 108 pots divided into 6 experimental runs of 18 pots per run.

Polyvinyl chloride pots (125 mm diameter, 100 mm deep and a volume 1 litre were packed with perlite and then 25–30 seeds of clover or ryegrass were sown in each pot. The pots were placed in aluminium trays and handed watered with demineralised water and a nutrient solution Table 4.3 for 21 days applied three days per week. The trays were filled with demineralised water to ensure pot dampness

The pots were then placed into black Polyvinyl chloride hydroponic tanks for 21 days (i.e. one experimental run). The pots were randomised in the tank (i.e. one treatment per tank) and three pots of each species per tank (i.e. 6 pots per tank) with three tanks per experimental run.

The concentration of P in the P treatments are listed in Table 4.2. Phosphorus treatments were applied as a single application at the beginning of the experiment at 25 kg/ha of P based on the combined surface area of the six pots. The P fertiliser treatments were weighted into two glass plates and placed at diagonally opposing corners of each tank. The SSP and LWSSP were made in Western Australia at CSBP Limited, Kwinana and the MCP, DCP and TCP were imported from China. The fertiliser was crushed and then sieved with particle sizes ranging from 75 to 150 microns. The same fertiliser treatments and applications were used in the leaching column experiment.

A complete liquid fertiliser solution (excluding P) was applied to 40 litres of demineralised water once at the beginning of the experiment to ensure that P was the only element limiting yield (see Table 4.3). The pots were watered for 11 hours per day (i.e. 7 am to 6 pm) for 30 min per hour and aerated 24 hours per day. The experiment contained six runs, with each treatment replicated three times and applied to each tank once to reduce any influence from slight differences in equipment and position within the glasshouse.

Table 4.3 Basal nutrient solution mixture composition applied to hydroponics experiment.

Basal nutrient solution in milligrams per element applied to each hydroponic tank before the start of each experiment run. The mixture was applied in two parts because its chemical analysis would cause precipitation of the elements if contained within a single part. The solution is modified from Marschner (2012) to remove phosphorus.

Element Amount applied to each tank (mg)

Boron 13 Calcium 4797 Copper 5 Iron 80 Magnesium 1461 Manganese 35 Nitrogen 79 Phosphorus 0.07 Potassium 7007 Sodium 131 Sulphur 1753 Zinc 6

The demineralised water, basal nutrient solution and dissolved P fertiliser were pumped on the surface of the pots facilitating flow through the growth medium and recycling within the system. The pH and electric conductivity (EC) were checked three times per week throughout the experiment and adjusted to a target pH of 6.5 in water (H₂O) and EC of 1.4 dS/cm. Water samples were taken four times during the experimental run of 21 days, at the begin of each week and the end of each run and subsequently analysed to determine P concentration (mg/L). The shoots of clover and ryegrass were harvested at the end of each run, dried, weighed, and then analysed for P concentration. Analysis for concentrations of P content in dry matter was conducted by bulking the replicates together.

4.2.2 Leaching column experiment

The design contained three replicates of six fertiliser treatments comprising SSP, LWSSP, MCP, DCP, TCP and a nil-P control. The fertiliser was ground and sieved to 75–150 microns to ensure equivalent particle sizes for all treatments. Polyvinyl chloride pots (152 mm diameter, 400 mm deep) were packed with perlite Table 4.4 to a total volume of 6 L.

Table 4.4 Analysis of perlite used in both hydroponics and leaching column experiments.

The critical chemical and physical properties of perlite relative to experimentation on phosphorus in hydroponics and leaching columns.

Analysis

pH (1:5 perlite:0.01 M CaCl2) 6.7

Bicarbonate-extractable P (Colwell 1965) (mg/kg) 4

Phosphorus buffering index 5.4

EC (dS/cm) 0.042

The columns were pre-leached with the equivalent of 164 mm of rainfall using demineralised water. Phosphorus treatments were applied at a rate equivalent to 120 kg P/ha to the surface of the columns as a single application at the beginning of the experiment. The columns were watered three times per week with 1.5 litres (equivalent to 84 mm of rainfall) of demineralised water and allowed to drain for a minimum of 48 hours between watering, throughout the four-week experiment. Leachate from the columns was collected in a clear plastic bag enclosing the base of the column. The total cumulative rainfall of 924 mm was designed to mimic above average annual rainfall at Mandurah (660–990 mm) on the SCP (Hanson and Foster 2012).

The rate of P leaching was measured by collecting leachate samples after each simulated rainfall event and analysed for total P (mg/L). The leachate collected was stirred and a sample taken, the bag was then emptied and replaced beneath the column. Residual undissolved mg P kg, determined post-experiment was analysed by removing the perlite from the column and sieving it into 3 fractions from the upper 90% of the column (> 2.2 mm, < 2.2–4 mm and > 4 mm). The remaining lower 10% was analysed for P (mg/kg), and the weights of these samples were recorded.

4.2.3 Analysis of data

Refer to Section 3.10 (Statistical analysis, calculations and data storage) for a detailed description of analysis.