Determination of total S in fertilizers

3.3. 7. 1 S> containing fertilizers

Solubility of S0 at 25 °C in toluene was about 2.0 1 8 g S 1 00 g-1 of saturated solution and 2.084 g S 1 00 mr1 of 1 00% acetone solution, respectively. The toluene extract can be directly added into a scintillation cocktail which is also toluene based. Acetone, on the other hand, produces some quenching if it was not evaporated before adding to the cocktail solution (Chatupote, 1 990).

A method developed by Chatupote (1 990) was employed. Approximately 0.050 g of 35so labelled S0 fertilizer (1 0-1 00% S) was mixed with 200 ml acetone in a 250 ml glass bottle and shaken o n an end-over-e nd shake r overnight. After diluting, aliquots of 1 -2 ml were evaporated in scintillation vials (Chatupote, 1 990) before measuring 35s activity as described in Section 3.3.8.

A suitable aliquot of each extract, containing 20-50 microgram of S, was used for determining the total s0 using the direct determination of S0 as described by Nguyen ( 1 988).

3.3. 7.2 Sulphate containing fertilizers

Total S in sulphate fertilizer materials was determined using a modified method described by AOAC ( 1 984). About 0.1 00 g of fertilizer was mixed with 200 ml of 1 :1 HCI:H20 and shaken on an end-over-end shaker overnight. A 0.1 ml aliquot was used for measuring 35s activity by mixing with 1 2 ml of the cocktail solution and 1 ml of deionized water (Section 3.3.8).

Aliquots of the HCI extract containing 1 0-50 microgram of S, were used for determining total S as described in Section 3.3.6.

3.3.8 RADIOASSA V OF 32p AND 35s ACTIVITIES

The radioassay of weak beta-emitters is best performed by liquid scintillation cou nting. A Beckman Liquid Scintillation System, model 3801 , Bench-top and microprocessor-controlled, was used for the radioassay. The instrument is also equipped with an automatic quench correction programme.

The automatic quench correction programme was used for single and dual label counting ( Beckman Liquid Scintillation System: Operation M anual) of 35s and 32p radioactivity in samples from experiments described in Chapter 4 and 5.

3.3.8. 1 Liquid scintillation counting

The scintillation cocktail used in these studies was prepared by m ixing 8 g of P PO (2,5- diphenyloxazole), 0.2 g of POPOP (1 ,4-di-[2-(5-phenyloxazole)]-benzene), 1 340 ml of toluene and 600 ml of Triton-X 1 00 (Faires and Boswell, 1 98 1 ) . The mixtures were stirred overnight by a magnetic stirrer and kept in a dark glass bottle. Twenty ml glass scintillation vials were used for counting. The various aliquots of digest (described above) were mixed with volumes of deionized water, where appropriate, to give aqueous phase:scintillation cocktail ratios of 1 :1 2. Commonly total liquid volume was 1 3 ml except when larger sample aliquots were required to give sufficient counts. The mixtures were mixed thoroughly and left overnight in a dark roo m in order to limit chemiluminescence before measurement of radioactivity.

3.3.8.2 Establishing quench curves

The liquid scintillation device is based on the direct relationship between the amount of light being emitted from the vial being linearly related to the number of beta particle emissions from the samples. In practice, a number of factors (beta particle not activating the fluor, chemicals deactivating fluor molecules, or light from the fluor being adsorbed by other chemicals I n the

mixture) act to reduce the amount of light being emitted per beta emission. The phenomenon is referred to as "quenching" and a sample in which it occurs is said to be "quenched." All samples prepared in the laboratory are quenched to some degree. Therefore, the number of counts recorded must be converted to a value that correctly reflects the number of beta e missions-disintegrations that actually occurred in the sample. In these experiments, the quench curves were generated using the "H-number" method, for single and dual isotope counting (Beckman Liquid Scintillation System: Operation Manual).

Separate sets of quenched samples were prepared for each counting system (e.g. soil extracts, herbage extracts, etc.) and both isotopes 35s and 32P. The following quench curves were generated:

1. Sulphate S extracts - after extraction with CaP-S and CaC12 extraction

Y = 96.09 + 0.086H - 0.0006H2, 2. HI-reducible S after methylene blue development

y _{95.20 + 0.079H - 0.0004H ,} 2 3. Herbage S digests (NaOBr-digestion)

y = 98.33 + o.o59H - o.ooo5u2, 4. _{Total S soil extracts (NaHC03 + Ag20 digestion)}

y = 97.46 ₊ 0.082H - 0.0006H , 2 5. Herbage P digests y = 94.7 + o.o74H - o.ooo5H2, 6. Extractable P (Olsen P) Where y = 95.0 ₊ 0.062H - 0.0008H , 2 Y = Counting efficiency (%) H = H-number R2 = 0.93 R2 = 0.94 R2 = 0.95 R2 = 0.94 R2 = 0.97 R2 = 0.94

After counting, all data were normalized to the day when the S labelled fertilizers were applied to experimental plots. Details of calculations of relevance to 32p and 35s activities in soil and herbage samples are described in the appropriate sections.

CHAPTER 4

EVALUATION OF THE ROLE OF SHEEP DUNG IN THE SHORT TERM I MMOBILIZATION OF SOIL AND FERTILIZER SULPHUR

4.1 INTRODUCTION

Any study which attempts to measure the immediate fate of fertilizer u nder pastoral field conditions needs to consider the whole nutrient cycle and factors, which in the short term, may influence the direction of nutrient flow. Once sulphate enters the plant root and is assimilated into shoot S, the above ground nutrient flows (as modelled by Saggar et al., _{1 990a, 1 990b) are}

similar whether S is applied as s0 or sulphate-S fertilizer ( Figure 4. 1 ) . Thus factors which are likely to play important roles in determining whether S0 is conserved more efficiently in soil than sulphate-S must influence the S transformations which generate plant available sulphate in the soil or affect the rate at which the sulphate pool is depleted by microbial immobilization, leaching and plant uptake. Some of these factors, which have a large influence on short term nutrient flow, are : climate, plant species, soil type and structure and defoliation frequency (grazing interval). These factors can be easily simulated and their effects studied in s mall plot or undisturbed soil core experiments. The effects of excretal return from the grazing animal are less easy to simulate. Some researchers, however (Smith R.G and M.J. Mclaughlin, personal communications), have used the return of dung (or dung products) on field plots and glasshouse pots to simulate excretal return. In general, however, little information is available on the immediate effect of sheep dung on the uptake of S by pasture plants.

The return of animal excreta is not easily simulated in small scale experiments. This is partly because o n the field scale the grazing animal returns excreta, containing large amounts of growth-limiting nutrients, u nevenly across a field. The irregular pattern of return depends on topographical and climatic influences upon the animals' grazing and camping behaviour (Hilder, 1 964; Gillingham, 1 980; Gillingham et al., 1 980; Rowarth, 1 987; Rowarth et al., 1 985, 1 988, 1 990; Saggar et al., 1 990a, 1 990b). Furthermore large nutrient (particularly nitrogen) returns in excreta applied to a small trial plot would have a major influe nce over nutrient availability and pasture production, probably confounding or obscuring the influence of the rate of a fertilizer S treatment. The return of S to soils as animal excreta, however, remains a major pathway of S in the S cycle of g razed pastures. Experiments conducted with sheep to measure the effect of their excreta on pasture production have shown that the return of sheep dung gave an 1 8% increase in dry matter production (Sears and Goodall, 1 948), whilst Watkin (1 954) found that sheep dung made no contribution to pasture production, and only observed an effect in combination with a high rate of nitrogen fertilizer. Site specific results such as these are expected because the responsiveness of pasture growth to dung return will partly

In a study in New South Wales, Australia, using 35s label gypsum, it was calculated that the excreta of 20 sheep per hectare could provide 20% of the S requirement of pasture plants over a grazing period of 1 20 days (Kennedy and Til l , 1 98 1 a) . Of the S excreted by sheep approximately 60% is excreted in the urine and 40% in dung (Till, 1 975). Dung has a C : S ratio at >200 : 1 . The return of excreta to a pasture soil may influence the immediate fate of fertilizer S in the following manner:

1 . I ncreased pasture and root growth, stimulated mainly by the excretal nitrogen, could increase the plant uptake of soil and fertilizer S, or accelerate the conversion of sulphate S to organic S forms (Barrow, 1 967b; Curll, 1 982; Boswell, 1 983; Goh and Nguyen, 1 990; Haynes and Williams, 1 99 1 ) .

2. The carbon added to soil as undigested herbage in dung has been shown to decay slowly (Barrow, 1 961b; Boswell, 1 983) in soil and therefore could act as a carbon source used by micro-organisms to immobilize free sulphate-S from the soil solution. Goh and Nguyen (1 990) have suggested that excretal returns could stimu late soil micro-and macro-flora and fauna such that S0 oxidation may increase.

Research on the spatial distribution of excreta has been undertaken for cattle (Petersen et al.,

1 956; Richard and Wolton, 1 976) and for sheep (Tallis and Donald, 1 964; Donald and Leslie, 1 969; Gillingham and During, 1 973; Gillingham, 1 978; G illingham, 1 980; Gillingham et al. ,

1 980; Thorrold et al., _{1 985). Recently, in New Zealand Morton and Baird ( 1 990) have shown}

that the spatial distribution of sheep dung in relation to stocking rates was best described by a negative binomial function. There was significantly more aggregation of dung patches at lower stocking densities than at higher stocking densities. These authors considered the returns of nitrogen from dung and u rine affected an insufficient area to influence pasture growth. I n addition, Rowarth, ( 1 987) and Rowarth e t al. ( 1 985, 1 988 and 1 990) have shown the major mechanisms controlling the movement of P from sheep dung into soil was the rate of p hysical break-down of sheep dung rather than the leaching of P from the dung sample. During the winter/spring period, the physical break-down take place within a month, as a result of high rainfall and biological activity, whereas in the summer/autumn period the dung persisted for approximately three months. During a short period (1 7 and 8 weeks for autumn and spring, respectively) dung P was less available to pasture than monocalcium phosphate. There is no information describing the influence of sheep dung on the subsequent fate of soil and fertilizer S in the zone of excreta-affected soil. Although Kennedy and Till (1 981 a) and Boswell ( 1 983) have studied the fate of dung S alone.

Thus, before proceeding with the main experiments to examine the immediate fate of fertilizer S in _{pastoral soils grazed} by sh_eep, preliminary experiments have had to be carried out to determine:

a. the likely area of pasture influenced by sheep dung return and b. the influence of dung return on the short term fate of fertilizer S.

These experiments also served to evaluate radioisotope handling and measuring techniques.