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Effect of centrifuge conditions on water and total dissolved phosphorus extraction from soil.

Introduction

Plants acquire their mineral nutrients from the water contained in the interstitial spaces within the soil known as soil water (Adams et al. 1980). This water provides a pathway through which pollutants can be exported via drainage or overland flow (Nash et al. 2001). Consequently analysis of soil water has been used to investigate pollutant mobilisation (Wolt and Graveel 1986; Elkhatib et al. 1987).

A difficulty with using soil water to investigate pollutant mobilisation has been obtaining samples that consistently and accurately reflect natural field conditions. Numerous methods have been proposed to extract soil water. These include centrifugation (Davies and Davies 1963; Gillman 1976), column displacement (Parker 1921; Adams et al. 1980), vacuum displacement (Adams 1974), and immiscible liquid displacement (Mubarak and Olsen 1976; Wheelan and Barrow 1980) through porous ceramic or plastic filters (Duke and Haise 1973; Hossner and Phillips 1973; De Jong 1976; Grossmann and Udluft 1991).

Most of the methods used for extracting soil water are impractical for routine analysis. Column displacement is constrained by the large sample sizes required (>500 g) and the slow sample turn-around time (Wolt and Graveel 1986). Immiscible liquid displacement uses toxic chemicals to displace the soil water and can alter the composition of the soil water obtained (Davies and Davies 1963; Elkhatib et al. 1987). For other extraction methods, the reproducibility of results has often been disappointing (Hansen and Harris 1975).

Centrifugation, one of the most widely used techniques for extracting water from soil, was first used in 1907. Since then, it has been progressively modified. A method reported in 1963 forms the foundation of the current techniques (Davies and Davies 1963). This method was further modified in 1976 to allow larger samples of up to 250 g of wet soil to be used (Gillman 1976). Subsequent modifications (Mubarak and Olsen 1976; Adams et al. 1980; Wolt and Graveel 1986) included improvements to sample handling (Slattery et al. 1998). Although centrifugation of soils to extract soil water is widely used, no single set of standard operating conditions in terms of centrifuge time and speed has been defined.

This paper presents the results of a study investigating the effects of varying centrifuge time and speed on the volume of soil water extracted and its total dissolved phosphorus (TDP) concentration using a modified centrifuge method based on that of Gillman (1976) and Slattery et al. (1998). As the soils were dried and rewet to a specific moisture content before centrifuging, the effects of varying soil-moisture content and time between rewetting and centrifuging (equilibration time) were investigated.

The aims of this study were to investigate the effects of varying centrifuge time and speed on the estimate of TDP and to determine the best combination of these factors to obtain sufficient volumes of soil water and reproducible samples for analysis for future use in routine extraction of soil water.

Materials and methods

The apparatus (Fig. 1) used for separating soil water from the soil was based on that of Gillman (1976) and the modifications of Slattery et al. (1998). The apparatus consisted of 4 polyethylene, machine-lathed cylindrical holders that fitted securely into Spintron GT-15F (radius 19.7 cm) centrifuge buckets (Spintron Pry Ltd, Australia). The vessels were hollow to accommodate polypropylene Nalgene[R]) bottles (Nalgene Pry Ltd, Australia) with the top and bottom removed to form an open cylinder. The bases of the polyethylene holders were drilled to enable each holder to act as a filter. Whatman No. 1 filter paper (Whatman International Ltd, England) was placed in the holders over the strainer. Wet soil (c. 200 g) was placed into the modified Nalgene cylinders for centrifugation. The soil water was collected beneath the polyethylene holder in the bottom of the Nalgene bottle (Fig. 1).

[FIGURE 1 OMITTED]

Whatman No. 1 filter papers (pore size 11[micro]m) were used on the polyethylene base. These filters were selected because of their flow characteristics and because they allowed the collection of both dissolved (<0.45 [micro]m) and particulate forms.

Soil characteritics

Soils from 2 sites in the Gippsland region of south-eastern Australia were used to optimise centrifuge conditions in these experiments. The first site was the Ellinbank Research Farm (38[degrees]24.8'S 145[degrees]55.6'E), where the soil was an acidic, mesotrophic, red Ferrosol (Isbell 1996), Gn4.11 (Northcote 1979), Haplustox (Anon. 1998). The second site was located in Arawata (38[degrees]10.2'S 146[degrees]2.7'E). Soil on this site was an acidic, eutrophic, grey Dermosol (Isbell 1996), Gn4.41 (Northcote 1979), Haplustalf (Anon. 1998). Soils were sampled to a depth of 0-20 mm, as this is where soil and surface runoff interact and potentially where phosphorus mobilisation and transport occurs (Ahuja et al. 1981; Sharpley et al. 1981). Soil properties and chemical characteristics are shown in Table 1.

Contrasting soils (a Ferrosol and a Dermosol) were selected to determine whether soil type affects the extraction of soil water and the phosphorus concentration in the soil water. Ferrosols are texturally classed as a fine sandy clay loam. They are generally very well-structured, well-drained soils with a high iron content so they adsorb phosphorus strongly. Dermosols are classed as heavy clay loans. These soils also tend to be well structured but are less well drained and do not have a high free iron content.

Experimental procedures

The aim of this study was to determine optimal centrifuge conditions for extracting soil water from soil. Therefore, it was necessary to use soils that had been dried, crushed, sieved, and rewet to achieve uniform soil moisture contents and to remove the variations in solute concentrations that are typically associated with heterogeneous soil cores (Giesler and Lundstrom 1993). The disadvantage of this approach is that the fewer soils may have different properties, especially chemical properties, from field-moist soils (Birch 1960; Nero and Hagin 1966; Bartlett and James 1980). It was expected that there would be some changes in the structure of rewet soils (i.e. through being dried, crushed, and sieved) that could lead to reduced collection times and yield larger volumes of filtrate than field moist soils. This hypothesis was tested by centrifuging some samples of field-moist soils for varying times and at varying centrifuge speeds.

Expt 1: Volume of soil water extracted at varying centrifuge times and speeds

In the initial experiments using only the Dermosol, the effects of centrifuge time and speed on the volume of soil water extracted were investigated. Soil (12.5 kg) that had been air-dried (40[degrees]C h crushed, and sieved (<2 mm) was re-wet (5.23 L of water) to a gravimetric moisture content of 42% approximately 1 h before commencement of the experiment.

The experiment used a factorial design comprising 4 centrifuge times (5, 10, 15, and 30 min) and 4 centrifuge speeds (1000, 2000, 3000, and 3500 r.p.m., corresponding to centrifugal forces of 220, 881, 1982, and 2698g, respectively). Four replicates (200.00 [+ or -] 0.05 g wet soil) were centrifuged at each time and speed combination. The soil water obtained from each of the centrifuge speed and time combinations was weighed to determine the volume of filtrate with an assumed density of 1 g/[cm.sup.3].

Expt 2: Volume and TDP concentrations extracted from soils at varying centrifuge times and speeds

Both Ferrosol and Dermosol soils were dried (40[degrees]C), crushed and sieved (<2 mm), and fewer to specific gravimetric water contents. The treatment design comprised 2 soil types, 2 soil water potentials (White 1987; Leeper and Uren 1997), 2 centrifuge times (5 and 30 min), 2 centrifuge speeds (1000 and 3000 r.p.m.), and 2 equilibration times prior to centrifugation in factorial combination, giving 32 different experimental combinations, performed in duplicate.

To obtain similar soil water potentials of 2 and 7.5 kPa for both soil types, the gravimetric soil water contents (as determined from soil moisture characteristic curves produced for these soils) were 55 and 64, and 46 and 49% for the Ferrosol and Dermosol treatments, respectively.

Two equilibration times before centrifugation, 6 h (short) and 24 h (long), were used to test the effects of sample holding time. Samples were stored at <4[degrees]C during this time. In all other respects, the experimental design was similar to the previous experiment. After weighing, the filtrate was filtered again using a 0.45 [micro]m Millex HV (Millipore, USA Cat. No. SLIIV 025 LS) syringe filter unit and prepared for TDP analysis (previous unpublished studies have shown similar trends between TDP and total P concentrations in soil water) using an alkaline persulfate digestion procedure adapted from Lachat method number 30-115-01-1-B (Anon. 1997). Alkaline persulfate digestion methods decompose most phosphates present and are a commonly used and accepted method. Samples were then analysed on a Lachat Quickchem 8000 (Zellweger Analytics Inc., USA) flow injection system at 880 nm using the phosphomolybdenum blue detection method.

Expt 3: Optimising centrifuge conditions for field moist soils

Samples of field-moist Ferrosol and Dermosol soil were collected to compare the results with those from the re-wet soils (Expts 1 and 21. Forty soil cores (20 mm depth) were collected from each site when the soils were near field capacity (c. 2 days after heavy rainfall). Soil cores from each site were bulked and hand mixed until visually homogeneous before being centrifuged. Four replicates of each soil were centrifuged at 3000 r.p.m. for 5 min, the optimum centrifuge conditions determined from Expt 2. However, to compare the effects of additional centrifugation, the same soil samples were then re-centrifuged for 25 min (i.e. 30 min total) and the soil water from this second extraction was also collected and weighed. The soil water samples were then analysed for TDP using the same procedure described above in Expt 2.

Statistical analysis

An analysis of variance (ANOVA) was performed for all the experiments using GENSTAT 5.41 (1998). As the spread of residual values was quite small, no transformations were required for any of the datasets. Least significant differences (l.s.d.) at 0.05 were also calculated for each dataset.

Results and discussion

Effects of centrifuge time and speed on the volume of soil water extracted

As expected, increasing the centrifuge time from 5 to 30 min significantly increased (P < 0.001) the volume of soil water extracted (Fig. 2). The choice of maximum centrifuge time (30 min) was a compromise. From a practical point of view, 30 min was considered the maximum time that could be allowed if a sufficient number of replicate samples were to be processed. On the other hand, a sufficient amount of filtrate was needed for subsequent analyses. Given that Fig. 2 shows that the volume of filtrate did not reach a plateau even after 30 min, a greater volume of filtrate could be expected if the centrifuge time were increased further.

[FIGURE 2 OMITTED]

Increasing centrifuge speed also significantly (P < 0.001) increased the volume of soil water extracted by ~15%. It was found that 3000 r.p.m, was the maximum speed that this equipment could sustain. A higher centrifuge speed would be expected to increase the volume of soil water extracted as water from the smaller pores should be extracted. Centrifuge speed was again an important factor (P < 0.001) in Expt 2 affecting the volume of soil water extracted and accounting for 29% of the total variance. Soil moisture content, soil type, and centrifuge time were also highly significant (P < 0.001), accounting for 28%, 22%, and 9.7% of the variance, respectively (Figs 3 and 4). Several second- and third-order interactions, although highly significant (P < 0.001), explained only a small proportion of the total sum of squares. Unlike Expt 1, the results suggest that centrifuge speed rather than centrifuge time had the major effect on filtrate volume.

[FIGURES 3-4 OMITTED]

In Expt 2, the higher centrifuge speed (3000 r.p.m.) extracted the largest volumes of soil water (Fig. 3) regardless of the equilibration time allowed. Interestingly, increasing the centrifuge time for the Ferrosol resulted in only small changes in the volume of soil water extracted compared with the change observed for the Dermosol. As expected, the samples with the higher moisture contents yielded larger volumes of soil water than the samples with the lower moisture contents.

Centrifuging field-moist Ferrosol and Dermosol soils in Expt 3 for 30 min compared with 5 min at 3000 r.p.m. enabled the extraction of up to 55% more soil water from the replicate samples. The average volume of soil water extracted from the 4 replicate samples increased from 4.31 to 7.97 mL for the Ferrosol and from 4.41 to 8.02 mL for the Dermosol when the centrifuge time was increased from 5 to 30 min. Centrifuge time was highly significant (P < 0.001) in determining the amount of soil water that was extracted. Soil type was not an important factor (P - 0.8) in the amount of soil water that was extracted, as similar volumes were extracted from both soil types. Altering the centrifuge time and speed gave similar results to those obtained in the Expt 2, which used soils that had been dried and rewet. This was expected given that the same soil types were used. The volume of soil water extracted at a centrifuge time of 5 min was enough for analysis of TDR However, as it is more difficult to extract soil water from some field-moist soils, it is recommended that a slightly longer centrifuge time of 10 min be used to extract soil water. If large amounts of soil water are required then a longer centrifuge time can be used.

Effect of varying centrifuge time and speed on the TDP concentration extracted in the soil water

Contrasting soils were selected for Expt 2 in terms of their texture, particle size distribution, and adsorption characteristics in order to determine the effect of soil type on extracting soil water and TDP concentration from different soil types. Soil type was statistically significant (P < 0.001) and was the most important factor affecting the concentration of TDP observed in the soil water, accounting for 85% of the variance (1.0-1.3 and 1.6-2.0 mg/L for the Ferrosol and Dermosol, respectively). The significant difference in TDP concentration in the soil water for the different soil types was initially thought to be in part due to different P concentrations in the soils. A measure of soil-available P, Olsen P (Rayment and Higginson 1992), was, however, very similar, at 44 and 43 mg/kg for the Ferrosol and Dermosol, respectively. These results are consistent with data (M. Toiftl, unpublished data) from other recent experiments where neither TP nor TDP concentrations reflected the Olsen P levels in the different soils.

Centrifuge speed did not significantly affect the concentration of TDP extracted in the soil water (P = 0.140). To remove soil water that is tightly bound within the soil particles, a high centrifuge speed is needed. As the centrifuge speed increases, more soil water should be extracted until no further pore water is available. If the amount of P closely bound to the surface layers of the soil were extracted, an increase in P concentration might be expected. However, this hypothesis was unable to be tested as the maximum achievable speed with the equipment used was 3000 r.p.m. and the amount of water extracted had not started to reach a limit. The results from this experiment suggest that the concentration of TDP is reasonably homogenous in the water available at the centrifuge speeds used. These results are consistent with those of Shand et al. (1994), who used conditions different from those reported in this paper, to show that centrifugation forces from 750 to 1400g on the P composition of soil water were not critical. While centrifuge time would be expected to affect the volume of soil water extracted, it would not be expected to affect the concentration of P in the soil water. This expectation was confirmed by the results from the experiment. It was found that centrifuge time was not a highly significant factor (P < 0.05) affecting the concentration of TDP extracted in soil water within each soil type at either of the equilibration times.

Increasing the equilibration time from 6 to 24 h for the Ferrosol resulted in little change in the P concentration extracted in the soil water (Fig. 4). However, increasing the equilibration time by the same amount for the Dermosol led to a highly significant decrease (P < 0.001) in the concentration of P extracted in the soil water. Ferrosols are highly buffered and have high iron content, which is present as hydrous oxides. Hydrous oxides of iron in soil are known to fix and accumulate P in organic and inorganic forms (Wild 1988). Ferrosols are classed as 'high P fixing' soils in which P sorption from the soil water to the soil material occurs rapidly, whereas Dermosols are classed as 'low P fixing' soils, since P sorption occurs more slowly. The results of P sorption analysis of the 2 soils showed that the Fcrrosol had a higher P sorption capacity than the Dermosol (Table 1).

Soil moisture content was significant but not highly significant (at P = 0.01) for either soil type with respect to the concentration of TDP extracted in the soil water. At the high soil moisture contents for the Ferrosol, the concentration of TDP extracted was lower than for the low soil moisture content. Given that at the higher soil moisture content there was greater dilution, this result was expected. For the Dermosol there was little variation in the TDP extracted at the different soil moisture contents.

For the field-moist soils used in Expt 3, soil type was the most important factor affecting the TDP concentration (P < 0.001) extracted in the soil water. Centrifuge time was not a significant factor (P = 0.1) in the concentration of P extracted, which makes these results consistent with those obtained in the second experiment. The average TDP concentration extracted in the soil water from the Ferrosol was 0.79 and 0.78 mg/L for the 5 and 30 min centrifuge times, respectively. The average TDP concentrations for the Dermosol were 1.14 and 1.07 mg/L for the 5 and 30 min centrifuge times. The TDP concentrations extracted from the field-moist soils were much lower than those from the soils that had been dried and rewet as used in Expt 2. The reason for this was probably the drying and wetting cycle of the soil, which has previously been shown to cause a release of P from the soil into the soil water, often termed the 'microbial flush effect' (Turner and Haygarth 2001). The microbial flush effect has been suggested to be caused by the process of rewetting soils causing disruption of aggregates, which can then release organic matter, and also through microbial death, which occurs during the rewetting process by osmotic shock and cell lysis (Salema et al. 1982; Kieft et al. 1987; Van Gestel et al. 1993; Magid et al. 1999). This increases the amount of P in the soil water to concentrations that would not typically be seen in the field. It follows that TDP concentrations from the field-moist soils were lower than those from the dried and rewet soil, although similar trends were observed. This suggests that the centrifuge technique is applicable to field-moist soils, but for the reasons outlined above, dried and rewet soils provide a poor estimate of TDP concentrations in soil water in the field.

Conclusion

The results show that altering centrifuge time and speed can greatly affect the volume of soil water obtained from both the Dermosol and Ferrosol soils. However, neither centrifuge time nor centrifuge speed significantly affected the concentration of TDP measured in the soil water. Both centrifuge time and speed were, however important considerations in terms of obtaining enough soil water for analytical purposes.

Soil type was the most important factor affecting the concentration of TDP obtained in the soil water for both field-moist soils and those that had been dried and rewet. Equilibration time and soil moisture content were also found to have a highly significant effect on the concentration of TDP extracted in the soil water from rewet soils.

Low centrifuge times (5 min) and high centrifuge speed (3000 r.p.m.) allowed enough soil water for analysis (TDP) to be obtained from both soils (dried, crushed, and sieved) at the different moisture contents. To ensure sufficient soil water is collected from field-moist soil, a centrifuge speed of 3000 r.p.m. and a centrifuge time of 10 min is recommended.
Table 1. Soil properties and chemical characteristics

Characteristic Ellinbank (Ferrosol) Arawata (Dermosol)

Textural class Fine sandy clay loam Sandy clay loam
Colour Dark greyish brown Darkish grey brown
Gravel <5% <5%
Lime Negligible Negligible
pH([H.sub.2]O) (A) 5.6 5.4
pH(Ca[Cl.sub.2]) (B) 5.1 4.8
EC (dS/m) (C) 0.19 0.15
Organic matter (% W/W) (D) 23 15
Total C (% w/w) (E) 12 8.1
Olscn P (F) 44 43
Adsorbed P (mg/kg) (G) 3089 1613

Rayment and Higginson (1992) Methods: (A) 4A1; (B) 4B2;
(C) 3A1; (D) 6A1; (E) 6Al; (F) 9C2; (G) 9J1.


Acknowledgments

The authors thank Dr W. Slattery of Greenhouse Office, Canberra, for his assistance with the design of the centrifuge equipment, and Murray Hannah from PIRVic, for his help with statistical analysis. We also thank the Soils and Water Team at PIRVic for their assistance.

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Melissa Toifl (A,B,C), David Nash (B), Felicity Roddick (A), and Nichola Porter (C)

(A) RMIT University, School of Civil and Chemical Engineering, City Campus, GPO Box 2476V, Melbourne, Vic. 3001, Australia.

(B) Primary Industries Research Victoria (PIRVic), RMB 2460 Hazeldean Rd, Ellinbank, Vic. 3821, Australia.

(C) RMIT University, Department of Applied Chemistry, City Campus, GPO Box 2476V, Melbourne. Vic. 3001, Australia.
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Publication:Australian Journal of Soil Research
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Date:Dec 15, 2003
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