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Soil Science Society of America Journal 67:637-644 (2003)
© 2003 Soil Science Society of America

DIVISION S-6—SOIL & WATER MANAGEMENT & CONSERVATION

Influence of Long-term Cropping Systems on Soil Physical Properties Related to Soil Erodibility

Achmad Rachman*,a, S. H. Andersona, C. J. Gantzera and A. L. Thompsonb

a 302 Anheuser-Busch Natural Resources Building, Environmental Soil Science Program, University of Missouri, Columbia, MO 65211
b Dep. of Biological Engineering, Univ. of Missouri, Columbia, MO 65211

* Corresponding author (arbb1{at}mizzou.edu)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Crop rotations and manure application are thought to alter soil quality. This study was conducted to quantify the effects of over 100 yr of continuous crop management and annual manure applications on selected soil physical properties at Sanborn Field, Columbia, MO. Intact soil cores (76 mm i.d. by 76 mm) were collected from continuous corn (Zea mays L.), continuous wheat (Triticum aestivum L.), continuous timothy (Phleum pratense L.), and a rotation of corn–wheat–red clover (Trifolium pratense L.). The soil was Mexico silt loam (fine, smectitic, mesic, Aeric Vertic Epiaqualfs). Soil was tested throughout a 1-yr period for aggregate stability, single-drop rainfall splash detachment, and soil shear strength. Cropping systems affected aggregate stability (P < 0.01), soil strength (P < 0.01), and splash detachment (P < 0.01), but not bulk density. Continuous cropping to timothy produced soil that had three to four times greater aggregate stability, 21 to 27% greater soil strength, and 55 to 67% less soil splash compared with continuous wheat or continuous corn. Season significantly affected all measured soil properties, but the effect was inconsistent. The highest aggregate stability was found during July for all treatments. Splash detachment was more sensitive to cropping systems than other soil measures, and thereby the best measure for evaluating changes in soil erodibility. Cropping and soil management that accumulate plant residues can improve soil quality by increasing soil aggregate stability, shear strength, and resistance to splash detachment.


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
SOIL physical properties are important for favorable conditions for crop growth and maintaining soil quality. Tillage is used for planting and also to loosen surface and subsurface soil to decrease soil compaction. In some cases, however, continuous cultivation of row crops has detrimental effects on soil quality. Edwards (1982) reported that conventional tillage increased surface runoff in a watershed in Ohio by about 20 times compared with no tillage. The reduced surface runoff from no tillage was attributed to a greater number of macropores.

Many practices are known to influence soil physical properties. These include crop type (Harris et al., 1966; Alberts and Wendt, 1985; Scott et al., 1994), cultivation (Woodruff, 1939; Gantzer and Blake, 1978), and application of organic residues (Anderson et al., 1990; Gantzer et al., 1987; Ekwue, 1990). Effects of cropping systems on soil physical properties are often related to increases in soil organic matter (Dormaar, 1983; Ghidey and Alberts, 1997; Haynes, 2000). Harris et al. (1966) related the action of growing plant roots with both aggregate formation and breakdown. Cultivation generally tends to break down aggregates. The stability of soil aggregates often decreases for soil under annual crops, such as wheat or corn (Page and Willard, 1946; Low, 1972; Angers et al., 1999). Gantzer et al. (1987) reported that residue quantity had a larger effect on splash detachment, shear strength, and aggregate stability than residue type. MacRay and Mehuys (1985) concluded that long-term pastures are ideal for improving soil aggregation.

Soil-aggregate stability, shear strength, and soil erodibility are dynamic properties that change over time (Coote et al., 1988). Sanborn Field, located on the University of Missouri campus at Columbia, was established in the fall of 1888. The field originally consisted of nine cropping practices using corn, oat (Avena sativa L.), winter wheat, red clover, and timothy under three soil fertility treatments (Buyanovsky et al., 1997). During the past 100 yr, some plots had been managed with treatments consistent with the original with very minor alterations since 1888. It is hypothesized that soil physical properties have changed in these historical plots because of the effects of different cropping systems applied continuously for more than 100 yr. This study will provide a better understanding on how long-term soil and crop management affect bulk density, water aggregate stability, soil shear strength, and splash detachment. The objectives of this study were to evaluate the effects of long-term crop management on bulk density, wet-aggregate stability, soil shear strength, and splash detachment during a 1-yr period. In addition, aggregate stability for two shallow soil depths and the correlations between soil physical properties were evaluated.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Eight plots, located on Sanborn Field, University of Missouri-Columbia, were chosen as experimental units. The soil at the site is a Mexico silt loam. The plots compared in this study were all separated by a 1.5-m wide grass border. All plots were on a similar landscape position and had slopes ranging from 1.9 to 2.2%. Plots were 30.6 by 9.5 m. All have been continuously managed with the same treatments since 1888 (Upchurch et al., 1985; Anderson et al., 1990). The cropping and soil management treatments were continuous wheat unfertilized, continuous wheat with 13.5 Mg manure ha-1 yr-1, continuous corn unfertilized, continuous corn with 13.5 Mg manure ha-1 yr-1, continuous timothy unfertilized, continuous timothy with 13.5 Mg manure ha-1 yr-1, 3-yr corn–wheat–red clover rotation unfertilized, and 3-yr corn–wheat–red clover rotation with 13.5 Mg manure ha-1 yr-1. Manure was applied in August for the rotation and continuous wheat, October for continuous corn, and December for continuous timothy. The wheat treatments were moldboard plowed in August and planted in October after chisel plowing. The corn treatments were moldboard plowed in October, left bare until planting in April. The rotation treatments were moldboard plowed in August and then planted with red clover. During this study the rotation treatments were under red clover. The timothy treatments were not plowed during this study, but had been plowed and replanted in 1989. Table 1 indicates the soil and crop management history of the selected plots.


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  		Table 1. Crop and soil management history of selected Sanborn Field plots.{dagger}

 
Barnyard manure was used as fertilizer. Manure from a horse barn was used for the first 50 yr of this study. Subsequently, manure from a dairy barn was applied annually (Buyanovsky et al., 1997). Average properties and their standard errors on a dry weight basis of the manure applied during the years just prior and during this experiment were as follows: 18.6 ± 4.3 g kg-1 total N, 9.9 ± 5.9 g kg-1 P, 12.0 ± 9.1 g kg-1 K, 1.9 ± 1.9 g kg-1 Na, 4.0 ± 2.4 g kg-1 Mg, and 29.4 ± 22.8 g kg-1 Ca.

The soil studied is similar to soils in an area of approximately 4 million ha within Missouri, Illinois, Kansas, and Oklahoma (Jamison et al., 1968). Selected soil physical and chemical properties of the plots are shown in Table 2. The soil organic C ranged from 5.2 g kg-1 in the unfertilized corn to 23.4 g kg-1 in the manured timothy. Clay content ranged from 14.8% in the unfertilized rotation to 30.0% in the manured corn. This variation in surface soil texture was because of 100 yr of continuous cropping and subsequent erosion for the corn treatments (Gantzer et al., 1991).


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  		Table 2. Selected soil physical and chemical properties for the surface soil horizon of selected Sanborn Field plots.{dagger}

 
Intact soil samples were collected using a core sampler 76 mm i.d. by 76 mm (Blake and Hartge, 1986). Three sampling locations were chosen in each plot on points 7 m apart. The samples were taken in the crop row between plants for corn and wheat. For the timothy and rotation treatments, samples were taken at the sampling point in nontrafficked areas. Two soil cores were taken from each sampling location from the 20- to 96-mm depth. One soil core was used for aggregate stability analysis and the other core for splash detachment and soil shear strength determination. Samples were stored at 4°C until testing. Soil cores were collected on 5 Nov. 1992, 22 Jan. 1993, 24 Apr. 1993, and 23 Jul. 1993.

Bulk density was determined using the core method (Blake and Hartge, 1986). The bulk density was determined on the same samples used for fall-cone soil shear strength measurements.

Aggregate Stability Analysis
Soil from one core at each sampling location was removed, cut in half (20- to 58- and 58- to 96-mm depth increments), and spread out in a 3-mm thick layer to air dry. The air-dried samples were gently crumbled by hand and sieved to retain the 1- to 2-mm aggregates. These aggregates were stored at 4°C until tested using a wet sieving technique (Kemper and Rosenau, 1986). The two depths were analyzed separately.

Soil Splash Detachment Test
Soil splash detachment was conducted using a single-drop method (Gantzer et al., 1985). Gantzer et al. (1985) improved the two-stage baffle of Al-Durrah and Bradford (1981) by adding a drop discriminator to the system. The drop discriminator, which consists of two light-source sensors, an electronic controller, and an electromechanical shutter, maintains the same drop diameter throughout the study and prevents unaligned drops from hitting the soil surface.

The soil core was slowly satiated to 0 kPa for 24 h with de-aired deionized water. The deionized water was used to intensify the detachment of soil. The soil was drained to an equilibrated -2 kPa pressure with a tension table. A constant hydraulic head of 10 cm was maintained over the top of the drop former throughout the study. This head produced an average drop mass of 59.74 ± 0.29 mg. Assuming a spherical drop, the average drop diameter was 4.85 mm. Water drops were dropped a distance of 11.5 m onto a 16-mm diameter target area on the surface of the soil core. The velocity and the kinetic energy of drops were calculated from the results of Laws (1941) and found to be 9 m s-1 and 2.4 x 10-3 J, respectively. Before splash determinations were made, the surface of the soil core was carefully sprayed with a fine mist of water to remove any loose soil.

The splash measurement was conducted by first lowering the splash collector to just contact the soil surface. An aluminum disk with a hole of 16-mm diameter was attached to the bottom of the cylinder to capture the splash; the disk forms a 1-mm beveled edge around the hole. When a drop struck the soil surface, the shutter was automatically closed to prevent more drops from striking the soil surface. The splash collector was then removed; the soil was washed with distilled water into a previously weighed 100-mL beaker. The soil splash was then oven-dried at 105°C and weighed to the nearest 0.1 mg. Three splash determinations were collected at different locations on the surface so that the amount of splash was not affected by previous water drop impacts.

Fall-Cone Shear Strength Test
Immediately after soil splash determinations were conducted, the undrained soil shear strength was determined with a Geonor g-100 fall-cone device (Geonor Inc., Oslo Norway; Hansbo, 1957). The following equation was used to determine shear strength:

[1]
where {tau} is the undrained shear strength (kPa), Q is the mass of the cone (kg), h is the depth of penetration of the cone (m), and K is a proportionality factor which depends on the cone angle and soil texture. Since the soil tested in this study was silt loam, K = 10 ms-2 was used as reported by Towner (1973). Three fall-cone measurements were made on each soil core at least 20 mm from the edge of the core.

Statistical Analysis
Sanborn Field plots were established in 1888 without replication; statistical field replication concepts had not yet been developed. However, the long-term treatments have been continuously applied for more than 100 yr. To analyze the long-term effects of these treatments on soil physical properties, fertility treatments (manured and unfertilized) were used as replicates to obtain a pooled error term to test the effects of cropping systems (Anderson et al., 1990). Analysis of the soil property data indicated that fertility treatments had relatively smaller effects compared with cropping systems, supporting the idea that using fertility treatments would be appropriate for replication. Therefore, we sacrificed the effects of manure and the interactions of manure with cropping systems and focused on the effects of cropping treatments on soil physical properties. Analysis of variance was performed using SAS (SAS Institute, 1989) with P = 0.05. Statistical comparisons were not made among fertility treatments because of this decision. Single degree of freedom contrasts were developed before the study, which were used to compare differences in cropping treatments: timothy vs. others (rotation, corn, wheat); rotation vs. corn and wheat; and corn vs. wheat. Single degree of freedom contrasts were also developed before the study to compare the effect of season on the soil properties: July vs. others (April, November, January); April vs. November and January; and November vs. January. Linear regressions and correlations were also made between the measured soil physical properties.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Bulk Density
No effect of cropping systems and no significant interactions among cropping systems and season were observed for bulk density (Table 3 and Fig. 1) . Seasonal changes in bulk density were found in this study (Table 3, P < 0.01). Higher bulk density was typically found before spring tillage. Bulk density increased gradually from November to a high in April then decreased to a low in July. Single degree of freedom contrasts of July vs. Others, and April vs. November and January were highly significant (Table 4). These results agree with the results of Gantzer and Blake (1978) who found that the bulk density of a clay loam was higher in spring before tillage compared with fall. As time since tillage increased, consolidation occurs because of rainfall and subsequent drainage. Scott et al. (1994) reported a similar trend that bulk density decreased from May to November; then increased during winter at both the 0- to 50-mm and 50- to 100-mm depth intervals. Disking and weather fluctuations were responsible for temporal variations in bulk density.


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  		Table 3. Probability of F values (Pr > F) from analysis of variance for bulk density, aggregate stability, soil strength, and splash detachment as affected by long-term cropping systems and season (n = 32).

 


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  		Fig. 1. Average bulk density for selected cropping systems (n = 8). Bars represent the standard error of the mean.

 

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  		Table 4. Probability of F values (Pr > F) from single degree of freedom contrasts for bulk density, aggregate stability (20- to 58-mm depth), soil strength, and splash detachment as affected by long-term cropping systems and season (n = 32).

 
Aggregate Stability
Effects of cropping systems on aggregate stability were highly significant for both depths (Table 3, P < 0.01). As shown in Fig. 2 , aggregate stability was very low for corn and wheat, with the wheat treatment possessing the lowest value (17.85 ± 2.13% for the first depth and 21.81 ± 1.75% for the second depth). Increases in aggregate stability were observed for the 58- to 96-mm depth compared with the 20- to 58-mm depth for the corn, wheat, and rotation treatments. It is unknown why this occurred; it may be related to aggregate disruption from more intense wetting and drying of the surface soil. The contrasts of timothy vs. others and rotation vs. corn and wheat were both significant (P < 0.01). The timothy treatment had three times the aggregate stability compared with the other treatments (Fig. 2). We speculate that development of mechanical binding by roots that remain intact without annual tillage in the timothy treatments played an important role in enhancing the stability of soil aggregates. In the corn and wheat treatments, frequent tillage and exposure to raindrop impact during the fallow period from August to October for wheat and from October to April for corn may have increased the disruption of soil aggregates. Another possible reason is that the relatively high clay content and low organic matter content of the wheat and corn plots may have increased the wettability of soil aggregates causing the aggregates to suffer more slaking on sudden wetting (Chenu et al., 2000). Raindrop impacts on bare soils and slaking may be responsible for aggregate breakdown, therefore reducing the stability of soil aggregates.



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  		Fig. 2. Aggregate stability values of two soil depths, 20- to 58-mm and 58- to 96-mm depth, for selected cropping systems (n = 8). Bars represent the standard error of the mean.

 
Changing the cropping systems from continuous corn or wheat to a rotation of corn–wheat–red clover increased aggregate stability by 23 to 40% (relative differences, Fig. 2). Two possible reasons are suggested for the increase of aggregate stability in the rotation treatments as compared with the continuous corn and wheat: (i) canopy protection during the fallow period, and (ii) bonding material provided by red clover. The red clover in the rotation treatments was planted directly after tillage in August. The growing red clover protects the soil surface from the rain impact, which can detach soil aggregates, therefore providing protection for the aggregates. Red clover also fixes N that can speed decomposition of soil organic matter. The microbial mediated activity of organic matter decomposition produces organic polymers that bind soil particles together and may slow the rate of aggregate wetting and thus decrease the extent of slaking (Haynes, 2000; Chenu et al., 2000).

Season had a significant effect on aggregate stability for the 20- to 58-mm soil depth (Tables 3 and 5). This depth experienced wider variations in wetting and drying in addition to aggregate disruption from raindrop impact than the lower depth. Thus, aggregate stability at shallower depths would more likely be influenced by season.


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  		Table 5. Means of bulk density, aggregate stability, soil shear strength, and splash detachment as affected by cropping systems and season, Sanborn Field 1992-1993 (n = 2; subsamples = 3).

 
Splash Detachment
The analysis of variance for splash detachment data is shown in Table 3. Cropping systems, season, and their interaction significantly affected soil splash detachment (P < 0.01). Soil from the timothy treatments had 30 to 50% less splash detachment than the wheat and corn treatments (Fig. 3) . The rotation treatments also had 50 to 70% less soil splash than the corn and wheat treatments. In general, splash detachment decreased gradually from November to a low during January for the corn and wheat treatments and during April for the rotation and timothy treatments (Table 5). Soil splash then increased during July. Seasonal variation of splash detachment was more pronounced in the timothy and rotation treatments than in the corn and wheat treatments, which was statistically significant (P < 0.01; Table 3). Samples from the corn and wheat treatments experienced high splash detachment throughout the year with little change by season (Table 5).



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  		Fig. 3. Splash detachment for selected cropping systems (n = 8). Bars represent the standard error of the mean.

 
Gantzer et al. (1991) estimated soil erosion using the Universal Soil Loss Equation for the corn treatments to be 14.3 to 23.0 Mg ha-1 yr-1, and for the timothy treatments to be 0.2 to 0.4 Mg ha-1 yr-1. The difference is mostly related to roots and above ground plant material. These results emphasize the importance of selecting cropping systems that maintain plant material to control soil erosion. Table 3 also indicated that splash detachment was more sensitive than bulk density, aggregate stability, and soil strength as a parameter for evaluating soil and crop management effects as evidenced by its ability to detect significant interactions between cropping system and season. It is expected that soil splash detachment is more sensitive since it measured surface soil detachment on a very small area (equivalent to a raindrop diameter; splash was collected in a 16-mm diam. collector) while bulk density and aggregate stability were measured on bulk core samples.

Soil particles resist detachment until the raindrop impact force exceeds particle resistance to movement (Cruse et al., 2000). Two processes are involved in the detachment of soil particles by raindrop impact:(i) soil compression and cavity formation on impact and (ii) lateral jetting of water across the cavity boundaries (Nearing and Bradford, 1985). Splash detachment tests account for these two components while fall-cone measurements only evaluate the penetration resistance of a cone and do not evaluate the effects of lateral jetting from raindrop impact. Therefore, fall-cone measurements tend to over predict the resistance of a soil to detachment (Nearing and Bradford, 1985).

Soil Shear Strength
The timothy treatments had 10 to 27% greater soil strength and were significantly different than the other cropping treatments (P < 0.01, Table 4). Soil shear strength from the rotation treatments was significantly greater than the corn and wheat treatments (P < 0.01, Table 4, Fig. 4) .



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  		Fig. 4. Soil shear strength for selected cropping systems (n = 8). Bars represent the standard error of the mean.

 
Temporal variations in soil shear strength were also observed (Table 5). In general, soil shear strength decreased from November to January increased from January to April, and decreased again from April to July. Only the contrast of April vs. November and January was significant (P < 0.01; Table 4). Bradford and Grossman (1982) observed an increase of soil strength during April. This increase was attributed to compaction of the near-surface soil as a result of wetting and drying during late winter and early spring.

Correlations Between Soil Physical Properties
Soil shear strength increased as aggregate stability and organic C content increased (Fig. 5) . The best simple linear regression was found between soil strength and log aggregate stability (r = 0.90, Fig. 5). Inclusion of clay content as a second parameter in the regression relationship improved the coefficient of determination as follows:

[2]



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  		Fig. 5. Fall-cone soil shear strength vs. (a) the logarithm of aggregate stability (20- to 58-mm depth) and (b) organic C (n = 4). *, ** Significant at the 0.05 and 0.01 probability levels, respectively.

 
In contrast, soil shear strength was negatively correlated with bulk density (r = -0.73). Soil shear strength usually increases with increasing bulk density; however, in this study soils with higher bulk density, corn and wheat plots, had lower soil shear strength. Whereas, soils with low bulk density, timothy plots, had higher soil shear strength. These results indicated that the increase or decrease of soil shear strength was related to whether the soil management and cropping systems improved the stability of soil aggregates (Ekwue, 1990). A positive correlation between soil organic matter and aggregate stability (Tisdall and Oades, 1982; Angers, 1998; Haynes, 2000; Chenu et al., 2000) and soil shear strength (Gantzer et al., 1987; Ekwue, 1990; Ghidey and Alberts, 1997; Cruse et al., 2000) have been reported. Ghidey and Alberts (1997) found that the amount of dead root mass significantly affected soil shear strength, where mean soil shear strength of alfalfa (Medicago sativa L.) and bluegrass (Poa compressa L.) were approximately 22% higher that those for corn and soybeans. These findings may be a possible reason for the greater soil shear strength found in this study on timothy and rotation treatments compared with continuous corn and wheat treatments. Therefore, soil management and cropping systems that accumulate organic matter which, in turn, reduce the soil's vulnerability from slaking and dispersion on wetting, increase the soil's resistance to penetration by a dropped cone (Ekwue, 1990).

The relatively low coefficient of determination (r = -0.79, Fig. 6a) of soil splash detachment vs. aggregate stability indicates that aggregate stability alone is not a good predictor of splash detachment. The wet sieving procedure of aggregate stability analysis used in this study more closely approximates the slaking forces on a soil created by flowing runoff water rather than the forces of raindrop impact (Young, 1984). A high correlation (r = 0.97, Fig. 6b) was found between splash detachment and the ratio of raindrop kinetic energy to soil shear strength. Previous studies reported similar results (Al-Durrah and Bradford, 1981; Nearing and Bradford, 1985). The results of this study also indicate that as soil shear strength increases, the detachment of soil from raindrop impact decreases.



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  		Fig. 6. Soil splash detachment vs. (a) the logarithm of aggregate stability (20- to 58-mm depth) and (b) the ratio of kinetic energy and fall-cone soil shear strength (n = 4). *, ** Significant at the 0.05 and 0.01 probability levels, respectively.

 
Soil Properties Related to Soil Erodibility
Crop and soil management practices are important factors that control soil erosion in row-cropped land. Management practices that tend to accumulate organic matter on the soil surface modify soil surface properties such as bulk density, wet-aggregate stability, splash detachment, and soil shear strength (Clement and Williams, 1958; MacRay and Mehuys, 1985; Gantzer et al., 1987). Soil from continuous timothy was more resistant to splash detachment, had greater soil shear strength and wet-aggregate stability compared with the 3-yr rotation of corn–wheat–red clover, continuous wheat, and continuous corn cropping systems.

In this study, splash detachment was more sensitive than bulk density, wet-aggregate stability, and soil shear strength to the crop management treatments. These results are in agreement with studies conducted by Gantzer et al. (1987) and Bradford et al. (1986) supporting the concept that splash detachment is a more sensitive test than other measurements for evaluating changes in soil erodibility.

Summary
One hundred years of continuous corn, wheat, timothy, and a 3-yr rotation of corn–wheat–red clover significantly affected soil shear strength, splash detachment, and aggregate stability. The timothy treatments had the highest values of soil shear strength and aggregate stability and the lowest levels of splash detachment. The continuous wheat and corn treatments had the lowest soil shear strength and the highest splash detachment. Positive correlations were found between soil shear strength and log aggregate stability (r = 0.90). A negative correlation was found between splash detachment and aggregate stability (r = -0.79). These relationships suggest that some of the changes in the resistance of soil to detachment were related to soil management and cropping systems. Soil management (less frequent tillage) and cropping systems that provide protection to the soil surface from raindrop impact and accumulate organic matter increased stability of soil aggregates, soil shear strength, and resistance to splash detachment. This study also suggested that splash detachment is a more sensitive test than bulk density, aggregate stability, and soil shear strength for evaluating changes in soil erodibility.


   ACKNOWLEDGMENTS
 
The authors are grateful to the Agency for Agricultural Research and Development (AARD), Ministry of Agriculture, Indonesia, in providing the first author with financial support to study in the USA and conduct this study. This research was, in part, supported by the Missouri Agricultural Experiment Station project number MO-NRSL0117.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Contribution from the Center for Soil and Agroclimate Research, Indonesia and the Missouri Agricultural Experiment Station.

Received for publication April 9, 2002.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 




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