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TECHNICAL REPORT
Surface Water Quality

Bioavailable Phosphorus in Runoff from Alfalfa, Smooth Bromegrass, and Alfalfa–Smooth Bromegrass

^a CNH Soil Management Division, Rt. 150 E., P.O. Box 65, Goodfield, IL 61742
^b Soil Tech Inc., 5720 Smetana Dr., Minnetonka, MN 55343
^c Dep. of Agronomy, 1575 Linden Dr., Univ. of Wisconsin-Madison, Madison, WI 53706

* Corresponding author (rob.zemenchik{at}cnh.com)

Received for publication December 4, 2000.

	ABSTRACT

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

 
Runoff from sloping landscapes cropped with established alfalfa (Medicago sativa L.) may contain bioavailable P (BAP) which accelerates eutrophication of surface water bodies. Such BAP exists as either dissolved reactive P (DRP) or bioavailable reactive particulate P (BPP). We hypothesized that before and after harvest, sod-forming smooth bromegrass (Bromus inermis Leyss.) or alfalfa–smooth bromegrass mixtures would have less BAP, DRP, and BPP runoff losses than taprooted alfalfa. Swards established in 1992 near Lancaster, WI were subjected to a 72 mm simulated rainfall applied for 1 h in 1993 and 1994 to forage regrowth at 4 and 6 wk after first harvest and immediately (0 wk) after second harvest. Hourly BAP losses for all sward types were 82% less when 1.5 Mg ha^-1 of forage dry matter was present. High DRP losses (>0.050 kg ha^-1) were associated with high DRP concentrations (>7.1 µmol L^-1) and high surface soil P concentrations (>59 mg kg^-1) resulting from broadcast maintenance P fertilizer. High BPP losses (>0.035 kg ha^-1) were associated with high runoff volumes (>24 mm) and sediment concentrations (>2 g L^-1). Summed over all 6 rainfall simulations, total BAP loss was only 0.07 kg ha^-1 at the 6 wk stage of regrowth compared with 0.35 at 4 wk, and 0.41 at 0 wk. Moreover, there was no significant difference between sward types for DRP concentration, DRP loss, or BAP loss. We conclude that avoiding excessive defoliation was more effective at reducing BAP losses than specific forage species selection.

Abbreviations: BAP, bioavailable phosphorus • BPP, bioavailable reactive particulate phosphorus • DRP, dissolved reactive phosphorus • EDI, effective depth of interaction

	INTRODUCTION

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

 
RUNOFF FROM intensely managed agroecosystems may convey phosphorus (P) toward field depressions, waterways, and surface water bodies where it can accelerate eutrophication and promote the growth of aquatic weeds or algae (Schindler, 1977; Wendt and Corey, 1980; Sozogni et al., 1982). Phosphorus in runoff that promotes organism growth is referred to as bioavailable P (BAP) and consists of either dissolved reactive P (DRP) or bioavailable particulate P (BPP; Sharpley et al., 1993). Dissolved reactive P in runoff occurs by desorption, dissolution, and extraction of P from crop residues, manure, or recently applied P fertilizer (Daniel et al., 1998; Sauer et al., 1999; Sharpley et al., 1999). Bioavailable particulate P is represented by the portion of P bound to soil particles or to organic matter that enters surface water bodies and is potentially available for aquatic organisms.

Sod-forming grasses are used in field waterways because of their ability to retain P-laden runoff sediments and to convey agricultural runoff in concentrated flow paths while limiting gully erosion. These virtues have been presumed for other forage species such as alfalfa, that are grown on millions of hectares of U.S. cropland for high quality animal feed rations. However, surface runoff from alfalfa grown on loess may be greater than that from other forage swards when sloping soil form seals during intense precipitation events. Zemenchik et al. (1996) reported that soil loss and runoff volumes in swards that were mechanically harvested three times annually, were significantly less for smooth bromegrass compared with alfalfa in the presence of full or partial canopy. They suggested that rhizome development between smooth bromegrass plants beneath the plant canopy produced tillering that protected the soil. In the absence of plant canopy immediately after harvest, there was no significant difference in soil loss or runoff as both sward types were overwhelmed by the intensity of simulated rainfall. Surface runoff from established forages on silt loam or silty clay loam soils has been reported elsewhere for monoculture alfalfa (Converse et al., 1976; Young and Mutchler, 1976; Thomas et al., 1992) and for various grass or grass-legume pastures (Van Doren et al., 1940; Alderfer and Robinson, 1947; Knoll and Hopkins, 1959; Sauer et al., 1999). Converse et al. (1976) reported annual total P loss (i.e., DRP and total particulate P) of 0.75 kg ha^-1 in runoff from unmanured established alfalfa grown on Rozetta silt loam in southwest Wisconsin. Thomas et al. (1992) measured annual DRP losses in runoff from natural rainfall from a 3-year-old alfalfa stand grown on a Miamian silt loam in Ohio. They reported annual DRP losses of 0.4 kg ha^-1 with vegetative canopy and 0.1 kg ha^-1 without canopy. In grasslands, DRP represents the majority of BAP in runoff because sediment concentrations are typically quite low. Sharpley et al. (1993) showed that as sediment concentration decreases in runoff, the percentage of total BAP that is DRP increases while the proportion that is BPP decreases.

Water movement over soil that has received broadcast manure or fertilizer P applications, or that tests high in P, can further increase BAP loading (Sharpley et al., 1993), particularly as DRP that is immediately available for algal uptake (Walton and Lee, 1972; Peters, 1981). Sharpley et al. (1994) reviewed several studies that showed DRP in runoff to be positively correlated to plant-available P (i.e., Bray-1, Mehlich-3, and 0.1 M NaCl) measured in a shallow (i.e., 0 to 1 cm) soil sample. Rechcigl et al. (1992) showed that DRP in surface runoff was strongly correlated to P fertilizer surface-applied to bahiagrass (Paspalum notatum Flugge) pastures and contributed significantly to the formation of a 310 km² algae bloom in Lake Okeechobee, FL in 1986. They also reported that by reducing application rates from 48 to 12 kg P ha^-1, P concentrations in runoff decreased by 33 to 66% while total P losses in runoff decreased by a range of 17 to 78%.

Long-term fertilizer and livestock manure applications have resulted in accumulations of soil test P well beyond crop need in many agroecosystems in the USA (Motschall and Daniel, 1982; Combs and Burlington, 1992; Sims, 1993). Acutely high concentrations of Bray-1 P may accumulate in the top 1.0 cm of the profile (Sharpley et al., 1994). The depth to which rainfall interacts with the soil during interrill erosion has been referred to as the effective depth of interaction (EDI) which varies by soil type but is usually less than 2 cm (Sharpley, 1985). As a result, management practices that include broadcast manure or fertilizer on cropland of the Upper Mississippi Valley, including forage swards, may be increasing the risk for elevated levels of DRP, BPP, and BAP in runoff.

The objectives of this study were to (i) compare DRP, BPP, and BAP losses in runoff from loess soils managed with established alfalfa, smooth bromegrass, and an alfalfa–smooth bromegrass mixture at three stages of plant regrowth and (ii) determine how DRP and BPP concentrations and losses relate to soil losses and Bray-1 P in the top 1 cm of soil.

	MATERIALS AND METHODS

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

 
This study was conducted within the Upper Mississippi valley's driftless region at the University of Wisconsin, Lancaster Agricultural Research Station (lat., 42°50'; long., 90°47'; elev., 325 m) on a Rozetta silt loam soil (moderately well drained, superactive, mixed, mesic Typic Hapludalf). The top 15 cm of this soil had 22 g kg^-1 organic C and a particle-size distribution of 11% sand, 63% silt, and 26% clay (Bouyoucos, 1962). The bulk density of this soil averaged 1.3 Mg m^-3 during the study and an estimated soil erodibility K-value of 0.37 (Wischmeier and Smith, 1978). Fifteen plots with dimensions 10 m by 15 m were established in April 1992 on the contour of the south shoulder of a southwest facing hillside with slopes of 6 to 8%. The experimental design was a randomized complete block with five replications. The forage treatments were monoculture alfalfa, alfalfa–smooth bromegrass mixture, and monoculture smooth bromegrass.

Seedbed preparation in spring 1992 included shallow moldboard plowing (i.e., to 15 cm) and discing followed by a cultimulching pass to firm the seedbed and remove any remaining clods. All plots were sown on the contour with a Krause (Krause Corp, Hutchinson, KS) no-till drill set at 17.5 cm row spacing using a mixture of Dekalb ‘DK 122’ alfalfa at 17.3 kg ha^-1, ‘Badger’ bromegrass at 12.3 kg ha^-1, and an oat (Avena sativa L.) companion crop at 33.6 kg ha^-1. To achieve monoculture alfalfa, smooth bromegrass was removed from the alfalfa treatments with the postemergent herbicide sethoxydim (2-[1(ethoxyimino)butyl]-5-[2-ethylthio)propyl]-3-hydroxy-2-cyclohexene-1-one) applied at 1.76 L a.i. ha^-1 before each harvest in 1992 and first and second harvest in 1993. Similarly, to achieve monoculture smooth bromegrass, alfalfa was removed from the bromegrass plots with 2,4-D amine [(2,4-dichlorophenoxy)acetic acid] applied at 1.17 L a.i. ha^-1.

Phosphorus was broadcast at 22.7 kg ha^-1 and lime at 6.72 Mg ha^-1 as per soil test recommendations (Kelling et al., 1991) prior to seedbed preparation. A second similarly recommended P application was surface broadcast in September 1993 on established forage at 40.5 kg ha^-1 along with potassium at 69.6 kg ha^-1. Nitrogen (N) was surface broadcast at 112 kg N ha^-1 on all monoculture smooth bromegrass plots following first and second harvest in both 1993 and 1994. Neither alfalfa nor alfalfa–smooth bromegrass mixture received fertilizer N during the entire study period. All swards were managed in a three-harvest system in both years. Forage was mechanically harvested with a sicklebar mower for cutting to a 5.0-cm height, followed by a windrowing pass with a light rotary rake. After 24 h of wiltdown, forage was removed with a pull-behind chopper. First and second harvest were 11 June and 22 July in 1993, respectively, and 27 May and 6 July in 1994, respectively.

Rainfall Simulation
Simulated rainfall was applied with a portable, multiple-intensity apparatus (Meyer and Harmon, 1979), equipped with a Veejet 80150 single-nozzle (Spraying Systems, Wheaton, IL) located 3 m above a 0.84 m² test area. The test area was randomly located within each plot and then affixed to the soil surface as described by Zemenchik et al., 1996. Care was taken to avoid any obvious wheel tracks. In 1993 and 1994, simulated rainfall was applied to all plots after 4 and 6 wk regrowth following first harvest and immediately (0 wk) after second harvest. A different test area was located within each plot for simulated rainfall at each stage of regrowth. The 1-h rainfall delivered a 72 ± 6 mm rainfall with a corresponding energy of 0.278 MJ ha^-1 mm^-1. Such a storm is estimated to have a return period of 45 yr (Hershfield, 1961). The water supplied for all simulations came from the Research Station's groundwater well where potable water is drawn from a 25 m depth from within dolomitic limestone bedrock. Chemical analysis and solute content of the well water used for rainfall simulation in 1993 is shown in Table 1. Natural precipitation measured at Lancaster during 1993 was abnormally high with April at 146%, May at 132%, June at 246%, July at 228%, and August at 161% of normal when compared with 30 year averages (1951–1980; National and Oceanic Atmospheric Administration, 1990). Precipitation volume and distribution was normal in 1994.

The 0.6 L frozen runoff samples were subsequently thawed in a 20°C water bath in the laboratory. Concentrations of BPP were then measured by a modified version of the NaOH extraction used by Dorich et al. (1985) and Sharpley et al. (1991). Modifications included a reduction in the volume of the runoff sample from 20.0 to 2.0 mL and a reduction in the volume of 0.11 N NaOH reagent from 180 to 18 mL. The thawed samples were manually shaken so as to homogeneously resuspend all soil particles in the runoff sample. Immediately after shaking, a 2.0 mL subsample of the suspension was withdrawn and placed into a sterile polypropylene centrifuge tube (Fisher Scientific, Pittsburgh PA, cat no. 14-432-22) along with 18.0 mL of 0.11 N NaOH. This 20 mL BPP subsample suspension was then shaken at 160 to 165 revolutions per min for 17 h with an oscillating benchtop shaker. Immediately after shaking, the suspension was passed through a Whatman 13-mm diam. syringe filter with a 0.45 µm mesh opening (Fisher Scientific, Pittsburgh PA, cat no. 09-913-14). The malchite green colorimetric procedure (Van Veldhoven and Mannaerts, 1987; Ohno and Zibilski, 1991) was then used to determine BPP concentration of the aliquot. The malchite green method was chosen rather than the traditional molybdenum blue method of Murphy and Riley (1962) because it affords a decreased lower limit of P sensitivity to the nanomolar range needed for dilute concentrations of P in solution, such as those possible from grasslands. To determine DRP concentration, a similar pattern was followed where a separate 2.5 mL subsample of thawed runoff from each test area was withdrawn, filtered, and also subjected to the malchite green colorimetric procedure. After accounting for the reagent additions, total loss of DRP from each plot was calculated as the product of runoff volume and DRP concentration. A similar calculation was done for BPP. Total BAP losses for each plot were then calculated as the sum of BPP and DRP losses.

Forage within each simulated rainfall test area was hand harvested to a 5.0-cm cutting height 2 to 3 h after the rainfall. This forage was collected and oven dried at 60°C for 72 h to determine DM yield present at the time of rainfall on a dry weight basis. The 5.0-cm cutting height was used to set forage regrowth at the 0 wk stage to 0.0 Mg ha^-1. For the 4 and 6 wk growth stages, the 5.0-cm cutting height was the minimum height required for standing forage to contribute to DM yield.

Analysis of variance (ANOVA), correlation analysis, and regression analysis were done by Statistical Analysis Systems (SAS Inst., 1990). Concentrations and losses of DRP, BPP, and BAP in runoff were compared by sward type within each stage of regrowth for each year. Log₁₀ transformations were applied as needed because of a lack of homogeneity of variance as determined by Hartley's F_max test (Berenson et al., 1983). Means were separated by Fisher's protected Least Significant Difference (LSD; P = 0.10). Previously published runoff, sediment concentration, and soil loss data (Zemenchik et al., 1996) are presented to aid in interpreting P losses from the treatments during simulated rainfall.

	RESULTS AND DISCUSSION

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

 
Soil Bray-1 Phosphorus
Soil Bray-1 P (hereafter, soil P) concentrations in the surface 1 cm of soil increased dramatically from July 1993 to July 1994 as a result of maintenance P fertilizer that was broadcast on all plots in September 1993 (Table 2). Even though forages can sequester broadcast P from surface soil (Simons et al., 1995), severe soil P stratification from the broadcast fertilizer persisted until at least midway through the 1994 growing season. Averaged across all sward types and growth stages, mean soil P concentration in the top 1 cm of soil increased from 27 mg kg^-1 in July 1993 to 73 mg kg^-1 in July 1994. In contrast, mean soil P from the 0 to 7 cm depth in July 1993 was 21 mg kg^-1 and increased to only 27 mg kg^-1 by July 1994. Soil P stratification was present in all plots in 1994 regardless of sward type. When soil P concentrations for each year were plotted by sampling depth, there was evidence of stratification in most plots, even in July 1993 (Fig. 1) . Some of the surface soil P concentrations in 1993 and almost all in 1994, showed greater soil P at the shallow sampling depth as indicated by the 1:1 line. Nutrient cycling by decaying forage as well as earthworm castings (Sharpley and Syers, 1977) have been shown to enrich surface soils with P and possibly contribute to increased runoff P concentrations. The smaller increase in soil P concentration for the deeper 0 to 7 cm sampling depth between the two years, in conjunction with severe soil P stratification suggests that commercial soil sampling depths (e.g., 0 to 15 cm) may not detect soil P stratification in broadcast fertilizer management strategies common in forage production.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Concentration of soil Bray-1 P from 0 to 1 cm sampling depth compared with a 0 to 7 cm sampling depth. Samples taken at the time of simulated rainfall in July 1993 and July 1994. Recommended P fertilizer was broadcast in the fall of 1993.

In the presence of stratified soil P in 1994, forage regrowth for all sward types at 4 or 6 wk reduced DRP concentrations in runoff compared with 0 wk (Table 2). Forage DM production above the 5 cm cutting height for all sward types at the time of rainfall in either year ranged from 0.6 to 1.9 Mg ha^-1 after 4 wk regrowth, 1.8 to 3.3 after 6 wk, and were 0.0 after 0 wk. Concentrations of DRP in runoff at 0 wk regrowth in 1994 were at least 10 µmol L^-1. These high DRP concentrations were associated with high surface soil P and runoff sediment concentrations as well as low runoff volumes. A significantly reduced sediment concentration in runoff from smooth bromegrass in 1994 at 0 wk did not result in statistically significant reductions in DRP concentrations (Table 2). Generally, there was no significant difference in DRP concentration among sward types at any growth stage in either year. One exception was for 0 wk regrowth in 1993 where DRP concentration in runoff from smooth bromegrass was greater than that for alfalfa or alfalfa–smooth bromegrass. Although DRP concentration from smooth bromegrass was only 3.1 µmol L^-1, it exceeded the 2.0 µmol L^-1 DRP concentration threshold described above.

Bioavailable Particulate Phosphorus Concentration
In contrast to the results for DRP, increases in BPP concentration from 1993 to 1994 occurred in only two of nine comparisons (Table 2). One instance where BPP increased from 1993 to 1994 was at 0 wk regrowth where runoff sediment concentration from alfalfa was 2.5 g L^-1 while soil loss was 23 g m^-2 and was associated with a rather high BPP concentration of 9.8 µmol L^-1. Similarly, the alfalfa–smooth bromegrass mixture had runoff sediment concentration of 2.0 g L^-1, soil loss of 25 g m^-2 and a BPP concentration of 11.7 µmol L^-1. In contrast, monoculture smooth bromegrass had a sediment concentration of only 0.5 g L^-1, soil loss of 6 g m^-2 and BPP concentration of only 0.9 µmol L^-1. We believe that the ability of smooth bromegrass to expand its rhizome system into soil between the original seed rows not only restricts seal formation but also limits BPP concentrations and potential BPP runoff losses. In all other treatment and growth-stage combinations, BPP concentrations tended to be at least 33% less than 0 wk in 1994 and none were greater than 6.5 µmol L^-1. Protective canopies at the 4 and 6 wk stage of regrowth for all sward types limited sediment concentration to less than 1 g L^-1 in these cases. Maintaining canopy cover was as good a strategy as selecting sward type to reduce BPP concentration. Even in established forages, BPP from P-enriched surface soil particles may be a signficant vector for P losses once entrained in runoff on similar landscapes.

Phosphorus Losses in Runoff
Losses of DRP in runoff from established forage appear to be less affected by runoff volume and more by DRP concentration, which is in turn positively correlated to soil P as influenced by the practice of broadcast P fertilization. Consistently greater runoff volumes in 1993 compared with 1994 for all stages of regrowth did not result in greater DRP losses (Table 2). This is because the mean DRP concentration for all sward types and growth stages in 1993 was only 0.9 µmol L^-1 but increased to 7.3 µmol L^-1 in 1994. Averaged across all sward types and growth stages, soil P concentrations from 0 to 1 cm were 27 mg kg^-1 in July 1993 and resulted in DRP losses of 0.007 kg ha^-1. Meanwhile, soil P concentrations from 0 to 1 cm were 73 mg kg^-1 in July 1994 and resulted in DRP losses that averaged 0.035 kg ha^-1. These results are comparable to those reported by Wendt and Corey (1980) where DRP losses from 4-year-old established alfalfa on Onaway loam (fine-loamy, mixed, frigid Alfic Halorthod) were 0.049 kg ha^-1 for a similar 1-h simulated rainstorm.

For all sward types, BPP losses were greatest at 4 wk regrowth in 1993 and 0 wk regrowth in 1994. In contrast to DRP, losses of BPP were affected more by runoff volume and less by BPP concentration. For example, BPP loss averaged across sward types at 4 wk in 1993 was 0.064 kg ha^-1 before broadcast P fertilization and in 1994 decreased to 0.004 kg ha^-1 after broadcast P fertilization. This is in spite of surface soil P concentrations increasing on average by 170% from 1993 to 1994. Meanwhile, mean runoff volume averaged across sward types at 4 wk in 1993 was 38 mm and decreased to 23 mm in 1994. Soil moisture conditions at the time of rainfall between years did not likely affect the results. For example, antecedent surface soil moisture content from 0 to 0.5 cm deep immediately before simulated rainfall at the 4 wk regrowth stage were essentially the same at 30.5 in 1993 and 32.2 in 1994. A similar trend was present at the 6 wk stage of regrowth where runoff volume in 1994 averaged across sward types was only 6 mm and BPP loss was only 0.002 kg ha^-1 despite broadcast P fertilization.

In 1994, monoculture alfalfa at 4 wk regrowth had the greatest total BAP loss (0.099 kg ha^-1) mostly because of high BPP loss and high runoff volumes. In 1994, smooth bromegrass at 0 wk regrowth had significantly less BAP loss (0.055 kg ha^-1) than alfalfa (0.097 kg ha^-1) or alfalfa–smooth bromegrass (0.100 kg ha^-1), mostly because of low sediment concentrations and associated low soil losses. There was no statistical difference in BAP loss between treatments in either year at the 6 wk regrowth stage when vegetative canopies were at their maximum. We expect that direct BAP losses from within the plant canopies of these treatments are minimal midway through the growing season. However, Wendt and Corey (1980) report total BAP losses from monoculture alfalfa in October of 17.7 kg ha^-1 from a similar rainstorm and attribute much of this loss to P leaching from leaves after a killing frost.

Forage DM yield present during rainfall simulation was plotted against BAP losses without regard to year or soil P (Fig. 2) . Even though a regression model could not be developed to fit these data, it appears that BAP losses are consistently low when there is at least 1.5 Mg ha^-1 of standing forage in the field. We believe that a protective forage canopy limits the development of surface seals in these soils and therefore limits runoff and BAP losses. Furthermore, a protective canopy will limit the extent to which a high-energy rainstorm will interact with surface soils even if they are laden with P, and therefore limit P dissolution and associated DRP losses. Natural rainstorms with less kinetic energy would likely not require as much forage present to obtain such protection and preservation of water quality. However, a minimum of 1.5 Mg ha^-1 of forage DM is a reasonable estimate of vegetation required to reduce BAP losses for high-energy storms in this environment. Furthermore, many pastures and hay fields are on slopes greater than 6 to 8% as the plots in this experiment where sensitivity to limited canopy protection may increase.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Relationship between forage DM yield (present when a 72 ± 6 mm simulated rainfall was applied for 1 h) and total BAP loss in runoff. Values represent BAP loss from individual simulated rainfall events in 1993 and 1994 for alfalfa, alfalfa–smooth bromegrass, and smooth bromegrass at all stages of regrowth.

	CONCLUSIONS

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

Protective canopies at the 4 and 6 wk stage of regrowth for all sward types limited sediment concentration to less than 1 g L^-1 in all cases in this experiment. Hourly BAP losses for all sward types were 82% less when 1.5 Mg ha^-1 of forage DM was present, a level that is a reasonable estimate of vegetation required to reduce DRP and BPP losses for high-energy storms in this environment. Values for BAP losses reported in this experiment may be lower than those reported from other agroecosystems. However, forage producers, commercial fertilizer applicators, and livestock managers should implement strategies to avoid excessive defoliation and maintain this level of forage biomass on areas of the landscape where the potential for excessive levels of BAP in runoff are present.

	NOTES

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

 
Research was partially funded by Hatch Project No. 5168. Contribution of the Wisconsin Agric. Exp. Stn.

	REFERENCES

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

 

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