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Published online 20 February 2008
Published in J Environ Qual 37:718-724 (2008)
DOI: 10.2134/jeq2007.0191
© 2008 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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TECHNICAL REPORTS

Waste Management

Managing Broiler Litter Application Rate and Grazing to Decrease Watershed Runoff Losses

K. R. Sistania,*, G. E. Brinkb and J. L. Oldhamc

a USDA-ARS, Animal Waste Management Research Unit, Bowling Green, KY 42104
b USDA-ARS, U. S. Dairy Forage Research Center, Madison, WI 53707
c Dep. of Plant and Soil Sciences, Mississippi State Univ., Starkville, MS 39762

* Corresponding author (karamat.sistani{at}ars.usda.gov).

Received for publication April 18, 2007.

    ABSTRACT
 TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
 Conclusions
 REFERENCES
 
Pasture management and broiler litter application rate are critical factors influencing the magnitude of nutrients being transported by runoff from fields. We investigated the impact of pasture management and broiler litter application rate on nutrient runoff from bermudagrass (Cynodon dactylon) pastures. The experiment was conducted on a Ruston fine sandy loam with a factorial arrangement on 21 large paddocks. Runoff water was collected from natural rainfall events from 2001 to 2003. Runoff water and soil samples were analyzed for nutrients and sediments. Runoff was generally greater (29%) from grazed than hayed pastures regardless of the litter application rate. There was greater inorganic N in the runoff from grazed paddocks when litter rate was based on N rather than P. The mean total P loss per runoff event for all treatments ranged from 7 to 45 g ha–1 and the grazed treatment with litter applied on N basis had the greatest total P loss. Total dissolved P was the dominant P fraction in the runoff, ranging from 85% to 93% of the total P. The soluble reactive P was greater for treatments with litter applied on N basis regardless of pasture management. Runoff total sediments were greater for N-based litter application compared to those which received litter on P basis. Our results indicate that litter may be applied on N basis if the pasture is hayed and the soil P is low. In contrast, litter rates should be based on a P-basis if pasture is grazed.


    INTRODUCTION
 TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
 Conclusions
 REFERENCES
 
RUNOFF from manured pastures and hay fields has the potential to transport nutrients to nearby surface water. Phosphorus-enriched surface water may become eutrophic, leading to increased aquatic vegetation growth and an increase in biological oxygen demand (BOD) (Sharpley et al., 1994; Parry, 1998; Carpenter et al., 1998). Controlling eutrophication of the surface waters may require managing a number of factors (manure application rate and date and pasture management), particularly in diffuse landscapes such as the poultry production areas (Schindler, 2006; Dodds, 2006). Nonpoint-source pollution of water bodies during the past 20 yr has received global attention (Abu-Zreig et al., 2003; Gaston et al., 2003). Cabrera and Sims (2000) reported over 11.4 million tons of broiler litter (a mixture of manure, bedding materials, feathers, and feed) was generated in 1996, of which over 90% was land applied. Poultry litter is generally surface-applied to pastures and hay fields year-round to supply plant nutrients, particularly nitrogen (N) and phosphorus (P) and to improve soil physical conditions. Proper land application of animal manure is critical to water quality in watersheds with a significant number of poultry or livestock. Nutrient transport risk from land applied manure to surface water depends on the interaction of intrinsic site properties, climate, and pasture management (Gessel et al., 2004).

With regard to permanent pasture systems, the inability to incorporate manure or fertilizer leads to increased concentrations of nutrients such as P, Cu, and Zn near the soil surface. Long-term application of poultry litter often results in accumulation of soil P (Kingery et al., 1994). When soil P-sorption capacity is reached, potential P movement increases via runoff water at the field edge (Sharpley et al., 1994), leaching through the soil (Heckrath et al., 1995), or lateral transport of dissolved P within the soil (Walthall and Nolfe, 1998). Other parameters, such as precipitation and soil surface characteristics (slope, texture, and vegetative cover) may also influence P loss on a watershed scale (Gburek et al., 2002). In a simulated rainfall study, Edwards et al. (2000) showed that P loss magnitude was related to the proximity of the preceding rainfall. Pote et al. (1996, 1999) and McDowell and Sharpley (2002) reported that antecedent soil moisture affects runoff P transport.

Nutrient management planning now includes a risk assessment for potential P movement in the landscape (Sharpley et al., 2003). The risk assessment process allows scenario testing of various best management practices (BMPs) to reduce nutrient transport from grassland that has received animal manure (Oldham, 2007). Grassland management practices include grazing vs. haying, manure application based on P or N requirement of the crop, and double cropping. More research is needed to assess the impact of these practices on nutrient losses from agricultural lands.

Most nutrient runoff research has used small plots with rainfall simulators which exclude the influence of grazing animals and natural rainfall events. Information about P and other nutrient losses from watersheds with natural rainfall events under specific pasture management is lacking. The objective of this study was to determine the impact of pasture management and broiler litter application rate on the nutrient concentration of runoff water.


    Materials and Methods
 TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
 Conclusions
 REFERENCES
 
Site Description
The study was conducted from 2000 to 2002 at the Coastal Plain Branch of the Mississippi Agricultural and Forestry Experiment Station near Newton, MS (32.33°N, 89.08°W). Soil at the site was a Ruston fine sandy loam (fine-loamy, siliceous, semiactive, thermic Typic Paleudult). Common bermudagrass [Cynodon dactylon (L.) Pers.] was the dominant forage in the rolling 10-ha pasture which consisted of three small watersheds. A topographic map of the pasture was made on a 1 cm:6 m scale. Based on slope, natural drainage, and treatment requirements, the pasture was divided into 21 paddocks ranging from 0.10 to 0.70 ha. Soil berms 2 m wide and approximately 25 cm high were constructed to prevent runoff from one paddock to another. Within each paddock, a micro-flume runoff collector (Franklin et al., 2001) was installed on representative slopes (Table 1 ). Each flume was protected by a steel frame covered with wire mesh to prevent damage by grazing cattle or machinery. Above each collector, 5.30 m2 of soil surface was surrounded by landscape edging that directed all runoff water from the enclosed area into the flume. Incoming runoff was divided so that 1/10 of the flow was captured in a 3.78-liter plastic bottle. A second bottle was connected to the flume to capture extra runoff water in case of bigger runoff events. At each runoff event, total runoff volume from each paddock was recorded and the samples were transported to the lab for chemical analysis.


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Table 1. Mean slope of soil surface above each micro-flume runoff collector.

 
Experimental Design
The experimental design was a 2 x 3 factorial arranged in a randomized complete block design with three replicates and a single control treatment incorporated into each replicate. The first factor was broiler litter applied at rates to meet either the approximate annual N or P requirement (N- vs. P-basis) of the bermudagrass-annual ryegrass forage system. Where litter was applied on a P basis, inorganic N as ammonium nitrate (34% N) and K as commercial potash fertilizer (60% K2O) were applied at rates equivalent to that contained in litter applied on a N-basis to balance the forage productivity potential of both treatments. The second factor was pasture management: (i) grazed (G) paddocks continuously stocked throughout the year; (ii) managed to produce hay (H) throughout the year, and (iii) managed to graze ryegrass by continuous stocking and harvest bermudagrass as hay (G/H). Therefore, the treatment combinations were as follow: hayed and litter applied on a N or P basis (H-n or H-p), grazed and litter applied on a N or P basis (G-n or G-p), and grazed and hayed combinations and litter applied on a N or P basis (G/H-n or G/H-p), respectively. The control paddocks did not receive any litter or inorganic fertilizer. Each of the small watersheds within the pasture contained a complete replicate, and treatments were assigned to paddocks based on management requirements. The H treatments were assigned to smaller paddocks, while G and G/H treatments were assigned to larger paddocks to accommodate grazing.

The experiment was initiated in July 2000. Because approximately one half of the annual hay yield was expected to be produced after this date, enough litter (based on total litter N content) was applied to N-basis treatments to provide half the mean annual N requirement of common bermudagrass (270 kg ha–1), while litter was applied based on the litter P content to P-basis treatments to provide half the mean annual P requirement of common bermudagrass 45 kg P ha–1 (Brink et al., 2004). Inorganic N and K were applied to P-basis treatments at 100 kg ha–1. After litter application, cattle were stocked initially on G treatments at 1180 kg live weight ha–1, which maintained pastures at a 10- to 15-cm stubble height. In late October, G/H and H treatments were harvested for hay (10-cm stubble), and cattle were removed from all G treatments.

In early November, annual ryegrass was drilled at 34 kg pure live seed ha–1 (overseeding bermudagrass) on all paddocks except control treatments. Dry conditions delayed ryegrass emergence until February 2001, at which time litter was applied (N-basis) to supply 135 kg N ha–1 (Robinson, 1996) or (P-basis) to supply 22 kg P ha–1 (Brink et al., 2001). Inorganic N and K were applied to P-basis treatments at 100 kg ha–1, split equally between applications in February and April. Cattle were stocked on G and G/H treatments at 984 kg live weight ha–1, which maintained pastures at a 10- to 15-cm stubble height. In mid-May, cattle were removed from G/H treatments and ryegrass hay was harvested from H treatments. In late May, litter was applied to N and P-basis treatments. Inorganic N and K fertilizer were applied to P-basis treatments at 200 kg ha–1, split equally between three applications in May, June, and August. Applications in June and August were made after bermudagrass hay was harvested from G/H and H treatments. The third and final bermudagrass hay harvest from G/H and H treatments occurred in early October, and cattle were removed from G treatments. Before ryegrass was sown as described above, 20 composite soil samples were collected from within each paddock at 0 to 5 cm depth. This cycle of annual litter application and pasture management was continued until May, 2002. Cattle were replaced each spring so that initial stocking rate was approximately 1000 kg live weight ha–1.

Sampling and Data Analysis
After paddock areas were designated, background soil samples were collected at 0- to 5-, 5- to 10-, and 10- to 20-cm depth. Because of the inherent variability due to the size of each paddock, 20 soil samples were collected separately from each paddock. Initial soil chemical characteristics were determined using Mehlich-3 extractant (Mehlich, 1984) (Table 2 ). Soil and litter total N and C were measured by dry combustion using a CE Elantech CN analyzer (Lakewood, NJ).


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Table 2. Initial soil surface (0–5 cm) chemical properties of each paddock, collected July 2000.

 
All litter used in the experiment was obtained from a broiler house in the vicinity of the station. An 800- to 1000-g subsample was taken before application and ground to pass a 2-mm screen. Litter nutrient content was determined by ashing a 0.8-g subsample in a ceramic crucible at 500°C for 4 h followed by the dissolution of the ash in 1.0 mL of 6 mol L–1 HCl for one h, then dissolution in an additional 40 mL of a double acid solution of 0.0125 mol L–1 H2SO4 and 0.05 mol L–1 HCl for another h, and filtering through Whatman no. 1 paper (Southern Cooperative Series, 1983). The P and other nutrient determination was done using an inductively coupled plasma spectrophotometer (ICP). The pH of the litter was measured in 1:5 litter/water mixture. Broiler litter used during the study was collected from the same producer under the same house management; and the litter rate at each application was based on the individual N and P content. However, since there were no significant changes in the litter nutrient content at each analysis, only mean nutrient concentration is reported as follows: N 32.5 g kg–1, P 19.0 g kg–1, Ca 26.6 g kg–1, K 26.9 g kg–1, Mg 5.8 g kg–1, Cu 566 mg kg–1, Fe 1454 mg kg–1, Mn 606 mg kg–1, and Zn 452 mg kg–1.

Figure 1 shows the total monthly precipitation and air and soil temperatures during the study. After runoff producing events, total runoff volume was determined; and subsamples were collected and stored at 2°C until analyzed for pH, total P (TP), total dissolved P (TDP), soluble reactive P (SRP), NO3–N, NH4–N, and total sediment (TS). Subsamples filtered through a 0.45-µm membrane were used to analyze the soluble nutrients (TDP, SRP, NO3–N, and NH4–N). Samples for TP were digested according to method 4500-P A (APHA, 1992) and measured using Thermo Jarrell-Ash ICP, (Franklin, MA). Total dissolved P was also determined using ICP. Soluble reactive P, which refers to the orthophosphate concentration in filtered samples was measured by a colorimetric procedure (Southern Cooperative Series, 2000) using a Lachat instrument (Loveland, CO). A Lachat instrument was also used for NO3–N and NH4–N determination. Total sediment was determined by filtering 20 mL of runoff water through a pre-weighed dried filter paper (2V Whatman) and weighing again after drying.


Figure 1
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Fig. 1. Total monthly precipitation and air and soil temperatures recorded from 2001 to 2003.

 
Analysis of variance was used to analyze the data using the PROC GLM procedure of SAS (SAS Institute, 1999). Results are presented as averages of 48 rainfall events from August 2000 to spring 2002. Least significant differences (LSD) were used to separate means. The 0.05 probability level was used for all statistical analysis.


    Results and Discussion
 TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
 Conclusions
 REFERENCES
 
Runoff Volume
Regardless of litter application rate (N- vs. P-basis), the runoff volumes from H treatments were significantly lower than G treatments (Table 3 ). The difference between runoff volumes may be due to the height and thickness of the grass, particularly in haying vs. grazing paddocks. Animal trampling on grazed plots may have also caused soil compaction which resulted in lower infiltration and greater runoff volume. The runoff volume for G and G/H treatments was similar. These results were attributed to a majority of the runoff occurring during the winter when both G and G/H were grazed. Although runoff nutrient concentration is related to precipitation events, runoff volume is also important because an increase in runoff volume can lead to an increase in total nutrient loss. However, McLeod and Hegg (1984) reported that nutrient concentration in runoff from manure application to pasture watersheds grazed by dairy heifers was more dependent on the number of rainfall events and their timing with respect to application date than on quantity/volume of rainfall or runoff.


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Table 3. Mean runoff event volume (48 events), pH, total solid (TS), total P (TP), and TS and TP loads related to different treatment (TRT).

 
Runoff Water Quality
Effect of Pasture Management and Litter Rate
The mean runoff pH varied from 6.6 to 6.8. Runoff pH from combined G/H treatment with litter application on a N-basis was significantly lower than G-n or H-n treatments alone (6.8 vs. 6.6). Pasture management and litter application rate significantly influenced runoff inorganic N content (Fig. 2 ). The inorganic N (NO3–N and NH4–N) concentration of the runoff from G or G/H treatments with the litter applied on the N-basis was significantly greater than the same treatments with litter applied on the P-basis (1.64 and 1.38 vs. 0.70 and 0.62 mg L–1 for NH4–N; 1.25 and 1.11 vs. 0.65 and 0.47 mg L–1 for NO3–N) (Fig. 2). The inorganic N fertilizer applied with the P-based litter application may move more quickly into the soil and become less available for transport in runoff than N from litter sources, which remain on the surface for a longer time. However, within the H treatments, no differences were observed in inorganic N transport in N- or P-basis treatments. The lowest NO3–N and NH4–N concentrations in the runoff exiting the paddocks were with G or G/H treatments that received litter based on the P requirement of bermudagrass. Generally, it is expected that grazing animal traffic would likely decrease soil infiltration due to soil compaction, which results in greater nutrient losses by runoff water (Sauer et al., 1999). The lower inorganic N concentration of the runoff from H plots compared to G plots was attributed to N uptake by plant followed by hay removal. The overall effect of litter rate was greater than pasture management on runoff inorganic N content.


Figure 2
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Fig. 2. Inorganic N (NH4–N and NO3–N) content of runoff water collected from the following treatments: Control; Graze, with litter applied on N or P basis; Hay, with litter applied on N or P basis; and Graze/Hay, with litter applied on N or P basis. Bars within each parameter with the same letters are not significantly different, LSD 0.05 level.

 
Total runoff P (TP), which includes the total amount of dissolved and particulate P, was significantly different among treatments. Litter applied on a P-basis produced runoff water with lower TP content than litter applied on a N-basis (Fig. 3 ). The runoff water from G treatments with litter applied on a N-basis had the greatest TP content while G and G/H treatments with litter applied on the P-basis had the lowest TP content. The runoff TP concentrations from the H treatments were the same for both N- and P-based litter application; however, for the G/H treatments, significant differences were observed between the two rates. Regardless of pasture management, litter application on a N-basis produced high P concentrations in runoff. This was expected, since more litter was applied for the N than the P treatments. Udawatta et al. (2004) reported an average loss of 21 g ha–1 TP from three row-cropped watersheds during 66 runoff events. In our study, the mean TP loss (48 events) from pasture ranged from 7.3 (control) to 45 g ha–1 for G-n treatments (Table 3). The magnitude of TP loss for each treatment ranked as follows: G-n > G/H-n > H-n > H-p > G-p > G/H-p > control, but the differences were not always significant.


Figure 3
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Fig. 3. Total P, total dissolved P, and soluble reactive P content of runoff water collected from the following treatments: Control; Graze, with litter applied on N or P basis; Hay, with litter applied on N or P basis; and Graze/Hay, with litter applied on N or P basis. Bars within each parameter (e.g., total P) with the same letters are not significantly different, LSD 0.05 level.

 
The TDP, a measure of non-particulate P, followed the same trend as TP for all treatments. The TDP was the dominant P fraction in the runoff, ranging from 85 (control) to 93% of the runoff TP (H-n treatment). Similar results have been reported by other investigators (Edwards and Daniel, 1993; Sauer et al., 1999; Gaston et al., 2003). The runoff SRP content, a measure of soluble inorganic P (algal-available), was significantly different among treatments, with litter application on the N-basis consistently generating runoff with greater SRP content (Fig. 3). Pasture management had no significant effect on SRP regardless of the litter application rate, except when litter was applied on a P-basis for the combined G/H treatment, which was lower than the other treatments. Considering all three P fractions (TDP, SRP, and TP), the grazing and haying combination with litter being applied on the P-basis generated runoff with the lowest P content, and grazing year-round and litter application on a N-basis produced runoff with the greatest P content. Therefore, land management and litter rate greatly influenced nonpoint sources of P and other nutrient losses by surface runoff from watersheds fertilized with manure. According to Sharpley and Withers (1994), these losses are often representing a minor proportion of manure or fertilizer P applied (generally <5%), which may not have significant agronomic impact; however, they may contribute to the P-related eutrophication of surface waters, and thus have environmental importance.

Significant differences were observed in runoff TS from each treatment. Runoff from control paddocks had the greatest sediment content followed by H-n and G-n treatments. Based on calculation from mean runoff volume and total solids concentration (Table 3), G/H with litter applied on P-basis had the lowest TS load (7.4 kg ha–1) while the control and H-n had the greatest TS loads, 19.1 and 16.4 kg ha–1, respectively. We speculate the lack of lush grass vegetation in control paddocks resulted from nutrient deficiency which in turn contributed to increased soil erosion and TS in the runoff. However, no clear trend in regard to runoff TS was observed in relation to different pasture management (G, H, or G/H) or litter application rate (N- or P-basis).

Soil Surface Nutrient Dynamics
There were differences in soil pH among the paddocks receiving different treatments (Table 4 ). Surface (0–5 cm) soil pH at the beginning of the experiment ranged from 6.4 to 6.7 for all the paddocks (Table 2), while at the last soil sampling (May 2002), the soil pH was slightly lower (5.8 to 6.2). Litter application is generally expected to increase soil pH. However, the decrease in soil pH was more with the P-based litter treatments, which is not surprising because the inorganic N fertilizers applied with the P-based litter application are known to decrease soil pH. Other factors such as soil moisture and temperature at the time of soil sampling may have indirectly influenced soil pH. Mineralization of the litter N followed by nitrification may have also caused the decrease in soil pH (King et al., 1990; Sistani et al., 2004). There was a clear buildup of N in soil for all treatments except for the control. At the last soil sampling (May 2002), significant differences in N content were observed between the control and other treatments (Table 4). However, there were no differences between G or H treatments, regardless of litter application rate. Combined G/H with litter application on a N-basis had significantly greater soil TN than G/H-p treatment.


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Table 4. Soil surface (0–5 cm) chemical properties of each treatment (TRT) at different dates.

 
Compared to the initial soil C content (Table 2), all treatments increased surface soil (0–5 cm) TC. The initial surface soil TC was between 17.79 and 22.70 g kg–1, while in May 2002 (last soil sampling date), the TC of the same soil depth was between 29.6 and 40.6 g kg–1 for all treated soils (Table 4). More significant differences were observed in TC accumulation in G or G/H than in H alone. For example, the TC for GH-n was 40.6 g kg–1, while H-n was 29.6 g kg–1 (Table 4). The lower soil C in hayed treatments was probably due to the removal of a substantial quantity of C with the hay harvest.

Litter application on the N-basis caused greater buildup of P in the soil surface regardless of H, G, or G/H practices (Table 4), probably because N-based litter applications supplied more P than P-based litter applications. However, the impact of pasture management on the buildup of surface soil P was variable. For example, M3-P in May 2002 was significantly greater for G-n (298 mg kg–1) than for H-n (230 mg kg–1), but the difference between G-p (111 mg kg–1) and H-p (101 mg kg–1) was not significant, and differences in TP for the same treatments were not significant (Table 4). It is not surprising that soil P levels tended to be greater in grazed treatments than in hayed treatments because harvesting hay removes some of the applied P. Pasture management practices such as harvesting a winter cover crop (e.g., annual ryegrass) as hay have been proposed for remedial control of soil nutrient accumulation by many researchers (Brink et al., 2001; McLaughlin et al., 2005; Rowe et al., 2006). Rowe et al. (2006) reported that proper pasture management is critical in preventing nutrient accumulation in soil. In our study, grazing winter ryegrass and haying bermudagrass that received litter on a N basis produced the lowest soil concentration of Cu, Mn, and Zn. Similarly, Rowe et al. (2006) reported that inclusion of winter ryegrass increased the extraction of Cu (97%) and Zn (83%), which resulted in lower soil accumulation of these metals.

Soil surface metal content increased for most treatments compared to the initial soil analysis (Table 2). However, the significant differences were observed only with litter application on N-basis. Among all metals analyzed, the increases in soil Cu and Zn content on the May 2002 sampling date were much greater than the other metals (Tables 2, 4).


    Conclusions
 TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
 Conclusions
 REFERENCES
 
This study measured the impact of pasture management options and nutrient management philosophies such as litter application rate (N- vs. P-basis) on primary nutrient losses through surface runoff water from common bermudagrass pasture. The average runoff volume of the grazing treatments was greater than the haying treatments, regardless of the litter application rate. The inorganic N content of the runoff exiting the grazed or grazed/hayed paddocks that received broiler litter on N basis was greater than the same treatments that received litter on P basis. However, results indicated when pasture is hayed year-round, litter application rate on the N or P basis will have the same impact on runoff inorganic N content. In most cases, treatments that received broiler litter on a N basis generated runoff with greater total solids than those that received litter on a P basis.

Results of our study reinforce previous findings of a direct relationship between runoff total P and litter application rate. All the treatments that received broiler litter on a N basis had greater total P and total dissolved P in the surface runoff water. Total dissolved P was the dominant P fraction in the runoff, ranging from 85 to 93% of the total P. Soluble reactive P was greater in runoff water from treatments receiving litter on a N basis, regardless of pasture management. In most cases, results show a clear build up of nutrients (particularly C, N, P) and some of the metals in the soil surface. The build up was greatest with the litter application on the N basis.

The practical significance of our findings is that selection of pasture management and nutrient management options greatly impact the quality of the surface runoff water exiting fields. For example, this data may be used as a guide when a decision is needed as to whether to apply litter on N- or P-basis for fields grazed or hayed year-round. Our results indicate that litter may be applied on a N basis if the pasture is being hayed and the soil P is low. However, if grazing is the main usage of the pasture, litter should be applied based on the P requirement of the crop. Also, broiler litter application based on the P requirement of these crops was a superior litter management in regard to runoff water quality in this study. Further studies are warranted to gain more knowledge of spatial and temporal variability of the pastureland with animal interaction to reduce the potential loss of nutrients through surface runoff.


    NOTES
 TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
 Conclusions
 REFERENCES
 
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher.


    REFERENCES
 TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
 Conclusions
 REFERENCES
 




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P. A. Vadas, L. W. Good, P. A. Moore Jr., and N. Widman
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