Solid Manure As a Source of Fecal Indicator Microorganisms: Release

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Solid manure as a source of fecal indicator microorganisms: release under simulated rainfall Ryan Andrew Blaustein, Yakov Pachepsky, Robert L Hill, and Daniel Shelton Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b01095 • Publication Date (Web): 26 May 2015 Downloaded from http://pubs.acs.org on May 30, 2015

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Environmental Science & Technology

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Solid manure as a source of fecal indicator microorganisms: release

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under simulated rainfall

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Ryan A. Blausteina,b*, Yakov A. Pachepskyb, Robert L. Hilla, Daniel R. Sheltonb

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a

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College Park, MD, USA

Department of Environmental Science and Technology, University of Maryland at College Park,

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b

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Research Center, Beltsville, MD, USA

USDA-ARS Environmental Microbial and Food Safety Laboratory, Beltsville Agricultural

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GRAPHICAL ABSTRACT

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ABSTRACT Understanding and quantifying microbial release from manure is a precondition to

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estimation and management of microbial water quality. The objectives of this work were to

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determine the effects of rainfall intensity and surface slope on the release of Escherichia coli,

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enterococci, total coliforms, and dissolved chloride from solid dairy manure, and to assess the

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performance of the one-parametric exponential model and the two-parametric Bradford-Schijven

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model when simulating the observed release. A controlled-intensity rainfall simulator induced

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one hour of release in runoff/leachate partitioning boxes at three rainfall intensities (30, 60, and

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90 mm hr-1) and two surface slopes (5% and 20%). Bacterial concentrations in initial release

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were more than one order of magnitude lower than their starting concentrations in manure. As

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bacteria were released, they were partitioned into runoff and leachate at similar concentrations,

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but in different volumes, depending on slope. Bacterial release occurred in two stages that

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corresponded to mechanisms associated with release of manure liquid- and solid-phases.

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Parameters of the two models fitted to the bacterial release dependencies on rainfall depth were

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not significantly affected by rainfall intensity or slope. Based on two model performance tests,

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the Bradford-Schijven model is recommended for simulating bacterial release from solid manure.


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INTRODUCTION Agriculture generates animal waste in large quantities. In the United States, concentrated

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animal feeding operations (CAFOs) alone produce about 500 million tons of liquid and solid

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animal waste per year.1 The release of microorganisms from animal waste that is applied to

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agricultural land is induced by rainfall or irrigation events. Some released manure-borne

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microbes are pathogenic, and they can create contamination of recreational, irrigation, drinking,

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and other types of water if overland or subsurface flow that contains released suspensions can

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reach these water sources. Therefore, understanding and quantifying microbial release from

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manure becomes a precondition to estimation and management of microbial water quality.

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Microbial release from animal waste and its subsequent removal with runoff and leachate have

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been shown to be affected by application rates 2,3, vegetation status at the application area 4,5,

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method of animal waste application, e.g. surface application or soil incorporation 6,7, animal

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source 8-10, and waste age 11. Precipitation variability has also been found to impact microbial

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release.11,12,13

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Animal manures are classified by animal sources, by the method of collection on farms,

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and by consistency, i.e. content of solids. Solid manures are typically differentiated from manure

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slurries or liquid manures by having a dry solid content of above 20%.14,15 Farmyard manures

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commonly belong to the solid manure category.

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The majority of microbial release studies have been done with manure slurries, and

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relatively little is known about the release from solid manures. At the same time, solid manures

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as a microbial source are quite different from liquid/slurry manures. During rainfall over land-

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applied solid manure, some portion of rainwater water needs to travel within the manure’s

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complex pore space before it reaches the soil surface. Where the solid manure completely covers



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the soil surface, no direct transmission of rainfall energy to the soil surface occurs. While the

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rainwater simply dilutes liquid manure or slurry, solid manures often have rainwater flowing

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over the surfaces of the fecal material. The manure material enters runoff and infiltration only

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after being exposed to the sloughing action of rainwater rather than simply being suspended, as

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may be the case for liquid manure or slurry. The kinetics of protozoa oocysts released from solid

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animal waste under single-dripper raindrops have been described as a two-stage process with a

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fast initial release followed by a slower log-linear release.12,16 However, solid manure is different

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from solid animal waste in that it is a mixture of material of animal (solid and liquid excreta) and

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plant (bedding) origin that has a complex porous space and mechanical properties that are

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different from fecal material alone. No studies regarding the release of bacteria from solid

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manure under simulated rainfall were found in the scientific literature. In release studies that

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have incorporated rainfall simulation events, initial concentrations of fecal coliforms, E. coli, and

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enterococci in suspensions entering runoff and infiltration have been found to be similar to the

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concentrations found in slurry or liquid manure 17,18, but the concentrations could be quite

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different for solid manure since pathways may exist for rainwater to flow through the manure

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without substantial interactions with the fecal material. Models to simulate rainfall-induced

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microbial release from land-applied animal waste have been tested for slurries 17,18 and animal

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waste 12,16, but not for solid manures.

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Rainfall intensity has been suggested to have positive effects on the release manure-borne

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microorganisms from land-applied animal waste.6,8 Schijven et al. 12 conducted a laboratory

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study on protozoan release from solid animal waste using two ways of water application:

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dripping and misting to mimic a rain shower and a light drizzle, respectively. Cryptosporidium

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release efficiencies were reported to be greater in treatments with water applied via the dripping


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method due to greater impact energies exerted by the larger water droplets.12 The authors

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proposed that rainfall events with higher intensities should increase levels of microbial release

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from manure on farms. Although water application intensity may have positive effects on

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microbial release numbers, the concentrations of microorganisms in runoff during higher

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intensity rainfall events could actually end up being relatively low due to dilution by rainwater.

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In a study by Kress and Gifford 11, rainfall intensity had large impacts on the peak concentrations

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of fecal coliforms in runoff coming from 20-day-old cowpats, but the rainfall intensity had no

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significant effects on release from fresh cowpats. There are no known studies available that have

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evaluated the impacts of rainfall intensity on microbial release from solid manure when rainfall

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depth is the dependent variable.

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Land slope when combined with rainfall intensity has been shown to exert significant

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impacts on the depth of interactions between runoff and the soil surface layer (i.e., the mixing

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depth of runoff in the soil).19 The microtopography of a landscape was shown to cause

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differences in the runoff-recovery of E. coli and Salmonella released from swine slurry that had

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been applied on hillslope plots.20 Microorganisms that are released from solid manure can enter

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runoff from the outer surface of manure of the manure matrix, sides of the matrix, or through the

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base of the matrix. The slope that manure is positioned on may impact the lateral movement of

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water and bacteria through and out of the matrix. One can hypothesize that the slope steepness of

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applied manure may be a factor influencing the amounts of bacteria that are released from solid

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manure to runoff flowing over the manure surface and leachate flowing out of the base of the

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matrix.

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Microbial release from the same animal waste has been shown to differ among microbial species/groups for a variety of reasons.2,5,10 Detection of several microbial indicators for



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estimating microbial water quality effects by livestock wastewater runoff is beneficial, since one

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microorganism may not suitably serve as a suitable surrogate for every manure-borne

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pathogen.10 Release kinetic differences among manure constituents were established for slurries

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12,18

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, but have not yet been researched with solid manure. A review of the literature indicates that there are substantial gaps in our understanding of

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how bacteria are released from solid manure. The objectives of this work were to (a) determine

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the effects of rainfall intensity and slope steepness on the release of E. coli, enterococci, total

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coliforms, and chloride ion from solid cattle manure, and (b) to evaluate the applicability of

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known microbial release models to simulate release of the above indicator bacteria from solid

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manure.

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MATERIALS AND METHODS

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Rainfall simulator

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A multiple-intensity, intermittent rainfall simulator, based on the design of Meyer and

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Harmon 21, was used to perform simulated rainfall events. Different rainfall intensities were

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applied by controlling the time intervals for pauses between nozzle oscillation sweeps. The

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sprinkler nozzles (Veejet 80150; Spraying Systems Co., Wheaton, IL) were positioned to rain

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from a height of 3 m so the raindrops could approach terminal velocity upon landing. The

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pressure of water flowing into the nozzles was maintained at 41 N m-2 to control the rainfall

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intensity and the rain drop size distribution across a 1m2 area in the center of the spray pattern

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during each simulation event. This design allowed for raindrop impact energies to be

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approximately 275 kJ/ha-mm, which has been reported to be about the same as that of natural

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rainfall intensities greater than 2.5 cm hr-1.21 All rainfall simulations took place indoors to avoid 7 ACS Paragon Plus Environment

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the effects of wind or sunlight. The Christiansen coefficient of uniformity was measured for all

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intensities and varied between 84% and 86%.

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Partitioning boxes

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Partitioning boxes were designed to have solid manure applied on a fine mesh screen and

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to have both runoff, flowing over the manure surface, and leachate, flowing out of the base of the

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manure matrix, collected in PVC troughs under simulated rainfall. The partitioning boxes (70- x

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70-cm inside area) were made of wood and consisted of two components – an upper box with the

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bottom frame covered with nylon mesh with 185-µm openings (Component Supply Co./SKU

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Solutions, Fort Meade, FL) and a lower box with an impervious, plastic-covered base. The two

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boxes were secured together with c-clamps. During a rainfall simulation over a partitioning box

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loaded with manure, runoff could be collected in a trough installed below an opening at the

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upper box wall where runoff flow would be directed by the slope. Similarly, the leachate

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suspension could be collected in a trough installed below an opening at the lower box wall where

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this suspension would be directed by the slope. This design allowed for runoff and leachate to be

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collected simultaneously from the two troughs.

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Manure and rainwater composition

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The manure used in this study was obtained from dairy cattle at the USDA-ARS Dairy

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Research Facility, in Beltsville, MD. At this facility, the 2- to 5-year old dairy cattle are provided

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a corn silage-based TMR (total mixed ratio) diet in a free-stall barn. A synthetic manure mix,

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consisting of fresh cattle excreta combined with saw dust bedding, was used to represent

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farmyard manure that is similar to the manure produced at dairy concentrated animal feeding

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operations (CAFOs). To prepare the synthetic manure, cattle feces and urine were sampled from



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5 different cows in disinfected 5-gal buckets, stirred in their respective buckets, and then mixed

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together at a 6/1 volumetric ratio of feces/urine. This ratio was similar to proportions of excreta

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in CAFO dairy manure that were described in Van Horn et al. 22. The synthetic manure was

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stored at 4⁰ C until usage. On the morning of each experimental run, sawdust bedding was mixed

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into the synthetic manure to bring the manure-solids content up to approximately 30% by mass.

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New manure components were collected to prepare the solid manure each week to ensure high

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concentrations of indigenous bacteria and to maintain consistency of manure from week to week.

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Composite manure samples were collected on the day of each rainfall simulation event to obtain

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the average physical, chemical, and microbial contents of the manure that was used throughout

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the study and to measure the contents of for plant macro- and micro-nutrients (Supporting

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Information, Table SI-1).

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Synthetic rainwater was prepared to mimic the ion content and pH that are standard for

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rainfall in the Maryland, Pennsylvania, and Delaware region.4,23 The synthetic rainwater was

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made by adding reagent-grade chemicals to reverse-osmosis treated water to obtain

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concentrations of Ca2+, Mg2+, K+, Na+, NH4+, NO3-, Cl-, and SO42- at 0.08, 0.03, 0.02, 0.12, 0.34,

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1.36, 0.26, and 1.9 mg L-1, respectively. The rainwater solution was mixed in 500 gal holding

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tanks and pumped into a 100 gal tank that was connected to the rainfall simulator. Just before

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rainfall, the pH of the rainwater solution in the 100 gal tank was adjusted to 4.5 using variable

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amounts of HCl and/or NaOH.

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Experimentation

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Treatments followed a 2 x 3 factor design with two surface slopes (5% and 20%) and

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three rainfall intensities (30, 60, and 90 mm h-1). These rainfall intensities were chosen because

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of their linearity and correspondence to precipitation intensities in the mid-Atlantic region, where 9 ACS Paragon Plus Environment

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the one-year recurrence of rainfall for a 10 min duration is 87 mm h-1 and that for a 60 min

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duration is 31 mm h-1.24 All six treatments were performed in triplicate in randomized order.

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On each day of experimentation, before manure was added into the partitioning boxes,

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one composite sample of manure, which consisted of about five grab samples, was taken from

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the manure supply bucket. Manure was applied to the mesh layer of each partitioning box at the

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rate of 60 ton ha-1 wet weight (i.e., 2.94 kg box-1) by being dumped into the upper box and then

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evenly distributed, by hand, to fill any gap. The depth of manure that was spread across the mesh

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frames was a few cm thick (typically between 2 and 3 cm), and varied based on different-sized

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manure clumps in the matrix that were distributed across the plane. This application rate was

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chosen because it allowed for manure to completely cover the mesh frame. This same manure

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application rate was used in a four-year study (2004, 2005, 2007, 2009) that modeled the

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overland transport (i.e., release and subsequent runoff removal) of indicator bacteria from land-

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applied manure at the USDA-ARS OPE3 Field in Beltsville, MD 25. Three samples of manure

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were collected from each box before rainfall initiation to analyze for the starting contents of

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bacteria and Cl-. During rainfall, runoff and leachate were collected from their respective troughs

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in sterile 100-ml bottles upon their initial release (time 0) and then subsequently at 1, 2, 4, 7, 10,

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15, 20, 30, 40, 50, and 60 minutes. In order to collect both effluent types simultaneously, the

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sampling schedule of the 0-60 min release event would always follow the relative release time of

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the first effluent type released (e.g., if leachate appeared before runoff, then after the initial

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runoff sample was collected, the remaining runoff samples were collected on the relative time

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schedule of the leachate collection). All collection times and the duration time of each collection

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were recorded.



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Microbiological and chemical analyses

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The water contents of manure samples were determined by measuring water loss after

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drying the samples in an oven at 100⁰ C for 24 h to a constant dry weight. Wet manure samples

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were each blended with sterile deionized water (2 g manure in 200 ml of water) using a high

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speed blender (model 34BL97; Waring Laboratory, Torrington, CT) for 2 minutes to produce a

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homogenous slurry mixture. Slurry was allotted a1 hr settling time before processing. The

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manure slurries and the runoff and leachate samples were spread-plated on CHROMagarTM ECC

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(Chromagar, Paris, France) to enumerate E. coli and total coliforms and on m-Enterococcus agar

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(Neogen Corporation, Lansing, MI) to enumerate enterococci. Chloride ion content of each

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manure, runoff, and leachate sample was measured with the Waters 2695 Separations Module

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(Waters Corporation, Milford, MA).

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Release Modeling For each release event, the concentrations of E. coli, enterococci, total coliforms, and Cl-

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in the collected effluent samples - runoff and leachate - were converted, based on flow rate, to

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the cumulative total numbers of bacteria and masses of chloride that were removed with each

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effluent type. The cumulative amounts of the individual manure-constituents removed with

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runoff and leachate were interpolated to determine the rainfall depth-dependent and time-

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dependent total release from manure.

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The release of each bacterial group/species and Cl- were modeled as a function of

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rainfall-depth and as a function of time with the one-parametric exponential dependence model 26

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:

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= 1 − ( )

(1)


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and the two-parametric Bradford-Schijven model 16 :

= 1−

(2)

( )

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Here, N is the total number of bacteria or the mass of Cl- released per unit area of manure, [N] =

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CFU (for bacteria) or mg (for Cl-) m-2; is the initial number of bacteria or mass of Cl- per unit

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area of manure application, [ ] = CFU (for bacteria) or mg (for Cl-) m-2; W is rainfall depth,

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[W] = mm rainfall; and are constants, [ke] and [kp] = mm-1; β is a dimensionless shape

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parameter. We note that the original Bradford-Schijven model contained the product of time and

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rainfall rate in place of the rainfall depth in Eq. 2.

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Eq. 1 and Eq. 2 were fitted to the ‘rainfall depth-release’ and the ‘time-release’ data with

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a FORTRAN code REL_BACT 27, which was based on the Marquardt-Levenberg optimization

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algorithm as implemented by van Genuchten 28.

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Data Analysis

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The lag times between the start of rainfall and the generation of runoff and/or leachate

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were compared among treatments, and a two-factor ANOVA (α=0.05) was utilized to evaluate

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the effects of rainfall intensity and surface slope on the length of this lag period. The volumes of

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water partitioned into runoff and leachate were calculated based on flow rate and time. The

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concentrations of total coliforms, E. coli, enterococci, and Cl- in the manure liquid phase

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(CManure) were compared to the concentrations in the initial release (C0), and a two-factor

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ANOVA (α=0.05) was utilized to evaluate the effects of rainfall intensity and surface slope on

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the ratios of C0/CManure. The concentrations of the bacteria in synchronous runoff and leachate

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were analyzed with a one-to-one regression with the significance level set at 0.05. A two-factor



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ANOVA (α=0.05) was utilized to evaluate the effects of rainfall intensity and slope steepness on

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the release model parameters for each bacterial group/species and for Cl-.

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Release model performance was assessed for each model-fit by root-mean-squared-error (RMSE) and Akaike information criterion (AIC) values. RMSE were computed as: RMSE =

(3)

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where RSS is the residual sum of squares and n is the number of measurements.

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The RMSE units are dimensionless. A smaller RMSE indicates better model performance.

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The Akaike information criterion (AIC) provides another metric for model performance

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and accounts for the interplay between the model goodness-of-fit and model complexity.29 In this

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model comparison, the AIC test considers that Eq. 1 and Eq. 2 have a different number of fitting

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parameters (i.e., one and two, respectively). The corrected Akaike statistic is:

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AIC = $%$ &

' + 2 +

* ( )

(4)

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where RSS is the residual sum of squares, n is the number of measurements, and k is the number

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of model parameters. The AIC units are dimensionless. Having a more negative corrected

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Akaike statistic indicates better model performance.

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The correlations between bacterial concentrations and Cl- concentrations in release were

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determined. The Steiger’s Z-test 30, which is used to test whether one predictor (e.g., Cl-)

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correlates equally with two criterion variables (e.g., E. coli and enterococci), was utilized to

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compare how well each of the bacterial group/species concentrations correlated with Cl-

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concentrations in release.


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RESULTS

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Manure properties and concentrations of studied constituents

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The manure properties and the initial microbial contents did not substantially change

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throughout the experiment. The percentage of solids in the manure composite samples was 29.2

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± 0.6 %, pH was 7.9 ± 0.1, total carbon was 17.2 ± 1.9 %, and C:N ratio was 45.6 ± 3.3. The

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total coliform, E. coli, enterococci, and Cl- contents in the composite manure samples were 3.0 ±

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1.0 x 106 CFU, 1.9 ± 0.7 x 106 CFU, 1.6 ± 0.6 x 106 CFU, and 0.7 ± 0.2 mg per gram of wet

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manure, respectively. E. coli constituted over half of total coliforms, and the total contents of E.

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coli and enterococci in the manure were similar.

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Partitioning of water and bacteria between runoff and leachate

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A lag time was consistently observed between the start of rainfall and the initiations of

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runoff and leachate. The lag time was dependent on rainfall intensity (p

Solid Manure As a Source of Fecal Indicator Microorganisms: Release

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