Science of the Total Environment 472 (2014) 90–98

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Science of the Total Environment

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Does microbial centimeter-scale heterogeneity impact MCPAdegradation in and leaching from a loamy agricultural soil?

Annette E. Rosenbom a,⁎, Philip J. Binning b, Jens Aamand a, Arnaud Dechesne b,Barth F. Smets b, Anders R. Johnsen a

a Geological Survey of Denmark and Greenland, Department of Geochemistry, Øster Voldgade 10, DK-1350 Copenhagen, Denmarkb Technical University of Denmark, Department of Environmental Engineering, DK-2800 Kgs. Lyngby, Denmark

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Spatial distribution of MCPA-degradingbacteria in agricultural soil was assessed.

• Application of Monod-kinetics to de-scribe 3D-degradation of MCPA

• High degradation in plow layer elimi-nates impact of microbial heterogeneityon leaching.

• Wormhole facilitates rapid leachingwith bypass of microbially active lining.

⁎ Corresponding author. Tel.: +45 3814 2052; fax: +4E-mail address: aer@geus.dk (A.E. Rosenbom).

0048-9697/$ – see front matter © 2013 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.scitotenv.2013.11.009

a b s t r a c t

a r t i c l e i n f o

Article history:Received 17 September 2013Received in revised form 1 November 2013Accepted 1 November 2013Available online 28 November 2013

Keywords:Spatial biodegradationMonod-kineticsPreferential transportMCPA leachingCOMSOL simulations

The potential for pesticide degradation varies greatly at the centimeter-scale in agricultural soil. Three dimen-sional numerical simulations were conducted to evaluate how such small-scale spatial heterogeneity may affectthe leaching of the biodegradable pesticide 2-methyl-4-chlorophenoxyacetic acid (MCPA) in the uppermeter of avariably-saturated, loamy soil profile. To incorporate realistic spatial variation in degradation potential, we useddata from a site where 420 mineralization curves over 5 depths have been measured. Monod kinetics was fittedto the individual curves to derive initial degrader biomass values, whichwere incorporated in a reactive transportmodel to simulate heterogeneous biodegradation. Six scenarios were set up using COMSOLMultiphysics to eval-uate the difference between models having different degrader biomass distributions (homogeneous, heteroge-neous, or no biomass) and either matrix flow or preferential flow through a soil matrix with a wormhole.MCPA leached, within 250 days, below 1 m only when degrader biomass was absent and preferential flowoccurred. Both biodegradation in the plow layer and the microbially active lining of the wormhole contributedto reducingMCPA-leaching below1 m. The spatial distribution of initial degrader biomasswithin each soilmatrixlayer, however, had little effect on the overall MCPA-leaching.

© 2013 Elsevier B.V. All rights reserved.

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1. Introduction

The agricultural use of pesticides is of environmental concern be-cause of the many non-target side-effects it generates. Numerous

91A.E. Rosenbom et al. / Science of the Total Environment 472 (2014) 90–98

guidelines and models have been set up, to predict the general fate ofpesticides, and more specifically, the risk of pesticide leaching from ag-ricultural soil, e.g. Council-directive-91/414/EEC (1991). These guide-lines and models incorporate key processes like sorption anddegradation. None of these predictions, however, take into accountthat degradation in soil may vary in time and space. Instead, they relyon degradation parameters obtained from disturbed soil samples thatare homogeny (Beulke and Brown, 2001).

Spatial heterogeneity has an impact on degradation of pesticides insoil (Badawi et al., 2013b; Fredslund et al., 2008; Lopez-Blanco et al.,2005; Vieuble-Gonod et al., 2009; Vinther et al., 2008; Vinther et al.,2001). The largest and most homogeneous degradation potentials arefound in the plow layer, whereas deeper layers may have zones of fastand slow degradation (Badawi et al., 2013b; Sorensen et al., 2006).The active zones can often be linked to discontinuities in the soil matrixsuch as wormholes, which may facilitate rapid preferential transport ofsolutes deep into the ground (Badawi et al., 2013a; Lopez-Blanco et al.,2005; Morales et al., 2010; Ruamps et al., 2011; Singer et al., 2001;Young et al., 2008). This transport results in higher water-soluble car-bon concentrations, and larger bacterial densities and activities in thewormholes compared to the adjacent soil matrix (Bundt et al., 2001;Pivetz and Steenhuis, 1995; Vinther et al., 1999).

How such centimeter-scale microbial heterogeneity may impactpesticide leaching is, to our knowledge, lacking in the scientificliterature, which has partly been due to the lack of appropriate data.Recently, we quantified the centimeter-scale variation in potential4 chloro-2-methylphenoxyacetic acid (MCPA) mineralization in avariably-saturated, loamy soil (Badawi et al., 2013b). In the presentstudy, we have used this dataset for three-dimensional numericalmodeling to predict the impact of microbial heterogeneity on MCPA-biodegradation andMCPA-leaching through the upper meter of agricul-tural soil. The reported sigmoid degradation kinetics deviate significant-ly from the first-order description, which is commonly used innumerical modeling, and we therefore applied Monod-kinetics. Dataon MCPA degradation at five different concentrations in the plow layersoil enabled an estimation of the kinetic Monod parameters. Withthese parameters, it was possible to extract the initial degrader biomassfor all 420 mineralization curves, and these biomass data were used asinput for reactive transportmodeling. Themodel-simulations, hence, in-corporated the effect of growing, heterogeneously distributed degraderbiomass on the fate of MCPA as MCPA is used by the bacteria as energyand carbon sources.

More specifically, our aim was to determine the MCPA-leachingdown to 1 m below ground surface (b.g.s.) under the following condi-tions: 1) no degrader biomass and simple matrix flow; 2) averagedinitial degrader biomass resembling data from homogenized soil-samples and simple matrix flow; 3) random spatial variation in initialdegrader biomass and simple matrix flow; 4) no degrader biomassand preferential flow through a wormhole; 5) random spatial variationin initial degrader biomass and preferential flow through a matrix withawormhole; and 6) random spatial variation in initial degrader biomassbiased by placing the highest degrader biomass concentration along thewall of the wormhole, and preferential flow through a matrix with awormhole.

2. Materials and methods

To incorporate centimeter-scale degradation in a three dimensionalnumerical MCPA-transportmodel of the upper variably saturatedmeterof a loamy soil, two recently published mineralization-datasets havebeen applied to estimate the degradation kinetics (Monoddescription) and the matching three-dimensional distribution ofinitial degrader biomass. Dataset 1 describes the effect of MCPA-concentration on the degradation kinetics (Johnsen et al., 2013), andDataset 2 describes the spatial heterogeneities in MCPA (Badawi et al.,2013b).

2.1. Data

2.1.1. Dataset 1 — concentration effect on degradationData on the effect of MCPA-concentration on degradation kinetics

was obtained from a mineralization study (Johnsen et al., 2013),where soil from the A-horizon was thoroughly homogenized and 1-gsubsamples spiked with 14C-MCPA at concentrations of 0.1, 0.3, 1.0,3.0, and 10 mg kg−1. With a bulk density of approximately 1.7 g cm−3

(Nielsen, 2010), the MCPA-concentrations were 1.7 × 10−4,5.1 × 10−4, 1.7 × 10−3, 5.1 × 10−3, and 1.7 × 10−2 kg m−3. MCPAwas fully mineralized (N99.4%) when the evolution of 14CO2 eventu-ally leveled off for mineralization experiments conducted at both10 mg kg−1 and 0.1 mg kg−1 (Johnsen et al., 2013), i.e., there wasno detectable accumulation of MCPA metabolites. For each mineral-ization curve, the plateau was therefore assigned a value of 1 (fullmineralization) so that the normalized mineralization would pro-ceed from 0 to 1 and the corresponding normalized degradationfrom 1 to 0.

2.1.2. Dataset 2 — spatial distribution of degradationThe three-dimensional MCPA-biodegradation data incorporated into

the model was based on MCPA-mineralization in samples from a loamysoil profile (Badawi et al., 2013b). The profile (0–115 cm) was dividedinto five domains each representing a distinct soil horizon (A, B1, B2,B3, and C). For each horizon, 84mineralization curves were available, de-termined within x–y oriented grids of 6.5 cm × 10.5 cm. Each minerali-zation curve represented the potential of a 0.2 g soil sample that wasspiked with an initial 14C-MCPA-concentration of 1.7 × 10−2 kg m−3.The mineralization data were converted to normalized degradationdata by using the plateau-values as described for Dataset 1. Mostcurves showed a plateau of 40% mineralization. For some samples,the mineralization was too slow to reach a clear plateau within the120 day incubation. Mineralization for these samples was normalizedby assuming that they would also eventually reach a plateau-value of40%.

2.1.3. Dataset 3 — field-scale test of MCPA-leachingThe leaching of MCPA has previously been tested at the field scale

in the Danish Pesticide Leaching Assessment Program (http://pesticidvarsling.dk/om_os_uk/uk-forside.html). MCPA was sprayedin the maximum allowed dose (0.75 kg L−1) on two loamy fields(Silstrup and Faardrup) that geologically resemble the field (HøjeTåstrup) that was used for generating Datasets 1 and 2. Leaching ofMCPA was followed by sampling water in drains at a depth of1.1 m for two years. Initially, the drains were sampled once a week(time-proportional sampling), and later this was changed to one samplefor every 1500 L in summer and every 3000 L in winter (flow-propor-tional sampling). The detection limit for MCPA in the drain water was0.01 μg L−1.

2.2. Degradation kinetics

MCPA-biodegradation was described by Monod-kinetics (Eqs. (1)and (2)). The Monod-parameters were first estimated from Dataset 1with different initial MCPA-concentrations. These parameters werethen fixed and used to estimate the initial concentration of the degraderbiomass (X0) for each of the 420 degradation curves in Dataset 2, whichwas the input needed for Monod-simulation of MCPA-biodegradationand leaching.

The Monod-equations (Monod, 1949) as modified by Lawrence andMcCarty (1970) describe the effect of substrate concentration S [massper volume] on the rate dS/dt at which a growing microbial degraderconcentration X [mass per volume] removes the substrate.

dSdt

¼ − qmaxSXKs þ S

ð1Þ

Table 1Model input parameters (Rosenbom et al., 2009) used in the COMSOL Multiphysicssimulations.

Media Parameter Value

Clayey till Saturated conductivity, Ks [cm·s−1] 5.4 · 10−4

Porosity, θs [cm3·cm−3] 0.36Residual saturation, Sr [cm3·cm−3] 0.08van Genuchten parameter, α [1/cm] 0.00698van Genuchten parameter, n [−] 2.0Longitudinal dispersivity, αl [cm] 5.0Transverse dispersivity, αt [cm] 1.0Tortuosity, τ [−] 0.71Bulk density, ρ [kg·cm−3] 0.002

Wormhole Saturated conductivity, Ks [cm·s−1] 654Residual saturation, Sr [cm3·cm−3] 0.01van Genuchten parameter, α [1/cm] 0.1van Genuchten parameter, n [−] 2.0

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dXdt

¼ −YdSdt

¼ YqmaxSXKs þ S

ð2Þ

Here S is the concentration of MCPA, X is the degrader biomass, qmax

is themaximal, specificMCPA-degradation rate [(time)−1],Ks is the halfsaturation constant for MCPA [mass per volume], and Y is the growthyield [−] representing the mass of degrader biomass X synthesizedper unit of degraded substrate (S).

This version of theMonod-equations includes four unknownparam-eters (qmax, Ks, Y, and X0), which were estimated by jointly fitting thetopsoil degradation curves for five MCPA-concentrations (Dataset 1).The estimation was performed using a Shuffled Complex EvolutionMetropolis (SCEM) algorithm (Vrugt et al., 2003) with the followingcredible value-intervals:

X0 The average density of cultivable MCPA-degraders in thetopsoil used for the mineralizations was 610 cells cm−3

(Badawi et al., 2013b). This count was based on a highly sen-sitive, most-probable-number radio-respirometric method(Johnsen et al., 2009) with prolonged incubation (190 days)to detect even the most slow-growing MCPA degraders. Byassuming theweight of one cell to be 5 × 10−16 kg, the initialdegrader biomass concentration X0 would be approximately3 × 10−7 kg m−3. Growth-dependent estimates of degraderpopulations in soil are, however, always smaller than the totaldegrader populations estimated by direct counts or DNA-based techniques (Harwani, 2013). We therefore applied ahigh credible range of [5 × 10−7; 5 × 10−4] kg m−3 to takethis bias into account.

qmax and Ks The initial degradation rates (kg m−3 day−1) were esti-mated for each of the five MCPA-concentrations by linearregression (day 0 to day 2). These rates were then dividedby the initial degrader biomass, X0 (1.5 × 10−6 kg m−3) togive estimates of the specific degradation rate, q (day−1),which in Figure S1 is plotted against the initial MCPA-concentrations, S (kg m−3). Coarse estimates of qmax

(147 day−1) and Ks (6.7 × 10−3 kg m−3) were obtainedby fitting the Monod function (Figure S1). The credible in-tervals were set by multiplying and dividing these esti-mates by five.

Y In Dataset 1, 40–60% of the MCPA-carbon was mineralized toCO2, suggesting that the carbon-growth yield was 0.6 to 0.4.The net-yield factors would, however, be smaller becausethe need for cell maintenance is not included in Eqs. (1) and(2) and because the chlorine atom of the MCPA-moleculedoes not contribute to cell growth. The interval was set to[0.01; 0.6].

Based on the SCEM-estimated qmax, Ks, and Y from the topsoil(Dataset 1), Monod-curves were fitted to the sigmoid curves inDataset 2 to estimate the initial degrader biomass concentrationsX0 for each of the soil samples. Again the same SCEM-fitting methodwas employed, but with a lower credible X0 limit of 1 × 10−10 kg m−3,which corresponded to approximately one cell per cm3. For sampleswith no degradation or slow zero-order degradation, X0 was set tozero. The X0-values were used as input for the Monod-biodegradationof MCPA in the following COMSOL model.

2.3. Model setup

The numerical COMSOL Multiphysics 4.2a modeling toolbox(COMSOL Inc., Stockholm, Sweden) was used for the three-dimensional finite element simulations of MCPA-leaching presentedhere. With this tool it is possible to take into account the spatial miner-alization data from the variably-saturated upper meter of loamy soil.

The following interfaces were applied as governing equations (see SIfor details): (i) Richard's Equation Interface for the variably-saturatedwater flow, (ii) Solute Transport Interface — for transport of MCPA ac-counting for MCPA-biodegradation (Eq. (1) is included as a reactionterm), and iii) Coefficient FormPDE (Partial Differential Equation) Inter-face for the growth in biomass X described byMonod-kinetics (Eq. (2)).A time-dependent solver was applied using the Backward Differentia-tion Formula (BDF) method with maximum order of 5, minimumorder of 1, and free steps taken by solver with maximum time step ofone day.

MCPA-biodegradation and leaching were simulated for a three-dimensional soil column oriented in the xyz-plane (0.099 × 0.054 ×0.99 m)with a nodal spacing of 0.009 m resembling the distance in be-tween the sampling points of Dataset 2. MCPAwas “added” in the sameconcentration as in Dataset 3. In Scenarios 4–6, an artificial wormhole,in the form of a vertical, highly-permeable 0.9 × 0.9 cm zone withoutany MCPA-sorption, was placed in the center of the simulated soil col-umn. Hydraulic properties (“matrix zone 1” and “wormhole”) from asimilar clayey till were applied (Table 1). The description of the worm-hole was previously “validated” against field data on tracer leaching(Rosenbom et al., 2009).

The initial and boundary conditions for the flow and transport sim-ulations were defined from information regarding dates of MCPA-applications and simulated daily net-precipitation derived via theDanish Pesticide Leaching Assessment Programme, PLAP (Rosenbomet al., 2010). The date of MCPA-application was set to 2 May 2008,which lies within the period of application for normal agriculturalpractice in Denmark. On this date only, an MCPA-dose was applied asa flux and set to 2 × 10−5 kg m−2 day−1. This flux was estimatedfrom the maximum allowed use of the product MCPA750 in Denmark(2.7 × 10−4 L m−2, with concentration of the parent compound of0.75 kg L−1, Middeldatabasen (2010)). In May, the water table wassituated at a depth of 3–4 m, which at equilibrium corresponds to apressure head of approximately −0.99 m of water at the lower modelboundary. The initial pressure head was therefore set to −0.99 mat the lower boundary condition (z = 0.00 m) and decreasing to−1.98 m at the soil surface (z = 0.99 m). To achieve a realistic pre-cipitation input to the columnwithout accounting for evapotranspi-ration, plant interception and surface runoff, we applied a dailyMACRO-simulated net-precipitation of the period 1 May–31December 2008 from the Faardrup site, Denmark (Larsbo et al.,2005; Rosenbom et al., 2010) as upper boundary condition. Thetotal simulation period was 243 days. The net-precipitation appliedresembled the climate and soil conditions under which the mineral-ization data were collected. To simulate the worst case MCPA-leaching scenarios achievable with this net-precipitation, cropuptake via roots was neglected. MCPA-sorption data were obtainedfrom a comparable loamy soil profile (Sorensen et al., 2006), wherethe sorption coefficient Kd = 1.23 L kg−1 in the upper 0.1 m,

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1.18 L kg−1 0.25 m b.g.s., 0.04 L kg−1 0.55 m b.g.s., and 0.00 L kg−1

0.99 m b.g.s. (z = 0 m). A linear interpolation of Kd with depth wasassumed between these Kd-data-points. Kd is hence only varying withdepth and is not correlated with the MCPA-degradation. The moleculardiffusion coefficient (Dm = 6.77 × 10−10 m2s−1) of MCPA was speci-fied from a study in activated carbon reported by Cho et al. (2006).

Mineralization data was split up in five domains: the A-horizon rep-resented the plow layer (0–24 cm), the B1-horizon represented theupper B-horizon (25–31 cm), the B2-horizon the middle B-horizon(32–66 cm), the B3-horizon the lower B-horizon (67–90 cm), and theC-horizon the upper C-horizon (below 91 cm). MCPA-biodegradationand leaching were simulated for six scenarios of increasing complexitywith respect to water flow and the spatial distribution of degradationpotential.

• Scenario 1 had simple matrix flow of water and no biodegradation ofMCPA, thus resembling a situation where leaching is determinedonly by simple transport including sorption.

• Scenario 2 had simple matrix flow of water. MCPA-biodegradationwas introduced by an averaged initial degrader biomass (X0) at allnodes within each horizon (Fig. 3). Nodes with no biomass (X0 = 0)were included in the averages. Scenario 2 therefore corresponded tothe common situationwhere degradation data obtained fromhomog-enized soil samples are used as input for a model to simulate simpletransport including sorption.

• Scenario 3 had simple matrix flow of water, sorption, and variableMCPA-biodegradation that resembled the in-situ spatial distribution(Fig. 3). For each soil horizon, the initial degrader biomasses, i.e. the84 X0-values, were distributed randomly within each model layer byusing the Microsoft Excel RAND-function. The purpose of Scenario 3was to test if spatial heterogeneity in biodegradation would give dif-ferent model results than when applying biodegradation data fromhomogenized samples (Scenario 2).

• Scenario 4 In this scenario, simplematrix flowwith sorptionwas com-bined with preferential flow through a matrix with a wormhole. Thewormhole was simulated by placing a highly permeable, 9 × 9 mmcolumn in the center of the simulated soil column extending all theway through the soil profile. The wormhole had neither sorption norany MCPA-biodegradation. This scenario corresponded to a worst-case situation where MCPA could leach by rapid preferential flow.

• Scenario 5 combined the wormhole effect on water flow (preferentialflow as in Scenario 4), and the effect of spatially variable biodegrada-tion (as in Scenario 2, Fig. 3). Compared to Scenario 4, MCPA wasbiodegraded both in the matrix and along the wormhole.

Fig. 1. Kinetics of MCPA-degradation at five initial concentrations (symbols)(Johnsen et al., 2013), and the fitted Monod model (lines, Ks = 0.0016 kg m−3,Y = 0.0563, qmax = 4.1714 days−1, and X0 = 2.5702 × 10−5 kg m−3).

Fig. 2.Degradation kinetics of MCPA in 84 samples collected at five depths, correspondingto the five model layers. The degradation-curves were estimated from MCPA-mineralization curves obtained from soil samples (0.2 g) sampled in 6.5 × 10.5 cm gridsat depths of 8 cm (A), 28 cm (B1), 48 cm (B2), 85 cm (B3), and 115 cm (C) (Badawiet al., 2013b).

• Scenario 6 simulated a situation where the lining of a wormhole is amicrobiological hot-spot with increased capacity for MCPA-biodegradation. The model setup was as in Scenario 5 except thatthe four highest X0-values in each model layer were placed aroundthe wormhole (Fig. 3). Compared to Scenario 5, this tests whether ahotspot lining as previously observed for this field (Badawi et al.,2013a) would have any significant impact on MCPA-transportthrough the wormhole.

Fig. 3. The three dimensional distributions (0.099 × 0.054 × 0.99 m) of initial degraderbiomass X0 in the upper meter of a loamy soil. Averaged: The initial degrader biomasswas averagedwithin each soil horizon. Random: For each soil horizon, the initial degraderbiomass was distributed randomly within each model layer. Preferential: Identical to therandom distribution except for the introduction of a wormhole with increased degraderbiomass.

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3. Results

3.1. Estimation of the Monod-parameters — Dataset 1

By applying the SCEM-algorithm, a Monod-fit was obtained wherethe parameters could accurately describe MCPA-biodegradation in theA-horizon soil at the five initial MCPA-concentrations (Fig. 1). TheMonod parameters (Eqs. (1) and (2)) were as follows: Ks =0.0016 kg m−3, Y = 0.0563, qmax = 4.17 days−1, and X0 =2.57 × 10−5 kg m−3. As expected, the estimated size of the totalinitial degrader biomass (X0 = 2.57 × 10−5 kg m−3) was larger thanthe size of the cultivable initial degrader biomass (1.5 × 10−6 kg m−3).

3.2. Estimation of initial degrader biomass — Dataset 2

The 420MCPA-mineralization curves were converted to normalizeddegradation curves (Fig. 2). MCPA-degradation followed Monod-kinetics in all of the A-horizon-samples (R2

Average = 0.97; range =0.93–0.99), in 43% of the B1-horizon-samples (R2

Average = 0.94;range = 0.84–0.99), in 1% of the B2-horizon-samples (R2 = 0.96),and in 7% of the B3-horizon-samples (R2

Average = 0.93; range =0.82–0.99) (Figure S2). With the estimates of Ks, Y, and qmax fromDataset 1, it was possible to estimate the initial degrader biomassX0 for each of the samples that exhibited Monod-kinetics in Dataset2. The X0-values were high and relatively homogenous for the A-horizon, whereas they spanned orders of magnitude for the B1-horizon and they were low for the B3 horizons (Figure S3). Mostsigmoid curves could be fitted with high precision (Figure S2), butthree curves in the B1-horizon and one in the B3-horizon showed in-complete degradation and poor fits. These curves were thereforefitted manually by trial and error with various initial X0 values.

MCPA-degradation in the remaining samples (Figure S2) followedeither slow zero-order kinetic (B1: 30%, B2: 21%, B3: 4%, C: 0%) orMCPA was not degraded to any detectable extent (B1: 27%, B2: 77%,B3: 89%, C: 100%). X0 in these samples was set to zero.

The average initial degrader biomassX0, which includes the sam-ples devoid of degraders (X0 = 0), was 1.32 × 10−5 kg m−3 in theA-horizon, 8.55 × 10−7 kg m−3 in the B1-horizon, 8.35 × 10−9 kg m−3

in the B2-horizon, and 1.15 × 10−9 kg m−3 in the B3-horizon. Thenumbers corresponded roughly to 3 × 104 cells g−1 (A-horizon),2 × 103 cells g−1 (B1-horizon), 2 × 101 cells g−1 (B2-horizon),and 2 × 100 cells g−1 (B3-horizon). Considering the inherent uncer-tainties of the MPN-method, this is not far from the MPN-numbersreported in Dataset 2 (Badawi et al., 2013b).

The actual distribution of degradation potentials within the five ho-rizons showed very little or no spatial autocorrelation (Badawi et al.,2013b). A random distribution of X0-values within each model layer istherefore close to the real distributions.

3.3. Simulation of MCPA-leaching — Dataset 2

The results of the simulations are presented in a condensed form inFig. 4, and more visually as animations in the Supporting information(S4 through S9). Scenario 1–3 incorporated simple matrix flow and in-creasingly complex degradation. Without MCPA-degradation, MCPAleached bymatrix flow to the bottom of the A-horizon at concentrationsof 0.001–0.01 μg L−1 (Fig. 4, Scenario 1), which is well below the regu-latory threshold of 0.1 μg L−1. Biodegradation was introduced inScenarios 2 and 3. Both scenarios had identical initial total biomasses,but with different spatial distributions. In both cases the biodegradationprevented MCPA from leaching by matrix flow to the bottom of theA-horizon (Fig. 4, Scenarios 2 and 3).

Preferential flow was introduced in Scenarios 4, 5, and 6 with ahighly-permeablewormhole in the center of the low-permeablematrix.In the absence of degradation (Fig. 4, Scenario 4), MCPA leached fromthe bottom of the A-, B1- and B2-horizons with average concentrationsof 0.1 to 10 μg L−1 in the bulk matrix. The matrix concentration brieflyexceeded the 0.1 μg L−1 limit at one meter depth (horizon C). TheMCPA in the wormhole was following the bulk concentration, but at a10-fold higher level with maximum concentration of 2 μg L−1. Whenbiodegradation was introduced (Fig. 4, Scenarios 5 and 6), leachingwasminimized so thatMCPAwas undetectable belowhorizon B1. How-ever, in contrast to the simplematrix flow scenario, the degraders couldnot prevent MCPA from building up in horizons A and B1. The distribu-tion of the degrader biomasswithin each layer including the hotspot lin-ing in the wormhole had major effect on leaching to the bottom ofhorizons A and B1. The preferential localization of the biomass alongthe wormhole resulted in a two orders of magnitude reduction in theaverage MCPA concentration in both horizons.

Fig. 4. Temporal evolution of simulated average MCPA-leaching from the bottom of A-horizon, B1-horizon, B2-horizon, and soil profile (at one meter depth). BULK MATRIX shows theaverage MCPA-concentrations, whereas WORMHOLE shows the MCPA-concentration within the wormhole.

95A.E. Rosenbom et al. / Science of the Total Environment 472 (2014) 90–98

Overall, the simulation without MCPA degraders showed that pref-erential flow in a wormhole can lead to high MCPA-concentrations at1 meter depth, and that this transport can be counteracted by a highdegradation potential in the A-horizon. The specific distribution of theinitial degrader biomass within each layer is less important because allsamples from the A-horizon had high degrader populations. Theworm-hole as a microbial degradation hotspot did however significantly im-pact the leaching.

3.4. Field-scale test of MCPA-leaching — Dataset 3

Leaching ofMCPAwas not detected after thefirst application neitherat Silstrup, nor at Faardrup. The second application at Faardrup, whichwas followed bymore precipitation compared to the other two applica-tions and theprecipitation used in thismodel study, resulted in only onedetection of 0.28 μg L−1 43 days after application (Fig. 5). MCPA wasnot detected in groundwater samples collected at any of the two sites.

Fig. 5.Measured precipitation andMCPA-concentration indrainagewater sampled time- andflow-proportional from twoDanish loamyfields: Silstrup and Faardrup.Water sampleswith-out detections of MCPA are shown with the value of 0.01 μg L−1 (the detection limit). Arrows indicate application of MCPA.

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4. Discussion

The fate of pesticide in agricultural loamy soils is complex due topreferential flow and transport via discontinuities like wormholes andfractures (Rosenbom et al., 2008), which may represent biological‘hot-spots’ in soils (Jarvis, 2007). Preferential flow may, under certainclimatic conditions, facilitate rapid leaching of pesticides to greatdepth, via a network of discontinuities, ultimately reaching drinkingwater aquifers and affecting their water quality. The ability to predictpesticide leaching through these types of soils has received considerableattention over the last decades in order to authorize andmanage the useof pesticides. Few of the environmental fate models recommended foruse under the EU pesticide authorization procedure (e.g. MACRO,Larsbo et al. (2005)) take these discontinuities into account. Themodelsare one-dimensional and therefore cannot include spatial heterogene-ity. The impact of excluding the three-dimensional degradation poten-tial on pesticide leaching prediction has not hitherto been evaluated.This modeling study shows that:

• In this loamy soil, in the absence of preferential flow, MCPA-leachingto 1 meter depth is negligible within the simulated timeframe. Yearswill pass before MCPA gets into the drinking water aquifers evenif no degradation takes place (Scenario 1, Fig. 4). With degradation(Scenarios 2 and 3), a negligible amount of MCPA will pass theplow layer, which presents a rather homogenous high degradationpotential. As a consequence, the leaching predictions are similarwhether the natural spatial heterogeneity of degrading biomassor their spatial averages are used in the simulations. Hence, itseems unnecessary, at least for MCPA in this type of scenario, toconsider three-dimensional degradation potentials in the recommend-ed modeling practice of EU pesticide authorization procedure. Currentmodeling practice on the other hand (FOCUS, 2005) incorporates a dis-sipation factor (DT50-value— half time for degradation) often obtainedfrom batch experiments on disturbed homogenized soil and often esti-mated applying a first order degradation description. If such estimationis done for sigmoidmineralization-data, the initial simulated removal ofa compound by degradation can be overestimated and hereby lead toan underestimated leaching.

• If wormholes are present in loamy soils, MCPA will leach much morerapidly towards the groundwater. With no degradation, concentrationsin the wormhole at 1 meter depth will exceed the EU-drinking waterstandard of 0.1 μg L−1 already after 180 days (Scenario 4). This thresh-old will be exceeded in the bulk matrix 70 days later. Having naturalheterogeneous distribution of degradation potential in the soil, MCPA-leaching will be minimized and thereby limited to the horizons A andB1 within the 243 days simulation period (Scenario 5). The MCPA-

mass passing these two horizons can however pose a threat to thegroundwater aquifers in the long-term, since retardation will be lessin the deeper horizons. Introducing biological hot-spots on the wallsof the wormhole impacts MCPA-leaching through horizons A and B1.Average concentration in the bulk matrix was reduced by up to twoorders of magnitude. Areas covered with loamy soils are thereforevulnerable to pesticide leaching if the preferential flow and transportthrough the wormhole is more rapid than estimated (Nielsen, 2010;Rosenbom et al., 2009), so that degrader bacteria in the wormhole donot have sufficient time to respond to the pulse of MCPA.

The outcome of our study is, however, dependent on (i) the validityof the transformation of mineralization curves into degradation curves,(ii) the validity of the kinetics parameters, (iii) the date of MCPA-application in relation to the net precipitation, (iv) the conceptual de-scription of the loamy soil with or without wormhole, and (v) thechoices made in the description of the three-dimensional distributionof degradation potentials.

i. We used degradation data that was based on mineralization curves.The validity of the transformation from mineralization to degrada-tion was previously confirmed in Dataset 2 (Badawi et al., 2013b)where complete MCPA degradation, when mineralization curvesleveled off, was demonstrated by thin-layer chromatography fortopsoil spiked with 0.1 or 10 mg kg−1 of 14C-MCPA. Complete min-eralization of pesticides is, however, in many cases slower than sim-ple degradation because transient metabolites may accumulate,which would lead to underestimation of MCPA degradation in thesimulations.

ii. It was assumed that theMonod-parameters from anA-horizon sam-ple were representative for all samples at the five depths. This as-sumption seems valid since almost all degradation curves could befitted with a single set of Monod-parameters even though the typeof degrader cells and the nutrient availability etc. may vary betweensamples and with depth. This suggests that the initial density of de-grader cells (X0) was the most important parameter in defining thedegradation potential. The estimated yield from Dataset 1(Y = 0.0563) was much smaller than that suggested by Dataset2 where 40 to 60% of the 14C in MCPA was mineralized to 14CO2

suggesting yields of 0.6 to 0.4. The applied Monod model(Eqs. (1) and (2)) did not consider neither the death rate ofdegrader cells, nor the energy and carbon needed for cell mainte-nance. Also, the simple yield estimates from the 14CO2 plateauswere carbon ratios whereas the fitted yield Y took into accountthat part of the MCPA-molecule is comprised of chlorine, whichdoes not contribute to bacterial growth. The modeled Y-value

97A.E. Rosenbom et al. / Science of the Total Environment 472 (2014) 90–98

therefore represented a net-yield, being low due to these factors.Ignoring die-off of degraders after the MCPA substrate is exhaustedhas no consequence in the present study as the degrading bacteriaare not exposed to MCPA once the plume has been degraded orleached to deeper layers, but would lead to inaccurate results if asecond application of MCPA was included in the simulation.

iii. The upper boundary conditions represent the actual climate for theperiod atwhich Badawi et al. (2013b) collected soil samples. The de-gree of MCPA-transportmay however be higher if heavy rain eventsoccur shortly after application (Nolan et al., 2008).

iv. To conceptually represent awormhole in a hydrogeologicalmodel isa challenge. The physics of the unusually fast preferential transportoccurring in these conduits with the transport being at disequilibri-um with the soil medium is not currently well-defined (Beven andGermann, 2013; Nimmo, 2012; Rosenbom et al., 2008; Rosenbomet al., 2009). The physical description of the wormhole was takenfrom a previous study (Rosenbom et al., 2009), where rapid trans-port of two fluorescent tracers through a similar soil was describednumerically. The appliedmodel therefore allowed rapid preferentialMCPA transport. However, transport of MCPA in a real wormholemay under some circumstances be faster than simulated. Degrada-tion will then have less impact and the risk of leaching will beunderestimated. The inadequacy of the model under such transportconditions may explain why MCPA on few occasions has been de-tected in aquifers underlying loamy soils (Fig. 5).

v. The chosen description of the three dimensional distributions ofdegradation potentials has some inherent limitations. The simula-tion was based on two dimensional data from five depths in a6.5 × 10.5 cm soil column, but the representativeness of thedata is unknown. Furthermore, the samples were not exposedto the in situ changes in saturation, temperature, and geochemi-cal settings when 14C MCPA was mineralized. We also ignoreddegradation in the samples that showed zero-order kinetics be-cause the non-growth zero-order degradation proved very slowcompared to Monod-degradation and therefore added little tothe overall MCPA mineralization in Dataset 2. We cannot ruleout that the zero-order non-growth kinetics in the deeper layerswould in some cases be linked to incomplete MCPA degradationand thus production of metabolites that are not accounted for bythe model.

It was not possible to carry out reliable, leaching tests at the fieldthat was used for Datasets 1 and 2. Instead, we compared the simu-lations to results from two geologically similar fields that havebeen heavily instrumented and previously tested for MCPA leaching.The general absence of MCPA in the drainage water (Fig. 5) con-firmed the results of the simulations that included MCPA degraders.The detection of MCPA above the EU-threshold of 0.1 μg L−1 in onesample indicates that MCPA under rare conditions may by-pass themicrobiologically active layer and leach to 1.1 m, presumably throughlarge macropores.

4.1. Conclusions

This study suggests that microbial centimeter-scale heterogeneity inloamy agricultural soils has little effect on the leaching of easily degrad-able pesticides like MCPA. In assessing a pesticide's fate in the environ-ment it is, however, crucial to take into account the ability of themacropores to facilitate rapid transport through the upper meter andthereby by-pass the degradation in the plow layer and along the wallsof the wormhole.

Acknowledgments

This studywas funded primarily by theVillumKannRasmussen Foun-dation via the Center for Environmental and Agricultural Microbiology

(CREAM) and secondarily by the Geological Survey of Denmark andGreenland via the Danish Pesticide Leaching Assessment Programme.We are grateful to Morten Siwertsen and Anders Ekerot from COMSOLSupport.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.scitotenv.2013.11.009.

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