Journal of Environmental Science and Health, Part B (2012) 47, 495–504Copyright C© Taylor & Francis Group, LLCISSN: 0360-1234 (Print); 1532-4109 (Online)DOI: 10.1080/03601234.2012.665658

Solid beef cattle manure application impacts on soilproperties and 17β-estradiol fate in a clay loam soil

EMMANUELLE CARON1, ANNEMIEKE FARENHORST1, XIYING HAO2 and CLAUDIA SHEEDY2

1Department of Soil Science, University of Manitoba, Winnipeg, Manitoba, Canada2Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada

Livestock manure applied to agricultural land is one of the ways natural steroid estrogens enter soils. To examine the impact oflong-term solid beef cattle (Bos Taurus) manure on soil properties and 17β-estradiol sorption and mineralization, this study utilized asoil that had received beef cattle manure over 35 years. The 17β-estradiol was strongly sorbed and sorption significantly increased (P< 0.05) with increasing soil organic carbon content (SOC) and with an increasing annual rate of beef cattle manure. The 17β-estradiolmineralization half-life was significantly negatively correlated, and the total amount of 17β-estradiol mineralized at 90 days (MAX)was significantly positively correlated with 17β-estradiol sorption. The long-term rate of manure application had no significant effecton MAX, but the addition of fresh beef cattle manure in the laboratory resulted in significantly (P < 0.05) smaller MAX values. Noneof the treatments showed MAX values exceeding one-third of the 17β-estradiol applied.

Keywords: Estrogen, 17β-estradiol, long-term manure application, fate.

Introduction

Natural steroidal estrogens, of which 17β-estradiol is themost potent, are produced and excreted by all vertebrates.The estrogen 17β-estradiol is known to have adverseeffects on fish reproduction when present in surfacewater at part-per-trillion levels.[1] The concentration of17β-estradiol in urine and feces of livestock is species- andgender-dependent.[2,3] Runoff can transport 17β-estradiolin the soil to surface waters.[4,5] Concentrations of 17β-estradiol in runoff from plots which had received manurehave ranged from 0.6 to 2.5 µg L-1, compared with 0.1 µgL-1 in runoff from control soils.[6–8]

Alberta, the most important cattle [Bos taurus] produc-ing province in Canada, had 5.5 million heads on farmrepresenting 40% of the Canadian national total in 2010.[9]

In commercial beef cattle feedlots, 23% of N and 57% of Pdietary inputs end up in manure.[10] Consequently, this ma-nure is applied to soil to increase the fertility of Alberta’scrop land. Alberta’s crop land accounts for around 26% ofthe total area in crop land in Canada.[11] Most of Alberta’scrop land is under semi-arid climatic conditions and henceirrigation is part of some cropping systems. There are only

Address correspondence to Annemieke Farenhorst, Depart-ment of Soil Science, University of Manitoba, 362 Ellis Build-ing, Winnipeg, Manitoba, Canada R3T 2N2; E-mail: farenhor@cc.umanitoba.caReceived May 31, 2011.

limited studies on the fate of 17β-estradiol in Alberta cropland[12,13] and none of these studies have determined thepotential impact of manure applications on modifying soilproperties in association with 17β-estradiol fate parametersin soils.

The sorption of 17β-estradiol has been studied in a widerange of soils without considering the history of manure ap-plications to these soils.[12,14–16] Sorption of 17β-estradiolby soil is strongly influenced by soil organic matter, butsoil texture can also have a measurable effect.[17–19] Sev-eral studies have reported on a positive correlation between17β-estradiol sorption and mineralization in soils and othermedia.[13,14,20]

Stumpe and Marschner[21] found that long-term ma-nure applications (from 35 to 48 years depending on site)increased SOC by 3 to 15 g OC kg-1 in three differentsoils that had SOC ranging from 70 to 350 g OC kg-1.Consequently, the sorption of 17β-estradiol also increasedbut both increases and reductions were observed for 17β-estradiol mineralization. The one-time addition of sheep,swine, poultry or cattle manure on grassland and croplandsoils increased mineralization of 17β-estradiol.[21,22]

Parameters important to estimating the fate of 17β-estradiol in agricultural soils include those describingsorption by soil (e.g., Cs [µg/kg]) and those describingmineralization such as mineralization half-lives in days andthe maximum mineralizable amount [MAX, (% applied)].One objective of this study was to assess the impact of 35annual cattle feedlot manure applications on soil properties

496 Caron et al.

in a clay loam soil as well as on 17β-estradiol sorptionand mineralization parameters. A second objective was toassess the influence of a one-time fresh manure applicationin the laboratory on the rates and amounts of 17β-estradiolmineralization in the soil. This study is part of an on-goinglong-term field plot experiment on a well-drained clayloam soil subjected to both irrigated and non-irrigated(rainfed only) conditions, at Lethbridge, Alberta.[23–25]

Materials and methods

Experimental design

Soil samples (0–15 cm) were collected in October 2008 fromfield plots that had been established in 1973 at the Agri-culture and Agri-Food Canada Research Centre in Leth-bridge, Alberta, Canada. The experimental design includestwo adjacent blocks of land representing irrigated and non-irrigated conditions, respectively. The soil is a well-drainedDark Brown Chernozem Lethbridge clay loam [HaplicKastanozem in the FAO (Food and Agriculture Organi-zation of the United Nations) Soil Classification[25]] whichhad the following surface soil characteristics (0–15 cm) in1973: 37% sand, 39% clay, 14.6 g organic C kg-1 soil, 2.6 gN kg-1 soil, 690 mg total P kg-1 soil and a bulk density of1.22 Mg m-3.[23] Each block was originally part of a split-plot experiment with one of three tillage treatments as themain plot (cultivating, plowing and rototilling) and one offour manure rates as the subplot (annual manure applica-tions rates of 0, 60, 120 and 180 Mg ha-1 for the irrigatedblock, and of 0, 30, 60 and 90 Mg ha-1 for the non-irrigatedblock). However, since 1986, all manure is incorporated bythe same tillage operation (cultivating or disking to 10 cmdepth) because of the absence of significant tillage effectsfrom 1973 to 1986, leaving 24 plots in total (2 blocks X 4 ma-nure rates X 3 replicated plots). These manure applicationrates are respectively once, twice and three times those rec-ommended by the province in 1973 (N-based) for irrigatedand non-irrigated farmland, respectively, and since then,there have been no significant changes to these provincialrecommendations.[26,27]

The manure is always applied following crop harvest ineither September or October. The solid cattle manure con-tained per kg dry manure weight, 159.1 ± 237.9 mg NO3

--N, 1,602.9 ± 1,391.3 mg NH4

+-N, and 2,459.4± 957.6 mgPO4

3--P, 9,794.0 ± 3,540.2 mg Cl−, and 2,614.9±1,279.6 mgSO4, expressed as mean ± st.dev. of samples analyzed be-tween 1973 and 2007. On average, between 1973 and 2007,the solid beef manure applied had an electrical conductivityof 2.37±0.71 S m-1 and a pH of 7.0±0.4. For the irrigatedplots, annual irrigation has ranged from 0 to 432 mm withan annual mean of 160 mm. The water used for irrigationis slightly alkaline (pH of 8.4 between 1999 and 2008) andobtained from an irrigation dugout near the plots which

is filled in May each year with water from the St. Mary’sRiver Irrigation District (St. Mary’s reservoir and river sys-tem). The mean annual precipitation (rain plus snow) is 401mm.[28] Further details were previously presented on thislong-term experimental field study.[23–25, 28–35]

For the current study, 15 soil cores (0–15 cm) were col-lected in a grid pattern from each of 24 plots (7.62 m by15.5 m) after crop harvest but before the 2008 manure ap-plication. Samples were collected using an auger (2.54 cmdiameter) disinfected with ethanol and water between eachplots. The 15 cores for each plot were composited to obtainone bulk sample per plot for a total of 24 bulk samplesand soil was stored at 4◦C for three weeks prior to determi-nation of soil properties and 17β-estradiol mineralization,while a portion of soil was frozen for eight months and thenthawed, air-dried and sieved (<2 mm) prior to analysis of17β-estradiol sorption.

Soil properties

Soil texture was determined using the hydrometermethod.[36] Soil pH was determined using 5 g of soil and10 mL of 0.01M CaCl2.[37] SOC was determined after acid-ification using 6M HCl.[38] Total C (TC), SOC and total N(TN) were determined on a CN analyzer (2100 Soil, CarloErba instruments, Milan, Italy). PO4-P was determined us-ing the Olsen extraction with 2.5 g of soil.[39] NO3

−-N andNH4

+-N content in 5 g of soil were extracted using 25 mLof 2 M KCl.[40] PO4-P, NO3

−-N and NH4+-N concentra-

tions were determined using a continuous flow analyzer(Bran Luebbe autoanalyzer 3, Mequon, WI, USA). Micro-bial activity was determined by the fluorescein diacetatehydrolysis assay (FDA)[41] on soil samples (2 g dry weight)wetted to 80% field capacity and incubated in sterile Falcontubes at 20◦C for 7 days.

17β-estradiol sorption

Sorption of 17β-estradiol by soil was determined by batch-equilibrium experiments. Analytical grade 17β-estradiol(purity of 99%, from Sigma-Aldrich Chemical Company,St. Louis, MO) and 17β-estradiol [4–14C] (99% radiochem-ical purity, specific activity 1.67×1012–2.26×1012 Becq.mmol-1, from American Radiolabeled Chemicals, St. Louis,MO) (Fig. 1) were mixed in autoclaved water (30 minutesat 121◦C) to prepare the stock solution. The stock solu-tion was kept at 4◦C in glass amber bottles in the dark andwas used within one or two days of preparation. In orderto limit the potential impact of ethanol on 17β-estradiolsorption by soil, the percentage of ethanol contained inthe stock solution was less than 0.005%. Air-dried soil (5g) was weighed in glass tubes (in duplicate). Tubes werecapped with aluminum foil and autoclaved (30 minutes at121◦C). Sterilization of soils by autoclaving was necessarysince estrogen biodegradation during the sorption experi-ment would have lead to an underestimation of sorption.[42]

Long term manure impacts on 17β-estradiol fate 497

OHCH3

C14

OH

Fig. 1. Structure of 17β-estradiol [4–14C].

Previous studies have shown that autoclaving soil will notimpact its properties such as SOC, pH, cation exchange ca-pacity and soil surface area[42,43] nor the Kd values of somechlorinated hydrocarbons[43] and pesticides.[44]

Stock solution (10 mL with a specific activity of 16.67Becq. mL−1) was added to soil and the tubes were closedwith aluminum foil between the cap and the tube. The es-trogen concentration in soil was equivalent to 50 µg kg-1

of soil, assuming 100% sorption. This concentration is thatused by Caron et al.[12,13] and is within the mid-range of theconcentrations used by Casey et al.[14] The concentrationis also within the linear range of the 17β-estradiol sorp-tion isotherm for 17β-estradiol as determined by Casey etal.[45] and Kozarek et al.[46] Soil slurries were rotated at 5◦Cin the dark for 1h, 2h, 16h, 24h and 40h and then cen-trifuged for 30 min. at 7,000 rpm. Supernatant (1 mL) induplicates was added to 7 mL scintillation vials containing5 mL of scintillation cocktail (30% Scintisafe Fisher Scien-tific, Fairlawn, NJ) and the amount of radioactivity in thestock solutions and supernatant samples was quantifiedby Liquid Scintillation Counting (LSC) (LS 7500 Beck-man Instruments, Fullerton, CA). Concentration sorbed(Cs) was used in the statistical analysis and was obtainedby subtracting the concentration in the supernatant solu-tion from that initially applied. Cs was used because thisparameter is a better measure for evaluating equilibriumconditions, relative to using soil sorption coefficients suchas Kd that is calculated by Cs/Ce, where Ce is the concen-tration of the chemical in the supernatant at equilibrium.Controls consisting of glass tubes with solution only werealso included and demonstrated no loss of radioactivityduring the experiment, suggesting that there was no lossof 17β-estradiol due to sorption to glass. For the sorptiondata expressed in Cs, the coefficient of variation (CV) wason average 0.3% between laboratory replicates as well asbetween field replicates.

17β-estradiol mineralization

Mineralization of 17β-estradiol in soil was determined inmicrocosm experiments consisting of a sealed 1 L glassMason jar. Each soil microcosm consisted of a glass jarcontaining 25 g of soil or soil+manure (oven-dried weightbasis) (sieved to 2mm), a glass test tube with 3 mL of acid-ified water (pH∼3) to preserve humidity and a scintillation

vial with 5 mL of 0.5 M NaOH to trap evolved 14CO2. Ad-ditional microcosms were set-up as controls and contained25 g of autoclaved silica sand (triplicates) and 25 g of non-autoclaved silica sand (triplicates). Prior to the onset of theexperiment, soil moisture content was determined gravi-metrically. Soils were then brought to 80% of their fieldcapacity minus the 1 mL required for 17β-estradiol spik-ing (see below). Field capacity was measured for each soilusing the container method in which a mass of soil is sat-urated with water then drained under gravitational forcesand oven-dried to determine the mass of water left in thesoil.

In the first experiment, soil samples obtained from the24 experimental plots were incubated in triplicates in orderto assess whether the impact of 35 annual cattle feedlot ma-nure applications on soil properties had been large enoughto modify 17β-estradiol mineralization half-lives and MAXvalues in soil. A second mineralization experiment was runsimultaneously to determine whether a one-time fresh ma-nure application in the laboratory could also alter the ratesand amounts of 17β-estradiol mineralization parameters inthe soil. This second experiment consisted of adding 0.66g (dry weight) of solid cattle manure (moisture content of0.3435 g g-1) to 25 g of soil from the 0 and 60 Mg ha-1 fieldtreatments of both irrigated and non-irrigated plots. For0.89 g of manure (wet weight) at 0.3435 g g-1 moisture thevolume of water that was added is 0.31 mL. The amountof water added to the soil microcosms that did not receivemanure in the laboratory minus 0.31 mL was added to themicrocosms that received fresh manure in the laboratorybefore pre-incubation.

This one-time manure application was equivalent to awet weight manure application rate of 60 Mg ha-1 in thefield, assuming that this manure is incorporated into thesoil to 15 cm depth with a bulk density of 1,000 kg m−3 forboth the 0 and 60 Mg ha-1 soils. The bulk density used wasbased on field measurements using the ring method[47] andwas the same for each plot. The dry solid cattle manurecontained 250,100 mg C kg-1, 21,590 mg N kg-1, 30 mgof NO3

--N kg-1, 520 mg of NH4+-N kg-1 and 11,280 mg

kg-1 PO4-P. The soil properties for TN, NO3--N, NH4

+-N,FDA, Olsen P, TC, Inorganic C, SOC were determined formanure-amended and control soils, as described above.

Mason jars were incubated in the dark at 20◦C for apre-incubation period of seven days. Soil or silica sandwere then spiked with a 1 mL solution containing 1,667Becq. mL-1 17β-estradiol [4–14C] and analytical grade 17β-estradiol to yield a concentration of 50 µg kg-1 17β-estradiol in soil. This solution was deposited drop-wise ina grid pattern and then thoroughly mixed with the soil inorder to ensure a homogeneous distribution. Mason jarswere incubated in the dark at 20◦C with 0.5 M NaOHtraps being removed and replaced at 1, 4, 8, 10, 17, 35,49, 65, 77 and 90 days. Microcosms were aerated weeklyin order to avoid anaerobic conditions and moisture was

498 Caron et al.

maintained gravimetrically weekly throughout the exper-iment. The amount of radioactivity in the stock solutionand traps was determined by adding 8 mL of scintillationcocktail (30% Scintisafe scintillation cocktail; Fisher Sci-entific, Fairlawn, NJ) to vials and counting by LSC.

The cumulative amount of 14CO2 was fitted to the rise tothe max equation in Sigma Plot, 2000 (SPSS Inc, Chicago,Il.) to obtain mineralization rate (k) and the% of applied17β-estradiol mineralized at time infinity (MAX). The riseto the max equation is defined as Mt = MAX (1-e-kt), whereMt = % of applied 17β-estradiol mineralized at time t witht expressed in days. Mineralization half-lives were calcu-lated by ln (2) divided by k. For the samples that did notreceive manure in the laboratory, the average CV of miner-alization half-lives was 17.2 and 16.1% and that of MAXwas 8.4 and 8.1% for the laboratory and field replicates,respectively. For the samples that received manure in thelaboratory, the average CV of mineralization half-lives was10.7% and 12.5% and that of MAX was 8.9% and 7.8% forthe laboratory and field replicates, respectively.

Statistical analyses

Data of irrigated and non-irrigated plots were combinedto determine the Pearson correlation coefficient (P < 0.05)among the following parameters: Cs at each rotation time,mineralization half-lives, MAX and the soil properties TN,NO3

--N, NH4+-N, FDA, Olsen P, TC, SOC, Inorganic C,%

sand,% clay and% silt (1989–2002, JMP version 5.0, SASInstitute). Correlations were conducted on untransformeddata due to the robust nature of these analyses.[48] All cor-relations were checked visually to confirm that the correla-

tions were the result of general trends rather than extremeoutliners.

For irrigated and non-irrigated data separately, the ef-fects of the annual solid beef manure rate and the rotationtime on Cs was determined using a two-way ANOVA fol-lowed by the Tukey-Kramer HSD multiple comparison testwith α < 0.05 (SigmaStat for Windows version 3.1 SystatSoftware Inc.). Data respected normality (Shapiro-Wilksstatistic ≥ 0.90) and equality of variance. Furthermore,for irrigated and non-irrigated separately, the effect of theannual solid beef manure application rate on soil prop-erties (TN, NO3

--N, NH4+-N, FDA, Olsen P, TC, Inor-

ganic C, SOC,% sand,% clay and% silt) and 17β-estradiolmineralization parameters (mineralization half-lives andMAX) was determined using a one-way ANOVA followedby the Tukey-Kramer HSD multiple comparison test withα < 0.05. Normality was tested using the entire dataset(irrigated+non-irrigated) in order to increase n. All pa-rameters and soil properties respected normality except forOlsen P and% sand which were log-transformed. Equalityof variance was tested on irrigated and non-irrigated dataseparately and data respected this condition except in caseof log-transformed PO4 data for irrigated.

A paired t-test with α < 0.05 (SigmaStat for Windowsversion 3.1 Systat Software Inc.) was used to determinethe impact of a one-time fresh addition of solid beef ma-nure on soil properties (TN, NO3

--N, NH4+-N, FDA, Olsen

P, TC, Inorganic C, SOC) and mineralization parame-ters (mineralization half-lives and MAX). FDA, TN, TC,SOC, mineralization half-lives and MAX respected nor-mality (tested on irrigated and non-irrigated combined), aswell as Olsen P and Inorganic C after log-transformation.

Table 1. Pearson correlation coefficients (P < 0.05, n = 24) among 17β-estradiol sorption and mineralization parameters and soilproperties.

CS1 CS2 CS16 CS24 CS40 MHL MAX FDA Ols.P TotalN NO3– NH4

+ TotalC SOC SIC sand clay silt

CS1 1CS2 0.89∗∗∗ 1CS16 0.98∗∗∗ 0.88∗∗∗ 1CS24 0.59∗∗ 0.51∗ 0.62∗∗ 1CS40 0.88∗∗∗ 0.88∗∗∗ 0.88∗∗∗ 0.70∗∗∗ 1MHL −0.68∗∗∗ −0.77∗∗∗ −0.69∗∗∗ −0.47∗ −0.66∗∗∗ 1MAX 0.51∗ 0.59∗∗ 0.51∗ 0.40∗ 0.45∗ −0.77∗∗∗ 1FDA 0.49∗ 0.48∗ 0.53∗∗ — 0.48∗ −0.54∗∗∗ 0.41∗ 1Ols.P 0.44∗ — 0.41∗ — 0.55∗∗ — — — 1Total N 0.73∗∗∗ 0.65∗∗∗ 0.75∗∗∗ 0.66∗∗∗ 0.74∗∗∗ −0.46∗ 0.50∗ — 0.45∗ 1NO3

— 0.84∗∗∗ 0.75∗∗∗ 0.86∗∗∗ 0.71∗∗∗ 0.84∗∗∗ −0.52∗∗ 0.48∗ — — 0.90∗∗∗ 1NH4

+ 0.80∗∗∗ 0.68∗∗∗ 0.79∗∗∗ 0.56∗∗∗ 0.77∗∗∗ −0.50∗ 0.48∗ — 0.46∗ 0.83∗∗∗ 0.91∗∗∗ 1Total C 0.74∗∗∗ 0.66∗∗∗ 0.75∗∗∗ 0.64∗∗∗ 0.74∗∗∗ −0.46∗ 0.50∗ — 0.44∗ 1∗∗∗ 0.91∗∗∗ 0.84∗∗∗ 1SOC 0.74∗∗∗ 0.65∗∗∗ 0.71∗∗∗ 0.66∗∗∗ 0.75∗∗∗ −0.47∗ 0.50∗ — 0.47∗ 1∗∗∗ 0.91∗∗∗ 0.84∗∗∗ 1∗∗∗ 1SIC — — — — — — — — — — — — — — 1sand — — — −0.42∗ — — — — — — −0.48∗ 1clay — — — — — — — — — — — — — — — −0.80∗∗∗ 1silt — — — — — — — 0.45∗ — — — — — — — — 0.56∗∗ 1

Note: ∗, ∗∗, and ∗∗∗ refer to correlations being significant at the 0.05, 0.01 and 0.001 level, respectively. A dash (-) indicates the correlation is notsignificant at the 0.05 level. Abbreviations are Cs = concentration sorbed at each rotation time, MHL = mineralization half-life, MAX = maximummineralization, FDA = microbial activity, Ols. P = Olsen phosphorus, SOC = soil organic content, and SIC = soil inorganic carbon content.

Long term manure impacts on 17β-estradiol fate 499

Many transformations were considered for NO3--N and

NH4+-N with the optimum transformation being a square

transformation (Shapiro-Wilks statistic of 0.87 for NO3--

N and 0.82 for NH4+-N) and used in the t-test. The t-

tests were done on irrigated and non-irrigated data com-bined, but also on data from the irrigated and non-irrigatedseparately.

Results

In irrigated plots, CS significantly increased with rate ofannual cattle manure application in the order of 0 < (60 =120) < 180 Mg ha-1 (Fig. 2a). Cs was significantly smallerat 1h rotation time but there were no significant differencesacross the other rotation times indicating that equilibriumconditions were met at 2h (Fig. 2a). In non-irrigated plots,the interaction between the annual solid beef manure ap-plication rate and the rotation time was significant (Fig.2B). Within each rotation time, non-irrigated soils that hadreceived annual applications of solid beef manure alwaysdemonstrated significantly greater Cs values than controlsoils, except for the 24h rotation time that demonstratedstatistically similar Cs values across annual solid manureapplication rate treatments (Fig. 2b). Rotation time had noeffect on Cs values in case of an annual solid beef manurerate of 60 kg ha-1, but at all other manure treatments, the1h rotation time had significantly lesser Cs values than the40h rotation time (Fig. 2b).

Cs at each rotation time was significantly positively cor-related to soil organic carbon content (SOC) (Table 1).SOC, total C (TC), NO3

--N, NH4+-N, total N (TN), and

Olsen P were all significantly positively correlated with eachother, except for NO3

--N and Olsen P (Table 1). Mineral-ization half-life was significantly negatively correlated withSOC, Cs at each rotation time and FDA, whereas MAXwas significantly positively correlated with SOC, Cs at eachrotation time and FDA (Table 1). FDA had no significantcorrelation with SOC nor with any of the soil nutrient con-centration parameters (Table 1). Mineralization half-livesand MAX were significantly negatively correlated (Table 1).

In irrigated plots, SOC, TC and TN significantly in-creased in the order of 0 = 60 < 120 = 180 Mg ha-1,and NO3

--N and NH4+-N, concentrations significantly in-

creased with rate of cattle manure application in the orderof 0 < 60 < 120 = 180 Mg ha-1 (Table 2). The annualsolid manure application rate had no significant impact onany other soil property or MAX (Table 2). In non-irrigatedplots, NO3

--N, significantly increased in the order of 0 <

30 < 60 = 90 Mg ha-1, and FDA in the order of 0 ≤ 30= 60 < 90 Mg ha-1, but no other soil property nor MAXwas influenced by the rate of manure application (Table 2).For both irrigated and non-irrigated, mineralization half-lives were numerically shorter in soils with a history of ma-nure applications, relative to plots free of manure (Table 2).Mineralization half-lives were significantly shorter in the

47

48

49

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0 60 120 180 1 2 16 24 40

)h( emiT)1-ah gM( etaR

a

b

Main FactorsRate (Mg ha-1)

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(µg

kg-1

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a

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c

b bb

Time(h)

45

46

47

48

49

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0x1h

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Interactions

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(µg

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Fig. 2. Two-Way ANOVA results for the concentration of 17β-estradiol sorbed by soil (Cs) obtained from the a) irrigated and b)non-irrigated plots. Main factors are rotation time and manurerate and groups with the same letter are not statistically different.Error bars represent standard error. Note: Figures 2a and 2b aredifferent because there is a significant interaction between themain factors in case of the non-irrigated block (Fig. 2b), but notin case of the irrigated block (Fig. 2a).

60 Mg ha-1 manure plot for irrigated plots and in the 90Mg ha-1 manure plot for non-irrigated plots, relative to thecontrol soils, or soils that had received a greater rate of an-nual solid beef manure applications (Table 2). Regardlessof the manure treatment, irrigated soils demonstrated typ-ically lesser mineralization half-lives and typically largerMAX values than non-irrigated soils (Table 2).

The addition of fresh manure in the laboratory signifi-cantly increased NH4

−-N and significantly decreased MAXin both irrigated and non-irrigated plots (Table 3). In irri-gated plots, the addition of fresh manure in the laboratoryalso had a significant effect on some other soil propertiesand on increasing mineralization half-lives (Table 3).

Tab

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Mea

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(mg

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(mg

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2.4

(12.

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11.4

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3.1

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30.2

(6.3

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21.8

(17.

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8.4

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24.1

(15.

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29.3

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58.0

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51.2

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24.0

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46.9

(10.

2)A

29.2

(5.5

)A

120

2.5

(25.

4)A

B22

.32

(1.2

)A

3.5

(48.

6)A

509.

3(4

9.7)

A10

.0(2

3.0)

B11

3.9

(16.

5)C

6.3

(6.3

)C

97.7

(23.

5)B

91.8

(19.

8)B

5.9

(81.

4)A

20.6

(3.4

)A

48.6

(7.0

)A

30.8

(9.0

)A

180

3.5

(19.

6)B

25.4

(8.1

)A

3.2

(18.

8)A

425.

0(7

.6)

A11

.1(8

5.6)

B13

1.0

(5.4

)C

6.7

(7.5

)C

110.

6(8

.6)

B10

0.6

(9.4

)B

10.0

(15.

0)A

20.2

(5.0

)A

50.6

(9.5

)A

29.3

(13.

0)A

Non

-irr

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ed

08.

1(7

.3)

B16

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A1.

0(1

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A67

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7(4

0.7)

A9.

6(1

4.6)

A3.

2(2

8.1)

A31

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9.3)

A24

.1(4

3.6)

A7.

0(4

2.9)

A22

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.1)

A49

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A28

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A30

6.4

(32.

1)B

16.1

(11.

4)A

2.0

(25.

0)A

B55

4.8

(112

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9(3

6.7)

A35

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A51

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A44

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7.5)

A6.

5(2

8.6)

A25

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1.7)

A45

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A28

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A60

5.6

(9.8

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16.8

(6.0

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2.8

(10.

7)B

358.

3(5

0.6)

A5.

6(8

.9)

A64

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C4.

1(1

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A58

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A50

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A8.

0(2

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A22

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A49

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A49

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A90

3.6

(10.

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10.6

(16.

8)A

3.8

(15.

8)C

324.

0(4

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1(4

8.1)

A78

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C4.

6(1

0.9)

A52

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A46

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A22

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A47

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500

Long term manure impacts on 17β-estradiol fate 501

Table 3. Impact of manure application in the laboratory on 17β-estradiol mineralization parameters and soil properties. Means (andcoefficient of variation,%) are presented with numbers in bold indicating a significant impact of the fresh manure on the mineralizationparameter or soil property.

All data(irrigated+non-irrigated) Irrigated data only Non-irrigated data only

Parameter No fresh manure Fresh No fresh manure Fresh No fresh manure Fresh

Mineralization half-life (days) 4.8 (53.3) 5.5 (38.4) 2.7 (48.9) 3.6 (18.9) 6.9 (21.6) 7.0 (25.2)Maximum mineralization (%) 19.8 (18.5) 15.6 (17.5) 23.0 (10.1) 17.3 (17.1) 16.7 (5.5) 14.3 (11.6)Total N (g N kg−1) 4.1 (42.9) 4.5 (43.2) 4.0 (47.5) 4.8 (44.2) 4.1 (42.4) 4.2 (45.5)NH4

+-N (mg kg−1) 3.5 (22.8) 52.9 (19.4) 3.9 (24.1) 53.5 (20.5) 3.6 (23.0) 52.3 (20.1)NO3-N (mg kg−1) 33.1 (78.2) 53.6 (47.1) 34.7 (81.5) 54.0 (37.9) 37.0 (82.2) 53.3 (58.9)Total C (g Ckg−1) 44.6 (36.0) 49.9 (38.7) 44.1 (38.7) 53.3 (41.0) 45.0 (36.9) 46.7 (38.3)Soil Organic C (g OC kg−1) 37.0 (43.7) 40.6 (44.1) 36.5 (47.9) 43.2 (44.9) 37.5 (43.7) 38.0 (46.7)

Bold type: Significant impact on fresh manure.

Discussion

Previous studies utilizing the same experimental plots re-ported that TN, NO3

−-N, and SOC significantly increasedwith increasing annual solid beef manure application ratesto soil (Table 4). In agreement with what Stumpe andMarschner[21] reported, 17β-estradiol sorption significantlyincreased with increasing manure application rates be-cause of associated increases in SOC. Although SOC, 17β-estradiol sorption and 17β-estradiol mineralization weresignificantly positively correlated with each other, therewas no consistent impact of manure application on 17β-estradiol mineralization in soil, which was also the conclu-sion of Stumpe and Marschner.[21]

A range of other studies have reported that both17β-estradiol sorption and mineralization is positivelycorrelated to SOC.[12–14,18–20] The clay loam soil used in thisstudy had a greater SOC and finer texture than the soilsused in other laboratory studies on 17β-estradiol miner-alization. Concurrently, the clay loam soil demonstratedoften greater MAX values, ranging from 12.0 (includingsoils that received fresh manure) to 27.8% of that initiallyapplied, than the soils used in other studies which reportedMAX values ranging from 5 to 19.2%.[13,49–51] Since MAXis below one-third of that initially applied in this and otherlaboratory studies, it appears that 17β-estradiol and itsmetabolites are relatively persistent in the soil solution, inthe soil-bound form and/or fixed into microbial cells.

As observed in other studies,[49,52] the mineralization of17β-estradiol in soil was biologically mediated since theautoclaved silica sand and silica controls showed only 0.8%and 2.1% total mineralization. Soils that had never receivedmanure possessed the intrinsic capacity to mineralize es-trogens because microorganisms involved in 17β-estradioldegradation are commonly found in soils and other matri-ces.[53–55] Breakdown of 17β-estradiol in soil microorgan-isms is thought to occur through co-metabolic means.[50]

MAX was significantly reduced in soils that receiveda fresh application of manure in the laboratory. In other

studies, less than 6% of the applied 17β-estradiol was min-eralized in breeder litter after 17 weeks[56] also suggestingthat fresh manure is a poor media for 17β-estradiol miner-alization. In other studies, when 17β-estradiol was appliedto soils using urine as the solvent, 17β-estradiol mineraliza-tion in soils was less than when 17β-estradiol was appliedto soils using distilled water.[22] Hence, N containing con-stituents in manure may have contributed to the observeddecreases in MAX. Saison et al.[57] suggested that amend-ments (in this case grape skins and winery wastewaters) caninduce physicochemical changes in soils and thereby influ-ence the structure and functions of soil microbial commu-nities for some time after which the state of microbiologicalsystem returns. Perhaps the physicochemical changes thatoccurred upon the application of manure (e.g., significantincreases in NH4

+-N, NO3−-N TC and SOC were observed

in some cases) caused changes to the soil microbial com-munities and MAX was reduced because of correspondingchanges in the structure and function of soil microbial com-munities. However, the above findings and interpretationsare in contrast with those of Stumpe and Marschner[21]

who measured a 10- to 160-fold increase in mineralizationwhen manure, biosolids or wastewater amendments werefreshly applied to sand and 15 different soils from Israeland Germany. Although 17β-estradiol mineralization wasreduced by the fresh manure application to the clay loamsoil used in the current study, the reduction was short-lived because plots that had received 35 annual manureapplications in the field demonstrated significantly greatermineralization half-lives than control plots (no manure).

Regardless of manure treatment, long-term irrigationresults in soil conditions more conducive for 17β-estradiolmineralization because mineralization half-lives weretypically smaller and total amount of 17β-estradiol miner-alized at 90 days (MAX) were typically greater in irrigatedthan non-irrigated plots. Irrigation quality has beenshown to impact 17β-estradiol mineralization becauseStumpe and Marschner[50] reported that 17β-estradiolmineralization was significantly larger in soils that had

502 Caron et al.

Table 4. Comparison of the impact of manure rates on measured soil properties (0–15 cm) in this study with previous studies on thesame experimental plot.

Year Significant Not significant Reference

Irrigated

2008(this study) Total N, NO3−-N, NH4

+-N, total C andSOC

FDA, PO4, Inorganic C, texture –

1973 to1983 SOC, bulk density – Sommerfelt and Chang [23]

1973 to 1983 SOC, total N – Sommerfelt et al.[29]

1984 SOC, pH, electrical conductivity (EC),sodium adsorption ratio (SAR), total N,NO3

−-N,total P, PO43--P, Cl, HCO3,

Na, Ca+Mg, Zn

NH4+, SO4, Cu Chang et al.[24]

1973 to 1991 NO3−-N – Chang and Entz[30]

1973 to 1992 NO3−-N, organic N – Chang and Janzen[31]

1973 to 1991 % sand, SOC, total N, CEC Inorganic C Gao and Chang[32]

1973 to 1998 SOC, total N, NO3−-N NH4

+ Hao et al.[28]

1973 to 1998 Total P, soil test P, water-soluble P Aggregate size distribution Hao et al.[35]

1973 to 1998 – Total P and soil test P Chang et al.[25]

Non-irrigated

2008(this study) NO3−-N,FDA PO4-P, total N, NH4

+-N, total C SOC,Inorganic C, texture

–

1973 to1983 organic matter, bulk density – Sommerfelt and Chang[23]

1973 to 1983 SOC, total N – Sommerfelt et al.[29]

1984 SOC, pH, EC, SAR, total N, NO3−-N,

total P, PO43--P, Cl, SO4, HCO3, Na,

Ca+Mg, Zn

NH4+, Cu Chang et al.[24]

1973 to 1991 NO3−-N – Chang and Entz[30]

1973 to 1992 NO3--N, organic N Chang and Janzen[31]

1973 to 1991 % sand, SOC, total N, CEC Inorganic C Gao and Chang[32]

1973 to 1998 SOC, total N, NO3--N NH4+-N Hao et al.[28]

1973 to 1998 Total P, soil test P, water-soluble P Aggregate size distribution Hao et al.[35]

1973 to 1998 – Total P and soil test P Chang et al.[25]

Note: Abbreviations are SOC = soil organic carbon content, FDA = microbial activity, CEC = cation exchange capacity.

received long-term freshwater irrigation than wastewaterirrigation.

Conclusion

In surface soil samples collected from field plots with ahistory of 35 years of solid beef manure applications, 17β-estradiol sorption by soil significantly increased with in-creasing annual manure application rate, but regardless ofthe manure treatment between 93 to 100% of the applied17β-estradiol was sorbed by soil. Although 17β-estradiolsorption and total amount of 17β-estradiol mineralized at90 days (MAX) were significantly positively correlated, theannual manure application rate had no statistically signifi-cant impact on MAX, while the addition of fresh manureto soils in the laboratory reduced MAX in soils. The reduc-tion of MAX upon the application of fresh manure requiresfurther studies on the mechanisms causing this reduction.

Regardless of manure treatment, long-term irrigation re-sulted in soil conditions more conducive for 17β-estradiolmineralization and further studies are required to also ex-amine the mechanisms behind this latter observation. Weconclude that fresh manure applications can have a short-term significant effect on reducing 17β-estradiol mineral-ization, and that the long-term applications of manure tosoil increases soil organic carbon content and hence 17β-estradiol sorption in soil, without having a strong impact on17β-estradiol mineralization. We also conclude that MAXwas always less than 30% at 90 days, suggesting that resid-ual estrogenic activity could remain for more than a seasonin soils exposed to estrogens.

Acknowledgments

The authors acknowledge the financial support of the Nat-ural Sciences and Engineering Research Council of Canada

Long term manure impacts on 17β-estradiol fate 503

for research funding. We also thank the University ofManitoba Graduate Fellowship for financial support toE. Caron. The authors thank Mr. Paul Panich, Mr. BrettHill, Mrs. Pamela Caffin, Mr. Clarence Gilbertson andMr. Dan Inaba at AAFC Lethbridge for their assistanceto the laboratory experiments.

References

[1] Lai, K.M.; Scrimshaw, M.D.; Lester, J.N. The effects of naturaland synthetic steroid estrogens in relation to their environmentaloccurrence. Crit. Rev. Toxicol. 2002, 32 (2), 113–132.

[2] Ivie, G.W.; Christopher, R.J.; Munger, C.E.; Coppock, C.E.J. Fateand residues of [4-C-14] Estradiol-17-beta after intramuscular in-jection into Holstein steer calves. J. Anim. Sci. 1986, 62, 681–690.

[3] Palme, R.; Fischer, P.; Schildorfer, H.; Ismail, M.N. Excretion ofinfused C-14-steroid hormones via faeces and urine in domesticlivestock. Anim. Reprod. Sci. 1996, 43, 43–63.

[4] Matthiessen, P.; Arnold, D.; Johnson, A.C.; Pepper, T.J.; Pottinger,T.G.; Pulmam, K.G.T. Contamination of headwater streams in theUnited Kingdom by oestrogenic hormones from livestock farms.Sci. Tot. Environ. 2006, 367, 616–630.

[5] Kolodziej, E.P.; Sedlak, D.L. Rangeland grazing as a source ofsteroid hormones to surface water. Environ. Sci. Technol. 2007, 41,3514–3520.

[6] Nichols, D.J.; Daniel, T.C.; Moore, P.A. Jr.; Edwards, D.R.; Pote,D.H. Runoff of estrogen hormone 17β-Estradiol from poultry litterapplied to pasture. J. Environ. Qual. 1997, 26, 1002–1006.

[7] Bushee, E.L.; Edwards, D.R.; Moore, P.A. Jr. Quality of runofffrom plots treated with municipal sludge and horse bedding. Trans.ASAE. 1998, 41(4), 1035–1041.

[8] Finlay-Moore, O.; Hartel, P.G.; Cabrera, M.L. 17β-Estradiol andtestosterone in soil and runoff from grasslands amended with broilerlitter. J. Environ. Qual. 2000, 29, 1604–1611.

[9] Statistics Canada. Cattle statistics. 2011. Catalogue No. 23–012-X.http,//www.statcan.gc.ca/pub/23–012-x/23–012-x2010002-eng.pdf (accessed May 6, 2011).

[10] Kissinger, W.F.; Koelsch, R.K.; Erickson, G.E.; Klopfenstein, T.J.Characteristics of manure harvested from beef cattle feedlot. Appl.Eng. Agric. 2007, 23(3), 357–365.

[11] Alberta Agriculture and Food, Economics and CompetitivenessDivision, Statistics and Data Development Unit. Census of Agri-culture for Alberta, Provincial Highlights. 2006. AGDEX 852–5.

[12] Caron, E.; Farenhorst, A.; Gaultier, J.; Rank, N.; Goddard, T.;Sheedy, C. Sorption of four estrogens by surface soils from 41 cul-tivated fields in Alberta, Canada. Geoderma 2010, 155, 19–30.

[13] Caron, E.; Farenhorst, A.; Mc Queen, R.; Sheedy, C.; Goddard, T.;Gaultier, J. Mineralization of 17β-estradiol in 36 Surface Soils fromAlberta, Canada. Agric. Ecosyst. Environ. 2010, 139(4), 534–545.

[14] Casey, F.X.M.; Larsen, G.L.; Hakk, H.; Simunek, J. Fate and trans-port of 17β-estradiol in soil-water systems. Environ. Sci. Technol.2003, 37, 2400–2409.

[15] Lee, L.S.; Strock, T.J.; Sarmah, A.K.; Rao, P.S.C. Sorption and dissi-pation of testosterone, estrogens and their transformation productsin soil and sediment. Environ. Sci. Technol. 2003, 37, 4098–4105.

[16] Sangsupan, H.A.; Radcliffe, D.E.; Hartel, P.G.; Jenkins, M.B.; Ven-cil, W.K.; Cabrera, M.L. Sorption and transport of 17β-estradioland testosterone in undisturbed soil columns. J. Environ. Qual.2006, 35, 2261–2272.

[17] Yu, Z.; Xiao, B.; Huang, W.; Peng, P. Sorption of steroid estro-gens to soils and sediments. Environ. Toxicol. Chem. 2004, 23(3),531–539.

[18] Hildebrand, C.; Londry, K.L.; Farenhorst, A. Sorption and desorp-tion of three endocrine disrupters in soils. J. Environ. Sci. Health B.2006, 41, 907–921.

[19] Loffredo, E.; Senesi, N. Fate of organic pollutants in soils with em-phasis on adsorption/desorption processes of endocrine disruptorcompounds. Pure Appl. Chem. 2006, 78(5), 947–961.

[20] Layton, A.C.; Gregory, B.W.; Seward, J.R.; Schultz, T.W.; Sayler,G.S. Mineralization of steroidal hormones by biosolids in wastew-ater treatment systems in Tennessee U.S.A. Environ. Sci. Technol.2000, 34, 3925–3931.

[21] Stumpe, B.; Marschner, B. Organic waste effects on the behaviorof 17β-estradiol, estrone and 17α-ethinylestradiol in agriculturalsoils in long-and short-term setups. J. Environ. Qual. 2010, 39,907–916.

[22] Lucas, S.D.; Jones, D.L. Biodegradation of estrone and 17β-estradiol in grassland soils amended with animal wastes. Soil Biol.Biochem. 2006, 38, 2803–2815.

[23] Sommerfelt, T.G.; Chang, C. Changes in soil properties under an-nual application of feedlot manure and different tillage practices.Soil Sci. Soc. Am. J. 1985, 49, 983–987.

[24] Chang, C.; Sommerfelt, T.G.; Entz, T. Soil chemistry after 11 annualapplications of cattle feedlot manure. J. Environ. Qual. 1991, 20(2),475–480.

[25] Chang, C.; Whalen, J.K.; Hao, X. Increase in phosphorus concen-tration of a clay loam surface soil receiving repeated annual feedlotcattle manure applications in southern Alberta. Can. J. Soil Sci.2005, 85, 589–597.

[26] Alberta Environment and Alberta Agriculture. Confinement live-stock facilities waste management code of practice. 1973. Agdex538/710. Alberta Environment and Alberta Agriculture: Alberta.

[27] Alberta Agriculture, Food and Rural Development. Alberta Fer-tilizer Guide. 2004. Agdex 541–1, Alberta Agriculture, Food andRural Development: Alberta.

[28] Hao, X.; Chang, C.; Travis, G.R.; Zhang, F. Soil carbon and nitro-gen response to 25 annual cattle manure applications. J. Plant Nutr.Soil Sci. 2003, 166(2), 239–245.

[29] Sommerfelt, T.G.; Chang, C.; Entz, T. Long-term annual manureapplications increase soil organic matter and nitrogen and de-crease carbon to nitrogen ratio. Soil Sci. Soc. Am. J. 1988, 52,1668–1672.

[30] Chang, C.; Entz, T. Nitrate leaching losses under repeated cattlefeedlot manure applications in southern Alberta. J. Environ. Qual.1996, 25, 145–153.

[31] Chang, C.; Janzen, H.H. Long-term fate of nitrogen from an-nual feedlot manure applications. J. Environ. Qual. 1996, 25,785–790.

[32] Gao, G.; Chang, C. Changes in CEC and particle size distribution ofsoils associated with long-term annual applications of cattle feedlotmanure. Soil Sci. 1996, 161(2), 115–120.

[33] Hao, X.; Chang, C. Effect of 25 annual cattle manure applicationson soluble and exchangeable cations in soil. Soil Sci. 2002, 167(2),126–134.

[34] Hao, X.; Chang, C. Does long-term heavy cattle manure applicationincrease salinity of a clay loam soil in semi-arid southern Alberta?Agricult. Ecosys. Environ. 2003, 94, 89–103.

[35] Hao, X.; Chang, C.; Li, X. Long-term and residual effects of cattlemanure application on distribution of P in soil aggregates. Soil Sci.2004, 169(10), 715–728.

[36] Gee, G.W.; Bauder, J.W. Particle size analysis. In Methods of soilAnalysis. Part 1. Physical and Mineralogical Methods; Klute, A.,Ed.; ASA/SSSA: Madison, WI., 1986 383–412.

[37] McKeague, J.A. (Ed.) Manual on soil sampling and methods ofanalysis. Canadian Society of Soil Science: Ottawa, Canada, 1978;212 pp.

[38] Nelson, D.E.; Sommers, L.E. Total carbon, organic carbon, andorganic matter. In Methods of Soil Analysis, Part 2, Chemical andmicrobial properties; Page, A.L., Ed.; ASA/SSSA: Madison, WI,1982; 539–577.

[39] Schoenau, J.J.; O’Halloran, I.P. Sodium Bicarbonate-ExtractableInorganic P (Olsen et al. 1954). In Soil Sampling and Methods of

504 Caron et al.

Analysis, 2nd edition; Carter, M.R., Gregorich, E.G., Eds.; Cana-dian Society of Soil Science, CRC Press, Taylor & Francis GroupLLC, 2008; pp 90.

[40] Maynard, D.G.; Kalra, Y.P.; Crumbaugh, J.A. Extraction of NO3-N and NH4-N with 2.0 M KCl. In Soil Sampling and Methodsof Analysis 2nd edition; Carter, M.R., Gregorich, E.G, editors;Canadian Society of Soil Science, CRC Press, Taylor & FrancisGroup: Boca Raton, FL, USA, 2008; pp 72.

[41] Adam, G.; Duncan, H. Development of a sensitive and rapidmethod for the measurement of total microbial activity using flu-orescein diacetate (FDA) in a range of soils. Soil Biol. Biochem.2001, 33, 943–951.

[42] Wolf, D.C.; Hao, T.H.; Scott, H.D.; Lavy, T.L. Influence of steril-ization methods on selected soil microbial, physical and chemicalproperties. J. Environ. Qual. 1989, 18, 39–44.

[43] Lotrario, J.B.; Stuart, J.B.; Lam, T.; Arands, R.R.; O‘Connor, O.A.;Kosson, D.S. Effects of sterilization methods on the physical char-acteristics of soil: Implications for sorption isotherm analyses. Bull.Environ. Contam. Toxicol. 1995, 54, 668–675.

[44] Benoit, P.; Barriuso, E.; Houot, S.; Calvet, R. Influence of thenature of soil organic matter on the sorption desorption of 2,4-dichlorophenol and the herbicide, 2,4-dichlorophenoxyacetic acid(2,4-D). Eur. J. Soil Sci. 1996, 47, 567–578.

[45] Casey, F.X.M.; Simunek, J.; Lee. J.; Larsen, G.L.; Hakk, H. Sorp-tion, mobility, and transformation of estrogenic hormones in natu-ral soils. J. Environ. Qual. 2005, 34, 1372–1379.

[46] Kozarek, J.L.; Wolfe, M.L.; Love, N.G.; Knowlton, K.F. Sorptionof estrogen to three agricultural soils from Virginia, USA. TransASABE. 2008, 51(5), 1591–1597.

[47] Blake, G.R.; Hartge, K.H. Bulk Density. In Methods of Soil Anal-ysis. Part I. Physical and Mineralogical Methods, Agronomy No 9,2nd Ed.; Klute, A., editor; American Society of Agronomy: Madi-son, WI, 1986; 363–367.

[48] Legendre, P.; Legendre, L. Numerical Ecology, 2nd Ed. ElsevierB.V.: Amsterdam, The Netherlands, 1998.

[49] Colucci, M.S.; Bork, H.; Topp, E. Persistence of estrogenic hor-mones in agricultural soils: I. 17β-estradiol and estrone. J. Environ.Qual. 2001, 30, 2070–2076.

[50] Stumpe, B.; Marschner, B. Long-term sewage sludge applicationand wastewater irrigation on the mineralization and sorption of17β-estradiol and testosterone in soils. Sci. Total Environ. 2007,374, 282–291.

[51] Fan, Z.; Casey, F.X.M.; Hakk, H.; Larsen, G.L. Persistence and fateof 17β-estradiol and testosterone in agricultural soils. Chemosphere2007, 67, 886–895.

[52] Ying, G.-G.; Kookana, R.S. Sorption and degradation of estrogen-like–endocrine disrupting chemicals in soil. Environ. Toxicol. Chem.2005, 24(10), 2640–2645.

[53] Stumm-Zollinger, E.; Fair, G.M. Biodegradation of steroids hor-mones. Steroid Horm. 1965, 37(11), 1506–1511.

[54] Yoshimoto, T.; Nagai, F.; Fujimoto, J.; Watanabe, K.; Mizukoshi,H.; Makino, T.; Kimura, K.; Saino, H.; Sawada, H.; Omura, H.Degradation of estrogens by Rhodococcus zopfii and Rhodococcusequi isolates from activated sludge in wastewater treatment plants.Appl. Environ. Microbiol. 2004, 70(9), 5283–5289.

[55] Yu, C.-P.; Roh, H.; Chu, K.-H. 17 beta-estradiol-degrading bacteriaisolated from activated sludge. Environ. Sci. Technol. 2007, 41(2),486–492.

[56] Hemmings, S.J.N.; Hartel, P.G. Mineralization of hormones inbreeder and broiler litters at different water potentials and tem-perature. J. Environ. Qual. 2006, 35, 701–706.

[57] Saison, C.; Degrange, V.; Oliver, R.; Millard, P.; Commeaux, C.;Montange, D.; .Le Roux, X. Alteration and resilience of the soil mi-crobial community following compost amendment: effects of com-post level and compost-borne microbial community. Environ. Mi-crobiol. 2006, 8(2), 247–257.