Concentrations of Free and Conjugated Estrogens at Different Landscape Positions in an Agricultural Watershed Receiving Poultry Litter

Concentrations of Free and Conjugated Estrogensat Different Landscape Positions in an AgriculturalWatershed Receiving Poultry Litter

Sudarshan K. Dutta & Shreeram P. Inamdar &

Jerry Tso & Diana S. Aga

Received: 19 July 2011 /Accepted: 23 December 2011 /Published online: 21 January 2012# Springer Science+Business Media B.V. 2012

Abstract Animal hormones can enter the aquatic envi-ronment along with runoff as a result of manure or litterapplication on agricultural landscapes. Our understand-ing of the transport of these hormones and their concen-trations at various points along the watershed drainage ishowever limited. We investigated the transport of natu-rally produced poultry hormones in an agricultural wa-tershed located on coastal plain soils of Delawarereceiving land application of raw poultry manure. Theobjective of this study was to determine the concentra-tions of free and conjugated forms of estrogens in agri-cultural runoff at selected landscape positions in theagricultural watershed. Estrogen concentrations weredetermined for surface water, soil water, and runoffsediment. Estrogen forms that were analyzed were: Es-trone (E1), Estradiol (E2β and E2α), Estriol (E3), andtheir sulfate and glucuronide conjugates. Poultry litterapplication occurred at a rate of 9 Mg ha−1 in earlyspring (April 2010). Sampling was performed for sur-face runoff, subsurface drainage, and sediment for nine

storm events extending over 187 days before and aftermanure application (March–October 2010). Runoff wascollected from the field edge, upland and lowland ripar-ian positions and from the stream. Samples were ana-lyzed by for liquid chromatography with tandem massspectrometry (LC-MS/MS). Concentrations of estro-gens were low (<20 ng l−1) for most of the samplesand decreased from the field edge into the riparian zone.Estrogens were not detected in soil water and runoffsediments. Overall, this study suggests that manureapplication practices at our sites in Delaware such asincorporation of litter into the soil likely reduced theconcentrations of estrogens in runoff and reduced thethreat posed to aquatic ecosystems.

Keywords Estradiol . Agricultural runoff . Poultrymanure . Environmental pollution

1 Introduction

Animal manures such as poultry litter are often appliedto croplands as organic fertilizer. Recent studies suggestthat land application of animal manure could be animportant source of steroidal hormones for agriculturalrunoff (Jenkins et al. 2008; 2009; Lee et al. 2007). Thehormones of concern include estrogens such as estrone(E1), estradiol (E2), and estriol (E3) (Hanselman et al.2003; Lee et al. 2007) whose concentrations could be aslow as 40 ng l−1 to cause impairment in aquatic species(Yonkos 2005). The predicted no effects concentration

Water Air Soil Pollut (2012) 223:2821–2836DOI 10.1007/s11270-011-1069-1

S. K. Dutta (*) : S. P. InamdarDepartment of Plant and Soil Sciences,University of Delaware,152 Townsend Hall,Newark, DE 19716, USAe-mail: [email protected]

J. Tso :D. S. AgaDepartment of Chemistry, University at Buffalo,611 Natural Sciences Complex,Buffalo, NY 14260, USA

(PNEC) for E2 and E1 are even lower at 1 and 3 ng l−1,respectively (Young et al. 2004).

The bioactivity of estrogens varies depending on theirindividual chemical forms; for example, the unconjugat-ed or free forms of estrogens (e.g., E1, E2, and E3) havehigher endocrine disrupting effects than the conjugatedspecies (Hutchins et al. 2007; Lee et al. 2003). Further-more, the relative potency of E2 is 5–1,000 times highercompared to that of E1 and E3 (Lee et al. 2003; Legleret al. 2002). However, the conjugated forms may alsopose a potential threat since these forms can be convertedback to the toxic unconjugated species under certainenvironmental conditions (D’Ascenzo et al. 2003;Hanselman et al. 2003; Hutchins et al. 2007). To date,most agricultural studies have typically focused on con-centrations of E2 (Finlay-Moore et al. 2000; Jenkinset al. 2008; 2009), and few researchers have investigatedthe conjugated forms. To provide a comprehensive eval-uation of the potential threat posed to hormones, theconjugated as well as unconjugated forms of estrogensneed to be evaluated simultaneously.

The transport of estrogens in watersheds can occurthrough a variety of hydrologic flow-paths which includesurface runoff, infiltration, vertical drainage, groundwaterflow, and transport with sediment (Dutta et al. 2010a,2011; Finlay-Moore et al. 2000; Jenkins et al. 2009;Lægdsmand et al. 2009; Kuster et al. 2004).Most previousstudies have been conducted at the plot or field scale andhave focused on concentrations of estrogens in surfacerunoff (Dutta et al. 2010a, 2011; Haggard et al. 2005;Jenkins et al. 2008; 2009). Very few studies have investi-gated the fate and transport of estrogens in soil and ground-waters and at the watershed scale under typical agronomicconditions. Elevated concentrations of estrogens couldoccur in groundwaters if these contaminants are trans-ported via preferential flow paths and macropores (e.g.,biopores, cracks, and fissures) (Lægdsmand et al. 2009).Concentrations could also change at various points alongthe watershed drainage as runoff traverses grassed water-ways and vegetative and riparian buffers.

Our interest in this study was to characterize theconcentrations of estrogens in surface runoff and soilwaters at selected points along the watershed drainage.The study was conducted in an agricultural watershedon the coastal plain soil of Delaware and subject toapplication of poultry litter. Runoff sampling was per-formed for multiple storm events over the growingperiod. Samples were collected at the edge of the field,at upland and lowland riparian locations, and from the

receiving stream. Specific questions that were addressedin this study include: (1) What are the concentrations offree and conjugated forms of estrogens in agriculturalrunoff? (2) How do their concentrations vary acrossvarious landscape positions along the runoff flow pathswithin the agricultural watershed? (3) What are theconcentrations of free and conjugated estrogens in dif-ferent runoff components which include — surface wa-ter, soil water, and runoff sediment?

2 Materials and Methods

2.1 Site Description and Sampling Locationsin the Study Watershed

The study was conducted in an agricultural watershednear Middletown in New Castle County, Delaware(39.43°N, 75.67°W; Fig. 1). The watershed is locatedwithin the Appoquinimink River drainage basin and inthe vicinity of the Silver Lake. Predominant soil type inthe watershed is matapeake silt–loam with a slope gra-dient of 0–5%. The soil is classified as fine-silty, mixed,mesic typic hapludults (USDA-SCS 1970). Averageannual precipitation for the county is 1130 mm withhighest monthly precipitation typically occurring in July(USDA-SCS 1970). Precipitation during the summer isassociated with low-pressure systems from the southwhich produce high-intensity convective storm events.Average annual temperature is 54°F (12°C) with maxi-mum temperatures typically occurring during the latterpart of July. Corn (Zea mays L.) is the primary crop onthe agricultural fields with wheat as a cover crop duringthe winter. Planting of corn occurred during the firstweek of May with harvesting during the month ofSeptember. The agricultural land has received conven-tional tillage with disc-harrow every year for more than5 years (as of 2010). Raw poultry litter has been appliedto the fields once every 3 years and is incorporated intothe surface soil (5–10 cm) during application. In 2010,litter was applied on April 10 at the rate of 9 Mg ha−1.

The study watershed with the sampling locations isindicated in Fig. 1a. The total area of the cropland fieldsin the study watershed was 10 ha (Delaware DataMIL2011). The cropland drained towards a riparian forestand wetland along the northwestern edge of the water-shed adjoining Silver Lake. Runoff from the croplandalso drained via a grassed waterway located in themiddle of the study watershed (Fig. 1a).

2822 Water Air Soil Pollut (2012) 223:2821–2836

Sampling “nests” were established at three differentlocations along the watershed drainage — at the fieldedge (FE), and at the upland (UR) and lowland ripar-ian (LR) forest/wetland locations (Fig. 1a and b).Three separate sampling nests were established at thefield edge (FE 1–3) and one each at the upland andlowland riparian zones (Fig. 1a, b). Each nest had twowater sample collecting units — a surface runoff col-lector and a soil water collector. The surface runoffcollectors were PVC pipes with 13 cm (5 in) diameterand 46 cm (18 in) length. The pipes were screened fora length of ~15 cm above the soil surface to allowentry of surface runoff but capped with a lid to prevententry of rainfall. The lower portion of the pipe was

solid with a cap at the bottom so as to collect theincoming surface runoff. The soil water collector wasalso a PVC pipe (13 cm diameter and 60 cm length),but screened from 5 to 25 cm below the soil surface tocollect soil water from that soil depth. Again, thelower portion (below 25 cm) of the soil water samplerwas solid PVC with a cap at the bottom so that soilwater could be retained in the sampler. Essentially, thesoil water sampler was a zero-tension lysimeter thatcollected soil water under gravity flow. These sam-plers were inserted vertically below the A-horizonafter digging a soil pit. Both the surface runoff andsoil water sampling devices were capable of holdingmore than 2 l of runoff water. Two of the FE nests

MD

PA

DE

a

Fig. 1 a Location of study watershed in New Castle County inDelaware (inset) and a close-up aerial image of the study wa-tershed indicating the sampling nests and locations. The fieldedge sampling locations are indicated by FE 1, 2, and 3. Theupland riparian zone nest (UR) is situated after a 1-m grassbuffer. The lowland riparian zone nest (LR) is located at the

bottom of the riparian zone and is saturated year-round. Thestream sampling location is indicated by S. The topographiccontours at a 10-ft (3 m) elevation interval are indicated by thegrey lines. b Transect highlighting the topographic locations ofthe three sampling nests FE, UR, and LR and the stream locationS. The sampling points are marked with yellow circles

Water Air Soil Pollut (2012) 223:2821–2836 2823

(FE1 and FE2) were located along the cropland andriparian forest edges while the third (FE3) was posi-tioned at the edge of the grassed waterway. The uplandriparian zone was located inside the riparian zone butabove the valley bottom. The lowland riparian (LR)wetland location was completely saturated for theentire sampling period, likely due to upwelling ofshallow groundwaters in the valley bottom. Thus, the“soil water” sampler at this location was likely sam-pling some amounts of shallow groundwater in addi-tion to soil water. Grab surface water samples werealso collected from the main stream (S, Fig. 1) manu-ally following each storm event (storm sampling). Allinstrumentation was completed during the third weekof March 2010 and the first set of runoff samples werecollected on March 29, 2010. Since this samplingoccurred before poultry litter application (April 10),this data provides some idea of the background con-centrations of hormones in the watershed. After litterapplication, sampling was performed for a total ofeight natural storm events of varying magnitude andintensity (Fig. 2 and Table 1) extending from Aprilthrough October 2010 (a total of 187 days from March29 to October 2, 2010). Samples for UR, LR, and Slocations were available for all nine events; whereas,for the FE location data for six events was availablefor all three locations (FE 1–3), data for additional twoevents were available for (FE 3), and data for all nineevents were available for FE1.

2.2 Analyses for Free and Conjugated Estrogens

Allwater sampleswere collected in duplicate in 0.5-l darkamber glass bottles with fluoropolymer resin lined caps.The duplicate sampling was implemented so as to verifythe instrument accuracy for hormone analyses (describedbelow). Eventually, an average of these two values rep-resented the final concentration for the sampling location.The sampling bottles were rinsed with 10% nitric acid,washed with NANOpure™ water (Barnstead NanopureWater Purification System, Thermo Fisher Scientific Inc.,

Fig. 1 (continued)

Fig. 2 Rainfall amounts for storm events in the year 2010.Events sampled in this study during March–October 2010 areindicated by gray circles. The sampling numbers are indicatedwithin parentheses


Waltham, MA), and baked at 250°C for 4 h beforesampling to avoid any microbial contamination. Watersamples were brought back to laboratory and acidifiedwith sulfuric acid to lower the sample pH to 2.25 (±0.2).Subsequently, the samples were filtered with a 1.2-μmglass filter, followed by a 0.70-μm glass filter (WhatmanInternational Ltd.,Maidstone, Kent, UK) and stored at 0–4°C for liquid chromatography with tandem mass spec-trometry (LC-MS/MS) analysis. The sample portionpassing through the filter represented the dissolved phasewhile the sediment retained on the filter represented theparticulate phase moving with runoff. A detailed descrip-tion of the protocol used for hormone analysis has beenprovided by Tso et al. (2011). Filtered water sampleswere evaluated for twelve forms of estrogen (free andconjugates) using LC-MS/MS. The free forms includedestrone (E1), 17β-estradiol (E2 or E2β), 17α-estradiol(E2α), and estriol (E3); and the conjugate forms wereestrone 3-sulfate (E1-3S), estrone 3-glucuronide (E1-3G), 17β-estradiol 3-sulfate (E2β-3S), 17α -estradiol3-sulfate (E2α-3S), 17β-estradiol 17-sulfate (E2β-17S),17β-estradiol 3-glucuronide (E2β-3G), 17α -estradiol 3-glucuronide (E2α -3G), and 17α-ethinylestradiol (EE2).Details of the LC-MS/MS steps are reported by Tso et al.(2011). We also reported the details of the method quan-tification limit (MQL) and method detection limit(MDL), and the quality assurance parameters related tothe LC-MS/MS study in Tso et al. (2011). In short, watersamples were collected, acidified, filtered, spiked, andthen passed through solid phase extraction (SPE) car-tridge before it was ready for LC-MS/MS analyses (Tsoet al. 2011).

In addition to water and sediment analyses, the con-centrations of estrogens were also determined in the rawlitter samples prior to land application. The extraction ofhormones from sediments, soils and manure were per-formed using a Dionex 200 (Sunnyvale, CA) accelerat-ed solvent extraction (ASE) system. The procedures foranalyses of litter and soil samples have also been de-scribed by Tso et al. (2011). Concentrations of free andconjugated estrogens for raw litter and soil samplescollected after the April 13–14, 2010 event are reportedin Table 2. Soil samples after each event were notanalyzed due to the cost associated with analyzing thesamples. Selected physical and chemical characteristicsof the soil and manure samples have also been includedin Table 3. Analyses techniques used to measure thephysical and chemical properties of soil and manurehave been described by Dutta et al. (2010b).T

able

1Dates,am

ounts,days

sincelitterapplicationand7days

antecedent

rainfallof

thenine

naturalrainfalleventsmon

itoredin

2010

Param

eter

Storm

events

12

34

56

78

9

Date

March

28–2

9April13–1

4April25–2

7May

11–12

May

17–18

June

9–10

June

21–2

2July

9–11

Sep.30–

Oct.1

Total

rainfall(m

m)

35.6

6.0

38.6

13.5

25.7

6.35

19.8

62.5

130.8

Maxim

umrainfallintensity

(mm

h−1)

10.7

1.8

12.2

3.2

2.8

2.8

15.2

5.3

8.9

Dayssincelitterapplication

NA

417

3239

6174

9317

5

7-dayantecedent

rainfall(m

m)

43.5

20.3

45.7

13.5

25.7

6.9

24.4

62.5

153.2

NAno

tapplicable,as

themanurewas

appliedon

April10

,i.e.,12

days

afterthisevent


2.3 Data Analysis

The experimental design involved determining how hor-mone concentrations varied across the four landscapepositions — FE, LR, UR and S — for the nine eventssampled. Since the FE locations were replicated threetimes at FE1, 2, and 3, an average of the three locationswas taken to compare against respective values fromUR,LR, and S. Such comparisons were made for all the eightevents after manure application. Nonparametric statisti-cal analysis, Wilcoxon signed-rank test, was used todetermine if the estrogen concentrations were signifi-cantly different at an α level of 10% (p≤0.10). Allstatistical analyses were performed using the statisticalSoftware JMP (version 8, SAS Institute Inc., Cary, NC).

3 Results

3.1 Free Forms of Estrogens in Different RunoffComponents with Landscape Positions

No estrogens were detected in any of the backgroundsamples, i.e., the samples collected before manure appli-cation on March 29. Samples collected after manureapplication showed the presence of all the four free forms

of estrogens, i.e., estrone (E1), 17β-estradiol (E2β),17α-estradiol (E2α), and estriol (E3). The presence ofall these free forms was, however, not observed together;and except E1, presence of other forms was sporadic anddid not reveal any systematic trend. The number ofsamples for which E1 was detected was significantly(p≤0.10, n08) greater than the number of samples forall other free forms (E2β, E2α, and E3) (Table 3).Among all the free forms, concentrations for E1 alonewere high enough to display a specific trend with land-scape positions (Fig. 3).

The concentrations of E1 in surface runoff at the fieldedge (FE 1, 2, and 3) were significantly greater (p≤0.10)than the values recorded for other landscape positions(Fig. 3). Estrone (E1) concentrations in surface runoff atthe field edge varied from 2.2 to 57.5 ng l−1 (Fig. 3,Table 4). Surface runoff concentrations from E1 at theupland riparian location were significantly (p≤0.10)lower than the corresponding values at the field edgeand varied between 1.2 and 8.2 ng l−1 (Table 3). The E1concentrations decreased further for the downslope ri-parian zone and ranged from 1 to 2.2 ng l−1 (Table 4) butwere not significantly different (p≥0.10) from the up-land riparian surface runoff samples. Concentrations forE1 in stream water were below detection limit for thebackground conditions (March 29 event) but increased

Table 2 Mean (n04) concentrations of free and conjugatedestrogens in the raw poultry manure and soil sample collectedfrom the crop land (0–10 cm) on April 14, 2011. Standard

deviations of the concentrations are provided within parenthesis.The total amount of estrogen applied (load) to the field isincluded

Sample type Estrogens (ng g−1)

Free forms Conjugated forms

E1 E2β E2α E3 E2β-17S E2β-3S E2α-3S E1-3S

Manure 54.15 (18.6) 4.95 (0.88) 2.68 (0.22) 8.13 (1.75) 74.25 (1.06) 3.37 (0.48) 3.1 (0.58) 28.95 (2.61)

Cropland soil (04/14/2010) 16 (8.6) 5 (2.6) 8 (0.9) 11 (1.8) 8 (3.6) 10 (2.4) 7 (1.8) 4 (0.8)

Load (g ha−1) 0.49 0.05 0.02 0.73 0.67 0.03 0.03 0.26

Table 3 Selected physical and chemical characteristics of applied poultry manure soil samples collected after the first storm event

Sample types Physico-chemical properties measured

Ph CEC (meq 100 g−1) % Organic C

Manure 8.4 (0.2) 115.0 (2.5) >12 (NA)

Cropland Soil (04/14/2010) 7.1 (0.2) 14.0 (1.0) 2.2 (0.1)

NA not applicable


Fig. 3 Surface water concentrations for estrone (E1) at differentlandscape positions for the nine storm events. Event March 29occurred before manure application and therefore those sampleswere considered as background samples. The concentrations of

E1 having significant difference (p≤0.10) across all the nineevents among the different landscape positions, are presentedwith different letters (a and b)


Table 4 Concentrations of different free and conjugated estrogens observed in different runoff components across the storm events atdifferent landscape positions of an agricultural watershed

Sampling date Sampling location Concentrations of freeforms of estrogen(ng l−1)

Concentrations ofconjugated formsof estrogens (ng l−1)


4/14 Field edge surface runoff 15.7 0 12.0 0 0 26.8 0 29.2

Field edge soil water 0 0 0 0 0 0 0 0

Upland riparian surface runoff 8.2 0 0 0 2.5 22.2 0 0

Upland riparian soil water 1.9 0 0 0 0 0 0 0

Lowland riparian surface runoff 0 0 0 0 0 0 0 0

Lowland riparian soil water 0 0 0 0 0 0 0 0

Stream water 0 0 0 2.9 0 0 0 0

4/27 Field edge surface runoff 57.5 0 0 0 107 8 8.5 11.5

Field edge soil water 0 0 0 0 0 0.9 0 0

Upland riparian surface runoff 1.2 0 0 0 20 0 0 0

Upland riparian soil water 0 0 0 0 0 0 0 0


Lowland riparian soil water 0 0 0 19.2 0 0 0 0

Stream water 0 0 0 0 0 0 0 0

5/12 Field edge surface runoff 0 0 0 0 0 0 0 0

Field edge soil water 0 0 0 0 0 0 1.5 0

Upland riparian surface runoff 0 0 0 0 0 0 0 0


Lowland riparian surface runoff 1.0 0 0 0 0 0 0 0


Stream water 0 0 0 0 0 0 0 0

5/19 Field edge surface runoff 13.0 2.1 1.5 0 28.0 1.38 1.39 2.14


Upland riparian surface runoff 0 0 0 0 0 0 0 0




Stream water 0 0 0 0 0 0 0 0

6/10 Field edge surface runoff 3.5 0 0 0 0 0 0 0





Lowland riparian soil water 2.2 0 0 0 0 0 0 0

Stream water 2.0 0 0 0 0 0 0 0

6/23 Field edge surface runoff 2.2 0 0 0 0 0 0 1.5






to 2.1 ng l−1 for the mid to late summer storm events(June 10–June 23; Fig. 3). While concentrations for theother estrogens — estradiol (E2β, E2α) and estriol(E3)— were recorded at the field edge in surface runoff(1.5–12 ng L−1; Table 4), their presence at other locationswere minimal (only three out of 22 samples indicatedvalues above detection limit).

Soil water samples collected at the field edge didnot reveal any detectable concentrations for free formsof the estrogens. However, E1 was detected in soilwater at the upland riparian location and ranged up to2 ng l−1 (Table 4). Concentrations for E1 (0–2.2 ng l−1)as well as E3 (0–19 ng l−1) were also detected at thedownslope riparian location (LR, Table 4). Statisticalcomparisons among the locations for the free forms insoil water could not be performed because of insuffi-cient number of samples with concentrations abovethe detection limit. No free forms of estrogens weredetected in the sediment samples collected from sur-face runoff at the three landscape positions.

3.2 Conjugated Estrogens in Different RunoffComponents Across Landscape Positions

Among the conjugates of estrogens, only the sulfateforms were detected and that included E2β-17S, E2β-3S, E2α-3S, and E1-3S. The concentrations for theseforms varied from as low as 0.9 to as high as 100 ng l−1

for samples collected from different landscape positions(Table 4). The sum of conjugate estrogen concentrationsin surface runoff at the field edge ranged from 0.9 to135 ng l−1 (Fig. 4). The concentrations of the conjugatesdecreased significantly (p≤0.10) from field edge to theupland riparian. Concentrations of conjugates were notdetected for lowland riparian and stream locations(Table 4). Conjugates were not detected in any of the soilwater samples across all landscape positions. Similar tothe free forms, conjugated forms of estrogens were notdetected in the sediment samples.

Overall, the concentrations for conjugates (in surfaceas well as soil waters) for the first four events (until May19) were fairly high and in most cases exceeded thecorresponding concentrations for free forms. However,after the fourth event the conjugate concentrations werenot detected.

4 Discussion

4.1 Comparison Between Free and ConjugatedForms of Estrogens in Runoff

The concentrations of estrogens measured in our studyare within the range reported in other agricultural studies(Arikan et al. 2008; Fisher et al. 2005; Herman andMills2003; Table 5). Estrone (E1) was the dominant free formof estrogen in runoff for our watershed. Previous studiesin agricultural watersheds and farm waste treatmentplants have also reported higher concentrations of E1compared to other free forms (E2β and E3) (Arikan etal. 2008; Dutta et al. 2010a; Chen et al. 2010; Hutchinset al. 2007). For an agricultural watershed receiving rawpoultry manure, Arikan et al. (2008) found only the E1form (13 ng l−1) and E3 (12 ng l−1) in two out of the 82samples collected. E2 was not detected in any of thesamples collected. Similarly, Pailler et al. (2009) mea-sured the runoff water in a small mixed-land use, ruralcatchment (35 km2) in the southwestern part of Luxem-bourg and found E1 and E2β at concentrations of 27 and6 ng l−1, respectively. In another study, Hutchins et al.(2007) analyzed lagoon water samples near a concen-trated poultry farm operation and reported that E1 con-centrations were 10 to 100 folds higher than thecorresponding values for E2β, E2α, and E3. The ele-vated concentrations of E1 could be due to fact that E1 isa more stable form of estrogen compared to other freeforms under oxidized conditions (Hutchins et al. 2007).It should also be noted that E1 concentrations (40–

Table 4 (continued)

Sampling date Sampling location Concentrations of freeforms of estrogen(ng l−1)

Concentrations ofconjugated formsof estrogens (ng l−1)


Lowland riparian soil water 2.2 0 0 0 0 0 0 0

Stream water 2.1 0 0 0 0 0 0 0


Fig. 4 Surface water concentrations for arithmetic sum of allconjugates at different landscape positions for the nine stormevents. Event March 29 occurred before manure application andtherefore those samples were considered as background samples.

The concentrations of conjugates having significant difference (p≤0.10) across all the nine events among the different landscapepositions, are presented with different letters (a, b, and c)


Tab

le5

Con

centratio

nsof

differentestrog

ensin

differentruno

ffcompo

nentsrepo

rted

previously

invariou

swatershed,plot

scale,andlabo

ratory

stud

ies

Reference

Locationandland

useof

thestud

ySam

plingschemeandsampletype

Estrogens

concentrations

andmeasurementtechniqu

es

Watershed

scale

stud

yArikanetal.

2008

The

Cho

ptankRiver

Watershed

(175

6km

2)of

Delmarva

Peninsula.T

hewatershed

contains

62%

ofagricultu

ralland

which

ishaving

extensivepo

ultry

prod

uctio

nand/or

agricultu

ralland

sthat

receive

poultrymanure.

Surface

water

samples

werecollected

from

15subw

atershed

stations

and7stations

onthemajor

receivingriver.From

each

locatio

n,on

esamplewas

collected,overfour

season

s(A

pril–D

ecem

ber20

05).

Estradiol

was

notdetected

inanyof

thesamples.

Estrone

andEstriol

werefoun

din

only

1ou

tof

82samples

thatwerecollected;theconcentrations

were

13and12

ngl−1,respectiv

ely.Con

centratio

nswere

measuredwith

LC-M

S/M

S.

Fisheret

al.

2005

Raw

poultrylitterwas

appliedto

twoagricultu

rallands

(14ha)at

arate

of7.5Mgha

−1.One

fieldreceived

conv

entio

naltillage

andtheotherreceived

notill.

Manurewas

incorporated

intheconv

entio

naltillage

fieldandsurfaceappliedat

no-till

field.

Surface

runo

ffandgrou

ndwater

samples

were

collected

for10

storm

events(M

ay–N

ovem

ber20

02)

attheedge

ofthefields

near

apo

ndwhere

theruno

ffends

tothepo

nd.

E2concentrations

inthesurfaceruno

ffvaried

inthe

rang

efrom

37to

250ng

l−1.No-tillhadhigh

erconcentrations

comparedto

conv

entio

naltillage.No

grou

ndwater

samplehasshow

nthepresence

ofE2.

The

concentrations

weremeasuredby

immun

oassay

techniqu

e.

Herman

and

Mills

2003

1.2km

2agricultu

ralwatershed

receivingliq

uidcattle

manureanddrycompo

sted

poultrylitterin

the

pastureandcorn

fielddu

ring

mid–lateApril20

01.

Water

samples

werecollected

in7datesin

a2-week

interval

(not

basedon

anyrainfallevent)atdifferent

pointsfrom

astream

passingby

acorn

field.

Soil

water

samples

werecollected

in4differentdates

from

June

5to

Aug

ust23

.

The

E2concentrations

inthestream

samples

rang

edfrom

10to

120ng

l−1.T

hesoilwater

concentrations

varied

from

30to

160ng

l−1.The

concentrations

weremeasuredby

ELISA.

Field/plot

scale

stud

yDutta

etal.

2010

a;b

Raw

poultrylitterwas

appliedto

12×5m

agricultu

ral

plots(sum

mer

corn–w

interwheat)at

arate

of23

–32

Mgha

−1.The

manurewas

hand

appliedon

the

soilandwas

notincorpo

rated.The

plotsreceived

no-

tillandredu

cedconv

entio

naltillage.

Surface

runo

ffsamples

werecollected

attheedge

ofplot

during

thenaturalstorm

events.

Bothfree

andconjug

ated

estrog

enswereob

served

atsurfaceruno

ff.Estrogenconcentrations

werein

arang

efrom

1to

5ng

l−1.The

concentrations

were

measuredwith

LC-M

S/M

S.

Jenk

ins

etal.200

9Raw

poultrylitterwas

appliedto

10×30

magricultu

ral

plots(sum

mer

corn–w

interrye)

atarate

of7.4and

11Mgha

−1.The

plotswereno

-till

(NT)andcon-

ventionally

tilled(CT)andreceived

manureon

Oct

16,20

01andMay

21,20

02.In

CT,

manurewas

incorporated;whereas,in

NTmanurewas

surface

applied.

Surface

runo

ffsamples

werecollected

attheedge

ofplotsafterapplying

irrigatio

nson

Nov.1

4–15

,200

1;andon

June

4–5,

2002

.

The

flow

-weigh

tedconcentrationof

E2rang

edfrom

23to

389ng

l−1.Nosign

ificantdifference

was

ob-

served

inE2concentrations

betweenCTandNTplot

surfaceruno

ffsamples.E2concentrations

were

measuredby

ELISA.

Haggard

etal.200

5Raw

poultrylitterwas

appliedin

1.5×6.1m

plotsata

rate

of6.2Mgha

−1.The

manurewas

hand

applied

onthesoilandwas

notincorporated.

Surface

runo

ffsamples

werecollected

attheedge

oftheplot

fortworainfallevents,simulated

1dayand

30days

afterlitterapplication.

E2β

concentrations

inthesurfaceruno

ffwerein

the

rang

efrom

0to

200ng

l−1.E

2βconcentrations

were

measuredby

ELISA.


70 ng g−1) were much greater than the other free forms(2.7–5 ng g−1) in raw manure that was applied to ourwatershed (Table 2).

Our runoff data revealed the presence of only thesulfated conjugate forms with the glucuronide formsbeing completely absent in all samples. Glucuronideforms were also absent in the raw manure that wasapplied to our watershed soils (Table 2). Very few stud-ies have looked at the conjugated forms in agriculturalrunoff and thus we know very little about the presence/absence of these species for previous agricultural stud-ies. Work by Hutchins et al. (2007) for lagoon waterfrom concentrated animal feeding operations alsorevealed the presence of sulfated species (E1-3S, E2β-3S, E2α-3S, and E2β-17S) with a complete absence ofthe glucuronide forms. Similarly, our previous field-scale work in an adjacent watershed (Dutta et al.2010a) yielded only the sulfated forms (0.5–4.5 ng l−1)in surface runoff. Studies that have investigated thedissociation and stability of the various conjugated com-pounds in sewage waste have confirmed the higherstability of sulfated forms of estrogens versus the glu-curonide species (D’Ascenzo et al. 2003; Hanselman etal. 2003). Common fecal microorganisms such asEschericia coli are capable of hydrolyzing estrogen con-jugates via glucuronidase while a similar mechanism forsulfatase is not known (Hanselman et al. 2003).

Our data also revealed interesting differences in thetemporal persistence of individual free and conjugatedestrogens (Table 4). While concentrations for conju-gates were higher for the initial few events (especiallyfor the field-edge samples), they decreased dramati-cally for the later events. The same pattern, however,was not followed for E1, where the concentrationspersisted throughout the summer (April–June 2010).These differences could be attributed to the differencesin solubility, degradation, and transformation of theindividual forms of estrogens (Hanselman et al. 2003;Lee et al. 2007). Conjugated estrogens have a muchgreater aqueous solubility than the free forms due totheir polar sulfate functional groups (Hanselman et al.2003). Higher solubility of conjugated species mayexplain the high initial concentrations of conjugatesin earlier storm events followed by the sudden de-crease for the later events. In contrast, free estrogensare less soluble and have more potential to get sorbedonto sediments and soils and therefore have the potentialfor slower release throughout the summer (Hanselmanet al. 2003; Kuster et al. 2004).T

able

5(con

tinued)

Reference

Locationandland

useof

thestud

ySam

plingschemeandsampletype

Estrogens

concentrations

andmeasurementtechniqu

es

Lab

oratory

stud

yLægd

smand

etal.200

9Investigated

theleaching

ofE2β

throug

hintact

soil

corescollected

from

manuretreatedagricultu

ral

field.

Hog

manurewas

mixed

with

spiked

solutio

nsandappliedon

thetopof

thesoilcore.Estrogens

ofho

gmanurewereE1,αE2,

andE2andpresentatthe

ratesof

243,

115,

and9μgkg

−1,respectiv

ely.Soil

coreswerehaving

diam

eter

of0.6m,and

height

1m.

Artificialrainfallwas

appliedto

thesoilcoresat

10mm

h−1for12

h.Water

samples

werecollected

atthebo

ttom

ofthesoilcoresthroug

hout

theperiod

andup

to12

hafterirrigatio

nwas

ceased.

The

sum

ofconcentrations

ofnaturalestrog

ens(E2,

αE2,andE1)

leachedfrom

thefour

soilcoresrang

edup

to10

ngl−1.Estrogenconcentrations

were

measuredwith

GC-M

S/M

S.


4.2 Changes in Estrogen Concentrationswith Landscape Positions

Concentrations of E1 in surface runoff decreased pro-gressively with landscape positions from the field edgeto the stream. Similarly, while some conjugate concen-trations were detected in surface runoff at the field edge,the corresponding values for the riparian locations werebelow detection limits. This suggests some loss, degra-dation, and/or sorption of estrogens as they travelled inrunoff through the riparian buffer composed of trees,shrubs, herbs and grass species. The processes thatcould contribute to the decrease in estrogen concentra-tions include microbial and fungal degradation, sorptionto soil surfaces, and photolysis (Jurgens et al. 2002;Powers and Angel 2008). Some or all of these processescould be responsible for the decrease in estrogen con-centrations with landscape position observed in ourstudy. Previous plot-scale studies have also shown thatgrass buffers can help decrease the concentrations of E2in runoff due to infiltration (Nichols et al. 1997). Nicholset al. (1997) evaluated the effectiveness of grass filtersfor reducing E2β in runoff originating from pasturereceiving poultry litter application. They found thatrunoff concentrations of E2β were reduced by 58%,81%, and 94% for filter lengths of 6.1, 12.2, and18.3 m, respectively.

No free and conjugated forms of estrogens wereobserved in almost all of the soil water samples collect-ed at the field-edge and upland riparian zones. In aprevious agricultural study, Fisher et al. (2005) (Table 5)measured E2 concentrations in the groundwater for anagricultural field receiving raw poultry litter at the rateof 7.5 mg ha−1. They reported that the groundwater E2concentrations were below detection limit (the detectionlimit was 18 ng l−1 for their experiment) and that surfacerunoff was the primary mechanism of E2 transport fromthe agricultural fields.

In our study the concentrations of E1 in the streamwater samples increased from below detection limit to2 ng l−1 for two events in mid–late summer storm events(Table 4). This increase was however limited to onlytwo events and does not suggest any specific trend.Previous work by Herman and Mills (2003) (Table 5)has shown that subsurface drainage could transport E2to downstream locations in the watershed. Our observa-tions of low and non-detect concentrations of estrogensin soil waters of the upper and lower riparian locations,however, do not provide any definitive evidence for

subsurface transport of estrogens from the uplands tothe streams.

We did not find any free or conjugated forms ofestrogens in the runoff sediment (i.e., the sediment car-riedwith the runoff). This is similar to our previous study(Dutta et al. 2010a) where we did not find any sediment-bound estrogens and hypothesized that either there wereno sediment-bound estrogens or that the extracting sol-vent was unable to separate the strongly bound estrogensin the sample. Other studies have, however, observedsediment-bound estrogens for surface runoff and riverbed sediments (Kuster et al. 2004; Ternes et al. 2002).These studies have adopted varying protocols for extrac-tion. For example, Ternes et al. (2002) used a two-stepextraction which involved 4 ml methanol followed by3 ml acetone for two consecutive times. In our study weused water/methanol/acetone (50:25:25, v/v/v) with25 mM EDTA, 0.6 M sodium chloride, and 2% ammo-nium hydroxide as the extraction solvents (Tso et al.2011). The use of such extractant helps to alleviateionization interferences and sorption effects, for in-creased accuracy of analysis (Tso et al. 2011).

4.3 Environmental Significance and the Impactof Agronomic Practices on Estrogensin Watershed Runoff

While the predicted no-effect concentrations for theestrogens are very low (1–5 ng l−1 for various forms ofestrogens) the concentrations reported to cause impair-ment in aquatic species have generally been muchhigher (Nakamura 1984; Tyler and Routledge 1998;Yonkos 2005). Nakamura (1984) found that estradiolconcentration of 2,000 ng l−1 caused feminization ofmore than 84% of salmon exposed to the hormones.Similarly, Tyler and Routledge (1998) reported that anexposure period of 3 weeks to E1 concentration 20 to50 ng l−1 induced vitellogenin in male rainbow trout(Oncorhynchus mykiss). The concentrations of estro-gens measured in our stream water samples are wellbelow the concentrations reported to cause impairmentin aquatic species. In fact, majority of the concentrationsthat we reported for the stream and riparian locationswere below detection levels and also below the PNECthresholds. Furthermore, none of the soil water samplesyielded any estrogen concentrations. This was despitethe fact that our watershed-scale sampling was initiatedimmediately following manure application and thus hadthe potential to yield the highest concentrations of


hormones that could be expected at the site. Further-more, the farmer at our watershed site typically appliespoultry litter only once every 3 years to match theagronomic requirements of the corn crop. Thus, estro-gen concentrations should be expected to decrease fur-ther for runoff events during years 2 and 3.

The farmer in our watershed also incorporated thepoultry litter into the soil following litter application byusing a turbo till. This practice is common with mostfarmers in New Castle County in Delaware who preferto incorporate manure immediately after field applica-tion so as to reduce the loss of ammonia from themanure (personal communication with County Exten-sion personnel). It is very likely that incorporation oflitter into the soil reduced the losses of hormones withrunoff because of the potential sorption of these chem-icals on the soil particles and the enhanced opportunitiesfor microbial degradation in soils. Previous studies havedemonstrated that incorporation of litter can reduce thelosses of hormones in runoff while surface applicationcan yield high estrogen exports (Fisher et al. 2005).Thus, agronomic practices such as the rate, method,and timing of application of manure can have an impor-tant bearing on the losses of hormones from agriculturalwatersheds and should be considered when assessing thepotential for hormone loss from agricultural watersheds.

5 Conclusions

This study is one of the first studies to evaluate theconcentrations of estrogens in runoff at the watershedscale and across key landscape positions. Our study isalso among the very few agricultural studies that haveinvestigated both the free and conjugated forms of estro-gens in surface as well as soil waters. We also used themore rigorous, expensive, and accurate LC-MS/MS ap-proach for estrogen analyses while previous studieshave typically characterized estrogens using ELISAprocedures. Key conclusions that can be derived fromthis study are:

1. Among the free forms of estrogens, only E1 wasconsistently detected in runoff samples, whereassulfate species were the only conjugate species ob-served in runoff. Concentrations for the conjugatedforms were elevated for the initial storm events andthen declined dramatically for the later events. Incontrast, concentrations for E1, although very low,

persisted through the summer for surface runoffcollected at the field edge.

2. Concentrations of E1 in surface runoff displayed aprogressive decrease from the field edge to theriparian zone and the stream. Concentrations forother forms of estrogens in surface runoff were toolow at all landscape positions to reveal any distincttrend. Estrogens were not detected in soil water atany of the landscape positions.

3. The higher concentrations of estrogens in surfacerunoff versus soil water suggest that surface runoffis likely the primary flow path or mechanism fortransport of estrogens in agricultural watersheds.

4. This study suggests that the agronomic practicesadopted by the farmers such as application of poul-try manure once every 3 years at a rate of 9 Mg ha−1

and incorporation of manure within the soil at thetime of application can help reduce the potential forestrogen exports from agricultural watersheds.However, while point incorporation of manuremay be beneficial for reducing estrogen loss withrunoff, we do not recommend tillage or widespreaddisturbance of the soil surface since such practicesmay cause other problems such as soil erosion andenhanced loss of nutrients such as phosphorus(Andraski et al. 2003) with runoff.

Acknowledgements Funding for this study was provided bythe United Sates Department of Agriculture (USDA) throughGrant No.2009-02424. Any opinions, conclusion, or recommen-dations expressed in this material are those of the author(s) anddo not necessarily reflect the views of the USDA. We thank theSt. Andrews School at Middletown, DE, and the farmers forproviding access to the agricultural plots. We also thank Dr.Tom Sims, T.A. Baker professor, Department of Plant and Soil,and Mr. Michael D. Popovich, Conservation Planner, College ofAgriculture and Natural Resources, University of Delaware, forsupporting and acting as a liaison between the farmers of the St.Andrews School and the researchers. We also thank ShatrughanSingh, Gurbir Singh, Rachael Vaicunas, Weinan Pan, and PrithaDutta for their support with experimental plot setup and sam-pling. We also thank the editor and anonymous reviewers whosecomments and suggestions were very constructive and helpedfurther strengthen this manuscript.

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Concentrations of Free and Conjugated Estrogens at Different Landscape Positions in an Agricultural Watershed Receiving Poultry Litter