Emerging Contaminants (Hormones and Antibiotics) in

Emerging Contaminants Emerging Contaminants (Hormones and Antibiotics) (Hormones and Antibiotics) in Agricultural Watershedin Agricultural Watershed

Presenter: Sudarshan (Shan) DuttaPresenter: Sudarshan (Shan) Dutta

Lecture OutlineLecture Outline1.1.

Overview of Emerging ContaminantsOverview of Emerging Contaminants

2.2. Focus on Agricultural Watershed: Focus on Agricultural Watershed: Hormones and AntibioticsHormones and AntibioticsSourcesSourcesEnvironmental SignificanceEnvironmental SignificanceFate and Transport MechanismsFate and Transport Mechanisms

3.3. Analytical TechniquesAnalytical Techniques

4.4. Laboratory Illustration of ELISALaboratory Illustration of ELISA

OverviewOverview

What are Emerging Contaminants?What are Emerging Contaminants?

“…“…unregulated contaminantsunregulated contaminants, which , which may be may be candidates for future regulationcandidates for future regulation……....””

e.g. Steroidal Hormones,e.g. Steroidal Hormones,Antimicrobial disinfectant (Antibiotics),Antimicrobial disinfectant (Antibiotics),Insect repellents,Insect repellents,Detergent metabolites, etc.Detergent metabolites, etc.

––

BarcelBarcelóó

(2003).(2003).

Why they are Important?Why they are Important?Toxic at a very low concentrationsToxic at a very low concentrations(parts per trillions or ng/L)(parts per trillions or ng/L)

Develop female characteristics in male Develop female characteristics in male species resulting ecological disturbance. species resulting ecological disturbance. Also known as Endocrine Disruption.Also known as Endocrine Disruption.

Promote antibiotic resistance in pathogenic Promote antibiotic resistance in pathogenic microorganismsmicroorganisms

•• This is considered as This is considered as ““gender bendinggender bending””

pollution.pollution.

••

In 2004, 3 Colorado rivers have 50% of male fish In 2004, 3 Colorado rivers have 50% of male fish with both male and female characteristics dominant. with both male and female characteristics dominant.

(MSNBC, 2004)(MSNBC, 2004)

•• Every third fish in English Rivers are changing sex.Every third fish in English Rivers are changing sex.(Daily Mail, 2006)(Daily Mail, 2006)

Environmental Significance

Where in US?Where in US?

Koplin et al., 2002

Loftin, 2008

Sources in the EnvironmentSources in the Environment

(http://www.potomac.org/site/wp-content/uploads/images/diagram.jpg)

Animal Farms/CAFOs

Agriculture

Wastewater Treatment Plants

Industrial Byproducts

Residential Waste

Animal Waste

Hospital Waste

Focus on Focus on Agricultural Watershed:Agricultural Watershed:

Hormones and AntibioticsHormones and Antibiotics

HormonesHormones in Agriculture Landscapein Agriculture Landscape

Types of hormonesTypes of hormones

SourcesSources

Environmental SignificanceEnvironmental Significance

Fate and TransportFate and Transport

Analytical techniques for measurementAnalytical techniques for measurement

Types of HormonesTypes of Hormones

Estrone (E1)Estrone (E1)

Estradiol (E2Estradiol (E2ββ and E2and E2αα))

Estriol (E3)Estriol (E3)

Testosterone (T)Testosterone (T)

Progesterone (P)Progesterone (P)

EstrogensEstrogens

(Androgen)(Androgen)

Different Steroidal Hormones include:Different Steroidal Hormones include:

Sources of Hormones in Sources of Hormones in Agricultural LandscapeAgricultural Landscape

Farm animals (e.g.: Cattle, Swine, Poultry) Farm animals (e.g.: Cattle, Swine, Poultry) secrete different steroidal hormones through secrete different steroidal hormones through their feces and urinetheir feces and urine

Therefore, animal manure produced from Therefore, animal manure produced from these animals naturally contain steroidal these animals naturally contain steroidal hormones; like Estrogens, Testosterone, etc.hormones; like Estrogens, Testosterone, etc.

Concentration of hormones in Concentration of hormones in manure depends onmanure depends on

Animal and Species types , Sex (male vs. Animal and Species types , Sex (male vs. female), and Age of the animalsfemale), and Age of the animalsConcentrations vary between feces and Concentrations vary between feces and urineurineStorage conditionStorage condition

--

Aeration, Dryness, Temperature, SunlightAeration, Dryness, Temperature, Sunlight

Storage periodStorage period--

Hormone concentrations in manure decrease with time Hormone concentrations in manure decrease with time

Estrogen concentrations at Cattle manure:

(Hanselman et al., 2003)

Estrogen concentrations at Swine manure:

(Hanselman et al., 2003)

Estrogen concentrations at Poultry manure:

17β-estradiol: 14–904 µg kg-1, Estrone: NDRaw Poultry Litter

Estrogen (Estrone + Estradiol): 14 µg/kgImmature Broilers Male

Estrogen (Estrone + Estradiol): 65 µg/kgImmature Broilers Female

Estrogen (Estrone + Estradiol): 533 µg/kgLaying Hens (Dry)

Estrogen (Estrone + Estradiol): 133 µg/kgChicken Litter (Dry)

Types of HormonesType of Litter

We found (in LC/MS):We found (in LC/MS):E1: 1.98E1: 1.98µµg/kg; E2g/kg; E2ββ: 0.47: 0.47µµg/kg g/kg

E2E2ββ--17S: 11.4217S: 11.42µµg/kg; E1g/kg; E1--3S: 0.523S: 0.52µµg/kgg/kg

Storage ConditionStorage Condition

Agriculture field receiving animal manure Agriculture field receiving animal manure also receive steroidal hormones.also receive steroidal hormones.

ApplicationApplication of Manure in of Manure in Agricultural LandscapeAgricultural Landscape

Surface ApplicationSurface Application IncorporationIncorporation

Why should we be Why should we be concerned about concerned about

Hormones?Hormones?

••Animal Manure contain naturally producing Animal Manure contain naturally producing steroidal hormones; e.g. Estrogens, Androgens, etc. steroidal hormones; e.g. Estrogens, Androgens, etc.

••

Steroidal hormones are also known as Steroidal hormones are also known as Endocrine Endocrine Disrupting ChemicalsDisrupting Chemicals

(EDCs) (EDCs)

••

Endocrine disruption results abnormal reproductive Endocrine disruption results abnormal reproductive and physiological behaviors; e.g. Fish feminization.and physiological behaviors; e.g. Fish feminization.


Section of male testis showing the Section of male testis showing the presence of a large developing eggpresence of a large developing egg

Threshold Hormone Concentrations

Predicted NoPredicted No--effect Concentration :effect Concentration :––

1717ββ--Estradiol (E2Estradiol (E2ββ

or E2)or E2): : 1 ng/L !!1 ng/L !!

Estrone (E1)Estrone (E1): : 3 3 ––

5 ng/L !! 5 ng/L !!

1 ng/L = 0.000000001g in 1 L. 1 ng/L = 0.000000001g in 1 L.

(Yong, 2005)

Toxicity Levels of Hormones on Toxicity Levels of Hormones on Exposure AssaysExposure Assays

500 to 1000 ng/l of E2500 to 1000 ng/l of E2Feminization of 84 to 100% of the Feminization of 84 to 100% of the masumasu

salmon and chum salmon.salmon and chum salmon.

E2E2ββ

= 19= 19--26 ng/L; 26 ng/L; E1 = 60 ng/LE1 = 60 ng/L

Induction of vitellogenin (VTG) in juvenile Induction of vitellogenin (VTG) in juvenile female rainbow trout on 14 day exposurefemale rainbow trout on 14 day exposure

PredictedPredicted

NoNo--effects Concentration (PNEC)effects Concentration (PNEC)

E2E2ββ

= 40 ng/L= 40 ng/LEndocrine disruption in male fathead Endocrine disruption in male fathead minnow at 21 day exposureminnow at 21 day exposure

Effect of hormonesEffect of hormones

Toxicity depends on species and period of exposureToxicity depends on species and period of exposure

ConcentrationsConcentrations

E2E2ββ

= = 1 ng/l1 ng/l

40% of female fish showing male 40% of female fish showing male characteristics.characteristics.

10 ng/L Methyl Testosterone10 ng/L Methyl Testosterone

Management PracticesManagement Practices Hormone ConcentrationsHormone Concentrations

Raw & Pelletized Poultry LitterRaw & Pelletized Poultry Litter E2E2ββ: 200 : 200 ––

500 ng/L500 ng/L

Raw Poultry LitterRaw Poultry Litter E2E2ββ: 42 : 42 ––

350 ng/L350 ng/L

Observed Concentrations of Estrogens Observed Concentrations of Estrogens in Agricultural Surface Runoffin Agricultural Surface Runoff

(Haggard et al., 2005)

(Yonkos, 2005)

Hormone concentrations in surface runoff were Hormone concentrations in surface runoff were much higher than the concentrations required to much higher than the concentrations required to create endocrine disruption.create endocrine disruption.

(Finlay-Moore et al., 2000)


2530 ng/L2530 ng/LT: 10T: 10--1830 ng/L1830 ng/L

What happens to the What happens to the hormones applied to hormones applied to

agricultural landscape?agricultural landscape?

Fate and Transport of Hormones in Fate and Transport of Hormones in Agricultural LandscapeAgricultural Landscape

Sorption on the soilSorption on the soilDegradation by Degradation by temperature, sunlight, temperature, sunlight, and microbesand microbesSurface RunoffSurface RunoffMovement with Movement with sedimentssedimentsLeaching through Leaching through preferential flowpreferential flow--pathpath Lee, 2008

Lee, 2008

Sorption on SoilSorption on Soil

Lee, 2008

Sorption on Soil Depends onSorption on Soil Depends on

−− Soil organic matter content Soil organic matter content −− Soil texture, especially clay content Soil texture, especially clay content

−− Increases with increase in temperatureIncreases with increase in temperature

−− Photo degradation (Sunlight)Photo degradation (Sunlight)

−− Microbial degradation (Microbes) Microbial degradation (Microbes)

Degradation in SoilDegradation in Soil

Colucci et al., 2001

Hanselman et al., 2003

Management PracticesManagement Practices Hormone ConcentrationsHormone Concentrations


300 ng/L300 ng/L


2530 ng/L2530 ng/L

Movement with Surface RunoffMovement with Surface Runoff

(Jenkins et al., 2009)

(Finlay-Moore et al., 2000)

--

Large fractions of estrogens, received in Large fractions of estrogens, received in soil, move with the sedimentssoil, move with the sediments

Mechanism:Mechanism:

--

Estrogens are sorbed with soil particles Estrogens are sorbed with soil particles and particulate organic matter (POM) and particulate organic matter (POM) which control the movements with which control the movements with sedimentssediments

Movement with sediments:Movement with sediments:

Kuster et al., 2004

Lee et al., 2007

--

Estrogens can move Estrogens can move with preferential flow with preferential flow

Downward Movements:Downward Movements:

Figure: Weston and Seelig, 1994

--

Therefore, have Therefore, have potential to contaminate potential to contaminate groundwater groundwater

To Date, To Date, Environmentally Significant Environmentally Significant Transport Mechanism: Transport Mechanism:

Surface Runoff. It ends up in Surface Runoff. It ends up in streams streams

Estrogen have different forms !!!!Estrogen have different forms !!!!

However, to date, most previous However, to date, most previous studies have analyzed only E2studies have analyzed only E2ββ

Different forms of EstrogensDifferent forms of Estrogens

EstrogensEstrogens

are obtained in two different formsare obtained in two different forms--

i) i) FreeFree

FormsFormsEstrone (E1); 17Estrone (E1); 17ββ--Estradiol (E2Estradiol (E2ββ

/ E2); Estriol (E3) / E2); Estriol (E3)

--

ii) ii) ConjugatedConjugated

formsformsSulfate and Glucuronide conjugates of free estrogensSulfate and Glucuronide conjugates of free estrogens

Toxicity of free forms >>>> conjugated formsToxicity of free forms >>>> conjugated forms

Conjugates can be converted to free formsConjugates can be converted to free forms

Free and Conjugated Estrogens:Free and Conjugated Estrogens:

Hutchins

et al., 2007

Objective:Objective:

To investigate various forms of To investigate various forms of estrogens in surface runoff estrogens in surface runoff

Receiving different litter treatmentsReceiving different litter treatments

My Research:My Research:

Pelletized Poultry Pelletized Poultry LitterLitter

Raw Poultry Raw Poultry LitterLitter

Site Description and Site Description and Experimental Design:Experimental Design:

Experimental Design with Treatments

Raw Poultry Litter 2 (RPL2)


Control Plot

Pelletized Poultry Litter (PPL)



Control Plot

Pelletize d Poultry

Litter (PPL)

Farm Lane

Reduced Tillage

No–Tillage

Experimental Field (Plot Size: 12m X 5m)

FlowFlow--weighted concentrations of weighted concentrations of E1, E2E1, E2ββ

and E2and E2ββ--17S in surface runoff 17S in surface runoff

Treatments

Hor

mon

e C

once

ntra

tion

(ng/

L)

0

1

2

3

4

5

6

E1E2βE2β−17S

PPL

RPL2

RPL1

RPL2

RPL1

PPL

Reduced Tillage No Tillage

PNEC for E2β

PNEC for E1

Dutta et al., Submitted

R e d u c e d T illa g e

Mas

s Ex

port

s (μ

g/ha

)

0

1 0

2 0

3 0

4 0

5 0

6 0

P P LR P L 1R P L 2

N o T illa g e

E v e n t D a te s W ith E v e n t N u m b e rs

Mas

s Ex

port

s (μ

g/ha

)

0

1 0

2 0

3 0

4 0

5 0

6 0

P P LR P L 1R P L 2

(1 ) (2 ) (3 ) (4 ) (5 ) (6 ) (7 ) (8 ) (9 ) (1 0 )

04/29

05/21

05/13

05/10

05/17

06/0606/3007/0707/1007/24

Temporal pattern of mass exports (E1).

Rainfall intensity played important role in mass export.

Exports were high for some late events.

Estrogens in Surface RunoffEstrogens in Surface Runoff

Mainly controlled by:Mainly controlled by:

−− Amount of surface runoffAmount of surface runoff

−− Timing of the eventTiming of the event

−− Dissolved organic matter (DOM)Dissolved organic matter (DOM)Dutta et al., Submitted

Key Questions Key Questions Remain UnansweredRemain Unanswered

Fate and Transport of all free and conjugated Fate and Transport of all free and conjugated forms forms

Downward movements of different forms of Downward movements of different forms of Estrogens through preferential flowpathsEstrogens through preferential flowpaths

Watershed scale study: Watershed scale study: --

changes in forms and toxicity of estrogens at changes in forms and toxicity of estrogens at different watershed compartmentsdifferent watershed compartments

Edge of FieldEdge of Field Riparian WetlandRiparian Wetland

Edge of StreamEdge of Stream

Estrogens at Agricultural WatershedEstrogens at Agricultural Watershed

Inamdar et al., 2009

AntibioticsAntibiotics in Agriculture Watershedin Agriculture Watershed

Sources in Agriculture Watershed Sources in Agriculture Watershed

Environmental SignificanceEnvironmental Significance

Fate and TransportFate and Transport

Analytical techniques for measurementAnalytical techniques for measurement

Sources in Agricultural Landscape

Beatty, 2004

Practice of feeding to poultry, swine and cattlePractice of feeding to poultry, swine and cattleUsed to treat illnessUsed to treat illnessFeed additive to promote growth Feed additive to promote growth An estimated 60An estimated 60--80% of all 80% of all livestock and poultry receive antibioticslivestock and poultry receive antibiotics

Some are metabolizedSome are metabolizedSome are excreted with the manureSome are excreted with the manure

Major antibiotics used in agriculture farmMajor antibiotics used in agriculture farm−− Tetracycline; Sulfonamides; QuinolonesTetracycline; Sulfonamides; Quinolones

Why we should know about Why we should know about Antibiotics?Antibiotics?

→→ Promote evolution of antimicrobial resistant Promote evolution of antimicrobial resistant bacteria.bacteria.

→→ Compromising the efficacy of important Compromising the efficacy of important human medicines.human medicines.

→→ Reported concentrations in environment are Reported concentrations in environment are lower than Lowest Observable Adverse Effect lower than Lowest Observable Adverse Effect Level (LOAEL).Level (LOAEL).


Can be sorbed with soils.Can be sorbed with soils.

Can move with surface runoff.Can move with surface runoff.

Sorbed fraction can move with sedimentsSorbed fraction can move with sediments

Tetracycline mainly moves with sediment as it Tetracycline mainly moves with sediment as it gets sorbed strongly with soil. Sulfonamide gets sorbed strongly with soil. Sulfonamide sorption is less than Tetracycline.sorption is less than Tetracycline.

Fate and Transport of antimicrobialsFate and Transport of antimicrobials

−− Soil organic matter content Soil organic matter content

−− Clay ContentClay Content

−− Soil pHSoil pH

Lee, 2008

Sorption on Soil Depends on:Sorption on Soil Depends on:

Plant Uptake of Antimicrobials:Plant Uptake of Antimicrobials:

Lee, 2008

Transport of AntimicrobialsTransport of Antimicrobials

Lee et al., 2007

DOMDOM--FaciliatedFaciliated TransportTransport--

Through Surface Runoff (Major Process)Through Surface Runoff (Major Process)

--

Through Soil ProfileThrough Soil Profile

Mechanism:Mechanism:Antimicrobials bind to DOM by Antimicrobials bind to DOM by --

Cation ExchangeCation Exchange

--

Cation BridgingCation Bridging

Our new Watershed scale study funded Our new Watershed scale study funded by USDA AFRI (2010 by USDA AFRI (2010 -- 2012)2012)

We will monitorWe will monitorHow do watershed landscape positions How do watershed landscape positions

influence the amounts and forms of hormones influence the amounts and forms of hormones and antibiotics?and antibiotics?How do the seasonal changes over the year How do the seasonal changes over the year

influence the persistence and exports of influence the persistence and exports of hormones and antibioticshormones and antibiotics

Analytical TechniquesAnalytical Techniques

Two Major Techniques

Immunoassays

Chromatography

Enzyme Linked Immunosorbent Assay Enzyme Linked Immunosorbent Assay (ELISA)(ELISA)

BioassaysBioassays

RadioRadio--immunoassaysimmunoassays

Immunoassays

Competitive Immunoassay Competitive Immunoassay technique. technique.

Binding between specific antibody with specific Binding between specific antibody with specific antigen is measuredantigen is measured

Competition for antibody binding sites between Competition for antibody binding sites between antigen enzyme conjugates and the samples (specific antigen enzyme conjugates and the samples (specific hormones)hormones)

The binding between enzyme conjugates and The binding between enzyme conjugates and antibody is measured by coloring agentantibody is measured by coloring agent

ELISA

Pros.Pros.Easy and Quick to use. Easy and Quick to use. Less costlierLess costlier

Cons.Cons.Over estimation due to cross reactivity with organic Over estimation due to cross reactivity with organic substancessubstancesCan not measure conjugatesCan not measure conjugates

Pros and Cons of ELISA

Can be used as Can be used as initial screening toolinitial screening tool

followed by followed by chromatographic techniques. chromatographic techniques.

Over estimation by ELISA our study:Over estimation by ELISA our study:

MethodMethodReduced TillageReduced Tillage No TillageNo Tillage

PPL#PPL# RPL1RPL1 RPL2RPL2 PPLPPL RPL1RPL1 RPL2RPL2ELISA ELISA (ng/L)(ng/L) 77.15 77.15 53.71 53.71 49.97 49.97 41.03 41.03 39.67 39.67 26.41 26.41

LC/MS/MS LC/MS/MS (ng/L)(ng/L) 1.301.30 0.840.84 0.760.76 0.110.11 0.860.86 0.520.52

Dutta et al., submitted

These work based on the mechanism of These work based on the mechanism of activation of the estrogen receptor activation of the estrogen receptor

These are used for determining the estrogenic These are used for determining the estrogenic potencies of individual compoundspotencies of individual compounds

Total estrogenic activity of complex mixtures Total estrogenic activity of complex mixtures of compounds can be determinedof compounds can be determined

Bioassays (YES: Yeast Estrogen Screening)

Gas Chromatography Gas Chromatography –– Mass Spectrometry (GC Mass Spectrometry (GC --MS) MS)

Liquid Chromatography Liquid Chromatography –– Mass Spectrometry (LC Mass Spectrometry (LC --MS)MS)

Detection in mass spectrometry.Detection in mass spectrometry.

LCLC--MS is preferred over GCMS is preferred over GC--MS due to less requirement MS due to less requirement of samples.of samples.

Chromatographic Techniques

Questions?Questions?

Illustration of ELISA TechniqueIllustration of ELISA Technique

304 Worilow Hall 304 Worilow Hall

(The next building on the north)(The next building on the north)

33rdrd

FloorFloor

Documents

Emerging Contaminants (Hormones and Antibiotics) in