# Presentation: 060297

## Long Term Goals 1 and 2:Exposure Assessment and Risk Management of EDCs

- Marc A. Mills, PhD
- Land Remediation and Pollution Control Division
- National Risk Management Research Laboratory
-                                                           September 14, 2007

## Objective of Talk

- Provide an update on the progress of research in support of EDC LTG 1 and LTG 2
- Provide examples of research activities that support these LTGs
- Answer any questions

## Long Term Goal 1 - Provide a better understanding of the science underlying the effects, exposure, assessment, and management of endocrine disruptors 

*LTG 1 APGs covered in this presentation (from 2003 MYP):*
**Evaluate exposure methods, measurement protocols, and models for the assessment of risk management efficacy on EDCs (2008)**
**Identify risk management EDCs research (2008)**
**Evaluate at least three existing risk management tools to reduce exposure to EDCs (2008) **
**Develop at least two new risk management tools to reduce exposure to EDCs (2009)**

## Linkage and Timeline for APGs to Meet 

**                                              ****Long Term Goal 1 (2003)**

- *Receives input from FY04 and FY07 APGs in LTG 2
- ** Receive input from FY08 APG in LTG 2

## LTG 1 APG:Evaluate exposure methods, measurement protocols, and models for the assessment of risk management efficacy on EDCs (2008)

*Research to support this APG:*

## Bioinformatics Approach to Emerging Contaminants Research

**USEPA-ORD-NERL-Cincinnati**

_**Objective:**_
- Prioritize future ecological monitoring research by focusing on key aspects of analytical chemistry, occurrence, and monitoring, and ecotoxicity
_**Impact:**_
- Guiding future ORD, industry and science community research efforts in EDCs and emerging contaminants

- The Bioinformatic Process

- A Result of the Process

## Analytical Methods Development For Ecologically Relevant Pharmaceuticals& Metabolites/Degradation Products

**Analytical Methods Development For ****Ecologically Relevant Pharmaceuticals****& Metabolites/Degradation Products**
**USEPA-ORD-NERL-Cincinnati**

**Sample Collection**

- Sample volume – 250 mL, 500 mL, or 1-L, add isotope surrogates, Filter

**Sample Preservation**

**Preservation Options**
- Acid (pH 2) or alternative preservatives for labile compounds (sodium azide diazolidinyl urea or sodium pyrithione) to prevent microbial degradation
- Ascorbic acid or ammonium chloride will be added to remove residual chlorine
- Will investigate the effectiveness of enzyme hydrolysis (instead of acid)

**Dechlorination of Effluents**

**Hydrolysis of any remaining conjugates?**

**Extraction Options**
- Oasis HLB - reverse phase extraction cartridge
- Oasis MCX - mixed mode sorbent with reverse phase and cation exchange capacities

**UPLC-MS/MS**

_**Objective:**_
- Develop methods for waters, sediment, and tissues to conduct fate and occurrence studies.  Target analytes were identified using the bioinformatics approach and Program office priorities.
_**Impact:**_
- Assist the Program Offices, Regions, industry, and scientific community in designing and implementing exposure monitoring and assessment programs for selected emerging contaminants

## Use of Proteomics for Identifying Exposure Issues from MixturesEPA-ORD-NERL, EPA-ORD-NHEERL, Academia, Industry

_**Objective:**_
- To identify protein indicators resulting from exposures to mixtures of EDCs.  Link biomarkers of exposure with biomarkers of effects (Comp Tox Small Fish Study)
_**Impact:**_
- Provide Regional and State risk assessors with proven, rapid methods for characterizing early indicators of exposure to EDCs

## Assessment of the Occurrence and Potential Risks of EDCs in Discharges from Concentrated Animal Feeding OperationsEPA-ORD NHEERL, NERL, NRMRL, NCEA, Academic Co-operators

**Science Questions**:
- Determine how and to what degree human and wildlife populations are exposed to EDCs.
- Determine what effects are occurring in exposed human and wildlife populations.
- Determine what are the major sources and environmental fates of EDCs.
- Determine how unreasonable risk can be managed.

- Trenbolone Acetate

- Estradiol

## Possible EDC Exposures to Endangered and Declining Fish Populations (Pallid Sturgeon and Silvery Minnow)NERL Cincinnati, EPA-Reg 7, State of NM, US Fish and Wildlife Service 

_**Objective:**_
- Identify sources of EDCs to endangered species habitats
- If sources are found, deploy FHMs in endangered species habitats below sources
- Measure gene expression in indigenous fish in these habitats
_**Impact:**_
- Help the Federal and State services develop recovery program for endangered species
- Assist Federal and State service develop water quality standards for these EDCs

**Vtg Expression in male FHM livers following in-lab**
**exposure to Missouri River water and WWTP effluents**

## LTG 1 APG:Identify risk management EDCs research (2008)

*Research to support this APG:*
- Revision of RME for second round of EDCs of concern 2005 NRMRL
- Revision of RME for third round of EDCs of concern 2008 NRMRL

- X

- *This effort has been stopped due to funding reductions.  Planning is continuing as part of a larger, more collaborative effort between the ORD laboratories.  The need for separate NRMRL planning has also been reduced due to a growing body of literature for EDCs and RM.

## LTG 1 APG:Evaluate at least three existing risk management tools to reduce exposure to EDCs (2008) 

***Research to support this APG:***

## Evaluation of Drinking Water Treatment Technologies for Removal of Endocrine Disrupting Chemicals (EDCs)EPA-ORD-NRMRL

**Evaluation of Drinking Water Treatment Technologies for Removal of Endocrine Disrupting Chemicals (EDCs)****EPA-ORD-NRMRL**
- Objective**: ****Determine the ability of conventional and advanced drinking water treatment processes to remove EDCs from source waters**

- Project Design:
**Develop appropriate analytical methods to identify and quantify the target compounds in several water matrices.  **
**Evaluate the applicability of a bioassay to detect the presence/removal of endocrine activity in water samples pre- and post-treatment.**
**Conduct bench-scale evaluations of various drinking water treatment processes used in conventional and advanced drinking water treatment.  **

## Persistence Of Wastewater Compounds During Drinking Water Treatment: Removal & Potential ExposureEPA-ORD-NERL Cincinnati, USGS

**Objective:** Examine drinking water facilities impacted by human wastewater (due to proximity to WWTP discharges, or reclaimed water facilities) to determine the “worst case scenario” of persistence of wastewater compounds (esp. pharmaceuticals)  through drinking water treatment.

- USGS is developing two new methods.   The first will incorporate pharmaceuticals not currently included in their methods; the second will focus on disinfection/ degradation by products of compounds known to be present in the raw/ source waters
- Sampling is occurring in two rounds.  First Round: The raw and finished water of 10- 15 drinking water treatment facilities will be sampled to determine gross removal.  Second Round: At least quarterly for one year, 2- 4 drinking water treatment facilities will be sampled throughout the treatment process to gauge the effectiveness of each step, and determine any effects of seasonality on the compounds found in the water

## National Wastewater Treatment Plant Endocrine Disrupting Chemicals Screening Study

**Grab or Composite samples Collected from 50 Effluents, in 9 Regions/23 states**
- Fathead Minnow (FHM) exposed to effluents for 24 hours
- Vg measured by Quantitative Polymerase Chain Reaction (QPCR) on RNA extracted from livers of exposed fish
- Chemical analyses for natural and synthetic estrogens –NRMRL

**National Wastewater Treatment Plant Endocrine Disrupting Chemicals Screening** **Study**
**EPA-ORD-NERL, EPA-ORD-NRMRL, EPA Regions, States, WWTP operators**

**Findings**
- 26% of effluents caused up-regulation of Vg expression in male FHMs Estrogenic exposure
- 4% of effluents caused down-regulation of Vg expression in female FHMs Androgenic exposure
- Chemical analysis confirmed estrogenic compounds in WWTP effluents

## OW POTW Wastewater SurveyEPA-OW, EPA-ORD-NRMRL, EPA-ORD-NERL

- Integration of OW survey of POTW influent/effluents with ORD research on effluents and biosolids
- Collaboration with:
- EPA/OW/OST/EAD
- NRMRL – SSMB
- NERL – MIRB
- Information shared from NERL-NRMRL effluent screening study

- _Experimental Design_
- Survey up to 9 POTWs for operational data, demographics
- Characterize splits of 24-hour composite samples on influent and effluent samples
- OW - ~450 chemical and biological analytes
- NRMRL – Steroid hormones and APs
- NERL  - Exposure assays on effluents for ED activity

## Anaerobic Digester Project NRMRL- Cincinnati, EPA-Reg5, EPA-GLNPO, MWRDGC

***Anaerobic Digester Project ******NRMRL- Cincinnati, EPA-Reg5, EPA-GLNPO, MWRDGC***

**Objective:**
- To evaluate the removal efficiency of selected EDCs in sludge digesters**.**
- A series of parallel sludge digesters will be sampled over time for the influent and effluent concentrations of selected EDCs.
- In parallel, an inert, non-toxic tracer study will be performed to evaluate residence time distribution and effective reactor volume.
**Target analytes**
**Alkylphenols (APs) and Bisphenol **
**Hormones**
**Collaborations:**
**USEPA NRMRL**
**USEPA REG 5 Water Division**
**USEPA REG CRL**
**MWRDGC**

**Experimental Design:**
- Reactors
- Three replicate digesters
- Sampling over 3 solids retention times, ~ 60 days
- Digester operations
- Digester design parameters
- Daily operational parameters
- Feed schedules, flows, and loadings
- Operating characteristics (temp, pH, VSS, etc)
**Sampling **
- Daily sampling:  one 1-liter grab samples collected during each 8-hr shift (3/day) and composited
**Status**
- QAPP in progress
- Expected to start study soon

- Calumet Water
- Reclamation Plant

## LTG 1 APG:Develop at least two new risk management tools to reduce exposure to EDCs (2009)

*Research to support this APG:*

## Strategies to Suggest Substitutes for Endocrine Active SubstancesEPA-ORD-NRMRL, Academia

_**Objectives:**_
- Substitutes for existing EDCs require:
- Chemical properties that maintain desirable performance
- Significantly lower undesirable endocrine activity
- Computational approach _developed_
- Methodology tested with alkylphenol ethoxylate surfactants
- Satisfactory substitute found
_**Impact:**_
- Process may be used by EPA and industry to suggest suitable chemical substitutes for replacing endocrine active compounds

_**Status:**_
- Research area has been discontinued under the EDC MYP due to funding limitations

## On-site WWT ResearchEPA-ORD-NRMRL, EPA-ORD-NERL

**Grailville Constructed Wetland**
- Objective:  Constructed Wetland designed/operated for nutrient/pathogen removal will be evaluated for efficacy at managing EDCs
- Collaborators
- NRMRL WSWRD
- NRMRL LRPCDNERL - MIRB

**Status**
- Samples collected for screening the EDC concentrations
- Evaluating matrix interferences
- QAPP in progress based on EDC levels in influent/effluents

**Future planned research** for On-site WWT
- Septic systems (pilot systems under construction)
- Innovative treatment

## Future Work on Exposure and Risk Management of EDCs for LTG 1

_**Exposure **_
- Occurrence of EDCs and Pharmaceuticals from CAFOs
- Develop -omic and chemical measures of exposure
- Characterize fate and transport of EDCs
- Enhance collaborations with STAR Coops
- Source identification of EDCs and Pharmaceuticals
- Analytical Chemistry
- Biomarkers of exposure
- Endangered Species exposure to EDCs and emerging contaminants
- Develop an bioinformatics approach(es) to:
- Identify veterinary pharmaceuticals potentially discharged from CAFOs and over the counter (OTCs) pharmaceuticals in wastewater
- Further evaluate bioavailability and clearance in wildlife

## Future Work on Exposure and Risk Management of EDCs for LTG 1

_**Risk Management**_
- CAFO
- Characterization of existing BMPs and innovative treatment
- Further collaborations with Coops
- On- site wastewater treatment
- Characterization of existing technologies
- Evaluation of innovative technologies
- Centralized WWT
- Unit operations
- Biosolids management
- Drinking water
- Unit operations
- Large scale systems

## Long Term Goal 2 - Determine the extent of the impact of endocrine disruptors on humans, wildlife, and the environment

*LTG 2 APG covered in this presentation **(from 2003 MYP)**:*
**Characterize sources of exposure and environmental fates of EDCs (2008)**

## Linkage and Timeline for APGs 

**Linkage and Timeline for APGs **
**to Meet Long Term Goal 2 (2003)**

## LTG 2 APG:Characterize sources of exposure and environmental fates of EDCs (2008)

*Research to support this APG:*

## Ohio River EDC Instream Effects  NERL-Cincinnati, NRMRL-Cincinnati, ORSANCO, USGS

**Objective:**
- Establish linkages between EDC occurrence and negative effects in wild fish
- Characterize the impact of EDCs from WWTP effluents on indigenous and deployed fish
- Determine the extent of EDC exposure/effects using probabilistic study design in a Great River system
- Integrate molecular/cellular indicators with traditional water quality metrics

-                **Experimental Design**
**   **At selected POTW locations on the Ohio River, sampling stations upstream and downstream of discharge will be selected for the following:
- Collect water for lab exposure and gene expression assays
- Collect indigenous fish for measuring gene expression and histopathology
- Deploy FHM for gene expression assay
- Collect water samples for chemical characterization of hormones
- Evaluate passive sampling devices for characterizing EDC exposures for deployed and indigenous fish

## Land Application of BiosolidsEPA-ORD-NRMRL, Academia 

**North Carolina Study**
- Anaerobically digested and lightly limed biosolids
- Applied to fescue field in Sept 2004
- Measure APEO concentration
- Background: -5 wks, -1day
- After application: 0 day, 2 wks, 4 wks, 9 wks, 14 wks
- depths: 0-5 cm, 10-15 cm, 20-25 cm
**Future Biosolids and EDCs research**
- Evaluate other biosolids types
- Include steroid hormones along with the alkylphenols
- Continue to leverage against microbial biosolids studies

## Evaluating the potential for EDCs from CAFOs to contaminate GWEPA-ORD-NRMRL

- We have limited field data that show that swine CAFOs can contaminate ground water with estrogens, but:
- field sites represent worst case scenario, therefore, need to investigate additional sites
- need to monitor long-term fate in ground water at this site
- QA/QC concerns regarding swine lagoon waste sample preservation and storage have been addressed:
- formaldehyde preservation required in field sampling
- estrogen concentrations in refrigerated, centrifuged, filtered samples stable for up to 14 days
- estrogen concentrations in refrigerated SPE extracts also 		stable for up to 14 days; much better reproducibility

## LTG 2 .....to be continued in the next presentation

**Any questions at this point?**