# Perspectives, Research and Development

on “Difficult to Test” Substances Lawrence Burkhard NHEERL, MED

# • Focus MCCs = Methodology challenging chemicals



## – Log K ow 

## s > 7

– Low aqueous solubility

Log K OW estimates EPI - Suite Decabromodiphenyl ether 12.1 Decabromodiphenyl ethane 13.6 Aqueous solubility estimates EPI - Suite Decabromodiphenyl ether 2.6 ng /L or 28 fg /L Decabromodiphenyl ethane 0.97 ng /L or 1.2 fg /L

# Aqueous Solubility Measurement for 

# MCCs

• Aqueous Solubility

– OCSPP 830.7840 Column Elution, Shake Flask

– OECD 105 Column Elution, Shake Flask

– OCSPP 830.7860 Generator Column

• Challenges

• Micro

- emulsions, micro

- particulates, adsorption to testing equip ...

• Measurements can be done

– Generator column

– Assume

• 1 ng /L Analytically doable

• 1 pg/L Analytically possible

• 1 fg /L In theory, analytically possible Very difficult

• Shake Flask & Column Elution

• For MCCs, waste of time and resources!

# K ow 

# Measurement for MCCs

• OCSPP 830.7750 Shake Flask

• OECD 107 Shake Flask

• OCSPP 830.7570 Estimation via HLPC

• OECD 117 Estimation via HLPC

• OCSPP 830.7560 Generator Column



## • OECD 123 Slow Stir

• Shake Flask & Estimation via HPLC

– For MCCs, waste of time and resources!

# K ow 

# Measurement for MCCs

• OCSPP 830.7560 Generator Column

• OECD 123 Slow Stir

• Challenge

• Large volumes of water phase

• Micro

- emulsions, micro

- particulates, adsorption to testing equip ...



### • Slow Stir & Generator Column

– PCB 209 exp. Log K ow = 8.23 0.2 L verified

– 9.23 2 L

– 10.23 20 L

– 11.23 200L

– 12.23 2,000 L



### • Not practical

# K ow 

# Measurement for MCCs

• Research Issues & Challenges

– Generator column (OCSPP 830.7560 )

• K ow - developed and validated

– 4.6 mm ID column 1 ml/min

• Higher flows, bigger columns

– 10 mm ID 4.7 ml/min

– 25 mm ID 29.5 ml/min



### – Higher temperatures

– Radiolabeled chemicals

– Increasing solubility in water phase

• Addition of ??? & extrapolation to 0% Days of Collection Column ID mm Volume 4.6 10 25 20 L 14 2.9 0.47 200 L 140 29.4 4.7 2,000 L 1,400 294 47

# BCF Testing

• BCF testing (OECD 305, OCSPP 850.1710 & 850.1730)

– Waterborne exposure

• Log K ow < 5

– Bioavailability not a problem (Freely dissolved = Total)

– Testing fairly easy

• Log K ow 5

- 7

– Testing becomes difficult

– Bioavailability starts to limit chemical uptake (Freely dissolved < Total)

• Log K ow > 7

– Almost impossible to perform

• Low aqueous solubility

• Binding to DOC and POC, adsorption to testing apparatus, fish slime

... • Freely dissolved << Total

– Little environmental relevance For MCCs, waste of time and resources!

# BCF Testing for MCCs

• Updated OECD 305

– Dietary exposure (ring test done)

– Provides BMF

– High environmental relevance



### • Research Issues & Challenges

– Measuring elimination rate constant (k 2 

### )

– Complications from growth dilution



### – Understand impacts of

• Feeding rate

• Food composition

• Dosing form (spiked fish chow vs naturally incorporated)



### – No guidance for regulatory decisions

# Toxicity Testing for MCCs

• Aqueous exposures

– In theory, can be done

– Generating solutions extremely difficult

– Time to accumulate enough chemical for an effect often exceeds duration of the test

– Use of solvents and/or surfactants

• Exceed aqueous solubility

• Unanticipated toxic effects of solvents/surfactants

• Hard to know bioavailable (freely dissolved) concentration of the chemical

– Is partitioning to DOC and POC different?

– Acute Toxicity Testing Protocols (e.g., 96 hr EC50) may substantially underestimate the true toxicity of these compounds

# Toxicity Testing for MCCs

• Recommendation

– Start here

– Dietary and/or longer term aqueous exposures



### • Research Issues & Challenges

– What effects

• Chronic endpoints: Growth, reproduction ...

– Adverse Outcome Pathway ( AOP )

– More complex outcomes and many potential endpoints



### – Understand impacts of

• Dietary

– Dosing form (spiked vs naturally incorporated)

– Feeding rate

– Food composition

• Water

– Freely dissolved ( bioavailable chemical) in water

# Research Recommendations 

# for MCCs:

• Bioaccumulation

• Conduct dietary exposures with MCCs

– Resolve uptake and depuration time windows

– Relevance of feeding rate, food composition, and dosing form

– Measure assimilation efficiencies



### • Determine if existing dietary (GI) assimilation models 

### are adequate

• Identify meaningful BMF decision point

• Terrestrial species

# Research Recommendations 

# for MCCs:

• Persistence Testing

– Improve understanding of persistence in the environment based on:

• Molecular structure (Inherent persistence)

• Environmental factors (Situational persistence)

• Ongoing loadings (Pseudo persistence)



### – Improve understanding of biodegradation processes

– Evaluate ready biodegradation tests

• Bioavailability and spiking issues



### – Generate high quality data for model compounds

# Research Recommendations 

# for MCCs

• Biotransformation Testing

– Conduct in vitro depletion assays

– Evaluate dosing issues (solvents vs passive sampling)

– Establish model

- based methods to estimate K Met 

## from in vitro test data

– Compare in vitro estimates to in vivo K Met 

## measurements

# Research Recommendations 

# for MCCs

• Exposure Analysis

– Improve models

• Training sets for models: Log K ow 

### < 7



### • MCC predictions: Huge extrapolations



## – Validate models with field measurements

– Improve environmental characterization

• What loadings?

• What environments?

• What biodegradation rates?



### • Food web transfer

# ORD’s Chemical Safety for 

# Sustainability Research Plan (CSS)

• Project: Integrated Modeling for Ecological Risk Assessment CSS11.02

– Project Lead: Sandy Raimondo, NHEERL

• 3 Tasks:

– Internal Dose

– Improving assessments of internal dose for methodologically challenging compounds (MCC)

– Ecological Effects

– Developing models to transcribe effects into ecological context

– External Dose

- accounting for external dose across various spatial scale and diverse scenarios.

• 3 Case Studies

– Ecological models for endangered wildlife populations exposed to pesticide

– Integrated Exposure and Effects Modeling of MCCs

– Pesticide impacts for aquatic endangered species

# • Questions?