Perspectives, 

Research and Development 

on 

“Difficult to Test” Substances

Lawrence Burkhard

NHEERL, MED
• Focus MCCs = Methodology challenging chemicals

– Log Kows > 7

– Low aqueous solubility 

Log KOW 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!
Kow 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!
Kow 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 Kow = 8.23 0.2 L verified 

– 9.23 2 L

– 10.23 20 L

– 11.23 200L

– 12.23 2,000 L 

• Not practical
Kow Measurement for MCCs

• Research Issues & Challenges 

– Generator column (OCSPP 830.7560 )

• Kow - 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 Kow < 5

– Bioavailability not a problem (Freely dissolved = Total)

– Testing fairly easy

• Log Kow 5-7

– Testing becomes difficult

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

• Log Kow > 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 (k2)

– 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 KMet

from in vitro test data

– Compare in vitro estimates to in vivo KMet

measurements
Research Recommendations

for MCCs

• Exposure Analysis

– Improve models

• Training sets for models: Log Kow < 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?