Scott Dyer

The Procter & Gamble Company

Christian Schlechtriem and Christoph Schaefe

Fraunhofer IME

Gordon Cash

USEPA Contributor

Difficult to Test Subs

1

Mention of trade names, products, or services does not convey official Eapproval, endorsement, or recommendation.
Contents

 High Production Volume (HPV) Program – OE

 U.K. = Competent Authority

 Defining the class of materials

 Chainlengths Environmental Sources

 Phys/Chem Properties and Ecotoxicity

 Effect of Alkyl Chain length: Water Sol. and Ec Trials and tribulations Solving the program: Fraunhofer Institute
Aliphatic Alcohol Family

 Aliphatic alcohols category covering linear and 

essentially-linear monohydric alcohols with cachainlengths of C6 to C22. 

 Essentially

-linear alcohols are primarily mono

branched at the 2-position with the branching predominantly methyl.

C

n-C-C-C-C-C-OH

C

m

n=1-16m is predo
Sources of Alcohols in the Environme

Anionic and Nonionic Alcohol-based surfactants

Other consumer products

Other potential inputs containing wax esters or fatty acids

Free fatty alcohols

Water column production

In situ synthesis

WWTP

Terrestrial runoff Plant waxes

Free fatty alcohols

Wax esters Fatty acids

In situ bacterial synthesis

Free fatmeasurwater corisk ass
Water Solubility: SPARC vs. Measured 

10

100

1000

10000

Solubility (mg/l) 

Solubility (mg/L)

0.00001

0.0001

0.001

0.01

0.1

1

10

6 8 10 12 14 16 18 20 

Carbon #

Solubility (mg/l) 

Solubility (mg/L)
Acute Toxicity: Effect of Water Solubi

0.001

0.01

0.1

1

Acute toxicity (mol/l)

Fish DapDapOtheAlga

FisDaDaOth

0.00000001

0.0000001

0.000001

0.00001

0.0001

1 2 3 4 5 6 7 8

logKow

Acute toxicity (mol/l) Alga

AlgaFish InveDapKoneSolu

C13 (Kow 5.3)

Estimated Chronic SAR

AlgAlgFisInvDaKoSo
Trials and Tribulations

 Development of a chronic toxicity program

 Daphnia magna

 Task 1 

 generation of stable solutions

 Contractor #1: well known European contract labor

 Highly experienced in highly hydrophobic chemicals (e.g

 Use of a flow-through generator column

 Results

 Lack of ability to achieve stable concentrations

 Large variability and increased loss through time 

 Evidence of biodegradation

 Task 2 

 seek another laboratory’s expert help

 Fraunhofer 
Initial Dialogue with Fraunhofer

How to maintain concentrations of highly hydroph

quickly biodegrading substances?

How to achieve test solutions as close to the water s

limit as possible? 

Potential chronic effects ~ water solublity limits 

chainlengths >C13

Flow-through failed (why, issues, etc.)

Microbial contamination in the test vessels

Potential Sources: Air, Water, Food, Test Org

High exchange rate would be necessary having 

significant impact on pelagial crustaceans (e.g., 
Performing Daphnia Reproduction 

(OECD 211) with Long

-Chain Fatty Al

© Fraunhofer IME 

Christian Schlechtriem, Uwe Boshof, Heinrich Jürling, Christop
D. Magna Reproduction Test Overvie

The test was performed in tight100 mL - vessels (single exposur

A saturated solution daily generated by st

the test substance in test media under sloconditions under sterile conditions, servehighest concentration and was serially dilprepare the other concentrations. 

The test was performed in tight100 mL - vessels (single exposurto OECD 211). Each day the test organisms wertransferred to fresh media conta

Analysis for the test concentrati

Saturated solution, all freshly psolutions and pooled aged solut
Analysis of Test Solutions 

• All test concentrations were sampled for chemical analy

times a week 

• A 500 mL aliquot of the fresh solutions • After 24 h, at the next renewal, the aged test liquids w

(vessels 1-5 and 6-10)

• The analyte was extracted liquid-liquid partitioning with • Samples were derivatized with MSTFA, duplicate measu

were performed by GC-MS using deuterated n-decanol o

dodecanol as internal standard. 

• The method was validated in Daphnia test medium in th

appropriate concentration range according to guideline SANCO/-825/00 and SANCO/3029/99 (specificity, lineaaccuracy, precision, identity and limit of quantification 

N-Methyl-N-(trimethylsilyl)trifluoroacetamide(MSTFA)
Preparation of Test Solutions: WAF Ap

Add test compound to WAF bottle (loading = mg/L)

Dissolve test compound in solvent (e.g. ethanol) Place aliquot of solution (e.g. 1 mL) into sterilized, still 

(> 50°C) 2 L mixing bottle (wet bottle sidewall only

)

Slowly rotate horizontally to cover a max. area of gl

continue rotation until solvent has evaporated und

Add sterile water obtained by autoclaving or sterile filtr

Note: pH may change by autoclaving

Close bottle, then stir slowly for up to 24h at room temAfter stirring the vessels will be left to settle for at least Saturated aqueous phase were taken out of the drain poThe first fraction (0-100 mL) was discardedPrepare dilution series for lower test concentrationsSaturate the surfaces of the test flasks with eluant, then Fill test flasks, add daphnia and feed algaeRelate toxicity to measured test concentrations (not loa
Pilot Trial I (Dodecanol)

Prior to the OECD 211 studies, several pre-studies includi

GLP Daphnia reproduction pilot study with Dodecanol aPentadcanol were performed to demonstrate the feasibiliof) the study plan.C12 Dodecanol: First Alcohol Studied in the Projec

t

• Initial trials of C12 solutions checked for acute D. magn

and for the concentrations after different loadings

o Concentrations

o Consistent preparation of test solutionso Highest conentration ~ water solubilityo Maintenance of test concentrations over time

o Screen/range-find sensitivities of Daphnia reprod
• Rapid loss of Dodecanol over time

• Provoked a discussion about its kinetics. Kinetics Study Purpose: Determine potential factors responsible f

Dodecanol

Pilot Trial I (Dodecanol): Results, Nex

• Factors Investigated

• Adsorption to glassware• Source of biodegradation

• Air • Algae• Daphnia 
0

20406080

100120

dodecanol concentration (% of 0 4 8 12 16 20 24

initial)

Kinetic 1

AlgaeAlgaeAlgae+DaphnidAlgae+Daphnid

..

Dodecanol Kinetics Study

dodecanol concentration (% of 0 4 8 12 16 20 24

time (h)

0

20406080

100120

0 4 8 12 16 20 
60

80

100120

Kinetic 

Algae Algae Algae Algae

Dodecanol Kinetics Study: Sources of 

0

2040

0 4 8 12 16 20 24

AlgaeDaphDaphWAF WAF 
C12 Kinetics Study: Conclusions

1. Sterile WAF = no loss over time2. For C12-OH, algae provided the primary source of alco

through time due to adsorption and/or biodegradatio

n

Autoclaving improved the situation; initial loss was 

observed

3. Next Steps

⇒Daily renewal of test media⇒At each transfer rinsing the daphnids with sterilized 

water

⇒Decision to perform the C15-OH pre-study using aut

algae 
Pre-Study: Pentadecanol

Objectives: same as with C12

o Concentrations

o Consistent preparation of test solutionso Highest concentration ~ water solubilityo Maintenance of test concentrations over time

o Screen/range-find sensitivities of Daphnia reprod

o Special Notes:

 Lower water solubility  potential for oil drop

resulting in greater variability in concentration

 Discussed used of filtration* to produce 

uniform/saturated solutions 

 Created test solutions with and without filtrati Art: also investigated effect of technician on co

of sample preps

* Apolar 0.2 μm filter (Millex FG, 50 mm, Millipore), which was wetted 

with methanol prior to use to allow the passage of the aqueous solutio
Results of Pre-Study with C15 (Pentad

• Preparations without filtration provided considerably h

concentrations than model predictions.

• This observation raised the question concerning the rela

between the water soluble and non-water soluble test m(droplets) which were analytically detected in the test mcould not be distinguished 

• Could the higher concentrations affect toxicity (acute vs

to Daphnia via physical effects (droplets)? 

Acute non-GLP toxicity tests were performed at higher lowithout filtration (above saturation limit) 

Results: 

• Higher loadings = higher the measured concentrations 

saturation was observed)

• No acute effects were observed (no droplet effect) 
Chronic Pre-Study with Pentadecanol 

Results• All daphnids survived in the controls. However, the reprodu

output was very low (11 per female << 60 as given by OECD 2

• All daphnids seemed to be in bad condition, indicated by we

pigmentation and very small sizes. The time until the first b

very high (13.3 d >> 9 d as given by OECD 211)

Relationship between test substance losses and 

bacteria concentrations end of 24 h intervals

• Test concent

0

102030405060708090

100

1 10 100 1000 10000 100000 100000

0

1E+07

bacteria/mL

losses (%)

• Test concent

correlated wdepletion

• Bacteria > 10

not found up 

• 50-70% loss

adsorption t

• No correlation between substance losses and bacteria density• High densities are always associated with high losses,• Losses below 60% only at low densities (but high losses can occur due to other reasons)
Conclusions of Pre-Study with Pentad

Feeding autoclaved algae to Daphnia did not meet survival reproduction criteriaFinal considerations: live feed, renew test media daily and mbacterial contamination by:

A. Performing the entire test in a clean bench

B. Ensuring an axenic feed algae cultureC. At each transfer rinse daphnids with sterilized dilution 

Using sterile filtered artificial air for gentle aeration at the w

to avoid adverse oxygen depletion

Due to stressful conditions definitive tests should be avoide

winter season (reduced reproductive output)

Differences between C12-OH and C15-OH

• C15-OH adsorbs much more to glass. New glassware redu• C15-OH has to be tested in critical loadings above water s

potentially causing physical stress
Definitive Chronic Studies 

Resulting deviations of the test protocol from OECD 2111. Minimizing degradation of test substance

• Conduct tests under sterile conditions• Daily renewal of test solutions including feed alga• Rinsing Daphnia with sterilized water at transfer

2. Adapted guideline to cope with stress induced by 

degradation of the test substance and counter-measu

• Gentle aeration with sterile-filtered air near the s

the test medium to avoid oxygen depletion

• Modify OECD 211 criteria for 60 offspring/survivi

female by 21 days to utilize historic OECD 202b cr(60 neonates/control)

• Extend study for up to 3 extra days 
Day of Daphnia Study

0 5 10 15 20

Measured Concentration (μg/L)

Running Geometric Mean

0.1

1

10

100

1000

Day of Daphnia Study

0 5 10 15 20

Measured Concentration (μg/L)

0.1

1

10

100

1000

25 μg/L (nominal)500 μg/L (nominal)

Dodecanol

Schäfers C, et al. Environmental p

Definitive Studies: Concentrations/Ti

Day of Daphnia StudyDay of Daphnia Study

0 5 10 15 20

Measured Concentration (μg/L)

Running Geometric Mean

0.1

1

10

100

1000

Day of Daphnia StudyDay of Daphnia Study

0 5 10 15 20

Measured Concentration (μg/L)

0.1

1

10

100

1000

24.4 μg/L (nominal)500 μg/L (nominal)

Tetradecanol Environmental p

long chain alcohPart 2: Structurerelationship for ctoxicity of long cEcotoxicology anEnvironmental S996–1005.
Chronic toxicity of long chain alcohols

Level 1 Level 2 Level 3 Level 4 

C10 Nominal 152 411 1110 3000 

Measured initial % 80.2 80.9 86.7 93.3 Measured total % 15.3 25.3 33.1 40.9% effect on Survival 0 0 0 100% effect on Reproduction 0 8 14 

C12 Nominal 25 69 185 500

Measured initial % 80.8 80.7 88.2 106.8Measured total % 13.5 7.7 7.4 19.1% effect on Survival 0 0 0 60% effect on Reproduction Reproduction

0

8 10

6 

C14 Nominal 24 69 185 500

Measured initial % 40.4 73.9 74.7 73.3Measured total % 6.4 5.2 6.9 15.3% reduction of survival 0 0 0 30% effect on Reproduction 3 8 15 19 

C15 Nominal 30 65 139 300

Measured initial % 50.4 86 75 80Measured total % 6.7 12.0 13.3 21.0% effect on Survival 0 0 0 0% effect on Reproduction 4.3 6.5 13.6 19.9 

Significant effect on SurvivalSignificant effect on Reproduction >=10 %)

Significant effect on Reproduction (< No significant effect
Chronic Toxicity of Long Chain Alcoho

Exposure to multiple measures (21),related to: Arithmetic mean measured initial 

concentration 

10000

(μg/L)Water solubility

 Arithmetic mean m

geometric means oconcentrations 

10000

Water solu

10

100

1000

10 11 12 13 14 15 16

Concentration (μg/L)

Chain length (#C)

EC10 EC50 >NOEC imm EC10 

Ecx...

1

10

100

1000

10 11 12 13 14 

Dark Blue: ImmobilityPink: Reproduction
Conclusions

• Cookbook methods for highly insolubles was not succe

• Alcohols confirmed Narcosis/Non-Polar organic QSAR• The batch-renewals = best approach for highly bioegrad

• Minimize bacterial growth• Heroics (in view of the sponsors)
Publications Telling the Full Story

Series of articles in Ecotoxicology and Environmental Safety, Reprints of the Schaefers et al, 2009 are available at the Work
Fraunhofer Institute for Molecular Biology 

and Applied Ecology IME 57392 Schmallenberg, Germanychristian.schlechtriem@ime.fraunhofer.dewww.ime.fraunhofer.de

Thank You