Confidential and Privileged 

 

 )O 

Common Synonyms: 

Physical Properties

Log Kow measured - OPPTS 830.7560 

Water Solubility mg/L measured - OPPTS 830.7840/7860 

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Confidential and Privileged 

Endpoint Assay Toxicity Value

*

Degradability

Chemical Oxygen Demand: ppm O2 / mg measured - dichromate oxidation 

Ready Biodegradability OECD 301B % ThCO2 OECD 301B, silica addition 

Treatability study in progress –

Biodegradability study in progress –

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 DETERMINATION OF THE n-OCTANOL/WATER 

PARTITION COEFFICIENT USING THE GENERATOR COLUMN METHOD 

U.S. EPA Product Properties Test Guidelines 

OPPTS 830.7560, Partition Coefficient (n-Octanol/Water), Generator Column Method 

AUTHORS: 

STUDY INITIATION DATE: 

STUDY COMPLETION DATE: 

SUBMITTED TO: 

Page 1 of 43 

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TABLE OF CONTENTS 

Title/Cover Page ..................................................................................................................................... 1 

Good Laboratory Practice Compliance Statement ................................................................................. 2 

Quality Assurance Statement ................................................................................................................. 3 

Report Approval ..................................................................................................................................... 4 

Table of Contents ................................................................................................................................... 5 

Summary ................................................................................................................................................ 8 

Introduction ............................................................................................................................................ 9 

Objective ................................................................................................................................................ 9 

Experimental Design .............................................................................................................................. 9 

Materials and Methods ........................................................................................................................... 9 

 Test Substance ......................................................................................................................... 10 

Solvents and Reagents ............................................................................................................. 10 

Saturated Solvent Preparation ................................................................................................. 10 

 Stock Preparations ................................................................................................................... 10 

 Standard Preparations .............................................................................................................. 11 

Preparation of Generator Column ........................................................................................... 11

 Apparatus Configuration ......................................................................................................... 12 

 Analytical Method ................................................................................................................... 12 

Calibration Curve and Limit of Quantitation .......................................................................... 13 

Matrix Blank and Matrix Fortification Samples ..................................................................... 14 

 Example Calculations .............................................................................................................. 14 

Results and Discussion ......................................................................................................................... 15 

Generator Column and Measured pH ...................................................................................... 15 

Measured Phase Concentrations and Calculated Partition Coefficient ................................... 16 

Conclusions .......................................................................................................................................... 16 

References ............................................................................................................................................ 17 

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TABLE OF CONTENTS 

- Continued - 

TABLES

Table 1 HPLC Operational Parameters .......................................................................................... 18 

Table 2 Quality Control Samples of in n-Octanol and Aqueous Matrices ...................... 19 

Table 3 Measured Concentrations of in n-Octanol Solution Supernatant ....................... 20 

Table 4 Measured Concentrations of in Aqueous Generator Column Samples ............... 21 

Table 5 Calculated n-Octanol/Water Partition Coefficients (Pow) for at 25oC ................. 22 

FIGURES

Figure 1. Diagram of generator column ........................................................................................... 23 

Figure 2. Diagram of apparatus configuration ................................................................................. 24 

Figure 3. Method outline for the processing of in n-octanol/water generator column 

aqueous samples ............................................................................................................... 25 

Figure 4. Calibration curve for the analysis of by HPLC/UV ............................................ 26 

Figure 5. Representative chromatograms of low- and high-level calibration 

standards for HPLC/UV analysis ...................................................................................... 27 

Figure 6. Chromatogram of the n-octanol matrix blank sample ( .......... 28 

Figure 7. Chromatograms of the aqueous matrix blank and matrix fortification samples . 29 

Figure 8. A representative chromatogram of an n-octanol test solution sample 30 

Figure 9. A representative chromatogram aqueous generator column sample ....... 31 

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SUMMARY 

STUDY TITLE: Determination of the 

n-Octanol/Water Partition Coefficient Using the Generator 

Column Method 

SPONSOR:

SPONSOR’S REFERENCE NO. 

STUDY MONITOR

TESTING FACILITY:

LOCATION OF STUDY, RAW

DATA AND A COPY OF THE FINAL 

REPORT: 

TEST SUBSTANCE: 

TEST DATES: Experimental OECD Start –

Experimental EPA Start – 

Experimental Termination – 

TEST SYSTEM: n-Octanol and HPLC-grade reagent water 

RESULT: The n-octanol/water partition coefficient (Pow) of 

at 25oC was determined by the generator column method. The concentration of 

 in the supernatant of the centrifuged n-octanol solution used to coat the 

generator column was determined by taking triplicate aliquots of the supernatant for 

volumetric dilution initially in acetonitrile (ACN) and finally in 1:1 (v/v) acetonitrile 

(ACN):H2O into the calibration range of the high performance liquid 

chromatography with ultraviolet detection (HPLC/UV) methodology. Three aqueous 

samples were collected from the generator column into an equal volume of ACN and 

directly analyzed by HPLC/UV. had an approximate retention time of 

min. The mean calculated partition coefficient (Pow) was

which gives a log10 Pow of . 

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INTRODUCTION 

A study was performed to determine the n-octanol/water partition coefficient (Pow) of 

 at 25°C. The study was conducted by for 

, at the analytical chemistry facility in . The 

test was performed using HPLC-grade reagent water saturated with n-octanol and the generator column 

method. The generator column was prepared on and definitive samples were collected 

on the same day. Raw data generated by and a copy of the final report are filed 

under Project Number archives located on the site. The original 

signed report will be maintained by 

OBJECTIVE 

The objective of this study was to experimentally determine the n-octanol/water partition 

coefficient of using the generator column method. 

EXPERIMENTAL DESIGN 

A single generator column was prepared for the definitive test. The column was packed with an 

inert support and loaded with a nominal 1.0% (w/w) solution of the test substance in n-octanol. Dilutions 

of the n-octanol stock supernatant used to coat the generator column support were analyzed to determine 

the actual concentration of in the n-octanol phase. The column temperature was maintained at 

25 ± 0.1°C and reagent water (saturated with n-octanol) was pumped through the same prepared column 

at approximately 1.0 mL per minute to partition with the n-octanol phase. Three samples of the aqueous 

phase generator column effluent were collected and analyzed to determine the concentration of 

in the aqueous phase fractions. The Pow for was calculated from the ratio of the concentration of 

the solute in the n-octanol and aqueous phases. 

MATERIALS AND METHODS 

The study was conducted according to the procedures outlined in the protocol, “ 

Determination of the n-Octanol/Water Partition Coefficient Using the Generator 

Column Method.” The study was also performed based on procedures in U.S. EPA Product Properties 

Test Guidelines, OPPTS 830.7560, Partition Coefficient (n-Octanol/Water Generator Column Method

(1). 

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Test Substance 

The test substance was received from , and was 

assigned . The test substance was identified as 

 Batch number – An expiration date of was provided by the Sponsor. A 

purity of % was obtained from the Certificate of Analysis provided by the Sponsor (Appendix 2). 

The test substance was stored under ambient conditions in the dark. 

Solvents and Reagents 

All solvents and reagents were ACS reagent-grade or better. 

Solvent/Reagent Manufacturer/Lot 

Reagent Water: HPLC-Grade, Burdick & Jackson, Lot Nos. DH268-E and DH268-F 

(equivalent to ASTM Type II Designation D1193-06 (2)) 

n-Octanol Sigma-Aldrich, Lot No. SHBB3592V 

Methanol (MeOH): HPLC-Grade, Burdick & Jackson, Lot No. DH300 

Acetonitrile (ACN): HPLC-Grade, Burdick & Jackson, Lot No. DG852 

Chromosorb WHP: 100-120 mesh, Supelco, Lot No. 345 

Phosphoric Acid (H3PO4): Aldrich/Sigma-Aldrich, Lot No. MKBH9674V 

Saturated Solvent Preparation 

Mutually saturated n-octanol and reagent water were prepared prior to conducting the test. The 

solvents were mutually saturated by combining 200 mL of n-octanol and 700 mL of reagent water in a 

French square bottle. The bottle was placed in an agitating water bath (Boekel, Cat. No. 290400) and 

gently agitated for approximately 0.7 days at a temperature of 25°C. After the shake period, the contents 

of the bottle were decanted into a separatory funnel and the phases were allowed to separate. The 

saturated n-octanol phase and water phases were then each drained into separate glass bottles. The 

separated n-octanol and water saturated solvents were stored under ambient conditions. Before use in the 

generator column, the saturated water was equilibrated at the test temperature (25oC) in a constant 

temperature water bath. 

Stock Preparations 

A primary stock solution of the test substance was prepared at a nominal concentration 

of 1.00 mg a.i./mL by accurately weighing grams (weight adjusted for % purity) of the test 

substance in a beaker using an analytical balance (Sartorius CPA225D). The test substance was dissolved 

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in methanol, quantitatively transferred to a 100-mL volumetric flask and brought to final volume with 

methanol. The 1.00 mg a.i./mL stock solution, identified as , was used to prepare a set of test 

substance secondary stock solutions in methanol using the following dilution scheme: 

Stock 

Concentration 

(mg a.i./mL) 

Aliquot 

(mL) 

Final 

Volume 

(mL) 

Secondary Stock 

Concentration 

(mg a.i./mL) 

1.00 

0.100 

0.0100 

10.0 

10.0 

10.0 

100 

100 

100 

0.100 

0.0100 

0.00100 

Standard Preparations 

The 1.00 mg a.i./mL (1000 mg a.i./L) stock solution was used to prepare a set of 

HPLC/UV calibration standards in 50:50 (v/v) ACN: reagent water using the following dilution scheme: 

Stock 

Concentration 

(mg a.i./L) 

Aliquot 

(μL) 

Final 

Volume 

(mL) 

Standard 

Concentration 

(mg a.i./L) 

1000 

1000 

1000 

1000 

1000 

1000 

25.0 

50.0 

100 

250 

500 

1000 

100 

100 

100 

100 

100 

100 

0.250 

0.500 

1.00 

2.50 

5.00 

10.0 

Preparation of Generator Column 

The generator column was supplied by At-Mar Glass Co. (Kennett Square, PA) and was 

manufactured based upon the specifications listed in OPPTS Guideline 830.7560 (1). The glass column 

consisted of three sections of fused tubing of the following dimensions (approximate length x outer 

diameter): (1) 20 cm x 6 mm; joined to (2) 5 cm x 9 mm; joined to (3) 3 cm x 6 mm. The entire column 

was enclosed in a water jacket for temperature control. A diagram of the generator column is presented in 

Figure 1. 

The inert support material used in the generator column was Chromosorb WHP (100-120 mesh) 

(Supelco, Lot 345). A plug of silanized glass wool (Supelco, Lot V0446) was placed in one end of the 

20-cm long 6 mm diameter section of the generator column (just above the enlarged section, Figure 1). A 

sufficient quantity of the support material was poured into the column with gentle tapping/vibration, to 

fill most of the 20-cm long section of the column. Another plug of glass wool was used to retain the 

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support material. The column’s water jacket was connected to a pump immersed in a constant 

temperature water bath and the column was allowed to thermally equilibrate for a minimum of 30 minutes 

at 25°C 

An approximately 1% (w/w) solution of test substance in n-octanol was prepared by 

combining g of test substance with g of n-octanol, both weighed using an analytical 

balance, for a total combined mass of g. The solution was sonicated for approximately 

five minutes to ensure dissolution of the test substance and centrifuged at a setting of 14,000 rpm for 

10 minutes to minimize any undissolved test substance in the supernatant. To load the generator column, 

an aliquot of the n-octanol test substance solution supernatant was poured into the packed column and 

gentle suction was applied at the bottom of the column. After letting excess solution drain from the 

column, the column was then back-flushed with reagent water saturated with n-octanol to remove any 

entrapped air and excess n-octanol. The column was then re-plumbed to the definitive configuration 

(Figure 2) and allowed to equilibrate at a flow rate of approximately 0.5 mL/min for a minimum of 

30 minutes before collecting samples. 

Apparatus Configuration 

A constant temperature water bath was used to maintain the test temperature (25.0 ± 0.1°C) 

throughout the experiment. The temperature of the water bath was monitored using a min/max 

thermometer. A submersible pump placed in the constant temperature water bath was used to direct bath 

water maintained at 25.0°C into the jacket surrounding the generator column. 

An Erlenmeyer flask was used as a reservoir for the reagent water saturated with n-octanol that 

was pumped through the generator column. The flask was immersed in the constant temperature water 

bath. The inlet line for a Jasco PU-1580 solvent delivery pump was placed in the Erlenmeyer flask, and 

this pump was used to control the flow of reagent water through the generator column. A diagram of the 

apparatus configuration is presented in Figure 2. 

Analytical Method 

The analytical methods for the determination of in n-octanol and water were developed 

by . To determine the concentration of in the supernatant of the 1% 

nominal (w/w) solution in n-octanol used to coat the column and partition with the aqueous phase, three 

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aliquots of the supernatant were volumetrically diluted in ACN followed by dilution in 1:1 ACN:water 

using gas-tight syringes and volumetric flasks. Aliquots from each of the final dilutions were submitted 

for determination of by HPLC/UV. 

For the aqueous phase analysis, three generator column aqueous samples pumped at a known and 

fixed flow rate (1.0 mL/min) were collected from the generator column directly into 10.0-mL volumetric 

flasks pre-filled with 5.00-mL of ACN. The resulting diluted aqueous samples had an overall dilution 

factor of 2.00. An aliquot from each diluted aqueous sample was transferred to an autosampler vial and 

submitted for determination of by HPLC/UV. An additional aqueous sample was collected 

without dilution in ACN for measurement of pH. The minimum and maximum temperatures of the 

constant temperature water bath were recorded for each aqueous sample collected. Details of the aqueous 

sample collection and processing are provided in Figure 3. 

Concentrations of in the samples were determined using an Agilent Series 1100/1200 

HPLC equipped with an Agilent Series 1200 variable wavelength UV detector. Chromatographic 

separations were achieved using a Poroshell 120 EC-C18 column (50 x 3.0 mm, 2.7-m particle size). 

The instrument operational parameters are summarized in Table 1. 

Calibration Curve and Limit of Quantitation 

 calibration standards of in 1:1 ACN: water, ranging in concentration from 0.250 to 

10.0 mg a.i./L were analyzed concurrently with the diluted n-octanol and water samples. The calibration 

set was injected at the beginning and end of the analytical sequence with one calibration standard injected 

after every test samples. A linear regression equation was generated for the analytical sequence 

using the peak area response (Figure 4) versus the respective concentrations of the calibration 

standards. The concentration of in the samples was determined by substituting the peak area 

response of the samples into the linear regression equation. The calibration curve is presented in 

Figure 4. Representative chromatograms of low- and high-level calibration standards are presented in 

Figure 5. 

The limit of quantitation (LOQ) for this analysis was defined using the low-level calibration 

standard equivalent definition, however, the method was capable of quantitation of at 

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significantly lower concentrations in n-octanol solutions. The LOQ for the determination of in 

the n-octanol phase was , calculated as the product of the concentration of the low-level 

calibration standard (0.250 mg a.i./L) and the dilution factor of the n-octanol phase matrix blank sample 

(2000). The method LOQ for the determination of in the aqueous phase was 

calculated as the product of the concentration of the low-level calibration standard (0.250 mg a.i./L) and 

the dilution factor of the aqueous phase matrix blank sample (2.00). 

Matrix Blank and Matrix Fortification Samples 

One matrix blank of n-octanol and one matrix blank of reagent water (saturated with n-octanol) 

were prepared and analyzed concurrently with the definitive test samples to determine possible 

interferences. No chromatographic interferences were observed in the n-octanol and aqueous blank 

samples. Chromatograms of the n-octanol and aqueous phase matrix blank samples are presented in 

Figures 6 and 7, respectively. 

An aqueous matrix fortification sample was prepared at a concentration of 1.00 mg a.i./L and 

analyzed concurrently with the generator column samples to assess method efficiency. The 

1.00-mg a.i./L aqueous matrix fortification sample was prepared by transferring 50.0 μL of the 0.100-mg 

a.i./mL stock solution into a 5.00-mL aliquot of reagent water saturated with n-octanol measured into a

10.0-mL volumetric flask. This matrix fortification sample was diluted two-fold by bringing the flask to 

final volume with acetonitrile, resulting in an overall dilution factor of 2.00. The diluted aqueous matrix 

fortification sample was analyzed concurrently with the generator column samples. The matrix 

fortification sample had a measured concentration of corresponding to a procedural 

recovery of of the nominal concentration (Table 2). The aqueous generator column results were 

not corrected for this procedural recovery. The chromatogram of the aqueous matrix fortification sample 

is presented in Figure 7. 

Example Calculations 

The measured concentration of in sample number was calculated as follows: 

 (mg a.i./L) in sample = Peak Area - (Y-intercept)

Slope x Dilution Factor

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Peak area = First Initial (Sample) Volume (Vi) = mL 

Y-intercept = First Final Volume (Vf) = mL 

Slope = Dilution Factor (Vf / Vi) = 

Molecular Weight g/mol 

 (mg a.i./L) in sample = x 

 (mg a.i./L) in sample =

Calculations performed using Microsoft® Excel 2010 in the full precision mode. Manual calculations may differ slightly. 

The molar concentration for the n-octanol solution was calculated in the same manner as the 

molar concentration calculation for the aqueous samples. The Pow for this aqueous sample (sample 

number ) was calculated from the sample’s molar concentration and the mean molar 

concentration the n-octanol solution using the following equations: 

Mean (M) concentration in n-octanol solution = 

 concentration in aqueous sample (M) = 

Partition Coefficient (Pow) = Mean Concentration in n-Octanol Solution (M) 

 in Aqueous Sample (M) 

Partition Coefficient (Pow) = 

 

Partition Coefficient (Pow) = 

 log10 Pow = 

Calculations performed using Microsoft® Excel 2010 in the full precision mode. Manual calculations may differ slightly. 

RESULTS AND DISCUSSION 

Generator Column and Measured pH 

The temperature of the water bath remained constant at 25.0 ± 0.1°C throughout the experiment 

and the nominal flow rate of reagent water (saturated with n-octanol) through the generator column was 

verified at the 1.0 ml/min flow rate used to collect the aqueous samples. The measured pH of reagent 

water (saturated with n-octanol) pumped through the generator column was 7.12. The measured pH of an 

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un-diluted aqueous sample collected from the generator column was . The pH of the 

generator column water sample indicates in pure water in the 

absence of any . 

Measured Phase Concentrations and Calculated Partition Coefficient 

The results from the analyses of the n-octanol solution supernatant are presented in Table 3. A 

representative test solution sample chromatogram is shown in Figure 8. The mean measured 

concentration of in the n-octanol phase was mg a.i./L, or 

The results from the analysis of the aqueous phase generator column samples are presented in 

Table 4. The mean measured concentration of in these samples was mg a.i./L which is 

equivalent to M. A representative chromatogram of an aqueous sample is shown in Figure 9. 

From the measured concentrations in the aqueous samples and in the n-octanol solution 

supernatant, the mean Pow for 25oC was determined to be 

The corresponding log10 Pow value was calculated as . The partition 

coefficients are presented in Table 5. 

CONCLUSIONS 

The mean calculated n-octanol/water partition coefficient for at 25oC in reagent water, 

expressed as the logarithm (base 10) (log Pow), was determined to be 

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REFERENCES 

1. U.S. EPA Product Properties Test Guidelines. 1996. OPPTS 830.7560. Partition Coefficient

(n-Octanol/Water), Generator Column Method.

2. American Society for Testing and Materials. 2006. Standard Specification for Reagent Water.

D1193-06, ASTM Section II Water and Environmental Technology, Vol. 11.01: 45-47.

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Table 1 

HPLC Operational Parameters 

INSTRUMENT: Agilent Series 1100/1200 High Performance Liquid Chromatograph (HPLC) 

with an Agilent Series 1200 Variable Wavelength Detector 

ANALYTICAL COLUMN: Agilent Poroshell 120 EC-C18 (50 mm x 3.0 mm, 2.7-m particle size) 

STOP TIME: 7.00 minutes 

FLOW RATE: 0.470 mL/minute 

OVEN TEMPERATURE: 40.0°C 

MOBILE PHASE: Solvent A (90.0%): 0.1% H3PO4 in HPLC-grade Reagent Water 

Solvent B (10.0%): Acetonitrile (ACN) 

GRADIENT ELUTION 

PROFILE: TIME (min.) SOLVENT A (%) SOLVENT B (%) FLOW (mL/min.) 

 0.00 90.0 10.0 0.470 

 0.10 90.0 10.0 0.470 

1.50 5.0 95.0 0.470 

3.60 5.0 95.0 0.470 

 3.70 90.0 10.0 0.470 

 7.00 90.0 10.0 0.470 

INJECTION VOLUME: 10.0 μL 

 RETENTION TIME: Approx. minutes 

UV WAVELENGTH: 

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Table 2 

Quality Control Samples of in n-Octanol and Aqueous Matrices 

Sample Number Sample 

Type Matrix 

Concentration of 

Percent 

 Recovery 3 Fortified 

(mg a.i./L) 

Measured 3

(mg a.i./L) 

Matrix Blank n-octanol -- 

Matrix Blank 

Matrix Fortification 

reagent water 

reagent water 

-- 

1

 The limit of quantitation (LOQ) for n-octanol samples was 500 mg a.i./L, calculated as the product of the concentration of 

the low-level calibration standard (0.250 mg a.i./L) and the dilution factor of the n-octanol matrix blank sample (2000). 2

 The LOQ for aqueous samples was 0.500 mg a.i./L, calculated as the product of the concentration of the low-level 

calibration standard (0.250 mg a.i./L) and the dilution factor of the aqueous matrix blank sample (2.00). 3

 Results were generated by Microsoft Excel 2010 in full precision mode. Manual calculations may differ slightly.

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Table 3 

Measured Concentrations of in n-Octanol Solution Supernatant 

Sample ID 

Measured Concentration of 

 mg a.i./L 1 molar (M) 1, 2

Mean n-Octanol Phase = 

Standard Deviation = 

CV = 

1

 Results were generated using Microsoft® Excel 2010 in full precision mode. Manual 

calculations may differ slightly. 2

 Molar concentration calculated using mg a.i./L concentration and molecular weight of 

). 

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Table 4 

Measured Concentrations of in Aqueous Generator Column Samples 

Sample ID 

( -) 

Water Bath / Column 

Min/Max Temperature 

(oC) 

Concentration in mg a.i./L 1 Concentration i

Measured 

Concentration Mean ± Std. Dev. (CV) Measured Concentration 

1 2 3 

1 Results were generated using Microsoft® Excel 2010 in full precision mode. Manual calculations may differ slightly. 2 Molar concentration calculated using mg a.i./L concentration and molecular weight of l).
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Table 5 

Calculated n-Octanol/Water Partition Coefficients (Pow) for at 25oC 

Sample ID 

( ) 

Partition Coefficient (Pow) 1 log10 (Partition Coefficient) 1

Pow

Mean ± Std. Dev. 

(CV) log 10 Pow 

Mean ± Std. Dev. 

(CV) 

1 

2 

3 

 

1

 Results were generated using Microsoft® Excel 2010 in full precision mode. Manual calculations may differ slightly.

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Silanized glass wool 

Silanized glass wool 

aqueous solution

Chromosorb WHP 

aqueous eluate 

water from 

constant 

temperature 

bath 

Figure 1. Diagram of generator column. 

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Figure 2. Diagram of apparatus configuration. 

JASCO 

PU-1580 

Pump

Recirculating 

Chiller

Collection 

Flask

Generator 

Column

Heated 

Circulator

Reagent 

Water 

Reservoir

Constant 

Temperature 

Bath

Submersible 

Pump

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1. Set up the constant temperature water bath in a clear plastic reservoir by filling with ballast water (tap water),

install a heated circulator, and submerge a copper coil connected to a recirculating chiller. Set the heated

circulator and recirculating chiller to establish a temperature of 25.0 ± 0.1°C in the water bath. Immerse an

Erlenmeyer flask (covered with aluminum foil) containing reagent water (saturated with n-octanol) in the water

bath to allow the aqueous solution to equilibrate at the test temperature. Analyze the pH of an aliquot of the

equilibrated saturated aqueous solution.

2. Prepare the generator column (based upon procedures in method OPPTS 830.7560). To pack the generator

column, first insert a plug of silanized glass wool into one end of the longer (20 cm) 6 mm diameter section of the 

column (just above the larger diameter section). Pour an appropriate quantity of Chromosorb WHP (100-120

mesh) chromatographic support material into the column with gentle vibration/tapping (sufficient quantity to fill 

most of the column). Retain the packing material with a second plug of silanized glass wool. Connect the jacket 

of the packed generator column assembly to a submersible pump immersed in the constant temperature water bath 

and allow the generator column to thermally equilibrate for at least 30 minutes. Label the column with the project 

number and generator column ID.

3. In a Teflon Oak Ridge centrifuge tube, prepare an approximately 1% (w/w) solution of the test substance (T.S.) in 

n-octanol. Sonicate the solution for approximately 5 minutes. Centrifuge the solution at a setting of 14000 RPM 

for 10 minutes. Transfer an aliquot of the supernatant to a glass vial or beaker. Dilute triplicate aliquots of the

supernatant as appropriate for HPLC-UV analysis (appending solution ID with '-1,' '-2' and '-3'), with final

dilutions in 1:1 ACN:H2O (v/v). Prepare an n-octanol blank sample (MAB) and process in the same manner as

the n-octanol T.S. solution. Store initial dilutions of the n-octanol samples in PC-R.

4. Load the n-octanol T.S. solution supernatant onto the generator column by applying gentle suction at the bottom 

of the generator column and allow any excess solution to drain from the column. Place the inlet line from a Jasco 

solvent delivery pump into the immersed Erlenmeyer flask containing the aqueous solution, and pump aqueous

solution up from the bottom of the generator column to backflush and rinse the column and to remove trapped air 

and excess n-octanol. Re-plumb the aqueous solution line to the top of the generator column, and pump the

aqueous solution at a flow rate of approximately 0.5 mL/min for at least 30 minutes to equilibrate the system.

5. Adjust the flow rate to approximately 1.0 mL/min and allow the system to equilibrate for at least 15 minutes

before collecting a minimum of three consecutive aqueous solution samples. Collect appropriate volumes of

aqueous samples by submerging the drain end of the aqueous solute line (from the column) in an appropriate

volume of dilution solvent pre-loaded in a volumetric flask, and collecting to volume. Record temperature at time 

of each sample collection. If necessary, perform additional serial dilutions of the aqueous samples for HPLC-UV 

analysis. Prepare aqueous matrix blank and matrix fortification samples, and dilute as appropriate for HPLC-UV 

analysis. Collect a separate aqueous sample (without solvent dilution) for pH analysis.

Figure 3. Method outline for the processing of in n-octanol/water generator column aqueous 

samples. 

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Figure 5. Representative chromatograms of low- and high-level calibration standards for 

HPLC/UV analysis. 

(A): Calibration Standard I.D.: 10794-001-1 (0.250 mg a.i./L) 

(B): Calibration Standard I.D.: 10794-001-6 (10.0 mg a.i./L) 

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Figure 6. Chromatogram of the n-octanol matrix blank sample 

Dilution factor = 2000 

The arrow indicates the approximate retention time of the test substance. 

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Figure 7. Chromatograms of the aqueous matrix blank and matrix fortification samples. 

(A): Matrix Blank Sample 

(B): Matrix Fortification Sample, 1.00 mg a.i./L

Dilution factor = 2.00 

The arrow indicates the approximate retention time of the test substance. 

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Figure 8. A representative chromatogram of an n-octanol test solution sample 

Dilution factor = 2000 

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Figure 9. A representative chromatogram of an aqueous generator column sample

Dilution factor = 2.00 

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 DETERMINATION OF WATER SOLUBILITY 

USING THE COLUMN ELUTION METHOD 

U.S. EPA Product Properties Test Guidelines 

OPPTS 830.7840, Water Solubility: Column Elution Method; Shake Flask Method 

OPPTS 830.7860, Water Solubility (Generator Column Method) 

and 

OECD Guideline for the Testing of Chemicals 

OECD 105, Water Solubility 

AUTHORS: 

STUDY INITIATION DATE: 

STUDY COMPLETION DATE:

SUBMITTED TO: 

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TABLE OF CONTENTS 

Title/Cover Page ..................................................................................................................................... 1 

Good Laboratory Practice Compliance Statement ................................................................................. 2 

Quality Assurance Statement ................................................................................................................. 3 

Report Approval ..................................................................................................................................... 4 

Table of Contents ................................................................................................................................... 5 

Summary ................................................................................................................................................ 7 

Introduction ............................................................................................................................................ 8 

Objective ................................................................................................................................................ 8 

Experimental Design .............................................................................................................................. 8 

Materials and Methods ........................................................................................................................... 8 

 Test Substance ........................................................................................................................... 9 

Solvents and Reagents ............................................................................................................... 9 

Preparation of Generator Column ............................................................................................. 9 

 Apparatus Configuration ......................................................................................................... 10 

 Stock Preparation .................................................................................................................... 11 

 Standard Preparations .............................................................................................................. 11 

 Analytical Method ................................................................................................................... 11 

Calibration Curve and Limit of Quantitation .......................................................................... 12 

Definitive Test Procedure ........................................................................................................ 12 

Reagent Blank Sample ............................................................................................................ 13 

 Example Calculations .............................................................................................................. 13 

Results and Discussion ......................................................................................................................... 13 

Conclusions .......................................................................................................................................... 14 

References ............................................................................................................................................ 15 

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TABLE OF CONTENTS 

- Continued - 

TABLES

Table 1. HPLC Operational Parameters. ......................................................................................... 16 

Table 2. Quality Control Sample of in Reagent Water ..................................................... 17

Table 3. Measured Concentrations of in Aqueous Samples 

Collected from the Generator Column at 20oC ................................................................. 18 

FIGURES

Figure 1. Diagram of generator column ........................................................................................... 19 

Figure 2. Diagram of apparatus configuration ................................................................................. 20

Figure 3. Method outline for the processing of solubility samples .................................... 21 

Figure 4. Calibration curve for .......................................................................................... 22 

Figure 5. Representative chromatograms of low- and high-level 

 calibration standards ......................................................................................................... 23 

Figure 6. Representative chromatogram of reagent blank sample ..................................... 24 

Figure 7. Representative chromatograms of solubility samples ......................................... 25 

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SUMMARY 

STUDY TITLE: Determination of Water 

Solubility Using the Column Elution Method 

PROJECT NUMBER: 

SPONSOR: 

TESTING FACILITY: 

SPONSOR’S REPRESENTATIVE: 

STUDY MONITOR: 

SPONSOR’S REFERENCE NO.: 

LOCATION OF STUDY, RAW

DATA AND A COPY OF THE 

FINAL REPORT: 

TEST SUBSTANCE: 

TEST DATES: Experimental OECD Start – 

Experimental EPA Start –

Experimental Termination – 

TEST SYSTEM: HPLC-grade reagent water 

SUMMARY: The water solubility of the test substance in reagent water at 20oC was 

determined using the column elution method. The mean water 

solubility for at was 

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INTRODUCTION 

A study was performed to determine the water solubility of in reagent-grade water. 

 conducted this study for at the 

analytical chemistry facility in . The test was performed using reagent-grade water 

and the column elution method as modified by use of a generator column apparatus. The generator 

column was prepared on and definitive samples were collected on 

 Raw data generated by and a copy of the final report are filed under Project 

Number archives located on the site. The original signed report will 

be maintained by

OBJECTIVE 

The objective of this study was to experimentally determine the water solubility of at 

20oC using the column elution method. 

EXPERIMENTAL DESIGN 

The column elution method is generally applicable to liquid or solid test substances that are stable 

in water and are soluble in the range from 1 μg/L to 5000 mg/L. However, aqueous solubilities below 

1 μg/L may be reported if analytical methods allow. The test consisted of generating aqueous solutions 

of the test substance by pumping HPLC-grade reagent water through a generator column packed with a 

solid support coated with the test substance. The concentration of test substance in the saturated aqueous 

solutions eluted from the column represented the water solubility at the test temperature. 

MATERIALS AND METHODS 

The study was conducted according to the procedures outlined in the protocol, “ 

: Determination of Water Solubility Using the Column Elution Method” 

(Appendix 1). The test was performed based on procedures in the U.S. EPA Product Properties Test 

Guidelines, OPPTS 830.7840, Water Solubility: Column Elution Method; Shake Flask Method (1), 

U.S. EPA Product Properties Test Guidelines, OPPTS 830.7860, Water Solubility (Generator Column 

Method) (2), and OECD Guideline for the Testing of Chemicals, 105: Water Solubility (3). 

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Test Substance 

The test substance was received from on and was assigned 

identification number . The test substance was identified as Batch 

number – An expiration date of was provided by the Sponsor. A purity of 

was obtained from the Certificate of Analysis provided by the Sponsor (Appendix 2). The test substance 

was stored . 

Solvents and Reagents 

All solvents were HPLC-grade or higher quality and reagents were ACS reagent-grade or better. 

Solvent/Reagent Manufacturer/Lot 

Reagent Water: HPLC-Grade, Burdick & Jackson 

 (equivalent to ASTM Type II Designation D1193-06 (4)). 

Acetonitrile (ACN): HPLC-Grade, Burdick & Jackson, Lot No. DG852 

Chromosorb WHP: 100-120 mesh, Supelco, Lot No. 345 

Methanol (MeOH): HPLC-Grade, Burdick & Jackson, Lot No. DH300 

Acetone: HPLC-Grade, Burdick & Jackson, Lot No. DH050 

Preparation of Generator Column 

The generator column was supplied by At-Mar Glass Co. (Kennett Square, PA) and was 

manufactured based upon the specifications listed in OPPTS Guideline 830.7860 (2). The glass column 

consisted of three sections of fused tubing of the following dimensions (approximate length x outer 

diameter): (1) 20 cm x 6 mm; joined to (2) 5 cm x 9 mm; joined to (3) 3 cm x 6 mm. The entire column 

was enclosed in a water jacket for temperature control. The inlet and outlet were fitted with adapters for 

connection to chemically-inert tubing. A diagram of the generator column is presented in Figure 1. 

The inert support material used in the generator column was Chromosorb WHP (100-120 mesh). 

To coat the support material with the following procedure was employed: Approximately 

1 gram of support material was weighed (Sartorius/CPA225D) in a 250-mL round-bottom flask. A 

10.0-mg/mL solution of was prepared in acetone and was used to coat the inert column support 

material. The solution was prepared by accurately weighing 0.5000 grams of into a scintillation 

vial. The test substance aliquot was then quantitatively transferred into a 50.0-mL volumetric flask and 

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was brought to final volume using acetone. This solution was added to the 250-mL round-bottom flask 

containing the Chromosorb support material. The 50.0-mL volumetric flask used to prepare the 

10.0-mg/mL solution was then rinsed with acetone and the rinsate was transferred to the 250-mL roundbottom flask. The round-bottom flask was placed onto a rotary evaporator and the acetone carrier solvent 

was rotary evaporated at a temperature of 35 to 40oC until the removal of the acetone solvent was 

complete. 

A plug of silanized glass wool (Supelco, Lot V0446) was placed in one end of the 20-cm long 

6 mm diameter section of the generator column (just above the enlarged section, Figure 1). Next, coated 

support material was transferred from the round-bottom flask directly into the column with the aid of 

gentle tapping and vibration. A second plug of silanized glass wool was then inserted into the top of the 

column to retain the coated support material. The packed generator column was labeled with the project 

number and a column ID and was allowed to thermally equilibrate at 20.0 ± 0.1oC for at least 30 minutes. 

 The column was back flushed with reagent-grade water to remove entrapped air. The column was then 

re-plumbed to the definitive configuration (Figure 2) and allowed to equilibrate at a reagent water flow 

rate of approximately 0.5 mL/min for a minimum of 30 minutes. After the equilibration period, the 

solvent delivery pump flow rate was adjusted to 0.1 mL/min until the first set of samples was collected. 

On the day of sample collection, the flow rate was adjusted to approximately 1.0 mL/min and allowed to 

drain to waste for at least 10 minutes before the collection of the first definitive sample. 

Apparatus Configuration 

A constant temperature water bath was setup in a clear plastic reservoir by filling the reservoir 

with tap water, installing a heated circulator (Fisher/IC-2150), and submerging a copper coil connected to 

a recirculating chiller (Fisher/IsoTemp 3016D). The heated circulator and recirculating chiller were set to 

maintain the test temperature (20 ± 0.1°C) throughout the experiment. The temperature of the water bath 

was monitored using a N.I.S.T. Traceable Digital Thermometer. A submersible pump (Little Giant Pump 

Company/PE-2H) placed in the constant temperature water bath was used to direct bath water maintained 

at 20 ± 0.1°C into the jacket surrounding the generator column. 

A 2-L Erlenmeyer flask was used as a reservoir for the reagent water that was pumped through 

the generator column. The flask was immersed in the constant temperature water bath. The inlet line of a 

Jasco PU-2080 solvent delivery pumping system was placed into the 2-L flask and the top of the flask 

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was covered with aluminum foil. The pump was used to accurately control the flow rate of the aqueous 

solution through the generator column. A diagram of the apparatus configuration is presented in 

Figure 2. 

Stock Preparation 

A primary stock solution of the test substance was prepared at a nominal concentration 

of 1.00 mg a.i./mL by accurately weighing grams of the test substance (weight adjusted for 

 purity = a.i.) in a scintillation vial using an analytical balance. The test substance was dissolved 

in methanol (MeOH), quantitatively transferred to a 100-mL volumetric flask, brought to final volume 

with MeOH and identified as 

Standard Preparations 

The 1.00 mg a.i./mL (1000 mg a.i./L) stock solution, identified as was used to 

prepare a set of calibration standards in 50:50 (v/v) acetonitrile (ACN): reagent water using the 

following dilution scheme: 

Stock 

Concentration 

(mg a.i./mL) 

Aliquot 

(mL) 

Final 

Volume 

(mL) 

Standard 

Concentration 

(mg a.i./L) 

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

0.0250 

0.0500 

0.100 

0.250 

0.500 

1.00 

100 

100 

100 

100 

100 

100 

0.250 

0.500 

1.00 

2.50 

5.00 

10.0 

Analytical Method 

The analytical method for the determination of in reagent water was developed at 

 (Figure 3). Each sample consisted of a 5.00-mL aqueous fraction collected from 

the generator column directly into a 10.0-mL volumetric flask pre-filled with 5.00-mL of ACN. The 

resulting generator column samples had an overall dilution factor of 2.00. Details of the analytical 

sample processing are provided in Figure 3. 

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Concentrations of in the samples were determined using an Agilent Series 1100/1200 

HPLC equipped with an Agilent Series 1200 variable wavelength UV detector. Chromatographic 

separations were achieved using a Poroshell 120 EC-C18 column (50 x 3.0 mm, 2.7-m particle size). 

The instrument operational parameters are summarized in Table 1. 

Calibration Curve and Limit of Quantitation 

calibration standards of in 50:50 (v/v) ACN: reagent water, ranging in concentration 

from mg a.i./L were analyzed concurrently with the solubility samples. The calibration set 

was injected at the beginning and end of the analytical sequence with one calibration standard injected 

after a maximum of every test samples. A linear regression equation was generated for the analytical 

sequence using the peak area response versus the respective concentrations of the calibration 

standards. The concentration of in the samples was determined by substituting the peak area 

responses of the samples into the linear regression equation. The calibration curve for is 

presented in Figure 4. Representative chromatograms of low- and high-level calibration 

standards are presented in Figure 5. 

The limit of quantitation (LOQ) was defined using the low-level calibration standard equivalent 

definition. For this analysis, the method LOQ for in reagent water was defined as 

0.500 mg a.i./L, calculated as the product of the concentration of the lowest-level calibration standard 

(0.250 mg a.i./L) and the method dilution factor for the reagent blank sample (2.00). 

Definitive Test Procedure 

The volume of each aqueous generator column sample collected was 5.00 mL. Samples were 

collected at a flow rate of approximately 1.0 mL/minute until an equilibrium concentration was reached, 

as defined by the protocol as at least consecutive generator column sample collections where the 

measured sample concentrations were in statistical agreement of each other (e.g., CV <10% for solubility 

determinations of 1 ppb or greater, as per Guideline (2)). Following this equilibrium, a second trial was 

performed at approximately half the initial flow rate setting, until an equilibrium concentration was 

reached. The test was terminated when the mean measured concentrations of at the two flow 

rates were determined to be within of each other. An additional aqueous sample was collected at 

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1.0 mL/min for measurement of pH along with a reagent water sample using a pH meter 

(Thermo Orion/4 Star Plus). 

Reagent Blank Sample 

One reagent blank sample, consisting of unfortified reagent water, was analyzed to determine 

possible interferences. No interferences were observed at or above the low standard or LOQ for the test 

substance during analysis of the reagent blank sample. The chromatogram of the reagent blank sample is 

presented in Figure 6. 

Example Calculations 

The measured concentration of the test substance in sample number was calculated 

as follows: 

 (mg a.i./L) in sample = Peak Area - (Y-intercept)

Slope x Dilution Factor 

Peak Area = Sample (Initial) Volume (Vi) = mL 

Y-intercept = - Final Volume (Vf) = mL 

Slope = Dilution Factor (Vf/Vi) = (10.0/5.00) = 

 

 (mg a.i./L) in sample = x 

 (mg a.i./L) in sample = 

Calculations performed using Microsoft® Excel 2010 in the full precision mode. Manual calculations may differ slightly. 

RESULTS AND DISCUSSION 

The reagent blank sample (Figure 6) did not show any chromatographic interferences and 

background levels were significantly below the low-level calibration standard. 

The temperature of the water bath remained constant at 20.0 ± 0.1°C throughout the experiment. 

Under the instrumental conditions presented, the mean measured reagent water solubility of at 

was mg a.i./L at a flow rate of approximately /min and 

was a.i./L ) at a flow rate of approximately min (Table 3). 

The mean concentration for the approximately mL/min flow rate settings differed by 

which was well within the ± test criterion. The overall mean measured water solubility at 

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 was mg a.i./L ). Representative chromatograms of solubility 

samples at flow rates of approximately and mL/min are presented in Figure 7. The pH was 

measured for an aqueous sample collected at a flow rate of mL/min. The measured pH of reagent 

water and an aqueous sample was and respectively. 

CONCLUSIONS 

The water solubility of in reagent water at determined to be mg a.i./L 

). 

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REFERENCES 

1. U.S. EPA Product Properties Test Guidelines. 1998. OPPTS 830.7840. Water Solubility: 

Column Elution Method; Shake Flask Method. 

2. U.S. EPA Product Properties Test Guidelines. 1998. OPPTS 830.7860. Water Solubility 

(Generator Column Method). 

3. Organisation for Economic Cooperation and Development. 1995. Guideline for Testing of 

Chemicals, 105: Water Solubility. 

4. American Society for Testing and Materials. 2006. Standard Specification for Reagent Water. 

D1193-06, ASTM Section II Water and Environmental Technology, Vol. 11.01: 45-47. 

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Table 1 

HPLC Operational Parameters 

INSTRUMENT: Agilent Series 1100/1200 High Performance Liquid Chromatograph (HPLC) 

with an Agilent Series 1200 Variable Wavelength Detector 

ANALYTICAL COLUMN: Agilent Poroshell 120 EC-C18 (50 mm x 3.0 mm, 2.7-m particle size) 

STOP TIME: 7.00 minutes 

FLOW RATE: 0.470 mL/minute 

OVEN TEMPERATURE: 40.0°C 

MOBILE PHASE: Solvent A (90.0%): 0.1% H3PO4 in HPLC-grade Reagent Water 

Solvent B (10.0%): Acetonitrile (ACN) 

GRADIENT ELUTION 

PROFILE: TIME (min.) SOLVENT A (%) SOLVENT B (%) FLOW (mL/min.) 

 0.00 90.0 10.0 0.470 

 0.10 90.0 10.0 0.470 

 1.50 5.0 95.0 0.470 

 3.60 5.0 95.0 0.470 

 3.70 90.0 10.0 0.470 

 7.00 90.0 10.0 0.470 

INJECTION VOLUME: 10.0 μL 

RETENTION TIME: Approx. 

UV WAVELENGTH: 

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Table 2 

Quality Control Sample of in Reagent Water 

Sample Number Sample 

Type 

 Concentration 

Percent 2

Recovered 

Fortified 

(mg a.i./L) 

Measured 1, 2

(mg a.i./L) 

REB-1 Reagent Blank < LOQ -- 

1

 The limit of quantitation (LOQ) for the method was defined as 0.500 mg a.i./L, calculated as the product of the lowlevel standard (0.250 mg a.i./L) and the dilution factor of the reagent blank sample (2.00). 

2 Results were generated using Microsoft® Excel 2010 in full precision mode. Manual calculations may differ 

slightly.

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Table 3 

Measured Concentrations of in Aqueous Samples 

Collected from the Generator Column at 20oC 

Sample ID 

Measured 

Flow Rate 

(mL/min.) 

Water Bath / Column 

Min/Max Temperature 

(oC) 

Calculated 

Concentration 

(mg a.i./L) 1

Mean Calculated 

Concentration 

(mg a.i./L) 1,2

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

 

1

 Results were generated using Microsoft® Excel 2010 in full precision mode. Manual calculations may differ slightly.

2

 CV = Coefficient of Variation. 

 % Diff. = Percent difference of mean measured concentrations from the two flow rate settings: 

 = (absolute value of the difference of means  average of means) x 100.

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Figure 1. Diagram of generator column. 

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JASCO 

PU-2080 

Pump

Recirculating

Chiller

Collection 

Flask

Generator 

Column

Heated 

Circulator

Reagent 

Water 

Reservoir

Constant

Temperature

Bath

Submersible

Pump

Figure 2. Diagram of apparatus configuration. 

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Method Outline for the Processing of Column Elution Aqueous Samples 

for Determination of Water Solubility 

1. Adjust the flow rate to approximately 1.0 mL/minute (first definitive flow rate) and drain to waste for at 

least 10 minutes before collecting aqueous solute samples. 

2. Determine the flow rate of the column by measuring the amount of time required to fill a 5.00-mL or 

10.0-mL volumetric flask to volume. 

3. Collect aqueous samples from the generator column directly into 10.0-mL volumetric flasks pre-filled with 

5.00-mL of acetonitrile (ACN), measured with a volumetric pipette. For each sample, place the drain end of 

the line in the neck of the volumetric flask, fill the flask to final volume and remove the line from the flask. 

Stopper and mix. Record temperature minimum and maximum (min/max) during each sample collection 

time. Repeat for each of a minimum of five consecutive replicate aqueous samples. 

4. Collect an aqueous sample from the generator column in a vial or small beaker and measure the pH of the 

un-diluted sample. 

5. If necessary, perform additional dilutions of samples in dilution solvent composed of 1:1 v/v ACN:H2O. 

Transfer an aliquot of each sample's final dilution to an autosampler vial, seal and submit for HPLC/UV 

analysis. Place any remaining portion of the initial sample dilutions in refrigerated storage. 

6. Perform a second trial at approximately half the flow rate of the first. Adjust the flow rate to approximately 

0.5 mL/minute (confirmatory flow rate) and drain to waste for at least 10 minutes before collecting aqueous 

solute samples. After collecting all samples at the confirmatory flow rate, adjust to a setting of 0.1 to 

0.2 mL/min. 

Note: Rinse all glassware with ACN prior to use 

Figure 3. Method outline for the processing of solubility samples. 

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Figure 4. Calibration curve for 

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Figure 5. Representative chromatograms of low- and high-level calibration standards. 

(A): Calibration Standard I.D.: 10794-001-1 (0.250 mg a.i./L) 

(B): Calibration Standard I.D.: 10794-001-6 (10.0 mg a.i./L) 

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Figure 6. Representative chromatograms of reagent blank sample. 

Reagent Blank Sample ID , Dilution factor = 2.00 

Note: The arrow indicates the approximate retention time of 

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Figure 7. Representative chromatograms of solubility samples. 

(A): Solubility Sample I.D.: (Column elution flow rate setting = ~ 1.0 mL/min, 

Dilution factor = 2.00) 

(B): Solubility Sample I.D.: Column elution flow rate setting = ~ 0.5 mL/min, 

Dilution factor = 2.00) 

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Estrogen Receptor Activation by Test Articles

Final Report

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CONFIDENTIAL

Page 2 of 8

TABLE OF CONTENTS

1 Objective ................................................................................................................................................. 3

1.1 Regulatory Guidelines ................................................................................................................... 3

2 Test Articles ............................................................................................................................................ 3

3 Test Methods .......................................................................................................................................... 4

3.1 In vitro ADME-Tox Experimental Conditions ................................................................................. 4

3.1.1 Cytotoxicity experimental conditions ............................................................................................. 4

3.1.2 ER Alpha Foci Formation experimental conditions ...................................................................4

4 Results .................................................................................................................................................... 5

4.1 In vitro ADME-Tox Summary ......................................................................................................... 5

4.1.1 Estrogen Receptor Activation Assay Results ............................................................................ 5

4.1.2 AC50 Curves ............................................................................................................................. 6

4.1.2.1 Estrogen Receptor Alpha Foci Formation ............................................................................. 6

4.1.2.2 Cytotoxicity ............................................................................................................................ 7

5 Storage and Retention of Records ......................................................................................................... 8

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1 Objective

The objective of this study was to determine whether test compounds were capable of activating

Estrogen Receptor alpha.

1.1 Regulatory Guidelines 

This study was not conducted under US FDA Good Laboratory Practice Regulations (GLPs).

Standard operating procedures of were used throughout the study.

2 Test Articles

Client ID

Physical

Form

Submitted

FW

Parent

MW Stock solutions

 were stored at -20 °C. Stock solutions were stored at -20 °C.

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3 Test Methods

Testing was performed at .

3.1 In vitro ADME-Tox Experimental Conditions 

3.1.1 Cytotoxicity experimental conditions

Client ID

Test conc.

(μM) Assay time

Cell

lines Readout

Analytical

method

3.1.2 ER Alpha Foci Formation experimental conditions

Client ID

Test conc.

(μM)

Assay

time

Cell

lines Readout

Reference

compound

Analytical

method

The U2OS reporter assays were purchased from ThermoFisher Scientific. 3000 cells are plated

in each well of a 384 well plate. The cells are seeded in 25 μl of medium and placed overnight in

an incubator. The next day, compound is serial diluted in DMSO in a 384 well compound plate.

The serially diluted 384 well compound plate is then diluted in medium to make a 2x concentrated

stock plate. 25 μl of each well is added to each well of cells in the test plates. The Estrogen

receptor cells are incubated with compound for 20 hours for the receptor assay and 48 hours for

the cytotoxicity test. The plates are then imaged using the Thermo ArrayScan VTi imagers and

images are analyzed using the Thermo ArrayScan software.

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4 Results

4.1 In vitro ADME-Tox Summary 

4.1.1 Estrogen Receptor Activation Assay Results

Client ID

ER Activation

AC50

Cytotoxicity

AC50 comment

positive Control ER

NR= no response seen

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4.1.2 AC50 Curves 

4.1.2.1 Estrogen Receptor Alpha Foci Formation 

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4.1.2.2 Cytotoxicity

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5 Storage and Retention of Records

All documents generated in this study (raw data, the study plan, a copy of this report, etc.) will be

stored for three years from the date of this document. Only authorized employees will

have access to the archives.

The original final report will be provided to the sponsor and will be kept by the sponsor at its

facility.

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Final Report

Approved:

Date:

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Page 2 of 8

TABLE OF CONTENTS

1 Objective ................................................................................................................................................. 3

1.1 Regulatory Guidelines ................................................................................................................... 3

2 Test Articles ............................................................................................................................................ 3

3 Test Methods .......................................................................................................................................... 4

3.1 In vitro ADME-Tox Experimental Conditions ................................................................................. 4

3.1.1 Cytotoxicity experimental conditions ............................................................................................. 4

3.1.2 AR Translocation experimental conditions ............................................................................... 4

4 Results .................................................................................................................................................... 5

4.1 In vitro ADME-Tox Summary ......................................................................................................... 5

4.1.1 Androgen Receptor Translocation Assay Results ..................................................................... 5

4.1.2 AC50 Curves ............................................................................................................................. 6

4.1.2.1 Androgen Receptor Translocation ......................................................................................... 6

4.1.2.2 Cytotoxicity ............................................................................................................................ 7

5 Storage and Retention of Records ......................................................................................................... 8

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1 Objective

The objective of this study was to determine whether test compounds were capable of activating

the Androgen Receptor.

1.1 Regulatory Guidelines 

This study was not conducted under US FDA Good Laboratory Practice Regulations (GLPs).

Standard operating procedures of were used throughout the study.

2 Test Articles

Client ID

Physical

Form

Submitted

FW

Parent

MW Stock solutions

 were stored at -20 °C. Stock solutions were stored at -20 °C.

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3 Test Methods

Testing was performed at 

3.1 In vitro ADME-Tox Experimental Conditions 

3.1.1 Cytotoxicity experimental conditions

Client ID

Test conc.

(μM) Assay time

Cell

lines Readout

Analytical

method

Cell

3.1.2 AR Translocation experimental conditions

Client ID

Test conc.

(μM)

Assay 

time

Cell

lines Readout

Reference

compound

Analytical

method

The U2OS reporter assays were purchased from ThermoFisher Scientific. 3000 cells are plated

in each well of a 384 well plate. The cells are seeded in 25 μl of medium and placed overnight in

an incubator. The next day, compound is serial diluted in DMSO in a 384 well compound plate.

The serially diluted 384 well compound plate is then diluted in medium to make a 2x concentrated

stock plate. 25 μl of each well is added to each well of cells in the test plates. The Androgen

receptor cells are incubated for 4 hours for the receptor assay and 48 hours for the cytotoxicity.

The plates are then imaged using the Thermo ArrayScan VTi imagers and images are analyzed

using the Thermo ArrayScan software.

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4 Results

4.1 In vitro ADME-Tox Summary 

4.1.1 Androgen Receptor Translocation Assay Results

Client ID

AR

Translocation

AC50 (Nuclear

Intensity)

Cytotoxicity

AC50 comment

NR= no response seen

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4.1.2 AC50 Curves

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4.1.2.2 Cytotoxicity

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Page 8 of 8 

5 Storage and Retention of Records 

All documents generated in this study (raw data, the study plan, a copy of this report, etc.) will be 

stored for three years from the date of this document. Only authorized employees will 

have access to the archives. 

The original final report will be provided to the sponsor and will be kept by the sponsor at its 

facility. 

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Page 1 of 21 

Recombinant Yeast Screen 

 for Oestrogenic and Androgenic Activity 

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2 

Sponsor and Test Facility Details 

Sponsor 

Test facility 

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3 

Table of Contents 

Sponsor and Test Facility Details ..........................................................................................2

Study Director Statement ......................................................................................................5

Contributing Scientists...........................................................................................................6

Summary...............................................................................................................................7

1. Introduction...................................................................................................................8

1.1 Objective...........................................................................................................8

1.2 Test procedure..................................................................................................8

2. Experimental Procedure ...............................................................................................9

2.1 Study Schedule.................................................................................................9

2.2 Test substances................................................................................................9

2.3 Test organisms .................................................................................................9

2.4 Preparation of test organism inoculum ..............................................................9

2.5 Preparation of test substances........................................................................10

2.6 Preparation of oestrogen positive reference substance...................................10

2.7 Preparation of androgen positive reference substance....................................10

2.8 Preparation of screen plates ...........................................................................10

3. Assessment of Results ...............................................................................................11

4. Maintenance of Records.............................................................................................12

5. Results .......................................................................................................................13

6. Reference...................................................................................................................14 

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4 

Figures 

Figure 1 Estrogen assay: ......................................................15

Figure 2 Estrogen assay: ......................................................16

Figure 3 Estrogen assay: .......................................................................17

Figure 4 Estrogen assay: I ..........................................................................18

Figure 5 Estrogen assay: ...................................................................19

Annex 

Annex 1 Media ...........................................................................................................20

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5 

Study Director Statement 

Recombinant Yeast Screen for Oestrogenic and Androgenic Activity: 

6 compounds 

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6 

Contributing Scientists 

Recombinant Yeast Screen for Oestrogenic and Androgenic Activity: 

Study management 

Head, Department of In Vitro Technologies 

Department of In Vitro Technologies 

Assistant 

Department of In Vitro Technologies 

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7 

Summary

This study was performed to screen for estrogenic and androgenic activity. 

The test was performed using a 96-well microtitre plate serial dilution procedure. Dilutions of 

the test substance in the range 100 mg/L – 50 μg/L were incubated in the presence of the 

recombinant yeast strains, Saccharomyces cerevisiae hER, containing the human estrogen 

receptor gene and Saccharomyces cerevisiae hAR, containing the human androgen receptor 

gene. Two positive reference substances, 17-β-estradiol and dihydrotestosterone, were tested in 

this study. 

High levels of estrogenic and androgenic activity were demonstrated by the positive reference 

substances, 17-β-estradiol and d sistent with the responses 

expected for these substances. demonstrated estrogenic 

activity. 

 

 

 

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1. Introduction

1.1 Objective 

The obje for estrogenic and androgenic 

activity. , s a positive reference substance. 

1.2 Test procedure 

The test was performed using a 96-well microtitre plate serial dilution procedure based on 

that described by Routledge and Sumpter (1996). Dilutions of the test substance were 

incubated in the presence of the recombinant yeast, Saccharomyces cerevisiae hER, 

containing the human oestrogen receptor gene, and Saccharomyces cerevisiae hAR, 

containing the human androgen receptor gene. Detection of estrogenic and androgenic 

activity (binding of the test substance to the human receptor genes) was indicated by the 

activation of a reporter gene (lac-Z, encoding the enzyme β-galactosidase) and was measured 

by photometric measurement of the conversion of the chromogenic substrate, CPRG, from 

yellow to red. 

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2. Experimental Procedure

2.1 Study Schedule 

Th s approved by and the Study Director 

on and by t tal phase of the study 

wa between a . 

2.2 Test substances 

Lab reference 

number 

Test Substance Name 

 

Expiry:

Purity: Not supplied 

Storage conditions:

Date received: 

2.3 Test organisms 

Saccharomyces cerevisiae hER * 

Saccharomyces cerevisiae hAR * 

* Developed by GlaxoWellcome Research and Development, Ware, England and 

supplied by Brunel University, Uxbridge, England. 

2.4 Preparation of test organism inoculum 

Stock cultures of the test organisms were stored frozen under liquid nitrogen. One cryovial 

of each stock culture was allowed to thaw to room temperature and then 0.1 - 0.2ml of the 

contents were inoculated into 50 mL sterile growth medium (Annex 1) and incubated in a 

shaking water bath at 30 ± 2°C for approximately 24 hours to provide the test inoculum. 

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2.5 Preparation of test substances 

The test substances were dissolved in ethanol to give a stock solutions containing 1 g/L. 

These stock solutions were diluted with ethanol in serial two-fold steps to give a range of 

concentrations down to 1 mg/L, equivalent to 20-fold the final test concentrations. 

2.6 Preparation of oestrogen positive reference substance 

17-β-Estradiol (Sigma, Dorset, England, code E8875-1G) was dissolved in ethanol to give a 

stock solution containing 100 μg/L. This stock solution was diluted with ethanol in serial 

two-fold steps to give a range of concentrations down to 100 ng/L, equivalent to 20-fold the 

final test concentrations. 

2.7 Preparation of androgen positive reference substance 

Dihydrotestosterone (5α-Androstan-17β-ol-3-one, Sigma, Dorset, England, code A8380-1G) 

was dissolved in ethanol to give a stock solution containing 200 μg/L. This stock solution 

was diluted with ethanol in serial two-fold steps to give a range of concentrations down to 

200 ng/L, equivalent to 20-fold the final test concentrations. 

2.8 Preparation of screen plates 

Separate 96 well microtitre plates (Linbro®/Titertek®, code 76-232-05, ICN Biomedicals 

Inc., Ohio, USA) were used to screen each test substance for estrogenic and androgenic 

activity. The appropriate positive reference substances were tested on each plate. Duplicate 

10 μL aliquots of each dilution of the test substance and reference substance dilution series 

were transferred into duplicate rows of wells on the screen plate using an automatic multichannel pipette to give a descending concentration series from left to right. The highest 

concentration of each series was prepared by transferring 20 μL aliquots of the stock solution. 

The duplicate rows were separated by a single “blank” row containing 10 μL ethanol in each 

well. The screen plates were then placed into an incubator set at 37 ± 2°C for approximately 

30 minutes to allow the ethanol solvent to evaporate to dryness. 

Following evaporation, 200 μL assay medium containing a chromogenic substrate 

(chlorophenol red-β-D-galactopyranoside, CPRG) and the test organism inoculum was 

transferred into every well of the screen plate using an automatic multi-channel pipette. This 

addition represented a 20-fold dilution (10-fold dilution for the highest concentration) of the 

original test and reference substance concentrations such that the final test concentrations 

were in the range 100 mg/L - 50 μg/L for the test substances, 10 μg/L - 5 ng/L for the 17-βestradiol reference substance and 20 μg/L - 10 ng/L for the dihydrotestosterone reference 

substance. 

The lid and base of each plate were then sealed together using masking tape and the plates 

secured to an automatic plate shaker and shaken for 2 minutes at high speed. The plates were 

incubated at 30 ± 2°C for a minimum of 3 days. 

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3. Assessment of Results

After 3 days incubation at 30 ± 2°C the screen plates were read using a FLUOstar OPTIMA 

plate reader and the colour development of the assay m ium (conversion of CPRG from 

yellow to red) in each well measured at an absorbance o nm. To correct for turbidity the 

screen plates were read at a second absorbance level of nm. Calculation of the corrected 

absorbance value was as follows: 

Corrected value = Abs’ at nm - (Abs’ at nm - Abs’ of blank at nm). 

The presence of estrogenic and/o ndrogenic activity was depicted graphically by plotting 

corrected absorbance values at nm against test concentration (mg/L) for each test 

substance and reference substance

If no estrogenic response was observed for test substances after 3 days incubation, the plates 

were then incubated for a further 4 days at room temperature, thereby allowing a total of 7 

days incubation for the development of any slow acting estrogenic response shown by the test 

substances. After this time the plates were observed visually and if any wells containing test 

substance showed a further development of colour change from yellow to red, the plates were 

then read for a second time as described above. Androgen assay plates were not incubated 

beyond 3 days due to the rapid colour development in all wells post 3 days incubation. 

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4. Maintenance of Records

ion relating to this study will be maintained in the archives of 

 for a period of one year from the date on which the Study Director 

his will include but may not be limited to the Study Protocol, raw 

data, a copy of the final report, test material a cimens generated 

during the course of this study. If requested, will continue to 

retain the materials at additional cost. 

Samples and specimens that no longer afford evaluation will be discarded in accordance with 

Standard Operating Procedures. 

 will retain a copy of the final report in its archive indefinitely. 

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5. Results

The results of the endocrine disrupter assessments for estrog activity conduc

substances in this study are presented in Figures 1 – 5. The test substance, 

was tested on each of the assay plates used for the testing of other test subst

 

 tested as a reference substance, demonstrated a significant estrogenic response 

ncubation, consistent with the expected response for this chemical. 

 

. 

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6. Reference

ROUTLEDGE, E. J. & SUMPTER, J. P. (1996 ) Estrogenic activity of surfactants and some of 

their degradation products assessed using a recombinant yeast screen. Environmental 

Toxicology and Chemistry, 15 (3), 241-248. 

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Figure 2 Estrogen assay: 

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Figure 3 Estrogen assay:

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Figure 4 Estrogen assay: 

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Figure 5 Estrogen assay: 

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Annex 1 Media 

GROWTH MEDIUM 

Minimal Medium 

The basal component of the growth medium was “Minimal Medium” comprising the 

following: 

KH2PO4 13.61 g 

(NH4)2SO4 1.98 g 

KOH pellets 4.2 g 

MgSO4 0.2 g 

Fe2(SO4)3 solution (40 mg/50 mL H2O) 1 mL 

L-leucine 50 mg 

L-histidine 50 mg 

Adenine 50 mg 

L-arginine-HCl 20 mg 

L-methionine 20 mg 

L-tyrosine 30 mg 

L-isoleucine 30 mg 

L-lysine-HCl 30 mg 

L-phenylalanine 25 mg 

L-glutamic acid 100 mg 

L-valine 150 mg 

L-serine 375 mg 

The above constituents were suspended in 1 litre of HPLC grade water, placed onto a heated 

stirrer to dissolve, dispensed in 45 mL aliquots into glass containers and then sterilised at 

121oC for 10 minutes. The sterilised minimal medium was then stored at room temperature. 

Additional components of the growth medium were prepared as follows: 

Vitamin Solution 

Thiamine 4 mg 

Pyridoxine 4 mg 

D-Pantothenic acid 4 mg 

Inositol 20 mg 

Biotin solution 2 mg 

The above constituents were suspended in 100 mL HPLC grade water, sterilised by filtering 

through a 0.2 μm pore size disposable filter into sterile glass bottles, and then stored at 4oC. 

 

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Annex 1 Media (continued) 

D-(+)-Glucose 

A 20% w/v solution was prepared, sterilised by autoclaving at 121oC for 10 minutes and then 

stored at room temperature. 

L-Aspartic Acid 

A stock solution of 4 mg/mL was prepared, sterilised by autoclaving at 121oC for 10 minutes 

and then stored at room temperature. 

L-Threonine 

A solution of 24 mg/mL was prepared, sterilised by autoclaving at 121oC for 10 minutes and 

then stored at 4oC. 

Copper (II) Sulphate 

A 20 mM solution was prepared, sterilised by filtering through a 0.2 μm pore size filter into 

sterile glass bottles and then stored at room temperature. 

Summary of Growth Medium constituents 

Minimal medium 45 mL 

Glucose solution 5 mL 

L-aspartic acid solution 1.25 mL 

Vitamin solution 0.5 mL 

L-threonine solution 0.4 mL 

Copper (II) sulphate solution 125 μL 

The above constituents were combined on the day of use to give a nominal volume of 50 mL 

growth medium. 

ASSAY MEDIUM 

Growth medium 50 mL 

CPRG (see below) 0.5 mL 

Test organism inoculum 0.2 mL 

The above constituents were combined immediately prior to addition to the screen plates. 

Chlorophenol red-ß-D-galactopyranoside (CPRG) 

A 10 mg/mL stock solution was prepared, sterilised by filtering through a 0.2 μm pore size 

filter into sterile glass bottles and then stored at 4oC. 

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Toxicity Testing for

Using Daphnia magna, Pimephales promelas, and

Pseudokirchneriella subcapitata

Report

Submitted to:

Prepared by:

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EXECUTIVE SUMMARY

Environmental Protection Agency (EPA) Office of Pollution Prevention and Toxics (OPPT)

Toxic Substances Control Act (TSCA) test guidelines (40 CFR 797) and good laboratory

practice (GLP) (40 CFR 792) were followed to develop acute toxicity data for

. The required acute toxicity testing included a 96-hour test with

Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), a 48-hour Daphnia

magna acute test, and a 96-hour Pimephales promelas (fathead minnow) acute test. The testing

regime was comprised of a range-finding test for each species as outlined by TSCA methodology

followed by definitive testing.

The results of the range-finding testing indicated no observed toxicity at treatments below the

reported solubility for mg/L) for all three species. Observed toxicity at treatments

above the reported water solubility level may be due to undissolved in the test solution.

The results of the range finding tests were used to determine the appropriate testing regime in the

definitive testing. Definitive testing was conducted with the highest test treatment equal to the

reported water solubility of The results of the definitive testing indicated no observed

toxicity below the water solubility for to all three test organism.

Table ES-1. Results of range finding and definitive toxicity testing using Daphnia magna,

Pimephales promelas, and Pseudokirchneriella subcapitata with

Species Lowest Tested Treatment with

Toxic Response in Range

Finding Test (mg/L)

Definitive Test EC50 (mg/L)

Daphnia magna

Pimephales promelas

Pseudokirchneriella

subcapitata

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TABLE OF CONTENTS

EXECUTIVE SUMMARY ............................................................................................................ ii

1.0 INTRODUCTION ............................................................................................................... 1

2.0 METHODS .......................................................................................................................... 1

2.1 Overview ...................................................................................................................... 1

2.2 Sample Collection and Handling.................................................................................. 1

2.3 Test Species .................................................................................................................. 1

2.4 Test Organism Culturing/Acclimation ......................................................................... 2

2.5 Toxicity Test Methods.................................................................................................. 2

2.6 Laboratory Dilution Water ........................................................................................... 9

2.7 Test Solution Preparation and Analysis........................................................................ 9

2.8 Data Analyses and Interpretation ............................................................................... 10

2.9 Quality Assurance/Quality Control Procedures – Toxicity Data ............................... 11

3.0 RESULTS AND DISCUSSION........................................................................................ 11

3.1 Results of Range-Finding Tests using ........................................................ 11

3.2 Results of Definitive Tests using .................................................................. 12

4.0 CONCLUSIONS ............................................................................................................... 14

5.0 LITERATURE CITED...................................................................................................... 14

APPENDICES

C LABORATORY REPORT

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LIST OF TABLES

Table ES-1. Results of range finding and definitive toxicity testing using Daphnia magna,

Pimephales promelas, and Pseudokirchneriella subcapitata with ................................... ii

Table 1. Test conditions for Daphnia magna range-finding tests using .........................3

Table 2. Test conditions for Pimephales promelas range-finding tests using .................4

Table 3. Test conditions for Pseudokirchneriella subcapitata range-finding tests using

............................................................................................................................................5

Table 4. Test conditions for Daphnia magna definitive tests using ................................6

Table 5. Test conditions for Pimephales promelas definitive tests using ........................7

Table 6. Test conditions for Pseudokirchneriella subcapitata definitive tests using ......8

Table 7. Summary of survival of Daphnia magna in range finding test using .............11

Table 8. Summary of survival of Pimephales promelas in range finding test using .....12

Table 9. Summary of Pseudokirchneriella subcapitata growth in range finding test using

..........................................................................................................................................12

Table 10. Summary of survival of Daphnia magna in definitive testing using .............13

Table 11. Summary of survival of Pimephales promelas in definitive testing using ....13

Table 12. Summary of Pseudokirchneriella subcapitata growth in definitive testing using

..........................................................................................................................................14

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1.0 INTRODUCTION

Acute toxicity data was developed for following test

regulations under the Toxic Substances Control Act (TSCA) as outlined in 40 CFR 797.

is a

mg/L. As per TSCA guidelines the study was conducted in two rounds of

testing with three aquatic test species; an invertebrate (Daphnia magna), a vertebrate (fathead

minnow, Pimephales promelas), and an algal species (Pseudokirchneriella subcapitata, formerly

Selenastrum capricornutum). The first round of testing was conducted utilizing a very broad

range of treatments in order to accurately define a possible toxic level of in an aqueous

solution. Based on results from these range-finding tests, definitive testing was conducted to

determine if at or below the water solubility concentration was toxic to test organisms.

2.0 METHODS

2.1 Overview

This study utilized EPA’s TSCA testing procedure and reflects the guidance given in 40 CFR

792 and 797. Aquatic toxicity values for three species groups (i.e., an invertebrate, a vertebrate

and a plant) were calculated in this study as required in the protocol. The following sections

summarize the specific methods used in this study. The toxicity of was tested using 48-

hour Daphnia magna, 96-hour Pimephales promelas (fathead minnow), and 96-hour

Pseudokirchneriella subcapitata tests as per the TSCA guidelines

The D. magna and fathead minnow studies were performed at

. The Pseudokirchneriella subcapitata tests were

conducted at

2.2 Test Material and Handling

Approximately 100 g of were provided by and shipped to for

use in this study. The sample was shipped in a glass jar contained in a steel container.

Upon arrival, the sample was logged into the chemical inventory. The sample of

was stored in the same glass jar and steel container in chemical storage

locker when not in use. A Material Safety Data Sheet (MSDS) accompanied the sample and is

included in Appendix A. provided a sub-sample of to for use

in the Pseudokirchneriella subcapitata testing.

2.3 Test Species

Toxicity tests were performed using D. magna (<24-hour old), P. promelas (5 day old), and P.

subcapitata. D. magna is a freshwater, planktonic invertebrate. P. promelas is a freshwater

minnow widely distributed throughout North America with a diet consisting of invertebrates and

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detritus. P. subcapitata is a green algae species that is routinely used in toxicity assessments.

All three species are EPA-approved indicator species for the demonstration of toxicity and are

currently used for assessments under the TSCA program.

2.4 Test Organism Culturing/Acclimation

D. magna used in both the range-finding and definitive testing were cultured at

in moderately hard reconstituted water (Appendix B). No acclimation was necessary. Original

source organisms were received from

provides annual taxonomic verification of all stocks. For both the range-finding and

definitive testing, five-day old fathead minnows were shipped from

in moderately hard synthetic water (Appendix B).

provides annual taxonomic verification of all organisms stocks. Pseudokirchneriella

subcapitata is routinely cultured at and was used for testing in the range-finding and

definitive testing using (Appendix D).

2.5 Toxicity Test Methods

For the range finding invertebrate and vertebrate toxicity tests that were conducted at

in , one replicate treatment and control were used for each test

treatment in a series bracketing the solubility limit for the test material The

definitive tests consisted of four replicate test chambers for each treatment and the control. The

D. magna tests used 35 mL polyethylene containers, while the fathead minnow tests utilized 473

mL polystyrene containers. The Pseudokirchneriella subcapitata range-finding test consisted of

one test chamber while the definitive testing utilized four test chambers. Pseudokirchneriella

subcapitata test chambers consisted of 250 mL flasks that were oscillated at a rate of 100

revolutions per minute for the entire test.

Test conditions (Tables 1 through 6) conform to those specified in the EPA OPPT TSCA test

guidelines and good laboratory practices (GLP) as outlined in 40 CFR 797 and 792, respectively.

Organisms were not fed during the static 48 and 96 hour acute tests. Test treatments consisted of

amending laboratory water with nominal treatments of that bracketed the water

solubility limit of the test material. Five test treatments and a control were prepared for each

test. Fathead minnow testing was conducted in a climate controlled incubator at 222

oC with a

16:8 hour light:dark photoperiod, while D. magna testing was conducted in a separate

temperature controlled incubate at 202

oC with a 16:8 hour light:dark photoperiod. P.

subcapitata was conducted using four test treatments and a control for the range-finding test. Six

test treatments and a control were utilized in the definitive test. Test chambers were held on a

shaker table in a constant temperature water bath at 242

oC with a 24:0 hour light:dark

photoperiod.

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Table 1. Test conditions for Daphnia magna range-finding tests using

PARAMETER CONDITIONS

1. Test type: Static

2. Test duration: 48 hours

3. Temperature: 20C ± 2C

4. Light quality: Ambient laboratory illumination

5. Light intensity: 10-20 μE/m2/s (50-100 ft-c) (ambient laboratory

levels)

6. Photoperiod: 16 hours light, 8 hours dark

7. Test chamber size: 35 ml Nalgene cups

8. Test solution volume: 25 ml

9. Renewal of test solutions: None

10. Age of test organisms: <24 hours, released within 8 hours of each other

11. Number organisms per test chamber: 5

12. Number replicate chambers per treatment:

1

13. Number organisms per treatment: 5

14. Feeding regime: None

15. Test chamber cleaning: Cleaned before use; utilize standard good laboratory

practices

16. Test solution aeration: Only if dissolved oxygen concentration is below 60

percent saturation; aeration must be achieved before

addition of test chemical

17. Dilution water: Moderately hard synthetic freshwater or other

appropriate water

18. Test treatments: Five test treatments and a control

19. Dilution series: 0.01, 0.1, 1, 10, 100 mg/L

20. Endpoint: Significant mortality

21. Test acceptability: 90% or greater survival in controls

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Table 2. Test conditions for Pimephales promelas range-finding tests using

PARAMETER CONDITIONS

1. Test type: Static

2. Test duration: 96 hours

3. Temperature: 22C ± 2 C

4. Light quality: Ambient laboratory illumination

5. Light intensity:

10-20 μE/m2/s (50-100 ft-c) (ambient laboratory

levels)

6. Photoperiod: 16 hours light, 8 hours dark

7. Test chamber size: 473 ml polypropylene cups

8. Test solution volume: 250 ml

9. Renewal of test solutions: None

10. Age of test organisms: 1-14 days; 24 hours range in age

11. Number organisms per test chamber: 10

12. Number replicate chambers per treatment: 1

13. Number organisms per treatment: 10

14. Feeding regime:

Artemia nauplii are made available while holding

prior to the test

15. Test chamber cleaning:

Cleaned before use; washed with detergent, then

rinsed in sequence with acetone, clean water, 5

percent nitric acid, two or more changes of dilution

water

16. Test solution aeration:

Only if dissolved oxygen concentration falls below

4.5 mg/L; rate should not exceed 100 bubbles/minute

17. Dilution water:

Moderately hard synthetic freshwater or other

appropriate water

18. Test treatments: Five test treatments and a control

19. Dilution series: 0.01, 0.1, 1, 10, 100 mg/L

20. Endpoint: Significant mortality

21. Test acceptability: 90% or greater survival in controls

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Table 3. Test conditions for Pseudokirchneriella subcapitata range-finding tests using

PARAMETER CONDITIONS

1. Test type: Static

2. Test duration: 96 hours

3. Temperature: 24C ± 2 C

4. Light quality: Ambient laboratory illumination

5. Light intensity: 4,000 lux

6. Photoperiod: 24 hours light, 0 hours dark

7. Test chamber size: 250 mL

8. Test solution volume: 100 mL

9. Renewal of test solutions: None

10. Age of test organisms: 4 days

11. Number organisms per test chamber: 1x104cells/mL

12. Number replicate chambers per treatment: 1

13. Number organisms per treatment: 1x104cells/mL

14. Feeding regime: None

15. Test chamber cleaning: Cleaned before use; utilize standard good laboratory

practices

16. Test solution aeration: None

17. Dilution water: EPA Culture

18. Test treatments: Four test treatments and a control

19. Dilution series: 0.006, 0.06, 0.6, 6.0 mg/L

20. Endpoint: Algal growth

21. Test acceptability: Demonstrated logarithmic growth in control

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Table 4. Test conditions for Daphnia magna definitive tests using

PARAMETER CONDITIONS

1. Test type: Static Non-renewal

2. Test duration: 48 hours

3. Temperature: 20C ± 2C

4. Light quality: Ambient laboratory illumination

5. Light intensity: 10-20 μE/m2/s (50-100 ft-c) (ambient laboratory

levels)

6. Photoperiod: 16 hours light, 8 hours darkness

7. Test chamber size: 35 ml Nalgene cups

8. Test solution volume: 25 ml

9. Renewal of test solutions: None

10. Age of test organisms: <24 hours, released within 8 hours of each other

11. Number organisms per test chamber: 5

12. Number replicate chambers per treatment:

4

13. Number organisms per treatment: 20

14. Feeding regime: None

15. Test chamber cleaning: Cleaned before use; utilize standard good laboratory

practices

16. Test solution aeration: Only if dissolved oxygen concentration is below 60

percent saturation; aeration must be achieved before

addition of test chemical

17. Dilution water: Moderately hard synthetic freshwater or other

appropriate water

18. Test treatments: Five test treatments and a control

19. Dilution series: 0.25, 0.5, 1, 2, 4 mg/L

20. Endpoint: Significant mortality at 24 and 48 hours

21. Test acceptability: 90% or greater survival in controls

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Table 5. Test conditions for Pimephales promelas definitive tests using

PARAMETER CONDITIONS

1. Test type: Static Non-renewal

2. Test duration: 96 hours

3. Temperature: 22C ± 2 C

4. Light quality: Ambient laboratory illumination

5. Light intensity:

10-20 μE/m2/s (50-100 ft-c) (ambient laboratory

levels)

6. Photoperiod: 16 hours light, 8 hours darkness

7. Test chamber size: 473 ml polypropylene cups

8. Test solution volume: 250 ml

9. Renewal of test solutions: None

10. Age of test organisms: 1-14 days; 24 hours range in age

11. Number organisms per test chamber: 10

12. Number replicate chambers per treatment: 2

13. Number organisms per treatment: 20

14. Feeding regime:

Artemia nauplii are made available while holding

prior to the test

15. Test chamber cleaning:

Cleaned before use; washed with detergent, then

rinsed in sequence with acetone, clean water, 5

percent nitric acid, two or more changes of dilution

water

16. Test solution aeration:

Only if dissolved oxygen concentration falls below

4.5 mg/L; rate should not exceed 100 bubbles/minute

17. Dilution water:

Moderately hard synthetic freshwater or other

appropriate water

18. Test treatments: Five test treatments and a control

19. Dilution series: 0.25, 0.5, 1, 2, 4 mg/L

20. Endpoint: Significant mortality at 48 and 96 hours

21. Test acceptability: 90% or greater survival in controls

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Table 6. Test conditions for Pseudokirchneriella subcapitata definitive tests using

PARAMETER CONDITIONS

1. Test type: Static Non-renewal

2. Test duration: 96 hours

3. Temperature: 24 C ± 2 C

4. Light quality: Ambient laboratory illumination

5. Light intensity: 4,000 lux

6. Photoperiod: 24 hours light, 0 hours darkness

7. Test chamber size: 250 mL

8. Test solution volume: 100 mL

9. Renewal of test solutions:

None

10. Age of test organisms: 4 days

11. Number organisms per test chamber: 1x104cells/mL

12. Number replicate chambers per treatment: 4

13. Number organisms per treatment: 4x104cells/mL

14. Feeding regime: None

15. Test chamber cleaning: Cleaned before use; utilize standard good laboratory

practices

16. Test solution aeration: None

17. Dilution water: EPA Culture

18. Test treatments: Six test treatments and a control

19. Dilution series: 0.25, 0.5, 1, 2, 4, 15 mg/L

20. Endpoint: Algal growth

21. Test acceptability: Demonstrated logarithmic growth in control

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2.6 Laboratory Dilution Water

Laboratory dilution water for testing was moderately-hard reconstituted water typically used for

culturing at . culture water is formulated from deionized water

produced from an E-Pure deionization system that is amended with reagent grade chemicals

following the procedure outlined in EPA’s Acute Whole Effluent Toxicity Test Methods (EPA

2002a). For both range-finding and definitive tests, the dilution water consisted of reconstituted

water with batch number . This water was formulated on and had a

pH of 8.1 su, dissolved oxygen (DO) of 7.9 mg/L, specific conductance 341 umhos/cm, total

residual chlorine of <0.01 mg/L, total ammonia of <0.01 mg/L, and with a hardness and

alkalinity of 98 and 72 mg/L as CaCO3, respectively. This dilution water has total organic carbon

concentration < 1.0 mg/L and all priority pollutants are below 1.0 μg/L. USEPA (2002b) algae

culture and test medium was the control water and dilution water used by for both the

range-finding and definitive tests.

2.7 General Test Solution Preparation and Analysis

In both the range-finding and definitive testing for fathead minnows and D. magna, was

added without a carrier or solvent to laboratory culture water at the highest test treatment. This

solution was stirred using a mechanical stirrer for up to four hours. The reported solubility of

/L in deionized water. Test solutions were made in moderately hard

reconstituted water, consistent with the test method, and therefore maximum water solubility of

was not achievable and thus undissolved was present in test chambers at

treatments above and below the reported solubility limit.

In tests with Pseudokirchneriella subcapitata, stock solutions were prepared by mechanically

stirring the highest test treatment into the algal test medium for approximately two hours. The

stock solution was decanted to remove undissolved product and then used in testing with P.

subcapitata.

For all tests, nominal concentrations were obtained through serial dilution and

concentrations were not verified analytically.

2.7.1 Test Solution Preparation - Range-Finding Tests

The D. magna and fathead minnow range finding test treatments were made by amending 500

mL of laboratory water with 50 mg of to make a nominal 100 mg/L test treatment. This

was the highest tested treatment for D. magna and fathead minnow. As noted in Section 2.7, the

maximum test solution was mechanically stirred to dissolve as much product as possible and

obtain a homogenous suspension of the undissolved A serial dilution series was used

with the amended homogenous suspension to make all other test treatments (10, 1, 0.1, 0.01

mg/L).

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As noted in Section 2.7, for testing with Pseudokirchneriella subcapitata stock solutions were

prepared by mechanically stirring the highest test treatment into the algal test medium (prior to

the addition of algal cells) for approximately two hours. The stock solution was then decanted to

remove undissolved product and used in a serial dilution to prepare the other test concentrations.

The nominal range-finding treatments for Pseudokirchneriella subcapitata were 6, 0.6, 0.06, and

0.006 mg/L

Approximately 500 mL of each test treatment was made, which was sufficient volume to provide

one replicate and general water chemistry (specific conductance, DO, pH, and temperature) for

each of the test treatments. Laboratory benchsheets are included in Appendices C and D.

2.7.2 Test Solution Preparation - Definitive Tests

Based on results of the range-finding tests, definitive tests for all three species (D. magna, P.

promelas, and P. subcapitata) targeted nominal test concentrations slightly above and below the

solubility limit of As noted in Section 2.7, the maximum solution was mechanically

stirred to dissolve as much product as possible to obtain a homogenous suspension of the

undissolved A serial dilution was used with the homogenous suspension to obtain each

treatment used in the definitive testing. For the definitive testing for D. magna and P.

promelas consisted of mg/L test treatments, as well as a control. For P.

subcapitata definitive testing with consisted of mg/L test treatments

and a control. For all treatments and tests, sufficient volume was made to provide the required

number of replicates and general water chemistry (specific conductance, DO, pH, and

temperature). For the definitive testing, all concentrations had some undissolved

present as a precipitate in the test chambers. Laboratory benchsheets are included in Appendices

C and D.

All physicochemical monitoring equipment used in water quality analyses (meters, titration

indicators, probes) were calibrated daily in accordance with standard operating procedures

(SOPs).

2.8 Data Analyses and Interpretation

Point estimates of the potential significant toxicity values for each test including 50% mortality

based on nominal test treatments for were computed using the software CETIS®

(Tidepool Scientific Software) at . The linear

interpolation method was used to calculate the point estimates. also utilized CETIS®

(Tidepool Scientific Software) and methods to calculate point estimates, which included the

growth inhibition treatment to 25% of tested organisms. Statistical datasheets are included in

Appendices C and D.

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TSCA Testing

11

2.9 Quality Assurance/Quality Control Procedures – Toxicity Data

In addition to routine use of laboratory controls for each type of water and test species,

nd rely on reference toxicant test data to ensure quality control of toxicity tests

organisms. Reference toxicant tests, using reagent grade sodium chloride, are used by

for Daphnia magna and fathead minnow testing. Quality control charts are used to

determine whether species are responding within normal bounds. Reference toxicant tests are

conducted once a month with organisms cultured in the laboratory or run concurrently with

organisms ordered from outside suppliers. D. magna, which are cultured at

required a monthly reference toxicant test. P. promelas, which were ordered from Aquatic

Research Organisms, required concurrent reference toxicant tests using the same batch of

organisms used in testing. Reference toxicant test data are reported for D. magna and P.

promelas in Appendix E and are included for Pseudokirchneriella subcapitata in Appendix C

( Laboratory Report).

3.0 RESULTS AND DISCUSSION

3.1 Results of Range-Finding Tests using

Daphnia magna

The D. magna 48-hour acute range-finding test was terminated with 100% survival in the

control. This control survival is acceptable (minimum acceptable survival = 90%). The survival

of test organisms indicated toxicity at treatments above the water solubility of

Table 7 summarizes the toxicity at 24 and 48 hours of the range finding test in each test

treatment.

Table 7. Summary of survival of Daphnia magna in range finding test using

Test Treatment (mg/L Number of Surviving Test Organisms (%)

24 Hours 48 Hours

(

* Solubility limit =

Pimephales promelas (Fathead Minnow)

The fathead minnow 96-hour acute range-finding test was terminated with 100% survival in the

control. This control survival is acceptable (minimum acceptable survival = 90%). The survival

***

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TSCA Testing

12

of test organisms indicated toxicity at test treatments above the water solubility of

Table 8 summarizes the toxicity at 24, 48, 72, and 96 hours of the range finding test in

each test treatment.

Table 8. Summary of survival of Pimephales promelas in range-finding test using

Test Treatment

(mg/L )

Number Surviving Test Organisms (%)

24 Hours 48 Hours 72 Hours 96 Hours

* Solubility limit = mg/L

Pseudokirchneriella subcapitata

The Pseudokirchneriella subcapitata 96-hour range-finding test was terminated with 0.107

absorbance at 750 nm wavelength in the control. An acceptable technique for measuring growth

of algal cells involves the use of a spectrophotometer to determine the absorbance of the cultures

at wavelength of 750 nm (USEPA 2002b). The control met USEPA test acceptability criteria.

The absorbance of test organisms indicated toxicity at that was above

the water solubility of Table 9 summarizes the toxicity at 96 hours of the

range finding test in each test treatment.

Table 9. Summary of Pseudokirchneriella subcapitata growth in range-finding test using

Test Treatment

(mg/L

Absorbance at 96

Hours

* Solubility limit = mg/L

3.2 Results of Definitive Tests using

Daphnia magna

The D. magna 48-hour acute definitive test was terminated with 100% survival in the control.

This control survival is acceptable (minimum acceptable survival = 90%). The survival of test

*

*

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TSCA Testing

13

organisms indicated no toxicity at treatments at or below the water solubility of

. Table 10 summarizes the toxicity at 24 and 48 hours of the definitive test in each test

treatment. Statistical analysis of the 24-hour and 48-hour survival resulted in EC50 values greater

than . Although the treatment resulted in survival, this is not

considered significant because a 50% reduction would be required and survival improved at the

mg/L treatment.

Table 10. Summary of survival of Daphnia magna in definitive testing using

Test Treatment (mg/L Number of Surviving Test Organisms (%)

24 Hours 48 Hours

* Solubility limit = mg/L

Pimephales promelas (Fathead Minnow)

The fathead minnow 96-hour acute definitive test was terminated with 100% survival in the

control. This control survival is acceptable (minimum acceptable survival = 90%). The survival

of test organisms indicated no toxicity at test treatments at or below the water solubility of

. Table 11 summarizes the toxicity at 24, 48, 72, and 96 hours of the

definitive test in each test treatment. Statistical analysis of the 24-hour, 48-hour, 72-hour, and

96-hour survival resulted in EC50 values greater than mg/L

Table 11. Summary of survival of Pimephales promelas in definitive testing using

Test Treatment

(mg/L

Number Surviving Test Organisms (%)

24 Hours 48 Hours 72 Hours 96 Hours

* Solubility limit = mg/L

Pseudokirchneriella subcapitata

The Pseudokirchneriella subcapitata 96-hour definitive test was terminated with 0.116

*

*

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TSCA Testing

14

absorbance at 750 wavelength in the control. The controls were above test acceptability criteria

for cell density (minimum cell density = 1x106). The absorbance of test organisms indicated no

toxicity at test treatments below the water solubility of Table 12

summarizes the toxicity at 96 hours of the definitive test in each test treatment.

Table 12. Summary of Pseudokirchneriella subcapitata growth in definitive testing

using

Test Treatment

(mg/L

Absorbance at 96

Hours

* Solubility limit = mg/L

4.0 CONCLUSIONS

The toxicity of was evaluated using three species including an invertebrate (Daphnia

magna), a vertebrate (Pimephales promelas) and an algal species (Pseudokirchneriella

subcapitata). Range finding testing indicated toxicity at treatments greater than the reported

water solubility limit of At these levels undissolved was present in the test

solution. Definitive toxicity testing using treatments in slight excess of the water solubility limit

indicated no acute toxicity to any test species.

5.0 LITERATURE CITED

USEPA. 2002a. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters

to Freshwater and Marine Organisms. EPA-821-R-02-012. October 2002, Fifth Edition. U.S.

Environmental Protection Agency.

USEPA. 2002b. Short Term Methods for Estimating the Chronic Toxicity of Effluents to

Freshwater Organisms. EPA-821-R-02-013. October, 2002, Fourth Edition. U.S. Environmental

Protection Agency.

*

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C u s t o m e r   R e p o r t

The OECD 301B method is designed to provide the screening of chemicals for ready biodegradability in an 

aerobic aqueous medium. Samples are required to achieve a threshold of 60% degradation based on the 

maximum available carbon from a given sample formulation. Total carbon is determined analytically for each 

sample and used as the reference for the determination of the percentage of carbon dioxide (% ThCO2) produced 

by microbiological degradation. 

For the purpose of determining biological degradation, two criteria can be achieved. Ready Biodegradability can 

be achieved by obtaining the 60% threshold within a 10 day window within a total of 28 days of testing. The 

second criteria, Ultimate Biodegradation, can be achieved if the amount of biodegradation meets or exceeds the 

60% threshold at a time point determined in the test (e.g. when the rate of degradation reaches a plateau). 

Two test samples were submitted for OECD 301B biodegradation testing . Neither test 

sample achieved the required level of degradation based on the total accumulated carbon dioxide over the 31 

days of the test. Due the low rate of apparent respiration within the test samples, chamber pH values were 

checked at day 17 and on the final day of the test and were within normal ranges. The microorganism 

concentrations were also checked on day 17 of the test, and were determined to exceed 1E7 CFU/ml per sample 

chamber.

COD determination was conducted on each test sample was performed by dichromate oxidation and 

spectrophotometric absorbance analysis. Lack of sample solubility and agglomeration may affect the results of 

this analysis. 

Project Summary

.

Cover

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Sample List

Method Name

Sample # Sample Name Sample Notes

OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

1 Apply to silica gel as indicated in com.

2

3 Control ‐ Sodium Acetate

Chemical Oxygen Demand (COD) Determination

7

Cover ‐‐ 1

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Title Biodegradation Test

Project ID ‐‐ Test Start Date

Contact

Entry Date

R e s u l t   T a b l e

r.

Result Table *

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Sample # 1

Interval Result

Inoculum Mixed Environmental Organisms  ()

31 day

Image: Test Sample

Figure - Test chamber carbon dioxide (CO2) measurement as the percent 

of theoretical maximum (% ThCO2) derived from the test sample. 

Average values are plotted with the standard deviation (+/- SD) for the 

time course of the test. Curve fit is applied to calculate the statistical 

confidence for 95% (blue) and predicted 95% (red) boundary lines. 

Shading below the curve fit applies to the 10 day biodegradation window 

required for the determination of Ready Biodegradability.

Sample # 2

Interval Result

Inoculum Mixed Environmental Organisms  ()

31 day

Image: Test Sample

Figure - Test chamber carbon dioxide (CO2) measurement as the percent 

of theoretical maximum (% ThCO2) derived from the test sample. 

Average values are plotted with the standard deviation (+/- SD) for the 

time course of the test. Curve fit is applied to calculate the statistical 

confidence for 95% (blue) and predicted 95% (red) boundary lines. 

Shading below the curve fit applies to the 10 day biodegradation window 

required for the determination of Ready Biodegradability.

Result Table 1 of 4 ‐‐

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Result Table *

Sample # 3 Control ‐ Sodium Acetate

Interval Result

Inoculum Mixed Environmental Organisms  ()

14 day 60 % ThCO2

Image: Control Sample

Figure - Test chamber carbon dioxide (CO2) measurement as the percent 

of theoretical maximum (% ThCO2) derived from the test sample. 

Average values are plotted with the standard deviation (+/- SD) for the 

time course of the test. Curve fit is applied to calculate the statistical 

confidence for 95% (blue) and predicted 95% (red) boundary lines. 

Shading below the curve fit applies to the 10 day biodegradation window 

required for the determination of Ready Biodegradability.

Test Method Chemical Oxygen Demand (COD) Determination

Sample # 7

Interval Result

Inoculum None  ()

dichromate oxidation ppm O2 consumed/mg

Sample # 8

Interval Result

Inoculum None  ()

dichromate oxidation ppm O2 consumed/mg

Result Table 2 of 4 ‐‐ 1 r.

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Result Table *

OECD 301 B - Solution Biodegradation

Test conditions:

- inoculum: Surface water from Skokie, IL water district.

- proportion and nature of industrial waste water in sewage: unknown, discharge from waste treatment facility 

within 1 mile.

- test duration and temperature: 28 days or as indicated, 22C +/- 2C

- bacterial inoculum ~1E5 cfu/ml 

Legend

Sample Analysis

TC - Total Carbon determined by catalytic oxidation of the test sample.

IC - Inorganic Carbon 

TOC - Total Organic Carbon - determined by the subtraction of TC from IC.

TN - Total Nitrogen determined by chemical luminescence.

%S - Percent Solids- is the dry (non-volatile) percent of the test sample. 

For the sample analysis, percent solids is determined when estimating the weight of material to test. For 

biodegradable materials, the best degradability will be obtained with sample compositions that are linear organic 

(carbon containing) molecules lacking carbon to carbon double bonds. The total carbon (TC) provides an 

indication of the material composition, but does not provide information on chemical structure or function. Inorganic 

carbon is typically low in most biodegradable materials, and increases over the course of the test due to the action 

of the microorganisms in creating waste, or biological compounds that are generated from the consumption of the 

carbon based test sample. Total nitrogen can be an indication of nutrient abundance, but is not typically used as 

part of the test sample assessment. 

Result Table 3 of 4

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Result Table *

* This report is governed by and incorporates by reference, the conditions of testing as posted on the date of issuance and is intended for your exclusive use.

Any Copying or replication of this report to or for any other person or entity, or use of our company name or Service Mark is permitted only with our prior written

consent.  This report sets forth our findings solely with respect to test samples identified herein.  The results set forth in this report are not indicative or

representative of the quality or characteristics of the lot from which a test sample was taken or any similar identical product unless specifically and expressly

noted.  Our report includes all tests requested and the results there of based upon the information provided.  You have 60 days from the date of issuance of this

report to notify us of any material error or omission caused by our handling of the samples, provided however that such notice shall be in writing and shall

specifically address the issue you wish to raise.  A failure to raise such issue within the prescribed time shall constitute your unqualified acceptance of the

completeness of this report, the test conducted and the correctness of the report contents.

Result Table 4 of 4 ‐‐

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R e p o r t   A d d e n d u m

Project ID Entry Date Test Start Date

Image Table

Sample # 1

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Inoculum Mixed Environmental Organisms

Image: Test Sample

Figure - Test chamber carbon dioxide (CO2) measurement as the percent of 

theoretical maximum (% ThCO2) derived from the test sample. Average values 

are plotted with the standard deviation (+/- SD) for the time course of the test. 

Curve fit is applied to calculate the statistical confidence for 95% (blue) and 

predicted 95% (red) boundary lines. Shading below the curve fit applies to the 

10 day biodegradation window required for the determination of Ready 

Biodegradability.

Addendum 1 of 3

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Image Table

Sample # 2

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Inoculum Mixed Environmental Organisms

Image: Test Sample

Figure - Test chamber carbon dioxide (CO2) measurement as the percent of 

theoretical maximum (% ThCO2) derived from the test sample. Average values 

are plotted with the standard deviation (+/- SD) for the time course of the test. 

Curve fit is applied to calculate the statistical confidence for 95% (blue) and 

predicted 95% (red) boundary lines. Shading below the curve fit applies to the 

10 day biodegradation window required for the determination of Ready 

Biodegradability.

Addendum 2 of 3

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Image Table

Sample # 3 Control ‐ Sodium Acetate

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Inoculum Mixed Environmental Organisms

Image: Control Sample

Figure - Test chamber carbon dioxide (CO2) measurement as the percent of 

theoretical maximum (% ThCO2) derived from the test sample. Average values 

are plotted with the standard deviation (+/- SD) for the time course of the test. 

Curve fit is applied to calculate the statistical confidence for 95% (blue) and 

predicted 95% (red) boundary lines. Shading below the curve fit applies to the 

10 day biodegradation window required for the determination of Ready 

Biodegradability.

Addendum 3 of 3

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C u s t o m e r   R e p o r t

ID Entry Date

Project Title

Biodegradation Test

The OECD 301B method is designed to provide the screening of chemicals for ready 

biodegradability in an aerobic aqueous medium. Samples are required to achieve a threshold 

of 60% degradation based on the maximum available carbon from a given sample formulation. 

Total carbon is determined analytically for each sample and used as the reference for the 

determination of the percentage of carbon dioxide (% ThCO2) produced by microbiological 

degradation. 

For the purpose of determining biological degradation, two criteria can be achieved. Ready 

Biodegradability can be achieved by obtaining the 60% threshold within a 10 day window within 

a total of 28 days of testing. The second criteria, Ultimate Biodegradation, can be achieved if 

the amount of biodegradation meets or exceeds the 60% threshold at a time point determined 

in the test (e.g. when the rate of degradation reaches a plateau). 

Two test samples were submitted for OECD 301B biodegradation re- testing (

 using a customer supplied industrial inoculum. Following arrival of the inoculum, 

the material was coarse filtered to remove particulate material. OECD 301B buffer salts were 

added to the inoculum filtrate and then incubated with constant agitation and normalized for 7 

days at 24C prior to setup of the test chambers. Each test sample (and control) was introduced 

into the test chambers by homogenizing the sample material with silica gel in a manner 

described by the customer supplied method. 

Project Summ

r.

Cover

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Cover ‐‐

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Sample List

Method Name

Sample # Sample Name Sample Notes

OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

1 Apply to silica gel as indicated in com.

2

3 Control ‐ Sodium Acetate

4 Silica gel blank

Cover

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Title Biodegradation Test

‐‐

Result Table *

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Sample # 1

Interval Result

Inoculum Mixed Environmental Organisms  ()

35 day 0 % ThCO2

Image: Chart

Figure - Test chamber carbon dioxide (CO2) measurement as the percent 

of theoretical maximum (% ThCO2) derived from the test sample. 

Average values are plotted with the standard deviation (+/- SD) for the 

time course of the test. Curve fit is applied to calculate the statistical 

confidence for 90% (blue line) and predicted 90% (red) boundary lines.

Sample # 2

Interval Result

Inoculum Mixed Environmental Organisms  ()

35 day 0 % ThCO2

Image: Chart

Figure - Test chamber carbon dioxide (CO2) measurement as the percent 

of theoretical maximum (% ThCO2) derived from the test sample. 

Average values are plotted with the standard deviation (+/- SD) for the 

time course of the test. Curve fit is applied to calculate the statistical 

confidence for 90% (blue line) and predicted 90% (red) boundary lines. 

Result Table 1 of 3

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Result Table *

Sample # 3 Control ‐ Sodium Acetate

Interval Result

Inoculum Mixed Environmental Organisms  ()

17 day

35 day

Image: Chart

Figure - Test chamber carbon dioxide (CO2) measurement as the percent 

of theoretical maximum (% ThCO2) derived from the test sample. 

Average values are plotted with the standard deviation (+/- SD) for the 

time course of the test. Curve fit is applied to calculate the statistical 

confidence for 90% (blue line) and predicted 90% (red) boundary lines. 

Shading below the curve fit applies to the 10 day biodegradation window 

required for the determination of Ready Biodegradability.

Sample # 4 Silica gel blank

Interval Result

Inoculum Mixed Environmental Organisms  ()

35 day 0 % ThCO2

Result Table 2 of 3 ‐‐

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Result Table *

* This report is governed by and incorporates by reference, the conditions of testing as posted on the date of issuance and is intended for your exclusive use.

Any Copying or replication of this report to or for any other person or entity, or use of our company name or Service Mark is permitted only with our prior written

consent.  This report sets forth our findings solely with respect to test samples identified herein.  The results set forth in this report are not indicative or

representative of the quality or characteristics of the lot from which a test sample was taken or any similar identical product unless specifically and expressly

noted.  Our report includes all tests requested and the results there of based upon the information provided.  You have 60 days from the date of issuance of this

report to notify us of any material error or omission caused by our handling of the samples, provided however that such notice shall be in writing and shall

specifically address the issue you wish to raise.  A failure to raise such issue within the prescribed time shall constitute your unqualified acceptance of the

completeness of this report, the test conducted and the correctness of the report contents.

OECD 301 B - Solution Biodegradation

Test conditions:

- inoculum: Surface water from Skokie, IL water district.

- proportion and nature of industrial waste water in sewage: unknown, discharge from waste treatment facility 

within 1 mile.

- test duration and temperature: 28 days or as indicated, 22C +/- 2C

- bacterial inoculum ~1E5 cfu/ml 

Legend

Sample Analysis

TC - Total Carbon determined by catalytic oxidation of the test sample.

IC - Inorganic Carbon 

TOC - Total Organic Carbon - determined by the subtraction of TC from IC.

TN - Total Nitrogen determined by chemical luminescence.

%S - Percent Solids- is the dry (non-volatile) percent of the test sample. 

For the sample analysis, percent solids is determined when estimating the weight of material to test. For 

biodegradable materials, the best degradability will be obtained with sample compositions that are linear organic 

(carbon containing) molecules lacking carbon to carbon double bonds. The total carbon (TC) provides an 

indication of the material composition, but does not provide information on chemical structure or function. Inorganic 

carbon is typically low in most biodegradable materials, and increases over the course of the test due to the action 

of the microorganisms in creating waste, or biological compounds that are generated from the consumption of the 

carbon based test sample. Total nitrogen can be an indication of nutrient abundance, but is not typically used as 

part of the test sample assessment. 

Result Table 3 of 3

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R e p o r t   A d d e n d u m

Project ID Entry Date Test Start Date

Image Table

Sample # 1

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Inoculum Mixed Environmental Organisms

Image: Chart

Figure - Test chamber carbon dioxide (CO2) measurement as the percent of 

theoretical maximum (% ThCO2) derived from the test sample. Average values 

are plotted with the standard deviation (+/- SD) for the time course of the test. 

Curve fit is applied to calculate the statistical confidence for 90% (blue line) and 

predicted 90% (red) boundary lines.

Addendum 1 of 3 ‐‐

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Image Table

Sample # 2

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Inoculum Mixed Environmental Organisms

Image: Chart

Figure - Test chamber carbon dioxide (CO2) measurement as the percent of 

theoretical maximum (% ThCO2) derived from the test sample. Average values 

are plotted with the standard deviation (+/- SD) for the time course of the test. 

Curve fit is applied to calculate the statistical confidence for 90% (blue line) and 

predicted 90% (red) boundary lines. 

Addendum 2 of 3

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Image Table

Sample # 3 Control ‐ Sodium Acetate

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Inoculum Mixed Environmental Organisms

Image: Chart

Figure - Test chamber carbon dioxide (CO2) measurement as the percent of 

theoretical maximum (% ThCO2) derived from the test sample. Average values 

are plotted with the standard deviation (+/- SD) for the time course of the test. 

Curve fit is applied to calculate the statistical confidence for 90% (blue line) and 

predicted 90% (red) boundary lines. Shading below the curve fit applies to the 

10 day biodegradation window required for the determination of Ready 

Biodegradability.

Addendum 3 of 3

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