### Confidential and Privileged )O

Common Synonyms:

Physical Properties
Log K ow 

### measured - OPPTS 830.7560 Water Solubility mg/L measured - OPPTS 830.7840/7860

### Confidential and Privileged

Endpoint Assay Toxicity Value

*
Degradability
Chemical Oxygen Demand: pp m O 2 

### / mg measured - dichromate oxidation Ready Biodegradability OECD 301B % ThCO 2 

### OECD 301B, silica addition Treatability study in progress

– Biodegradability study in progress

–

### 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

### - 5 -

TABLE OF CONTENTS
Title/Cover Page .............................................................................................................. ....................... 1
Good Laboratory Practice Compliance Statement ................................................................................. 2
Quality Assuran ce 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 Ca lculated Partition Coefficient ................................... 16

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

Refere nces .................................................................................................................... ........................ 17

### - 6 -

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 (P ow 

### ) 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 ge nerator column sample ....... 31

### - 8 -

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 (P ow 

### ) 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 (A CN) and finally in 1:1 (v/v) acetonitrile (ACN):H 2 

### O into the calibration range of the high performance liquid chromatography with ultraviolet detec tion (HPLC/UV) methodology. Three aqueous samples were collected from the generator co lumn into an equal volume of ACN and directly analyzed by HPLC/UV. had an approximate retention time of min. The mean calculated partition coefficient (P ow 

### ) was which gives a log 10 

### P ow 

### of

.

### - 9 -

INTRODUCTION A study was performed to determine the n- octanol/water partition coefficient (P ow 

### ) of
at 25°C. The study was conducted by for , at the anal ytical chemistry facility in . The test was performed using HPLC-grade reagent water saturated with n- octanol and the generator column method. The gene rator 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 de finitive 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 gene rator 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 P ow 

### 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 proce dures 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).

### - 10 -

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 (H 3 PO 4 ): 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 (Boe kel, 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 funne l 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 stor ed under ambient conditions. Before use in the generator column, the saturated water was equilibrate d at the test temperature (25oC) in a constant temperature water bath.

Stock Preparations A primary stock solution of the test subs tance 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 (S artorius CPA225D). The test substance was dissolved

### - 11 -

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, iden tified as , was used to prepare a set of test substance secondary stock solutions in meth anol using the following dilution scheme:
Stock Concentration (mg a.i./mL) Aliquot (m L) 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-M ar 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 th e 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 genera tor 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 in to 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

### - 12 -

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 supernatan t 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-flushe d with reagent water saturated with n- octanol to remove any entrapped air and excess n- octanol. The column was then re-plu mbed to the definitive configuration (Figure 2) and allowed to equilibrate at a flow ra te 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 reservoi r 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 deliv ery 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

### - 13 -

aliquots of the supernatant were volumetrically dilu ted 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 th e generator column directly into 10.0-mL volumetric flasks pre-filled with 5.00-mL of ACN. The resu lting 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 variab le wavelength UV detector. Chromatographic separations were achieved using a Porosh ell 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 be ginning 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 e quation. The calibration curve is presented in Figure 4. Representative chromatograms of low- a nd 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

### - 14 -

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 for tification 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 m easured 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 dilu tion factor of 2.00. The diluted aqueous matrix fortification sample was analyzed concurrently w ith the generator column samples. The matrix fortification sample had a measured concentration of corresponding to a procedural recovery of of the nominal concentration (Tab le 2). The aqueous generator column results were not corrected for this procedural recovery. The chro matogram of the aqueous matrix fortification sample is presented in Figure 7.

Example Calculations The measured concentration of in samp le number was calculated as follows:
(mg a.i./L) in sample = Peak Area - (Y-intercept) Slope x Dilution Factor

### - 15 -

Peak area = First Initial (Sample) Volume (V i ) = mL Y-intercept = First Final Volume (V f ) = mL Slope = Dilution Factor (V f / V i ) = Molecular Weight g/mol

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

(mg a.i./L) in sam ple =

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 P ow 

### 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 (P ow ) = Mean Concentration in n -Octanol Solution (M) in Aqueous Sample (M)

Partition Coefficient (P ow ) =

Partition Coefficient (P ow ) =
log 10 P ow =
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 temp erature of the water bath rem ained c onstant 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 coll ect 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

### - 16 -

un-diluted aqueous sample collected from the generato r 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 show n in Figure 9.

From the measured concentrations in the aqueous samples and in the n- octanol solution supernatant, the mean P ow 

### for 25oC was determined to be The corresponding log 10 

### P ow 

### 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 P ow 

### ), was determined to be

### - 17 -

REFERENCES

1. U.S. EPA Product Properties Test Guidelines. 1996. OPPTS 830.7560. Partition Coefficient ( n -Octanol/W ater), Generator Column Method.

2. American Society for Testing and Materials. 2006. Standard Specification for Reagent Water. D1193-06, ASTM Section II Water and Envi ronmental Technology, Vol. 11.01: 45-47.

### - 18 -

Table 1
HPLC Operational Parameters

INSTRUMENT: Agilent Series 1100/1200 High Performance Liquid Chroma tograph (HPLC) with an Agilent Series 1200 Variable W avelength Detector

ANALYTICAL COLUMN: Agilent Poroshell 120 EC-C18 (50 mm x 3.0 m m , 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% H 3 PO 4 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:

### - 19 -

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

Samp l e 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, calculate d 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.

### - 20 -

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 ).- 21 -Table 4Measured Concentrations of inAqueous Generator Column SamplesSample ID( -)Water Bath / ColumnMin/Max Temperature(oC)Concentration in mg a.i./L1Concentration in mol/L (M)1, 2MeasuredConcentrationMean ± Std. Dev. (CV)MeasuredConcentrationMean ± Std. Dev. (CV)1231Results were generated using Microsoft®Excel 2010 in full precision mode. Manual calculations may differ slightly.2Molar concentration calculated using mg a.i./L concentration and molecular weightofl).

### - 22 -

Table 5

Calculated n- Octanol/Water Partition Coefficients (P ow 

### ) for at 25oC

Sample ID ( ) Partition Coefficient (P ow ) 1 log 10 (Partition Coefficient) 1

P ow Mean ± Std. Dev. (CV) log 10 P ow Mean ± Std. Dev. (CV)

1 2 3

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

### - 23 -

Silanized glass wool Silanized glass wool aqueous solution

Chromosorb WHP

aqueous eluate water from

constant temperature bath



### Figure 1 . Diagram of generator colum n

.

### - 24 -

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

### - 25 -

1. Set up the constant temperature water bath in a clear pl astic 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 temper ature 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 te st 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). P our 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 fo r 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:H 2 O (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 generato r 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 fl ask containing the aqueous solution, and pum p 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 aque ous 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 additiona l serial dilutions of the aqueous samples for HPLC-UV analysis. Prepare aqueous matrix blank and matrix fortif ication 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.

### - 26 -

### - 27 -

Figure 5. Representative chrom a tograms 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)

### - 28 -

Figure 6. Chroma togram of the n -octanol matrix blank sample

Dilution factor = 2000 The arrow indicates the approximate retention time of the test substance.

### - 29 -

Figure 7. Chroma togram s 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.

### - 30 -

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

Dilution factor = 2000

### - 31 -

Figure 9. A representative chroma togram of an aque ous generator column sample

Dilution factor = 2.00

### 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:

Page 1 of 37

### - 5 -

TABLE OF CONTENTS
Title/Cover Page .............................................................................................................. ....................... 1
Good Laboratory Practice Compliance Statement ................................................................................. 2
Quality Assuran ce 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

Refere nces .................................................................................................................... ........................ 15

### - 6 -

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

### - 7 -

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 C OPY OF THE FINAL REPORT 

### :

TEST SUBSTANCE 

### :

TEST DATES 

### : Experimental OECD Start – Experimental EPA Start – Experimental Termination –

TEST SYSTEM 

### : HPLC-grade reagent water

S UMMARY 

### : The water solubility of the test s ubstance in reagent water at 20oC was determined using the column elution method. The mean water solubility for at was

### - 8 -

INTRODUCTION A study was performed to determine the water so lubility 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 ge nerating aqueous solutions of the test substance by pumping HPLC-grade reagen t 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 th e water solubility at the test temperature.

MATERIALS AND METHODS The study was conducted according to the proce dures 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).

### - 9 -

Test Substance
The test substance was received from on and was assigned
identification number . The test substance was identified as Batch number – An expiration da te 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 a nd 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-M ar 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 th e 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 generato r 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 (Sarto rius/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 quantitativ ely transferred into a 50.0-mL volumetric flask and

### - 10 -

was brought to final volume using acetone. This so lution 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 a nd 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 V 0446) 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 sec ond 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 entr apped 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 ad justed 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 (Fis her/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 wate r 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

### - 11 -

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 subs tance 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 anal ytical 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 cons isted 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 diluti on factor of 2.00. Details of the analytical sample processing are provided in Figure 3.

### - 12 -

Concentrations of in the samples were determined using an Agilent Series 1100/1200 HPLC equipped with an Agilent Series 1200 variab le wavelength UV detector. Chromatographic separations were achieved using a Porosh ell 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 concurren tly with the solubility samples. The calibration set was injected at the beginning and end of the analyti cal 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 r espective 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 chromatogr ams 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 L OQ for in reagent water was defined as 0.500 mg a.i./L, calculated as the product of the concen tration 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 consecu tive generator column sample collections where the measured sample concentratio ns 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 ra te 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 ot her. An additional aqueous sample was collected at

### - 13 -

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 unfortifie d 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 subs tance 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 (V i ) = mL Y-intercept = - Final Volume (V f ) = mL Slope = Dilution Factor (V f /V i ) = (10.0/5.00) =

(mg a.i./L) in sample = x (mg a.i./L) in sam ple =
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 cons tant at 20.0 ± 0.1°C throughout the experiment. Under the instrumental conditions presented, the m ean 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 th e approximately mL/min flow rate settings differed by
which was well within the ± test criteri on. The overall mean measured water solubility at

### - 14 -

was mg a.i./L ). Representative chromatograms of solubility samples at flow rates of approxima tely 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 ).

### - 15 -

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 Envi ronmental Technology, Vol. 11.01: 45-47.

### - 16 -

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% H 3 PO 4 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:

### - 17 -

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.

### - 18 -

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.

### - 19 -

Figure 1 . Diagram of generator column.

### - 20 -

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.

### - 21 -

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 dir ectly into 10.0-mL volumetric flasks pre-filled with 5.00-mL of acetonitrile (ACN), measured with a volumet ric 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:H 2 O. 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 wa ste for at least 10 minutes before collecting aqueous solute samples. After collecting all samples at the c onfirmatory 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.

### - 22 -

Figure 4. Calibration curve for

### - 23 -

Figure 5. Representative chrom a tograms of low- a nd 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)

### - 24 -

Figure 6. Representative chrom a tograms of reagent blank sample.
Reagent Blank Sample ID , Dilution factor = 2.00
Note: The arrow indicates the approximate retention time of

### - 25 -

Figure 7. Representative chrom a tograms 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.: Col umn elution flow rate setting = ~ 0.5 mL/min, Dilution factor = 2.00)Estrogen Receptor Activation by Test Articles

Final ReportCONFIDENTIAL

Page 2 of 8 TABLE OF CONTENTS 1 Objective

................................................................................................................................................. 3

Regulatory Guidelines

................................................................ ................................................... 3 1.1 2 Test Articles

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

.......................................................................................................................................... 4

In vitro ADME-Tox Experimental Conditions

................................................................ ................. 4 3.1 3.1.1 Cytotoxicity experimental conditions

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

................................ ................................ ... 4 4 Resul ts

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

In vitro ADME-Tox Summary

......................................................................................................... 5 4.1 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

......................................................................................................... 8CONFIDENTIAL

Page 3 of 8 

# 1 Objective The objective of this study was to determine whether test compounds were capable of activating Estrogen Receptor alpha

. 

## Regulatory Guidelines 1.1 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.CONFIDENTIAL

Page 4 of 8 

# 3 Test Methods Testing was performed at

. 

## In vitro ADME

-Tox Experimental Conditions 3.1 3.1.1 Cytotoxicity experimental conditions

Client ID Test conc. ( μ M) Assay time Cell lines Readout Analytical method



### 3.1.2 ER Alpha Foci Formation experimental conditions

Client ID Test conc.

( μ M) 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.CONFIDENTIAL

Page 5 of 8 

# 4 Results

In vitro ADME

-Tox Summary 4.1
4.1.1 Estrogen Receptor Activation Assay Results

Client ID ER Activation AC50 Cytotoxicity

AC50 comment

p ositive Control ER

NR= no response seenCONFIDENTIAL

Page 6 of 8 

### 4.1.2 AC50 Curves

4.1.2.1 Estrogen Receptor Alpha Foci FormationCONFIDENTIAL

Page 7 of 8 

### 4.1.2.2 CytotoxicityCONFIDENTIAL

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.Final Report

Approved:

Date:CONFIDENTIAL

Page 2 of 8 TABLE OF CONTENTS 1 Objective

................................................................................................................................................. 3

Regulatory Guidelines

................................................................ ................................................... 3 1.1 2 Test Articles

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

.......................................................................................................................................... 4

In vitro ADME-Tox Experimental Conditions

................................................................ ................. 4 3.1 3.1.1 Cytotoxicity experimental conditions

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

............................................................................... 4 4 Resul ts

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

In vitro ADME-Tox Summary

......................................................................................................... 5 4.1 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

......................................................................................................... 8CONFIDENTIAL

Page 3 of 8 

# 1 Objective The objective of this study was to determine whether test compounds were capable of activating the Androgen Receptor. 

## Regulatory Guidelines 1.1 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.CONFIDENTIAL

Page 4 of 8 

# 3 Test Methods Testing was performed at 

## In vitro ADME

-Tox Experimental Conditions 3.1 3.1.1 Cytotoxicity experimental conditions

Client ID Test conc. ( μ M) Assay time Cell lines Readout Analytical method Cell



### 3.1.2 AR Translocation experimental conditions

Client ID Test conc.

( μ M) 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.CONFIDENTIAL

Page 5 of 8 

# 4 Results

In vitro ADME

-Tox Summary 4.1
4.1.1 Androgen Receptor Translocation Assay Results

Client ID AR

Translocation AC50 (Nuclear Intensity) Cytotoxicity

AC50 comment

NR= no response seenCONFIDENTIAL

Page 6 of 8 

### 4.1.2 AC50 CurvesCONFIDENTIAL

Page 7 of 8 

### 4.1.2.2 CytotoxicityCONFIDENTIAL

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.Page 1 of 21 

## Recombinant Yea st Sc reen for Oestrogenic and Androgenic Activity

### 2



# Spons or an d Test Facility Details

Sponsor

Test facili ty

### 3



# T able 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

### 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

### 5



# Stud y Director Statement

Recombinant Yeast Sc reen fo r Oestrogenic and Androgenic Activity: 6 compounds

### 6



# Con tribu ting Scientists

Recombinant Yea st Sc reen for Oestrogenic and Androgenic Activity:

Study management

Head, Department of In Vitro Technologies

Department of In Vitro Technologies

Assistant Department of In Vitro Technologies

### 7



# Summar y

Thi s 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 μ g/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.

### 8



# 1. Intro duc tion

1.1 Object ive
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.

### 9



# 2. Experime n tal Procedure

2.1 Stud y 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:

P u rity: 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.

### 10



## 2.5 Prepara tion of test substances

The test substanc es 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 μ g/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 (5 α -Androstan-17 β -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 μ L aliquots of each dilution of the test substance and reference substance dilution serieswere 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 μ L aliquots of the stock solution. The duplicate rows were separated by a single “blank” row containing 10 μ L 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 μ L 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 μ g/L for the test substances, 10 μ g/L - 5 ng/L for the 17- β

estradiol 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.

### 11



# 3. A s sessment of Results

After 3 d ays 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.

### 12



# 4. Mainte nan ce 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.

### 13



# 5. Resul t

s

The results of the endocri ne disrupter ass essments 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.

.

# 6. Refere nce

R OU TLED GE, 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.

### 15

### 16



### Figure 2 Estrogen a ssay:

### 17



### Figure 3 Estrogen assay:

### 18



### Figure 4 Estrogen assay:

### 19



### Figure 5 Estrogen a ssay:

### 20



## An nex 1 Media

GROWTH MEDIUM

Minimal Medium

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

KH 2 

### PO 4 

### 13.61 g ( NH 4 

### ) 2 

### SO 4 

### 1.98 g KOH pellets 4.2 g MgSO 4 

### 0.2 g Fe 2 

### (SO 4 

### ) 3 

### solution (40 mg/50 m L H 2 

### O) 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 μ m pore size disposable filter into sterile glass bottles, and then stored at 4oC.

### 21



## Annex 1 M edia (continued)

D-(+)-Glucose
A 20% w/v solution was prepared, sterilised b y 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 μ m 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 μ L
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 μ m pore size filter into sterile glass bottles and then stored at 4oC.

# Toxicity Testing for

Using Daphnia magna , Pimephales promelas , and Pseudokirchneriella subcapitata

Report

Submitted to:

Prepared by:TSCA Testing

ii 

## 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 EC 50 

### (mg/L)

Daphnia magna Pimephales promelas Pseudokirchneriella subcapitataTSCA Testing

iii 

### 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 REPORTTSCA Testing

iv 

### 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 ..........................................................................................................................................14TSCA Testing

1 

### 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. magn a 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 i s a freshwater, planktonic invertebrate. P. promelas is a freshwater minnow widely distributed throughout North America with a diet consisting of invertebrates andTSCA Testing

2



### 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 o 

### C with a 16:8 hour light:dark photoperiod, while D. magna testing was conducted in a separate temperature controlled incubate at 20  2 o 

### C 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 o 

### C with a 24:0 hour light:dark photoperiod.TSCA Testing

3



### 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/m 2 /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 controlsTSCA Testing

4



### 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/m 2 /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 controlsTSCA Testing

Inc. 5 

### 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: 1x10 4 cells/mL 12. Number replicate chambers per treatment: 1 13. Number organisms per treatment: 1x10 4 cells/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 controlTSCA Testing

6



### 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/m 2 /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 controlsTSCA Testing

. 7 

### 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/m 2 /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 controlsTSCA Testing

8



### 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: 1x10 4 cells/mL 12. Number replicate chambers per treatment: 4 13. Number organisms per treatment: 4x10 4 cells/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 controlTSCA Testing April 24, 2013

9 

### 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 CaCO 3, 

### 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).TSCA Testing

10



### 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.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 

## * * 

## *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 

## *

*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 EC 50 

### 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 EC 50 

### 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 

## * *TSCA Testing

14



### absorbance at 750 wavelength in the control. The controls were above test acceptability criteria for cell density (minimum cell density = 1x10 6 

### ). 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. 

## *

# C u s t o m e r R e p o r t

The OECD 301B method is designed to provide the screeni ng of chemicals for ready biodegradability in an aerobic aqueous medium. Samples are required to ac hieve a threshold of 60% degradation based on the maximum available carbon from a given sample formulatio n. 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 cr iteria 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 respirati on 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 tes t, and were determined to exceed 1E7 CFU/ml per sample chamber.
COD determination was conducted on each test samp le was performed by dichromate oxidation and spectrophotometric absorbance analysis. Lack of sample so lubility and agglomeration may affect the results of this analysis. Pro j ect Summar y 

### .

CoverSam p le List Method Name

S ample # S ample Name S ample 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

‐‐

### 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 *

OECD 301 B ‐ Solution Biodegradation by CO2 Evolution Test Method

1 Sample # Interval Result

Inoculum Mixed Environmental Or g anisms ()

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.

2 Sample # Interval Result

Inoculum Mixed Environmental Or g anisms ()

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 ‐‐

## Result Table *

3 Control ‐ Sodium Acetate Sample # Interval Result

Inoculum Mixed Environmental Or g anisms ()

60 14 % ThCO2 day

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.

Chemical Oxygen Demand (COD) Determination Test Method

7 Sample # Interval Result

Inoculum None ()

dichromate oxidation ppm O2 consumed/mg

8 Sample # Interval Result

Inoculum None ()

dichromate oxidation ppm O2 consumed/mg



### Result Table 2 of 4 1

‐‐ r.

## 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 sew age: unknown, discharge from waste treatment facility within 1 mile. - test duration and temperature: 28 day s or as indicated, 22C +/- 2C - bacterial inoculum ~1E5 cfu/ml Legend Sample Analysis TC - Total Carbon determined by catalyti c 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-vola tile) 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 wi th sample compositions that are linear organic (carbon containing) molecules lacking carbon to car bon double bonds. The total carbon (TC) provides an indication of the material composition, but does not provide information on chemic al structure or function. Inorganic carbon is typically low in most biodegradable materials, and incr eases 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 indicati on of nutrient abundance, but is not typically used as part of the test sample assessment.



### Result Table 3 of 4

## 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 wi th 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 an y similar ide n tical 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 sa mples, provid ed however that such notice sh all 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 content s.



### Result Table 4 of 4 ‐‐

# R e p o r t A d d e n d u m

Project ID Test Start Date Entry Date Image Table

1 Sample #

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Mixed Environmental Organisms Inoculum

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

### Image Table

2 Sample #

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Mixed Environmental Organisms Inoculum

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

### Image Table

3 Control ‐ Sodium Acetate Sample #

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Mixed Environmental Organisms Inoculum

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

# 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. Samp les 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 diox ide (% ThCO2) produced by microbiological degradation. For the purpose of determining biological degradati on, 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 cr iteria, Ultimate Biodegradati on, 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 inoc ulum. Following arrival of the inoculum, the material was coarse filtered to remove par ticulate material. OE CD 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 sa mple material with silica gel in a manner described by the customer supplied method. Pro j ect Summ 

### r.

Cover

### Cover ‐‐Sam p le List Method Name

S ample # S ample Name S ample 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

### Title Biodegradation Test



## ‐‐
Result Table *

OECD 301 B ‐ Solution Biodegradation by CO2 Evolution Test Method

1 Sample # Interval Result

Inoculum Mixed Environmental Or g anisms ()

0 35 % ThCO2 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.

2 Sample # Interval Result

Inoculum Mixed Environmental Or g anisms ()

0 35 % ThCO2 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.



### Result Table 1 of 3

## Result Table *

3 Control ‐ Sodium Acetate Sample # Interval Result

Inoculum Mixed Environmental Or g anisms ()

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.

4 Silica gel blank Sample # Interval Result

Inoculum Mixed Environmental Or g anisms ()

0 35 % ThCO2 day



### Result Table 2 of 3 ‐‐

## 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 wi th 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 identi cal 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 sa mples, provide d how ever 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 re port conte n ts. 

### OECD 301 B - Solution Biodegradation

Test conditions: - inoculum: Surface water from Skokie, IL water district. - proportion and nature of industrial waste water in sew age: unknown, discharge from waste treatment facility within 1 mile. - test duration and temperature: 28 day s or as indicated, 22C +/- 2C - bacterial inoculum ~1E5 cfu/ml Legend Sample Analysis TC - Total Carbon determined by catalyti c 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-vola tile) 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 car bon double bonds. The total carbon (TC) provides an indication of the material composition, but does not provide information on che mical 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 g enerated from the consumption of the carbon based test sample. Total nitrogen can be an indicati on of nutrient abundance, but is not typically used as part of the test sample assessment.



### Result Table 3 of 3

# R e p o r t A d d e n d u m

Project ID Test Start Date Entry Date Image Table

1 Sample #

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Mixed Environmental Organisms Inoculum
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 stat istical confidence for 90% (blue line) and predicted 90% (red) boundary lines.



### Addendum 1 of 3 ‐‐

### Image Table

2 Sample #

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Mixed Environmental Organisms Inoculum
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 stat istical confidence for 90% (blue line) and predicted 90% (red) boundary lines.



### Addendum 2 of 3

### Image Table

3 Control ‐ Sodium Acetate Sample #

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Mixed Environmental Organisms Inoculum

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 stat istical 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