THE DOW CHEMICAL COMPANY STUDY ID: PAGE 1 OF 40

- 

### STUDY TITLE

GROWTH INHIBITION TEST WITH THE GREEN ALGA Pseudokirchneriella subcapitata

Test Guidelines
OECD, Guideline 201 (1984) EEC Directive 92/69/EEC, Part C.3 (1992)

Author(s)

Study Completion Date

01 March 2012

Sponsor
The Dow Chemical Company

Midland, Michigan 48674

Performing Laboratory
Toxicology & Environmental Research and Consulting The Dow Chemical Company

Midland, Michigan 48674

La boratory Project Study IDTHE DO\V CHEMICAL COMPANY
STUDY ID: PAGE



### COMPLIANCE GOOD LABORATORY PRACTICE STANDARDS

Title: \VITH



### this were**v'J~LL"'-"'v~,",'-'** 

### in**"',",LL'iJ"UU.,"~vv** 

### with**hA1"q~"'~'J** 

### Practice Standards:

Organisation Economic Co-Operation and Development Series on Principles of Good Laboratory Practice and Compliance Monitoring, 1. OECD

Principles on Good Laboratory (as revised 1997) ENV/MC/CHEM(98) 17.

US Environmental Protection Agency

-- TSCA GLPs Title 40

Substances Control Act**TSCA)~** 

### Good Laboratory Practice Final Rule.THE DOW CHEMICAL COMPANY STUDY ID: 1 11131 PAGE 3



### QUALITY ASSURANCE STATElVlENT



### Compound:

Title: GROWTH INHillITION TEST WITH
THE GREEN ALGA 

### Pseudokirchneriella subcapitata

This study was examined for conformance with Good Laboratory Practices as published by the USEPA TSCA and OECD. The final report was determined to be an accurate reflection of the data obtained. The dates of Quality Assurance activities on this study

are listed below.

Study Initiation Date: 9 June 2011

Final protocol
Study conduct
Protocol, data, and draft report DATE OF AUDIT:



### 10 June 2011

24 August 2011

14 October 2011

- 16 February 2012 DATE FINDINGS REPORTED TO STUDY DIRECTORJMANAGEMENT:



### 10 June 2011

24 August 2011

20 February 2012

Final report The date of the signature below is the date of the final report audit.

The final report accurately reflects the raw data of the study.

Quality Assurance Toxicology & 

### Environmental Research and Consulting The Dow Chemical Company 1803 Building Midland, Michigan 48674

### SIGNATURE PAGE THE DOW CHEMICAL COMPANY STUDY ID: PAGE 4



### WITHTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 5



### TABLE OF CONTENTS

Page

- 

### STUDY TITLE ....................................................................................................................1

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS ..................2

QUALITY ASSURANCE STATEMENT ..........................................................................3

SIGNATURE PAGE ...........................................................................................................4

SUMMARY .........................................................................................................................7

INTRODUCTION ...............................................................................................................8 Purpose ............................................................................................................................8 Test Guidelines ................................................................................................................8 Quality Assurance ...........................................................................................................8 Archiving ........................................................................................................................8 Safety

............................................................................................................................... 8

MATERIALS AND METHODS .........................................................................................8 Test Material ...................................................................................................................8 Test Organism .................................................................................................................9 Culture and Test Medium ...............................................................................................9 Test Vessels .....................................................................................................................9 Selection of Test Concentrations ....................................................................................9 Preparation of Test Solutions ........................................................................................10 Definitive Test ...............................................................................................................10 Analytical Measurements ..............................................................................................11 Statistical Analysis ........................................................................................................12

RESULTS AND DISCUSSION ........................................................................................12 Analytical Results .........................................................................................................12 Test Conditions .............................................................................................................12 Biological Results .........................................................................................................13 Acceptance Criteria .......................................................................................................13

CONCLUSIONS ................................................................................................................14

REFERENCES ..................................................................................................................14

TABLES ............................................................................................................................15
Table 1. Test Material Information ..............................................................................15 Table 2. Typical Culturing Conditions.........................................................................16 Table 3. Summary of Results from Analysis of Test Solutions for ....................17 Table 4. Summary of Temperatures, Light Intensities, and pH Values During the Exposure Phase .................................................................................................18 Table 5. Mean Cell Density (x 10 4 

### cells/mL) after 24, 48, and 72 Hours of Exposure ............................................................................................................19 Table 6. Mean Specific Growth Rate (day 1 

### ) after 72 Hours of Exposure ....................20THE DOW CHEMICAL COMPANY STUDY ID: PAGE 6



### TABLE OF CONTENTS

Page

- 

### FIGURE .............................................................................................................................21 Figure 1. Graphical Representation of the Mean Cell Density Over Time ..................21

APPENDICES ...................................................................................................................22 APPENDIX A. Certificate of Analysis for Test Substance .........................................22 APPENDIX B. Growth Medium Composition ............................................................24 APPENDIX C. Analytical Report ................................................................................25
APPENDIX D. 24 -, 48- and 72- Hour Cell Density (Cells x 10 4 

### /mL) Values for Replicate Test Flasks for Each Test Level ........................................................39
APPENDIX E. 72

- Hour Mean Specific Growth Rate (Day -1 

### ) Values for Replicate Test Flasks for Each Test Level ........................................................................40THE DOW CHEMICAL COMPANY STUDY ID: PAGE 7

- 

### SUMMARY

TITLE: 

### GROW TH
INHIBITION TEST WITH THE GREEN ALGA Pseudokirchneriella subcapitata

STUDY NUMBER:

TEST SUBSTANCE: 

### Lot # Batch purity of ±

TES T DATES: 23 to 26 August 2011

TEST ORGANISM: Pseudokirchneriella subcapitat a

TEST CONDITIONS: Exposure Scenario: 72-hour, static
Temperature Range: 22.1 – 23.8 °C Light Intensity Range: 4440 -5810 lux pH Range: 7.2 -7.3 Shaking Rate: 100 rpm. Photoperiod: 24 hour light

NOMINAL TEST

CONCENTRATIONS: 0 ( AAP medium control), 100 mg L

MEASURED TEST

CONCENTRATIONS: < LLQ ( AAP medium control), 0.00253 mg L

RESULTS: Based on nominal loading rates, both the 72-hour E
r 

### C 50 

### for specific growth rate and the E y 

### C 50 

### for cell density were >100 mg/L and the NOECs were 100 mg/L 

### Based on mean measured concentrations, the 72-hour
calculated E r 

### C 50 

### for specific growth rate was >0.00253 mg L and the NOEC was 0.00253 mg
L. The 7 2- hour calculated E y 

### C 50 

### cell density was >0.00253 mg L and the NOEC for cell density

was determined to be 0.00253 mg/L.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 8

- 

### INTRODUCTION

Purpose

The purpose of this study was to evaluate the acute toxicity of (

to the unicellular green alga Pseudokirchneriella subcapitata , over a 72-hour static exposure period. Reduction in algal growth rates and yield in test substance treatments relative to the controls were used to evaluate the phytotoxicity of the tes t substance. The

data collected were used to calculate the 72 - hour E r 

### C 50 

### (based on growth rate) and E y 

### C 50 

### (based on cell density) values, and no- observed

- effect concentration (NOEC) values for each endpoint.

Test Guidelines

OECD Organisation for Economic Cooperation and Development (1984). OECD Guideline for Testing of Chemicals No. 201, Alga, Growth Inhibition Test. Adopted 7 June 1984.

EC Official Journal of the European Communities (1992). Annex to Commission Directive 92/69/EEC, C.3 Algal Inhibition Test, Vol. 35, 29 December 1992.

Quality Assurance

The study conduct, data, protocol, protocol changes/revisions, and final report will be inspected by the Quality Assurance Unit, Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

Archiving

The data, protocol, protocol changes/revisions, and final report will be archived by the Toxicology & Environmental Research and Consulting archivist and stored at The Dow Chemical Company, Midland, Michigan.

Safet y
Health and safety exposure data on the test material will be reviewed prior to initiation of the study and appropriate safety precautions followed.

MATERIALS AND METHODS

Test Material

The test material, ( was received on 22 Marc h 2010 (Lot # Batch and stored under ambient conditions until use. The total purity was ± 0.03% by gas chromatography with identification by Fourier transformTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 9

- 

### infrared spectroscopy and gas chromatography mass spectometry ( et al., 2010 ). Detailed information on the test chemical is presented in Table 1 , and the certificate analysis for the lot used in the study is presented in Appendix A

.

T est Organism

Pseudokirchneriella subcapitata from in -house cultures were initially obtained from the University of Texas at Austin Culture Collection (UTEX1648; lot # 090810) in September 2010. This species is widely accepted and recommended for toxicity testing by the test guidelines. Stock cultures of this organism were maintained aseptically by periodic transfer into sterile medium. Typical culture conditions are listed in Table 2

. The algal inoculum for the test was prepared from a 4 -day old stock culture. A Coulter Multisizer 3 was used to determine the cell density of the stock culture. This evaluation

determined that a 0.0762 mL aliquot of the culture was required to inoculate each test

vessel at an initial cell densit y of approximately 10,000 cells/mL.

Culture and Test Medium

The algae were cultured in freshwater algal nutrient medium (i.e., AAP medium), prepared with filtered (0.45 μm) and sterile deionized water and reagent grade chemicals. The source of the deionized water was Lake Huron water supplied to The Dow Chemical

C ompany by the City of Midland Water Treatment Plant prior to treatment. The base water used to prepare the media is analyzed periodically to verify that no contaminants are present at levels that may interfere with the test results. Algae were cultured under continuous illumination of approximately 5,200 ± 780 lux at a temperature of 23 ± 2oC. All culturing is done axenically in 250-mL Erlenmeyer flasks by pipetting 0.5 mL of cultured cells into 50 mL sterile media. All flasks are continually oscillated. A complete description of the medium is provided in Appendix B

.

Test Vessels

Test vessels were sterilized 250

- mL borosilicate Erlenmeyer flasks with foam stoppers each containing 50 mL test medium. Each flask was uniquel y labeled for identification

purposes.

Selection of Test Concentrations

Because the solubility of is low (i.e., 0.003 mg L in AAP ; et al

., 2011 ) and the toxicity is also suspected to be low, a limit test was conducted at a nominal concentration of 100 mg L.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 10

- 

### Preparation of Test Solutions

Bu lk test solutions were prepared a day prior to test initiation (day 0) at nominal (target)

concent rations of 0 (water control; AAP ), and 100 mg L . After adding the to the AAP media, the flask was covered with parafilm and left to stir for appoximately 24

hours.

Preliminary analysis showed that filtering with both 0.45 and 0.22 μm filters removed all of

the from the solution ( et al. , 2011 ) . Therefore, in an attempt to reduce the amount of suspended particulates while maximizing exposure concentrations, solutions were centrifuged prior to use. Approximately 250 mL of each solution were added to polypropylene 250- mL bottles which were centrifuged for 45 minutes at approximately 10,000 rpm. Solutions were carefully decanted into collection vessels which were sampled for analytical confirmation of dose.

While centrifuging and decanting removed much of the particulate matter, s ome small particulate matter was still visible suspended in the AAP. T herefore, test solutions were

sh aken vigorously prior to dosing test vessels and prior to analytical sampling to make the test solutions as homegeneous as possible.

Definitive Test

The definitive test was conducted a s a limit test, aseptically under static conditions for approximately 72 hours from 23 August to 26 August 2011. S even replicate test vessels were prepared for the 100 mg L treatment and seven replicate test vessels were prepared at the medium control level. Each replicate contained 50 mL of the appropriate

test solution. S ix replicates for each the AAP medium control and 100 mg L test
level were inoculated with approximately 10,000 cells 

### /mL. Inoculations were mad e within 30 minutes of sampling for analytical verification of test solution concentrations.

An additional replicate for the AAP control and treatment were prepared but not inoculated with algae to serve as a counting blank. These blanks were used to correct the daily counts for the interference of the test material and to monitor pH and concentration of the test material without the algal biomass. At test initiation and following sampling

for cell densities at 24 and 48 hours the replicate test vessels were placed in a walk -in environmental chamber (Lab -Line Environmental Chamber, Lab- Line Inc., Melrose, Illinois) on a shaker table (set at 100 rpm) according to a computer- generated

randomization. All vessels were uniquely labeled for identification purposes. TheTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 11

- 

### incubator was set at 23 ± 2 °C with continuous light at approximately 5200 ± 780 lux. The photoperiod was set at 24 hours of light per day.

Algal cell densities of the initial inoculum and test cultures were determined by electron particle counting using a Coulter Multisizer 3 fitted with a 70 μm aperture tube. Cells

were cumulatively counted at a lower threshold e quivalent spherical diameter of approximately 2.6 μm to a higher threshold equivalent spherical diameter of approximately 8.7 μm. Two separate cell count readings were made per replicate. Total cell counts were determined at approximately 24, 48, and 72 hours. Two readings were

ma de per replicate. The readings for the blank replicates were used to correct for background in daily cell count calculations. The adjusted cell counts were converted to cell density (cells/mL) for statistical analysis and reporting. In addition, at test termination, morphological observations were done on compiled s ample s of all inoculated replicates from all the controls and test vessels

. At test initiation, samples were collected from each bulk test solution for measurement of pH. At test termin ation, pH was measured in aliquots collecteded from pooled inoculated replicates (control, and blanks). Temperature was continuously monitored with a minimum/maximum thermometer placed in a surrogate vessel. Light intensity was

measured at test initiation at each position where inoculated replicate vessels were placed during the in- life phase.

Morphological observa tions were made on a composited sample of the inoculated replicates at each test concentration at test terminati on. The cells were observed under a microscope (Olympus BHB-DO System Microscope (Olympus Corporation, Tokyo, Japan); 20x or 40x objective lens; WF10x eyepiece; 1.25x Dual Observation Deck) using a Bright Line Hemacytometer Counting Chamber.

Analytical Measurements

Samples w ere collected from the bulk test solutions on day 0 and from individual test
vessels on day 3. To assess analytical method precision and solution homogeneity, three additional samples w ere collected on day 0 from the 100 mg L concentration bulk

treatm ent solutions. To confirm concentrations, the collected samples were analyzed using liquid chromatography with positive ion electrospray ionization tandem mass spectrometry detection (HPLC/ESI

- MS/MS). For a full description of the analytical sampling procedure and method refer to Appendix C

.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 12

- 

### Statistical Analysis

The primary endpoint of this test was the response of the alga e to expressed as a function of average specific growth rate (per unit time). Results are also expressed in terms of cell density, expressed cells/mL (x 10,000). For each endpoint, mean values were calculated for the exposure concentration level and the control level. Percent inhibition values were calcaulted based on differences in mean exposure concentration values compared to control values.

The following formula was used to calculate growth rate:

Where: **μ** = mean specific growth rate from moment i to j (days -1 

### ) ln = natural logarithm Ni = initial cell density at time i Nj = cell density at time j ti = the moment time for the start of the period tj = the moment time for the end of the period

The response variables ( i.e., growth rate, cell density) were analyzed using a student t- test which was adjusted for unequal variances, if necessary. All analyses were conducted using SAS version 9.2 (SAS Institute, Cary, North Carolina).

RESULTS AND DISCUSSION

Analytical Results

Detailed results from the analytical confirmation of the test solutions, analytical method precision, solution homogeneity, and linearity, as well as supporting tables an d figures,

can be found in Appendix C

. A summary of the results obtained from the analyses of test solutions is presented in

Table 3 . Measured concentrations were 0.00227% of nominal at 0 hours and

0.00152-0.00368% of nominal at 72 hours. Control solutions were less than the lowest standard quantified, 0.000559 mg L.

Test Conditions

Temperature, light intensity, and pH results are presented in Table 4 . Temperatures during the exposure period ranged from 22.1 to 23.8 oC. Light intensity ranged from ij i j ji 

### tt NN

**− −** 

**=**

**−** 

### lnln **μ**THE DOW CHEMICAL COMPANY STUDY ID: PAGE 13

- 

### 4440 - 5810 lux. The pH was 7.2 at test initiation, and ranged from 7.2 to 7.3 in replicates both with and without algae at test termination.

Biological Results

All biological results are expressed in terms of both nominal and mean measured

concentrations of

Mean cell density (biomass) results are presented in Table 5 (individual data are in Appendix D ). Ther e were no significant differences between the control and

treatment (p = 0.9094). Therefore, the EC 50 

### and the NOEC for cell density are > 0.00253

and 0.00253 m g L , respectively

. Mean specific growth rate results are presented in Table 6 (individual data are presented

in Appendix E ) and a graphical representation of these data ( i.e. , growth curve) is presented in Figure 1 . There were no significant diffe rences between the control and treatment (p = 0.8651 ). T herefore, the E r 

### C 50 

### and the NOEC for specific growth rate

are > 0.00253 m g and 0.00253 L , respectively

. Microscopic evaluation of cell s in both the control and test vessels revealed no abnormal observations at any test level.

Acceptance Criteria

The following acceptance criteria set forth in the OECD 201 guideline (2006) were met:

1) The biomass in the control cultures increased exponentially by a factor of at l east 16 within 72 hours. The 72-hour mean cell density increased by approximately 184-fold, thereby exceeding the minimal criteria.

2) The mean coefficient of variation for section

- by

- section specific growth rates (days 0 -1, 1-2 , etc) was 1. 6%, which is bel ow the limit of 7% set by the guideline.

3) The mean coefficient of variation for section

- by

- section specific growth rates (days 0 -1, 1-2 and 2-3, for 72- hour tests). The growth rate (per day) for each control replicate was calculated for each time period. A mean, standard deviation, and coefficient of variation was calculated for each replicate based on each daily growth rate. The mean coefficient of variation for the control replicates was 34%,

which was below the maximum criterion of 35%.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 14

- 

### CONCLUSION S

The acute toxicity ranges for with Pseudokirchneriella subcapitata were empirically determined in a 72 -hour exposure. No toxicity was noted at nominal loading rates of 100 mg/L Based on mean measured concentrations, the 72- hour calculated

E r 

### C 50 

### for specific growth rate was >0.00253 mg L and the NOEC was 0.00253 mg

L. The 7 2- hour calculated E y 

### C 50 

### cell density was >0.00253 mg L and the NOEC for cell density was determined to be 0.00253 mg/L.

REFERENCES

&

Organisation for Economic Cooperation and Development (2006 ). OECD Guideline for Testing of Chemicals No. 201, Freshwater Alga and Cyanobacteria, Growth Inhibition
Test. Adopted 23 March 206.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 15

GROWTH INHIBITION TEST WITH THE GREEN ALGA Pseudo kirchneriella subcapitata

- Table 1. Test Material Information



### Test Material Common Name Benzoylated

Chemical Name

CAS number

Molecular Formula

Molecular Weight
Supplier The Dow Chemical Company, Midland, Michigan
(Lot # Batch

Purity/Characterization The p urity of the test material was determined to be area by gas chromatography with identification by Fourier transform infrared spectroscopy and gas chromatography mass
spectronomy

Chemical StructureTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 16

GROWTH INHIBITION TEST WITH THE GREEN ALGA Pseudo kirchneriella subcapitata

- Table 2 . Typical Culturing Conditions



### Organism: Pseudokirchneriella subcapitata

Temperature: 23 ± 2 **°** C

Light (lux): 8000 ± 1600 (test subculture)

Photoperiod: Continuous (24 hours light/day)

Medium: Algal assay procedure medium (AAP)

pH: Range: approximately 7.0 –7.5

Culture Conditions: Axenic

Culture Volume: 50 mL

Culture Vessel: 250- mL Erlenmeyer flask

Culture Vessel Cap: Foam capTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 17

GROWTH INHIBITION TEST WITH THE GREEN ALGA Pseudo kirchneriella subcapitata

- Table 3

. Summary of Results from Analysis of Test Solutions forTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 18

GROWTH INHIBITION TEST WITH THE GREEN ALGA Pseudo kirchneriella subcapitata

- Table 4 . Summary of Temperatures, Light Intensities, and pH Values During the Exposure Phase

Temperature :

Temperature( o C) a

0 hr b 24 hr 48 hr 72 hr

Minimum NA 22.5 22.3 22.1
Maximum 23.7 23.8 22.9 22.9
Overall Min: 2 2.1
Overall Max: 2 3.8

a Temperature was recorded using a Minimum/Maximum thermometer placed in a surrogate vessel.

b The temperature recorded for Day 0 was the start temperature at test initiation. NA = not applicable

Light Intensity Range : 4 440 lux
– 5810 lux

pH :

Test Material Concentration (mg/L) Vessel IDs 0 hr 72 hr

Bulk Solution with algae (pooled) without algae (Blank)

Medi um Control (<LLQ a ) A,B,C, D,E,F 7.2 7.2 7.3 Blank: G

0.00253 mg/L A,B,C, D,E,F 7.2 7.3 7.3 Blank: G
Min 7.2
Max 7.3

a Lowest level quantified = 0.000559 mg LTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 19

GROWTH INHIBITION TEST WITH THE GREEN ALGA Pseudo kirchneriella subcapitata

- Table 5 . Mean Cell Density (x 10 4 cells/mL) after 24, 48, and 72 Hours of Exposure

Mean Measured Concentrations

(m g L) Cell Density (x 10,000 cells/mL) Exposure Hour

24 48 72 % Inhibition a
Medium Control (<LLQ b ) Mean 2.933 26.61 184.8

---- c

SD d 0.272 1.15 15.1

0.00253 mg/L Mean 2.716 26.32 183.5 1
SD 0.356 3.49 22.4

Results ( m g L)

72

- Hour

E y C 50 >0.00253
NOEC 0.00253

a % Inhibition = Percent inhibition compared to control

b Lowest level quantified = 0.000559 mg L

c

---- = Not Applicable

d SD = Standard DeviationTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 20

GROWTH INHIBITION TEST WITH THE GREEN ALGA Pseudo kirchneriella subcapitata

- Table 6 . Mean Specific Growth Rate (day 1 ) after 72 Hours of Exposure

Mean Mesured Concentrations

(μg L) 72

- Hour Specific
Growth Rate ( day -1 ) % Inhibition a
Medium Control (<LLQ b ) Mean 1.73 9

---- c

SD d 0.0 27

0.00253 mg/L Mean 1.735 0
SD 0.040

Results ( mg L)

72

- Hour

E r C 50 > 0.00253
NOEC 0.00253
The following formula was used to calculate growth rate:

Where: **μ** = mean specific growth rate from moment i to j (days -1 ) ln = natural logarithm
Ni = initial cell density at time i Nj = cell density at time j
ti = the moment time for the start of the period tj = the moment time for the end of the period

a % Inhibition = Percent inhibition compared to control

b Lowest level quantified = 0.000559 mg L

c

---- = Not Applicable

d SD = Standard Deviation ij i j ji 

### tt NN

**− −** 

**=**

**−** 

### lnln **μ**THE DOW CHEMICAL COMPANY STUDY ID: PAGE 21

GROWTH INHIBITION TEST WITH THE GREEN ALGA Pseudo kirchneriella subcapitata

- Figure 1 . Graphical Representation of the Mean Cell Density Over Time



### Time (hours)Cell Density (x104)



### 0 24 48 72 96 1 10 100 1000 AAP Control
100 mg/LTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 22

GROWTH INHIBITION TEST WITH THE GREEN ALGA Pseudo kirchneriella subcapitata

- APPENDIX A . Certificate of Analysis for Test SubstanceTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 24

GROWTH INHIBITION TEST WITH THE GREEN ALGA Pseudo kirchneriella subcapitata

- APPENDIX B . Growth Medium Composition



### Algal Assay Procuedure Medium (AAP) Composition

Nutrient Concentration (mg/L)

NaNO 3 

### 25.5 MgCl 2 

### * 6H 2 

### O 12.2

CaCl 2 

### * 2H 2 

### O 4.4 MgSO 4 

### * 7H 2 

### O 14.7

NaHCO 3 

### 15.0

K 2 

### HPO 4 

### 1.044

H 3 

### BO 3 

### 0.186

MnCl 2 

### * 4H 2 

### O 0.417

ZnCL 2 

### 0.003 32

NaMoO 4 

### * 2H 2 

### O 0.00726

CoCl 2 

### * 6H 2 

### O 0.00143

CuCl 2 

### * 2H 2 

### O 0.000011

NaEDTA * 2H 2 

### O 0.3

FeCl 3 

### * 6H 2 

### O 0.16

Note: pH for medium use on tests was adjusted to 7.5 ± 0.1 prior to solution preparation.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 25

GROWTH INHIBITION TEST WITH THE GREEN ALGA Pseudo kirchneriella subcapitata

- APPENDIX C . Analytical Report

### R rt 

### Title:

N

. America 

### Toxicology Infonnation Collection (TIq 

### Report
The Dow Chemical Company

TERC nla



### Study number: Study Title: GROW T H IN HI BITI O N TEST WITH THE G REEN AL GA P suedok irchn erie ll a s u bca pit ata 

### File number: Project#: 50840 

### Task/rep: 3 1 8 -1 Project / Sample Description (optional):

Test material(s): Test materiallot #:

Report recipient(s): Analysis request date: Date submittorprepared samples: Numberofsamples or estimate:

Date samples prepared for analys is: Analysis date: Number of samples analyzed: Analys is method: Method reference:

T H E D O W CH E MICA L COM P ANY 

### 9 / 8 / 2011 8 / 23 / 2011 2

8

/

23 / 2011,8 / 26/2011 8

/

23 / 2011

, 8 /2 6 /

2011 

### 16 HPLC /E S1- MSIMS

s ee R e ference Section

PAGE 1THE DOW CHEMICAL COMPANY PAGE 2 

### INTRODUCTION

This report summarizes the analytical support provided for study The study was

conducted to evaluate the acute toxicity of ( to the unicel lular green algae Pseudokirchner iella subcapitata. The test solutions were set at target concentrations of 0 (control algal growth media) and 100 mg L algal growth media (AAP) in a sta tic exposure scenario. The test solution concentrations were confirmed at 0-hrs and 72-hours of the study. The samples were analyzed for using liquid chromatogra phy with positive ion el ectrospray ionization tandem mass spectrometry de tection (HPLC/ESI-MS/MS).

EXPERIMENTAL

Test Material/Analytical Standard

Chemical Name:

Synonyms:

Lot Number:

CAS Number:

Molecular Formula:

Molecular Weight:

Purity: [Standards were not corrected for purity]

Internal Standard (IS)

Chemical Name: 1,2-Benzenediol

Identification: Asinex Corporation BAS 14278248

Molecular Formula: C 20 

### H 14 

### O 4


### Nominal Concentration: 10.8 ppm in methanol

Molecular Weight: 318.3228THE DOW CHEMICAL COMPANY PAGE 3 

### Test Vessel Sample Collection & Preparation

Sample aliquots (~3.00 mL) were collected from each test vessel using Eppendorf pipettes and transferred to glass vials. The sample s were prepared for analysis as follows:

**** AAP control samples:

o 1 mL sample + 25 μL internal sta ndard (dilution factor = 1)

**** 100 mg/L samples:

o 1 mL sample + 25 μL internal sta ndard (dilution factor = 1)

Method Precision/Solution Homogeneity

To assess analytical method precision and solu tion homogeneity, four samples were collected at 0-hr from the 100 mg/L test vessel. Thes e samples were collected and prepared for analysis along with the other da y 0 samples as described above.

Standard Preparation/Detector Calibration

A stock solution containing a nominal concen tration of approximately 500 μg mL methanol was used to prepare analytical standards over a con centration range of approximately 0.0005 to 0.01 mg L 10% meth anol/90% AAP for analysis with each sample set.

A standard regression equation was derive d from MassHunter software (Agilent Technologies) plotting the standard concentrations versus the corresponding peak area ratios (analyte peak area/internal st andard peak area) to genera te an equation of the form:

Y = mX + b

where:

Y represents the peak area ratio (analyte peak area/internal standard peak area)

m represents the slope of the regression

X represents the analyte concentration (μg/mL = mg/L)

b represents the Y-interceptTHE DOW CHEMICAL COMPANY PAGE 4 

### Calculation of Sample Concentrations

Measured Concentration (mg/L) = [(peak area ratio - b) / m] * dilution factor

Representative Lowest Level Quantified (LLQ)

LLQ = Lowest Standard Analyzed (0.000559 mg L 10% methanol/90% AAP) * lowest dilution factor (1) = 0.000559 mg L AAP

Instrument and Conditions for HPLC/ESI-MS/MS Analysis

Spectra can be found in “ An Acute Toxicity Study with the Fresh Water Cladoceran, Daphnia magna”

Pump: Agilent 1200 Series; G1312B

Autosampler: Agilent 1200 Series; G1367C

Column Heater: Agile nt 1200 Series; G1316B

MSD: Agilent Triple Quad; G6410A

Positive ion electrospray ionization, no split

Multiple reaction monitoring (MRM) mode; Q1 m/z 389, Q3 m/z 105

Benzendiol Q1 m/z 319, Q3 m/z 105

Column Heater: 40 °C

Fragmentor: 150 (V)

Collision Energy: 2V

Dwell: 250 msec

Gas Temperature: 300 °C

Drying Gas: 6 L/min

Nebulizer Pressure: 15 psi

Capillary Voltage: 1,000 VTHE DOW CHEMICAL COMPANY PAGE 5 

### Injection Volume: 50 μL

Analytical Column: Phenomenex Polar RP 2.0 x 50 mm, 2.5 μm

HPLC Eluent: A: Milli-Q water + 0.1% formic acid

B: Methanol + 0.1% formic acid

Gradient: Time %A %B Flow (mL/min)

0 95 5 0.50

2 0 100 0.50

5 0 100 0.50

6 95 5 0.50

8 95 5 0.50



## RESULTS

The results and mean concentrations from th e analyses of test solutions for are presented in Table 1.

The test solution homogeneity data are summarized in Table 2.

The HPLC/ESI –MS/MS instrumentation exhib ited a linear response ove r the concentration range extending from 0.000559 to 0.0112 mg L 10% methanol/90% AAP.

A typical standard curve is show n in Figure 1. The supporting st andard response data used to generate the curve are presented in Table 3.

Typical chromatograms from the analysis of a standard, sample, and AAP control are found in Figures 2, 3 and 4, respectively.THE DOW CHEMICAL COMPANY PAGE 6 

## REFERENCESTHE DOW CHEMICAL COMPANYPAGE 7Table 1. Summary of Results from Analysis of Test Solutions for0 (AAP Control) A<LLQ(NA)<LLQ(NA)0 (AAP Control) B<LLQ(NA)<LLQ(NA)0 (AAP Control) C<LLQ(NA)<LLQ(NA)0 (AAP Control) D<LLQ(NA)<LLQ(NA)1 (AAP Control) E<LLQ(NA)<LLQ(NA)2 (AAP Control) F<LLQ(NA)<LLQ(NA)3 (AAP Control) G<LLQ(NA)<LLQ(NA)100A0.00368(0.00368%)0.00298(0.00298%)100B0.00332(0.00332%)0.00280(0.00280%)100C0.00267(0.00267%)0.00247(0.00247%)100D0.00275(0.00275%)0.00251(0.00251%)100E0.00235(0.00235%)0.00231(0.00231%)100F0.00319(0.00319%)0.00273(0.00273%)100G0.00152(0.00152%)0.00189(0.00189%)Note: All values were copied and pasted in full precision but values are displayed using only 3 significant figuresNA: Not applicable<LLQ = less than the lowest level quantitated = 0.000559 mg L AAPMean Concentration of Vessel = (0 hr conc. + 72 hr conc.) / 2Relative Standard Deviation = Standard Deviation / Mean Concentration of Dose Level * 100a,baSamples were collected from bulk dose solutions at study initiation (0 hr).0.00227(0.00227%)b0 hr value for this dose level represents the mean of 4 injections (4 samples, 1 injection/sample). Four replicate samples were collected toevaluate method precision and solution homogeneity.<LLQa(NA)Dose Level(mg/L)Test V essel IDMeasured Concentration, mg/L**(Percent of Target)**0 hr72 hrMean Concentration of Vessel,mg/L**(Percent of Target)**Mean Concentration of Dose Level,mg/L**(Percent of Target)**0.00253(0.00253%)<LLQ(NA)StandardDeviation0.000358RelativeStandardDeviation14.2%NANATHE DOW CHEMICAL COMPANYPAGE 8Table 2. Summary of Homogeneity Data for 0 hr SamplesNote: All values were copied and pasted using full precision but values are displayed using only 3 significant figures100mg/L‐A‐Day00.00215100mg/L‐B‐Day00.00211100mg/L‐C‐Day00.00223100mg/L‐D‐Day00.00258AverageAnalyzedConc.(mg/L)0.00227StandardDeviation0.000216RelativeStandardDeviation(%)9.50%SampleIDAnalyzedConcentration(mg/L)THE DOW CHEMICAL COMPANYPAGE 9Table 3. Representative Standards/Refit Table forSample Name File NameAnalyteConcentration(mg/L)Analyte Peak Area(counts)IS Peak Area(counts)Area RatioCalculatedConcentration(mg/L)Accuracy(%)Standard A082611b07.d 0.000559742.51339850.02180.000547 97.8Standard B082611b08.d0.001121222.67336860.03630.00111 99.0Standard C082611b09.d0.002802539.2334420.07590.00265 94.6Standard D082611b10.d0.005595217330300.1580.00584 104Standard E082611b11.d0.01129876328580.3010.0114 102Standard A082611b21.d 0.000559739337060.02190.000550 98.4Standard B082611b22.d0.001121222.65339730.03600.00110 97.9Standard C082611b23.d0.002802635.02328920.08010.00281 100Standard D082611b24.d0.005595057.3337970.1500.00552 98.7Standard E082611b25.d0.01129725330970.2940.0111 99.3Standard A082611b43.d 0.000559761.53320930.02370.000620 111Standard B082611b44.d0.001121175.18326680.03600.00110 97.9Standard C082611b45.d0.002802609.5327710.07960.00279 99.8Standard D082611b46.d0.005594840317930.1520.00562 100Standard E082611b47.d0.01129319319090.2920.0111 98.7Area Ratio = Analyte Peak Area / IS Peak AreaCalculated Concentration = (Area Ratio - b) / mAccuracy = Calculated Concentration / Analyte Concentration x 100Regression equation is y = mx + bRefer to figure 1 for value of m and bTHE DOW CHEMICAL COMPANYPAGE 10Figure 1. Typical calibration curve fromHPLC/ESI-MS/MS analysis ofTHE DOW CHEMICAL COMPANYPAGE 11Figure 2. Typical chromatogram (HPLC/ESI-MS/MS) of solvent standard prepared ata nominal concentration of 0.00280 mgL 10% methanol/90% AAP, m/z 389/105 (top, shaded peak) with0.263 mg internal standard/L, m/z 319/105(bottom, shaded peak).THE DOW CHEMICAL COMPANYPAGE 12Figure 3. Typical chromatogram (HPLC/ESI-MS/MS) of test solution prepared ata target concentration of 100 mg LAAP, m/z 389/105 (top, shaded peak) with 0.263 mginternal standard/L, m/z 319/105 (bottom, shaded peak).THE DOW CHEMICAL COMPANYPAGE 13Figure 4. Typical chromatogram (HPLC/ESI-MS/MS) of a control AAP sample, analyzed for (top, no quantifiable peak) withno internal standard added (bottom, no quantifiable peak).THE DOW CHEMICAL COMPANY STUDY ID: PAGE 39

GROWTH INHIBITION TEST WITH THE GREEN ALGA Pseudo kirchneriella subcapitata

- APPENDIX D . 24

- , 48 - and 72

- Hour Cell Density (Cells x 10 4 /mL) Values for Replicate Test Flasks for

Each Test Level

Mean Measured
Concentrations

(μg L) Replicate Cell Density (Biomass) (Cells x 10 4 /mL)

72

- Hour % Inhibition a 24 48 72

Medium Control A 2.564 24.62 167.1 (<LLQ b ) B 3.270 27.94 178.9
C 2.982 27.00 204.4
D 3.174 26.98 171.7
E 2.692 26.03 187.0
F 2.916 27.10 199.8
Mean 2.933 26.61 184.8

---- c
SD d 0.272 1.15 15.1 0.00253 mg/L A 3.106 26.72 219.0
B 2.974 31.41 195.3
C 2.518 24.49 162.5
D 2.124 22.29 163.7
E 2.700 29.27 191.9
F 2.872 23.75 168.8
Mean 2.716 26.32 183.5 1

SD 0.356 3.49 22.4

a % Inhibition = Percent inhibition from control

b Lowest Level Quantified= 0.000559 mg L

c

---- = Not Applicable

d SD = Standard Deviation Note: Means, standard deviations, and percent inhibitions are calculated from the unrounded numbers, not the rounded numbers in the table. Hand calculated values may differ slightly.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 40

GROWTH INHIBITION TEST WITH THE GREEN ALGA Pseudo kirchneriella subcapitata

- APPENDIX E . 72

- Hour Mean Specific Growth Rate (Day -1 ) Values for Replicate Test Flasks for Each

Test Level

Mean Measured
Concentrations

(μg L) Replicate 72

- Hour Specific Growth Rate (hour -1 ) 72

- Hour % Inhibition a
Medium Control A 1.706 (<LLQ b ) B 1.729
C 1.773
D 1.715
E 1.743
F 1.766 Mean 1.73 9

---- c

SD d 0.0 27 0.00253 mg/L A 1.796
B 1.758
C 1.697
D 1.699
E 1.752
F 1.710 Mean 1.735 0

SD 0.0 4 0

a % Inhibition = Percent inhibition from control

b Lowest Level Quantified= 0.000559 mg L

c

---- = Not Applicable

d SD = Standard Deviation Note: Means, standard deviations, and percent inhibitions are calculated from unrounded numbers, not the rounded numbers in the table. Hand calculated values may differ slightly.
The following formula was used to calculate growth rate:

Where: **μ** = mean specific growth rate from moment i to j (days -1 ) ln = natural logarithm
Ni = initial cell density at time i Nj = cell density at time j
ti = the moment time for the start of the period tj = the moment time for the end of the period ij i j ji 

### tt NN

**− −** 

**=**

**−** 

### lnln **μ**