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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 

Laboratory Project Study ID 
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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. 
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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 

WITH 
THE DOW CHEMICAL COMPANY

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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 104 cells/mL) after 24, 48, and 72 Hours of 

Exposure ............................................................................................................19

Table 6. Mean Specific Growth Rate (day1) after 72 Hours of Exposure ....................20
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TABLE OF CONTENTS

Page

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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 104/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 ........................................................................40
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SUMMARY

TITLE: GROWTH 

INHIBITION TEST WITH THE GREEN ALGA 

Pseudokirchneriella subcapitata

STUDY NUMBER:

TEST SUBSTANCE: Lot # Batch purity of ± 

TEST DATES: 23 to 26 August 2011 

TEST ORGANISM: Pseudokirchneriella subcapitata

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 ErC50 for 

specific growth rate and the EyC50 for cell density were 

>100 mg/L and the NOECs were 100 mg/L 

Based on mean measured concentrations, the 72-hour 

calculated ErC50 for specific growth rate was 

>0.00253 mg L and the NOEC was 0.00253 mg 

L. The 72-hour calculated EyC50 cell density was 

>0.00253 mg L and the NOEC for cell density 

was determined to be 0.00253 mg/L.
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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 test substance. The 

data collected were used to calculate the 72- hour ErC50 (based on growth rate) and EyC50

(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.

Safety 

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 March 2010 (Lot # 

Batch and stored under ambient conditions until use. The total purity 

was ± 0.03% by gas chromatography with identification by Fourier transform 
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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. 

Test 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 density 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 

Company 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 uniquely 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. 
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Preparation of Test Solutions 

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

concentrations 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, some small 

particulate matter was still visible suspended in the AAP. Therefore, test solutions were 

shaken 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 as a limit test, aseptically under static conditions for 

approximately 72 hours from 23 August to 26 August 2011. Seven 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. Six replicates for each the AAP medium control and 100 mg L test 

level were inoculated with approximately 10,000 cells/mL. Inoculations were made 

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. The 
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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 equivalent 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 

made 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 samples 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 termination, 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 observations were made on a composited sample of the inoculated 

replicates at each test concentration at test termination. 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 were 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 were collected on day 0 from the 100 mg L concentration bulk 

treatment 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. 
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Statistical Analysis

The primary endpoint of this test was the response of the algae 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 and 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

j i

ji tt

NN

−

−

− = lnln

μ
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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). There were no significant differences between the control and 

treatment (p = 0.9094). Therefore, the EC50 and the NOEC for cell density are >0.00253 

and 0.00253 mg 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 differences between the control and 

treatment (p = 0.8651). Therefore, the ErC50 and the NOEC for specific growth rate 

are >0.00253 mg and 0.00253 L, respectively. 

Microscopic evaluation of cells 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 least 

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 below 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%. 
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CONCLUSIONS 

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 

ErC50 for specific growth rate was >0.00253 mg L and the NOEC was 0.00253 mg 

L. The 72-hour calculated EyC50 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. 
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 GROWTH INHIBITION TEST WITH THE GREEN ALGA 

Pseudokirchneriella subcapitata

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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 purity 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 Structure
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Pseudokirchneriella subcapitata

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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 cap
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Table 3. Summary of Results from Analysis of Test Solutions for 

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

Temperature: 

 

Temperature(oC)a

0 hrb 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: 22.1

Overall Max: 23.8

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: 4440 lux – 5810 lux

pH: 

Test Material 

Concentration

(mg/L)

Vessel IDs

0 hr 72 hr

Bulk Solution with algae

(pooled)

without algae

(Blank)

Medium Control

(<LLQa

) 

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 L

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Table 5. Mean Cell Density (x 104 cells/mL) after 24, 48, and 72 Hours of Exposure

Mean Measured 

Concentrations

(mg L)

Cell Density (x 10,000 cells/mL)

Exposure Hour

24 48 72 % Inhibitiona

Medium Control

(<LLQb) Mean 2.933 26.61 184.8 ----c

SDd 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 (mg L)

72-Hour

EyC50 >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 Deviation

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Table 6. Mean Specific Growth Rate (day1

) after 72 Hours of Exposure

Mean Mesured

Concentrations

(μg L)

72-Hour Specific

Growth Rate

(day-1) % Inhibitiona

Medium Control

(<LLQb) Mean 1.739 ----c 

SDd 0.027

0.00253 mg/L Mean 1.735 0

SD 0.040

Results (mg L)

72-Hour

ErC50 >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

j i

ji tt

NN

−

−

− = lnln

μ

- 
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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/L

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APPENDIX A. Certificate of Analysis for Test Substance

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APPENDIX B. Growth Medium Composition

Algal Assay Procuedure Medium (AAP) Composition 

Nutrient Concentration (mg/L)

NaNO3 25.5

MgCl2 * 6H2O 12.2

CaCl2 * 2H2O 4.4

MgSO4 * 7H2O 14.7

NaHCO3 15.0

K2HPO4 1.044

H3BO3 0.186

MnCl2 * 4H2O 0.417

ZnCL2 0.00332

NaMoO4 * 2H2O 0.00726

CoCl2 * 6H2O 0.00143

CuCl2 * 2H2O 0.000011

NaEDTA * 2H2O 0.3

FeCl3 * 6H2O 0.16

Note: pH for medium use on tests was adjusted to 7.5 ± 0.1 prior to solution preparation.

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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 INHIBITION TEST WITH THE GREEN ALGA Psuedokirchn erie lla subcapit ata 

File number: Project#: 50840 Task/rep: 318-1 

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: 

THE DOW CH EMICA L COMPANY 

9/8/2011 

8/23/2011 

2 

8/23/2011,8/26/2011 

8/23/2011 , 8/26/2011 

16 

HPLC/ES1-MSIMS 

see Reference Section 

PAGE 1 

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INTRODUCTION 

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

conducted to evaluate the acute toxicity of 

 ( to the unicellular green algae Pseudokirchneriella 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 static exposure scenario. The test solution 

concentrations were confirmed at 0-hrs and 72-hours of the study. The samples were 

analyzed for using liquid chromatography with positive ion electrospray ionization 

tandem mass spectrometry detection (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: C20H14O4

Nominal Concentration: 10.8 ppm in methanol 

Molecular Weight: 318.3228 

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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 samples were prepared for analysis as follows: 

 AAP control samples: 

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

 100 mg/L samples: 

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

Method Precision/Solution Homogeneity

To assess analytical method precision and solution homogeneity, four samples were collected 

at 0-hr from the 100 mg/L test vessel. These samples were collected and prepared for 

analysis along with the other day 0 samples as described above. 

Standard Preparation/Detector Calibration 

A stock solution containing a nominal concentration of approximately 500 μg mL 

methanol was used to prepare analytical standards over a concentration range of 

approximately 0.0005 to 0.01 mg L 10% methanol/90% AAP for analysis with each 

sample set. 

A standard regression equation was derived from MassHunter software (Agilent 

Technologies) plotting the standard concentrations versus the corresponding peak area ratios 

(analyte peak area/internal standard peak area) to generate 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-intercept 

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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: Agilent 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 V 

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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 the 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 exhibited a linear response over the concentration 

range extending from 0.000559 to 0.0112 mg L 10% methanol/90% AAP. 

A typical standard curve is shown in Figure 1. The supporting standard 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. 

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REFERENCES 

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Table 1. Summary of Results from Analysis of Test Solutions for 

0 (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)

100 A 0.00368 (0.00368%) 0.00298 (0.00298%)

100 B 0.00332 (0.00332%) 0.00280 (0.00280%)

100 C 0.00267 (0.00267%) 0.00247 (0.00247%)

100 D 0.00275 (0.00275%) 0.00251 (0.00251%)

100 E 0.00235 (0.00235%) 0.00231 (0.00231%)

100 F 0.00319 (0.00319%) 0.00273 (0.00273%)

100 G 0.00152 (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 * 100

a,b

a Samples were collected from bulk dose solutions at study initiation (0 hr).

0.00227 (0.00227%)

b 0 hr value for this dose level represents the mean of 4 injections (4 samples, 1 injection/sample). Four replicate samples were collected to 

evaluate method precision and solution homogeneity. 

<LLQ a (NA)

Dose Level 

(mg/L) Test V essel ID

Measured Concentration, mg/L (Percent of Target)

0 hr 72 hr

Mean Concentration of Vessel, 

mg/L (Percent of Target)

Mean Concentration of Dose Lev

mg/L (Percent of Target)

0.00253 (0.00253%)

<LLQ (NA)

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Table 2. Summary of Homogeneity Data for 0 hr Samples 

Note: All values were copied and pasted using full precision but values are displayed using only 3 signi

100mg/L‐A‐Day0 0.00215

100 mg/L‐B‐Day0 0.00211

100 mg/L‐C‐Day0 0.00223

100mg/L‐D‐Day0 0.00258

Average Analyzed Conc. (mg/L) 0.00227

Standard Deviation 0.000216

Relative Standard Deviation (%) 9.50%

Sample ID Analyzed Concentration (mg/L)

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Table 3. Representative Standards/Refit Table for 

Sample Name File Name

Analyte 

Concentration 

(mg/L)

Analyte Peak Area 

(counts)

IS Peak Area 

(counts) Area Ratio

C

Con

Standard A 082611b07.d 0.000559 742.51 33985 0.0218 

Standard B 082611b08.d 0.00112 1222.67 33686 0.0363 Standard C 082611b09.d 0.00280 2539.2 33442 0.0759 Standard D 082611b10.d 0.00559 5217 33030 0.158 Standard E 082611b11.d 0.0112 9876 32858 0.301 Standard A 082611b21.d 0.000559 739 33706 0.0219 

Standard B 082611b22.d 0.00112 1222.65 33973 0.0360 Standard C 082611b23.d 0.00280 2635.02 32892 0.0801 Standard D 082611b24.d 0.00559 5057.3 33797 0.150 Standard E 082611b25.d 0.0112 9725 33097 0.294 Standard A 082611b43.d 0.000559 761.53 32093 0.0237 

Standard B 082611b44.d 0.00112 1175.18 32668 0.0360 Standard C 082611b45.d 0.00280 2609.5 32771 0.0796 Standard D 082611b46.d 0.00559 4840 31793 0.152 Standard E 082611b47.d 0.0112 9319 31909 0.292 

Area Ratio = Analyte Peak Area / IS Peak Area

Calculated Concentration = (Area Ratio - b) / m

Accuracy = Calculated Concentration / Analyte Concentration x 100

Regression equation is y = mx + b 

Refer to figure 1 for value of m and b

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Figure 1. Typical calibration curve from HPLC/ESI-MS/MS analysis of 

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Figure 2. Typical chromatogram (HPLC/ESI-MS/MS) of solvent standard prepared at a nominal concentrati

 L 10% methanol/90% AAP, m/z 389/105 (top, shaded peak) with 0.263 mg internal standard/ (bottom, shaded peak). 

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Figure 3. Typical chromatogram (HPLC/ESI-MS/MS) of test solution prepared at a target concentration of 

 AAP, m/z 389/105 (top, shaded peak) with 0.263 mg internal standard/L, m/z 319/105 (bottom, sh

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Figure 4. Typical chromatogram (HPLC/ESI-MS/MS) of a control AAP sample, analyzed for (top, no qu

no internal standard added (bottom, no quantifiable peak). 

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 GROWTH INHIBITION TEST WITH THE GREEN ALGA 

Pseudokirchneriella subcapitata

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APPENDIX D. 24-, 48- and 72-Hour Cell Density (Cells x 104

/mL) Values for Replicate Test Flasks for 

Each Test Level

Mean Measured 

Concentrations

(μg L) Replicate

Cell Density (Biomass) (Cells x 104/mL)

72-Hour % Inhibitiona 24 48 72

Medium Control A 2.564 24.62 167.1

(<LLQb) 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

SDd 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.

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 GROWTH INHIBITION TEST WITH THE GREEN ALGA 

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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

% Inhibitiona

Medium Control A 1.706

(<LLQb) B 1.729

C 1.773

D 1.715

E 1.743

F 1.766

Mean 1.739 ----c

SDd 0.027

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 0.040

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

j i

ji tt

NN

−

−

− = lnln

μ

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