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nt 
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON D.C., 20460 

VIA FACSIMILE: 201-437-4801 

April 19,2012 

Attention: Glenn Merritt (201-437-4000) 

OFFICE OF 

PREVENTION, PESTICIDES AND 

TOXIC SUBSTANCES 

Subject: Follow-up to fax transmission of 4/13/12 (ePMA removal ofCBI claims regarding 

identified studies) 

Dear Mr. Merritt: 

This is sent to you pursuant our conversation of several minutes ago. In Larry Robinson's fax of 

April 13th while there is a waiver of confidential business information (CBI) claims in the 

identified studies there is a an assertion of copyright protections and some additional text that 

provides in part ... "Reproduction, reference, summarization, retransmission or other use of this 

copyrighted document. .. including electronic transmission, using the internet or email .... is 

strictly prohibited .... " 

While we understand the basis for the copyright protections, we have a concern that the language 

in the fax might be interpreted to mean that ePMA is asserting that the studies submitted per the 

TSCA activity are not to be made available in an Agency e-docket (electronic docket) or be 

included in EPA websites among the collection of TSCA health and safety studies. 

We are thinking that the identified limitations noted in the fax are not intended to apply to EPA's 

normal procedures for the handling of non-CBI materials submitted by entities subject to TSCA 

regulations (including the posting of such materials on government websites) and submitted by 

such entities in order to comply with a TSCA requirement, but are rather intended to limit 

entities outside the government from the unauthorized use or appropriation of the studies. 

Can you please confirm CPMA's intent? Many thanks. 

Scott M. Sherlock, Attorney Advisor 

Environmental Assistance Division 

Office of Pollution Prevention and Toxics 


CONFIDENTIAL BUSINESS INFORMATION

REPORT

Study Title

FRESH WATER ALGAL GROWTH INHIBITION TEST

WITH

C.I. PIGMENT BLUE 61

Author

Ir. L.M. Bouwman

Study completion date

23 October 2008

Test Facility

NOTOX B.V.

Hambakenwetering 7

5231 DD 's-Hertogenbosch

The Netherlands

Laboratory Proiect Identification

NOTOX Project 488234

NOTOX Substance 183591/A

-Pagel1of 29-
--ASK- ~ ~

C.I. Pigment Blue 61 NOTOX Project 488234

1. CONTENTS

1. CONTENTS.............................................................................. 2

2. STATEMENT OF GLP COMPLIANCE ...................................................................... 3

3. QUALITY ASSURANCE STATEMENT ..................................................................... 4

4. SUMMARY.................................................................................................. 5

5. INTRODUCTION ........................................................................................... 6

5.1. Preface ................ ....................... 6

5.2. Aim of the study ......................................................................................... 6

5.3. Guidelines............................................................................................. 6

5.4. Storage and retention of records and materials ...................................................... 6

5.5. Definitions............................................ ................................................... 7

6. MATERIALS AND METHODS ............................................................................... 7

6.1. Test Substance.......................................................................................... 7

6.1.1. Test substance information .................................................................... 7

6.1.2. Study specific test substance information ..................................................... 7

6.1.3. Reference substance ........................................................................... 8

6.1.4. Preliminary data....................................... .......................................... 8

6.2. Test System........................................................................................... 8

6.3. Fresh water algae culture............................................................................... 8

6.4. Preparation of test solutions ........................................................................ .9.

6.5. Limit test .............................................................................................. 9

6.5. 1. Test concentrations............................................................................

6.5.2. Test procedures and conditions............................................................... 10

6.5.3. Sampling for analysis of test concentrations................................................. 10

6.5.4. Measurements ............... .................. 10

6.5.5. Recording of cell densities .................................................................... 11

6.6. Electronic data capture ............................................................................... 11

6.7. Interpretation............................................................................................ 11

6.7.1. Data handling ................................................................................. 11

6.7.2. Acceptability of the test ........................................................................ 12

6.8. List of deviations ... I.................................................................................... 12

6.8.1. List of protocol deviations ..................................................................... 12

6.8.2. List of standard operating procedures deviations ........................................... 12

7. RESULTS .......................................................... ........................................ 12

7.1. Measured test substance concentrations............................................................. 12

7.2. Mean cell densities................................................................................... 13

7.3. Reduction of growth rate and inhibition of yield ..................................................... 13

7.4. Determination of effect concentrations ............................................................... 14

7.5. Experimental conditions ............................................................................... 14

8. CONCLUSION............................................................................................. 15

TABLES

Table 1 Mean cell densities (x 10 cells/mI) ........................................................... 13

Table 2 Percentage reduction of growth rate (total test period) and percentage inhibition of

yield .......................................................................................... 14

Table 3 Percentage reduction of growth rate at different time intervals............................ 14

Table 4 Effect parameters .............................................................................. 14

Table 5 pH levels ........................................................................................ 14

Table 6 Individual cell densities ........................................................................ 16

Table 7 Calculation of growth rate and yield .......................................................... 16

Table 8 Calculation of growth rate (section-by-section.) .............................................. 17

FIGURES

Figure 1 Growth curves at different concentrations of C.l. Pigment Blue 61 ....................... 13

APPENDICES

APPENDIX I WORKSHEET DATA........................................................................... 16

APPENDIX It STATISTICS: YIELD (0-72 HOURS) ......................................................... 18

APPENDIX III REFERENCE TEST ........................................................................... 20

APPENDIX IV ANALYTICAL REPORT

-Page 2 -
0.1. Pigment Blue 61 NOTOX Project 488234

2. STATEMENT OF GLP COMPLIANCE

NOTOX B.V., s-Hertogenbosch, The Netherlands

The study described in this report has been correctly reported and was conducted in compliance

with:

The Organization for Economic Cooperation and Development (OECD) Good Laboratory

Practice Guidelines (1997).

Which essentially conform to:

The United States Food and Drug Administration Good Laboratory Practice Regulations.

The United States Environmental Protection Agency Good Laboratory Practice Regulations.

The sponsor is responsible for Good Laboratory Practice (GLP) compliance for all test

substance information unless determined by NOTOX.

NOTOX B.V.

Ir. L.M. Bouwman Ing. E.J. van de Waart, M.Sc.

Study Director Head of In Vitro & Environmental Toxicology

Date: ...... .. Date: ..... / / j d.........

-Page 3 -
0.1. Pigment Blue 61 NOTOX Project 488234

3. QUALITY ASSURANCE STATEMENT

NOTOX B.V., 's-Hertogenbosch, The Netherlands

This report was inspected by the NOTOX Quality Assurance Unit to confirm that the methods

and results accurately and completely reflect the raw data.

The dates of Quality Assurance inspections are given below.

During the on-site process inspections procedures applicable to this type of study were

inspected.

The reporting date is the date of reporting to the Study Director. The QAU report was then

forwarded to the Test Facility Management.

Typ ofStart EndReotn

Typecons Phase/Process Inspection Inspection Reotn

inspetionsdate date dt

Study Protocol 09-Sep-08 09-Sep-08 09-Sep-08

Report 07-Oct-08 07-Oct-08 07-Oct-08

Process Environmental toxicology 14-Jul-08 18-Jul-08 18-Jul-08

Test substance handling

Exposure

Observations/Measurements

Analytical and physical

chemistry 25-Aug-08 28-Aug-08 01-Sep-08

Test substance handling

Observations/Measurements

Head of Quality Assurance

C.J.Mitchell B.Sc.

Date: ...... V~~

-Page 4 -
C.!. Pigment Blue 61 NOTOX Project 488234

4. SUMMARY

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with C.I. Pigment

Blue 61.

The study procedures described in this report were based on the OECD guideline No. 201,

2006. In addition, the procedures were designed to meet the test methods of the EEC directive

92/69, Part C.3, 1992, the ISO International Standard 8692, 2004 and the OECD series on

testing and assessment number 23, 2000.

The batch of C.lI. Pigment Blue 61 tested was a dark blue powder with granules with a purity of

96.830% and not completely soluble in test medium at the loading rate prepared.

Preparation of test solutions started with a loading rate of 100 mg/I applying a 10-minute

treatment period with ultrasonic waves followed by a 3 days of magnetic stirring to reach

maximum solubility of the test substance in the test medium. Subsequently, the aqueous

mixture was filtered through a membrane filter (0.45 pm). The resulting filtrate was observed to

be clear and light blue.

A limit test was performed, exposing exponentially growing algal cultures (six replicates) to the

filtered solution prepared at a loading rate of 100 mg/I and a control group. The initial cell

density was 10~4 cells/mI and the total test period was 72 hours. Samples for analytical

confirmation of actual exposure concentrations were taken at the start, after 24 hours of

exposure and at the end of the test.

Analysis of the samples taken at the start of the test showed a measured concentration of

549 pg/I at the loading rate of 100 mg/I. During the exposure period the measured concentration

decreased to 342 pg/l (i.e. 62% of initial). Based on these results, the average exposure

concentration was calculated to be 422 pg/I.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

No reduction of growth rate and less than 50% inhibition of yield was recorded at the

concentration of C.I. Pigment Blue 61 tested. However, it can not be excluded that the inhibition

was caused by light absorption due to the colouring of the test substance rather than by toxicity.

The ECw for both growth rate reduction (ERC5C: 0-72h) and yield inhibition (EyCw: 0-72h) was

beyond 422 pg/I.

The NOEC for growth rate reduction was 422 pg/I and the NOEC for yield inhibition was below

422 pg/I. This concentration was well above the given water solubility limit of C.I. Pigment Blue

61 (i.e. 2.5 pg/I).

-Page 5 -
C.I. Pigment Blue 61 NOTOX Project 488234

5. INTRODUCTION

5.1. Preface

Sponsor Color Pigments Manufacturers Association, Inc.

300 N. Washington St. Ste. 102,

ALEXANDRIA, VA 22314

USA

Study Monitor Mr. L. Robinson

Test Facility NOTOX B.V.

Hambakenwetering 7

5231 DD s-Hertogenbosch

The Netherlands

Study Director Ir. L.M. Bouwman

Technical Coordinator W.J. Koolen

Principal Scientist Ir. M.J C. Brekelmans

Study Plan Start 15 September 2008

Completion 18 September 2008

5.2. Aim of the study

The purpose of the study was to evaluate the test substance for its ability to generate toxic

effects in Pseudokirchneriella subcapitata during an exposure period of at least 48 and at most

96 hours and, if possible, to determine the EC50 for both reduction of growth rate and inhibition

of yield.

5.3. Guidelines

The study procedures described in this report were based on the Organization for Economic Cooperation and Development (OECD), OECD guidelines for Testing of Chemicals, guideline No.

201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", Adopted March 23, 2006.

In addition, the procedures were designed to meet the test methods prescribed by the following

guidelines and guidance documents:

* European Economic Community (EEC), EEC directive 92/69, Part C: Methods for the

determination of ecotoxicity, Publication No. L383, December 1992, C-3: "Algal Inhibition

Test'.

" ISO International Standard 8692: 'Water quality - Freshwater algal growth inhibition test with

unicellular green algae", Second edition, 01 October 2004.

* Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD

series on testing and assessment number 23, December 14, 2000.

5.4. Storage and retention of records and materials

Records and materials pertaining to the study including protocol, raw data, specimens (except

specimens requiring refrigeration or freezing) and the final report are retained in the NOTOX

archives for a period of at least 2 years after finalization of the report. After this period, the

sponsor will be contacted to determine how the records and materials should be handled.

NOTOX will retain information concerning decisions made.

Those specimens requiring refrigeration or freezing will be retained by NOTOX for as long as

the quality of the specimens permits evaluation but no longer than three months after finalization

- Page 6 -
ClI. Pigment Blue 61 NOTOX Project 488234

of the report.

NOTOX will retain a test substance sample until the expiry date, but no longer than 10 years

after finalization of the report. After this period the sample will be destroyed.

5.5. Definitions

Cell density is the number of cells per millilitre.

Growth rate is the increase in cell density per unit time. It is derived from the slope of the

growth curve in a logarithmic plot. Following from the mathematical nature of exponential

growth, the measure of the specific growth rate is preferable over biomass or yield. The ERCW0 is

the concentration of test substance that results in a 50% reduction in growth rate relative to the

control.

Yield is defined as the biomass at the end of the exposure period minus the biomass at the start

of the exposure period. The EyC50 is the concentration of test substance that results in a 50%

inhibition of yield relative to the control.

No Observed Effect Concentration (NOEC) is the highest concentration tested at which the

measured parameter(s) show(s) no significant effect on algal growth relative to control values.

6. MATERIALS AND METHODS

6.1. Test Substance

6.1.1. Test substance information

Identification C.l. Pigment Blue 61

Structure

N

S03H

Molecular formula C37H29N303S

Molecular weight 596

CAS Number 1324-76-1

Description Dark blue powder with granules

Batch 2084701

Purity 96.830%

Test substance storage At room temperature in the dark

Stability under storage conditions Stable

Expiry date 02 January 2018

6.1.2. Study specific test substance information

Stability in water At least 96 hours

Solubility in water No

-Page 7 -
C.I. Pigment Blue 61 NOTOX Project 488234

6.1.3. Reference substance

This report includes the results of the most recent reference test with potassium dichromate

(Appendix 1ll).

6.1.4. Preliminary data

The solubility of Pigment Blue 61 in water (bi dest.) at ambient temperature is 2.5 pg/I (Clariant,

SU-Technology & Innovation Management, Pigment Analytics).

6.2. Test System

Species Pseudokirchneriella subcapitata, strain: NIVA CHL 1

Source In-house laboratory culture.

Reason for selection This system is an unicellular algal species sensitive to

toxic substances in the aquatic ecosystem and has

been selected as an internationally accepted species.

6.3. Fresh water algae culture

Stock culture Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on

agar. The suspensions were continuously aerated and

exposed to light in a climate room at a temperature of

21 -24oC.

Light intensity 60 to 120 pE/m 2

/s when measured in the

photosynthetically effective wavelength range of 400

to 700 nm.

Stock culture medium M1; according to the NPR 6505, formulated using

Milli-Ro water (tap-water purified by reverse osmosis;

Millipore Corp., Bedford, Mass., USA) with the

following composition:

NaNO3 500 mg/I

K2HP0 4 40 mg/I

MgSO 4.7H-20 76 mg/I

Na2C0 3. 1 OH20 54 mg/I

CsiHSO?7.H-20 6 mg/I

NH4NO3 330 mg/I

CaCI 2.H-2o 36 mg/I C.HsFeo 7.xH20 6 mg/i

1-31303 2.9 mg/I

MnC12.41-120 1.81 mg/I

ZnCI 2 0.11 mg/i

CuSO4 .5H20 0.08 mg/i

(NH4)8M07024.4H20 0.018 mg/I

Pre-culture 3 days before the start of the test, cells from the algal

stock culture were inoculated in culture medium at a

cell density of 10 cells/mi. The pre-culture was

maintained under the same conditions as used in the

test. The cell density was measured immediately

before use.

-Page 8 -
ClI. Pigment Blue 61 NOTOX Project 488234

Pre-culture medium M2; according to the OECD 201 Guideline, formulated

using Milli-Q water (tap water purified by reverse

osmosis (milli-RO) and subsequently passed over

activated carbon and ion-exchange cartridges: Milli-Q

water; Millipore Corp., Bedford, Mass., USA)

preventing precipitation and with the following

composition:

NH4CI 15 mng/ MgcI 2.6H20 12 mng/

CaCl 2.2H20 18 mngI MgSO4.7H20 15 mg/I KH2PO4 1.6 mg/I FeCI 3.6H20 64 Pg/I Na2EDTA.2H 20 100 pg/i

H3B03 185 pig/I MnC12.4H20 415 pg/I ZnC12 3 pg/i cod2 6H20 1.5 Pg/I CUC12.2H20 0.01 pg/i Na2MoO4.2H20 7 pg/i NaHCO3 50 mg/I Hardness (ca+Mg) 0.24 mmoVl (24 mg CaCO3/1)

pH 8.1±t.0.2

6.4. Preparation of test solutions

The standard test procedures required generation of test solutions, which contained completely

dissolved test substance concentrations or stable and homogeneous mixtures or dispersions.

The testing of concentrations that would disturb the test system was prevented as much as

possible (e.g. film of the test substance on the water surface).

The batch of ClI. Pigment Blue 61 tested was a dark blue powder with granules with a purity of

96.830% and not completely soluble in test medium at the loading rate prepared.

Preparation of test solutions started with a loading rate of 100 mg/I applying a 10-minute

treatment period with ultrasonic waves followed by a 3 days of magnetic stirring to reach

maximum solubility of the test substance in the test medium. Subsequently, the aqueous

mixture was filtered through a membrane filter (Schleicher & Schuell, 0.45 pm). The resulting

filtrate was observed to be clear and light blue.

After preparation, volumes of 50 ml were added to each replicate of the respective test

concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing

a cell density of 104 cells/mi.

6.5. Limit test

6.5.1. Test concentrations

ClI. Pigment Blue 61 Filtered solution prepared at a loading rate of 100 mg/I

Controls Test medium without test substance or other additives

Replicates 6 replicates of both test groups

In addition, 1 extra replicate for both test groups for

sampling purposes and 2 replicates of the limit

concentration without algae

-Page 9 -
C.I. Pigment Blue 61 NOTOX Project 488234

6.5.2. Test procedures and conditions

Test duration 72 hours

Test type Static

Test vessels 100 ml, all-glass, containing 50 ml of test solution

Medium M2

Cell density An initial cell density of 1 X 104 cells/mi.

Illumination Continuously using TLD-lamps of the type 'Cool-white'

of 30 Watt, with a light intensity within the range of 78

to 82 pE.M-2.s-1

Incubation Vessels were distributed at random in the incubator.

During incubation the algal cells were kept in

suspension by continuous shaking.

6.5.3. Sampling for analysis of test concentrations

During the limit test samples for possible analysis were taken from both the control and the limit

concentration according to the schedule below. In addition, the filter used for preparation of the

limit concentration was stored for possible analysis of the residue. The method of analysis is

described in the appended Analytical Report (Appendix IV).

Frequency at t0- h, t--24 h and t=72 h

Volume 2 ml

Storage Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration

before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was

demonstrated by running a test vessel at the highest substance concentration but without algae

and samples for analysis were taken at the start, after 24 hours of exposure and at the end of

the test period.

Additionally, reserve samples of 2 ml were taken from all test solutions for possible analysis. If

not already used, these samples were stored in a freezer for a maximum of three months after

delivery of the draft report, pending on the decision of the sponsor for additional analysis.

6.5.4. Measurements

pH At the beginning and at the end of the test.

The pH of the solutions should preferably not deviate

by more than 1.5 units during the test.

Temperature of medium Continuously in a temperature control vessel.

Appearance of the cells At the end of the limit test microscopic observations

were performed to verify a normal and healthy

appearance of the inoculum culture and to observe for

any abnormal appearance of the algae.

- Page 10 -
C.l. Pigment Blue 61 NOTOX Project 488234

6.5.5. Recording of cell densities

At the beginning of the test, cells were counted using a microscope and a counting chamber.

Thereafter cell densities were determined by spectrophotometric measurement of samples at

720 nm using a Varian Cary 50 single beam spectrophotomneter with immersion probe

(pathlength =20 mm). Algal medium was used as blank and the extra replicate without algae as

background for the treated solution.

6.6. Electronic data capture

Observations/measurements in the study were recorded electronically using the following

programme(s):

Cary 50 single beam spectrophotometer including Cary UWVIS Pharma Upgrade Version 3.1

software (Varian, Mulgrave, Australia): Algal cell density.

REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton,

NJ, USA): Temperature

6.7. Interpretation

6.7.1. Data handling

Calibration curve

Quantification of cell densities was based on a calibration curve. Cell density was plotted versus

extinction using spectrophotometric measurements of a minimum of six dilutions of an algal

suspension with different cell densities. The calibration curve was composed using linear

regression. The software automatically calculates the cell densities based on this curve for the

spectrophotometric measurements at the various points in time during the test period.

Comparison of average -growth rates

The average specific growth rate for a specific period is calculated as the logarithmic increase in

the biomass from the equation for each single vessel of controls and treatments:

In Xi-In X

P/q = -t (day)

ti-ti

Where: liq = the average specific growth rate from time i toj

Xi= the biomass at time

Xi= the biomass at time

The average growth rate at each test substance concentration is then compared with the control

value and the percentage reduction in growth rate is calculated:

%/ C- PTX100

PC

Where: %Ir = percent inhibition in average specific growth rate

Pic = mean value for average specific growth rate in the control group

PT = average specific growth rate for the treatment replicate

Yield

The percent inhibition in yield is calculated for each treatment replicate as follows:

Y

-Page 11 -
C.I. Pigment Blue 61 NOTOX Project 488234

Where: %Iy = percent inhibition of yield

Yc = mean value for yield in the control group

Yr = value for yield for the treatment replicate

Growth inhibition is calculated for the total period of exposure.

Determination of the average exposure concentrations

The average exposure concentration was calculated as:

24 x JC .' xC -t24 + 48 x VCt24 xC t=, 2

_72 b eing the Time Weight Average (TWA) of the

concentrations of 0.1. Pigment Blue 61 measured in the samples taken at the start (Ct=(), after

24 hours (Ct-24) and the end of the test (Ct-72).

Determination of the NOEC and the EC~o

Statistical analysis of the data was not needed for growth rate reduction as the effects recorded

were not significant (<1 0%).

No EC50-values could be calculated because the test substance did not induce 50% effect (EC50

> maximum soluble concentration tested).

6.7.2. Acceptability of the test

1 . In the controls, cell density increased by an average factor of > 16 within 2 days.

2. The mean coefficient of variation for section-by-section specific growth rates in the control

cultures did not exceed 35%.

3. The coefficient of variation of average specific growth rates during the whole test period in

replicate control cultures did not exceed 7%.

6.8. List of deviations

6.8.1. List of protocol deviations

There were no deviations from the protocol.

6.8.2. List of standard operating procedures deviations

Any deviations from standard operating procedures were evaluated and filed in the study file.

There were no deviations from standard operating procedures that affected the integrity of the

study.

7. RESULTS

7.1. Measured test substance concentrations

The results of analysis of the samples taken during the final test are described in Table 2 of the

appended Analytical Report.

Analysis of the samples taken at the start of the test showed a measured concentration of

549 pg/I at the loading rate of 100 mg/I. During the exposure period the measured concentration

decreased to 342 pg/I (i.e. 62% of initial). Based on these results, the average exposure

concentration was calculated to be 422 pg/I. This concentration was relatively high when

compared to the water solubility limit and the concentrations measured during the acute toxicity

study with Daphnia magna (NOTOX Project 488233). It may therefore be possible that the test

substance was not completely dissolved although no undissolved particles were observed (due

to the low concentration).

-Page 12 -
C.l. Pigment Blue 61 NOTOX Project 488234

7.2. Mean cell densities

Table 1 shows mean cell densities measured at 24-hour intervals at the different concentrations

of C.l. Pigment Blue 61. The respective growth curves are shown in Figure 1 (see Appendix I for

the cell densities per replicate).

Table I Mean cell densities (X 10 ceils/mI)

Concentration Exposure time (hours)

C.l. Pigment

Blue 61 _______________________

W/0gil 0 24 I 48 I 72

control 1.0 9.6 52.0 226.6

422 - 1.0 7.4 40.8 179.4

250

Concentration

ClI. Pigment Blue 61

200 - MYI)

~' 150

50 .--- control

0 Ii

0 24 48 72

Exposure time (hours)

Figure 1 Growth curves at different concentrations of C.l. Pigment Blue 61

7.3. Reduction of growth rate and inhibition of yield

Table 2 shows the calculation of the percentages of growth rate reduction (total test period) and

the percentages of yield inhibition. Table 3 shows the calculation of the percentages of growth

rate reduction at different time intervals (see Appendix I for the values of growth rate and yield

per replicate). Statistical analysis of the data for yield inhibition is shown in Appendix 11.

Growth rate was in the range of the control at a ClI. Pigment Blue 61 concentration of 422 pg/I

during the 72-hour test period, while yield was statistically inhibited by 20% (2 Sample t-Test,

a = 0.05). However, it can not be excluded that the inhibition was caused by light absorption due

to the colouring of the test substance rather than by toxicity.

Microscopic observations at the end of the test revealed a normal and healthy appearance of

the exposed cells when compared to the control.

-Page 13 -
C.l. Pigment Blue 61 NOTOX Project 488234

Table 2 Percentage reduction of growth rate (total test period) and percentage

inhibition of yield

Concentration Mean growth rate Yield (0-72 h)

C.l. Pigment Blue 61

(lpg/I) _p (-72 hq Reduction (%) X10 4 cells/mI Inhibition %

control 0.07530 225.62

422 0.07204 4.3 178.41 20.9

Table 3 Percentage reduction of growth rate at different time intervals

Concentration Mean growth rate

C.I. Pigment

Blue 61 ___

(jig/I) i (0-24 h) Reduction (%) p24-48 h) Reduction (%) p48-72 hJ Reduction %

control -0.09389 I 0.07069 I0.06130

422 0.08340 11.2 0.07108 -0.5 0.06163 -0.5

7.4. Determination of effect concentrations

Table 4 shows the effect parameters based on average exposure concentrations.

Table 4 Effect parameters

7.5 E Perimental conditionsio

Table ~ h ~ ~ Hrcre ~ ~ ~~~~CI 5Phw ttebgn ignt Blen oftets.TepHwswtih

limtsprscibd y heprtool(60-.0 pefralyno vryngbymoe ha 15 ni).Th

Temerue reaie the within limioredatts prrbedibyan the prdof etocole consn pH-C withinth

2-C).

Table 5 pH levels

Concentration Exposure time (hours) CAl. Pigment

Blue 61

W/1) 0 2Z

control8.7.

4228.7. _ _

-Page 14 -
C.I. Pigment Blue 61 NOTOX Project 488234

8. CONCLUSION

Under the conditions of the present study with Pseudokirchneriella subcapitata, no reduction of

growth rate and less than 50% inhibition of yield was recorded at the concentration of CAl.

Pigment Blue 61 tested. However, it can not be excluded that the inhibition was caused by light

absorption due to the colouring of the test substance rather than by toxicity.

The EC50 for both growth rate reduction (ERC50: 0-72h) and yield inhibition (EyC50: 0-72h) was

beyond 422 pg/I.

The NOEC for growth rate reduction was 422 pg/I and the NOEC for yield inhibition was below

422 pg/I. This concentration was well above the given water solubility limit of C.I. Pigment Blue

61 (i.e. 2.5 pg/I).

-Page 15 -
C.l. Pigment Blue 61 NOTOX Project 488234

APPENDIX I WORKSHEET DATA

Table 6 Individual cell densities

Number of inoculated cells at t=-O: I X1O4 cells/mi

Concentration Vessel Exposure time (hours)

CA. Pigment Blue 61 number ______

(Pg/I) 0 24 48 72

control 1 1.00 9.10 54.15 232.71

2 1.00 8.29 53.26 246.22

3 1.00 9.00 51.40 223.41

4 1.00 9.66 51.19 227.82

5 1.00 12.33 47.63 201.20

_____________ 6 1.00 9.21 54.34 228.33

422 1 1.00 7.12 43.07 183.93

2 1.00 6.81 39.72 162.88

3 1.00 7.12 38.96 182.61

4 1.00 7.37 38.38 165.95

5 1.00 7.88 39.55 176.22

1 1_________ 6 1 1.00 1 8.20 1 45.29 1 204.87 1

Table 7 Calculation of growth rate and yield

Concentration Vessel Growth rate Yield Growth rate red. Yield inhib.

CAl. Pigment Blue 61 number Mp (X10 4 cells/mi) M% M%

Wp/O) 0-72 h 0-72 h 0-72 h 0-72 h

control 1 0.07569 231.71

2 0.07648 245.22

3 0.07513 222.41

4 0.07540 226.82

5 0.07367 200.20

6 0.07543 227.33

mean 0.07530 225.62

CV 1%

422 1 0.07242 182.93 4 19

2 0.07074 161.88 6 28

3 0.07232 181.61 4 20

4 0.07100 164.95 6 27

5 0.07183 175.22 5 22

__________ 6 0.07392 203.87 2 10

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0.1. Pigment Blue 61 NOTOX Project 488234

APPENDIX I WORKSHEET DATA - continued -

Table 8 Calculation of growth rate (section-by-section)

Concentration Vessel Growth rate (ps) Growth rate reduction()

C.I. Pigment Blue 61 number

(uigh) 0-24 h 24-48 h 48-72 h 0-24 h 24-48 h 48-72 h

control 1 0.09201 0.07431 0.06075

2 0.8813 0.07751 0.06379

3 009155 0.07260 0.06122

4 0.09450 0.06948 0,06221

5 0.10467 0.05631 0.06003

6 0.09251 0.07396 0.05981

mean 0.09389 0.07069 0.06130

CV 6% 11% 2% _ ___

The mean CV for section-by-section specific rowth rate was. 20% ____

422 1 0.08179 0.07500 0.06049 13 -6 1

2 0.07993 0.07348 0.05880 15 -44

3 0.08179 j 0.07082 0,06437 13 0 -5

4 0.08323 j 0.06875 0.06101 11 3 0

5 0 08601 0.06722 j 0.06226 8 5 -2

__________ 6 0.08767 0.07121 0.06289 7 -1 -3

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C.I. Pigment Blue 61 NOTOX Project 488234

APPENDIX 11 STATISTICS: YIELD (0-72 HOURS)

Chi-Square Test for Normality

Actual and Expected Frequencies

INTERVAL <-1.5 -1.5 to <-0.5 -0.5 to 0.5 >0.5 to 1.5 >1.5

EXPECTED 0.8040 2.9040 4.5840 2.9040 0.8040

OBSERVED 1 2 7 1 1

Chi-Square = 2.8987 (p-value =0.5749)

Critical Chi-Square = 13.277 (alpha = 0.01 ,df = 4)

= 9.488 (alpha = 0.05 *df = 4)

Data PASS normality test (alpha = 0.01). Continue analysis.

F-Test for Equali.ty of Two Variances

GROUP IDENTIFICATION VARIANCE F

1 control 216.4185

2 100 228.6941 1.0567

(p-value -0.9532)

Critical F = 14.9396 (P=0.01, 5, 5)

7.1464 (P=0. 05, 5, 5)

Since F <= Critical F, FAIL TO REJECT Ho: Equal Variances (alpha =0.01).

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0.1. Pigment Blue 61 NOTOX Project 488234

APPENDIX 11 STATISTICS: YIELD (0-72 HOURS) - continued -

ANOVA Table

SOURCE DF SS MS F

Between 1 6684.9361 6684.9361 30.0371

within (Error) 10 2225.5632 222.5563

Total 11 8910.4992

(p-value =0.0003)

Critical F = 10.0443 (alpha 0.01, df -1,10)

= 4.9646 (alpha =0.05, df = 1,10)

Since F > Critical F REJECT Ho: All equal (alpha = 0.05)

2 Sample t-Test - TABLE 1 or 2 Ho: Control=Treatment

TRANSFORMED MEAN CALCULATED IN SIG

GROUP IDENTIFICATION MEAN ORIGINAL UNITS t STAT 0.05

1 control 225.6150 225.6150

2 100 178.4100 178.4100 5.4806*

Equal Var: t critical value = 2.2281 (2 Tailed, alpha -0.05, df = 10)

(p-value = 0.0001)

2 Sample t-Test - TABLE 2 OF 2 Ho: Control=Treatment

Equal Variances:

MUM OF MIN SIG DIFF % OF DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

1 control 6

2 100 6 19.1912 8.5 47.2050

-Page 19 -
C.I. Pigment Blue 61 NOTOX Project 488234

APPENDIX III REFERENCE TEST

Pseudokirchneriella subcapitata, strain: NIVA Cf-L-i. Fresh water algal growth inhibition test

with potassium dichromate (NOTOX Project 489063).

Start of first exposure: 04 August 2008

Completion last exposure: 07 August 2008

The study procedures described in this report were based on the EEC Directive 92/69,

Publication No. L383 Part C-3 adopted December, 1992; OECD guideline No. 201, Adopted

March 23, 2006; and ISO Standard 8692, Second edition, 01 October 2004.

This reference test was carried out to check the sensitivity of the test system used by NOTOX to

Potassium dichromate (Merck, Art. 4864, Batch K34869764 607).

Algae were exposed for a period of 72 hours to K2Cr2O7 (Potassium dichromate) concentrations

of 0. 18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/I and to a blank-control. The initial cell density was

1.0 X 10 cells/mi.

Results:

Overview of % reduction of growth rate and % inhibition of yield in the reference test:

Nominal conc. Mean growth rate Yield (0-72 h)

K2Cr2O7

Jmg/p)(0-72 h) Reduction (%) X104 cells/mi Inhibition(%

control 0.07596 ______ 237.05

0.18 0.07513 1.1 222.51 6.1

0.32 0.07411 2.4 206.73 12.8

0.56 0.06944 8.6 147.61 37.7

1.0 0.05454 28.2 49.77 79.0

1.8 0.03007 60.4 7.73 96.7

3.2 0.02505 67.0 5.07 97.9

Under the conditions of the reference study with Pseudokirchneniella subcapitata, potassium

dichromate reduced growth rate of this fresh water algae species at nominal concentrations of

1.0 mg/I and higher.

The ECr, for growth rate reduction (ERCSO: 0-72h) was 1.7 mg/I with a 95% confidence interval

ranging from 1.1 to 2.7 mg/I. The historical ranges for growth rate reduction lie between 0.82

and 2.3 mg/I. Hence, the ERC50: 0-72h for the present batch corresponds with this range.

The EC~o for yield inhibition (EyC 5a: 0-72h) was 0.61 mg/I with a 95% confidence interval

ranging from 0.39 to 0.96 mg/I. Historical ranges are not yet available.

The protocol, raw data and report of this study are kept in the NOTOX archives. The test

described above was performed under GLP conditions with a QA-check.

-Page 20 -
APPENDIX IV

FRESH WATER ALGAL GROWTH INHIBITION TEST

WITH C.I. PIGMENT BLUE 61;

DETERMINATION OF THE CONCENTRATIONS

Author

Ir. M.J.C. Brekelmans

Study completion date

October 21, 2008

Laboratory Proiect Identification

NOTOX Project 488234

NOTOX Substance 14335911A

-Page Iof 9-
C.I. Pigment Blue 61 NOTOX Project 488234

1. CONTENTS

1 . CONTENTS............................2

2. REPORT APPROVAL........................3

3. INTRODUCTION......................... .............................................. 4

3.1. Preface............................4

3.2. Aim of the study.................................................................... ..... 4

4. MATERIALS AND METHODS ....................................................... 4

4.1. Reagents..................................................................................... 4

4.2. Samples................................................ 4

4.3. Analytical method............................................................... 5

4.3.1. Analytical conditions ................................................................... 5

4.3.2. Preparation of the calibration solutions .............................................. 5

4.3.3. Sample injections ...................................................................... 6

4.4. Electronic data capture........................................................................ 6

4.5. Formulas ..................................................................................... 6

RESULTS..............................7

4.6. Calibration curves ............................................................................. 7

4.7. Samples ........................................................................................ 7

4.7.1. Procedural recovery samples......................................................... 7

4.7.2. Test samples... ....................................................................... 7

5. TABLES ............................................................................................. 8

6. FIGURES............................................................................................ 9

-Page 2 -
C.I. Pigment Blue 61 NOTOX Project 488234

2. REPORT APPROVAL

PRINCIPAL SCIENTIST: Ir. M.J.C. Brekelmans

(Analytical Chemistry)

li

Date: Oi,4~s'

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C.l. Pigment Blue 61 NOTOX Project 488234

3. INTRODUCTION

3.1. Preface

Study plan Start 23 September 2008

(analytical study) Completion 24 September 2008

3.2. Aim of the study

The purpose of the analytical study was to determine the actual concentrations in samples taken

from the test solutions used during the ecotoxicity test.

4. MATERIALS AND METHODS

4.1. Reagents

Water Tap water purified by a Milli-Q water purification system

(Millipore, Bedford, MA, USA)

Methanol VWR International, Leuven, Belgium

Sodium I -heptanesulfonate Fluka Chemie, Buchs, Switzerland

monohydrate (HSA)

Acetic acid Merck, Darmstadt, Germany

Dimethyl sulfoxide (DMSO) Merck

M2-medium see main report

All reagents were of analytical grade, unless specified otherwise.

4.2. Samples

The samples were stored in the freezer. Storage stability of samples under these conditions was

determined in NOTOX project 488235. On the day of analysis, the frozen samples were defrosted

at room temperature. The test samples were diluted in a 1:1 (v:v) ratio with methanol and

analysed.

Concentrations in the test solution were expected to be low. To demonstrate proper preparation

of the test solution, it was decided to analyse the residue on a filter used during preparation of

this solution. An aliquot of the residue was transferred quantitatively into a 5 ml volumetric flask

using DMSO. This solution was ultrasonicated for 10 minutes, diluted by a factor of 100 with

50/50 (v/v) methanol/water and analysed.

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C.I. Pigment Blue 61 NOTOX Project 488234

4.3. Analytical method

4.3.1. Analytical conditions

Quantitative analysis was based on the analytical method validated for the test substance in

NOTOX project 488235

Analytical conditions:

Instrument Alliance Separation Module 2695

(Waters, Milford, MA, USA)

Detector Dual A Absorbance Detector 2487 (Waters)

Column Symmetry Shield C18, 150 mmn x 4.6 mmn i.d., dp =5 pm

(Waters)

Column temperature 50*C ± 1VC

Injection volume 100 PI

Mobile phase A - 5/0.5/94.5 (v/vlv) water/acetic acid/methanol,

contain ing 1.2 g/l HSA

B - 5/0.5/94.5 (vlvlv) water/acetic acid/[50/50 (v/v)

methanol/water], containing 1.2 g/l HSA

C - methanol

Gradient ______ _____

Time %A %B %C

[minutes]

0 0 100 0

20 100 0 0

20.1 0 100 0

23 0 100 0

23.1 0 0 100

28 0 0 100

28.1 0 100 0

35 0 100 0

Flow 2 mI/min

UV detection 313 nm

4.3.2. Preparation of the calibration solutions

Stock- and spiking solutions

Stock solutions of the test substance were prepared in dimethyl sulfoxide at concentrations of

985 and 1240 mg/I. In order to dissolve the test substance the solutions were ultrasonicated for

10 minutes.

Spiking solutions were made up from a stock solution and/or dilutions of this solution. The solvent

of the spiking solutions was dimethyl sulfoxide.

Calibration solutions

Calibration solutions in the concentration range 0. 04 - 10 mg/I were prepared from two stock

solutions. The end solution of the calibration solutions was 50/50 (v/v) methanol/water.

Procedural recovery samples

2 ml blank medium was spiked with the test substance at a target concentration of 0. 1 or 10 mg/I.

The accuracy samples were treated similarly as the test samples (see paragraph 4.2 'Samples').

-Page 5 -
C. . Pigment Blue 61 NOTOX Project 488234

4.3.3. Sample injections

Calibration solutions were injected in duplicate. Test samples and procedural recovery samples

were analysed by single injection.

4.4. Electronic data capture

System control, data acquisition and data processing were performed using the following

programme:

- Empower version 5.00 (Waters, Milford, MA, USA).

Temperature and/or relative humidity during sample storage and/or performance of the studies

were monitored continuously using the following programme:

- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton,

NJ, USA).

4.5. Formulas

Response (R) Peak area test substance (units]

Calibration curve R =a CN + b

where:

CN = nominal concentration (mg/I]

a = slope (units x I/mg]

b = intercept [units]

Analysed concentration (CA) CA = (R -b) xd (mg/I] a

where:

d = dilution factor

Recovery CA x1OO%

CN

Relative to initial concentration CA (t = x hours) x 100%

CA (t =O0hours)

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C.l. Pigment Blue 61 NOTOX Project 488234

RESULTS

4.6. Calibration curves

Several peaks were observed in the chromatogram of the test substance solution. The peak area

of the largest peak (i.e. the peak at a retention time of 10 minutes) was used as response in the

calculations.

A calibration curve was constructed using four concentrations. For each concentration, two

responses were used. Linear regression analysis was performed using the least squares method

with a 1/concentration2 weighting factor. The coefficient of correlation (r) was > 0.99.

Representative chromatograms of a test substance solution and blank solution are shown in

Figure 1.

4.7. Samples

4.7.1. Procedural recovery samples

The results for the procedural recovery samples are given in Table 1.

Mean recoveries of the procedural recovery samples were 102% and 103%. Because the

criterion that mean recoveries should be between 70% and 110% was met, the results for the test

samples were accepted.

4.7.2. Test samples

The results for the test samples are given in Table 2. In the chromatograms of samples taken

from the solutions prepared at a loading rate of 100 mg/I at t=24 and 72 hours, no extra peaks

were observed. The decrease of concentration in time is therefore assumed to be due to the very

limited solubility of the test substance in the test medium.

In order to demonstrate proper preparation of the test solution, it was decided to analyse the

residue on the 0.45 pm filter used for preparation of the nominal 100 mg/I test solution. Based on

retention times of the peaks in the chromatogram, the residue was identified as test substance

indicating proper preparation of the test solution.

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C.l. Pigment Blue 61 NOTOX Project 488234

5. TABLES

Table I Procedural recovery samples

D--ate of Date of Target Nominal Analysed Recovery Mean

preparation analysis concentration concentration concentration rcvr

fdd-mm-yy [dd-mm-yyl [m/ec[goveryl11~. %

23-09-08 23-09-08 0.1 0.100 0.102 102 103

0.100 0.104 104

23-09-08 23-09-08 10 10.0 10.2 102 102

10.0 10.2 102

Table 2 Concentrations of the test substance in test medium (limit test)

Time of Date of Date of Concentration

sampling sampling analysis 1 Loading rate 2 Analysed Relative to

[hours] [dd-mm-yy] [dd-mm-yy] [mg/I] [mg/I] initial

0 15-09-08 23-09-08 0 n.d.

100 0.549

1003 0.559

24 16-09-08 23-09-08 0 n.d.

100 0.439 80

1003 0.460 82

72 18-09-08 23-09-08 0 nd.

100 0.342 62

1003 0.331 59

2 Samples were stored in the freezer until the day of analysis.

2 A filtered test solution prepared at the loading rate.

3 Without algae.

n.d. Not detected.

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C.I. Pigment Blue 61 NOTOX Project 488234

6. FIGURES

Figure 1 Chromatograms of a 10.0 mgII test substance solution [top; res. id. 1526] and

corresponding blank [bottom; res. id. 1502].

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