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### 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, PESTICIDESAND TOXIC SUBSTANCES



### Subject: Follow-upto fax transmission of4/13/12 (ePMA

### removal ofCBI claimsregarding identified studies) 

## DearMr. Merritt: This is sentto you pursuantourconversationofseveral minutes ago. In Larry Robinson's fax of April 13th 

### while there is a waiverofconfidential business information (CBI)claims in the identified studiesthere is a an assertion ofcopyrightprotections and some additional text that provides in part
... "Reproduction, reference, summarization, retransmission orotheruse ofthis copyrighted document.
..includingelectronic transmission, using the internet oremail
....is strictlyprohibited
...." While we understand the basisforthe copyrightprotections, we have a concernthat the language in the fax mightbe interpreted to meanthat ePMA is asserting that the studies submitted perthe TSCA activity arenot tobemade available in an Agencye-docket (electronic docket) orbe included in EPA websites amongthe collectionofTSCAhealth and safety studies. We are thinkingthat the identified limitations noted in the fax are not intended to apply to EPA's normal procedures for the handling ofnon-CBI materials submitted by entities subjectto TSCA regulations (includingtheposting ofsuch materials ongovernmentwebsites) and submittedby such entities in orderto complywith a TSCA requirement, but are rather intended to limit entities outside the government from the unauthorized use orappropriation ofthe studies. Canyou pleaseconfirm CPMA's intent? Manythanks.

Scott M. Sherlock,Attorney Advisor Environmental AssistanceDivision Office ofPollution Prevention andToxics

### CONFIDENTIAL BUSINESS INFORMATION



## REPORT

StudyTitle 

## FRESH WATER ALGALGROWTH 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 specifictest 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 oftest 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 oftest concentrations................................................. 10 6.5.4. Measurements ............... .................. 10 6.5.5. Recording ofcell densities.................................................................... 11 6.6. Electronic data capture............................................................................... 11 6.7. Interpretation............................................................................................ 11 6.7.1. Data handling ................................................................................. 11 6.7.2. Acceptability of thetest........................................................................ 12 6.8. List of deviations... I.................................................................................... 12 6.8.1. Listof protocol deviations..................................................................... 12 6.8.2. Listof 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 ofyield ..................................................... 13 7.4. Determination ofeffect concentrations............................................................... 14 7.5. Experimental conditions............................................................................... 14 8. CONCLUSION............................................................................................. 15 TABLES Table 1 Mean celldensities (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 Table4 Effect parameters .............................................................................. 14 Table 5 pH levels........................................................................................ 14 Table 6 Individual cell densities........................................................................ 16 Table 7 Calculation ofgrowth rate and yield.......................................................... 16 Table 8 Calculation ofgrowth 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 inthis report has been correctly reported and was conducted incompliance 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 InVitro &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 Unitto confirm thatthe 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 thistype of study were inspected. The reporting date isthe 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 QualityAssurance C.J.Mitchell B.Sc.



### Date: 

### ...... V~~

-Page4-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 inthis report were based on the OECD guideline No. 201, 2006. In addition, the procedures were designed to meet the test methods ofthe 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 adark blue powder with granules with a purity of 96.830% and not completely soluble intest 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 bya 3days of magnetic stirring to reach maximum solubility of the test substance inthe 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 aloading rate of 100 mg/I and acontrol 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 thesamples taken at the startof 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 acceptabilitycriteria prescribed by the protocol and was considered valid. No reduction of growth rate and less than 50% inhibition ofyield was recorded at the concentration ofC.I. Pigment Blue 61 tested. However, itcan not be excluded that the inhibition was caused by light absorption due to the colouring of the test substance ratherthan 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 foryield 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 ofthe study The purpose of the studywas to evaluate the test substancefor its ability to generate toxic effects in Pseudokirchneriella subcapitata during an exposure period ofat least 48 and at most 96 hours and, if possible, to determine the EC 5 0 for both reduction of growth rate and inhibition of yield. 5.3. Guidelines The study procedures described inthis reportwere based on the Organization for Economic Cooperation and Development (OECD), OECD guidelines forTesting of Chemicals, guideline No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", Adopted March 23, 2006. Inaddition, 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 ofdifficult 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 thefinal report are retained inthe NOTOX archives for aperiod of at least 2years after finalization ofthe 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 ofthe 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 atest substance sample until the expiry date, but no longer than 10 years after finalization ofthe report. After this period the sample will be destroyed. 5.5. Definitions Cell density isthe number ofcells per millilitre. Growth rate isthe increase incell density per unittime. It isderived from the slope of the growth curve ina 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 ina50% reduction ingrowth rate relative to the control. Yield isdefined as the biomass at the end of the exposure period minus the biomass at the start of the exposure period. The EyC50 isthe concentration of test substance that results ina50% inhibition ofyield relative to the control. No Observed Effect Concentration (NOEC) isthe highest concentration tested at which the measured parameter(s) show(s) no significant effect on algal growth relativeto control values.
6. MATERIALS AND METHODS 6.1. Test Substance 6.1.1. Test substance information Identification C.l. Pigment Blue 61 Structure

N

S0 3H 

### Molecularformula C37H2 9N303S Molecularweight 596 CAS Number 1324-76-1 Description Dark blue powder with granules Batch 2084701 Purity 96.830% Test substance storage At room temperature inthe dark Stability understorage conditions Stable Expiry date 02 January 2018 6.1.2. Study specific test substance information Stability inwater At least 96 hours Solubility inwater No

-Page 7
-C.I. Pigment Blue 61 NOTOX Project 488234 6.1.3. Reference substance This report includes the results ofthe most recent reference test with potassium dichromate (Appendix 1ll). 6.1.4. Preliminary data The solubility of Pigment Blue 61 inwater (bi dest.) at ambient temperature is2.5 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 sensitiveto toxic substances inthe 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 apure culture on agar. The suspensions were continuously aerated and exposed to light ina climate room at atemperature of 21 -24oC. Light intensity 60 to 120 pE/m2 /s when measured inthe photosynthetically effective wavelength range of400 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 K 2HP0 4 40 mg/I MgSO4.7H- 20 76 mg/I Na2C03. 1 OH

20 54 mg/I CsiHSO?7.H- 20 6 mg/I NH

4NO 3 330 mg/I CaCI2 .H-

2o 36 mg/I C.HsFeo7 .xH 20 6 mg/i 1-

3130 3 2.9 mg/I MnC12.41-120 1.81 mg/I ZnCI 2 0.11 mg/i CuSO4 .5H20 0.08 mg/i (NH4)8M0 7024.4H 20 0.018 mg/I Pre-culture 3days before the start ofthe test, cellsfrom the algal stock culturewere inoculated inculture medium at a cell density of 10 cells/mi. The pre-culture was maintained under the same conditions as used inthe 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: NH

4CI 

### 15 mng/ MgcI 2.6H 2 0 12 mng/ CaCl 2.2H 2 0 

### 18 mngI MgSO4.7H 2 0 

### 15 mg/I KH

2PO 4 

### 1.6 mg/I FeCI 3 .6H 2 0 64 Pg/I Na

2 EDTA.2H2 0 100 pg/i H

3B0 3 

### 185 pig/I MnC12.4H 2 0 415 pg/I ZnC12 3 pg/i cod2 6H 2 0 1.5 Pg/I CUC12.2H 20 

### 0.01 pg/i Na

2MoO4 .2H 2 0 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 oftestsolutions The standard test procedures required generation of test solutions, which contained completely dissolvedtest substance concentrations or stable and homogeneous mixtures ordispersions. The testing of concentrations that would disturb the test system was prevented as much as possible (e.g. film ofthe test substanceon the water surface). The batch of ClI. Pigment Blue 61 tested was adark blue powder with granules with a purity of 96.830% and notcompletely soluble intest medium at the loading rate prepared. Preparation oftest solutions started with a loading rate of 100 mg/I applying a 10-minute treatment period with ultrasonic waves followed by a3daysof magnetic stirring to reach maximum solubility ofthe test substance inthe 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 of50 mlwere added to each replicate ofthe respectivetest concentration. Subsequently, 1ml of an algal suspension was added to each replicate providing acell densityof 104 cells/mi. 

### 6.5. Limittest 6.5.1. Test concentrations ClI. Pigment Blue 61 Filtered solution prepared ata loading rate of 100 mg/I Controls Test medium without test substance or other additives Replicates 6 replicates of both test groups Inaddition, 1extra 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 oftest solution Medium M2 Cell density An initial cell density of 1X104 cells/mi. Illumination Continuously using TLD-lamps of the type 'Cool-white' of30 Watt, with a light intensity within the range of 78 

### to 82 pE.M- 2.s-1 Incubation Vessels were distributed at random inthe 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. Inaddition, the filter used for preparation of the limit concentration was stored for possible analysis ofthe residue. The method of analysis is described inthe appended Analytical Report (Appendix IV). Frequency att0- h,t--24 hand t=72 h Volume 2 ml Storage Samples were stored inafreezer 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 atestvessel at the highest substance concentration butwithout algae and samples for analysis were taken at the start, after24 hours ofexposure and at the end of the test period. Additionally, reserve samples of2 ml were taken from all test solutions for possible analysis. If not already used, these samples were stored inafreezer for a maximum ofthree months after delivery of the draft report, pending on the decision ofthe sponsor foradditional analysis. 6.5.4. Measurements pH At the beginning and at the end ofthe test. The pH of the solutions should preferably not deviate by more than 1.5 units during the test. Temperature of medium Continuously inatemperature control vessel. Appearance ofthe cells At the end of the limit test microscopic observations were performed to verify a normal and healthy appearance ofthe inoculum culture and toobserve for any abnormal appearance ofthe algae.

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

### 6.5.5. Recording of cell densities At the beginning ofthe 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 aVarian 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 inthe 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 ofcell densities was based on acalibration curve. Cell densitywas 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 intime during the test period. Comparison of average -growth rates The average specific growth rate for aspecific period iscalculated asthe logarithmic increase in the biomass from the equation for each single vessel of controls and treatments: InXi-InX P/q = -t (day) ti-ti Where: liq = the average specific growth ratefrom time itoj 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 ingrowth rate is calculated:

%/C- PTX100 PC Where: %Ir =percent inhibition inaverage specific growth rate Pic= mean value for average specific growth rate inthe control group PT =average specific growth ratefor the treatment replicate Yield The percent inhibition inyield iscalculated for each treatment replicate asfollows: Y

-Page 11 -C.I. Pigment Blue 61 NOTOX Project 488234 Where: %Iy = percent inhibition of yield Yc = mean value for yield inthe control group Yr= value for yield for the treatment replicate Growth inhibition is calculated for the total period ofexposure. Determination of the average exposure concentrations The average exposure concentration was calculated as: 24xJC .' xC -t 24 +48xVCt24 xCt=, 2

_72 being the Time Weight Average (TWA) of the concentrations of 0.1. Pigment Blue 61 measured inthe samples taken at the start (Ct=(), after 

### 24 hours (Ct-24) 

### and theend ofthe test (Ct-72). Determination ofthe NOEC and the EC~o Statistical analysis of the data was not needed for growth rate reduction as the effects recorded were not significant (<10%). No EC 50-values could be calculated because the test substance did not induce 50% effect (EC50 > maximum soluble concentration tested). 6.7.2. Acceptability ofthe test 1. Inthe controls, cell density increased by an average factor of> 16 within 2days. 2. The mean coefficient ofvariation for section-by-section specific growth rates inthe control cultures did not exceed 35%. 3. The coefficient ofvariation ofaverage specificgrowth rates during the whole test period in replicate control cultures did notexceed 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 inthe studyfile. There were no deviationsfrom standard operating procedures that affected the integrity ofthe study. 

### 7. RESULTS 7.1. Measured test substance concentrations The results ofanalysis ofthe samples taken duringthe final test are described inTable 2ofthe appended Analytical Report. Analysis ofthe samples taken at the start ofthe test showed ameasured concentration of 549 pg/I atthe loading rate of 100 mg/I. During the exposure period the measured concentration decreased to 342 pg/I (i.e. 62% of initial). Based onthese 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 studywith Daphnia magna (NOTOX Project488233). Itmay therefore be possible thatthe test substancewas notcompletely dissolved although no undissolved particles were observed (due tothe low concentration).

-Page 12

-

### C.l. Pigment Blue 61 NOTOX Project 488234 7.2. Mean cell densities Table 1shows mean cell densities measured at 24-hour intervalsat the different concentrations of C.l. Pigment Blue 61. The respective growth curves are shown inFigure 1(seeAppendix Ifor the cell densities per replicate). Table I Mean cell densities (X10 

### ceils/mI)

Concentration Exposuretime (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 ofC.l. Pigment Blue 61 7.3. Reduction of growth rate and inhibitionofyield Table 2shows the calculation of the percentages ofgrowth rate reduction (total test period) and the percentages ofyield inhibition. Table 3shows the calculation ofthe percentages of growth rate reduction at different time intervals (seeAppendix Ifor the values ofgrowth rate and yield per replicate). Statistical analysis of the data for yield inhibition is shown inAppendix 11. Growth rate was inthe range of the control at a ClI. Pigment Blue 61 concentration of422 pg/I during the 72-hour test period, while yield was statistically inhibited by 20% (2Sample t-Test, 

### a=

### 0.05). However, itcan not be excluded that the inhibitionwas caused by light absorption due to the colouring ofthe test substance ratherthan bytoxicity. Microscopicobservations atthe end ofthe test revealed anormal and healthyappearance of the exposed cellswhen compared to the control.

-Page 

### 13-C.l. Pigment Blue 61 NOTOX Project 488234 Table 2 Percentage reduction ofgrowth 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 4cells/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 ofeffect concentrations Table 4 shows the effect parameters based on average exposure concentrations. Table 4 Effect parameters

7.5 E Perimental conditionsio Table 

### ~

~ ~ ~ ~ ~~~~CI 5Phw h Hrcre ttebgn ignt Blen oftets.TepHwswtih limtsprscibd y heprtool(60-.0 pefralyno vryngbymoe ha 15 ni).Th

Temerue reaie within the limioredatts prrbedibyan the prdof etocole pH-C consn 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 Project488234 8. CONCLUSION Under the conditions ofthe present study with 

### Pseudokirchneriella subcapitata, no reduction of growth rate and less than 50% inhibition ofyield was recorded at the concentration of CAl. Pigment Blue 61 tested. However, itcan notbe excluded that the inhibition was caused by light absorption due to the colouring of the test substance rather than bytoxicity. The EC 50for 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

-Page 16

-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 forsection-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 008601 0.06722 

# j 0.06226 8 5 -2

__________ 6 0.08767 0.07121 0.06289 7 -1 -3

-Page 17-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
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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

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APPENDIX III REFERENCE TEST
Pseudokirchneriella subcapitata, strain: NIVA Cf-L-i. Fresh water algal growth inhibition test with potassium dichromate (NOTOX Project 489063). Start offirst exposure: 04 August 2008 Completion last exposure: 07 August 2008 The study procedures described inthis reportwere 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 checkthe sensitivity ofthe test system used by NOTOX to Potassium dichromate (Merck, Art.4864, Batch K34869764 607). Algae were exposed for a period of72 hours to K 2Cr2O 7 (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 densitywas 1.0 X 10 

### cells/mi. Results: Overview of% reduction of growth rate and %inhibition ofyield inthe reference test:

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

2 Cr2O7 

### 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 ofthe reference studywith Pseudokirchneniella subcapitata, potassium dichromate reduced growth rate ofthis 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 to2.7 mg/I. The historical rangesfor 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 (EyC5 a: 0-72h) was 0.61 mg/I with a95% confidence interval ranging from 0.39 to 0.96 mg/I. Historical ranges are not yetavailable. The protocol, raw data and report of this study are kept inthe NOTOX archives. The test described above was performed under GLP conditions with aQA-check.

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## 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 Iof9-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

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-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 studywas to determine the actual concentrations insamples 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 inthe freezer. Storage stability of samples under these conditions was determined inNOTOX project 488235. On the day of analysis, the frozen samples were defrosted at room temperature. The test samples were diluted ina 1:1 (v:v) ratio with methanol and analysed. Concentrations inthe test solution were expected to be low. To demonstrate proper preparation ofthe test solution, itwas decided to analysethe residue on afilter used during preparation of this solution. An aliquot of the residue was transferred quantitatively into a5 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 x4.6 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, containing 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 

### ofthe calibration solutions Stock- and spiking solutions Stock solutions of the testsubstance were prepared indimethyl sulfoxide at concentrations of 985 and 1240 mg/I. Inorder to dissolve the test substance the solutions were ultrasonicated for 10 minutes. Spiking solutions were made up from astock solution and/or dilutions ofthis solution. The solvent of the spiking solutionswas dimethyl sulfoxide. Calibration solutions Calibration solutions inthe concentration range 0.04
- 10 mg/I were prepared from two stock solutions. The end solution of the calibration solutionswas 50/50 (v/v) methanol/water. Procedural recovery samples 2 ml blank medium was spiked with the test substance at atarget concentration of 0.1or 10 mg/I. The accuracy samples were treated similarly as the test samples (see paragraph 4.2 'Samples').

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-C. . Pigment Blue 61 NOTOX Project 488234 4.3.3. Sample injections Calibration solutions were injected induplicate. 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 ofthe studies were monitored continuously using the following programme:

- REES Centron Environmental Monitoring system version SQL2.0 (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 =xhours) x 100% CA (t=O0hours)

-Page 6-C.l. Pigment Blue 61 NOTOX Project 488234 RESULTS 4.6. Calibration curves Several peaks were observed inthe chromatogram ofthe 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. Acalibration 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/concentration 2 weighting factor. The coefficient of correlation (r)was >0.99. Representative chromatograms of atest 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 inTable 1. Mean recoveries ofthe 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 sampleswere accepted. 4.7.2. Test samples The results for the test samples are given inTable 2. Inthe chromatograms of samples taken from the solutions prepared at a loading rate of 100 mg/I att=24 and 72 hours, no extra peaks were observed. The decrease of concentration intime istherefore assumed to be due to the very limited solubility of the test substance inthe test medium. Inorder todemonstrate proper preparation ofthe test solution, itwas 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 inthe 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 intest medium (limittest) 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 inthe 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 testsubstance solution [top; res. id. 1526] and corresponding blank [bottom; res. id. 1502].

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