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### REPORT : Algal Growth Inhibition Assay

HLS study number: CEF0008 Version ID: Audited Draft 1 Report Issue date: 30 September 2013Huntingdon Life Sciences: CEF0008

2 

## Sponsor and Te st Facility Details Sponsor

Test facility Huntingdon Life Sciences Eye Research Centre Eye Suffolk IP23 7PX UKHuntingdon Life Sciences: CEF0008

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## Ta ble of Content

s Sponsor and Test Facility Details

................................ ................................ .......................... 2 Compliance with Good Laboratory Practice

................................ ................................ .......... 5 Quality Assurance Statement

................................ ................................ ................................ 6 Contributing Scientists

................................ ................................ ................................ .......... 7 Summary

................................ ................................ ................................ .............................. 8 1. Introduction

................................ ................................ ................................ ................... 9 2. Test Substance

................................ ................................ ................................ ........... 10 3. Experimental Procedure

................................ ................................ .............................. 11 3.1 Test species

................................ ................................ ................................ .......... 11 3.1.1 Name

................................ ................................ ................................ ............. 11 3.1.2 Source

................................ ................................ ................................ ........... 11 3.1.3 Pre
- culture

................................ ................................ ................................ ..... 11 3.2 Culture medium

................................ ................................ ................................ ..... 11 3.3 Test substance preparation

................................ ................................ ................... 11 3.4 Exposure conditions

................................ ................................ .............................. 12 3.4.1 Experimental design

................................ ................................ ...................... 12 3.4.2 Test concentrations

................................ ................................ ........................ 12 3.4.3 Stability of test concentrations

................................ ................................ ....... 12 3.4.4 Environmental conditions

................................ ................................ ............... 12 3.5 Measurement of growth

................................ ................................ ........................ 13 3.6 Evaluation of data

................................ ................................ ................................ . 13 3.7 Validity criteria

................................ ................................ ................................ ...... 14 3.8 Protocol deviations

................................ ................................ ................................ 14 4. Maintenance of Records

................................ ................................ .............................. 15 5. Result s ................................ ................................ ................................ ........................ 16 5.1 Chemical analysis

................................ ................................ ................................ . 16 5.2 Algal growth

................................ ................................ ................................ .......... 16 5.2.1 Observations

................................ ................................ ................................ .. 17 5.3 Environmental parameters

................................ ................................ .................... 17 6. Conclusions

................................ ................................ ................................ ................. 18 7. References

................................ ................................ ................................ .................. 19 

## List of Figures Figure 1 Inhibition of growth

................................ ................................ ....................... 20Huntingdon Life Sciences: CEF0008

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## List of T ables Table 1 Measured concentrations

............................................................................. 21 Table 2 Cell densities

............................................................................................... 22 Table 3 Inhibition of growth

....................................................................................... 23 Table 4 Environmental parameters

........................................................................... 24 

## List of Appendices Appendix 1 Certificate of analysis

................................................................................... 25 Appendix 2 Algal nutrient medium (OECD)

..................................................................... 26 Appendix 3 The Determination of in OE CD medium

...................................... 27 Appendix 4 Compliance statement

................................................................................. 37Huntingdon Life Sciences: CEF0008

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## Compliance with Good Laboratory Practice
: A lgal G rowth Inhibition Assay The study described in this report was conducted in co mp liance wi th the f ol lo wing Good Laboratory

Practice standards and I consider the data generated to be valid. The UK Good Laboratory Practice Regulat io ns (Statutory Instrument 1999 No. 3106, as amended by Statutory Instrument 2004 No. 994). OECD Principles of Good La boratory Practice (as revised in 1997), ENV/MC/CHEM (98) 17. EC Commissio n Di

rective 2004/10/EC of 11 Febru ary 2004 (Official Journal No L 50/44). These principles of Good Laboratory Practice are accepted by

the Regulatory

A uthori ti es of the United States of

America and Japan on the basis of Intergovernmental Agreements.

________________________________ ___________ ________________________ Romalie Allen BSc Date Study Di rector Hunt ingdon Life SciencesHuntingdon Life Sciences: CEF0008

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## Quality Assurance Statement
: A lgal Growth Inhibition Assay The fo llo wing inspect io ns and audits have been carri ed out in relation to thi s study : Study Phase Date(s) of Inspection Date of Reporting to Study Director and Management Protocol Audit 13 Jun 2013 13 Jun 2013 Report Audit 17 Sep 2013 17 Sep 2013 Process based inspections : Ato r about th e time t his study w as in progress inspect io ns of procedures em

ploy ed on this ty

pe of study

w ere carri ed out. T hese were conducted and reported to appropriate Company Management as indicated below: Process Based Inspections Date(s) of Inspection Date of Reporting to Management Envi ronmental

Measurements & Moni toring/Ammonia Analysis 07 Mar 2013 07 Mar 2013 Counting of Cells (Haemocy

tometer/Coul

ter Counter/Spectrophotometer) 09 Apr 2013 09 Apr 2013
Sam pling of Test Media for Chemical Analysis 09 Jul 2013 09 Jul 2013 Dose Form

ulation 09 Jul 2013 09 Jul 2013 Study Management & Conduct 09 Jul 2013 09 Jul 2013 Experimental Set
- Up 11 Jul 2013 11 Jul 2013 Sam ple Preparation (To Include Sample Management & Tracking) 27 Feb 2013 01 Mar 2013 Standard Preparation & Fo rtificat io n Procedures 05 Aug 2013

– 06 Aug 2013 07 Aug 2013 Result Processing 30 Aug 2013 30 Aug 2013 Chro mat ography 30 Aug 2013 30 Aug 2013 In addit io n, an inspection o f the f acili ty where thi s study

was conducted was carried out on an annual basis. These inspect io ns were promptly reported to Company Management.

________________________________ ___________ ________________________ T om Pullinger MRQA Date Group Manager Department of Qualit y Assurance Hunt ingdon Life SciencesHuntingdon Life Sciences: CEF0008

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## Contributing Scientist s
: A lgal Growth Inhibition Assay 

### Study management Aquatic Ecotoxicology and Biodegradation Romalie Allen BSc Study Di rector 

### Expert department Environmental Analysis Simo n Tate Higher National Certificate (Applied Chemistry) Senior Study

ManagerHuntingdon Life Sciences: CEF0008

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## Summary The effect of on the growth of the unicellular gre en alga Pseudokirchneriella subcapitata was assessed under non
- axenic condit ions. The study was conducted in accordance wit h EC Methods for Determinat io n of Ecotoxicit y, Annex IV to Commissio n Regulat io n (EC) No 761/2009 Part C, Method 3 “Freshwater Algae and Cyanobacteria, Growth Inhibit io n Test” and Procedure 201 of the “Guidelines for Testing of Chemicals” of the Organisat io n fo r Econo mic Co
- operation and Development: Freshwater Alga and Cyanobacteria, Growth Inhibit io n Test” (2006 , corrected 2011 ). Tri plicate algal

cul tures, wi th an ini ti al cell densi

tyof ca . 1 x 10 4 /mL, were exposed to dissolved in algal nutrient medium at nominal concentrations of 0.008, 0.030, 0.080, 0.260, 0.820, 2.64, 8.44 and 27.0 mg/L. The test media were prepared in OEC D medium fro m an aqueous stock solut io n, which was made by the di rect addi tion of the test substance to the dilution medium. The cultures were incubated in an orbital incubator under continuous illuminat io n at tem peratures ranging from 21.4 to 23. 0  C for 7 2 hours. Atthe start of the test, the measured levels of in samples of the test cultures ranged between 88 and 113 % of thei r no minal values. After 72 hours, the measured levels ranged between 60 and 10 2 % of thei r no minal values (between 59 and 9 0 % of their starting values). The greatest losses were observed at the lower test concentrations. Based on the geometric me an , the overall measured levels of were 0.00668, 0.0221, 0.0589, 0.212, 0.719, 2.39, 7.56 and 28.9 mg/L. These values wer e used in the calculat io n of study end
- points. Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the growth rate and yield. The fo llo wing values were derived from the data:

Parameter Endpoint 72 hours (mg/L)
Growth rate EC 10 25.8 [25.5 & 28.3] EC 50 > 28.9 [ 14% inhibition ] NOEC 7.56 LOEC 28.9

Yield EC 10 9.69 [ 7.91 & 11.1$ ]

EC 50 28.2 [ 25.8 & 31.2$ ] NOEC 7.56 LOEC 28.9 [ ] 95% confidence limits $ C onfidence interval may be unreliable due to goodness of curve fit NOEC No observed effect concentration LOEC Lowest observed effect concentrationHuntingdon Life Sciences: CEF0008

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## 1. Introduction This study

was designed to assess the effect of on the growth of the unicellular green alga Pseudokirchneriella subcapitata
. The study was conducted in accordance wit h EC Methods for Determinat io n of Ecotoxicit y, Annex IV to Commissio n Regulat io n (EC) No 761/2009 Part C, Method 3 “Freshwater Algae and Cyanobacteria, Growth Inhibit io n Test” and Procedure 201 of the “Guidelines for Testing of Chemicals” of the Organisat io n fo r Econo mic Co
- operation and Development: Freshwater Alga and Cyanobacteria, Growth Inhibit io n Test” (2006 , corrected 2011 ). The protocol was approved by Huntingdon Life Sciences Management and by th e Study Di rector on 17 June 2013
. The experimental start and complet io n dates of the study were 9 July 2013 and 02 August 2013 , respectively. The study was conducted at Huntingdon Life Sciences, Eye Research Centre, Eye, Suffo lk, IP23 7PX. A separate physiochemical property study (CEF0001) indicated that the limit of so lubilit y of the test substance was 27.5 mg/ L at 20oC
.Huntingdon Life Sciences: CEF0008

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## 2. Te st Substance
Name Chemical name CAS No. Physical state White c ry stalline p owder Batch number 12342 Puri ty To be provided Storage Room tem perature in the dark Expi ry Date To be provided Date received: 4 March 2013 Certificate of analysis: Appendix 1Huntingdon Life Sciences: CEF0008

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## 3. Experimental Procedure 3.1 Test species 3.1.1 Name Pseudokirchneriella subcapitata , Strain No. CCAP 278/4. 3.1.2 Source Axenic, uni
- cellular, liquid slope cultures of algae were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland and arrived on 10 July 2013
. 3.1.3 Pre - culture The liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium (Appendix 2) was inoculated with cells aseptically removed from the slope culture; these primary

liquid cul

tures (100 m

L) were incubated for approximately three days in an orbi tal incubator under continuous illuminat io n at no minal temperatures in the range 21 to 25  C. Subsequent ly, appropriate vo lu mes of these primary cul tures were asept ic ally transferred to fresh sterile algal nutrient medium to prepare secondary l iquid cultures; these cul tures were incubated, as stated above, for a further three days to provide an inocul um in the log phase of growth, characterised by a cell densit y of 8.3 x 10 5 cells/mL. 

### 3.2 Culture medium Sterile algal nutri ent medium as recommended in Official Journal No. L383A Part C.3 and OECD Procedure 201 (see Appendix 2 ). 

### 3.3 Test substance preparation The test substance (54 .3 mg) was ground into a smooth pourable paste on an agate mortar wi th the dropwi se addit io n of OECD medium. The paste was then added to a volumetric flask (2 L) containing OECD medium (1500 mL), which was then shaken before being made up to vol ume and sti

rred f or ca . one hour until no particulate matter r emained. The fo rm ulat io n was then filtered (0.45 μm pore size ) to provide an aqueous sol ut io n wi th a no minal concentration of 27.0 m g/L that was used di

rect ly fo r the highest test concentration and subsequent dilut io n t o nominally 8.44 mg/L
. A liquots (313, 313, 310, 317, 308, 375 and 267 mL) were serially diluted in vo lu metric fla s ks (1 L) co nt aining OECD med ium to provide test media wi th nominal concentrations of 8.44, 2.64, 0.82, 0.26, 0.08, 0.03 and 0.008 mg/L. An a liquot ( 4.8 mL) of the secondary algal inoculum was added to a portion ( 400 mL) of the test medium

at each concentration, to give an init ial cell densi ty of ca . 1 x 10 4 cells/mL. An aliquot of the appropriate inoculated test medium was added to each of the test vessels.Huntingdon Life Sciences: CEF0008

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### 3.4 Exposure conditions 3.4.1 Experimental design A range finding test was fo llo wed by a definit ive test wi th eight test concentrations, plus an algal nutri ent medium

control group . Six flasks were established and incubated for the control group and three flasks for each test group, pl us an addi

tional flask at nominal concentrations of 0.008 and 27.0 mg/L, which contained test medium but no algal cells. The media remaining in the preparation flasks were used for water qualit y measurements at the s tart. Before the start of the test, the required number of empt y test vessels (250 mL conical flasks), were loosely stoppered with foam bungs, covered with aluminium fo il that was secured by autoclave tape and sterilised by autoclaving (121  C for at least 15 minutes). After the addi tion of the inoculated test medium, each flask was then loosely plugged with a foam bung. The control cultures were prepared as for the test med a except that no test substance was added and a larger vo lu me (700 m L) of medium was made using 8.4 m L of inoculum
. 3.4.2 Test concentrations The range finding test emplo yed no minal test concentrations of 0.1, 1.0, 10 and 25 mg/L
. After 72 hours, algal growth was inhibited by 39% at 25 m g/L and 15% at 10 mg/L , no significant inhibit io n was o bserved at 1 or 0.1 mg/L. Based on this result, t he definit ive test concentrations emplo yed were 0.008, 0.03, 0.08, 0.26, 0.82, 2.64, 8.44 and 27.0 mg/L as

; wi th the highest concentration approximately the limi t of sol ubilit y of the test substance
. 3.4.3 Stability of test concentrations The test concentrations of were measured using liquid chromatography wi th tandem m ass spectrometric detection (LC
- MS/MS , see Appendix 3 ). At the start of the definit ive test, two s am

ples ( 10 mL) were taken from the freshly
- prepared control and test media and each 10 m

L sample was added to a vial containing 10 mL o f water:acetonitrile:formic acid (95:5:0.1)
. After 72 hours, the contents of the replicate flasks fo r each group were po oled and further samples taken for analysis. Addit io nal samples were also taken fro m a flask containing at 0.008 and 27.0 mg/L but with no algal cells, in order to obtain informat io n on the extent of adsorption/absorption of the test substance by the algal cells. On each occasio n, one of the samples was analysed and the other was stored in a freezer in case further analysis was required. 3.4.4 Environmental conditions Conical flasks (250 mL) each containing control or test culture (100 mL) were placed in an illuminated orbital incubator according to a random number sequence. The cultures were incubated, without renewal o f medium for 72 hours under continuous illuminat io n (no minallyHuntingdon Life Sciences: CEF0008

13 4440 to 8880 lux.) provided by 6 x 30 W “cool white” 1 metre fluorescent tubes. The tem perature was maintained at 23 ± 2°C. Tem perature and pH of control and test flasks at the start and end of the test were recorded. Gaseous exchange and suspensio n of the algal cells were ensured by the act io n of the orbital shaker, oscill at ing at a nominal 1 3 0 cycles per minute. The minimum and maximum tem perature and light intensit y (four corner positions and in a central position of the rando m block design) within the test area were determined each day . To minimise the impact of differe nces in light intensit y across the test area on algal growth, control and test flasks were re - posi tioned in the test area each day during the definit ive test. 

### 3.5 Measurement of growth Sam ples were taken from control and test flasks at 24, 48 and 72 hours and the cell densit ies measured using a Coulter Z Series Particle Count and Size Analyser. The estimate of cell numbers in each sample was based on the mean of three consecutive counts, corrected for background counts of uninoculated dilut io n med ium . The p resence of any abnormal cells was also noted during screening of each test level. 

### 3.6 Evaluation of data The data were compiled in an Excel spreadsheet and analysed using SAS 9.1 (SAS Inst itut e 2002), using mean measured concentrations. The area under the curve was divided by init ial count and total time to give AUCP (Area Under the Curve expressed as a Proportion of the init ial cell count), where a value of 1 represents growth and a value of 0 represents complete toxicit y (all algae inhibited). In order to e stimate the concentration at which 50% inhibit ion of growth occurred (EC 50 ), sigmo idal curves were fitted to AUCP and growth rate. For both variables, 0% inhibit io n was defined as the control mean and 100% inhibit io n was defined as no growth. The minimum of the curve (for infinite concentration) was bounded between 0 and 1 for AUCP and between
- 1000 and 0 for growth rate. The formulae for these curves are given below:



##                  

         

     

      

Otherwise Min Con ep Con Min Con Min Control Con EC ion concentrat s ep Otherwise Min Con ep Con Min Con Min Control Con

100

50 50 1 rate Growth 50 log exp where 100 1 50 1 50 1 AUCP

Con = an est imate of the control mean Min = an est imate of the min imum o f th e curve s = slope estimateHuntingdon Life Sciences: CEF0008

14 Yi eld was calculated for each test vessel as the final cell densit y (af ter 72 hours) minus the presumed ini

ti al cell densi

ty of 1 x 10 4 cells/mL. A mean yield value for each test concentration was calculated and the percentage inhibit io n was determined using the formula below: % Inhibit io n = ((Y C - Y T ) / Y C ) x 100 Where Y C = mean value for yield in the control group Y T = val ue f or yield in treatment replicate All 95% confidence intervals for EC 50 were calculated using the likelihood ratio method (Donaldson and Schnabel, 1985). Ho wever, no EC 50 was calculable for growth rate as there was insignificant inhibit io n observed at all levels. The EC 10 (wi th 95% confidence interval) was also estimated by

reparameterising th e above formulae. For yield and growth rate, Williams’ test (1971, 1972) was also used to compare each treated group with the control

unless there was evidence of a non
- mo notonic dose
- response relat io nship, in which case Dunnett’s test (1955, 1964) was used. 

### 3.7 Validity criteria For the test to be valid, the fo llo wing criteria must be met:

 C ell concentration in control cultures should have increased by a factor of at least 16 wi thin 72 hours. Thi s corresponds to a specific growth rate of 0.92 day- 1 ;

 T he me an coefficient of variat io n fo r daily growth rates (Days 0
- 1, 1
- 2 and 2
- 3) in the control cul tures m ust not exceed 35%;

 T he coefficient of variat io n of the average specific growth rate in replicate control cul tures m ust not exceed 7%. 

### 3.8 Protocol deviations There were no protocol deviat io ns.Huntingdon Life Sciences: CEF0008

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## 4. Ma intenance of Records This report has been compiled fro m original data, which will be stored, together with a copy of the final report, a copy

of the protocol

and amendments in Hunt ingdon Life Sciences’ Archives, Hunting don Life Sciences Ltd, Eye, Suffol k, UK. Records will be retained for a minimum period of at least one year from the date of issue of the final report. At the end of the one year retention period the Sponsor will be contacted and advice sought on the future requi rements. Under no ci

rcumstances will any

i tem be di scarded wi thout the Sponsor’s knowledge. Hunt ingdon Life Sciences will retain the Qualit y Assurance records relevant to this study f or twenty y ears, and a copy

of the final report in its archives indefinitely.Huntingdon Life Sciences: CEF0008

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## 5. Results 5.1 Chemical analysis The results of chemical analysis are given in Table 1 and example chromatograms are illustrated in Appendix 3
. Atthe start of the test, the measured levels of in samples of the test cultures ranged between 88 and 113 % of thei r no minal values. After 72 hours, the measured levels ranged between 60 and 102 % of thei r no minal values (between 59 and 90 % of their starting values). The greatest losses were observed at the lower test concentrations. Based on the geometric me an , the overall measured levels of were 0.00668, 0.0221, 0.0589, 0.212, 0.719, 2.39, 7.56 and 28.9 mg/L.
. After 72 hours, analysis o f samples of media containing at 27.0 mg/L, which had been incubated without algal cells gave similar results to test media incubated in the presence of algal cells. These results indicate that the presence of algal cells did not affect the stability of the test substance. However, for th e lowest concentration (no minally 0.008mg/L) the concentration without the presence of algal cells was 0.00762 mg/L (95% of no minal) com pared to 0.00583 mg/L (73% of no minal) in those flasks with algal cells indicating so me effect of the algal cells on stabilit y of the test material. 

### 5.2 Algal growth Individual cell densit ies for each culture and the mean values are given in Table 2. The calculated average specific growth rate and yield values are given in Table 3 and are expressed in terms of percentage inhibit io n by co mparing the test group value with that of the control curve. The test results have been expressed in terms of mean measured concentrations o f

. The following values were derived from the data:
Parameter Endpoint 72 hours (mg/L)
Growth rate EC 10 25.8 [25.5 & 28.3] EC 50 > 28.9 [14% inhibition] NOEC 7.56 LOEC 28.9

Yield EC 10 9.69 [7.91 & 11.1$]

EC 50 28.2 [25.8 & 31.2$] NOEC 7.56 LOEC 28.9 [ ] 95% confidence limits $ C onfidence interval may be unreliable due to goodness of curve fit NOEC No observed effect concentration LOEC Lowest observed effect concentrationHuntingdon Life Sciences: CEF0008

17 The mean coefficient of variat io n (CoV) for section by sect io n daily growth rates in th e control cul tures was 14.4 during the definit ive test and the CoV for the average specific growth rates of the control culture was 1.25 during the 72
- hour exposure period. 5.2.1 Observations No microscopic abnormalit ies of the cells were detected. 

### 5.3 E nvironmental parameters The measurements of water qualit y (tem perature and pH) in control and test flasks are summarised in Table 4 ; tem perature remained wit hin acceptable limit s throughout the study, al though the pH of the con

trol cul tures increased by

more than 1.5 pH uni

ts during the 72
- hour exposure period. The increase in pH observed during the definit ive test is not thought to have affected either the validit y or integri ty of the study b ecause the validit y cri

teria f or thi s ty pe of test were met by

the control

cul tures. The tem perature of the incubator ranged between 21.4 and 23.0  C. Atthe start of the test, the test media at all concentrations were colourless.Huntingdon Life Sciences: CEF0008

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## 6. Conclusions After 72 hours of exposure of the unicellular gr een alga Pseudokirchneriella subcapitata to , the E r C 50 and E y C 50 , based on mean measured concentrations, were >28.9 mg/L (14% inhibit io n) and 28.2 m g/L, respect ively. T he “no observed effect concentration” (NOEC) at 72 hours fo r growth rate and yi eld was 7.56 m g/L, and the “lowest observed effect concentration” (LOEC) was 28.9 mg/L based on mean measured concentrations,Huntingdon Life Sciences: CEF0008

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## 7. References DONALDSON, J.R. and SCHNABEL, R.B. (1985) Computational experience wit h confidence regions and confidence intervals fo r nonlinear least squares. National Bureau of Standards , Boulder, Colorado. DUNNETT, C.W. (1955) A mult iple co mpar ison procedure for comparing several treatments wi th a control
. Journal of the American Statistical Association , 50 , 1096
- 1121. DUNNETT, C.W. (1964) New tables for mult iple com parisons wi

th a control
. Biometrics , 20 , 482
- 491. SAS INSTITUTE (2002) SAS OnlineDoc  Version Nine . SAS Institute Inc., Cary, NC, USA. WILLIAMS, D.A. (1971) A test for differences between means when several dose levels are com pared wi th a zero dose control
. Biometrics, 27 , 103
- 117. WILLIAMS, D.A. (1972) The comparison of several dose levels with a zero dose control. Biometrics, 28 , 519
- 531.Huntingdon Life Sciences: CEF0008

20 Figure 1 Inhibition of growth a) Growth rate 0 – 72 h ours

b) Yield 0 – 72 hoursHuntingdon Life Sciences: CEF0008

21 Table 1 Measured concentrations

Nominal concentration (m g/L) Measured concentrations ( m g/L) Overall mean*

0 hours %N 72 hours %N %t0
Control nd
- nd
- - nd 0.008 0.00766 96 0.00583 73 76 0.00668 0.008A
- - 0.00762 95 -
- 0.030 0.0264 88 0.0185 62 70 0.0221 0.080 0.0719 90 0.0483 60 67 0.0589 0.260 0.276 106 0.163 63 59 0.212 0.820 0.818 100 0.632 77 77 0.719 2.64 2.54 96 2.24 85 88 2.39 8.44 8.11 96 7.04 83 87 7.56 2 7.0 30.4 113 27.5 102 90 28.9 27.0A
- - 28.4 105 -
- nd None detected (less than the limit of detection of 0.0004 mg/L) %N Measured concentration expressed as a percentage of the nominal concentration (calculated using unrounded values but expr essed to 3 significant figures). %t0 Measured concentration after 72 hours expressed as a percentage of the starting concentration. A Culture medium incubated under test conditions without algal cells. * Geometric mean.Huntingdon Life Sciences: CEF0008

22 Table 2 Cell densities Exposure concentrations

( mg/L ) Replicate

number Cell densities (cells/mL) Nominal Measured 24 hours 48 hours 72 hours

Control nd R 1 66850 349283 1947550 R 2 83883 392550 1921550 R 3 82517 449850 2254050 R 4 71417 3 86717 2021717 R 5 85917 413750 1875883 R 6 81850 429183 2078017 Mean 78739 403556 2016461 0.008 0.00668 R 1 76183 439817 1963950 R 2 70517 406550 1900517 R 3 73783 391717 1920383 Mean 73494 412694 1928283 0.030 0.0221 R 1 84317 434850 2091717 R 2 85883 420317 2056483 R 3 88117 465183 2062117 Mean 86106 440117 2070106 0.080 0.0589 R 1 83883 467950 2143117 R 2 81683 432750 2135583 R 3 78650 422450 2048050 Mean 81406 441050 2108917 0.260 0.212 R 1 80383 440850 1958750 R 2 83983 44 3283 2170383 R 3 86750 440917 2138217 Mean 83706 441683 2089117 0.820 0.719 R 1 75583 412183 2185317 R 2 69483 397283 1967817 R 3 72150 443550 2199850 Mean 72406 417672 2117661 2.64 2.39 R 1 74250 384383 2033417 R 2 69917 365583 1820217 R 3 61083 318217 1805517 Mean 68417 356061 1886383 8.44 7.56 R 1 83017 382850 2065417 R 2 76283 349883 2048250 R 3 86883 402383 1912550 Mean 82061 378372 2008739 27.0 28.9 R 1 64583 273717 1004317 R 2 64017 256883 933250 R 3 66917 243617 960317 Mean 65172 258072 965961
nd None detected (less than the limit of detection of 0.0004 mg/L) R 1
- R 6 Replicate number.Huntingdon Life Sciences: CEF0008

23 Table 3 Inhibition of growth

Parameter Exposure concentrations ( mg/L ) Sample

size Mean % Inhib ition p value Nominal Measured

Growth rate to 72 hours Control nd 6 0.0737 0.0
-

0.008 0.00668 3 0.0731 0.8 0.816 0.030 0.0221 3 0.0741
- 0.5 0.976 0.080 0.0589 3 0.0743
- 0.9 0.754 0.260 0.212 3 0.0742
- 0.7 0.910 0.820 0.719 3 0.074 4
- 0.9 0.697 2.64 2.39 3 0.0728 1.2 0.386 8.44 7.56 3 0.0736 0.0 1.000 27.0 28.9 3 0.0635 13.8 <0.001***

0
- 24 hours Control nd 6 0.0858 0.0
-

0.008 0.00668 3 0.0831 3.2 0.706 0.030 0.0221 3 0.0897
- 4.5 0.325 0.080 0.0589 3 0.0874
- 1.8 0.976 0.260 0.212 3 0.0885
- 3.1 0.711 0.820 0.719 3 0.0825 3.9 0.490 2.64 2.39 3 0.0800 6.8 0.050 8.44 7.56 3 0.0876
- 2.1 0.941 27.0 28.9 3 0.0781 9.0 0.006**

24
- 48 hours Control nd 6 0.0681 0.0
-

0.008 0.00668 3 0.0719
- 5.5 0.110 0.030 0.0221 3 0.0 679 0.3 1.000 0.080 0.0589 3 0.0704
- 3.3 0.590 0.260 0.212 3 0.0693
- 1.8 0.969 0.820 0.719 3 0.0730
- 7.1 0.021* 2.64 2.39 3 0.0687
- 0.9 1.000 8.44 7.56 3 0.0637 6.5 0.039* 27.0 28.9 3 0.0573 15.9 <0.001***

48
- 72 hours Control nd 6 0.0671 0.0

0.008 0.00668 3 0.0643 4.2 0.601 0.030 0.0221 3 0.0645 3.8 0.701 0.080 0.0589 3 0.0652 2.8 0.911 0.260 0.212 3 0.0647 3.6 0.757 0.820 0.719 3 0.0676
- 0.8 1.000 2.64 2.39 3 0.0695
- 3.7 0.735 8.44 7.56 3 0.0696
- 3.7 0.712 27.0 28.9 3 0.0550 1 8.0 <0.001***

Yield Control nd 6 2006461 0.0
-

0.008 0.00668 3 1918283 4.4 0.794 0.030 0.0221 3 206010 6
- 2.7 0.981 0.080 0.0589 3 209891 7
- 4.6 0.756 0.260 0.212 3 207911 7
- 3.6 0.907 0.820 0.719 3 2107661
- 5.0 0.674 2.64 2.39 3 187638 4 6.5 0.40 4 8.44 7.56 3 1998739 0.4 1.000 27.0 28.9 3 955961 52.4 <0.001*** nd None detected (less than the limit of detection of 0.0004 mg/L) p values are for the comparison with Control using Dunnett's test * p <0.05, ** p <0.01, *** p <0.001 Note: Negative values indicate an increase in algal growth above that of Control growth.Huntingdon Life Sciences: CEF0008

24 Table 4 Environmental parameters a) Temperature and pH

Exposure concentrations

( mg/L ) Temperature  C pH Nominal Measured 0 h 72 h 0 h 72 h Control nd 22.3 22.8 7.74 9.32 0.008 0.00668 22.5 23.0 7.84 9.13 0.030 0.0221 22.5 22.7 7.86 9.49 0.080 0.0589 22.5 23.1 7.87 9.54 0.260 0.212 22.4 22.8 7.88 9.54 0.820 0.719 22.3 23.1 7.90 9.28 2.64 2.39 22.3 23.4 7.89 8.92 8.44 7.56 22.3 22.8 7.84 8.55 27.0 28.9 22.3 23.0 7.70 8.07
nd None detected (less than the limit of detection of 0.0004 mg/L)

b) Light intensity

Incubator positions Light intensity (lux)

Day 0 Day 1 Day 2 Day 3 Top, left 5960 6210 6190 6160 Bottom, left 5840 5790 5930 5900 Centre 7010 7040 6990 6860 Top, right 5850 6000 6000 5870 Bottom, right 5910 5990 5900 5890 M ean 6114 6206 6202 6136 R ange %

- 4.3/+ 14.7

- 6.7 /+ 13.4

- 4.9

/+ 12.7

- 4.3/+ 11.8Huntingdon Life Sciences: CEF0008

25 Appendix 1 Certifi cate of analysis Aw aited from Sponsor, to be included prior to finalisationHuntingdon Life Sciences: CEF0008

26 Appendix 2 A lgal n utrient m edium ( OECD ) Four stock solut io ns were prepared according to the fo llo wing table, using filtered, dechlorinated tap water which had been so ft ened and treated by reverse osmosi s, before microfiltrat io n and purificat io n (resistivit y of 18 Megohm/cm). Stock solut io ns were sterilised by autoclaving (solut io ns 1
- 3) or by membrane filtration (so lut io n 4) before being stored at 4  C in the dark. Aliquots of stock solut io ns 1
- 3 were further diluted with the same diluent and autoclaved again to produce the working strength nutrient medium. Stock solut io n 4 was added to the medium on the day

of use. The pH of the medium after equilibrat ion wi th ai r is approximately

8.

Nutrient Concentration in stock solution (g/L) Volume of stock

sol ution per litre of final medium (mL) Final

concentration in test solution (mg/L) Stock solution 1: macro - nutrients NH 4 Cl MgCl 2 .6H2 O CaCl 2 .2H2 O MgSO 4 .7H2 O KH2 PO 4 1.5 1.2 1.8 1.5 0.16 10 15 12 18 15 1.6 Stock solution 2: Fe - EDTA FeCl 3 .6H2 O Na 2 EDTA.2H2 O 0.064 0.1 1 0.064 0.1 Stock solution 3: trace elements H 3 BO 3 MnCl 2 .4H2 O ZnCl 2 CoCl 2 .6H2 O CuCl 2 .2H2 O Na 2 MoO 4 .2H2 O 0.185 0.415 3 .0 x 10 - 3 1.5 x 10 - 3 1.0 x 1 0 - 5 7 .0 x 10 - 3 1 0.185 0.415 3 .0 x 10 - 3 1.5 x 10 - 3 1.0 x 10 - 5 7 .0 x 10 - 3 Stock solution 4: NaHCO 3 NaHCO 3 50 1 50Huntingdon Life Sciences: CEF0008

27 Appendix 3 The Determination of in OECD medium 

## 1. Method 1.1 General principl e Test sol ut io ns were diluted with water:acetonitrile:formic acid (95:5:0.1 v:v:v) prior to quant it ation by liquid chromatography wi th tandem m ass spectrometric detection system (LC
- MS/MS). Materials Grade Ac etonitrile HPLC Ammonium formate AR Formic acid AR Methanol HPLC Water HPLC 1.1.1 Test article fortifying and calibration solutions Some standard sol

utions used in this study

have been shared wit h other GLP studies being performed f

or the same sponsor. This is considered to have no impact o n the integrit y or validit y of

this study
. A weighed amount of was di ssolved in acetonitrile to produce a 1 mg/mL stock standard solut io n. The stock standard was diluted progressively wit h acetoni trile to give fo rtificat io n standard solut io ns. Th e appropriate fortificat io n standard so lut io n was further diluted with water:acetoni trile:formic acid (95:5:0.1 v:v:v) to produce a series of calibrat io n sol ut io ns in the range 0.1 to 5 ng/mL. 1.1.2 Ty pical preparation of reagents Preparation of water:acet oni tri le:formic acid (95:5:0.1 v:v:v) : Acetonitrile (5 0 mL) and formic acid (1 mL) w ere thoroughly mixed with water ( 95 0 mL) prior to use. Preparation of

water:methano l: fo rmic acid (90:10:0.1 v:v:v) + 0.01M ammo nium formate: Water (900 mL) was thoroughly m ixed wit h met hano l (100 mL), formic acid (1 mL) and ammo nium formate (0.63 g) prior to use. Preparation of

0.1% formic acid in methano l: Methano l (1000 mL) was thoroughly mixed with formic acid (1 mL) prior to use. NOTE

– to prepare different volumes of th e above reagents, the ratio of the individual com ponents was maintained.Huntingdon Life Sciences: CEF0008

28 

### 1.2 Procedure An aliquot (10 mL) of the sample was taken and fortified if required. Water:acetonitrile:formic acid (95:5:0.1 v:v:v) (10 mL) was added and mixed well . The sample was diluted by

a factor of 2 wi

th water:acetonitrile:formic acid (95:5:0.1 v:v:v) . Any further dilut io ns, where required, were performed with water:acetoni trile:formic acid (95:5:0.1 v:v:v) prior to quanti tation by L C
- MS/MS. 1.2.1 Instrumentation LC - MS/MS co nditions Instrument: AB Sciex API 4000 Data system: Analyst v.1.4.2 Ioni sat io n mo de: Ionspray posi tive Ion moni toring details: MRM m/z 186>57 Col umn: Pheno me nex aqua C 18 (5 cm  2 mm) Mobile phase A: Water:methano l: fo rmic acid (90:10:0.1 v:v:v) + 0.01M ammonium

f orm ate Mobile phase B: Methano l + 0.1% formic acid (by v ol ume) Pum p programme: 60:40 A:B isocratic Cycle time: 8 minutes Flow rate: 0.3 m L/min Inject io n vo lu me: 20 μL Retention t ime: Approximately 1.3 minutes LOQ: 0.001 m g/L LOD: 0.1 ng/m L (equivale ntto 0.0004 mg/L in aqueous media)Huntingdon Life Sciences: CEF0008

29 1.2.2 Calculation of results Test samples were quant ified using the fo llo wing equation: Residue found (mg/L) = 1000 1 x D x M 1 x x Where x (residue concentration in final so lut io n) was calculated using the linear re gression:
y = m x + c where x (concentration in ng/mL) = y  c m c = int ercept m = slope y = peak area of sample M = matri

x concentration (mL/mL) D = dilut io n factorHuntingdon Life Sciences: CEF0008

30 

## 2.0 Results and Discussion 2.1 Calibration The chro mat ographic response t o was shown to be linear over the range of concentrations 0.1 to 5 ng/mL. Ty pical calibrat io n data is presented in Table 1 and Figure 1. Ty pical chromatograms of calibrat io n standards are presented in Figures 2 and 3. 

### 2.2 Validation The methodolo gy

f or the determinat io n of residues in OECD medium was validated at 0.001 m

g/L and 3 0 m

g/L. The validat ion data are presented in Table 2 and summarised in the fo llo wing table:

Fortification level (mg/L) Mean recovery (%) CV (%) 0.001 99 6.6 3 0 100 7.0 ( 99 ) ( 6.4 ) ( ) O verall values 

### 2.3 Limit of quantitation (LOQ) The limit of quant it ation is defined as the lowest fortificat io n level at which acceptable recovery data are obtained. The validation of the methodology f or the determination of residues of in OECD medium, demonstrated that i t can be accurately determined at a limit of quantitation (LOQ) of 0.001 m g/L. 

### 2.4 Limit of detection (LOD) The limit of detection of the method is defined as the value of the lowest calibrat io n standard chro mat ographed that gave a peak height to baseline no ise ratio  3. For this study the limit of detection was 0.1 ng/m

L (equivalent to 0. 0004 mg/L in OECD medium ). 

### 2.5 Samples Sam ples o f OECD medium fro m the test were analysed in suitably siz ed batches alo ng with control samples fortified wit h which acted as procedural recovery samples. Procedural

recoveri es performed on control OECD medium were in the range 70 to 110% confirming the validit y of the methodology

on each day

of analysis . Ty

pical chromatograms of untreated, untreated fortified wit h and treated sam ples are presented in Figures 4 to 7.Huntingdon Life Sciences: CEF0008

31 Table 1 Ty pical standard calibration for by LC - MS/MS

Standard concentration (ng/mL ) Peak area

0 0 0.1 1.24419e+004 0. 2 2.26717e+004 0.4 3.43865e+004 0.8 6.81640e+004 1 9.29424e+004 2 1.88240e+005 3 2.47370e+005 4 3.34724e+005 5 4.27935e+005 Calibration curve: y

= 84070.6 x +4170.97 (batch 4 ) r = 0.99 89 Where x = concentration y = peak area r = regression coefficientHuntingdon Life Sciences: CEF0008

32 Table 2 Validation of in OECD medium by LC - MS/MS
Fortification level (mg/L) Replicate Analyte Detected (mg/L) Recovery

% Mean

% Standard

Deviation % Relative

Standard Deviation % Control A ND
- Control B ND
- 0.001 A 0.00102 102 0.001 B 0.000991 99 0.001 C 0.00103 103 99 6.5 6.6 0.001 D 0.000875 88 0.001 E 0.00104 104 30 A 30.1 100 30 B 32.3 108 30 C 28.9 96 100 7.0 7.0 30 D 31.2 104 30 E 27.0 90 Overall mea

n (%): 99 Overall standard deviation (%): 6.4 Overall relative standard deviation (%): 6.4
ND

– Not detected (<LOD of 0.0004 mg/L) Control aquatic media sample used

– 13/00/A2759 Data system: Analyst v.1.4.2Huntingdon Life Sciences: CEF0008

33 Figure 1 Ty pical standard chem ical calibration for by

LC - MS/MS

cef0008_b4.rdb ( ): "Linear" Regression ("No" weighting): y = 84070.6 x + 4170.97 (r = 0.9989044)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 Concentration, ng/mL 0.0 5.0e4 1.0e5 1.5e5 2.0e5 2.5e5 3.0e5 3.5e5 4.0e5 4.4e5Area, countsHuntingdon Life Sciences: CEF0008

34 Figure 2 Ty pical chromatogram of a 0.1 ng/mL calibration

standard

Sample Name: "Cal 0.1 ng/mL" Sample ID: "" File: "CEF0008_B417.wiff" Peak Name: " " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Standard Concentration: 0.100 ng/mL Calculated Conc: 0.0984 ng/mL Acq. Date: 30/07/2013 Acq. Time: 17:48:47 Modified: No Proc. Algorithm: IntelliQuan - IQA Min. Peak Height:1000.00 cps Min. Peak Width: 0.00 sec Smoothing Width: 9 points RT Window: 6.00 sec Expected RT: 1.30 min Use Relative RT: No Int. Type: Valley Retention Time: 1.32 min Area: 1.24419e+004 counts Height: 2.55e+003 cps Start Time: 1.21 min End Time: 1.44 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 5000.0 1.0e4 1.5e4 2.0e4 2.5e4 3.0e4 3.5e4 4.0e4 4.5e4 5.0e4 5.5e4 6.0e4 6.5e4 7.0e4 7.5e4 8.0e4Intensity, cps

1.32

Figure 3 Ty pical chromatogram of a 5 ng/mL calibration

standard

Sample Name: "Cal 5 ng/mL" Sample ID: "" File: "CEF0008_B423.wiff" Peak Name: " " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Standard Concentration: 5.00 ng/mL Calculated Conc: 5.04 ng/mL Acq. Date: 30/07/2013 Acq. Time: 18:40:48 Modified: No Proc. Algorithm: IntelliQuan - IQA Min. Peak Height:1000.00 cps Min. Peak Width: 0.00 sec Smoothing Width: 9 points RT Window: 6.00 sec Expected RT: 1.30 min Use Relative RT: No Int. Type: Valley Retention Time: 1.32 min Area: 4.27935e+005 counts Height: 7.95e+004 cps Start Time: 1.17 min End Time: 1.58 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 5000.0 1.0e4 1.5e4 2.0e4 2.5e4 3.0e4 3.5e4 4.0e4 4.5e4 5.0e4 5.5e4 6.0e4 6.5e4 7.0e4 7.5e4 8.0e4Intensity, cps 1.32

1.60 2.05 1.20 5.73 5.89 3.41 3.00 2.32 3.29 5.67 5.08 2.94 0.10 0.61 2.39 7.50 3.90 0.98 7.36 6.18 4.52 4.21 6.84

4.65 7.98 6.96Huntingdon Life Sciences: CEF0008

35 Figure 4 Chromatogram of an untreated OECD medium sample (an alytical

identification 13/00/A3041 Con)

Sample Name: "A3041 Con" Sample ID: "" File: "CEF0008_B422.wiff" Peak Name: " " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.00 ng/mL Acq. Date: 30/07/2013 Acq. Time: 18:32:08 Modified: No

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 5000.0 1.0e4 1.5e4 2.0e4 2.5e4 3.0e4 3.5e4 4.0e4 4.5e4 5.0e4 5.5e4 6.0e4 6.5e4 7.0e4 7.5e4 8.0e4Intensity, cps

1.30 1.41 2.09

0.42 1.00 0.71 2.60 5.14 2.03 3.12 3.34 3.04 3.40 3.69 0.15 4.25 4.01 4.76 1.85 2.21 5.69 4.44 7.98 6.19 4.85 7.35 7.59 7.11 6.73 4.72 5.60 6.53 5.83 6.89

6.36 5.42

Figure 5 Chromatogram of an untreated OECD medium sample fortified

with at 0.1 m g/L (analytical identification 13/00/A3041 F 0.1 )

Sample Name: "A3041 F0.1" Sample ID: "" File: "CEF0008_B424.wiff" Peak Name: " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Unknown Concentration: N/A Calculated Conc: 25.9 ng/mL Acq. Date: 30/07/2013 Acq. Time: 18:49:29 Modified: Yes Proc. Algorithm: IntelliQuan - IQA Min. Peak Height:1000.00 cps Min. Peak Width: 0.00 sec Smoothing Width: 9 points RT Window: 6.00 sec Expected RT: 1.30 min Use Relative RT: No Int. Type: Manual Retention Time: 1.32 min Area: 2.22127e+005 counts Height: 4.18e+004 cps Start Time: 1.14 min End Time: 1.61 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 5000.0 1.0e4 1.5e4 2.0e4 2.5e4 3.0e4 3.5e4 4.0e4 4.5e4 5.0e4 5.5e4 6.0e4 6.5e4 7.0e4 7.5e4 8.0e4Intensity, cps 1.32Huntingdon Life Sciences: CEF0008

36 Figure 6 Chromatogram of a test sample at 0.82 m g/L of on

Day 0 (analytical identification 13/ CEF /A3053 )

Sample Name: "A3053" Sample ID: "" File: "CEF0008_B420.wiff" Peak Name: " " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Unknown Concentration: N/A Calculated Conc: 205. ng/mL Acq. Date: 30/07/2013 Acq. Time: 18:14:49 Modified: Yes Proc. Algorithm: IntelliQuan - IQA Min. Peak Height:1000.00 cps Min. Peak Width: 0.00 sec Smoothing Width: 9 points RT Window: 6.00 sec Expected RT: 1.30 min Use Relative RT: No Int. Type: Manual Retention Time: 1.32 min Area: 1.76155e+005 counts Height: 3.30e+004 cps Start Time: 1.13 min End Time: 1.78 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 5000.0 1.0e4 1.5e4 2.0e4 2.5e4 3.0e4 3.5e4 4.0e4 4.5e4 5.0e4 5.5e4 6.0e4 6.5e4 7.0e4 7.5e4 8.0e4Intensity, cps

1.32

Figure 7 Chromatogram of a test sample at 0.82 m g/L of on

Day 3 (analytical identification 13/ CEF /A3087 )

Sample Name: "A3087" Sample ID: "" File: "CEF0008_B521.wiff" Peak Name: " " Mass(es): "186.0/57.0 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Unknown Concentration: N/A Calculated Conc: 158. ng/mL Acq. Date: 02/08/2013 Acq. Time: 18:49:34 Modified: No Proc. Algorithm: IntelliQuan - IQA Min. Peak Height:1100.00 cps Min. Peak Width: 0.00 sec Smoothing Width: 9 points RT Window: 6.00 sec Expected RT: 1.34 min Use Relative RT: No Int. Type: Valley Retention Time: 1.35 min Area: 1.30212e+005 counts Height: 2.30e+004 cps Start Time: 1.15 min End Time: 1.61 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time, min 0.0 5000.0 1.0e4 1.5e4 2.0e4 2.5e4 3.0e4 3.5e4 4.0e4 4.5e4 5.0e4 5.5e4 6.0e4 6.5e4 7.0e4Intensity, cps

1.34Huntingdon Life Sciences: CEF0008

37 Appendix 4 C ompliance statement