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Working for a better future

REPORT

: Algal Growth Inhibition Assay

HLS study number: CEF0008

Version ID: Audited Draft 1 Report

Issue date: 30 September 2013
Huntingdon Life Sciences: CEF0008

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Sponsor and Test Facility Details

Sponsor

Test facility Huntingdon Life Sciences

Eye Research Centre

Eye

Suffolk

IP23 7PX

UK
Huntingdon Life Sciences: CEF0008

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Table of Contents

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. Results........................................................................................................................ 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.......................................................................................20
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List of Tables

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 OECD medium ......................................27

Appendix 4 Compliance statement .................................................................................37
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Compliance with Good Laboratory Practice

: Algal Growth Inhibition Assay

The study described in this report was conducted in compliance with the following Good 

Laboratory Practice standards and I consider the data generated to be valid.

The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 No. 

3106, as amended by Statutory Instrument 2004 No. 994).

OECD Principles of Good Laboratory Practice (as revised in 1997), 

ENV/MC/CHEM (98) 17.

EC Commission Directive 2004/10/EC of 11 February 2004 (Official Journal No 

L 50/44).

These principles of Good Laboratory Practice are accepted by the Regulatory Authorities of

the United States of America and Japan on the basis of Intergovernmental Agreements.

___________________________________________ ________________________

Romalie Allen BSc Date

Study Director

Huntingdon Life Sciences
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Quality Assurance Statement

: Algal Growth Inhibition Assay

The following inspections and audits have been carried out in relation to this 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: At or about the time this study was in progress inspections of

procedures employed on this type of study were carried out. These were conducted and 

reported to appropriate Company Management as indicated below:

Process Based Inspections Date(s) of Inspection Date of Reporting to 

Management

Environmental Measurements & 

Monitoring/Ammonia Analysis

07 Mar 2013 07 Mar 2013

Counting of Cells 

(Haemocytometer/Coulter 

Counter/Spectrophotometer)

09 Apr 2013 09 Apr 2013

Sampling of Test Media for 

Chemical Analysis

09 Jul 2013 09 Jul 2013

Dose Formulation 09 Jul 2013 09 Jul 2013

Study Management & 

Conduct

09 Jul 2013 09 Jul 2013

Experimental Set-Up 11 Jul 2013 11 Jul 2013

Sample Preparation (To 

Include Sample 

Management & Tracking)

27 Feb 2013 01 Mar 2013

Standard Preparation & 

Fortification Procedures

05 Aug 2013 – 06 Aug 2013 07 Aug 2013

Result Processing 30 Aug 2013 30 Aug 2013

Chromatography 30 Aug 2013 30 Aug 2013

In addition, an inspection of the facility where this study was conducted was carried out on an 

annual basis. These inspections were promptly reported to Company Management.

___________________________________________ ________________________

Tom Pullinger MRQA Date

Group Manager

Department of Quality Assurance

Huntingdon Life Sciences
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Contributing Scientists

: Algal Growth Inhibition Assay

Study management 

Aquatic Ecotoxicology and Biodegradation

Romalie Allen BSc

Study Director

Expert department

Environmental Analysis

Simon Tate Higher National Certificate (Applied Chemistry)

Senior Study Manager
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Summary

The effect of on the growth of the unicellular green alga Pseudokirchneriella 

subcapitata was assessed under non-axenic conditions. 

The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, 

Annex IV to Commission Regulation (EC) No 761/2009 Part C, Method 3 “Freshwater Algae

and Cyanobacteria, Growth Inhibition Test” and Procedure 201 of the “Guidelines for 

Testing of Chemicals” of the Organisation for Economic Co-operation and Development: 

Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (2006, corrected 2011).

Triplicate algal cultures, with an initial cell density of ca. 1 x 104/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 OECD 

medium from an aqueous stock solution, which was made by the direct addition of the test 

substance to the dilution medium. The cultures were incubated in an orbital incubator under 

continuous illumination at temperatures ranging from 21.4 to 23.0C for 72 hours. 

At the start of the test, the measured levels of in samples of the test cultures 

ranged between 88 and 113% of their nominal values. After 72 hours, the measured levels 

ranged between 60 and 102% of their nominal values (between 59 and 90% of their starting 

values). The greatest losses were observed at the lower test concentrations. Based on the

geometric mean, 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 were used in the calculation 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 following values were derived from the data:

Parameter Endpoint

72 hours (mg/L)

Growth rate

EC10 25.8 [25.5 & 28.3]

EC50 > 28.9 [14% inhibition]

NOEC 7.56

LOEC 28.9

Yield

EC10 9.69 [7.91 & 11.1$]

EC50 28.2 [25.8 & 31.2$]

NOEC 7.56

LOEC 28.9

[ ] 95% confidence limits

$ Confidence interval may be unreliable due to goodness of curve fit

NOEC No observed effect concentration

LOEC Lowest observed effect concentration
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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 with EC Methods for Determination of Ecotoxicity, 

Annex IV to Commission Regulation (EC) No 761/2009 Part C, Method 3 “Freshwater Algae 

and Cyanobacteria, Growth Inhibition Test” and Procedure 201 of the “Guidelines for 

Testing of Chemicals” of the Organisation for Economic Co-operation and Development: 

Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (2006, corrected 2011).

The protocol was approved by Huntingdon Life Sciences Management and by the Study

Director on 17 June 2013.

The experimental start and completion 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, Suffolk, 

IP23 7PX.

A separate physiochemical property study (CEF0001) indicated that the limit of solubility of

the test substance was 27.5 mg/L at 20oC.
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2. Test Substance

Name

Chemical name

CAS No.

Physical state White crystalline powder

Batch number 12342

Purity To be provided

Storage Room temperature in the dark

Expiry Date To be provided

Date received: 4 March 2013

Certificate of analysis: Appendix 1
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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 cultures (100 mL) were incubated for approximately three days in an 

orbital incubator under continuous illumination at nominal temperatures in the range 21 to 

25C. Subsequently, appropriate volumes of these primary cultures were aseptically

transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these 

cultures were incubated, as stated above, for a further three days to provide an inoculum in 

the log phase of growth, characterised by a cell density of 8.3 x 105cells/mL. 

3.2 Culture medium

Sterile algal nutrient 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 

with the dropwise addition 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 volume and stirred for ca. one hour until no particulate matter remained. The 

formulation was then filtered (0.45 μm pore size) to provide an aqueous solution with a 

nominal concentration of 27.0 mg/L that was used directly for the highest test concentration 

and subsequent dilution to nominally 8.44 mg/L.

Aliquots (313, 313, 310, 317, 308, 375 and 267 mL) were serially diluted in volumetric flasks 

(1 L) containing OECD medium to provide test media with nominal concentrations of 8.44, 

2.64, 0.82, 0.26, 0.08, 0.03 and 0.008 mg/L. 

An aliquot (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 initial cell density of ca. 1 x 104cells/mL. An 

aliquot of the appropriate inoculated test medium was added to each of the test vessels.
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3.4 Exposure conditions

3.4.1 Experimental design 

A range finding test was followed by a definitive test with eight test concentrations, plus an 

algal nutrient medium control group.

Six flasks were established and incubated for the control group and three flasks for each test 

group, plus an additional 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 quality measurements at the start.

Before the start of the test, the required number of empty test vessels (250 mL conical flasks), 

were loosely stoppered with foam bungs, covered with aluminium foil that was secured by 

autoclave tape and sterilised by autoclaving (121C for at least 15 minutes). After the 

addition of the inoculated test medium, each flask was then loosely plugged with a foam 

bung.

The control cultures were prepared as for the test meda except that no test substance was 

added and a larger volume (700 mL) of medium was made using 8.4 mL of inoculum. 

3.4.2 Test concentrations 

The range finding test employed nominal test concentrations of 0.1, 1.0, 10 and 25 mg/L. 

After 72 hours, algal growth was inhibited by 39% at 25 mg/L and 15% at 10 mg/L, no 

significant inhibition was observed at 1 or 0.1 mg/L. Based on this result, the definitive test 

concentrations employed were 0.008, 0.03, 0.08, 0.26, 0.82, 2.64, 8.44 and 27.0 mg/L as 

; with the highest concentration approximately the limit of solubility of the test 

substance.

3.4.3 Stability of test concentrations

The test concentrations of were measured using liquid chromatography with

tandem mass spectrometric detection (LC-MS/MS, see Appendix 3). At the start of the 

definitive test, two samples (10 mL) were taken from the freshly-prepared control and test 

media and each 10 mL sample was added to a vial containing 10 mL of 

water:acetonitrile:formic acid (95:5:0.1). After 72 hours, the contents of the replicate flasks 

for each group were pooled and further samples taken for analysis. Additional samples were 

also taken from a flask containing at 0.008 and 27.0 mg/L but with no algal cells, 

in order to obtain information on the extent of adsorption/absorption of the test substance by 

the algal cells.

On each occasion, 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 of medium for 72 hours under continuous illumination (nominally 
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4440 to 8880 lux.) provided by 6 x 30 W “cool white” 1 metre fluorescent tubes. The 

temperature was maintained at 23 ± 2°C. 

Temperature and pH of control and test flasks at the start and end of the test were recorded. 

Gaseous exchange and suspension of the algal cells were ensured by the action of the orbital 

shaker, oscillating at a nominal 130 cycles per minute. The minimum and maximum 

temperature and light intensity (four corner positions and in a central position of the random 

block design) within the test area were determined each day. To minimise the impact of 

differences in light intensity across the test area on algal growth, control and test flasks were 

re-positioned in the test area each day during the definitive test. 

3.5 Measurement of growth

Samples were taken from control and test flasks at 24, 48 and 72 hours and the cell densities 

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 dilution medium. The presence 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 Institute 

2002), using mean measured concentrations.

The area under the curve was divided by initial count and total time to give AUCP (Area 

Under the Curve expressed as a Proportion of the initial cell count), where a value of 1 

represents growth and a value of 0 represents complete toxicity (all algae inhibited). In order 

to estimate the concentration at which 50% inhibition of growth occurred (EC50), sigmoidal 

curves were fitted to AUCP and growth rate. For both variables, 0% inhibition was defined 

as the control mean and 100% inhibition 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

Con Min

Con ep

Con Min

Min

Con Control

EC

concentration Otherwise ep s

Con Min

Con ep

Con Min

Min

Con Control

50 100

50

1

Growth rate

50

 where exp log

50 1 100

50 1

1

AUCP

Con = an estimate of the control mean

Min = an estimate of the minimum of the curve

s = slope estimate
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Yield was calculated for each test vessel as the final cell density (after 72 hours) minus the 

presumed initial cell density of 1 x 104cells/mL. A mean yield value for each test 

concentration was calculated and the percentage inhibition was determined using the formula 

below:

% Inhibition = ((YC - YT) / YC) x 100

Where YC = mean value for yield in the control group

YT = value for yield in treatment replicate

All 95% confidence intervals for EC50 were calculated using the likelihood ratio method 

(Donaldson and Schnabel, 1985). However, no EC50 was calculable for growth rate as there 

was insignificant inhibition observed at all levels. The EC10 (with 95% confidence interval) 

was also estimated by reparameterising the 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-monotonic dose-response 

relationship, in which case Dunnett’s test (1955, 1964) was used.

3.7 Validity criteria

For the test to be valid, the following criteria must be met:

 Cell concentration in control cultures should have increased by a factor of at least 16 

within 72 hours. This corresponds to a specific growth rate of 0.92 day-1;

 The mean coefficient of variation for daily growth rates (Days 0-1, 1-2 and 2-3) in the 

control cultures must not exceed 35%;

 The coefficient of variation of the average specific growth rate in replicate control 

cultures must not exceed 7%.

3.8 Protocol deviations

There were no protocol deviations.
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4. Maintenance of Records

This report has been compiled from original data, which will be stored, together with a copy

of the final report, a copy of the protocol and amendments in Huntingdon Life Sciences’ 

Archives, Huntingdon Life Sciences Ltd, Eye, Suffolk, 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 requirements. Under no circumstances will any item be discarded 

without the Sponsor’s knowledge.

Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study for 

twenty years, and a copy of the final report in its archives indefinitely.
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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. 

At the start of the test, the measured levels of in samples of the test cultures 

ranged between 88 and 113% of their nominal values. After 72 hours, the measured levels 

ranged between 60 and 102% of their nominal values (between 59 and 90% of their starting 

values). The greatest losses were observed at the lower test concentrations. Based on the

geometric mean, 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 of 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 the lowest concentration (nominally 0.008mg/L) the 

concentration without the presence of algal cells was 0.00762 mg/L (95% of nominal) 

compared to 0.00583 mg/L (73% of nominal) in those flasks with algal cells indicating some 

effect of the algal cells on stability of the test material. 

5.2 Algal growth

Individual cell densities 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 inhibition by comparing the test group value with that of the 

control curve.

The test results have been expressed in terms of mean measured concentrations of 

. The following values were derived from the data:

Parameter Endpoint

72 hours (mg/L)

Growth rate

EC10 25.8 [25.5 & 28.3]

EC50 > 28.9 [14% inhibition]

NOEC 7.56

LOEC 28.9

Yield

EC10 9.69 [7.91 & 11.1$]

EC50 28.2 [25.8 & 31.2$]

NOEC 7.56

LOEC 28.9

[ ] 95% confidence limits

$ Confidence interval may be unreliable due to goodness of curve fit

NOEC No observed effect concentration

LOEC Lowest observed effect concentration
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The mean coefficient of variation (CoV) for section by section daily growth rates in the

control cultures was 14.4 during the definitive 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 abnormalities of the cells were detected.

5.3 Environmental parameters

The measurements of water quality (temperature and pH) in control and test flasks are 

summarised in Table 4; temperature remained within acceptable limits throughout the study,

although the pH of the control cultures increased by more than 1.5 pH units during the 72-

hour exposure period. The increase in pH observed during the definitive test is not thought to 

have affected either the validity or integrity of the study because the validity criteria for this 

type of test were met by the control cultures.

The temperature of the incubator ranged between 21.4 and 23.0C.

At the start of the test, the test media at all concentrations were colourless.
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6. Conclusions

After 72 hours of exposure of the unicellular green alga Pseudokirchneriella subcapitata to 

, the ErC50 and EyC50, based on mean measured concentrations, were >28.9 mg/L 

(14% inhibition) and 28.2 mg/L, respectively.

The “no observed effect concentration” (NOEC) at 72 hours for growth rate and yield was 

7.56 mg/L, and the “lowest observed effect concentration” (LOEC) was 28.9 mg/L based on 

mean measured concentrations,
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7. References

DONALDSON, J.R. and SCHNABEL, R.B. (1985) Computational experience with 

confidence regions and confidence intervals for nonlinear least squares. National Bureau of 

Standards, Boulder, Colorado.

DUNNETT, C.W. (1955) A multiple comparison procedure for comparing several treatments 

with a control. Journal of the American Statistical Association, 50, 1096-1121.

DUNNETT, C.W. (1964) New tables for multiple comparisons with 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 

compared with 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.
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Figure 1 Inhibition of growth

a) Growth rate 0 – 72 hours

b) Yield 0 – 72 hours
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Table 1 Measured concentrations

Nominal

concentration

(mg/L)

Measured concentrations (mg/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

27.0 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 expressed 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.
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Table 2 Cell densities

Exposure concentrations

(mg/L) Replicate

number

Cell densities (cells/mL)

Nominal Measured 24 hours 48 hours 72 hours

Control nd

R1 66850 349283 1947550

R2 83883 392550 1921550

R3 82517 449850 2254050

R4 71417 386717 2021717

R5 85917 413750 1875883

R6 81850 429183 2078017

Mean 78739 403556 2016461

0.008 0.00668

R1 76183 439817 1963950

R2 70517 406550 1900517

R3 73783 391717 1920383

Mean 73494 412694 1928283

0.030 0.0221

R1 84317 434850 2091717

R2 85883 420317 2056483

R3 88117 465183 2062117

Mean 86106 440117 2070106

0.080 0.0589

R1 83883 467950 2143117

R2 81683 432750 2135583

R3 78650 422450 2048050

Mean 81406 441050 2108917

0.260 0.212

R1 80383 440850 1958750

R2 83983 443283 2170383

R3 86750 440917 2138217

Mean 83706 441683 2089117

0.820 0.719

R1 75583 412183 2185317

R2 69483 397283 1967817

R3 72150 443550 2199850

Mean 72406 417672 2117661

2.64 2.39

R1 74250 384383 2033417

R2 69917 365583 1820217

R3 61083 318217 1805517

Mean 68417 356061 1886383

8.44 7.56

R1 83017 382850 2065417

R2 76283 349883 2048250

R3 86883 402383 1912550

Mean 82061 378372 2008739

27.0 28.9

R1 64583 273717 1004317

R2 64017 256883 933250

R3 66917 243617 960317

Mean 65172 258072 965961

nd None detected (less than the limit of detection of 0.0004 mg/L)

R1-R6 Replicate number.
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Table 3 Inhibition of growth

Parameter

Exposure concentrations

(mg/L)

Sample 

size Mean % Inhibition 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 0.0744 -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 0.0679 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 18.0 <0.001***

Yield

Control nd 6 2006461 0.0 -

0.008 0.00668 3 1918283 4.4 0.794

0.030 0.0221 3 2060106 -2.7 0.981

0.080 0.0589 3 2098917 -4.6 0.756

0.260 0.212 3 2079117 -3.6 0.907

0.820 0.719 3 2107661 -5.0 0.674

2.64 2.39 3 1876384 6.5 0.404

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. 
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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

Mean 6114 6206 6202 6136

Range % -4.3/+14.7 -6.7/+13.4 -4.9/+12.7 -4.3/+11.8
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Appendix 1 Certificate of analysis

Awaited from Sponsor, to be included prior to finalisation
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Appendix 2 Algal nutrient medium (OECD)

Four stock solutions were prepared according to the following table, using filtered, 

dechlorinated tap water which had been softened and treated by reverse osmosis, before 

microfiltration and purification (resistivity of 18 Megohm/cm). Stock solutions were 

sterilised by autoclaving (solutions 1-3) or by membrane filtration (solution 4) before being 

stored at 4C in the dark.

Aliquots of stock solutions 1-3 were further diluted with the same diluent and autoclaved 

again to produce the working strength nutrient medium. Stock solution 4 was added to the 

medium on the day of use. The pH of the medium after equilibration with air is 

approximately 8.

Nutrient

Concentration

in stock solution

(g/L)

Volume of stock

solution per litre

of final medium

(mL)

Final

concentration in

test solution

(mg/L)

Stock solution 1: macro-nutrients

NH4Cl

MgCl2.6H2O

CaCl2.2H2O

MgSO4.7H2O

KH2PO4

1.5

1.2

1.8

1.5

0.16

10 15

12

18

15

1.6

Stock solution 2: Fe-EDTA

FeCl3.6H2O

Na2EDTA.2H2O

0.064

0.1

1 0.064

0.1

Stock solution 3: trace elements

H3BO3

MnCl2.4H2O

ZnCl2

CoCl2.6H2O

CuCl2.2H2O

Na2MoO4.2H2O

0.185

0.415

3.0 x 10-3

1.5 x 10-3

1.0 x 10

-5

7.0 x 10-3

1 0.185

0.415

3.0 x 10-3

1.5 x 10-3

1.0 x10-5

7.0 x 10-3

Stock solution 4: NaHCO3

NaHCO3 50 1 50
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Appendix 3 The Determination of in OECD medium

1. Method

1.1 General principle

Test solutions were diluted with water:acetonitrile:formic acid (95:5:0.1 v:v:v) prior to 

quantitation by liquid chromatography with tandem mass spectrometric detection system

(LC-MS/MS).

Materials Grade

Acetonitrile HPLC

Ammonium formate AR

Formic acid AR

Methanol HPLC

Water HPLC

1.1.1 Test article fortifying and calibration solutions

Some standard solutions used in this study have been shared with other GLP studies being 

performed for the same sponsor. This is considered to have no impact on the integrity or 

validity of this study.

A weighed amount of was dissolved in acetonitrile to produce a 1 mg/mL stock 

standard solution. The stock standard was diluted progressively with acetonitrile to give 

fortification standard solutions. The appropriate fortification standard solution was further 

diluted with water:acetonitrile:formic acid (95:5:0.1 v:v:v) to produce a series of calibration 

solutions in the range 0.1 to 5 ng/mL.

1.1.2 Typical preparation of reagents

Preparation of water:acetonitrile:formic acid (95:5:0.1 v:v:v):

Acetonitrile (50 mL) and formic acid (1 mL) were thoroughly mixed with water (950 mL) 

prior to use.

Preparation of water:methanol:formic acid (90:10:0.1 v:v:v) + 0.01M ammonium formate:

Water (900 mL) was thoroughly mixed with methanol (100 mL), formic acid (1 mL) and 

ammonium formate (0.63 g) prior to use.

Preparation of 0.1% formic acid in methanol:

Methanol (1000 mL) was thoroughly mixed with formic acid (1 mL) prior to use.

NOTE – to prepare different volumes of the above reagents, the ratio of the individual 

components was maintained.
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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 with water:acetonitrile:formic acid (95:5:0.1 v:v:v). Any 

further dilutions, where required, were performed with water:acetonitrile:formic acid 

(95:5:0.1 v:v:v) prior to quantitation by LC-MS/MS.

1.2.1 Instrumentation

LC-MS/MS conditions

Instrument: AB Sciex API 4000

Data system: Analyst v.1.4.2

Ionisation mode: Ionspray positive

Ion monitoring details: MRM m/z 186>57

Column: Phenomenex aqua C18 (5 cm  2 mm)

Mobile phase A: Water:methanol:formic acid (90:10:0.1 v:v:v) + 0.01M 

ammonium formate

Mobile phase B: Methanol + 0.1% formic acid (by volume)

Pump programme: 60:40 A:B isocratic

Cycle time: 8 minutes

Flow rate: 0.3 mL/min

Injection volume: 20 μL

Retention time: Approximately 1.3 minutes

LOQ: 0.001 mg/L

LOD: 0.1 ng/mL (equivalent to 0.0004 mg/L in aqueous 

media)
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1.2.2 Calculation of results

Test samples were quantified using the following equation:

Residue found (mg/L) = 

1000

1

 x D x 

M

1

x x 

Where x (residue concentration in final solution) was calculated using the linear regression:

y = m x + c where x (concentration in ng/mL) = 

y  c

m

c = intercept

m = slope

y = peak area of sample

M = matrix concentration (mL/mL)

D = dilution factor
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2.0 Results and Discussion

2.1 Calibration

The chromatographic response to was shown to be linear over the range of 

concentrations 0.1 to 5 ng/mL. Typical calibration data is presented in Table 1 and Figure 1. 

Typical chromatograms of calibration standards are presented in Figures 2 and 3.

2.2 Validation

The methodology for the determination of residues in OECD medium was 

validated at 0.001 mg/L and 30 mg/L. The validation data are presented in Table 2 and 

summarised in the following table:

Fortification level

(mg/L) Mean recovery (%) CV (%)

0.001 99 6.6

30 100 7.0

(99) (6.4)

( ) Overall values

2.3 Limit of quantitation (LOQ)

The limit of quantitation is defined as the lowest fortification level at which acceptable 

recovery data are obtained. The validation of the methodology for the determination of

residues of in OECD medium, demonstrated that it can be accurately determined 

at a limit of quantitation (LOQ) of 0.001 mg/L.

2.4 Limit of detection (LOD)

The limit of detection of the method is defined as the value of the lowest calibration standard 

chromatographed that gave a peak height to baseline noise ratio 3. For this study the limit 

of detection was 0.1 ng/mL (equivalent to 0.0004 mg/L in OECD medium).

2.5 Samples

Samples of OECD medium from the test were analysed in suitably sized batches along with 

control samples fortified with which acted as procedural recovery samples. 

Procedural recoveries performed on control OECD medium were in the range 70 to 110% 

confirming the validity of the methodology on each day of analysis. Typical chromatograms 

of untreated, untreated fortified with and treated samples are presented in 

Figures 4 to 7.
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Table 1 Typical 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 = 0.9989

Where x = concentration

y = peak area

r = regression coefficient
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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 mean (%): 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.1.4.2
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Figure 1 Typical standard chemical 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

4.4e5

Area, counts
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Figure 2 Typical 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

8.0e4

Intensity, cps

1.32

Figure 3 Typical 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

8.0e4

Intensity, cps

1.32

0.10 0.61 0.98 1.20 1.60 2.05 2.32 2.39 2.94 3.003.29 3.41 3.90 4.21 4.52 4.65 5.08 5.67 5.73 5.89 6.18 6.84 6.967.36 7.50 7.98
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Figure 4 Chromatogram of an untreated OECD medium sample (analytical 

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

8.0e4

Intensity, cps

1.30 0.15 0.42 0.71 1.00 1.41 1.85 2.03 2.09 2.21 2.60 3.04 3.12 3.34 3.40 3.69 4.01 4.25 4.444.724.76 4.855.145.42 5.60 5.69 5.83 6.19 6.366.53 6.736.89 7.11 7.35 7.59 7.98

Figure 5 Chromatogram of an untreated OECD medium sample fortified 

with at 0.1 mg/L (analytical identification 13/00/A3041

F0.1)

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

8.0e4

Intensity, cps

1.32
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Figure 6 Chromatogram of a test sample at 0.82 mg/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

8.0e4

Intensity, cps

1.32

Figure 7 Chromatogram of a test sample at 0.82 mg/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

7.0e4

Intensity, cps

1.34
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Appendix 4 Compliance statement