Endpoint study record: Toxicity to aquatic algae and 

cyanobacteria.1,3,5-Triazine-1,3,5(2H,4H,6H)-Triethanol 

(CAS No. 4719-04-4) SOCMA 41103115 

Administrative Data 

Data source 

Reference 

Materials and methods 

Test guideline 

Principles of method if other than guideline 

GLP compliance 

Test materials 

Test material identity 

UUID IUC5-a973cfea-cc82-4c03-ae54-1f2a683feb82 

Dossier UUID 0 

Author hvryenhoef / Harlan Laboratories Ltd. / Other / other: 

Date 2012-07-24 10:31:29 BST 

Remarks 

 

Purpose flag robust study summary 

Study result type experimental result Study period 27 February 2012 - 4 May 2012 

Reliability 1 (reliable without restriction) 

Rationale for reliability 

incl. deficiencies 

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test 

guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant 

results. 

Reference 

type 

Author Year Title Bibliographic 

source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Company 

study no. 

Report 

date 

study 

report 

Vryenhoef 

H Mullee 

DM 

2012 Algal 

Growth 

Inhibition 

Test 

 Harlan 

Laboratories 

Ltd Shardlow 

Business 

Park 

Shardlow 

Derbyshire 

DE72 2GD 

UK 

41103115 SOCMA Society 

of Chemical 

Manufacturers & 

Affiliates 1850 

M St NW Suite 

700 

Washington, DC 

20036-5810 

USA 

 2012-

06-28 

Qualifier Guideline Deviations 

according to EU Method C.3 (Algal Inhibition test) no 

according to OECD Guideline 201 (Alga, Growth Inhibition Test) no 

according to EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) no 

according to other guideline: no 

The test item was known to be volatile and hence testing was conducted in completely filled, stoppered test vessels in order to

minimise possible losses due to volatilisation. Following the recommendations of published data (Herman et al 1990) in order to

prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture

medium to provide a source of carbon dioxide for algal growth. 

yes (incl. certificate) 

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Details on test material 

Details on properties of test surrogate or analogue material 

Analytical monitoring 

Details on sampling 

Details on analytical methods 

Identifier Identity 

Common name 1,3,5-Triazine-1,3,5(2H,4H,6H)-Triethanol (CAS No. 4719-04-4) 

Sponsor's identification : 1,3,5-Triazine-1,3,5(2H,4H,6H)-Triethanol

(CAS No. 4719-04-4) 

Supplier : Q2 Technologies 

Description : yellow slightly viscous liquid 

Purity : 77.5% 

Batch number : 3951-91 

Expiry date : 18 October 2012 

Storage conditions : room temperature in the dark 

Not applicable 

yes 

- Concentrations: 

1.0, 3.2, 10, 32 and 100 mg active ingredient (ai)/l testing groups 

- Sampling method: 

Samples were taken from the control (replicates R1 – R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 96 

hours for quantitative analysis. Duplicate samples were taken at 0 and 96 hours and stored at approximately -20oC for further 

analysis if necessary. 

Given the volatile nature of the test item, an additional test replicate was prepared for each test concentration at 0 hours and 

incubated alongside the study remaining unopened. Samples of these additional replicates were taken for unopened vessel 

analysis at 96 hours. 

The test item contains 22.5% water, at the request of the Sponsor all test concentrations were corrected for the water content.

The test item was known to degrade immediately in water to form ethanolamine (0.84 mg/l ethanolamine ≡ to 1 mg ai/l) and 

formaldehyde (0.41 mg/l formaldehyde ≡ to 1 mg ai/l). Therefore analysis of the test preparations was preformed for the 2 

degradation products only. The ethanolamine concentration in the test samples was determined by high performance liquid 

chromatography coupled to mass spectrometry (HPLC-MS) using an external standard. The formaldehyde concentration in the 

test samples was determined by derivatisation with 2,4-dinitrophenylhydrazine phosphoric acid solution with high performance 

liquid chromatography (HPLC) using an external standard. The ethanolamine and the formaldehyde gave a chromatographic 

profile consisting of a single peak. 

A volume of test sample was diluted (if required) with test medium to give a theoretical concentration of 1 mg ai/l. To 100 ml of 

this was added 1 ml of 2,4-dinitrophenylhydrazine phosphoric acid solution and 50 μl of pentafluoropropionic acid. The test 

sample was then shaken to mix and allowed to stand for 15 minutes before analysis for both ethanolamine and formaldehyde. 

Standard solutions of ethanolamine (Sigma Aldrich, ACS reagent, >99.0%) were prepared in test medium at nominal 

concentrations of 0.10, 0.20, 0.50, 0.75, 1.0 and 1.5 mg ai/l. 

Standard solutions of formaldehyde (Sigma Aldrich, ACS reagent, 37% wt in water) were prepared in test medium at a nominal 

concentration of 1.0 mg ai/l. 

Procedure 

The ethanolamine standards and samples were analysed by HPLC-MS using the following conditions: 

HPLC System : Agilent Technologies 1200 MSD incorporating autosampler and workstation 

Mass selective detector 

Source : electrospray 

Fragmentation energy : 60 V 

Polarity : positive 

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Vehicle 

Details on test solutions 

Mode : single ion mode with 62.1 m/z 

Gas temperature : 350oC 

Drying gas : 10 litre/minute 

Nebuliser pressure : 40 psi 

Capillary voltage : 1000 V 

Gain : 1 

Column : Synergi Hydro RP, 4 μm, (75 x 4.6 mm i.d.) 

Column temperature : ambient 

Mobile phase : ultra-gradient water:methanol: 

pentafluoropropionic acid: (950:50:0.5 v/v/v) 

Flow rate : 0.75 ml/min 

Injection volume : 10 μl 

Retention time : approximately 1.4 minutes 

The formaldehyde standards and samples were analysed by HPLC using the following conditions: 

HPLC System : Agilent Technologies 1100 incorporating autosampler and workstation 

Column : Zorbax SB-C18, 3.5 μm, (75 x 4.6 mm i.d.) 

Mobile phase : ultra-gradient water:acetonitrile: formic acid: 

(400:600:1.0 v/v/v) 

Flow rate : 0.75 ml/min 

UV/Vis detector wavelength : 360 nm 

Injection volume : 50 μl 

Retention time : approximately 2.3 minutes 

no 

Rabge-Finding Test 

- Method: 

The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding 

test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 

10, 100 and 1000 mg ai/l for a period of 96 hours. All test concentrations were corrected for a test item water content of 22.5%. 

The test was conducted in 250 ml glass conical flasks each completely filled with test preparation and sealed with ground glass

stoppers to reduce losses due to volatilisation. Two replicate flasks were used for each control and test concentration. The test 

item was dissolved directly in culture medium. 

An amount of test item (1290 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 1000 mg ai/l 

stock solution from which a series of dilutions was made to give further stock solutions of 100, 10, 1.0 and 0.10 mg ai/l. An 

aliquot (900 ml) of each of the stock solutions was separately inoculated with algal suspension (20.4 ml) to give the required 

test concentrations of 0.10, 1.0, 10, 100 and 1000 mg ai/l. 

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and 

homogeneity. 

The control group was maintained under identical conditions but not exposed to the test item. 

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined 

using a Coulter® Multisizer Particle Counter. The flasks were then sealed with ground glass stoppers and incubated (INFORS 

Multitron Version 2 incubator) at 24 ± 1oC under continuous illumination (intensity approximately 7000 lux) provided by warm 

white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 96 hours. 

After 96 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter. 

A sample of each test concentration was taken for chemical analysis at 0 and 96 hours in order to determine the stability of the 

test item under test conditions. All samples were stored at approximately -20°C prior to analysis. 

Definitive Test 

Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 

32 and 100 mg ai/l. All test concentrations were corrected for a test item water content of 22.5%. 

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Test organisms 

For the purpose of the definitive test, the test item was dissolved directly in culture medium. 

Amounts of test item (258 and 82 mg) were each separately dissolved in culture medium and the volume adjusted to 2 litres to 

give 100 and 32 mg ai/l stock solutions respectively. A series of dilutions was made from these stock solutions to give further

stock solutions of 10, 3.2 and 1.0 mg ai/l. An aliquot (1800 ml) of each of the stock solutions was separately inoculated with 

algal suspension (37 ml) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg ai/l. 

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and 

homogeneity. 

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 96 hours.

As in the range-finding test 250 ml glass conical flasks were used. Six flasks each completely filled with test preparation were 

used for the control and three flasks each completely filled were used for each treatment group. 

The control group was maintained under identical conditions but not exposed to the test item. 

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 4.92E+05 cells per ml. 

Inoculation of 1800 ml of test medium with 37 ml of this algal suspension gave an initial nominal cell density of 1.00E+04 cells 

per ml and had no significant dilution effect on the final test concentration. 

The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1°C under 

continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly 

shaken at approximately 150 rpm for 96 hours. 

Samples were taken at 0, 24, 48, 72 and 96 hours and the cell densities determined using a Coulter® Multisizer Particle 

Counter. 

The pH of the control and each test concentration was determined at initiation of the test and after 48 and 96 hours exposure. 

The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded hourly using the 

incubators’ internal pt-100 temperature probe and Multicom Software Package. 

- Eluate: 

Not applicable 

- Controls: 

A positive control (Safepharm Laboratories Project Number: 0039/0994) used potassium dichromate as the reference material. 

An amount of reference material (100 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/l 

stock solution from which a series of dilutions was made to give further stock solutions of 10, 2.0, 1.0, 0.50, 0.25 and 0.125 

mg/l. An aliquot (250 ml) of each of the 0.125, 0.25, 0.50, 1.0 and 2.0 mg/l stock solutions was separately mixed with algal 

suspension (250 ml) to give the required test concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l. 

The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with 

polyurethane foam bungs to reduce evaporation. Six replicate flasks were prepared for the control and three replicate flasks 

prepared for each test concentration. 

The flasks were incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity 

approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 

72 hours. 

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter. 

- Chemical name of vehicle : 

Not applicable 

- Concentration of vehicle in test medium: 

Not applicable 

- Evidence of undissolved material : 

At both the start and end of the mixing period and following the 1-hour settlement period the WAF was observed to have formed 

a clear colourless media column with an oily layer of test material floating at the surface. 

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period 

all control, 10 and 20 mg/l test cultures were observed to be green dispersions, the 40 mg/l test cultures were observed to be 

pale green dispersions whilst the 80 and 160 mg/l test cultures were observed to be extremely pale green dispersions. 

EXAMPLE 

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Test organisms (species) 

Details on test organisms 

Study design 

Test type 

Water media type 

Limit test 

Pseudokirchnerella subcapitata 

TEST ORGANISM 

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella 

subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish 

Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of 

culture medium (Section 3.2). The master cultures were maintained in the laboratory under constant aeration and illumination at

21 ± 1°C. 

Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in 

conical flasks to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam 

stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1°C until the 

algal cell density was approximately 104 - 105 cells/ml. 

A positive control (Harlan Laboratories Ltd Project Number: 41200591) used zinc chloride as the reference item. The positive 

control was conducted between 13 February 2012 and 17 February 2012. 

ACCLIMATION 

- Acclimation period: 

Not recorded. 

- Culturing media and conditions: 

The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture

with the addition of 500 mg/l of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed system 

(Herman et al 1990). 

Culture medium: 

NaNO3 25.5 mg/l 

MgCl2.6H2O 12.164 mg/l 

CaCl2.2H2O 4.41 mg/l 

MgSO4.7H2O 14.7 mg/l 

K2HPO4 1.044 mg/l 

NaHCO3 15.0 mg/l 

H3BO3 0.1855 mg/l 

MnCl2.4H2O 0.415 mg/l 

ZnCl2 0.00327 mg/l 

FeCl3.6H2O 0.159 mg/l 

CoCl2.6H2O 0.00143 mg/l 

Na2MoO4.2H2O 0.00726 mg/l 

CuCl2.2H2O 0.000012 mg/l 

Na2EDTA.2H2O 0.30 mg/l 

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 

7.5 ± 0.1 with 0.1N NaOH or HCl. 

For the purposes of the range-finding and definitive test, additional sodium bicarbonate (500 mg/l) was added to the prepared 

culture medium prior to use. 

static 

freshwater 

no 

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Total exposure duration 

Post exposure observation period 

Test conditions 

Hardness 

Test temperature 

pH 

Dissolved oxygen 

Salinity 

Nominal and measured concentrations 

Details on test conditions 

96 h 

A re-growth test was performed after 96 hours to determine the algicidal or algistatic effect of the test item. Aliquots were 

removed from each replicate control and test culture (0.25 ml and 0.50 ml respectively) and the replicates pooled. Fresh sterile 

culture medium (100 ml) was added to each to ensure that the test concentration was reduced to below the inhibiting level. 

The flasks were plugged with polyurethane foam bungs are incubated (INFORS Multitron® Version 2 Incubator) at 24 ± 1°C 

under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 150 rpm for 168 hours.

Re-growth was observed to have occurred in the control, 1.0 and 3.2 mg ai/l test cultures after 48 hours and in the 10, 32 and 

100 mg ai/l test cultures after 168 hours. These results indicate that the test item was algistatic in effect. 

Not applicable 

Temperature was maintained at 24 ± 1oC throughout the test. The temperature within the incubator was recorded daily.

The pH value of the control cultures (see Table 6) was observed to increase from pH 7.7 at 0 hours to pH 8.8 at 48 hours and 

pH 10.2 at 96 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of 

algal cells in the log phase of growth (see Figure 1). Due to the need to test in completely filled and sealed test vessels there 

was minimal transfer of CO2 from the gaseous phase to the aqueous phase and hence CO2 required for photosynthesis and 

growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. Despite the addition 

of 500 mg/l of sodium bicarbonate to the culture medium the increase in pH after 96 hours was in excess of that recommended 

in the Test Guidelines (1.5 pH units after 96 hours). This was considered to have had no adverse effect on the results of the 

study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test

Guidelines. 

Not applicable 

Not applicable -freshwater used 

The test item was known to degrade rapidly in water forming ethanolamine and formaldehyde. It was therefore considered 

appropriate to analyse the test samples for the presence of both degradation products. 

Analysis of the test preparations at 0 hours showed measured concentrations of ethanolamine to range from 0.52 to 92 mg/l 

and concentrations of formaldehyde to range from 0.48 to 44 mg/l. A decline in measured concentrations was observed at 96 

hours in the range of 0.34 to 57 mg ethanolamine/l and 0.11 to 31 mg formaldehyde/l. 

Given the volatile nature of the test item additional test vessels were prepared at the start of the test and incubated alongside 

the study to provide samples for unopened analysis at 96 hours. Analysis of these additional samples showed measured 

concentrations of ethanolamine to range from 0.37 to 58 mg/l and concentrations of formaldehyde to range from 0.13 to 27 

mg/l. These results were similar to the results obtained from test samples which had been opened on a daily basis and hence it 

was considered that the decline in concentration observed over the test duration was due to instability rather than volatility.

Given that toxicity cannot be attributed to a single degradation product or mixture of degradation products it was considered 

appropriate to calculate the results based on nominal test concentrations only. 

250 ml glass conical flasks were used. Six flasks each completely filled with test preparation were used for the control and three 

flasks each completely filled were used for each treatment group. 

The control group was maintained under identical conditions but not exposed to the test item. 

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 4.9E+05 cells per ml. 

Inoculation of 1800 ml of test medium with 37 ml of this algal suspension gave an initial nominal cell density of 1.00E+04 cells 

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Reference substance (positive control) 

Any other information on materials and methods incl. tables 

Results and discussions 

Effect concentrations 

per ml and had no significant dilution effect on the final test concentration. 

The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1°C under 

continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly 

shaken at approximately 150 rpm for 96 hours. 

Samples were taken at 0, 24, 48, 72 and 96 hours and the cell densities determined using a Coulter® Multisizer Particle 

Counter. 

- Control end cells density: algal cell density was approximately 1.29E+06 cells/ml 

OTHER TEST CONDITIONS 

- Sterile test conditions: No 

- Adjustment of pH: 

No adjustments recorded 

- Photoperiod: 

Not applicable 

EFFECT PARAMETERS MEASURED : 

- Determination of cell concentrations: 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, 

using a Coulter® Multisizer Particle Counter. 

Determination of ECx values 

For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test 

concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package 

(IDBS). ECxvalues were then determined from the equation for the fitted line. 

Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating doseeffect experiments of Litchfield and Wilcoxon (1949). 

VALIDATION: 

The results of the test are considered valid if the following performance criteria are met: 

The cell concentration of the control cultures must increase by a factor of at least 16 after 72 hours (OECD/EC Guidelines) and

by a factor of at least 100 after 96 hours (EPA/OPPTS Guidelines). 

The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course 

of the test (0-24, 24-48 and 48-72 hours) must not exceed 35% (OECD Guidelines). 

The coefficient of variation of the average specific growth rate in replicate control cultures after 72 hours must not exceed 7% 

(OECD Guidelines). 

The coefficient of variation of the cell density values in replicate control cultures must not exceed 20% after 72 and 96 hours

(OPPTS Guidelines). 

yes (Zinc chloride) 

See Appendix 1 for data evaluation.

Duration Endpoint Effect 

conc. 

Nominal/Measured Conc. 

based 

on 

Basis for 

effect 

Remarks (e.g. 95% CL) 

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Details on results 

72 h LOEC 3.2 nominal act. ingr. other: growth 

rate, yield and 

biomass 

integral 

 

72 h NOEC 1 mg/L nominal act. ingr. other: growth 

rate, yield and 

biomass 

integral 

 

72 h EC50 5.2 

mg/L 

nominal act. ingr. growth rate It was not possible to calculate 95% 

confidence limits for the ErC50 value as the 

data generated did not fit the models 

available for the calculation of confidence 

limits. 

72 h EC50 3.5 

mg/L 

nominal act. ingr. cell number 

(Yield) 

95% confidence limits 3.2 - 3.8 mg ai/l 

72 h EC50 3.6 

mg/L 

nominal act. ingr. biomass 95% confidence limits 3.3- 3.9 mg ai/l 

96 h EC50 4.2 

mg/L 

nominal act. ingr. growth rate 95% confidence limits 3.9 - 4.6 mg ai/l 

96 h EC50 3.2 

mg/L 

nominal act. ingr. cell number 

(Yield) 

95% confidence limits 3.0 - 3.4 mg ai/l 

96 h EC50 3.3 

mg/L 

nominal act. ingr. biomass 95% confidence limits 3.1 - 3.6 mg ai/l 

96 h LOEC 3.2 

mg/L 

nominal act. ingr. other: growth 

rate, yiled and 

biomass 

 

96 h NOEC 1 mg/L nominal act. ingr. other: growth 

rate, yield and 

biomass 

integral 

 

- Exponential growth in the control (for algal test): yes 

Observations on cultures 

All test and control cultures were inspected microscopically at 96 hours. There were no abnormalities detected in any of the 

control or test cultures. 

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 96-Hour test period 

all control and 1.0 mg ai/l test cultures were observed to be bright green dispersions. The 3.2 mg ai/l test cultures were 

observed to be green dispersions, the 10 mg ai/l test cultures were observed to be extremely pale green dispersions whilst the 

32 and 100 mg ai/l test cultures were observed to be clear colourless solutions. 

Range-finding Test 

The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata the test item 

during the range-finding test are given in Table 1. 

Validation criteria 

The following data show that the cell concentration of the control cultures increased by a factor of 73 after 72 hours and by a

factor of 150 after 96 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by

a factor of 16 after 72 hours and the EPA/OPPTS Guidelines that states the enhancement must be at least by a factor of 100 

after 96 hours. 

Mean cell density of control at 0 hours : 8.64E+03 cells per ml 

Mean cell density of control at 72 hours : 6.34E+05 cells per ml 

Mean cell density of control at 96 hours : 1.29E+06 cells per ml 

The mean coefficient of variation for section by section specific growth rate for the control cultures over the test period (0-72 

hours) was 15% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not 

exceed 35%. 

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Results with reference substance (positive control) 

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 - 72 h) was 3% and 

hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%. 

The coefficient of variation of the cell density values in replicate control cultures was 14% after 72 hours and 7% after 96 hours 

and hence satisfied the validation criterion which states that this must not exceed 20%. 

Cell density values determined at each sampling time and pH values at 0 and 96 hours are given in Table 2. Daily specific 

growth rates for the control cultures are given in Table 3. Growth rate, yield and biomass integral values for the control and test 

cultures after 72 hours and percentage inhibition values are given in Table 4. Growth rate, yield and biomass integral values for 

the control and test cultures after 96 hours and percentage inhibition values are given in Table 5. 

The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage inhibition values are plotted

against test concentration in Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7. 

From the data given in Tables 2 and 4, it is clear that the growth rate (r), yield (y) and biomass integral (b) of 

Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 96-Hour exposure 

period. 

Accordingly the following results were determined from the data: 

nhibition of growth rate 

ErC10 (0 - 72 h) : 3.0 mg ai/l 

ErC20 (0 - 72 h) : 3.7 mg ai/l 

ErC50 (0 - 72 h) : 5.2 mg ai/l* 

ErC90 (0 - 72 h) : 8.9 mg ai/l 

ErC10 (0 - 96 h) : 3.0 mg ai/l 

ErC20 (0 - 96 h) : 3.4 mg ai/l 

ErC50 (0 - 96 h) : 4.2 mg ai/l; 95% confidence limits 3.9 – 4.6 mg ai/l 

ErC90 (0 - 96 h) : 5.9 mg ai/l 

where ErCx is the test concentration that reduced growth rate by x%. 

Statistical analysis of the growth rate data at 72 and 96 hours was carried out for the control and all test concentrations using 

one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's 

multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically 

significant differences between the control and 1.0 mg ai/l test concentration (P<0.05), however all other test concentrations 

were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 

1.0 mg ai/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 3.2 mg ai/l. 

A positive control (Harlan Laboratories Ltd Project No: 41200591) used zinc chloride as the reference item at concentrations of

0.010, 0.032, 0.10, 0.32 and 1.0 mg/l. 

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test. 

Accordingly the following results were determined from the data: 

ErC50 (0 - 72 h) : 0.31 mg/l; 95% confidence limits 0.28 - 0.35 mg/l 

EyC50 (0 - 72 h) : 0.26 mg/l* 

EbC50 (0 - 72 h) : 0.27 mg/l* 

ErC50 (0 - 96 h) : 0.35 mg/l; 95% confidence limits 0.30 - 0.40 mg/l 

EyC50 (0 - 96 h) : 0.27 mg/l* 

EbC50 (0 - 96 h) : 0.26 mg/l* 

*It was not possible to calculate 95% confidence limits for these EC50 values as the data generated did not fit the models 

available for the calculation of confidence limits. 

No Observed Effect Concentration (NOEC) based on growth rate : 0.10 mg/l 

No Observed Effect Concentration (NOEC) based on yield : 0.10 mg/l 

No Observed Effect Concentration (NOEC) based on biomass integral : 0.10 mg/l 

Lowest Observed Effect Concentration (LOEC) based on growth rate : 0.32 mg/l 

Lowest Observed Effect Concentration (LOEC) based on yield : 0.32 mg/l 

Lowest Observed Effect Concentration (LOEC) based on biomass integral : 0.32 mg/l 

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Reported statistics and error estimates 

Any other information on results incl. tables 

Overall remarks, attachments 

Overall remarks 

Attached background material 

Applicant's summary and conclusion 

Validity criteria fulfilled 

Conclusions 

The resutls of the positive control were within the normal ranges for this reference item. 

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's 

multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth 

rate, yield and biomass integral data after 72 and 96 hours for the control and all test concentrations to determine any 

statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS 

computer software package (SAS 1999 - 2001). 

See Appenidix 3 for Tables 

This study is classified as acceptable and satisfies the guideline requriemetns for this study end point.REFERENCES

Dunnett, C W (1955) A Multiple Comparison Procedure for Comparing Several Treatments With a Control. J Am Stat Assoc50, 

1096-1121. 

Hermanet al(1990)Impact of volatile aromatic hydrocarbons, alone and in combination, on growth of the freshwater 

algaSelenastrum capricornutum.Aquatic Toxicology18, 87 - 100 

Litchfield, J T and Wilcoxon, F (1949) A Simplified Method of Evaluating Dose-Effect Experiments. J Pharmacol Exp Ther96, 

99-113. 

SAS/STAT Proprietary Software Release 8.02 (1999 - 2001), SAS Institute Inc,,,. 

Sokal, R R and Rohlf, F J (1981) Biometry.: W H Freeman and Company. 

Xlfit, ID Business Solutions Ltd 

Attached document Remarks 

41103115 Appendix 1.docx / 21.91 KB (application/vnd.openxmlformatsofficedocument.wordprocessingml.document) Appendix 1 (Data Analysis) 

41103115 Appendix 2.docx / 31.26 KB (application/vnd.openxmlformatsofficedocument.wordprocessingml.document) Appendix 2 (Verification of Test 

Concentrations) 

41103115 Appendix 3.docx / 33.01 KB (application/vnd.openxmlformatsofficedocument.wordprocessingml.document) Appendix 3 (Tables 1 - 6) 

41103115 Appendix 4.docx / 95.7 KB (application/vnd.openxmlformatsofficedocument.wordprocessingml.document) Appendix 4 (Figures 1 - 7) 

yes 

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 96-Hour period and 

gave the following results based on nominal test concentrations: 

Growth Rate (0-72 h) 5.2 mg ai/l* 

Yield (0-72 h) 3.5 mg ai/l; 95% confidence limits 3.2 - 3.8 mg ai/l 

Biomass (0-72 h) 3.6 mg ai/l; 95% confidence limits 3.3 - 3.9 mg ai/l 

Growth Rate (0-96 h) 4.2 mg ai/l; 95% confidence limits 3.9 - 4.6 mg ai/l 

Yield (0-96 h) 3.2 mg ai/l; 95% confidence limits 3.0 - 3.4 mg ai/l 

Biomass (0-96 h) 3.3 mg ai/l; 95% confidence limits 3.1 - 3.6 mg ai/l 

*It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models 

available for the calculation of confidence limits. 

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Executive summary 

No Observed Effect Concentration (NEOC) = 1.0 mg ai/l 

Lowest Observed Effect Concentration (LOEC) = 3.2 mg ai/l 

A re-growth test was performed which showed the test item to be algistatic in effect. 

A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella 

subcapitata. The method followed was designed to be compatible with the OECD 

Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and 

Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission 

Regulation (EC) No 761/2008, USTitle 40 Part 797 Section 1050, US EPA Pesticide 

Assessment Guidelines, Sub-Division J, Section 122-2, the US EPA Draft Ecological 

Effects Test Guideline OPPTS 850.5400 and the ASTM E1218-04, Standard Guide 

for Conducting Static Toxicity Tests with Microalgae.

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to an aqueous solution of the test item 

at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg active ingredient (ai)/l (three replicate flasks per concentration) for 96 

hours, under constant illumination and shaking at a temperature of 24 ± 1°C. 

The test item was known to be volatile and hence testing was conducted in completely filled, stoppered test vessels in order to

minimise possible losses due to volatilisation. Following the recommendations of published data (Hermanet al1990) in order to 

prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture

medium to provide a source of carbon dioxide for algal growth. 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, 

using a Coulter®Multisizer Particle Counter.

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 96-Hour period and 

gave the following results based on nominal test concentrations: 

The test item was known to degrade rapidly in water forming ethanolamine and formaldehyde. It was therefore considered 

appropriate to analyse the test samples for the presence of both degradation products. 

Analysis of the test preparations at 0 hours showed measured concentrations of ethanolamine to range from 0.52 to 92 mg/l 

and concentrations of formaldehyde to range from 0.48 to 44 mg/l. A decline in measured concentrations was observed at 96 

hours in the range of 0.34 to 57 mg ethanolamine/l and 0.11 to 31 mg formaldehyde/l. 

Given the volatile nature of the test item additional test vessels were prepared at the start of the test and incubated alongside 

the study to provide samples for unopened analysis at 96 hours. Analysis of these additional samples showed measured 

concentrations of ethanolamine to range from 0.37 to 58 mg/l and concentrations of formaldehyde to range from 0.13 to 27 

mg/l. These results were similar to the results obtained from test samples which had been opened on a daily basis and hence it 

was considered that the decline in concentration observed over the test duration was due to instability rather than volatility.

Given that toxicity cannot be attributed to a single degradation product or mixture of degradation products it was considered 

appropriate to calculate the results based on nominal test concentrations only. 

Exposure ofPseudokirchneriella subcapitatato the test item gave the following results based on the nominal test concentrations: 

A re-growth test was performed which showed the test item to be algistatic in effect. 

TiPoint

(Hours)

Response 

Variable EC50(mg ai/l) 95% Confidence Limits (mg ai/l)

No Observed 

Effect 

Concentration 

(NOEC)

(mg ai/l)

Lowest Observed 

Effect 

Concentration 

(LOEC)

(mg ai/l)

72

Growth Rate 5.2 * 1.0 3.2

Yield 3.5 3.2 - 3.8 1.0 3.2

Biomass 3.6 3.3 - 3.9 1.0 3.2

96

Growth Rate 4.2 3.9 - 4.6 1.0 3.2

Yield 3.2 3.0 - 3.4 1.0 3.2

Biomass 3.3 3.1 - 3.6 1.0 3.2

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*It was not possible to calculate 95% confidence limits for the Er

C50value as the data generated did not fit the models available 

for the calculation of confidence limits. 

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