# Endpoint study record: Toxic ity 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 protocol s, with no or minor deviations from standard test guidelines and/or minor methodologic al 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 (Al ga, Growth Inhibition Test) no

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

according to other guideline: no

The test item was known to be volatile and hence testing was c onducted 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 re striction 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) 

# Pa g e 1 of 12 End p oint stud y record: Toxicit y to a q uatic al g ae and c y anobacteria.1,3,5-Triazine-1,3,5 ( 2H,4H,...

24/07/2012 file://\\hl750-nas-1\UserData$\hvr y enhoef\index.html

### 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 s lightly 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 pool ed) and each test group (replicates R1 - R3 pooled) at 0 and 96 hours for quantitative analysis. D uplicate 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 te st concentration at 0 hours an d 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 Spon sor 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 ethanolami ne concentration in the test samples wa s determined by high performance liquid chromatography coupled to mass spectrometry (HPLC-MS) usin g an external standard. The form aldehyde concentration in the test samples was determined by derivatisation with 2,4-dinitr ophenylhydrazine phosphoric acid solution with high performance liquid chromatography (HPLC) using an ex ternal 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 me dium to give a theoretical concentration of 1 mg ai/l. To 100 ml of this was added 1 ml of 2,4-dinitrophenylh ydrazine 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 re agent, >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 analys ed by HPLC-MS using the following conditions:

HPLC System : Agilent Technologies 1200 MS D incorporating autosampler and workstation

Mass selective detector Source : electrospray Fragmentation energy : 60 V Polarity : positive 

# Pa g e 2 of 12 End p oint stud y record: Toxicit y to a q uatic al g ae and c y anobacteria.1,3,5-Triazine-1,3,5 ( 2H,4H,...

24/07/2012 file://\\hl750-nas-1\UserData$\hvr y enhoef\index.html

### 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 analys ed 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 r ange-finding test. The range-findin g test was conducted by exposing Pseudokirchnerie lla subcapitata cells to a series of nom inal test concentrations of 0.10, 1.0, 10, 100 and 1000 mg ai/l for a period of 96 hours. All test concent rations were corrected for a test item water content of 22.5 %.

The test was conducted in 250 ml glass c onical flasks each completely filled with test preparation and sealed with ground glass stoppers to reduce losses due to volatilisation. Two replicate fl asks were used for each control and test concentration. The te st item was dissolved directly in culture medium.

An amount of test item (1290 mg) was dissolved in culture medi um 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 gi ve further stock solutions of 100, 10, 1.0 and 0.10 mg ai/l. An aliquot (900 ml) of each of the stock solu tions was separately inoculat ed 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 prepa red concentrations were inverted seve ral 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 we re then sealed with ground glass stoppers and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1oC under continuous ill umination (intensity approximatel y 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly s haken at approximately 150 rpm for 96 hours.

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

A sample of each test concentration was taken for chemical anal ysis at 0 and 96 hours in order to determine the stability of th e test item under test conditions. All samples we re 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 co rrected for a test item water content of 22.5%. 

# Pa g e 3 of 12 End p oint stud y record: Toxicit y to a q uatic al g ae and c y anobacteria.1,3,5-Triazine-1,3,5 ( 2H,4H,...

24/07/2012 file://\\hl750-nas-1\UserData$\hvr y enhoef\index.html

# 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 dissolv ed 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 solution s 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 prepa red concentrations were inverted seve ral times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test pre parations were verified by c hemical analysis at 0 and 96 hours.

As in the range-finding test 250 ml glass conical flasks were us ed. Six flasks each completely filled with test preparation wer e used for the control and three flasks each complete ly 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 suspen sion 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 al gal suspension gave an initial nominal cell density of 1.00E+04 cell s per ml and had no significant dilution effe ct on the final test concentration.

The flasks were sealed with ground gl ass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1°C under continuous illumination (intensit y 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 determi ned at initiation of the test and after 48 and 96 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temper ature 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 cultur e 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 gi ve 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 mi xed with algal suspension (250 ml) to give the required test conc entrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l. The test was conducted in 250 ml gl ass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Six replicate fl asks were prepared for the control and three replicate flasks prepared for each test concentration. The flasks were incubated (INFORS Multitron Version 2 inc ubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constant ly shaken at approximately 150 rpm for 72 hours. Samples were taken at 0, 24, 48 and 72 hours and the cell densit ies 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 la yer of test material floating at the surface.

At the start of the test all control and test cultures were obse rved to be clear colourless solutions. After the 72-Hour test p eriod all control, 10 and 20 mg/l test cultures were observed to be gr een dispersions, the 40 mg/l test cultures were observed to be pale green dispersions whilst the 80 and 160 mg/l test cultur es were observed to be extr emely pale green dispersions. EXAMPLE 

# Pa g e 4 of 12 End p oint stud y record: Toxicit y to a q uatic al g ae and c y anobacteria.1,3,5-Triazine-1,3,5 ( 2H,4H,...

24/07/2012 file://\\hl750-nas-1\UserData$\hvr y enhoef\index.html

### 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 subcapitat a strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Al gae and Protozoa (CCAP), SAMS Resear ch 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 mainta ined 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 i n conical flasks to give an initial cell density of approximately 103 ce lls/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and cons tant illumination at 24 ± 1°C until the algal cell density was approximately 104 - 105 cells/ml.

A positive control (Harlan Laboratories Lt d Project Number: 41200591) used zinc chlori de 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 te sts was the same as that used to maintain the stock culture with the addition of 500 mg/l of sodium bicarbonate to counterac t 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, addi tional sodium bicarbonate (500 mg/l) was added to the prepared culture medium prior to use.

static

freshwater

no 

# Pa g e 5 of 12 End p oint stud y record: Toxicit y to a q uatic al g ae and c y anobacteria.1,3,5-Triazine-1,3,5 ( 2H,4H,...

24/07/2012 file://\\hl750-nas-1\UserData$\hvr y enhoef\index.html

### 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 algi cidal 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 steril e culture medium (100 ml) was added to each to ensure that the te st 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 (int ensity approximately 7000 lux) and constant ly 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 i ndicate 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 t he amount of carbon dioxide required by the large number of algal cells in the log phase of growth (see Figure 1). Due to t he need to test in completely filled and sealed test vessels the re was minimal transfer of CO2 from the gaseous phase to t he aqueous phase and hence CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which result s in an increase in the pH of the culture. Despite the additio n of 500 mg/l of sodium bicarbonate to the culture medium the in crease in pH after 96 hours was in excess of that recommended in the Test Guidelines (1.5 pH units after 96 hours). This wa s considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the cont rol cultures exceeded the validat ion criterion given in the Test Guidelines.

Not applicable

Not applicable -freshwater used

The test item was known to degrade rapidly in water fo rming 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 vess els were prepared at the start of the test and incubated alongsi de 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 conc entration 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 te st preparation were used for the control and th ree flasks each completely filled we re 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 suspen sion 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 al gal suspension gave an initial nominal cell density of 1.00E+04 cell s 

# Pa g e 6 of 12 End p oint stud y record: Toxicit y to a q uatic al g ae and c y anobacteria.1,3,5-Triazine-1,3,5 ( 2H,4H,...

24/07/2012 file://\\hl750-nas-1\UserData$\hvr y enhoef\index.html

### 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 effe ct on the final test concentration.

The flasks were sealed with ground gl ass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1°C under continuous illumination (intensit y 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), pe rcentage inhibition (arithmetic ax is) was plotted against test concentration (logarithmic axis) and a li ne fitted by computerised interpolat ion using the Xlfit software package (IDBS). ECxvalues were then determined from the equation for the fitted line.

Where appropriate 95% confidence limits fo r 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 cultur es 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 sect ion specific growth rates in the control cultures during the co urse 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 fo r data evaluation.

Duration Endpoint Effect

conc. Nominal/Measured Conc. based

on Basis for effect Remarks (e.g. 95% CL) 

# Pa g e 7 of 12 End p oint stud y record: Toxicit y to a q uatic al g ae and c y anobacteria.1,3,5-Triazine-1,3,5 ( 2H,4H,...

24/07/2012 file://\\hl750-nas-1\UserData$\hvr y enhoef\index.html

### 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% conf idence 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% conf idence 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 inspect ed 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 obse rved to be clear colourless solutions. After the 96-Hour test p eriod 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 fr om the exposure of Pseudokirchneriella subcapitata the test ite m during the range-finding test are given in Table 1.

Validation criteria

The following data show that the cell concentration of the cont rol 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 th e 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 secti on by section specific growth rate for the control cultures over the test period (0- 72 hours) was 15% and hence satisfied the validation criterion gi ven in the OECD Guideline which states the mean must not exceed 35%. 

# Pa g e 8 of 12 End p oint stud y record: Toxicit y to a q uatic al g ae and c y anobacteria.1,3,5-Triazine-1,3,5 ( 2H,4H,...

24/07/2012 file://\\hl750-nas-1\UserData$\hvr y enhoef\index.html

### Results with reference substance (positive control) The coefficient of variation for average specific growth rate fo r the control cultures over the test period (0 - 72 h) was 3% a nd 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 ho urs and hence satisfied the validation criterion whic h states that this must not exceed 20%.

Cell density values determined at each samp ling 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. Gr owth rate, yield and biomass int egral values for the control and test cultures after 72 hours and percentage inhibiti on values are given in Table 4. Growth rate, yield and biomass integral values f or the control and test cultures after 96 hours and per centage 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 t he 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 wa s carried out for the control and all test concentrations usi ng one way analysis of variance incorporating Bartlett's test fo r homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatment s 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 Obse rved Effect Concentration" (NOEC) based on growth rate was 1.0 mg ai/l. Correspondingly the "Lowest Observed Effect Conc entration" (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 simila r 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% c onfidence limits for these EC 50 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 

# Pa g e 9 of 12 End p oint stud y record: Toxicit y to a q uatic al g ae and c y anobacteria.1,3,5-Triazine-1,3,5 ( 2H,4H,...

24/07/2012 file://\\hl750-nas-1\UserData$\hvr y enhoef\index.html

### 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 with in the normal ranges for this reference item.

One way analysis of variance incorporating Bartlett's test fo r homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatment s with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after 72 and 96 hours fo r the control and all test c oncentrations to determine any statistically significant differ ences between the test and control groups. All st atistical analyses were performed using the SA S computer software package (SAS 1999 - 2001).

See Appenidix 3 for Tables

This study is classified as acceptable and satisfies the guideli ne 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 Assoc 50

, 1096-1121.

Herman et al (1990)Impact of volatile aromatic hydrocarbons, alone and in combination, on growth of the freshwater alga Selenastrum capricornutum

. Aquatic Toxicology 18 , 87 - 100 Litchfield, J T and Wilcoxon, F (1949) A Simplified Method of Evaluating Dose-Effect Experiments. J Pharmacol Exp Ther 96

, 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 Pseudokirchneri ella 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% c onfidence limits 3.2 - 3.8 mg ai/l Biomass (0-72 h) 3.6 mg ai/l; 95% c onfidence 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% conf idence limits 3.0 - 3.4 mg ai/l Biomass (0-96 h) 3.3 mg ai/l; 95% c onfidence limits 3.1 - 3.6 mg ai/l

*It was not possible to calculate 95% confidence limits fo r the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits. 

# Pa g e 10 of 12 End p oint stud y record: Toxicit y to a q uatic al g ae and c y anobacteria.1,3,5-Triazine-1,3,5 ( 2H,4...

24/07/2012 file://\\hl750-nas-1\UserData$\hvr y enhoef\index.html

### 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 th e 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 alga Pseudokirchneriella

subcapitata

. 

# The method followed wa s designed to be compat ible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibi tion Test" referenced as Me thod C.3 of Commission Regulation (EC) No 761/2008, USTitle 40 Part 797 Section 1050, US EPA Pesticide Assessment Guidelines, Sub-Division J, Sect ion 122-2, the US E PA Draft Ecological Effects Test Guideline OP PTS 850.5400 and t he ASTM E1218-04, Standard Guide for Conducting Static To xicity Tests with Microalgae.
Following a preliminary range-finding test, Pseudokirchneriella subcapitata was 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 replic ate flasks per concentration) fo r 96 hours, under constant illumination and s haking at a temperature of 24 ± 1°C.

The test item was known to be volatile and hence testing was c onducted 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 re striction 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 Pseudokirchneri ella 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 fo rming 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 vess els were prepared at the start of the test and incubated alongsi de 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 conc entration 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 of Pseudokirchneriella subcapitata to the test item gave the following resu lts based on the nominal test concentrations:

A re-growth test was performed which showed th e test item to be algistatic in effect. TiPoint (Hours) Response Variable EC 50 (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 

# Pa g e 11 of 12 End p oint stud y record: Toxicit y to a q uatic al g ae and c y anobacteria.1,3,5-Triazine-1,3,5 ( 2H,4...

24/07/2012 file://\\hl750-nas-1\UserData$\hvr y enhoef\index.html*It was not possible to calculate 95% confidence limits for the E r C 50 value as the data generated did not fit the models available
for the calculation of confidence limits. 

# Pa g e 12 of 12 End p oint stud y record: Toxicit y to a q uatic al g ae and c y anobacteria.1,3,5-Triazine-1,3,5 ( 2H,4...

24/07/2012 file://\\hl750-nas-1\UserData$\hvr y enhoef\index.html