2-(4,6-bis-(2,4-dimethylphenyl)-1,3,5-triazin-2-yl)-5-(octyloxy)-phenol

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test"

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

412-440-4

EC Name:

-

Cas Number:

2725-22-6

Molecular formula:

C33H39N3O2

IUPAC Name:

2-(4,6-bis-(2,4-dimethylphenyl)-1,3,5-triazin-2-yl)-5-(octyloxy)-phenol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Description: Pale yellow powder
Batch: S36913601
Purity: Not provided
Expiry date: Not provided
Storage conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control (replicates R1 - R6 pooled) and the 100 % v/v saturated solution (replicates R1 - R6 pooled) at 0 and 72 hours for quantitative analysis. Additional replicates incubated under identical conditions to the test replicates provided media for analysis at 24 and 48 hours. 0, 24 and 48 hour samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Test solutions

Details on test solutions:

An amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.
An aliquot (2 liters) of this saturated solution was inoculated with algal suspension (10.1 mL) to give the required test concentration of 100 % v/v saturated solution.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

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

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ± 1 °C

pH:

7.5 ± 0.1

Nominal and measured concentrations:

Analysis of the test preparations at 0, 24, 48 and 72 hours showed all measured test concentrations to be below the limit of quantification which was determined to be 0.00010 mg/L.

Details on test conditions:

Exposure Conditions
Six flasks each containing 100 mL of solution were used for the control and 100% v/v saturated solution 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 characterized by a cell density of 9.90 x 105 cells per mL. Inoculation of 2 liters of test medium with 10.1 mL of this algal suspension gave an initial nominal cell density of 5 x 103 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs 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 72 hours.

Test Organism Observations
Samples were taken at 0, 25, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Water Quality Criteria
The pH of the control and 100 % v/v saturated solution test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.

Verification of Test Concentrations
Samples were taken from the control (replicates R1 - R6 pooled) and the 100 % v/v saturated solution (replicates R1 - R6 pooled) at 0 and 72 hours for quantitative analysis. Additional replicates incubated under identical conditions to the test replicates provided media for analysis at 24 and 48 hours. 0, 24 and 48 hour samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 8

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The test concentration was the highest attainable test concentration that could be prepared due to the limited solubility of the test item in water.
Accordingly the following results were determined from the data:
Inhibition of Growth Rate
ErC10 (0 - 72 h) : >100 % v/v saturated solution
ErC20 (0 - 72 h) : >100 % v/v saturated solution *
ErC50 (0 - 72 h) : >100 % v/v saturated solution
where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and 100 % v/v saturated solution test group using a Student’s t-test. There were no statistically significant differences (P≥0.05), between the control and 100 % v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100 % v/v saturated solution.
Inhibition of Yield
EyC10 (0 - 72 h) : >100 % v/v saturated solution
EyC20 (0 - 72 h) : >100 % v/v saturated solution
EyC50 (0 - 72 h) : >100 % v/v saturated solution
Where:
EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out as in Section 4.2.2.1. There were no statistically significant differences (P≥0.05), between the control and 100 % v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100 % v/v saturated solution.
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.

Water Quality Criteria
The pH values of the control and each test preparation are given in Table 2. Temperature was maintained at 24 ± 1 ºC throughout the test.
The pH value of the control cultures (see Table 2) was observed to increase from pH 7.5 at 0 hours to pH 8.5 at 72 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) exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Observations on Test Item Solubility
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.

Results with reference substance (positive control):

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 – 72 h) : 1.2 mg/L; 95% confidence limits 1.0 – 1.3 mg/L
EyC50 (0 – 72 h) : 0.52 mg/L; 95% confidence limits 0.45 – 0.62 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Satisfied the validation criterion given in the OECD Guideline

Conclusions:

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values of greater than 100 % v/v saturated solution. The No Observed Effect Concentration (NOEC) was 100 % v/v saturated solution.
This study showed that there were no toxic effects at saturation.