A mixture of: isomers of 2-(2H-benzotriazol-2-yl)-4-methyl-(n)-dodecylphenol; isomers of 2-(2H-benzotriazol-2-yl)-4-methyl-(n)-tetracosylphenol; isomers of 2-(2H-benzotriazol-2-yl)-4-methyl-5,6-didodecyl-phenol. n=5 or 6

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24.08. - 27.08-1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study, GLP. The coefficient of analysis of the daily growth rate in the control replicates and the coefficient of analysis of the average specific growth rates in the control replicates were not reported.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

yes

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Nominal and measured concentrations:

Nominal: 0.1, 1 and 10 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

OECD guideline study, GLP. The coefficient of analysis of the daily growth rate in the control replicates and the coefficient of analysis of the average specific growth rates in the control replicates were not reported. The effects are based on biomass instead of growth rate.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Water samples were taken from the solvent control and each test group (replicates R1 - R3 and R4 - R6 pooled) at 0 and 72 hours
for quantitative analysis.
- Sample storage conditions before analysis: storage at ambient temperatures in light and dark conditions for approximately 72 hours.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
For the purpose of the definitive study the test material was prepared using a preliminary solution in 1% v/v Tween 80-tetrahydrofuran. An amount of test material (500 mg) was dissolved in solvent and the volume adjusted to 10 mL to give a 500 mg/10 mL solvent stock solution. An aliquot (200 µL) of this solvent stock solution was dispersed in 2 litres of algal suspension to give the required test concentration of 5.0 mg/L.
- Controls: A solvent control (containing 100 µL solvent per litre) and an untreated control were prepared.
- Chemical name of vehicle: Tween 80-dimethylformamide
- Concentration of vehicle in test medium: 5 % v/v
- Evidence of undissolved material: at concentrations in excess of 5 mg/L. there was a marked precipitation of the test material on addition of the solvent stock solution to culture medium.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Centre for Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria.
- Age of inoculum: At initiation of the study the culture contained a nominal cell density of 10E4 cells per mL.

ACCLIMATION
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 °C

pH:

8.0 at the beginning and 10.1 - 10.3 at the end

Nominal and measured concentrations:

nominal: 5 mg/L
mean measured: 3.56 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: the flasks were covered with aluminium foil
- Fill volume: 100 mL
- Aeration: constant
- Initial cells density: 2.47x10E4 cells/mL (nominal: 10E4 cells/mL)
- Control end cells density: 1.53x10E6 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH of each control, solvent control and test flask was determined at initiation of the study and after 72 hours exposure. The temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continous illumination
- Light intensity and quality: approximately 7000 lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: from samples at at 0, 24, 48 and 72 hours, using counting chamber
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: The use of a limit test is in agreement with the current EEC Test Guidelines.
- Range finding study
- Results used to determine the conditions for the definitive study:
The results of the initial range-finding study showed no significant effect on grovdh at the concentrations of 1.0 and 10 mg/L. The small reduction in the cell density after 72 hours for the 10 mg/L test group as compared to the solvent control is not considered to be significant given that the test material was present in the test vessels as a cloudy dispersion above the limit of water solubility for the test material under these test conditions. Additional solubility work conducted showed that the highest attainable test concentration that resulted in a solution of the test material under these test conditions was 5 mg/L and therefore a second rangefinding study was conducted at this concentration. The results of the second range-finding study showed no effect on growth at the test concentration of 5 mg/L.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 3.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 3.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: not observed
- Colour differences: not observed
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: At concentrations in excess of 5.0 mg/L. there was a marked
precipitation of the test material on addition of the solvent stock
solution to culture medium.

Description of key information

There is a high probability that the product is not harmful to aquatic algae and cyanobacteria.

Additional information

The toxicity to algae was assessed in two tests according to OECD guideline 201. In the first test Scenedesmus subspicatus was exposed to the test item at nominal concentrations of 0.1, 1 and 10 mg/L. The ErC50 based on nominal test concentrations was determined to be > 10 mg/L, the NOEC was determined to be 1 mg/L (ABC Inc. 1993).  In the second study Scenedesmus subspicatus was exposed to the test item at a nominal concentration of 5 mg/L. The EbC50 based on measured test concentrations was determined to be > 3.5 mg/L the NOEC was determined to be >= 3.5 mg/L (Safepharm Laboratories, 1995).  All values are clearly above the water solubility of the compound (0.13 µg/L). In conclusion, no effects on algae could be recorded up to the water solubility.