Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and Ethanol, 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4th - 7th February, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(2011)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(2009)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Version / remarks:

(2004)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations sampled for analysis: 0 (blank-control) and 100 mg/L (with and without algae)
- Sampling
Frequency: at t=0 h, t=24 h and t=72 h
Volume: 2 ml
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis (≤ -15°C). On the day of analysis, the samples were defrosted at room temperature. If necessary, the samples were diluted with M2-medium to obtain concentrations within the calibration range.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The preparation of test solutions started with the highest test concentration. No special treatment other than a short magnetic stirring period was applied to fully dissolve the test substance in test medium. The lower test concentrations were prepared by subsequent dilutions in test medium. After preparation, volumes of 50 ml were added to each replicate of the respective test concentration.
- Controls: blank control (test medium without test substance or other additives)
- Evidence of undissolved material: The test substance was completely soluble in test medium at the concentrations tested. The final test solutions were all clear and colourless.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green microalgae
- Source: In-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2) at a cell density of 1 x 10^4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Culturing media and conditions (same as test or not): yes

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

- Start of the test: 20.8°C
- During the exposure period the temperature was maintained between 21.3 and 22.9°C.

pH:

- Start: 8.0-8.2
- End: 7.7-7.9

Nominal and measured concentrations:

- Nominal: 0 (blank-control), 0.10, 1.0, 10 and 100 mg/L
- Measured: See the "Remarks on results including figures and tables" field for details.

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass, with 100 ml capacity and filled with 50 ml of test solution
- Type: closed (capped)
- Aeration: No
- Initial cells density:of 1 x 10^4 cells/ml
- No. of vessels per concentration (replicates): 3 replicates of 0.10, 1.0 and 10 mg/L and 6 replicates of 100 mg/L
- No. of vessels per control (replicates): 6 replicates

GROWTH MEDIUM
- Standard medium used: yes (M2)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap-water purified by reverse osmosis
- Culture medium different from test medium: No. Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2)
- Intervals of water quality measurement: The pH was measured at the beginning and at the end of the test.
Temperature of medium: Continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Photoperiod: Continuously
- Light intensity and quality: 99 to 108 µE.m-2.s-1; TLD-lamps of the type ‘Cool-white’ (30 Watt)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.
- Appearance of the cells: At the end of the test microscopic observations were performed on the highest test concentration to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x10

Reference substance (positive control):

yes

Remarks:

K2Cr2O7 (Potassium dichromate)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- No significant differences were recorded between the values for growth rate at any of the test concentrations when compared to the control group. For yield, a slight effect of 16% inhibition was recorded at the highest test group. For yield, the 72h-NOEC was between analytically confirmed concentrations of 10 and 100 mg/L.
- Microscopic observations of algae exposed to the highest test group at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

- Acceptability of the test:
1. In the controls, cell density increased by an average factor of > 16 within two days.
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%.
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.

Results with reference substance (positive control):

- K2Cr2O7 (Potassium dichromate): The EC50 for growth rate reduction (ErC50: 0-72h) was 1.7 mg/L with a 95% confidence interval ranging from 1.2 to 2.3 mg/L.

- Percentage reduction of growth rate (total test period) and percentage inhibition of yield

Concentration Accelerator (PT 25E or PT 25E/2) (mg/L)	Mean growth rate		Yield (0-72 h)
	µ (0-72 h)	Reduction (%)	x10^4cells/ml	Inhibition (%)
Control	0.07076		162.31
0.10	0.07094	-0.3	165.18	-1.8
1.0	0.06950	1.8	148.21	8.7
10	0.07173	-1.4	173.99	-7.2
100	0.06818	3.6	136.61	15.8

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, no reduction of growth rate was recorded at any of the concentrations of Accelerator (PT 25E or PT 25E/2) tested. Both the EC50 for growth rate reduction (72h-ErC50) and the EC50 for yield inhibition (72h-EyC50) exceeded an analytically confirmed concentration of 100 mg/L. The 72h-NOEC for growth rate reduction was 100 mg/L. For yield, the 72h-NOEC was between analytically confirmed concentrations of 10 and 100 mg/L.

Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201, freshwater algae (Pseudokirchneriella subcapitata) were exposed to Accelerator (PT 25E or PT 25E/2) at the following nominal concentrations: 0 (blank-control), 0.1, 1, 10 and 100 mg/L.

Both the EC50 for growth rate reduction (72h-ErC50) and the EC50 for yield inhibition (72h-EyC50) exceeded an analytically confirmed concentration of 100 mg/L. The 72h-NOEC for growth rate reduction was 100 mg/L. For yield, the 72h-NOEC was between analytically confirmed concentrations of 10 and 100 mg/L. The study is considered to be reliable without restrictions.

Description of key information

In a 72 h toxicity study conducted according to OECD guideline 201, freshwater algae (Pseudokirchneriella

subcapitata) were exposed to Accelerator (PT 25E or PT 25E/2). The EC50 for growth rate reduction (72h-

ErC50) exceeded an analytically confirmed concentration of 100 mg/L. The 72h-NOEC for growth rate

reduction was 100 mg/L. The study is considered to be reliable without restrictions.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

In a 72 h toxicity studyconducted according to OECD guideline 201, freshwater algae (Pseudokirchneriella subcapitata) were exposed to Accelerator (PT 25E or PT 25E/2) at the following nominal concentrations: 0 (blank-control), 0.1, 1, 10 and 100 mg/L.

Both the EC50 for growth rate reduction (72h-ErC50) and the EC50 for yield inhibition (72h-EyC50) exceeded an analytically confirmed concentration of 100 mg/L. The 72h-NOEC for growth rate reduction was 100 mg/L. For yield, the 72h-NOEC was between analytically confirmed concentrations of 10 and 100 mg/L. The study is considered to be reliable without restrictions.