4,4'-methylenebis[2,6-diethylaniline]

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:

November 2014 - March 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TEST MATERIAL
- Molecular formula: C21H30N2
- Analysis date: August 12, 2014
- Date of production: August 11, 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Controlled room temperature (15-25ºC, <70 RH%), protected from light and humidity.
- Stability under test conditions: stable for min. 3 years from manufacturing date
- Expiry date: August 10, 2017
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Analytical monitoring:

yes

Details on sampling:

For determination of the test item concentration, samples were taken from each testing concentration and from the control at the start and at the end of the experiment. Three replicate samples were taken from the test solutions and one control sample on both analytical occasions.

Vehicle:

no

Details on test solutions:

The test solutions used in the test were prepared by mechanical dispersion without using of any solubilising agent. The test item is poorly water soluble material. In order to achieve maximum dissolved concentration, a supersaturated solution (100 mg/L nominal loading) was prepared by dispersing/dissolving an amount of 0.1 g test item into 1000 mL test medium (OECD medium) approx. one day before the start of the test. This solution was agitated by orbital shaker (~350 rpm) for a period of approx. one day and then the non-dissolved test material was separated by filtration through a fine (0.22 μm) filter in order to obtain the saturated test solution (i.e. 100 % v/v saturated solution). The test solutions of the chosen test concentrations were prepared by appropriate diluting of this stock solution. The test solutions were prepared just before introduction of the algae (start of the experiment).

Test organisms (species):

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

Details on test organisms:

- Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
- Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Origin: The algae were supplied by the SAG: Collection of Algal Cultures, D-37073 Göttingen, Germany
- Breeding Conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardized conditions according to the test guidelines.
- Pre-culturing: The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The preculture was incubated for four days at this test.) The algal cultures used in this study did not contain deformed or abnormal cells.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

The cell concentration was determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. The cell morphology was examined in parallel.

Post exposure observation period:

None

Hardness:

Not given in report; standard hardness recommended by OECD for algal toxicity studies is approx. 24 mg/l as CaCO3

Test temperature:

Culture temperature was checked at the beginning of the study and each day thereafter in a flask filled with water, in the climatic chamber. In addition temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.4 – 22.6 °C measured in the flask and between 21.4 and 24.0°C measured within the climate chamber.

pH:

The pH was measured in each test group at the start and in each test vessel at the end of the test. The pH of the control cultures did not vary by more than 1.5 units during the experiment. The range of the pH was 7.98 – 9.60 during the experiment.

Dissolved oxygen:

Not measured

Salinity:

Freshwater used

Conductivity:

No data

Nominal and measured concentrations:

Based on the results of the preliminary range-finding test five test concentrations in a geometric series with a spacing factor of 2 and a concurrent untreated control were used in the main test. The following nominal concentrations were tested: 6.3, 12.5, 25.0, 50.0 and 100.0 % v/v saturated solution.
The corresponding calculated geometric mean test item concentrations (based on the analytical measurements) were 0.10, 0.19, 0.41, 0.91, 1.65 mg/L.

Details on test conditions:

LIGHT INTENSITY:
The algal culture flasks were continuously illuminated with fluorescent lamps (with a spectral range of 400-700 nm). The light intensity was checked and recorded at the position occupied by algal culture flasks at the start of the test. The average measured light intensity was 8200 lux. The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

Reference substance (positive control):

yes

Remarks:

For the evaluation of the reliability of the applied test system and the experimental conditions potassium dichromate is tested at least twice a year.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.65 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: expressed as ErC50

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.65 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: expressed as EyC50

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.41 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.41 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

MICROSCOPIC EXAMINATIONS:

Shape of the algal cells:
No morphological abnormalities were observed in the control and in the two lowest test item concentrations (0.10 and 0.19 mg/L measured) during the experiment. :
Swollen and thin cells were observed amongst the mostly normal algal cells in the three highest concentrations (0.41, 0.91 and 1.65 mg/L measured)

Cell density in control:
In the control the cell density has increased from nominal N= 1 × 104 cells/mL at the start of the test (0 hours) to N= 64.17 × 104 cells/mL (mean value) after 72 hours. Thus, the algal growth in the control was high enough to pass the validity criteria in this assa

Results with reference substance (positive control):

The date of the last study with the reference item Potassium dichromate was: 23 – 26 September 2014.
The 72h ErC50: 1.48 mg/L (95 % confidence limits: 1.30 – 1.69 mg/L)
The 72h EyC50: 0.69 mg/L (95 % confidence limits: 0.61 – 0.78 mg/L

Reported statistics and error estimates:

Mean values and standard deviations were calculated for each treatment at the start, and at the end of the test using Excel for Windows software.
For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test (α = 0.05) using TOXSTAT software. The data were checked for normality by Chi-Square Test, for homogeneity of variance by Hartley’s and Bartlett’s Test. The ErCx and EyCx values were determined directly from the raw data.

Validity criteria fulfilled:

yes

Conclusions:

The 72-h EC50 value based on growth rate and yield was determined to be higher than the saturation concentration (1.65 mg/L measured). The overall NOEC was determined to be 0.19 mg/L. The results are based on the measured concentrations.

Executive summary:

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours. The test was performed according to EC-method C.3 and OECD no. 201. The test design included three replicates at each test concentration and six replicates for the untreated control. Based upon the results from the preliminary experiment, nominal concentrations of 6.3, 12.5, 25.0, 50.0 and 100.0 % v/v saturated solution and an untreated concurrent control was tested in the main study. The corresponding calculated geometric mean test item concentrations (based on the analytical measurements) were 0.10, 0.19, 0.41, 0.91, and 1.65 mg/L. All biological results were based on the measured concentrations. With respect to the inhibitory effect of the test item, the 0-72h average specific growth rates and yield were significantly different from that of the control group in the concentrations of 0.41, 0.91, and 1.65 mg/L (measured). Accordingly the No Observed Effect Concentration (NOEC) was determined to be 0.19 mg/L. The Lowest Observed Effect Concentration (LOEC) was determined to be 0.41 mg/L. In conclusion, the 72-h EC50 value based on growth rate and yield was determined to be higher than the saturation concentration (1.65 mg/L measured).

Description of key information

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours. The test was perfomed according to EC-method C.3 and OECD no. 201. With respect to the inhibitory effect of the test item, the 0-72h average specific growth rates and yield were significantly different from that of the control group in the concentrations of 0.41, 0.91, and 1.65 mg/L (measured). Accordingly the No Observed Effect Concentration (NOEC) was determined to be 0.19 mg/L. The Lowest Observed Effect Concentration (LOEC) was determined to be 0.41 mg/L. In conclusion, the 72-h EC50 value based on growth rate and yield was determined to be higher than the saturation concentration (1.65 mg/L measured).

Key value for chemical safety assessment

EC50 for freshwater algae:

1.65 mg/L

EC10 or NOEC for freshwater algae:

0.19 mg/L

Additional information

Source: GLP-report