Reaction products of 3-aminomethyl-3,5,5-trimethylcyclohexylamine with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

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:

from 2014-06-02 to 2014-07-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Toxic potential was analysed by addition of humic acid

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

yes

Remarks:

addition of humic acid

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

yes

Remarks:

addition of humic acid

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

1996

Deviations:

yes

Remarks:

addition of humic acid

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling method: For determination of the test item concentration, three replicate samples were taken from each testing concentration and from the controls at the start and at the end of the experiment.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A supersaturated solution (nominal loading: 100 mg/L) was prepared by adding an excess of test item in OECD medium, then the mixture agitated in ultrasonic bath for approx. 2×15 minutes. Between the two separate ultrasonic bath periods mild heating was used in water bath for about one hour at 37 °C. After that the mixture was stirred for approx. 24 hours at room temperature. The non-dissolved part of the test item was separated by filtration through a fine membrane filter (0.2 μm) to obtain the saturated stock solution (i.e. 100 % v/v saturated solution). The concentration of the saturated stock solution was analytically determined and then the test solutions of the chosen test concentrations were accordingly diluted from this stock solution. The concentration of the stock solution was determined to be 9.21 mg/L, which was slightly lower than the highest planned nominal test concentration (i.e. 10 mg/L), however it was considered to be acceptable as the highest test concentration and for diluting the lower concentrations. Nevertheless the dilution was based on the measured concentration of the stock solution (i.e. 9.21 mg/L).
- Differential loading: An appropriate amount of humic acid (nominal concentration: 10 mg/L) was dissolved in each individual test concentration.
- Control: Untreated control, Humic acid control, toxic reference control

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum) (Printz-Starr).
- Strain: 61.81 SAG
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany
- Method of cultivation: 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 standardised conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Test temperature:

22.5 - 22.7 °C measured in the flask
22.1 and 23.3 °C in the climate chamber

pH:

7.81 to 9.95

Nominal and measured concentrations:

nominal: 0.6, 1.3, 2.5, 5.0 and 10.0 mg/L
measured: 0.40, 0.85 2.07, 4.55 and 7.15 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Fill volume: 100 mL
- Initial cells density: The test was started by inoculation of 0.1 mL algal biomass to 100 mL test item solution. The initial cell density was about 10^4 cells/mL in each test flask.
- Replicates: The test was performed with three replicates at each test concentration and six replicates were included in the untreated- and humic acid-control respectively.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to OECD medium, according to OECD 201

OTHER TEST CONDITIONS
- Adjustement of humic acid: An appropriate amount of humic acid (nominal concentration: 10 mg/L) was then dissolved in each individual test concentration.
- Light intensity and quality: 7046 lux

EFFECT PARAMETERS MEASURED: 24, 48 and 72 hours after starting the test
- Determination of cell concentrations: The cell numbers were determined after starting the test by manual cell counting using a microscope with counting chamber.
- Morphological changes of algal cells: The morphological changes of algal cells compared to the control were examined after starting the test using a microscope.
- Toxic reference control: For the evaluation of the quality of the algae and the experimental conditions, potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
- Humic acid control: Algal Mineral Salts Test Medium (OECD medium) with addition of humic acid (nominal concentration: 10 mg/L (5 mg humic acid was dissolved in 500 mL OECD medium)) was inoculated (without test item) and examined in parallel in the study.

TEST CONCENTRATIONS
- Range finding study: In order to select appropriate test concentrations for use in the definitive test, a non-GLP preliminary range-finding test was conducted to determine the approximate toxicity of the test item.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.99 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.13 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.07 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.07 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Observation of abnormalities: Pinches on cells in the concentrations of 0.85, 2.07, 4.55 and 7.15 mg/L (measured), swollen cells were observed at the 48 h and 72 h observation periods.
- Unusual cell shape: in the concentration of 2.07 mg/L at the 48 h and 72 h observation periods.
- Aggregation of algal cells: Algal cells were observed in masses in all test item concetrations and in the humic acid control during the experiment.
- Any stimulation of growth found in any treatment: Stimulation probably due to the presence of humic acid.

Results with reference substance (positive control):

For the evaluation of the quality of the algae and the experimental conditions, potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Reported statistics and error estimates:

Mean values and standard deviations of cell concentrations were calculated for each treatment at the start, after 0 h, 24 h and 48 h and at the end of the test (72 hours after the start of the test) using Excel for Windows software. Percentage inhibition of growth rate (μ) and yield (y) were calculated using EXCEL for Windows software. The EC values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software (based on the measured geometric mean concentrations). For the determination of the LOEC and NOEC, the calculated mean growth rate (μ) and yield (y) at the test concentrations were tested on significant differences to the Humic acid control values by Bonferroni t-Test using TOXSTAT software.

Validity criteria fulfilled:

yes

Conclusions:

In a study with the test item the ErC50 value was determined to be 3.13 mg/L (growth rate) and 2.50 mg/L (yield).The ErC10 was determined to be 1.99 mg/L and 1.40 mg/L (yield).

Executive summary:

A study according to OECD guideline 201, EC Regulation C.3 and OPPTS 850.5400 was conducted to determine the aquatic toxic potential of the test item on the growth of a unicellular green algal species Pseudokirchneriella subcapitata by addition of humic acid. Humic acid is formed by biodegradation of dead organic matter. It is a principal component in the environment, for example in lakes and in the soil. Exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to various concentrations of the test item over several generations under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and thus, over several algal generations. The test method of application and the test species Pseudokirchneriella subcapitata are recommended by the test guidelines. Based on the results of non-GLP Preliminary Range-Finding Test the following five test concentrations in a geometric series (with a separation factor of 2.0) were tested: 0.6, 1.3, 2.5, 5.0 and 10.0 mg/L (nominal). Each test group contained dissolved humic acid in 10 mg/L nominal concentration. Untreated- and humic acid-control (10 mg/L) ran parallel in the test. The analytically measured concentrations deviated more than 20 % from the nominal during the experiment therefore the geometric mean of the measured concentrations were calculated in order to determine exposure concentrations. The corresponding calculated geometric mean concentrations were the followings: 0.40, 0.85, 2.07, 4.55 and 7.15 mg/L. All biological results are based on the measured geometric mean test item concentrations. 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 using TOXSTAT software. The test item had a statistically significant inhibitory effect on the growth of Pseudokirchneriella subcapitata after the exposure period of 72 hours based on the average specific growth rate and yield in the two highest test concentrations of 4.55 and 7.15 mg/L (Bonferroni t-Test, 1 tailed, α=0.05) when compared to the humic acid-control. Accordingly, the 72-hour NOEC related to growth rate and yield was determined to be 2.07 mg/L. The EC values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software. The 72-h EyC10 based on yield was determined to be 1.40 mg/L and the 72-h EyC50 as 2.50 mg/L. The 72 hour ErC10 was determined to be 1.99 mg/L and ErC50 value was determined to be 3.13 mg/L.

Description of key information

In a 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with the test item according to EU Method C.3 (Algal Inhibition test), the 72-h EC50 based on growth rate was determined to be 3.13 mg/L and the EC10 based on growth rate was determined to be 1.99 mg/L. The NOEC was determined to be 2.07 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

3.13 mg/L

EC10 or NOEC for freshwater algae:

1.99 mg/L

Additional information

The aquatic toxic potential in this study was determined by the addition of humic acid. Humic acid is a principal component in the environment, it is formed by biodegradation of dead organic matter. Therefore, it simulates natural conditions much more realistic than without the addition of humic acid.

A study according to OECD guideline 201, EC Regulation C.3 and OPPTS 850.5400 was conducted to determine the aquatic toxic potential of the test item on the growth of a unicellular green algal species Pseudokirchneriella subcapitata by addition of humic acid. Humic acid is formed by biodegradation of dead organic matter. It is a principal component in the environment, for example in lakes and in the soil. Exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to various concentrations of the test item over several generations under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and thus, over several algal generations. The test method of application and the test species Pseudokirchneriella subcapitata are recommended by the test guidelines. Based on the results of non-GLP Preliminary Range-Finding Test the following five test concentrations in a geometric series (with a separation factor of 2.0) were tested: 0.6, 1.3, 2.5, 5.0 and 10.0 mg/L (nominal). Each test group contained dissolved humic acid in 10 mg/L nominal concentration. Untreated- and humic acid-control (10 mg/L) ran parallel in the test. The analytically measured concentrations deviated more than 20 % from the nominal during the experiment therefore the geometric mean of the measured concentrations were calculated in order to determine exposure concentrations. The corresponding calculated geometric mean concentrations were the followings: 0.40, 0.85, 2.07, 4.55 and 7.15 mg/L. All biological results are based on the measured geometric mean test item concentrations. 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 using TOXSTAT software. The test item had a statistically significant inhibitory effect on the growth of Pseudokirchneriella subcapitata after the exposure period of 72 hours based on the average specific growth rate and yield in the two highest test concentrations of 4.55 and 7.15 mg/L (Bonferroni t-Test, 1 tailed, α=0.05) when compared to the humic acid-control. Accordingly, the 72-hour NOEC related to growth rate and yield was determined to be 2.07 mg/L. The EC values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software. The 72-h EyC10 based on yield was determined to be 1.40 mg/L and the 72-h EyC50 as 2.50 mg/L. The 72 hour ErC10 was determined to be 1.99 mg/L and ErC50 value was determined to be 3.13 mg/L.