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

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:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For read-across justification please refer to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.55 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.36 mg/L

Nominal / measured:

meas. (arithm. mean)

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:

weight of evidence

Study period:

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline and GLP study, 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:

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 (0.1 g test item was added to 1000 mL OECD medium), then 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 (0.22 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 57.1 mg/L. The test solutions were prepared immediately before introduction of algae (start of the experiment).
- Differential loading: An appropriate amount of humic acid (nominal concentration: 10 mg/L) was then dissolved in each individual test concentration.
- Controls: 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

Test temperature:

22.5 - 22.8 °C measured in the flasks
22.1 - 23.7 °C measured within the climate chamber

pH:

7.67 to 10.47

Nominal and measured concentrations:

Nominal: 0.2, 0.7, 1.8, 5.1, 14.3 and 40.0 mg/L
Measured: 0.14, 0.36, 1.68, 3.10, 8.23 and 30.60 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 dissolved in each individual test concentration.
- Light intensity and quality: 8046 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:

EC50

Effect conc.:

2.11 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.:

1.83 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.55 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.:

1.26 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.:

0.36 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.:

0.36 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Observation of abnormalities: Swollen cells were detected in the concentration of 1.68 mg/L at the 48 h and 72 h observation periods. Deformation of algal cells were observed in the concentration of 3.10 mg/L at the 72 h observation period and at the concentration of 8.23 mg/L at the 48 h and 72 h observation periods. The number of observed algal cells was not sufficient to determine the morphological abnormalities in the highest test concentration.
- Aggregation of algal cells: Algal cells were observed in masses in all test item concentrations and in the humic acid control during the expriment.
- Any stimulation of growth found in any treatment: Probably due to 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 SPSS PC+ 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 read-across study with Reaction products of MXDA with bisphenol A diglycidylether (BADGE) the ErC50 was determined to be 2.11 mg/L and EyC50 based on yield was 1.83 mg/L. The EC10 was determined to be 1.55 mg/L (growth rate) and 1.22 mg/L (yield).

Executive summary:

A read-across study according to OECD guideline 201, EC Regulation C.3 and OPPTS 850.5400 was conducted to determine the aquatic toxic potential of the Reaction products of MXDA with bisphenol A diglycidylether (BADGE) on the growth of a unicellular green algal species Pseudokirchneriella subcapitata. The purpose of this study was to determine the effect of the test item with 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 six test concentrations in a geometric series (with a separation factor of 2.8) were tested: 0.2, 0.7, 1.8, 5.1, 14.3 and 40.0 mg/L (nominal). Untreated- and humic acid-control (10 mg/L) was run 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: 0.14, 0.36, 1.68, 3.10, 8.23 and 30.60 mg/L. All biological results are based on the measured geometric mean 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 concentrations of 1.68, 3.10, 8.23 and 30.60 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 0.36 mg/L. The EC values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software. The 72-h ErC10 was determined to be 1.55 mg/L and the 72 -h ErC50 as 2.11 mg/L. The 72 -h EyC10 was determined to be 1.26 mg/L and the 72 -h EyC50 as 1.83 mg/L.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For read-across justification please refer to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

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

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

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

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline and GLP study, 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

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 read-across study with Reaction products of IPDA with bisphenol A diglycidylether (BADGE) the EC50 value was determined to be 3.13 mg/L (growth rate) and 2.50 mg/L (yield). The EC10 was determined to be 1.99 mg/L (growth rate) and 1.40 mg/L (yield).

Executive summary:

A read-across study according to OECD guideline 201, EC Regulation C.3 and OPPTS 850.5400 was conducted to determine the aquatic toxic potential of the Reaction products of IPDA with bisphenol A diglycidylether (BADGE) 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.

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

from 2009-03-19 to 2009-09-13

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The study is regarded as reliable without restrictions because it was conducted in accordance with GLP regulation and guideline.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

440/2008/EC; 31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The concentrations of the testing solutions (nominal S.S./16 – S.S./1) were analysed using HPLC-UV method at the beginning and at the end of the test.

Vehicle:

no

Details on test solutions:

The measured test item concentrations were out of the ± 20 % range of the nominal concentrations during the test.
The corresponding measured mean test item concentrations were:
0.03; 0.08; 0.28; 0.86 and 3.78 mg/L.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum) (Printz-Starr).

Origin: The algae were supplied by the Georg-August-Universität Göttingen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften Experimentelle Phykologie und Sammlung von Algenkulturen (SAG), Nikolausberger Weg 18, 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 standardised conditions according to the test guidelines.
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 pre-culture was incubated for three days at this test.)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Determination of Cell Number
The cell number were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber.

Behaviour of the Test Item
The behaviour of the test item in test medium was determined daily in all replicates of all test concentrations.

Test temperature:

The cultures were maintained at a temperature in the range of 21 – 24 °C ± 2 °C, which was checked and recorded at the beginning of the study and every 24 hours in a flask filled with water. In addition, the temperature was continuously measured (with a min/max thermometer) within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the study in the controls and every concentration.

Nominal and measured concentrations:

The corresponding measured mean test item concentrations were: 0.03; 0.08; 0.28; 0.86 and 3.78 mg/L.
Analysis showed that the deviation from the nominal concentrations was higher than ± 20 % during the study. Therefore, all reported biological results are related to the geometric mean of the measured test item concentrations at the start and at the end of the test calculated by statistical software.

Details on test conditions:

Temperature
The cultures were maintained at a temperature in the range of 21 – 24 °C ± 2 °C, which was checked and recorded at the beginning of the study and every 24 hours in a flask filled with water. In addition, the temperature was continuously measured (with a min/max thermometer) within the climate chamber.

pH
The pH was checked at the beginning and at the end of the study in the controls and every concentration.

Light Intensity
The light intensity at the position occupied by algal culture flasks during the test was about 8127 lux, which was ensured with fluorescent lamps (with a spectral range of 400 - 700 nm).

Equipment
Normal laboratory equipment and the following were necessary for determination of the parameters of the test:
- pH meter
- thermometer
- light-meter
- laboratory orbital shaker
- microscope with counting chamber
- climate chamber
- balance

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.08 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.28 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.28 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.08 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.28 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Purpose
The purpose of this study was to determine the effect of the test item on the growth of an unicellular green algal species Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum).
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.

Test Concentrations
The following nominal concentrations were used in the main study: 0.05; 0.15; 0.44; 1.33 and 4.00 mg /L.

Results
Analytical results
The test item concentration of the test solutions was measured by HPLC-UV method at the start and at the end of the test.

The corresponding measured mean test item concentrations were: 0.03; 0.08; 0.28; 0.86 and 3.78 mg/L.
Analysis showed that the deviation from the nominal concentrations was higher than ± 20 % during the study. Therefore, all reported biological results are related to the geometric mean of the measured test item concentrations at the start and at the end of the test calculated by statistical software.

The following table provides a summary of the influence of the test item on growth of algae:
(see Sect. "Remarks on results incl. tables and figures")

Conclusion
In this 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with the test item, the 72-h EC50 based on growth rate was determined as 0.40 mg/L. The 72-h EC50 based on yield was 0.29 mg/L. The overall NOEC was determined to be 0.08 mg/L.

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:

Statistical analysis:
Mean values and standard deviations of cell concentrations were calculated for each treatment at the start, after 0 h, 24 h, 48 h and at the end of the test (72 hours after the start of the test) using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).

Percentage inhibition of area A, growth rate r and yield Y were calculated using EXCEL for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The EC50 values of the test item and their confidence limits were calculated using Probit analysis. The analysis was done using the statistical software program “TOXSTAT 3.5”.

For the determination of the LOEC and NOEC, the calculated mean biomass b (area under the growth curve), growth rates r and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test by TOXSTAT software.

Table: Influence of the test item on the Growth of Pseudokirchneriella subcapitata

Parameter (0-72 h)	Biomass (b) [mg/L]	Growth rate (r) [mg/L]	Yield (y) [mg/L]
	Calculation based on geometric mean of measured concentrations
EC50	0.28	0.40	0.29
95 % conf. limits	0.24 – 0.32	0.36 – 0.45	0.26 – 0.33
NOEC	0.08	0.08	0.08
LOEC	0.28	0.28	0.28

The EC₅₀ values were determined by Probit Analysis (statistical software program TOXSTAT). The NOEC and LOEC values were determined directly from the raw data.

Validity criteria fulfilled:

yes

Conclusions:

The test item was tested for toxicity to algae in a 72-hr static test according to EU Guideline C.3 and OECD Guideline 201. The results showed an EC50 value of 0.28 mg/L based on Biomass and 0.40 mg/L based on Growth rate. The 72-hr ErC50 for algae was determined to be 0.4 mg/L.

Executive summary:

Analytical Results

Concentration of Test item:

The concentrations of the testing solutions (nominal S.S./16 – S.S./1) were analysed using HPLC-UV method at the beginning and at the end of the test. The measured test item concentrations were out of the ± 20 % range of the nominal concentrations during the test. The corresponding measured mean test item concentrations were: 0.03; 0.08; 0.28; 0.86 and 3.78 mg/L. Therefore, all reported biological results are related to the geometric mean of the measured test item concentrations at the start and at the end of the test calculated by EXCEL Software Program.

Biological Results

Growth Inhibition:

The influence of the test item on the growth of Pseudokirchneriella subcapitata is shown in Tables 1, 4-10 and Figure 1. The test item had a statistically significant inhibitory effect on the growth based on the average specific growth rate, yield and areas under the growth curves of Pseudokirchneriella subcapitata after the exposure period of 72 hours in the concentration range of 0.28 – 3.78 mg test item/L (based on the calculated concentrations).

The test concentration of 0.28 mg/L (calculated) was determined as the 72-hour LOEC.

The test concentration of 0.08 mg/L (calculated) was determined as the 72-hour NOEC.

The EC-values were calculated for the algal biomass b, for the growth rate r and for the yield y after 72-hour test duration (see Table below).

Table:   Influence of the test item on the Growth of Pseudokirchneriella subcapitata

Parameter (0-72 h) Biomass (b) [mg/L] Growth rate (r) [mg/L] Yield (y) [mg/L]   Calculation based on geometric mean of measured concentrations EC50 0.28 0.40 0.29 95 % conf. limits 0.24 – 0.32 0.36 – 0.45 0.26 – 0.33 NOEC 0.08 0.08 0.08 LOEC 0.28 0.28 0.28 The EC50values were determined by Probit Analysis (statistical software program TOXSTAT). The NOEC and LOEC values were determined directly from the raw data.

Conclusion:

In this 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with the test item, the 72-h EC50 based on growth rate was determined as 0.40 mg/L. The 72-h EC50 based on yield was 0.29 mg/L. The overall NOEC was determined to be 0.08 mg/L.

Description of key information

A read-across approach was applied for short-term toxicity to algae using data of symmetrical epoxy amine adducts reaction Reaction products of IPDA with bisphenol A diglycidylether (BADGE) and Reaction products of MXDA with bisphenol A diglycidylether (BADGE), as these substances were considered to show similar ecotoxicological properties as compared to the asymmetrical adduct BADGE with IPDA and MXDA. The ErC50 (based on growth rate) was determined to be 2.11 mg/L in the read-across study with Reaction products of MXDA with bisphenol A diglycidylether (BADGE) and 3.13 mg/L in the read-across study with IPDA. The worst case ErC10 was determined to be 1.55 mg/L (MXDA) and 1.99 mg/L (IPDA).

Key value for chemical safety assessment

EC50 for freshwater algae:

2.11 mg/L

EC10 or NOEC for freshwater algae:

1.55 mg/L

Additional information

A read-across approach was applied for short-term toxicity to algae using data of symmetrical epoxy amine adducts reaction Reaction products of IPDA with bisphenol A diglycidylether (BADGE) and Reaction products of MXDA with bisphenol A diglycidylether (BADGE), as these substances were considered to show similar ecotoxicological properties as compared to the asymmetrical adduct BADGE with IPDA and MXDA.

In addition a study with the registered substance itself is available. This study was not taken into account for risk assessment and classification as the read-across studies better simulate the natural aquatic environment. The aquatic toxic potential in these read-across studies 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.

Weight of evidence approach

Growth Inhibition Test of Reaction products of MXDA with bisphenol A diglycidylether (BADGE) on Algae

A read-across study according to OECD guideline 201, EC Regulation C.3 and OPPTS 850.5400 was conducted to determine the aquatic toxic potential of the Reaction products of MXDA with bisphenol A diglycidylether (BADGE) on the growth of a unicellular green algal species Pseudokirchneriella subcapitata. The purpose of this study was to determine the effect of the test item with 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 six test concentrations in a geometric series (with a separation factor of 2.8) were tested: 0.2, 0.7, 1.8, 5.1, 14.3 and 40.0 mg/L (nominal). Untreated- and humic acid-control (10 mg/L) was run 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: 0.14, 0.36, 1.68, 3.10, 8.23 and 30.60 mg/L. All biological results are based on the measured geometric mean 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 concentrations of 1.68, 3.10, 8.23 and 30.60 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 0.36 mg/L. The EC values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software. The 72-h ErC10 was determined to be 1.55 mg/L and the 72 -h ErC50 as 2.11 mg/L. The 72 -h EyC10 was determined to be 1.26 mg/L and the 72 -h EyC50 as 1.83 mg/L.

Growth Inhibition Test of Reaction products of IPDA with bisphenol A diglycidylether (BADGE) on Algae

A read-across study according to OECD guideline 201, EC Regulation C.3 and OPPTS 850.5400 was conducted to determine the aquatic toxic potential of the Reaction products of IPDA with bisphenol A diglycidylether (BADGE) 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.

Disregarded

Growth Inhibition test of Reaction Product of Bisphenol A diglycidylether (BADGE) with IPDA and MXDA on Algae

This study was not taken into account for risk assessment and classification as the read across studies better simulate the natural aquatic environment. Reaction Product of Bisphenol A diglycidylether (BADGE) with IPDA and MXDA was assessed in a toxicity to aquatic algae and cyanobacteria study, according to EU guideline C.3 Algal Inhibition test and OECD guideline 201 Alga, Growth Inhibition Test. Exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to a range of concentrations of the test item 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 measured test item concentrations were: 0.03; 0.08; 0.28; 0.86 and 3.78 mg/L. Results are reported as measured concentrations. The 72 -h EC50 based on growth rate was determined at 0.4 mg/L. The 72-h NOEC was determined to be 0.08 mg/L.