Reaction products of 4,4'-methylenebis(2,6-dimethylaniline) and 4-aminobenzenesulfonic acid with 2,4,6-trichloro-1,3,5-triazine, diazotised and subsequently coupled with N-(2-methoxyphenyl)-3-oxobutanamide

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:

21st July 2020 - 24th July 2020

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:

23 March 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1 March 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For determination of the test material concentrations, samples were taken from each concentration level and control at the start, on day 1, day 2 and at the end of the test. For these daily measurements, one extra replicate was included in each test group which was treated same as the replicates used for determination of the algal cells.
At the start and at the end of the experiment three replicate samples (5 mL) were taken from each test concentration and six replicate samples (5 mL) from the control group were taken. On days 1 and 2, two replicate samples (5mL) were taken for analytical measurements.

Vehicle:

no

Details on test solutions:

PRELIMINARY RANGE-FINDING TEST
The test material solutions (0.1, 1, 10 and 100 mg/L) were prepared by suspending the appropriate amount of the test material in OECD Medium and handled in the ultrasonic bath for 10 minutes. The test solutions were then mixed for 24 hours, and filtered through a membrane filter (0.45 μm). A test solution of 0.01 mg/L was prepared by the 10-fold dilution from the filtrate of the 0.1 mg/L test solution.

PREPARATION OF TEST SOLUTIONS FOR MAIN TEST
The test solutions (9.766, 31.25 and 100 mg/L) were prepared by mechanical dispersion without the use of a solubilising agent. Preparation of the test material solutions was performed using the WAF method. The test solutions (0.093, 0.298, 0.954 and 3.052 mg/L) were prepared by the appropriate dilution from the filtrate of the 9.766 mg/L test solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain number: 61.81 SAG
- Origin: SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany.
- Breeding: 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.
- Pre-culturing: The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing. The pre-culture was incubated for four days before 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

Test temperature:

The temperature was between 22.6 and 22.9°C measured in the flask and in the range of 21.8 – 23.8°C measured within the climate chamber.

pH:

7.83 – 9.77

Nominal and measured concentrations:

Nominal concentrations: 0.093, 0.298, 0.954, 3.052, 9.766, 31.25 and 100 mg/L
Measured concentrations: 0.0010, 0.0024, 0.0057, 0.0137, 0.0325, 0.0773, 0.1838 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask (250 mL) covered with air-permeable stoppers.
- Volume of test liquid in vessel: 100 mL
- Number of replicates per test concentration: 3
- Number of replicates per test concentration in control group: 6
- Initial cells density: 10^4 cells/mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water in the experiment. Separate stock solutions were first prepared in deionised water. The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations in the prepared algal growth medium.
- Stock solution 1: NH4Cl (15.0 mg/L), MgCl2 (5.6 mg/L), CaCl2 × 2 H2O (18.0 mg/L), MgSO4 × 7 H2O (15.0 mg/L) and KH2PO4 (1.6 mg/L).
- Stock solution 2: FeCl3 × 6 H2O (64.0 μg/L), Na2EDTA × 2H2O (100.0 μg/L).
- Stock solution 3: H3BO3 (185.0 μg/L), MnCl2 × 4 H2O (415.0 μg/L), ZnCl2 (3.0 μg/L), CoCl2 × 6 H2O (1.5 μg/L), CuCl2 × 2 H2O (0.01 μg/L), Na2MoO4 × 2 H2O (7.0 μg/L).
- Stock solution 4: NaHCO3 (50.0 mg/L).

OTHER CONDITIONS:
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity measured: 8092 lux.

EFFECT PARAMETERS MEASURED
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.

CALCULATIONS
The calculations were performed using Microsoft Excel for Windows software.
Average specific growth rate and yield were calculated for each test flask. Then the mean growth rate and mean yield were determined as arithmetic mean value over all test flasks per treatment.
The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test; days 0-1, 1-2 and 2-3) to demonstrate exponential growth for the entire study period.
The inhibition of algae growth was determined from the average specific growth rate and yield using the following equations:
- Average specific growth rate (μ):
The average specific growth rate (in day^-1) for a specific period is calculated as follows:

μi-j = (ln Xj - ln X i) / (tj - ti)

where:
μi-j = average specific growth rate from time i to j
Xj = biomass at time j
Xi = biomass at time i

Percent inhibition of growth rate (% Ir) = [(µC - µT) / µC] x 100

where:
μC = mean value for average specific growth rate in the control group
μT = average specific growth rate for the treatment replicate

- Yield (Y):
Yield is calculated as the biomass at the end of the test minus the starting biomass for each single vessel of controls and treatments. For each test concentration and control, mean yield value was calculated.

Percent inhibition in yield (% Iy): = [(Yc - YT) / Yc] x 100

where:
Yc = mean value for yield in the control group
YT = value for yield for the treatment replicate

TEST CONCENTRATIONS
Range finding study
- Test concentrations: 0.1, 1, 10, and 100 mg/L
- Results used to determine the conditions for the definitive study: yes. The algal cells were exposed to the test material solutions and a control for 72 hours. The test was performed with two replicates per test groups and three replicates in the control.
Based on the results of the preliminary range-finding test seven test concentrations in a geometric series with a spacing factor of 3.2 and one control were used in the main test.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.004 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.012 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.076 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.:

0.298 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.954 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

ANALYTICAL RESULTS
In the untreated control group the test material was not detected and the measured concentrations were not within ± 20 % during the experiment, therefore the biological results were based on the calculated test material concentrations. As the mean measured test material concentration in the five lowest test groups was < LOQ (limit of quantification), it was calculated by the factor between the two highest detected test concentrations.

BIOLOGICAL RESULTS
As the mean measured test material concentration in the five lowest test groups was < LOQ (limit of quantification), it was calculated by the factor between the two highest detected test concentrations. Biological results and endpoints were based on the calculated (measured) test material concentrations.
- Average Specific Growth Rate
The results of the statistical evaluation (Dunnett’s Test, α = 0.05) showed that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value at the concentrations of 0.0057, 0.0137, 0.0325, 0.0773 and 0.1838 (nominal 0.954, 3.052, 9.766, 31.25 and 100) mg/L.
Accordingly, the 72-h NOEC based on growth rate was determined to be 0.0024 (nominal 0.298) mg/L, while the 72-h LOEC based on growth rate was determined to be 0.0057(nominal 0.954) mg/L.
The 72-h ErC50 value was determined to be 0.076 mg/L (95 % confidence limits: 0.057 – 0.109 mg/L), (Probit analysis).
- Yield
The results of the statistical evaluation (Dunnett’s Test, α = 0.05) showed that the 0-72 h yield was statistically significantly different from the untreated control value at the concentrations of 0.0057, 0.0137, 0.0325, 0.0773 and 0.1838 (nominal 0.954, 3.052, 9.766, 31.25 and 100) mg/L.
Accordingly, the 72-h NOEC based on yield was determined to be 0.0024 (nominal 0.298) mg/L, while the 72-h LOEC based on yield was determined to be 0.0057(nominal 0.954) mg/L.
The 72 h EyC50 value was determined to be 0.010 mg/L (95 % confidence limits: 0.008 – 0.013 mg/L) (Probit analysis).
- Morphological Changes of Algal Cells
No abnormal shape of algal cells was noticed in the control and at the test material concentrations during the experiment. As sign of toxicity swollen cells were found between normal cells in the test groups of 0.0057 (nominal 0.954), 0.0137 (nominal 3.052), 0.0325 (nominal 9.766), 0.0773 (nominal 31.25) and 0.1838 (nominal 100) mg /L.

Results with reference substance (positive control):

For the evaluation of the reliability of the applied test system and the experimental conditions potassium dichromate is tested at least twice a year.
The date of the latest study performed in the laboratory of TOXI-COOP ZRT. (Study number: 392-201-5405) with the reference item Potassium dichromate was: 03 – 06 March 2020, Final Report issued: 16 April 2020.
The 72h ErC50: 0.52 mg/L, (95 % confidence limits: 0.46 – 0.58 mg/L)
The 72h EyC50: 0.31 mg/L, (95 % confidence limits: 0.28 – 0.35 mg/L)

Reported statistics and error estimates:

LOEC AND NOEC
The calculated mean growth rate and yield at the test concentrations were tested on significant differences to the control values using Dunnett t Test (2-sided, α = 0.05) by statistical software program SPSS PC+.

ECx VALUES
Probit analysis by statistical software program SPSS PC+ was performed.

Summary of the Biological Endpoints

Endpoints (0-72 h)	Growth rate (μ) (mg/L)	Yield (y) (mg/L)
	Calculation based on calculated (measured) concentrations
EC10 95 % conf. limits	0.004	0.001
	0.003 – 0.006	0.001 – 0.002
EC20 95 % conf. limits	0.012	0.002
	0.008 – 0.015	0.001 – 0.003
EC50 95 % conf. limits	0.076	0.010
	0.057 – 0.109	0.008 – 0.013
NOEC	0.0024 (nominal: 0.298)	0.0024 (nominal: 0.298)
LOEC	0.0057 (nominal: 0.954)	0.0057 (nominal: 0.954)

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, and based on growth rate, the 72 hour ErC10 was 0.004 mg/L. The 72 hour ErC20 was 0.012 mg/L. The 72 hour ErC50 was 0.076 mg/L. The NOErC was 0.0024 mg/L (nominal: 0.298 mg/L). The LOErC was 0.0057 (nominal: 0.954 mg/L).

Executive summary:

The acute toxicity of the test material on the growth of Raphidocelis subcapitata was assessed under GLP conditions and according to the OECD Guideline for Testing of Chemicals No. 201, Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals, OECD No. 23.

During the study, exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test material 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 design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 10^4 cells/mL at the start of the test in all of the test cultures. The alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals. The cell morphology was examined in parallel.

Under the conditions of the study, and based on growth rate, the 72 hour ErC10 was 0.004 mg/L. The 72 hour ErC20 was 0.012 mg/L. The 72 hour ErC50 was 0.076 mg/L. The NOErC was 0.0024 mg/L (nominal: 0.298 mg/L). The LOErC was 0.0057 (nominal: 0.954 mg/L).

All validity criteria were met.

Description of key information

Under the conditions of the study, and based on growth rate, the 72 hour ErC10 was 0.004 mg/L. The 72 hour ErC20 was 0.012 mg/L. The 72 hour ErC50 was 0.076 mg/L. The NOErC was 0.0024 mg/L (nominal: 0.298 mg/L). The LOErC was 0.0057 (nominal: 0.954 mg/L).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.076 mg/L

EC10 or NOEC for freshwater algae:

0.004 mg/L

Additional information

The acute toxicity of the test material on the growth of Raphidocelis subcapitata was assessed under GLP conditions and according to the OECD Guideline for Testing of Chemicals No. 201, Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals, OECD No. 23. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test material 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 design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 10^4 cells/mL at the start of the test in all of the test cultures. The alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals. The cell morphology was examined in parallel.

Under the conditions of the study, and based on growth rate, the 72 hour ErC10 was 0.004 mg/L. The 72 hour ErC20 was 0.012 mg/L. The 72 hour ErC50 was 0.076 mg/L. The NOErC was 0.0024 mg/L (nominal: 0.298 mg/L). The LOErC was 0.0057 (nominal: 0.954 mg/L).

All validity criteria were met.