Reaction mass of methyl 2-[[(E)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate and methyl 2-[[(Z)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

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:

06 March 2017 to 20 April 2017

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and the control (containing algae).
- Sampling method: Duplicate samples of the freshly prepared test media (containing algae) were taken. Samples were also taken after 6, 24 and 48 hours and at the end of the test (after the 72 hour test period).
- Sample storage conditions before analysis: All samples were diluted by a factor of two with acetonitrile and were stored in a freezer (≤ -20 °C), protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The highest test item concentration of nominal 100 mg test item/L was prepared by mixing 93.2 mg test item into 932 mL test water. The solution was carefully shaken for 1 hour to dissolve as much of the test item as possible. After cessation of shaking and a following period (30 min) of settling to allow phase separation, the aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium of the corresponding nominal test concentration. Adequate volumes of this water accommodated fraction were mixed into test water to obtain the desired dilutions. The test media were prepared just before introduction of the algae (= start of the test).
- Controls: Dilution water control

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: KORSHIKOV, Strain No. 61.81 SAG
- Age of inoculum: Exponentially growing pre-culture
- Method of cultivation: The algae were cultivated in the laboratories under standardised conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: Same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (24 mg/L) as CaCO3

Test temperature:

22.2 to 23.0 °C

pH:

8.1 to 8.2 at test start
9.7 to 10.1 at test end

Nominal and measured concentrations:

Water accommodated fractions of the oversaturated stock solution of nominal 100 mg/L and dilutions of 1:3, 1:9, 1:27 and 1:81 were tested, corresponding to initial mean measured test concentrations of 7.94, 2.38, 0.761, 0.250 and 0.0586 mg test item/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks.
- Type: Closed
- Material, size, headspace, fill volume: 50 mL test volume.
- Initial cells density: 5000 algal cells per mL test medium.
- Control end cells density: 664910 cells/mL.
- No. of vessels per concentration: 3 (plus 3 additional replicates of each test item concentration for analytical dose verification after 6, 24 and 48 hours and 1 additional replicate of each test concentration without algae to provide a "blank" for the spectrophotometric measurements).
- No. of vessels per control: 6 (plus 3 additional replicates of each test item concentration for analytical dose verification after 6, 24 and 48 hours and 1 additional replicate of each test concentration without algae to provide a "blank" for the spectrophotometric measurements).

GROWTH MEDIUM
- Standard medium used: Yes, OECD Medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water
- Intervals of water quality measurement: Temperature and appearance of the test item in the test media was recorded at the start of the test and after 24, 48 and 72 hours of exposure. pH was measured at the start and end of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: The pH was adjusted with 1 M HCl to pH 8.1.
- Photoperiod: Continuous illumination.
- Light intensity and quality: 6250 to 7170 lux (mean 6688 lux).

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: Spectrophotometer, cell density was measured after 24, 48 and 72 hours of exposure.
- Other: Algal cells were examined microscopically after 72 hours of exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Range finding study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 7.94 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 7.94 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 7.94 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence interval: 4.03 to >7.94 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.75 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.38 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

yield

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to the time weighted average test concentration of 7.94 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.
- Colour differences: In the highest test concentration of time weighted average test concentrations of 5.95 mg test item/L, the test item caused slightly yellowish coloration only at test start.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 0.549 mg/L for yield, 1.12 mg/L for growth rate and 0.567 mg/L for biomass, after 72 hours exposure.
- Other: Test performed in November/December 2016, project number 110664210.

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hour median effect concentration for growth rate (ErC50) and yield (EyC50), the corresponding 20% and 10% effect concentrations (EC20 and EC10, respectively) and where possible their 95 %-confidence limits were calculated with a 3-parametric normal distribution function. For the determination of the 72 hours LOEC and the 72 hours NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test (yield) and Step-down Jonckheere Terpstra test (growth rate). The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Table 1. Mean Algal Cell Densities during the Test Period of 72 Hours

Initial mean measured concentration (mg test item/L)	Mean density of algal cells (10000/mL)
	24 hours	48 hours	72 hours
Control	2.360 (SD 0.435)	14.313 (SD 4.105)	66.491 (SD 10.600)
0.0586	2.133 (SD 0.151)	17.210 (SD 2.115)	79.849 (SD 4.827)
0.250	1.277 (SD 0.087)	14.495 (SD 2.282)	75.529 (SD 13.200)
0.761	1.881 (SD 0.231)	13.921 (SD 3.613)	67.116 (SD 11.819)
2.38	1.478 (SD 0.349)	10.789 (SD 1.675)	61.830 (SD 1.805)
7.94	1.378 (SD 0.151)	8.440 (SD 1.455)	48.757 (SD 8.061)

SD: Standard Deviation

Table 2. Yield and Percentage Inhibition during the Test Period of 72 Hours

Initial mean measured concentration (mg test item/L)	24 hours		48 hours		72 hours
	Mean yield (10000 cells/mL)	Inhibition (%)	Mean yield (10000 cells/mL)	Inhibition (%)	Mean yield (10000 cells/mL)	Inhibition (%)
Control	1.860 (SD 0.435)	-	13.813 (SD 4.105)	-	65.991 (SD 10.600)	-
0.0586	1.633 (SD 0.151)	12.2	16.710 (SD 2.115)	-21.0	76.349 (SD 4.827)	-20.2
0.250	0.777 (SD 0.087)	58.2*	13.995 (SD 2.282)	-1.3	75.029 (SD 13.200)	-13.7
0.761	1.381 (SD 0.231)	25.7*	13.421 (SD 3.613)	2.8	66.616 (SD 11.819)	-0.9
2.38	0.978 (SD 0.349)	47.4*	10.289 (SD 1.675)	25.5	61.330 (SD 1.805)	7.1
7.94	0.878 (SD 0.151)	52.8*	7.940 (SD 1.455)	42.5*	48.257 (SD 8.061)	26.9*

SD: Standard Deviation

Negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control

* Mean value significantly different from the control (tested with Williams t-test, α = 0.05, one-sided)

Table 3. Growth Rate and Percentage Inhibition during the Test Period of 72 Hours

Initial mean measured concentration (mg test item/L)	24 hours		48 hours		72 hours
	Mean growth rate (1/day)	Inhibition (%)	Mean growth rate (1/day)	Inhibition (%)	Mean growth rate (1/day)	Inhibition (%)
Control	1.536 (SD 0.195)	-	1.661 (SD 0.139)	-	1.626 (SD 0.054)	-
0.0586	1.449 (SD 0.071)	5.7	1.767 (SD 0.062)	-6.4	1.691 (SD 0.021)	-3.9
0.250	0.936 (SD 0.067)	39.1*	1.679 (SD 0.082)	-1.1	1.669 (SD 0.060)	-2.6
0.761	1.320 (SD 0.121)	14.1*	1.653 (SD 0.124)	0.5	1.630 (SD 0.056)	-0.2
2.38	1.063 (SD 0.257)	30.8*	1.532 (SD 0.080)	7.8	1.606 (SD 0.010)	1.3
7.94	1.010 (SD 0.110)	34.3*	1.408 (SD 0.091)	15.2*	1.523 (SD 0.058)	6.3*

SD: Standard Deviation

Negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control

* Mean value significantly different from the control (tested with Step-down Jonckheere-Terpstra test, α = 0.05, one-sided)

Validity criteria fulfilled:

yes

Remarks:

In the controls, the 72-hour cell density increase was 133.0-fold (must be ≥ 16), the coefficient of variation (CV) of sectional (daily) growth rates was 13.1% (must be ≤ 35%) and the CV of average growth between replicates was 3.3% (must be ≤ 7%).

Conclusions:

The 72-hour EC50 and EC10 values for growth rate were determined to be > 7.94 mg test item/L, based on initial mean measured concentrations. The 72-hour EC50 and EC10 values for yield were calculated to be >7.94 and 1.75 mg test item/L, respectively. The 72-hour NOEC was determined to be 2.38 mg test item/L for both growth rate and yield.

Executive summary:

The influence of the test item on the growth of the freshwater green algae (Pseudokirchneriella subcapitata) was assessed in a 72 -hour static test according to OECD Guideline 201 and EU Method C.4. Water accommodated fractions of the oversaturated stock solution of nominal 100 mg/L test item and dilutions of 1:3, 1:9, 1:27 and 1:81 were tested, corresponding to initial mean measured test concentrations of 7.94, 2.38, 0.761, 0.250 and 0.0586 mg test item/L. The test was conducted under closed conditions with a dilution water control and additional replicates of the test item solutions and control for analytical dose verification and spectrophotometric blanks. The test item and its hydrolysis product were quantified by LC-MS/MS. The 72-hour EC50 and EC10 values for growth rate were determined to be > 7.94 mg test item/L. The 72-hour EC50 and EC10 values for yield were calculated to be >7.94 and 1.75 mg test item/L, respectively. The 72-hour NOEC was determined to be 2.38 mg test item/L for both growth rate and yield. The test item concentrations decrease significantly within 24 hours, whereas the concentrations of the hydrolysis product increase significantly over all test concentrations. Since the biological inhibition was observed mainly in the first 24 hours of the test, it is suggested the inhibition is caused mainly by the test item rather than the hydrolysis products. This study is considered to be reliable without restriction (Klimisch 1) as it was conducted according to guideline with no limitations in experimental design or reporting.

Description of key information

The 72-hour EC50 and EC10 values for growth rate were determined to be > 7.94 mg test item/L, based on initial mean measured concentrations. The 72-hour EC50 and EC10 values for yield were calculated to be >7.94 and 1.75 mg test item/L, respectively. The 72-hour NOEC was determined to be 2.38 mg test item/L for both growth rate and yield.

Key value for chemical safety assessment

EC50 for freshwater algae:

7.94 mg/L

EC10 or NOEC for freshwater algae:

7.94 mg/L

Additional information

The influence of the test item on the growth of the freshwater green algae (Pseudokirchneriella subcapitata) was assessed in a 72-hour static test according to OECD Guideline 201 and EU Method C.4 (2017). Water accommodated fractions of the oversaturated stock solution of nominal 100 mg/L test item and dilutions of 1:3, 1:9, 1:27 and 1:81 were tested, corresponding to initial mean measured test concentrations of 7.94, 2.38, 0.761, 0.250 and 0.0586 mg test item/L. The test was conducted under closed conditions with a dilution water control and additional replicates of the test item solutions and control for analytical dose verification and spectrophotometric blanks. The test item and its hydrolysis product were quantified by LC-MS/MS. The 72-hour EC50 and EC10 values for growth rate were determined to be > 7.94 mg test item/L. The 72-hour EC50 and EC10 values for yield were calculated to be >7.94 and 1.75 mg test item/L, respectively. The 72-hour NOEC was determined to be 2.38 mg test item/L for both growth rate and yield. The test item concentrations decrease significantly within 24 hours, whereas the concentrations of the hydrolysis product increase significantly over all test concentrations. Since the biological inhibition was observed mainly in the first 24 hours of the test, it is suggested the inhibition is caused mainly by the test item rather than the hydrolysis products. This study is considered to be reliable without restriction (Klimisch 1) as it was conducted according to guideline with no limitations in experimental design or reporting.