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 and oxydipropanol

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23 (2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: September 2015; signature: November 2015

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Definitive test: nominal concentrations: Solutions containing 0 (control), 10, 18, 32, 56 and 100 mg/L prepared as WAF: controls; Test medium without test substance or other additives.
- Sampling method: UPLC-analyses was conducted. See 'Details on analytical methods for more information'
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test item loadings were prepared as Water Accommodated Fraction (WAF). Before the preparation of test solutions, the test item was heated to 50 ⁰C in order to facilitate weighing. All test solutions were prepared separately at loading rates ranging from 1.0 to 100 mg/L (after correcting for the purity of the test item). After a 17-19 hour period of magnetic stirring to reach maximum solubility of the test item in test medium, the resulting mixtures were left to stabilize for one 60 – 92 minutes. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were siphoned off. The final test solutions were all clear and colourless (combined limit/range-finding test) or clear and ranged from colourless at the three lowest concentrations to slightly yellow at the two highest concentrations (final test).
- Controls: Algae was exposed to each loading level and a control. Which consisted of: test medium without test item or other additives tested under the same conditions as the test groups. Further a reference test was also conducted (non-concurrently) under static conditions to demonstrate the validity of the test system.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution): five Water Accommodated Fractions (WAF) of the test item with loading levels in the range of 0 (control), 10, 18, 32, 56 and 100 mg/L prepared in a series for each test item loading. All water accommodated fractions were analytically verified via analysis.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): 3 days (in pre-culture under the same conditions as the test).
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of
1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Culturing media and conditions (same as test or not): No.
Stock Culture Medium M1 ; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis) with the following composition: NaNO3 500 mg/L; K2HPO4.3H2O 52 mg/L; MgSO4.7H2O 75 mg/L; Na2CO3.10H2O 54 mg/L; C6H8O7.H2O 6 mg/L; NH4NO3 330 mg/L; CaCl2.2H2O 35 mg/L; C6H5FeO7.xH2O 6 mg/L; H3BO3 2.9 mg/L; MnCl2.4H2O 1.81 mg/L; ZnCl2 0.11 mg/L; CuSO4.5H2O 0.08 mg/L; (NH4)6Mo7O24.4H2O 0.018 mg/L
Pre-Culture and definitive test Medium M2 : NH4Cl 15 mg/L; MgCl2.6H2O 12 mg/L; CaCl2.2H2O 18 mg/L; MgSO4.7H2O 15 mg/L; KH2PO 1.6 mg/L; FeCl3.6H2O 64 µg/L; Na2EDTA.2H2O 100 µg/L; H3BO3 185 µg/L; MnCl2.4H2O 415 µg/L; ZnCl2 3 µg/L; CoCl2.6HO 1.5 µg/L; CuCl2.2H2O 0.01 µg/L; Na2MoO4.2H2O 7 µg/L; NaHCO3 50 mg/L; Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/l); pH 8.1 ± 0.2
- Any deformed or abnormal cells observed: None reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Ca+Mg: 0.24 mmol/l (24 mg CaCO3/l)

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 21.0 and 23.0 °C. Temperature remained within the limits prescribed (21-24°C, constant within 2°C).

pH:

0 hours: pH 8.1 (definitive test concentrations) and pH 8.1 (controls)
72 hours: pH 8.1-8.2 (definitive test concentrations) and pH 8.2 (controls).
pH did not vary more than 1.5 units.

Nominal and measured concentrations:

Combined range-finding / limit test: nominal concentrations: 1.0, 10.0 and 100.0 mg/L (prepared as WAF); plus Control containing no test substance or other additives.
Samples taken from test solutions containing WAFs prepared at 10 and 100 mg/L were analysed. The initial test item concentrations were 5.3 and 46 mg/L, respectively, and remained stable during the test period (92-95% of initial after 72 hours
Definitive Test: nominal concentrations: 0 (control), 10, 18, 32, 56 and 100 mg/L prepared as WAF: controls; Test medium without test substance or other additives.
Concentrations were measured to demonstrate the presence and stability of the test item in test medium. At the start of the test, the measured test concentrations were 4.9, 5.3, 17, 26 and 42 mg/L in WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L, respectively. The measured concentrations remained stable during the first 24 hours of exposure (88-103% of initial). At the end of the test, they had decreased to 0.20-55% of initial. In the WAF prepared at 18 mg/L, the measured concentration was below the limit of detection of the analytical method (LOD) at the end of the exposure period. Because the extent of decrease was random across the concentration range, it was decided to measure the reserve samples, which showed similar fluctuations. Given this result, the Time Weighted Average (TWA) measured exposure concentrations were calculated.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass.
- Type (delete if not applicable): Closed - Static
- Material, size, headspace, fill volume: Glass, 100 ml, containing 50 ml test solution
- Aeration: Vessel shaken continuously.
- Initial cells density: 1 x 10^4 cells/ml.
- Control end cells density: Mean (of replicates after 72 hours) 183 x10^4 cells/ml
- No. of vessels per concentration (replicates): 3 replicates of each test concentration. Further 1 or 2 replicates of each test group without algae.
- No. of vessels per control (replicates): 6 replicates of the control.
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: Yes. Medium M2.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis)
- Culture medium different from test medium: Yes. Medium M1 used for stock culture medium. Medium M2 used for pre-culture medium and test medium. See above for more information.
- Intervals of water quality measurement: Not reported.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 90 to 96 µE/m2/s.
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Spectrophotometer at 680 nm with immersion probe (pathlength = 20mm). Algal medium used as blank. The extra replicates without algae were used as turbidity control.
- Other: Initial cell density: Cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Concentrations were 1.0, 10.0 and 100.0 mg/L prepared as WAFs for a combined limit/range-finding test. The test concentrations in the definitive test were chosen based on the range finding study: at loading rates of 10, 18, 32, 56 and 100 mg/L prepared as WAFs.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes.
- Test concentrations: Three or six replicates per concentration were exposed to dilutions representing 1.0, 10 and 100 mg/L in the combined limit/range-finding test. 2 or 3 extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: N.D. ; loading rates based on WAF

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: n.d. ; loading rates based on WAF

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: n.d. ; loading rates based on WAF

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

44 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 19 - 58 mg/L; loading rates based on WAF

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): Yes (biomass parameter = 183)
- Observation of abnormalities (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the algae cells exposed to the 100 mg/L prepared as WAF when compared to the control.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: No.
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: Yes. At low concentrations 10, 18 and 56 mg/L nominal WAF loading a growth rate and yield inhibition was negative between 24h-48h. This low dose stimulation was limited and was taken into account in the data analysis and effect level calculations.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate inhibition (72h-ERC50) was 1.5 mg/L with a 95% confidence interval ranging from 1.4 to 1.6 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range. The EC50 for yield decrease (72h-EYC50) was 0.51 mg/L with a 95% confidence interval ranging from 0.49 to 0.53 mg/L. The historical ranges for yield decrease lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

For determination of the NOEL and the EL50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller). Additionally, the EL10 and EL20 values for yield inhibition were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. Calculation of ELx values were based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding TWA exposure concentrations of the test item.

Test for Normality
- Results Growth rate: Number of residues =21; Shapiro-Wilk's W = 0.926; p(W) = 0.114
p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution. Normality check passed (p > 0.01). Williams Multiple Sequential t-test Procedure gave a significant effect however judged to be biologically irrelevant (<10%) effect at 100 mg/L prepared as WAF.
- Results for Yield: Number of residues =21; Shapiro-Wilk's W = 0.950; p(W) = 0.334
p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution. Normality check passed (p > 0.01). Yield was statistically reduced at the highest concentration, i.e. was 36%.

Table 1. Mean cell densities (x10^4 cells/ml) during the combined limit/range-finding test

Time (h)	Test substance, loading rate (mg/L) prepared as WAF
	Control	1.0	10.0	100.0
0	1.0	1.0	1.0	1.0
24	5.3	n.d.	n.d.	4.5
48	24.2	n.d.	n.d.	21.3
72	138.7	167.9	147.1	102.0

 

Table 2. Percentage reduction of growth rate and inhibition of yield during the combined limit/range-finding test

Test item WAF loading rate (mg/L)	Percentage inhibition of growth rate at different time intervals during the final test							Percentage inhibition of yield (total test period) during the final test
		0-24h		24-48h		48-72h
	n	mean	% inhibition	mean	% inhibition	Mean	%inhibition	n	mean	Std. dev.	% inhib
Control	6	1.897	-	1.688	-	1.621	-	6	182.3	20.00	-
10	3	1.682	11.3	1.822	-7.9	1.621	0.0	3	167.7	15.55	8.0
18	3	1.843	2.9	1.766	-4.6	1.603	1.1	3	183.2	18.70	-0.5
32	3	1.822	4.0	1.656	1.9	1.596	1.6	3	159.6	17.77	13
56	3	1.797	5.3	1.744	-3.3	1.600	1.3	3	170.5	16.57	6.5
100	3	1.693	10.8	1.573	6.8	1.497	7.7	3	116.3	8.62	36*

*statistically significant

Validity criteria fulfilled:

yes

Conclusions:

The test substance 72h-EL50 was > 100 mg/L based on growth rate and nominal loadings into the WAF test system. The corresponding EL10 and NOELR were also > 100 mg/L.

Executive summary:

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines. Water Accommodated Fractions (WAF) were prepared individually at loading rates ranging from 10 to 100 mg/L in test medium (after correcting for the purity of the test item). Final test solutions were clear and ranged from colourless at the three lowest concentrations to slightly yellow at the two highest concentrations. A correction factor was applied to account for the purity/composition of the test item. A final test was performed based on the results of a combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. The initial cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure. Concentrations were measured to demonstrate the presence and stability of the test item in test medium. At the start of the test, the measured test item concentrations were 4.9, 5.3, 17, 26 and 42 mg/L in WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L, respectively. The measured concentrations remained stable during the first 24 hours of exposure (88-103% of initial). At the end of the test, they had decreased to 0.20-55% of initial. The study met the acceptability criteria prescribed and was considered valid. Under the conditions of the study with Pseudokirchneriella subcapitata, no inhibition of growth rate was recorded at any of the concentrations of the test item tested (up to 100 mg/L prepared as WAF). A statistically significant inhibition of Yield was observed at the highest concentration (36%). The EL10 for growth rate inhibition (72h-ERL10) was beyond the range tested, i.e. exceeded a concentration obtained at a WAF loading rate of 100 mg/L. Therefore, the 72h-ERL10 was > 100 mg/L. The EL10 for yield inhibition (72h-EYL10) was 44 mg/L with a 95% confidence interval ranging from 19 to 58 mg/L. The EL50 for growth rate inhibition (72h-ErL50) and the EL50 for yield inhibition (72h-EyL50) exceeded the loading rate of 100 mg/L. No EL50-values could be estimated because the test substance proved to be non-toxic (EL50 > maximum loading rate). The 72h-NOEL for growth rate inhibition was 100 mg/L based on biological relevance, while the 72h-NOEL for yield inhibition was 56 mg/L.

Description of key information

ErL50 (algae; growth rate) = > 100 mg/L ; WAF loading 72hour-freshwater, OECD TG 201, 2016

ErL10 (algae; growth rate)= > 100 mg/L ; WAF loading 72 hour-freshwater, OECD TG 201, 2016

NOELR (algae; growth rate) = > 100 mg/L; WAF loading, 72 hour-freshwater, OECD TG 201, 2016

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines. Water Accommodated Fractions (WAF) were prepared individually at loading rates ranging from 10 to 100 mg/L in test medium (after correcting for the purity of the test item). Final test solutions were clear and ranged from colourless at the three lowest concentrations to slightly yellow at the two highest concentrations. A correction factor was applied to account for the purity/composition of the test item. A final test was performed based on the results of a combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. The initial cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure. Concentrations were measured to demonstrate the presence and stability of the test item in test medium. At the start of the test, the measured test item concentrations were 4.9, 5.3, 17, 26 and 42 mg/L in WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L, respectively. The measured concentrations remained stable during the first 24 hours of exposure (88-103% of initial). At the end of the test, they had decreased to 0.20-55% of initial. The study met the acceptability criteria prescribed and was considered valid. Under the conditions of the study with Pseudokirchneriella subcapitata, no inhibition of growth rate was recorded at any of the concentrations of the test item tested (up to 100 mg/L prepared as WAF). A statistically significant inhibition of Yield was observed at the highest concentration (36%). The EL10 for growth rate inhibition (72h-ERL10) was beyond the range tested, i.e. exceeded a concentration obtained at a WAF loading rate of 100 mg/L. Therefore, the 72h-ERL10 was > 100 mg/L. The EL10 for yield inhibition (72h-EYL10) was 44 mg/L with a 95% confidence interval ranging from 19 to 58 mg/L. The EL50 for growth rate inhibition (72h-ErL50) and the EL50 for yield inhibition (72h-EyL50) exceeded the loading rate of 100 mg/L. No EL50-values could be estimated because the test substance proved to be non-toxic (EL50 > maximum loading rate). The 72h-NOEL for growth rate inhibition was 100 mg/L based on biological relevance, while the 72h-NOEL for yield inhibition was 56 mg/L.