1-methoxy-4-[3-methyl-4-(2-phenylethoxy)but-3-en-1-yl]benzene

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:

02-08-2021 to 05-08-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: November 2019 ; signature: August 2020

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All relevant concentration levels and the control were analytically verified via GC-MS at the start (0 hours) and at the end of the exposure (72 hours). A saturated solution with a nominal loading rate of 10 mg test item/L was prepared once at room temperature 72 ± 1 hour prior to the start of the exposure. Five dilutions were tested in a geometric series with a dilution factor of two (2) : 0 (control), 6.25, 12.5, 25.0, 50.0 and 100 % saturated solution. This corresponded to nominal loadings rates of: 0 (control), 0.625, 1.25, 2.5, 5.0 and 10 mg/L of the saturated solution and/or geometric mean measured test item concentrations of: 0 (control), 0.327, 0.696, 1.46, 3.77 and 10.1 μg/L of parent test item (used as a tracer). [Alternative units in mg/L are: 0 (control), 0.000327, 0.000696, 0.00146, 0.00377 and 0.0101 mg/L as geometric mean measured concentrations of parent test item used as a tracer].
- Sampling method: Analytical evaluation of the concentrations of the test item were carried out via GC-MS from freshly prepared media after 0 hours (with algae) and old test media after 72 hours (with algae) of exposure. Three replicates per concentration and six for the control (without test item) were prepared. Separate replicates for each measuring time were prepared. All samples for the test item analysis were taken from additionally prepared replicates with algae. The method was validated prior to this study with regard to linearity, accuracy, precision and specificity according to SANTE/2020/12830 rev.1 (2020).
- Sample storage conditions before analysis: All samples were immediately extracted and the extracts were stored at room temperature (ca. 20 ± 2 °C). Until the start of the analysis the prepared samples were stored on an autosampler at ambient room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution with a nominal loading rate of 10 mg test item/L was prepared once at room temperature 72 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out onto a curved glass slide. The glass slide with the test item was inserted in a glass flask with an appropriate amount of dilution water. A slow stirring procedure was applied for 72 ± 1 hour at room temperature. The magnetic stirrer bar was placed with a fish-clip system a few centimetres above the bottom of the flask to prevent direct contact with the test item on the bottom. After a separation phase of 1 hour, the saturated solution was removed by siphoning (from the approximate middle of the glass flask) and was used in the test. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. No presence of undissolved test item during the test was observed. Five dilutions were tested in a geometric series with a dilution factor of two (2) : 0 (control), 6.25, 12.5, 25.0, 50.0 and 100 % saturated solution. This corresponded to nominal loadings rates of: 0 (control), 0.625, 1.25, 2.5, 5.0 and 10 mg/L of the saturated solution and/or geometric mean measured test item concentrations of: 0 (control), 0.327, 0.696, 1.46, 3.77 and 10.1 μg/L of parent test item (used as a tracer). [Alternative units in mg/L are: 0 (control), 0.000327, 0.000696, 0.00146, 0.00377 and 0.0101 mg/L as geometric mean measured concentrations of parent test item used as a tracer]. For the negative control : Dilution water without test item incubated under the same conditions as the test groups was treated equivalent to the preparation of the saturated solution, above. The study was performed under static conditions. Due to the volatility of the test item, the study was performed in a closed system without headspace to reduce contact with air and losses of the test item.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: - Controls: For positive control - reference item: potassium dichromate were prepared in a separately conducted reference test (documented in the full study report). A negative/blank control without test item or reference item was also included.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Prior to start of the exposure, the test solutions were checked for undissolved test item. Presence of undissolved test item during preparation and during the test was not observed.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: HINDÁK, CCAP 278/4 (axenic)
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland, United Kingdom, PA37 1QA
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux for 24 hours per day.

ACCLIMATION
- Acclimation period: No. However, a three days old preculture, prepared in dilution water, was used as inoculum.
- Culturing media and conditions (same as test or not): No. Culture Medium: Nutrient medium Z according to LÜTTGE et al. (1994) Botanica Acta, Journal of the German Botanical Society, No. 3 Volume 107 page 111-186 (June 1994), THIEME-VERLAG.
Dilution water: OECD TG 201: (mg/L) - NH4Cl 15 ; MgCl2.6 H2O: 12 ; CaCl2.2 H2O: 18 ; MgSO4.7H2O: 15 ; KH2PO4: 1.6 ; FeCl3.6H2O: 0.064 ; Na2EDTA.2H2O: 0.1 ; H3BO3: 0.185 ; MnCl2.4H2O: 0.415 ; ZnCl2: 3x10-3 ; Na2MoO4.2H2O: 7x10-3 ; CoCl2.6H2O: 1.5x10-3 ; CuCl2.2H2O: 1x10-5 ; NaHCO3: 50 ; NaHCO3* : 250 ; MES monohydrate*: 2665.6 ; pH 8.1 +/- 0.2. This medium has a nominal hardness of 0.24 mmol Ca+Mg/L.
* additional compounds were added to enable sufficient growth under conditions without headspace.
- Any deformed or abnormal cells observed: None reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

In accordance with the OECD TG 201 guideline.

Hardness:

The test medium has a nominal hardness of 0.24 mmol Ca+Mg/L.
Dilution water: 42 mg/L CaCO3

Test temperature:

Nominal range: 21 - 24 °C, controlled at ± 2°C - measured room temperature values were min: 22.0 ; max 23.0 and mean 22.5 °C

pH:

0 hours: pH 8.1 ± 0.1 (and 8.22 in control); 72 hours: pH 9.57-9.73 (definitive test concentrations) and pH 9.74 (controls).

Conductivity:

Dilution water: 139 μS/cm

Nominal and measured concentrations:

- Preliminary test: Not applicable. See below
- First definitive test: 0 (control), and 100% of the saturated solution ; the results in the first definitive limit test showed a higher inhibition as expected. Therefore, the study had to be repeated as dose response test (Second definitive test)
- Second definitive test (dose-response test) : 0 (control), 6.25%, 12.5%, 25.0%, 50.0% and 100% of the saturated solution
- Equivalent geometric mean measured concentrations (with algae): 0 (control), 0.0179, 0.0431, 0.0481, 0.0597 and 0.221 µg/L (or 17.9, 43.1, 48.1, 59.7 and 221 ng/L) which were based on analysis during the definitive test period.
- Measured concentrations of the test item were in the samples with algae in the range of 0.0322 to 0.897 µg/L at the start of exposure (0 h) and in the range of: < LOQ (Limit of Quantification) to 0.0545 µg/L (and/or 0 to 6% and 20% of the initial test item concentrations) at the end of exposure (72 h). All effect values given were based on geometric mean measured concentrations after 72 hours for samples.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass.
- Type: Closed - Static. Sterile headspace flasks
- Material, size, headspace, fill volume: , volume: 59 mL, with aluminium tops with PFTE seals. Minimum headspace.
- Aeration: Vessel shaken continuously. Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: nominal: 5 x 10^3 - 10^4 and current: 6864 cells/mL
- Control end cells density: Mean (of replicates after 72 hours) 549397 cells/ml (or ca. x80 increase in cell density)
- No. of vessels per concentration (replicates): 3 replicates of each test concentration; 1 extra replicate of each test group for sampling purposes
- 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. OECD TG 201 medium. Additionally, compounds were added to enable sufficient growth under conditions without headspace. In accordance to OECD Guidance Document No. 23.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Prepared according to guidelines (see 'Details on test organisms' field for more details on composition). Ultrapure water was used to prepare the dilution water (conductivity max. = 0.1 μS/cm)
- Culture medium different from test medium: Yes. (see 'Details on test organisms' field)
- Intervals of water quality measurement: Start and end of the test period.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 60 - 120 µE/m2/s ; within ± 15 % over incubation area (or 4440 to 8880 lux)
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found at the highest preliminary test concentration level of 100% saturated solution (or 10 mg/L nominal loading rate).
- Other: Initial cell density: Microscopic evaluation of the cells was carried out at the start and end of the exposure. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation and adherence of algae to test containers or aggregation of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2 ; Five dilutions were tested in a geometric series with a dilution factor of two (2) : 0 (control), 6.25, 12.5, 25.0, 50.0 and 100 % saturated solution. This corresponded to nominal loadings rates of: 0 (control), 0.625, 1.25, 2.5, 5.0 and 10 mg/L of the saturated solution and/or geometric mean measured test item concentrations of: 0 (control), 0.327, 0.696, 1.46, 3.77 and 10.1 μg/L of parent test item (used as a tracer).
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes.
- Test concentrations: limit test : 0 (control), 10.0 and 100% of the saturated solution ; equivalent nominal loading rate: 0.1, 1.0 and 10.0 mg/L; geometric mean measured test item concentrations of: 0 (control), 0.712, 1.66 and 33.4 μg/L of parent test item (used as a tracer).
- Results used to determine the conditions for the definitive study: Yes. 14% growth rate inhibition and 44% yield inhibition was seen at the highest dose level.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: - µg/L ; based on geometric mean measured concentrations ; > 10 mg/L based on nominal loadings of the test item saturated solution

Remarks:

i.e. ErC50 is above the solubility limit

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 10.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: - µg/L ; based on geometric mean measured concentrations ; > 10 mg/L based on nominal loadings of the test item saturated solution

Remarks:

i.e. ErC10 is above the solubility limit

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.327 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 72-hour NOELR was 0.625 mg/L based on nominal loading rate of saturated solution (prepared at 10 mg/L).

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% C.I.: - µg/L ; based on geometric mean measured concentrations ; > 10 mg/L based on nominal loadings of the test item saturated solution

Remarks:

i.e. EyC50 is above the solubility limit

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.465 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% C.I.: 0.338 - 0.739 µg/L ; based on geometric mean measured concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.327 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 72-hour NOELR was 0.625 mg/L based on nominal loading rate of saturated solution (prepared at 10 mg/L)

Details on results:

- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the start of the incubation period and at test end revealed no morphological abnormalities in the test item concentration or control.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: None reported.
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: No (in average terms, there was an isolated incidence of low dose yield stimulation (limited to -2%) at 0.625 mg/L nominal loading rate in one single replicate)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No precipitate or similar observations reported. It was noted in the study, the test item is designed to break down under use conditions, meaning that in the aqueous conditions of this study, it is expected to be present as a mixture of the parent test item and the released components. In order to mimic those conditions, a saturated solution was prepared. The saturated solution was tested with a nominal loading rate of 10.0 mg/L of the test item. The analysis of parent test item was used as a tracer to provide an indication of its stability during the duration of the test. At the end of exposure after 72 hours, the measured parent test item concentrations were in the range of 3% to 12% of the initially measured concentrations. The geometric mean measured test item as parent concentration were calculated.
- Effect concentrations exceeding solubility of substance in test medium: Yes. See above and below.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate reduction (ERC50: 0-72h) was 0.851 mg/L with a 95% confidence interval ranging from 0.802 to 0.931 mg/L with headspace and 1.14 mg/L with a 95% confidence interval ranging from 1.06 to 1.22 mg/L without headspace. The EC50 for yield inhibition (EYC50: 0-72h) was 0.449 mg/L with a 95% confidence interval ranging from 0.396 to 0.506mg/L with headspace and 0.533 mg/L with a 95% confidence interval ranging from 0.480 to 0.586 mg/L without headspace.
The results with headspace and without headspace were within the test facility SOPs (historic values).
- Other: The sensitivity of the test system was in agreement with the historical data.

Reported statistics and error estimates:

EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression and/or third order polynomial regression, as appropriate. The NOEC / LOEC was determined by calculation of statistically significant differences of growth rate and yield using: As a standard, One Way Analysis of Variance (ANOVA) and Dunnett’s test. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.05.

Table 1. Results of the Definitive Test (0 - 72 hours)

Dilution level of the saturated test item solution [%]	Nominal test item loading rate [mg/L] #1	Geometric mean measured test item concentration [µg/L]	Growth Rate Inhibition [%]	Yield Inhibition [%]
100.0	10	10.1	9	33
50.0	5	3.77	5	21
25.0	2.5	1.46	4	16
12.5	1.25	0.696	4	15
6.25	0.625	0.327 #2	1	4
0.0	Control	< LOQ	-	-

#1 = 10.0 mg/L is the nominal loading rate of the saturated solution, the dilutions are calculated nominal loadings

#2 = value below LOQ taken with 1/2 LOQ (0.05 μg test item/L) into account for calculation of the geometric mean measured concentration

LOQ = = limit of quantification of the analytical method (0.1 μg/L of the test item)

All biological observations are documented in the full study report and all validity criteria were considered to be met.

 

Table 2. Percentage reduction in growth rate and inhibition of yield in the Definitive Test (at 72 hours)

Dilution level of the saturated test item solution [%]	Nominal test item loading rate #1	Geometric mean measured test item concentration	Replicate	Growth rate		Growth rate inhibition	Yield		Yield Inhibition
[%]	[mg/L]	[µg/L]	No.	[d-1]		[%]	[d-1]		[%]
100.0	100.0	10.1	1		1.30	11		336063	38
			2		1.34	8		376118	31
			3		1.35	8		386486	29
			Mean	(s)	1.33	9	(s)	366222	33
50.0	50.0	3.77	1		1.41	4		459578	15
			2		1.37	6		411450	24
			3		1.38	6		421435	22
			Mean	(s)	1.39	5	(s)	430821	21
25.0	25.0	1.46	1		1.40	4		453236	16
			2		1.40	4		454271	16
			3		1.41	4		457622	16
			Mean	(s)	1.41	4	(s)	455043	16
12.5	12.5	0.696	1		1.41	3		467607	14
			2		1.41	4		459533	15
			3		1.41	4		462120	15
			Mean	(s)	1.41	4	(s)	463087	15
6.25	6.25	0.327	1		1.47	0		552552	-2
			2		1.45	1		524664	3
			3		1.42	3		480539	11
			Mean	(ns)	1.45	1	(ns)	519252	4
0% (Control)	0 (Control)	0 (Control)	1		1.43			497969
			2		1.48			582823
			3		1.48			576009
			4		1.46			545288
			5		1.45			531029
			6		1.45			522078
			Mean		1.46			542533

#1 = 10.0 mg/L is the nominal loading rate of the saturated solution, the dilutions are calculated nominal loadings

Negative inhibition = increase in growth

Statistically significant differences of growth rates and yield compared to control values are marked (s) and non-statistically significant are marked (ns)

 

Table 3. Measured Concentration of test item in the Definitive Test with Algae

Sampling		Fresh Media, 0 hours	Old Media, 72 hours		Geometric mean measured test item concentration
Dilution level of the saturated test item solution [%]	Nominal test item loading rate [mg/L] #1	Meas. conc. [µg/L]	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]
100.0	10	29.7	3.44	12	10.1
50.0	5	14.9	0.953	6	3.77
25.0	2.5	7.32	0.293	4	1.46
12.5	1.25	3.77	0.129	3	0.696
6.25	0.625	2.14	< LOQ	-	0.327
0.0 (Control)	0.0 (Control)	< LOQ	< LOQ		< LOQ

#1 = 10.0 mg/L is the nominal loading rate of the saturated solution, the dilutions are calculated nominal loadings

#2 = value below LOQ taken with 1/2 LOQ (0.05 μg test item/L) into account for calculation of the geometric mean measured concentration

Meas. conc. = measured concentration of the test item, dilution factors taken into account

% = percent of the initially measured concentration of the test item

LOQ = limit of quantification of the analytical method (0.02 µg/L of the test item).

 

Table 4. Section-by-Section and Average Specific Growth Rates of the Control Group (0 - 72 hours) in the Second Definitive (dose response) Test

	Replicate No.	Specific growth rate [d-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.33	1.55	1.42	1.43	0.109	7.58
	2	1.33	1.41	1.71	1.48	0.198	13.3
	3	1.26	1.62	1.57	1.48	0.195	13.2
	4	1.19	1.67	1.53	1.46	0.249	17.0
	5	1.39	1.66	1.32	1.45	0.180	12.4
	6	1.42	1.49	1.43	1.45	0.040	2.73	11.0
				Mean	1.46
				SD ±	0.02
				CV [%]	1.35

SD = Standard deviation

CV = Coefficient of variation

Validity criteria fulfilled:

yes

Conclusions:

The test item 72h-EyL50 (yield) was > 10 mg/L based on nominal loading rate. The 72h-EyC50 was > 10.1 µg/L based on geometric mean measured concentrations. The corresponding EyL10 was 0.840 (C.I. 0.646 – 1.32) mg/L and the EyC10 was 0.465 (C.I. 0.338- 0.739) µg/L. The test item 72h-ErL50 (growth rate reduction) was > 10 mg/L based on nominal loading rate. 72h-ErC50 was > 10.1 µg/L based on geometric mean measured concentrations. The corresponding ErL10 was > 10 mg/L and the ErC10 was > 10.1 µg/L and the NOErL was 0.625 mg/L nominal loading and the NOErC was 0.327 µg/L.

Executive summary:

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6864 cells/mL. The test item is designed to break down under use conditions, meaning that in the aqueous conditions of this study, it is expected to be present as a mixture of the parent test item and the released components. In order to mimic those conditions, a saturated solution was prepared and subsequently tested and with further dilutions. The analysis of parent test item was used as a tracer to provide an indication of its stability during the duration of the test. The saturated solution was prepared with a nominal loading rate of 10 mg test item/L once at room temperature 72 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out onto a curved glass slide. The glass slide with the test item was inserted in a glass flask with an appropriate amount of dilution water. A slow stirring procedure was applied for 72 ± 1 hour at room temperature. The magnetic stirrer bar was placed with a fish-clip system a few centimetres above the bottom of the flask to prevent direct contact with the test item on the bottom. After a separation phase of 1 hour, the saturated solution was removed by siphoning (from the approximate middle of the glass flask) and was used in the test. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. No presence of undissolved test item during the test was observed. Five dilutions were tested in a geometric series with a dilution factor of two (2) : 0 (control), 6.25, 12.5, 25.0, 50.0 and 100 % saturated solution. This corresponded to nominal loadings rates of: 0 (control), 0.625, 1.25, 2.5, 5.0 and 10 mg/L of the saturated solution. Glass flasks without headspace were used to reduce losses of the test item. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were clear throughout the test period. Concentrations were analytically verified via GC-MS at test start and the end of exposure. At the start of the exposure the measured concentrations were in the range of 2.14 μg/L to 29.7 μg/L test item as parent concentration. At the end of exposure after 72 hours, the measured concentrations were in the range of 3% to 12% of the initially measured concentrations. The equivalent geometric mean measured test item concentrations were : 0 (control), 0.327, 0.696, 1.46, 3.77 and 10.1 μg/L of parent test item (used as a tracer). All the relevant validity criteria of the test guideline were fulfilled. The test item 72h-EyL50 (yield) was > 10 mg/L based on nominal loading rate. The 72h-EyC50 was > 10.1 µg/L based on geometric mean measured concentrations. The corresponding EyL10 was 0.840 (C.I. 0.646 – 1.32) mg/L and the EyC10 was 0.465 (C.I. 0.338- 0.739) µg/L. The test item 72h-ErL50 (growth rate reduction) was > 10 mg/L based on nominal loading rate. 72h-ErC50 was > 10.1 µg/L based on geometric mean measured concentrations. The corresponding ErL10 was > 10 mg/L and the ErC10 was > 10.1 µg/L and the NOErL was 0.625 mg/L nominal loading and the NOErC was 0.327 µg/L.

Applicant assessment indicates: the substance ErC10 was > 10.1 µg/L based on geometric mean measured concentration. This ErC10 value should be used in preference to any available NOErC in long-term toxicity studies for marine or freshwater organisms and any related hazard/risk assessment. Referenced in ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.11: PBT/vPvB assessment, June 2017) and citing the Report of the OECD Workshop on Statistical Analysis of Aquatic Toxicity Data (OECD, 1998) which stated: “NOECs are leading to misunderstandings, misinterpretations and NOECs are statistically unfounded”. Consequently, it can be considered that there are no significant effects below the limit of solubility. Since the ErC10 is greater than the saturation solubility.

Description of key information

ErC50 (algae; growth rate) = > 10.1 µg/L based on geometric mean measured concentrations, or > 10 mg/L based on nominal loading concentration, 72-hour, freshwater, OECD TG 201, 2022

ErC10 (algae; growth rate) = > 10.1 µg/L based on geometric mean measured concentrations, or > 10 mg/L based on nominal loading concentration, 72-hour, freshwater, OECD TG 201, 2022

NOEC (algae; growth rate) = 0.327 µg/L based on geometric mean measured concentrations, 72-hour, freshwater, OECD TG 201, 2022

 

Conclusion : The substance has apparent lower solubility in algal medium OECD TG 201 (2022), than in pure water within as measured in the flask method to EU Method A.6 (2021). This is potentially a result of differences in ionic strength and other parameters between the exposure matrix and that of pure water. the substance ErC50 and ErC10 was > 10.1 µg/L based on geometric mean measured concentration. This ErC10 value should be used in preference to any available NOErC in long-term toxicity studies for marine or freshwater organisms and any related hazard/risk assessment. Referenced in ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.11: PBT/vPvB assessment, June 2017) and citing the Report of the OECD Workshop on Statistical Analysis of Aquatic Toxicity Data (OECD, 1998) which stated: “NOECs are leading to misunderstandings, misinterpretations and NOECs are statistically unfounded”. Consequently, it can be considered that there are no significant effects below the limit of solubility. Since the ErC10 is greater than the saturation solubility.

Additional information

Key study : OECD TG 202, 2022 : The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6864 cells/mL. The test item is designed to break down under use conditions, meaning that in the aqueous conditions of this study, it is expected to be present as a mixture of the parent test item and the released components. In order to mimic those conditions, a saturated solution was prepared and subsequently tested and with further dilutions. The analysis of parent test item was used as a tracer to provide an indication of its stability during the duration of the test. The saturated solution was prepared with a nominal loading rate of 10 mg test item/L once at room temperature 72 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out onto a curved glass slide. The glass slide with the test item was inserted in a glass flask with an appropriate amount of dilution water. A slow stirring procedure was applied for 72 ± 1 hour at room temperature. The magnetic stirrer bar was placed with a fish-clip system a few centimetres above the bottom of the flask to prevent direct contact with the test item on the bottom. After a separation phase of 1 hour, the saturated solution was removed by siphoning (from the approximate middle of the glass flask) and was used in the test. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. No presence of undissolved test item during the test was observed. Five dilutions were tested in a geometric series with a dilution factor of two (2) : 0 (control), 6.25, 12.5, 25.0, 50.0 and 100 % saturated solution. This corresponded to nominal loadings rates of: 0 (control), 0.625, 1.25, 2.5, 5.0 and 10 mg/L of the saturated solution. Glass flasks without headspace were used to reduce losses of the test item. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were clear throughout the test period. Concentrations were analytically verified via GC-MS at test start and the end of exposure. At the start of the exposure the measured concentrations were in the range of 2.14 μg/L to 29.7 μg/L test item as parent concentration. At the end of exposure after 72 hours, the measured concentrations were in the range of 3% to 12% of the initially measured concentrations. The equivalent geometric mean measured test item concentrations were : 0 (control), 0.327, 0.696, 1.46, 3.77 and 10.1 μg/L of parent test item (used as a tracer). All the relevant validity criteria of the test guideline were fulfilled. The test item 72h-EyL50 (yield) was > 10 mg/L based on nominal loading rate. The 72h-EyC50 was > 10.1 µg/L based on geometric mean measured concentrations. The corresponding EyL10 was 0.840 (C.I. 0.646 – 1.32) mg/L and the EyC10 was 0.465 (C.I. 0.338- 0.739) µg/L. The test item 72h-ErL50 (growth rate reduction) was > 10 mg/L based on nominal loading rate. 72h-ErC50 was > 10.1 µg/L based on geometric mean measured concentrations. The corresponding ErL10 was > 10 mg/L and the ErC10 was > 10.1 µg/L and the NOErL was 0.625 mg/L nominal loading and the NOErC was 0.327 µg/L.

Applicant assessment indicates: the substance ErC10 was > 10.1 µg/L based on geometric mean measured concentration. This ErC10 value should be used in preference to any available NOErC in long-term toxicity studies for marine or freshwater organisms and any related hazard/risk assessment. Referenced in ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.11: PBT/vPvB assessment, June 2017) and citing the Report of the OECD Workshop on Statistical Analysis of Aquatic Toxicity Data (OECD, 1998) which stated: “NOECs are leading to misunderstandings, misinterpretations and NOECs are statistically unfounded”. Consequently, it can be considered that there are no significant effects below the limit of solubility. Since the ErC10 is greater than the saturation solubility.