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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15-08-2021 to 20-08-2021

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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

An Acute Immobilisation Test to Daphnia magna STRAUS was carried out to determine the EC50-values of the test item after 24 and 48 hours of exposure under static conditions in a closed system

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: November 2019 ; signature: August 2020

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentration levels and the control were analytically verified via GC-MS in the fresh media at the start of exposure and at the renewal of the test solutions (0 and 24 hours) as well as in the 24-hours old media at the renewal and at the end of the exposure (24 and 48 hours). 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), 7.01, 11.7, 25.3, 50.3 and 107 μg/L of parent test item (used as a tracer). [Alternative units in mg/L are: 0 (control), 0.00701, 0.0117, 0.0253, 0.0503 and 0.107 mg/L as geometric mean measured concentrations of parent test item used as a tracer].
[Alternative units in mg/L are: 0 (control), 0.00701, 0.0117, 0.0253, 0.0503 and 0.107 mg/L as geometric mean measured concentrations of parent test item used as a tracer].
- Sampling method: At the start, samples were taken after preparation of each concentration level and the control and analysed. At the end of the renewal (24 hours) and the exposure (48 hours), samples of the old media were taken from the test vessels. The method was validated prior to this study 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), 7.01, 11.7, 25.3, 50.3 and 107 μg/L of parent test item (used as a tracer). [Alternative units in mg/L are: 0 (control), 0.00701, 0.0117, 0.0253, 0.0503 and 0.107 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 semi-static conditions with a renewal of the test solutions after 24 hours. 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 by evaporation.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: For positive control - reference item: 1.0, 2.0 and 4.0 mg/L 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):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphinds
- Strain: Daphnia magna STRAUS (Clone 5)
- Justification for species other than prescribed by test guideline: Not applicable.
- Age at study initiation (mean and range, SD): < 24 hours old daphnids from a healthy stock were used for the study
- Weight at study initiation (mean and range, SD): Not applicable.
- Length at study initiation (length definition, mean, range and SD): Not applicable.
- Stage and instar at study initiation: Juvenile ; < 24 hours
- Valve height at study initiation, for shell deposition study (mean and range, SD): Not applicable.
- Peripheral shell growth removed prior to test initiation: Not applicable.
- Method of breeding: Not reported. Although the breeder and culture conditions are given in the full study report. The Daphnia are from continuous in-house laboratory culture.
- Source: in-house laboratory cultures
- Age of parental stock (mean and range, SD): Not applicable.
- Feeding during test: No. The daphnids were not fed during the study. During culture: The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density typically of > 10^6 cells/mL. The algae are cultured at the test facility
- Food type: Not applicable.
- Amount: Not applicable.
- Frequency: Not applicable.

ACCLIMATION
- Acclimation period: Not applicable. Acclimatization was not necessary, because the composition of the dilution water is equivalent to the culture medium (Elendt M4)
- Acclimation conditions (same as test or not): Yes.
- Type and amount of food: Not applicable.
- Feeding frequency: Not applicable.
- Health during acclimation (any mortality observed): No mortality was reported, prior to and after introduction of the daphids to the test media from the culture medium and start of the exposure.

QUARANTINE (wild caught)
- Duration: Not applicable.
- Health/mortality: Not applicable.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: Not applicable.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

In accordance with the OECD TG 202 guideline.

Hardness:

Dilution water quality parameters: 0 hours: Total Hardness: 266 mg CaCO3/L ; 24 hours (renewal): 265 mg CaCO3/L

Test temperature:

Dilution water quality parameters: 0 hours: Temperature 21.4°C ; 24 hours (renewal): 21.1°C ; during the test period, the temperature in the incubator was 20 °C.

pH:

Dilution water quality parameters: 0 hours: pH 7.67 ; 24 hours (renewal): pH 7.45
Water quality parameters in fresh media and at renewal: 0 hours: pH 7.52 - 7.67 and control: pH 7.67 ; 24 hours (renewal): pH 7.49 - 7.56 and control: pH: 7.45 ; mean pH all replicates: did not differ by more than 1.5 units
Water quality parameters in old media 24 hours and 48 hours: 24 hours: pH 7.22 - 7.27 and control: pH 7.19 ; 48 hours: pH 7.63 - 7.73 and control: pH: 7.64 ; mean pH all replicates: did not differ by more than 1.5 units

Dissolved oxygen:

Dilution water quality parameters: O2 dissolved: 0 hours: 7.83 mg O2/L ; 24 hours (renewal) : 7.77 mg O2/L
Water quality parameters in fresh media and at renewal: O2 dissolved: 0 hours: 7.94 – 8.07 mg O2/L and control: 7.83 mg O2/L ; 24 hours (renewal): 7.81 – 8.11 mg O2/L and control: 7.77 mg O2/L ; dissolved oxygen in all replicates: did not decrease to less than 3 mg/L.
Water quality parameters in old media 24 hours and 48 hours: O2 dissolved: 24 hours: 7.87 – 7.96 mg O2/L and control: 7.79 mg O2/L ; 48 hours: 7.31 – 7.79 mg O2/L and control: 7.30 mg O2/L ; dissolved oxygen in all replicates: did not decrease to less than 3 mg/L.

Conductivity:

Dilution water quality parameters: 0 hours: 0 hours: 616 μS/cm ; 24 hours (renewal): 606 μS/cm

Nominal and measured concentrations:

Range finding test (non-GLP) prior to the definitive test: nominal concentrations: 1.0, 10.0 and 100.0% of the saturated solution prepared with dilution water ; equivalent geometric mean measured concentrations were: 0.970, 10.5 and 109.0 μg/L of parent test item, respectively.
Definitive test: 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), 7.01, 11.7, 25.3, 50.3 and 107 μg/L of parent test item (used as a tracer). [Alternative units in mg/L are: 0 (control), 0.00701, 0.0117, 0.0253, 0.0503 and 0.107 mg/L as geometric mean measured concentrations of parent test item used as a tracer].

Details on test conditions:

TEST SYSTEM
- Test vessel: 4.5 (ID) x 9.5 (H) cm, ca. 130 mL
- Type: A static test design in glass flasks sealed with screw caps (typically, made from polypropylene).
- Material, size, headspace, fill volume: Sealed glass flasks with screw caps. Fill volume ca. 130 mL ; no to minimum headspace
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: Control and test item: 20 per concentration, 5 per vessel (divided into 4 replicates).
- No. of vessels per concentration (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per control (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per vehicle control (replicates): Not applicable.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water / Elendt M4 according to OECD 202
- Culture medium different from test medium: No. Elendt M4, according to ELENDT (1990)
The test vessels were filled up with the test solutions. The daphnids were inserted with a small amount of dilution water (start of the exposure) by pipette. Thereafter, the test vessels were closed immediately with screw caps.

OTHER TEST CONDITIONS
- Adjustment of pH: No. The test vessels were not pH adjusted during or after test item exposure.
- Photoperiod: 16/8 h light/dark cycle during culture conditions. During testing: Light exclusion.
- Light intensity: Diffuse light, light intensity of max. 1500 lx during testing.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality), 24 hours and at 48 hours. An organism was considered to be immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Other (adverse) observations were not apparent within the study.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 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), 7.01, 11.7, 25.3, 50.3 and 107 μg/L of parent test item (used as a tracer). [Alternative units in mg/L are: 0 (control), 0.00701, 0.0117, 0.0253, 0.0503 and 0.107 mg/L as geometric mean measured concentrations of parent test item used as a tracer].
- Justification for using less concentrations than requested by guideline: Not applicable. See above and below.
- Range finding study
- Test concentrations: Non-GLP preliminary range finding test: Nominal concentrations of 1.0, 10.0 and 100.0% of the saturated solution prepared with dilution water ; equivalent geometric mean measured concentrations were: 0.970, 10.5 and 109.0 μg/L of parent test item, respectively. The preliminary range finding test was conducted under semi-static conditions without headspace over a period of 48 hours with three nominal test item concentrations of 1.0, 10.0 and 100.0% of the saturated solution prepared with dilution water. A saturated solution with a nominal loading rate of 10 mg/L was prepared (consistent with the definitive test method). From the saturated solution two dilution levels were prepared with the concentrations 1.00 and 10.0% of the saturated solution. The saturated test item solution showed no Tyndall effect. The saturated solution and the dilution levels were visually clear and colourless throughout the exposure period. Measured exposure concentrations using GC-MS were determined in fresh media 0 hours, 24 hours (old and new media) and 48 hours old media. The geometric mean measured concentrations were indicated.
- Results used to determine the conditions for the definitive study: Yes..

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate of saturated solution

Basis for effect:

mobility

Remarks on result:

other: 10.0 mg/L is the nominal loading rate of the saturated solution, dilutions are calculated nominal loadings

Remarks:

No immobilisation below solubility limit

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 107 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: - µg/L ; geometric mean measured concentration

Remarks:

No immobilisation below solubility limit

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate of saturated solution

Basis for effect:

mobility

Remarks on result:

other: 10.0 mg/L is the nominal loading rate of the saturated solution, dilutions are calculated nominal loadings

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

65.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 27.5-107.0 µg/L ; geometric mean measured concentration

Details on results:

- Behavioural abnormalities: None reported.
- Observations on body length and weight: Not applicable.
- Other biological observations: None reported.
- Mortality of control: No mortalities in control.
- Other adverse effects control: None reported.
- Abnormal responses: None reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None. Prior to start of the exposure intervals, the test solutions were checked for undissolved test item via laser beam (Tyndall effect). The saturated solution was visually clear. Presence of undissolved test item during preparation and during the test was not observed.
- Effect concentrations exceeding solubility of substance in test medium: Yes (at 24 hours only). The test item is known to break down (-lyse) under use conditions, meaning that in the aqueous conditions of this study, it is expected to be present as a mixture of the parent compound (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. The study was performed over a period of 48 hours under semi-static conditions in closed test vessels without headspace to reduce contact with air and losses of the test item by evaporation. 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), 7.01, 11.7, 25.3, 50.3 and 107 μg/L of parent test item (used as a tracer).

Results with reference substance (positive control):

- Results with reference substance valid?: Yes (in latest sensitivity check ; results reported in the full study report).
- Mortality: None reported. Acute immobilisation observed only.
- EC50/LC50: 24h-EC50 was 2.0 mg/L (C.I. 1.00 – 4.00 mg/L).
- Other: The EC50-value of the reference item potassium dichromate after 24 hours is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) listed in DIN 38412 - L 30 for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD Guideline TG 202

Reported statistics and error estimates:

The EC50-value after 24 and 48 hours of exposure is typically calculated by sigmoidal dose-response regression. The respective 95% confidence limits are calculated from the standard error and the t-distribution.

Table 1. Immobilisation after 24 and 48 h of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

Dilution level of the saturated test item solution [%]	Nominal Test item loadings #1 [mg/L]	Geometric mean measured test item concentrations [μg/L]	Number Immobilised										Immobilisation [%]
			24 hours					48 hours					24 hours					48 hours
			Replicates					Replicates					Replicate					Replicate
			1	2	3	4	Sum	1	2	3	4	Sum	1	2	3	4	MW		1	2	3	4	MW
100.0	10	107	0	0	0	0	0	2	2	4	4	12	0	0	0	0	0		40	40	80	80	60
50.0	5	50.3	0	0	1	0	1	0	0	1	1	2	0	0	20	0	5		0	0	20	20	10
25.0	2.5	25.3	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0	0	0	0	0
12.5	1.25	11.7	0	0	0	0	0	0	2	0	0	2	0	0	0	0	0		0	40	0	0	10
6.25	0.625	7.01	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0	0	0	0	0
0.0	Control		0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0	0	0	0	0

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

Table 2. Measured Concentrations of the Test Item during the Definitive Test

Dilution level of the saturated test item solution [%]	Nominal Test item loadings #1 [mg/L]	0 hours Start of the exposure (fresh media)	24 hours (old media)		24 hours (fresh media)	48 hours End of the exposure (old media)		Geometric mean measured test item concentrations
		Test item concentration (as parent)
		Meas. conc. [μg/L]	Meas. conc. [μg/L]	%	Meas. conc. [μg/L]	Meas. conc. [μg/L]	%	[μg/L]
100.0	10	92.4	84.6	92	134	124.0	93	107
50.0	5	46.0	37.6	82	68.7	54.0	79	50.3
25.0	2.5	23.4	18.5	79	35.9	26.4	73	25.3
12.5	1.25	10.9	8.92	82	15.8	12.4	79	11.7
6.25	0.625	6.70	4.96	74	9.96	7.29	73	7.01
0.0	Control	< LOQ	< LOQ		< LOQ	< LOQ		< LOQ

Meas. conc.= measured concentration of the test item (as parent), enrichment and dilution factors taken into account

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

LOQ = limit of quantification of the analytical method (0.2 μg/L of the test item)

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

Validity criteria fulfilled:

yes

Conclusions:

The test item 48-hour ELR50 was > 5 mg/L based on nominal loading rate of saturated solution (prepared at 10 mg/L). The 48h-EC50 was 65.5 (C.I: 27.5 – 107.0 ) µg/L based on geometric mean measured concentrations.

Executive summary:

The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. The study was conducted in a closed system (sealed glass flasks) without headspace under semi-static conditions over a period of 48 hours. The test item is known to break down (-lyse) 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 within Elendt M4 (according to OECD 202, Annex 3). The saturated solution with 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 (see Table 2). 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. The study was performed over a period of 48 hours under semi-static conditions in closed test vessels without headspace to reduce contact with air and losses of the test item by evaporation. All test concentrations were visually clear throughout the exposure intervals. Twenty daphnids were exposed to each concentration level and the control. The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette. 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. 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 concentrations of the parent test item were analytically verified via GC-MS in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) and in the 24-hours old media at the renewal and at the end of the test (24 and 48 hours) in all concentration levels and the control. The GC-MS method was analytically validated. The measured concentrations of the parent test item in the old media (24 and 48 hours) were in the range of 73 to 93% of the initially measured concentrations. The equivalent geometric mean measured test item concentrations were : 0 (control), 7.01, 11.7, 25.3, 50.3 and 107 μg/L of parent test item (used as a tracer). The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled. Under the conditions of this study, the 24-hour ELR50 was > 10 mg/L based on nominal loading rate of saturated solution (prepared at 10 mg/L). and the equivalent 24h-EC50 was > 107 (C.I: – ) µg/L based on geometric mean measured concentrations The test item 48-hour ELR50 was > 5 mg/L based on nominal loading rate of saturated solution (prepared at 10 mg/L). The equivalent 48h-EC50 was 65.5 (C.I: 27.5 – 107.0 ) µg/L based on geometric mean measured concentrations.

Description of key information

48-hour ELR50 (invertebrates) = > 5 mg/L based on nominal loading rate of saturated solution (prepared at 10 mg/L), 48-hour, freshwater, OECD TG 202, 2022

48h-EC50 (invertebrates) = 65.5 (C.I: 27.5 – 107.0 ) µg/L, 48-hour, freshwater, OECD TG 202, 2022

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

0.066 mg/L

Additional information

Key study : OECD TG 202, 2022 : The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. The study was conducted in a closed system (sealed glass flasks) without headspace under semi-static conditions over a period of 48 hours. The test item is known to break down (-lyse) 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 within Elendt M4 (according to OECD 202, Annex 3). The saturated solution with 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 (see Table 2). 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. The study was performed over a period of 48 hours under semi-static conditions in closed test vessels without headspace to reduce contact with air and losses of the test item by evaporation. All test concentrations were visually clear throughout the exposure intervals. Twenty daphnids were exposed to each concentration level and the control. The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette. 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. 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 concentrations of the parent test item were analytically verified via GC-MS in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) and in the 24-hours old media at the renewal and at the end of the test (24 and 48 hours) in all concentration levels and the control. The GC-MS method was analytically validated. The measured concentrations of the parent test item in the old media (24 and 48 hours) were in the range of 73 to 93% of the initially measured concentrations. The equivalent geometric mean measured test item concentrations were : 0 (control), 7.01, 11.7, 25.3, 50.3 and 107 μg/L of parent test item (used as a tracer). The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled. Under the conditions of this study, the 24-hour ELR50 was > 10 mg/L based on nominal loading rate of saturated solution (prepared at 10 mg/L). and the equivalent 24h-EC50 was > 107 (C.I: – ) µg/L based on geometric mean measured concentrations The test item 48-hour ELR50 was > 5 mg/L based on nominal loading rate of saturated solution (prepared at 10 mg/L). The equivalent 48h-EC50 was 65.5 (C.I: 27.5 – 107.0 ) µg/L based on geometric mean measured concentrations.