2-methylpentyl 2-hydroxybenzoate

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:

02-07-2020 to 04-09-2020

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
(i) In the first full test, five test item concentrations in a geometric series with a separation factor of 1.8, were prepared as follows: 0 (control), 1.0, 1.8, 3.2, 5.6 and 10% saturated solution (SS) prepared at a loading rate of 100 mg/L, was utilised. This test was disregarded as no EC50 could be determined reliably. Consequently, it was decided to not analyse any samples and, instead, to repeat the full test with a wider range of test concentrations.
(ii) In the second full test (definitive test), seven test item concentrations in geometric series with a separation factor of 2.2, were prepared as follows: 0 (control), 1.0, 2.2, 4.6, 10, 22, 46 and 100% SS prepared at a loading rate of 100 mg/L, was utilised. For the definitive test: equivalent average exposure concentrations (mean measured concentrations) were: 0 (control), 0.023, 0.063, 0.14, 0.30, 0.72, 1.5 and 3.2 mg/L which were based on analysis during the definitive test period. Controls: Test medium without test item or other additives. Spacing factor determined from range-finding test and physico-chemical properties of the substance.
- Sampling method: Singular samples for possible analysis were taken from all test concentrations and the control according to the schedule:
1. At the start of the test 0-hours and 24-hours from the freshly prepared solutions.
2. At the first renewal (t=24 hours)
3. The end of the test from the 24-hour old solutions, volume: 2.0 mL from the approximate centre of the test vessels.
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis (≤ -15°C). Reserve samples of 2.0 mL were taken for possible analysis if needed.
- Other: The preparation procedure for test solutions was based on a non-GLP saturation test and range-finder (cited in the full study report). Preparation of test solutions started with a loading rate of 100 mg/L applying a one-hour period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of one hour. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning through glass wool and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS with test medium. All test solutions were clear and colourless at the end of the preparation procedure. During the preparation, glassware was closed airtight with minimal headspace to prevent vaporization of test item during preparation. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration.
Definitions: Saturated Solution (SS): The soluble fraction of a pure test item obtained after removal of the undissolved fraction (e.g. by filtration, settlement, centrifugation or other removal steps) ; Loading rate: The ratio of test item to test medium (in mg/L) used in the preparation of a WAF, a saturated solution, dispersion of a poorly water-soluble test chemical (dosed above water solubility), or test solution. For unstable test chemicals, the loading rate refers to the parent test item (chemical).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The preparation procedure for test solutions was based on a non-GLP saturation test and range-finder (cited in the full study report). Preparation of test solutions started with a loading rate of 100 mg/L applying a one-hour period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of one hour. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning through glass wool and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS with test medium. All test solutions were clear and colourless at the end of the preparation procedure. During the preparation, glassware was closed airtight with minimal headspace to prevent vaporization of test item during preparation. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Thereafter, the replicates were closed airtight with minimal headspace to prevent vaporization of test item. In the first full test, five test item concentrations in a geometric series with a separation factor of 1.8, were prepared as follows: 0 (control), 1.0, 1.8, 3.2, 5.6 and 10% saturated solution (SS) prepared at a loading rate of 100 mg/L, was utilised. This test was disregarded as no EC50 could be determined reliably. Consequently, it was decided to not analyse any samples and, instead, to repeat the full test with a wider range of test concentrations. In the second full test (definitive test), seven test item concentrations in geometric series with a separation factor of 2.2, were prepared as follows: 0 (control), 1.0, 2.2, 4.6, 10, 22, 46 and 100% SS prepared at a loading rate of 100 mg/L, was utilised. For the definitive test: equivalent average exposure concentrations (mean measured concentrations) were: 0 (control), 0.023, 0.063, 0.14, 0.30, 0.72, 1.5 and 3.2 mg/L which were based on analysis during the definitive test period.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- 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. Samples taken from all test concentrations and the control were analysed. The concentrations measured at the start of the test were 0.016, 0.037, 0.084, 0.17, 0.38, 0.82 and 2.2 mg/L in solutions containing 1.0, 2.2, 4.6, 10, 22, 46 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. During the 24-hour renewal period, the concentrations remained stable, i.e. were at 102-120% relative to the initial concentrations. The measured concentrations at the start of the second renewal period were 0.024, 0.077, 0.19, 0.44, 1.2, 2.5 and 6.3 mg/L in solutions containing 1.0, 2.2, 4.6, 10, 22, 46 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. After the 24-hour renewal period, the concentrations were at 44 to 142% relative to the initial concentrations. The concentrations measured in the freshly prepared solutions at 24 hours of exposure indicate that the procedure of preparation is difficult to repeat, i.e. it is difficult to visually distinguish the test item from the test medium and thus to judge to which degree the phases are separated. In the present study, this is considered acceptable considering that the observed effects can be reliably related to the analytically confirmed results. Based on these results, the average exposure concentrations were calculated.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphinds
- Strain/clone: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- 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: Parthenogenesis
- Source: in-house laboratory cultures
- Age of parental stock (mean and range, SD): More than two weeks (upper range and mean details not provided
- Feeding during test: No. The daphnids were not fed during the study. During culture: Daily, a suspension of fresh water algae. The algae are cultured at the test facility
- Food type: Not applicable.
- Amount: Not applicable.
- Frequency: Not applicable.

ACCLIMATION
- Acclimation period: None.
- Acclimation conditions (same as test or not): Not applicable.
- Type and amount of food: Not applicable.
- Feeding frequency: Not applicable.
- Health during acclimation (any mortality observed): Not applicable.

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:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

In accordance with the OECD TG 202 guideline.

Hardness:

The ISO medium hardness: 180 mg/L expressed as CaCO3 and the pH: 7.7 ± 0.2

Test temperature:

The temperature continuously measured in a temperature control vessel varied between 19 and 21°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

pH:

Test conditions remained within the limits prescribed by the guideline; pH: 7.5-9.0, not varying by more than 1.5 unit at the end of the test (actual pH range = 7.4 – 7.6).

Dissolved oxygen:

Test conditions remained within the limits prescribed by the guideline; oxygen: > or = 3 mgO2/L at the end of the test (actual O2 = 8.7 to 9.1 mgO2/L).

Nominal and measured concentrations:

Range-finding test: 0 (control), 1.0, 1.0, 10 and 100% SS prepared at a loading rate of 100 mg/L
Definitive test: 0 (control), 1.0, 2.2, 4.6, 10, 22, 46 and 100% SS prepared at a loading rate of 100 mg/L, was utilised. For the definitive test: equivalent average exposure concentrations (mean measured concentrations) were: 0 (control), 0.023, 0.063, 0.14, 0.30, 0.72, 1.5 and 3.2 mg/L which were based on analysis during the definitive test period.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL, all glass, closed airtight without headspace.
- Type (delete if not applicable): Closed
- Material, size, headspace, fill volume: 50 mL, all glass, closed airtight without headspace.
- Volume of solution: ca. 50 mL
- Aeration: No.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): renewal after 24 hours intervals
- 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.
- Biomass loading rate: See above.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse Osmosis (RO-water, GEON Waterbehandeling, The Netherlands); used to prepare ISO Medium M7 (culture medium) and/or test medium
- Total organic carbon: Not applicable.
- Particulate matter: Not applicable.
- Metals: Not applicable.
- Pesticides: Not applicable.
- Chlorine: Not applicable.
- Alkalinity: Not applicable.
- Ca/mg ratio: See below.
- Conductivity: Not applicable.
- Salinity: Not applicable.
- Culture medium different from test medium: Yes. The following were added to Reverse Osmosis (RO-water, GEON), dilution water: CaCl2.2H2O 211.5 mg/L ; MgSO4.7H2O 88.8 mg/L ; NaHCO3 46.7 mg/L ; KCl 4.2 mg/L and the hardness of test medium expressed as CaCO3: 180 mg/L
- Intervals of water quality measurement: pH and dissolved oxygen: at the beginning, after 24 hours of exposure (fresh and spent solutions) and at the end of the test, for all concentrations and the control ; temperature : continuously.

OTHER TEST CONDITIONS
- Adjustment of pH: None.
- Photoperiod: The study was performed in the dark
- Light intensity: Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility (including mortality), 24 hours and at 48 hours.

VEHICLE CONTROL PERFORMED: Not applicable.

RANGE-FINDING STUDY
- Test concentrations: 0 (control), 1.0, 1.0, 10 and 100% SS prepared at a loading rate of 100 mg/L
- Results used to determine the conditions for the definitive study: Yes.
- Other: 0% immobilised at 24 to 48h in control, 0.10% and 1.0% SS. 100% immobilised at 24 to 48 h in 10% and 100% SS.
Spacing factor determined from range-finding test and physico-chemical properties of the substance. (i) In the first full test, five test item concentrations in a geometric series with a separation factor of 1.8, were prepared as follows: 0 (control), 1.0, 1.8, 3.2, 5.6 and 10% saturated solution (SS) prepared at a loading rate of 100 mg/L, was utilised. This test was disregarded as no EC50 could be determined reliably. Consequently, it was decided to not analyse any samples and, instead, to repeat the full test with a wider range of test concentrations. (ii) In the second full test (definitive test), seven test item concentrations in geometric series with a separation factor of 2.2, were prepared as follows: 0 (control), 1.0, 2.2, 4.6, 10, 22, 46 and 100% SS prepared at a loading rate of 100 mg/L, was utilised. For the definitive test: equivalent average exposure concentrations (mean measured concentrations) were: 0 (control), 0.023, 0.063, 0.14, 0.30, 0.72, 1.5 and 3.2 mg/L which were based on analysis during the definitive test period. Controls: Test medium without test item or other additives.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 0.72 to 1.50 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 0.31 - 0.49 mg/L

Details on results:

- Behavioural abnormalities: None
- Observations on body length and weight: Not applicable.
- Other biological observations: None.
- Mortality of control: No mortalities.
- Other adverse effects control: None.
- Immobilisation of control: None.
- Abnormal responses: None.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- Mortality: See below.
- EC50/LC50: 24h- and/or 48h- EC50 was within the expected range. The reference test was conducted in a separate study (cited in the full study report) under GLP.
- Other: The sensitivity of the test system was in agreement with the historical data.

Reported statistics and error estimates:

The 24 and 48h-EC50-values were calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations using the maximum likelihood estimation method.
ToxRat Professional was the software package utilised.

Table 1. Immobilisation Rates in the non GLP Preliminary Range Finding Test

Time (h)	Replicate	Test Item ; %SS prep. at 100 mg/L
		Control	0.10	1.0	10	100
0	A	5	5	5	5	5
	B	5	5	5	5	5
	Total introduced	10	10	10	10	10
24	A	0	0	0	5 (2)	5 (2)
	B	0	0	0	5	5 (2)
	Total immobilised	0	0	0	10	10
	Effect %	0	0	0	100	100
48	A	0	0	0	5 #	5
	B	0	0	0	5	5
	Total immobilised	0	0	0	10	10
	Effect %	0	0	0	100	100

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

# Microscopic observation revealed no test item attached to the daphnids.

 

Table 2. Immobilisation Rates after 24 and 48 h of exposure in the Definitive Test

Time (h)	Replicate	Test Item ; Average exposure concentration (mg/L)
		Control	0.023	0.063	0.14	0.30	0.72	1.5	3.2
0	A	5	5	5	5	5	5	5	5
	B	5	5	5	5	5	5	5	5
	C	5	5	5	5	5	5	5	5
	D	5	5	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20	20	20
24	A	0	0 (1)	0	0	0	0	5	5
	B	0	0	0	1	0	0	5	5
	C	0	0	0	0	0	0	5	5
	D	0	0	0	0	0 (1)	0	5 #	5
	Total immobilised	0	0	0	1	0	0	20	20
	Effect %	0	0	0	5	0	0	100	100
48	A	0	0 (2)	0	0	0 (2)	5	5	5
	B	0	0 (2)	0	1 (3)	5 (1)	5 (1)	5	5
	C	0	0	0	0 (4)	1	4	5	5
	D	0	0	0	0	0	5	5	5
	Total immobilised	0	0	0	1	6	19	20	20
	Effect %	0	0	0	5	30	95	100	100

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms werereimmersedinto the respective solutions before recording of mobility.

# Microscopic observation revealed no test item attached to the daphnids.

 

Table 3. Measured concentrations and average exposure concentrations of the Test Item during the Definitive Test

Test item % SS prep. at 100 mg/L	Measured concentrations (mg/L)				Average exposure conc. (mg/L)
	t=0h (fresh)	t=24h (old)	t=24h (fresh)	t=48 (old)
1.0	0.0160	0.0189	0.0237	0.0335	0.023
2.2	0.0371	0.0378	0.0769	0.102	0.063
4.6	0.0844	0.0890	0.188	0.190	0.14
10	0.166	0.172	0.441	0.420	0.30
22	0.378	0.392	1.17	0.938	0.72
46	0.818	0.984	2.48	1.63	1.5
100	2.21	2.51	6.25	2.73	3.2

 

Other: Samples taken from all test concentrations and the control were analysed. The concentrations measured at the start of the test were 0.016, 0.037, 0.084, 0.17, 0.38, 0.82 and 2.2 mg/L in solutions containing 1.0, 2.2, 4.6, 10, 22, 46 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. During the 24-hour renewal period, the concentrations remained stable, i.e. were at 102-120% relative to the initial concentrations. The measured concentrations at the start of the second renewal period were 0.024, 0.077, 0.19, 0.44, 1.2, 2.5 and 6.3 mg/L in solutions containing 1.0, 2.2, 4.6, 10, 22, 46 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. After the 24-hour renewal period, the concentrations were at 44 to 142% relative to the initial concentrations. The concentrations measured in the freshly prepared solutions at 24 hours of exposure indicate that the procedure of preparation is difficult to repeat, i.e. it is difficult to visually distinguish the test item from the test medium and thus to judge to which degree the phases are separated. In the present study, this is considered acceptable considering that the observed effects can be reliably related to the analytically confirmed results. Based on these results, the average exposure concentrations were calculated. See table 3.

Validity criteria fulfilled:

yes

Conclusions:

The test item 48h-EC50 was 0.40 (C.I: 0.31 – 0.49) mg/L based on average exposure concentrations.

Executive summary:

The acute toxicity to Daphnia magna was carried out according to OECD 202 Daphnia sp., Acute Immobilisation Test and/or was equivalent or similar to EU Method C.2 guideline under GLP. The study also followed the OECD Guidance Document number 23 on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals (2019). The preparation procedure for test solutions was based on a non-GLP saturation test and range finder (cited in the full study report). Following the preliminary range finding test in the 0.1 to 100 %SS (% saturated solution) preparation of the 100 mg/L loading rate, the definitive test was conducted at: 0 (control), 1.0, 2.2, 4.6, 10, 22, 46, 100 %SS preparation of the 100 mg/L loading rate in 50 mL, all glass, closed airtight vessels without headspace under semi-static conditions. Preparation of test solutions started with a loading rate of 100 mg/L applying a one-hour period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of one hour. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning through glass wool and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS with test medium. All test solutions were clear and colourless at the end of the preparation procedure. During the preparation, glassware was closed airtight with minimal headspace to prevent vaporization of test item during preparation. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Thereafter, the replicates were closed airtight with minimal headspace to prevent vaporization of test item. Previously, a definitive test was initially conducted using a 1.8 spacing factor but not further analysed, on the basis that no EC50 could be determined reliably from the results. In the definitive test, seven test item concentrations in geometric series with a separation factor of 2.2, were prepared as follows: 0 (control), 1.0, 2.2, 4.6, 10, 22, 46 and 100% SS prepared at a loading rate of 100 mg/L, was utilised. Samples taken from all test concentrations and the control were analysed. The concentrations measured at the start of the test were 0.016, 0.037, 0.084, 0.17, 0.38, 0.82 and 2.2 mg/L in solutions containing 1.0, 2.2, 4.6, 10, 22, 46 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. During the 24-hour renewal period, the concentrations remained stable, i.e. were at 102-120% relative to the initial concentrations. The measured concentrations at the start of the second renewal period were 0.024, 0.077, 0.19, 0.44, 1.2, 2.5 and 6.3 mg/L in solutions containing 1.0, 2.2, 4.6, 10, 22, 46 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. After the 24-hour renewal period, the concentrations were at 44 to 142% relative to the initial concentrations. The concentrations measured in the freshly prepared solutions at 24 hours of exposure indicate that the procedure of preparation is difficult to repeat, i.e. it is difficult to visually distinguish the test item from the test medium and thus to judge to which degree the phases are separated. In the present study, this is considered acceptable considering that the observed effects can be reliably related to the analytically confirmed results. Based on these results, the average exposure concentrations were calculated. The equivalent average exposure concentrations (mean measured concentrations) were: 0 (control), 0.023, 0.063, 0.14, 0.30, 0.72, 1.5 and 3.2 mg/L. The study met all the relevant acceptability criteria and was considered valid. Concentration-related immobility was found at average exposure concentrations of 0.14 mg/L and higher, with 30% immobility at 0.30 mg/L, 95% immobility at 0.72 mg/L and resulting in complete immobility at the two highest test concentrations after 48 hours of exposure. Under the conditions of this study, the 48h-EC50 was 0.40 (C.I: 0.31 – 0.49) mg/L based on average exposure concentrations.

Description of key information

48h-EC50 (invertebrates) = 0.40 (C.I: 0.31 – 0.49) mg/L based on average exposure concentrations, 48-hour, freshwater, OECD 202, 2020

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.4 mg/L

Additional information

Key study : OECD TG 202, 2020 : The acute toxicity to Daphnia magna was carried out according to OECD 202 Daphnia sp., Acute Immobilisation Test and/or was equivalent or similar to EU Method C.2 guideline under GLP. The study also followed the OECD Guidance Document number 23 on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals (2019). The preparation procedure for test solutions was based on a non-GLP saturation test and range finder (cited in the full study report). Following the preliminary range finding test in the 0.1 to 100 %SS (% saturated solution) preparation of the 100 mg/L loading rate, the definitive test was conducted at: 0 (control), 1.0, 2.2, 4.6, 10, 22, 46, 100 %SS preparation of the 100 mg/L loading rate in 50 mL, all glass, closed airtight vessels without headspace under semi-static conditions. Preparation of test solutions started with a loading rate of 100 mg/L applying a one-hour period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of one hour. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning through glass wool and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS with test medium. All test solutions were clear and colourless at the end of the preparation procedure. During the preparation, glassware was closed airtight with minimal headspace to prevent vaporization of test item during preparation. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Thereafter, the replicates were closed airtight with minimal headspace to prevent vaporization of test item. Previously, a definitive test was initially conducted using a 1.8 spacing factor but not further analysed, on the basis that no EC50 could be determined reliably from the results. In the definitive test, seven test item concentrations in geometric series with a separation factor of 2.2, were prepared as follows: 0 (control), 1.0, 2.2, 4.6, 10, 22, 46 and 100% SS prepared at a loading rate of 100 mg/L, was utilised. Samples taken from all test concentrations and the control were analysed. The concentrations measured at the start of the test were 0.016, 0.037, 0.084, 0.17, 0.38, 0.82 and 2.2 mg/L in solutions containing 1.0, 2.2, 4.6, 10, 22, 46 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. During the 24-hour renewal period, the concentrations remained stable, i.e. were at 102-120% relative to the initial concentrations. The measured concentrations at the start of the second renewal period were 0.024, 0.077, 0.19, 0.44, 1.2, 2.5 and 6.3 mg/L in solutions containing 1.0, 2.2, 4.6, 10, 22, 46 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. After the 24-hour renewal period, the concentrations were at 44 to 142% relative to the initial concentrations. The concentrations measured in the freshly prepared solutions at 24 hours of exposure indicate that the procedure of preparation is difficult to repeat, i.e. it is difficult to visually distinguish the test item from the test medium and thus to judge to which degree the phases are separated. In the present study, this is considered acceptable considering that the observed effects can be reliably related to the analytically confirmed results. Based on these results, the average exposure concentrations were calculated. The equivalent average exposure concentrations (mean measured concentrations) were: 0 (control), 0.023, 0.063, 0.14, 0.30, 0.72, 1.5 and 3.2 mg/L. The study met all the relevant acceptability criteria and was considered valid. Concentration-related immobility was found at average exposure concentrations of 0.14 mg/L and higher, with 30% immobility at 0.30 mg/L, 95% immobility at 0.72 mg/L and resulting in complete immobility at the two highest test concentrations after 48 hours of exposure. Under the conditions of this study, the 48h-EC50 was 0.40 (C.I: 0.31 – 0.49) mg/L based on average exposure concentrations.