[No public or meaningful name is available]

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:

09-03-2020 to 19-05-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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

Reason / purpose for cross-reference:

reference to other study

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 understatic 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: July 2016 ; signature: January 2017

Analytical monitoring:

yes

Details on sampling:

- Concentrations: the saturated solution and the control were analytically verified via GC-MS/MS in fresh media at the start of exposure (0 hours) and in corresponding old media at the end of the exposure (48 hours) in the limit test item loading rate and the control. The measured concentration of the test item was 0.125 μg/L at the test start and 0.0245 μg/L at test end. Equivalent Geometric mean measured concentrations were determined: 0 (control) and 0.0553 mg/L
- Sampling method: At the start of the exposure (0 hours), samples were taken after preparation of the saturated solution and analysed. At the end of 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 SANCO 3029/99 rev.4 (2000).
- Sample storage conditions before analysis: All original samples were stored at room temperature (ca. 20 ± 2 °C) before preparation. Prepared samples were stored in an autosampler at 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 prepared with of 100 mg/L of the test item was tested in a limit test. The limit concentration was selected based upon the very low solubility of the test item and in accordance with the OECD TG 202 Guideline for testing. Test item exposures was prepared from stock solution. A saturated solution with a nominal loading of 100 mg/L of the test item was prepared with dilution water (see below) one day before the start of the exposure (at day -1). A glass bottle was filled with an appropriate amount of the dilution water. The headspace in the glass flask was minimized. An appropriate volume of the test item was weighed out and placed by pipette onto the water surface and the bottle was closed with the screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of emulsion) was carried out for 24 ± 1 hour with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 1 hour at room temperature for separation of undissolved test item. Thereafter, the clear water phase was removed by siphoning from the bottom of the glass flask. The test item solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The saturated solution was visually clear. The Tyndall effect was negative. Presence of undissolved test item during preparation and during the test was not observed. The saturated solution was used as a limit concentration. 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.
- 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.
- 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
- 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:

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:

Dilution water quality parameters: 0 hours: Total Hardness: 249 mg CaCO3/L

Test temperature:

Dilution water quality parameters: 0 hours: Temperature 20.0 °C ; during the test period, the temperature in the incubator was 20 - 21 °C.

pH:

0 hours: pH 7.63 and control: pH 7.97 ; 48 hours: pH 7.17 and control: pH: 7.32; mean pH at 48 hours all replicates: did not differ by more than 1.5 units

Dissolved oxygen:

Dilution water quality parameters: 0 hours: O2 dissolved: 7.19 mg/L and control: 8.68 mg/L ; 48 hours: O2 dissolved: 5.76 mg/L and control: 5.05 mg/L; dissolved oxygen at 0 and 48 hours all replicates: did not decrease to less than 3 mg/L.

Conductivity:

0 hours: 581 μS/cm

Nominal and measured concentrations:

Range finding tests were not performed (non-GLP) prior to the definitive test: Not applicable. See below.
Definitive test: dilution level of saturated solutions: 0 (control), and 100 mg/L nominal ; in the definitive: a saturated solution prepared with of 100 mg/L of the test item was tested in a limit test. The limit concentration was selected based upon the very low solubility of the test item and in accordance with the OECD TG 202 Guideline for testing. The measured concentration of the test item was 0.125 μg/L at the test start and 0.0245 μg/L at test end. Equivalent Geometric mean measured concentrations were determined: 0 (control) and 0.0553 mg/L

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.
- 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: Not applicable. See below.
- Justification for using less concentrations than requested by guideline: 0 (control), and 100 mg/L nominal ; in the definitive: a saturated solution prepared with of 100 mg/L of the test item was tested in a limit test. The limit concentration was selected based upon the very low solubility of the test item and in accordance with the OECD TG 202 Guideline for testing. The measured concentration of the test item was 0.125 μg/L at the test start and 0.0245 μg/L at test end. Equivalent Geometric mean measured concentrations were determined: 0 (control) and 0.0553 mg/L
- Range finding study
- Test concentrations: Not applicable.
- Results used to determine the conditions for the definitive study: Not applicable.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 0.055 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:

EC50

Effect conc.:

> 0.055 µg/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

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. 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 100 mg/L of the test item in a limit test. The study was performed over a period of 48 hours under static conditions in closed test vessels without headspace to reduce contact with air and losses of the test item by evaporation. The measured concentration of the test item was 0.125 μg/L at the test start and 0.0245 μg/L at test end. Equivalent Geometric mean measured concentrations were determined: 0 (control) and 0.0553 mg/L. No immobilisation or other adverse effects were observed in any treatment or control group.

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 1.94 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 would typically be calculated by sigmoidal dose-response regression. The respective 95% confidence limits would be calculated from the standard error and the t-distribution. Since no immobilisation or other adverse effects were observed in any treatment or control group, in the limit test statistical analysis was not necessary.

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

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

Dilution level of saturated solution [ca. %]	Nominal concentrations [mg/L]	Geometric mean measured test item concentrations [μg/L]	Immobilisation [%]
			24 hours					48 hours
			Replicates					Replicates
			1	2	3	4	MV	1	2	3	4	MV
100	100	0.0553	0	0	0	0	0	0	0	0	0	0
0	Control		0	0	0	0	0	0	0	0	0	0

 

Table 2. Measured Concentrations and Percent of the Initially Measured Concentrations of the Test Item during the Definitive Test

Sampling	0 hours Start of the exposure (fresh media)	48 hours End of the exposure (old media)	Geometric mean measured test item concentration [μg/L]
Nominal concentrations [mg/L]	Test item concentration
	Meas. conc. [μg/L]	Meas. conc. [μg/L]
100	0.125 #1	0.0245 #2	0.0553
Control	< LOQ	< LOQ	< LOQ

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

LOQ = limit of quantification of the analytical method (0.025µg/L)

#1 mean value of three replicates

#2 mean value of two replicates, one < LOQ, but two > 70% of the LOQ, therefore taken into account

Validity criteria fulfilled:

yes

Conclusions:

The test item 48h-EC50 was > 0.0553 (C.I: – ) µg/L based on geometric mean measured concentrations. No effects and no immobilisation was observed on the Daphnia magna in the saturated solution of test item (i.e. the 48h-EC50 was found to be greater than the limit of solubility of the test item).

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 static conditions over a period of 48 hours within a limit test conducted at up to 100% saturated solution with Elendt M4 (according to OECD 202, Annex 3). The test item is known 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 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 100 mg/L of the test item in a limit test. The limit concentration was selected based upon the very low solubility of the test item and in accordance with the OECD TG 202 guideline for testing. Test item exposures was prepared from stock solution. A saturated solution with a nominal loading of 100 mg/L of the test item was prepared with dilution water (see below) one day before the start of the exposure (at day -1). A glass bottle was filled with an appropriate amount of the dilution water. The headspace in the glass flask was minimized. An appropriate volume of the test item was weighed out and placed by pipette onto the water surface and the bottle was closed with the screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of emulsion) was carried out for 24 ± 1 hour with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 1 hour at room temperature for separation of undissolved test item. Thereafter, the clear water phase was removed by siphoning from the bottom of the glass flask. The test item solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The saturated solution was visually clear. The Tyndall effect was negative. Presence of undissolved test item during preparation and during the test was not observed. Twenty daphnids were exposed to each concentration level and the control. The saturated solution and the control were analytically verified via GC-MS/MS in fresh media at the start of exposure (0 hours) and in corresponding old media at the end of the exposure (48 hours) in the limit test item loading rate and the control. The measured concentration of the test item was 0.125 μg/L at the test start and 0.0245 μg/L at test end. Equivalent Geometric mean measured concentrations were determined: 0 (control) and 0.0553 µg/L.This decrease in concentration with time is expected, given expected instability under the conditions of the test of the test item. No effects and no immobilisation were observed on the Daphnia magna in the saturated solution of test item. The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 hours) and at the end of the exposure (48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled. The 48h-EC50 was > 0.0553 µg/L based on geometric mean measured concentrations of the test item. Under the conditions of the study, the 48h-EC50 was found to be greater than the limit of solubility of the test item.

Description of key information

48h-EC50 (invertebrates) = > 0.0553 µg/L based on geometric mean measured concentrations or > 100 mg/L based on nominal loading concentration, the 48h-EC50 was found to be greater than the limit of solubility of the test item, 48-hour, freshwater, OECD TG 202, 2020

 

Conclusion : The substance has apparent lower solubility in aquatic Daphnia medium Elendt M4 or OECD TG 202 annex 3 (2004), than in pure water within as measured in the flask method to EU Method A.6 (2020). This is potentially a result of differences in ionic strength and other parameters between the Daphnia exposure matrix and that of pure water. The conclusion of the experimental study was that no effects were observed (0% immobilisation) at up to 100% saturation, with geometric mean measured concentration of 0.0553 µg/L. Consequently, it can be concluded the test item has no toxicity below the water solubility limit observed experimentally.

Additional information

Key study : OECD TG 202, 2020 : 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 static conditions over a period of 48 hours within a limit test conducted at up to 100% saturated solution with Elendt M4 (according to OECD 202, Annex 3). The test item is known 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 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 100 mg/L of the test item in a limit test. The limit concentration was selected based upon the very low solubility of the test item and in accordance with the OECD TG 202 guideline for testing. Test item exposures was prepared from stock solution. A saturated solution with a nominal loading of 100 mg/L of the test item was prepared with dilution water (see below) one day before the start of the exposure (at day -1). A glass bottle was filled with an appropriate amount of the dilution water. The headspace in the glass flask was minimized. An appropriate volume of the test item was weighed out and placed by pipette onto the water surface and the bottle was closed with the screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of emulsion) was carried out for 24 ± 1 hour with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 1 hour at room temperature for separation of undissolved test item. Thereafter, the clear water phase was removed by siphoning from the bottom of the glass flask. The test item solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The saturated solution was visually clear. The Tyndall effect was negative. Presence of undissolved test item during preparation and during the test was not observed. Twenty daphnids were exposed to each concentration level and the control. The saturated solution and the control were analytically verified via GC-MS/MS in fresh media at the start of exposure (0 hours) and in corresponding old media at the end of the exposure (48 hours) in the limit test item loading rate and the control. The measured concentration of the test item was 0.125 μg/L at the test start and 0.0245 μg/L at test end. Equivalent Geometric mean measured concentrations were determined: 0 (control) and 0.0553 µg/L.This decrease in concentration with time is expected, given expected instability under the conditions of the test of the test item. No effects and no immobilisation were observed on the Daphnia magna in the saturated solution of test item. The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 hours) and at the end of the exposure (48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled. The 48h-EC50 was > 0.0553 µg/L based on geometric mean measured concentrations of the test item. Under the conditions of the study, the 48h-EC50 was found to be greater than the limit of solubility of the test item.