(4E)-4-(3,3,4-trimethylcyclopentylidene)butanal

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:

From 23 September 2019 to 18 October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Temperatures were situated between 20.51 and 21.58°C throughout the test (average value: 20.93°C), which was slightly outwith the range recommended in the study plan (20°C ± 2°C, constant within 1°C). However, this minor deviation (1.07°C) was considered not to affect the results of the test as no impact was observed on the controls for the duration of the test (all validity criteria of the test have been fulfilled).

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

Temperatures 20.51 to 21.58°C throughout the test (average value: 20.93°C), which was slightly outwith the range recommended (20°C ± 2°C, constant within 1°C). However, this minor deviation (1.07°C) was considered not to affect the results of the test.

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

Temperatures 20.51 to 21.58°C throughout the test (average value: 20.93°C), which was slightly outwith the range recommended (20°C ± 2°C, constant within 1°C). However, this minor deviation (1.07°C) was considered not to affect the results of the test.

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspection dates: 29-30-31 May 2018 / Signature date: 15 November 2018

Specific details on test material used for the study:

- Appearance: From very light yellow to yellow liquid
- Storage condition of test material: Room temperature. Keep only in the original container in an appropriate place.
- Water solubility: 50.3 mg/L at 20°C (LPL 2020, OECD 105/EU A.6, slow-stirring method)
- Vapour pressure: 5.0 Pa at 20°C and 8.7 Pa at 25°C (LPL 2020, OECD 104/EU A.4, effusion method, Knudsen cell)
- Log Kow: 4.3 at 25°C (LPL 2020, OECD 123/EU A.23, slow-stirring method)

Analytical monitoring:

yes

Details on sampling:

Single samples for analysis were taken from the control and all test solutions at the start (t=0 h), at t=24 h (new and old solutions) and at the end of the test (t=48 h).
- Samples preparation: Specimen were injected directly after a dilution by a factor of two with acetonitrile (500 µL sample + 500 µL acetonitrile). If the sample concentrations were too high and not included in the concentration range of the calibration, the samples were diluted appropriately with a test water/acetonitrile (50/50 v/v) mixture.

Vehicle:

no

Details on test solutions:

- Preparation of stock and test solutions: The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the saturated stock solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and 1.2 L test water were added. Then an excess of the test substance (approx. 0.86 g for the t=0 h-stock solution preparation and approx. 1.15 g for the t=24 h-stock solution preparation) was carefully added directly to the surface of the test water. The mixing vessel was thereafter closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 23 hours of gentle stirring in the dark at room temperature, the contents of the vessel was allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port. Samples were taken from the remaining stock solution and chemically analysed. Then the stock solution was diluted with test water as necessary to obtain the required test concentrations into 500-mL volumetric flasks based on the measured concentration of the stock solution (53.10 mg/L at t=0 h and 51.15 mg/L at t=24 h). Each flask was inverted several times before filling the test vessels (without headspace) to ensure adequate mixing and homogeneity of test solutions. After filling and introduction of daphnids the vessels were sealed immediately. The test solutions in test vessels were observed to be clear and colourless. The test was carried out without adjustment of the pH.
- Test concentrations: Based on the results of range-finding tests, test solutions used in the definitive test were prepared by direct addition of the required amounts of stock solution to the test water to obtain the following nominal concentrations (spaced by a factor of approximately 1.78): 0.4, 0.7, 1.3, 2.2, and 4.0 mg/L.
- Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Species: Daphnia magna (Straus), clone 5
- Sex: Female
- Origin: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Reason for selection: Characteristic and common representative of freshwater zooplankton which has been selected as an internationally accepted invertebrate species.
- Validity of batch: Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured Daphnids.
- Age at test start: < 24 hours old
- Breeding conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C/Daphnia/day. The water was changed three times a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Feeding during test: No feeding

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

approximately 250 mg/L (as CaCO3).

Test temperature:

Temperatures were situated between 20.51 and 21.58°C throughout the test (average value: 20.93°C), which was slightly outwith the range recommended in the study plan (20°C ± 2°C, constant within 1°C). However, this minor deviation (1.07°C) was considered not to affect the results of the test as no impact was observed on the controls for the duration of the test (all validity criteria of the test have been fulfilled).

pH:

pH: 6.0-9.0, not varying by more than 1.5 units

Dissolved oxygen:

oxygen: ≥ 60% of the air-saturation value at the end of the test

Salinity:

not applicable

Nominal and measured concentrations:

- Nominal concentrations: 0.4, 0.7, 1.3, 2.2, and 4.0 mg/L
- Geometric mean measured concentrations: 0.55, 0.84, 1.55, 2.49, and 4.56 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 60-mL glass flasks sealed with assembled screw cap with hole and PTFE/silicone septum.
- Type (delete if not applicable): closed
- Aeration: No aeration of the test solutions occurred throughout the test.
- No. of organisms per vessel: 20 per treatment group (including controls), divided into 4 groups of 5 Daphnids
- No. of vessels per concentration (replicates): 4 replicates with daphnids per treatment group.
- No. of vessels per control (replicates): 4 replicates with daphnids per treatment group.
- Biomass loading rate: 5 daphnids per vessel each completely filled with test solution and without headspace
- Introduction of daphnids: Daphnids were introduced into the test vessels each completely filled with test solution and stoppered without headspace immediately after filling the test vessels with test solutions.

TEST MEDIUM / WATER PARAMETERS
Reconstituted water (Elendt M4 medium), as prescribed in OECD Guideline 202. The pH of this solution was in the range of 6 to 9 and the total water hardness was approximately 250 mg/L (as CaCO3).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h light : 8h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.
Additional measurements: Absence of undissolved test item in the stock solution was checked by determination of turbidity (ISO 7027, Water quality - Determination of turbidity). Turbidity analysis was not performed in compliance with the OECD GLP principles but in accordance with ISO 17025.

RANGE-FINDING STUDY
A range-finding test was conducted between September 2 and 5, 2019 to determine the range of concentrations for the definitive test.
The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the saturated stock solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and approx. 1 L test water (Elendt M4 medium) was added. Then an excess of the test item (approx. 0.93 g) was carefully added directly to the surface of the test water. The mixing vessel was thereafter closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 22 hours of gentle stirring in the dark and at room temperature, the contents of the vessel was allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port. Samples were taken from the saturated stock solution and chemically analysed. Then the stock solution was diluted with test water as necessary to obtain the required test concentrations into 500-mL volumetric flasks based on the measured concentration of the stock solution (55 mg/L). Each prepared concentration was inverted several times before filling the test tube (without headspace) to ensure adequate mixing and homogeneity. After filling and introduction of daphnids the vessels were sealed immediately. The test was carried out without adjustment of the pH. The test solution was observed to be clear and colourless at all concentrations.
In this range-finding test, ten daphnids per concentration (5 per vessel, in duplicate) were exposed to the nominal concentrations of 0.32, 1.0, 3.2, 10.0, 32.0 mg test item/L and to a concentration close to the solubility limit of the test item in test water (55.00 mg/L), and to a control. The test system was maintained in closed conditions, in a temperature controlled room (20°C ± 2°C) with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours. During the test the pH, oxygen concentrations and the temperature of the medium were within the optimal ranges for daphnids.
After 48 hours of exposure, immobilisation rates were 0% in the control and at 0.32 mg/L, 50% at 1.00 mg/L, 90% at 3.20 mg/L and 100% at 10, 32 and 55 mg/L
Based on the results of range-finding tests, test solutions used in the definitive test were prepared by direct addition of the required amounts of stock solution to the test water to obtain the following nominal concentrations (spaced by a factor of approximately 1.78): 0.4, 0.7, 1.3, 2.2, and 4.0 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.083 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 1.011 - 1.161 mg/L

Duration:

48 h

Dose descriptor:

other: The highest tested concentration without observed effect

Effect conc.:

0.55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Analytical results: See Table 6.1.3/2 in "Any other information on results incl. tables". The analytical results of this test showed that concentrations of test solutions prepared following the outlined procedures were overall reproducible. Indeed, analyses of samples taken from all fresh and old solutions revealed that test item levels found were overall stable, with losses of test item ≤ 20%. Besides, test item concentrations were satisfactorily maintained within ± 20% of the nominal concentrations throughout the test, except for the measured concentrations at 0.40 mg/L that were higher compared to their corresponding nominal concentration (160% at= 0h and 145% at t= 24 h). It would be difficult to determine the specific cause of this latter observation, since analytical results were in line with expectations, and that there was no dilution error in test solutions preparation. For this reason, the evaluation of the effects on Daphnia magna was based on the geometric means of the measured concentrations (as recommended by OECD Guideline 202).
- Biological results: See Table 6.1.3/1 in "Any other information on results incl. tables". After 24 hours of exposure, immobilisation rates were 0% at 0.55 and 0.84 mg/L, 35% at 1.55 mg/L, 55% at 2.49 mg/L and 65% at 4.56 mg/L. After 48 hours of exposure, immobilisation rates were 0% at 0.55 mg/L, 10% at 0.84 mg/L and 100% at 1.55, 2.49 and 4.56 mg/L.
- Turbidity measurements confirmed the absence of undissolved material in the stock solution (represented by turbidity analysis of the stock solution = 0.57 FNU, in comparison with the turbidity value of the control = 0.53 FNU).

Results with reference substance (positive control):

On May 14, 2019 (KD19-002; most recent test), the 24h-EC50 was 1.053 mg/L. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (expected 24h-EC50: 0.6 - 2.1 mg/L) at this time

Table 6.1.3/1: Acute immobilisation of daphnids after 24 and 48 hours in the final test

Nominal concentration(mg test item.L^-1)	Measured concentration (geometric mean) (mg test item.L^-1)	Replicate	Number of daphnids exposed	Immobilisation at 24 h		Immobilisation at 48 h
				Number	Total %	Number	Total %
Control	Control	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.40	0.55	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.70	0.84	1 2 3 4	5 5 5 5	0 0 0 0	0	0 1 1 0	10
1.30	1.55	1 2 3 4	5 5 5 5	3 1 2 1	35	5 5 5 5	100
2.20	2.49	1 2 3 4	5 5 5 5	3 3 3 2	55	5 5 5 5	100
4.00	4.56	1 2 3 4	5 5 5 5	4 3 3 3	65	5 5 5 5	100

Table 6.1.3/2: Concentrations of the test substance (mg/L) in test water - Final test

Nominal concentration (mg.L^-1)	Measured Concentration,mg.L^-1 (% to the nominal concentration) (% to the initial concentration)				Geometric mean measured concentrations
	Start (t=0 h)	t=24 hold	t=24 hfresh	End t=48 h	mg.L^-1	% nominal
Control	Abs.	Abs.	Abs.	Abs.	N.A.	N.A.
0.40	0.64 (160)	0.47 (118) (73)	0.58 (145)	0.53 (133) (91)	0.55	138
0.70	0.92 (131)	0.84 (120) (91)	0.87 (124)	0.73 (104) (84)	0.84	120
1.30	1.56 (120)	1.49 (115) (96)	1.62 (125)	1.52 (117) (94)	1.55	119
2.20	2.41 (110)	2.39 (109) (99)	2.64 (120)	2.52 (115) (95)	2.49	113
4.00	4.53 (113)	4.45 (111) (98)	4.84 (121)	4.45 (111) (92)	4.56	114

N.A.: not applicable

% = Percent of expected nominal concentration in test item.

Abs.= Absence: concentrations below the LOQ (0.10 mg/L) and the LOD (0.03 mg/L).

Validity criteria fulfilled:

yes

Remarks:

See "Overall remarks"

Conclusions:

The toxic effect of the test substance to the freshwater invertebrate Daphnia magna was investigated in a closed semi-static test. Under experimental conditions, the 48h-EC50 value was estimated to be 1.083 mg/L (95% CL: 1.011 - 1.161 mg/L), based upon geometric mean measured concentrations.

Executive summary:

This study was performed according to OECD Guideline 202 and EU Method C.2 with GLP compliance to assess the acute toxicity of test substance to Daphnia magna.

Based on the results of range-finding tests, twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solution ofthe test substance over 48 hours at the required nominal test concentrations 0.4, 0.7, 1.3, 2.2, and 4.0 mg/L, and to a control. The immobilisation of the daphnids was determined in a closed semi-static 48 -hours test by visual observation after 24 and 48 hours. Samples taken from the control and all test concentrations were analysed at the start (t=0 h), at t=24 h (new and old solutions) and at the end of the test (t=48 h) in order to determine if concentrations of the test substance were maintained.

Analyses of samples taken from all fresh and old solutions revealed that test substance levels found were overall stable, with losses of test substance ≤ 20%. Besides, test substance concentrations were satisfactorily maintained within ± 20% of the nominal concentrations throughout the test, except for the measured concentrations at 0.40 mg/L that were higher compared to their corresponding nominal concentration (160% at t= 0h and 145% at t= 24 h). For this reason, the evaluation of the effects on Daphnia magna was based on the geometric means of the measured concentrations. The evaluation of the effects on daphnids was based on the geometric means of the measured concentrations: 0.55, 0.84, 1.55, 2.49, and 4.56 mg/L.

After 48 hours of exposure, immobilisation rates were 0% at 0.55 mg/L, 10% at 0.84 mg/L and 100% at 1.55, 2.49 and 4.56 mg/L. Therefore, the highest tested concentration without observed effect was 0.55 mg/L and the 48h-EC50 value was estimated to be 1.083 mg/L (95% CL: 1.011 -1.161 mg/L), based upon geometric mean measured concentrations.

Description of key information

OECD Guideline 202, EU Method C.2, GLP, Key study, validity 1:

48h-EC50 (Daphnia magna) = 1.083 mg/L (95% CL: 1.011 - 1.161 mg/L) based on geometric mean measured concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.083 mg/L

Additional information

One key study is available to assess the short-term toxicity of the registered substance to aquatic invertebrates, Daphnia magna.

This study (LPL, 2020) was performed according to OECD Guideline 202 and EU Method C.2 with GLP compliance. Based on the results of range-finding tests, twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solution of the test substance over 48 hours at the required nominal test concentrations 0.4, 0.7, 1.3, 2.2, and 4.0 mg/L, and to a control. The immobilisation of the daphnids was determined in a closed semi-static 48h test by visual observation after 24 and 48 hours. Analyses of samples taken from all fresh and old solutions revealed that test substance levels found were overall stable, with losses of test substance ≤ 20%. Besides, test substance concentrations were satisfactorily maintained within ± 20% of the nominal concentrations throughout the test, except for the measured concentrations at 0.40 mg/L that were higher compared to their corresponding nominal concentration (160% at t= 0h and 145% at t= 24 h). For this reason, the evaluation of the effects on daphnids was based on the geometric means of the measured concentrations: 0.55, 0.84, 1.55, 2.49, and 4.56 mg/L. After 48 hours of exposure, immobilisation rates were 0% at 0.55 mg/L, 10% at 0.84 mg/L and 100% at 1.55, 2.49 and 4.56 mg/L. Therefore, the highest tested concentration without observed effect was 0.55 mg/L and the 48h-EC50 value was estimated to be 1.083 mg/L (95% CL: 1.011 -1.161 mg/L), based upon geometric mean measured concentrations.