Reaction mass of Benzenepropanal, 4-ethyl-α,α-dimethyl- and 3-(2-ethylphenyl)-2,2-dimethylpropanal

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:

20 Jun 2016 to 29 Oct 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April, 2004

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0, 24 and 48 hours. Samples were taken from the solvent control and each test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the old or expired pooled replicates at 24 and 48 hours for quantitative analysis. Duplicate samples were taken at and stored frozen for further analysis if necessary.

Vehicle:

yes

Remarks:

dimethylformamide

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed (OECD, 2000). A solvent stock solution was prepared by dissolving 200 mg of test item in a final volume of 10 mL of dimethylformamide (DMF). Dilutions were performed in DMF to give further solvent stock solutions of 10, 5.0, 2.5 and 1.3 mg/L. Aliquots of these solvent stock solutions were dispersed in test water with the aid of magnetic stirring for approximately 10 minutes to give 2.0, 1.0, 0.50, 0.25 and 0.13 mg/L stock solutions respectively. After the stirring period, each of the preparations were filtered (0.2 µm Gelman Acrocap filter, first approximate 100 mL discarded in order to pre-condition the filter) to ensure any undissolved test item was removed. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: in-house laboratory culture
- Feeding during test: no

ACCLIMATION
- Acclimation conditions: 16h light/8h darkness cycle with 20 minute dawn and dusk transition periods; temperature of 18 - 22 °C
- Type and amount of food: mixture of algal suspension (Desmodesmus subspicatus) and Tetramin flake food suspension
- Feeding frequency: Daily:

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/L (as CaCO3)

Test temperature:

21 - 22 °C

pH:

7.7 - 7.9

Dissolved oxygen:

8.8 - 9.5 mg O2/L

Nominal and measured concentrations:

- Nominal concentrations:0 (solvent), 0 (solvent control), 0.13, 0.25, 0.50, 1.0 and 2.0 mg/L (based on a range-finding study); see 'Any other information on materials and methods incl. tables' for test concentration considerations.
- Measured concentrations (t=0h): - Measured concentrations (t=48h):

Details on test conditions:

TEST SYSTEM
- Test vessel: glass stoppered conical flasks
- Type: closed
- Fill volume: 100 mL
- Aeration: none
- Renewal rate of test solution: daily
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

OTHER TEST CONDITIONS
- Adjustment of pH: if necessary with NaOH or HCl
- Photoperiod: 16/8 (light/dark), with 20 minute dawn to dusk transition
- Light intensity: 431 to 564 Lux

EFFECT PARAMETERS MEASURED
- Immobility after 24 and 48 h
- Water temperature, pH, dissolved oxygen and appearance of medium: daily. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 0.020, 0.20 and 2.0 mg/L
- Results used to determine the conditions for the definitive study: no significant immobilization was observed at the test concentrations of 0.020 and 0.20 mg/L. However, immobilization was observed at 2.0 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.87 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mobility: no immobility was observed in the negative control group, solvent control group, and up to a concentration of 0.5 mg/L throughout the test. At 1.0 and 2.0 mg/L, 80 and 95% of daphnids were immobile after 48 hours, respectively. See 'Any other information on results incl. tables'.
- Sub-lethal effects: sub-lethal effects of exposure were observed in the 1.0 and 2.0 mg/L test concentrations. This response was reduced movement
- Test item solubility: at the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Results with reference substance (positive control):

The 24-h EC50 value of the reference substance was determined to be 0.83 mg/L (experiment conducted between 29 February 2016 and 02 March 2016).

Reported statistics and error estimates:

The EC50 values and associated confidence limits (if applicable) at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. The Lowest Observed Effect Concentration and the No Observed Effect Concentration at 24 and 48 hours were calculated using the Step-down Cochran-Armitage Test Procedure. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Table: cumulative immobility measured after 24 and 48 hours

Nominal Concentration (mg/L)	24 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
Solvent Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.13	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.25	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.50	0	0	0	0	0	0	5 N	5 N	5 N	5 N
1.0	0	2	1	1	4	20	5 R	3 R	4 R	4 R
2.0	3	4	1	3	11	55	2 R	1 R	4 R	2 R
Nominal Concentration (mg/L)	48 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
Solvent Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.13	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.25	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.50	0	0	0	0	0	0	5 N	5 N	5 N	5 N
1.0	4	5	4	3	16	80	1 R	A/I	1 R	2 R
2.0	5	5	4	5	19	95	A/I	A/I	1 R	A/I

R1– R4= Replicates 1 to 4

N = No sub-lethal effects observed

R = Reduced movement

A/I = All daphnia immobilized

Validity criteria fulfilled:

yes

Remarks:

No mortality in the control and oxygen concentration and pH were according to the criteria.

Conclusions:

The 48-h Daphnia EC50 is 0.87 mg/L.

Executive summary:

The acute toxicity to aquatic invertebrates was determined in a study according to OECD TG 202 and in compliance with GLP criteria. In this study daphnids (D. magna, 20 per concentration) were exposed to nominal concentrations of 0 (control), 0 (vehicle control), 0.13, 0.25, 0.50, 1.0 and 2.0 mg/L for 48 hours under semi-static conditions. Test concentrations were analytically verified. The 0.13 mg/L test concentration at 0, 24 and 48 hours showed that measured concentrations of between 60 and 81% of nominal were obtained, however, as this concentration was below the No Observed Effect Concentration, this was considered not to have had an impact on the outcome of the test. The other test concentrations remained within 80 - 120% of the nominal test concentrations. Consequently, the effect values are based on nominal test concentrations. Immobility was recorded after 24 and 48 hours exposure. After 48 hours, immobility was determined to be 80 and 95% in the two highest test concentrations, whereas at the other (lower) test concentrations no effects were observed. Based on these findings the 48-h EC50 value was determined at 0.87 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.87 mg/L

Additional information

In this section first the Floralozone executive summary of the acute Daphnia test is presented and thereafter the Florhydral acute Daphnia tests. The latter study is used for deriving the acute chronic ratio for Floralozone from Florhydral, which is presented in Long-term toxicity to aquatic invertebrates Endpoint summary.

Floralozone: Acute Daphnia study of Floralozone

The acute toxicity to aquatic invertebrates was determined in a study according to OECD TG 202 and in compliance with GLP criteria. In this study daphnids (D. magna, 20 per concentration) were exposed to nominal concentrations of 0 (control), 0 (vehicle control),0.13, 0.25, 0.50, 1.0 and 2.0 mg/Lfor 48 hours under semi-static conditions. Test concentrations were analytically verified. The 0.13 mg/L test concentration at 0, 24 and 48 hours showed that measured concentrations of between 60 and 81% of nominal were obtained, however, as this concentration was below the No Observed Effect Concentration, this was considered not to have had an impact on the outcome of the test. The other test concentrations remained within 80 - 120% of the nominal test concentrations. Consequently, the effect values are based on nominal test concentrations. Immobility was recorded after 24 and 48 hours exposure.After 48 hours, immobility was determined to be 80 and 95% in the two highest test concentrations, whereas at the other (lower) test concentrations no effects were observed. Based on these findings the 48-h EC50 value was determined at 0.87 mg/L.

Florhydral: Acute Daphnia test

The acute toxicity to aquatic invertebrates was determined in a study according to OECD TG 202 and in compliance with GLP criteria. In this study daphnids (D. magna, 20 per concentration) were exposed to nominal concentrations of 0 (control), 0 (vehicle control),0.958, 1.63. 2.77. 4.71 amd 8 mg/l undersemi-static conditions. Test concentrations were analytically verified. The analytical concentrations remained > 80% of the nominal ones. The effect concentration is based on geometric mean concentrations resulting in an EC50 of 2 mg/l.