Reaction products of cyclohexanone with buta-1,3-diene and hydrogen peroxide and iron sulphate and methanol, hydrolysed, ammonium salts

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:

5 January to 5 May, 2021

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Study was performed according to a standard guideline.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch: 021015
Purity: 50.00% UB-20 ammonium salts; 50.00% Ethylene glycol
Physical State/Appearance: Clear brown liquid
Expiry Date: 24 November 2021
Storage Conditions: Approximately 4°C in the dark

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and the 100 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate sets of samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Reconstituted water (Elendt M4 medium) was used for test solution preparations. A preliminary media preparation trial was conducted which indicated that the test item was soluble in test media with the aid of prolonged stirring. The test concentrations used in the definitive test were determined by preliminary range-finding tests.

A nominal amount of test item (250 mg) was dispersed in 2.5 L of test water with the aid of propeller stirring (ca. 1500 rpm for 24 hours). After 24 hours stirring was stopped and undissolved material was removed by filtration through a 0.2 µm Gelman Acrocap filter to give the 100 mg/L test concentration.

A solvent control group was prepared containing 50 mg of ethylene glycol in order to determine whether effects were due to the presence of the solvent. A nominal amount of ethylene glycol (25 mg) was dissolved in test water and the volume adjusted to 500 mL to give a 50 mg/L ethylene glycol solvent control stock solution. The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

Tests were conducted using first instar Daphnia magna derived from in-house laboratory cultures.

Adult daphnids were maintained in 150 mL glass vessels containing 100 mL Elendt M4 medium in a temperature-controlled room maintaining the water temperature at 18 to 22°C. The lighting cycle was controlled to give a 16:8 hours light:darkness photoperiod, with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Raphidocelis subcapitata) and GEMMA Micro 300 fish food suspension.

Gravid adults were isolated the day before test initiation so that young daphnids produced overnight were < 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water were considered not to contain any contaminant that would affect the study integrity or outcome.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

No further remarks

Post exposure observation period:

Not applicable

Hardness:

250 mg/L as CaCO3

Test temperature:

20 to 21°C

pH:

7.8 to 8.0

Dissolved oxygen:

8.1 to 8.7 mg O2/L

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 100 mg test item/L, and 50 mg ethylene glycol/L (solvent control)

Details on test conditions:

Glass vessels (150 mL volume) containing test preparation (ca. 100 mL) were used. At test initiation, five daphnids were placed in each test and control vessel test containing preparations at random. Four replicate test, control and solvent control vessels were prepared. The test vessels were covered to reduce evaporation and kept in a temperature controlled room maintaining the water temperature at 18 to 22°C and a 16:8 hours light:darkness photoperiod (light intensity between 200 and 1200 Lux) with 20 minute dawn and dusk transition periods. Daphnids received no food during exposure and the contents of the test vessels were not aerated. The control group was maintained under identical conditions but not exposed to the test item or solvent.

Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a digital thermometer. The light intensity during the light period was measured using an ATP Instrumentation Lux meter. The appearance of the test media was recorded daily.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (tested twice in a 12 month period to demonstrate satisfactory conditions of the test)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Analysis of the 100 mg/L test preparations at 0 and 48 hours showed measured test concentrations of 93% and 80% of nominal respectively, thus results were based on the nominal test concentration.

The test was considered to be valid given that none of the control daphnids showed immobilisation or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Temperature was maintained at 20 to 21°C throughout the test, there were no treatment related differences for oxygen concentration or pH, and light intensity was in the range 770 to 794 Lux throughout the test. At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

The definitive test showed no immobilisation in the 20 daphnids exposed to the 100 mg/L test concentration for a period of 48 hours.

Results with reference substance (positive control):

The most recent positive control (potassium dichromate) test conducted prior to the current test (between 5 January and 29 January 2021) resulted in 48-h EC50, NOEC and LOEC values of 0.50 mg/L (95% confidence limits: 0.43 - 0.58 mg/L), 0.32 mg/L and 0.56 mg/L, respectively, and 24-h EC50, NOEC and LOEC values of 0.86 mg/L (95% confidence limits: 0.21 - 3.5 mg/L), 0.32 mg/L and 0.56 mg/L, respectively. Results were within the normal range for this reference item.

Reported statistics and error estimates:

No immobilisation was shown in all test organisms at the limit test concentration of 100 mg/L during the definitive test, therefore the 48-h EC50 was determined to be >100 mg/L.

Cumulative immobilisation data and observations in the definitive test

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	5N	5N	5N	5N
100	0	0	0	0	0	0	5N	5N	5N	5N
Solvent control / Ethylene glycol (50)	0	0	0	0	0	0	5N	5N	5N	5N
	48 hours
Control	0	0	0	0	0	0	5N	5N	5N	5N
100	0	0	0	0	0	0	5N	5N	5N	5N
Solvent control / Ethylene glycol (50)	0	0	0	0	0	0	5N	5N	5N	5N

R = Replicate; N = Normal

Analytical results for test samples

Time point (Hours)	Nominal concentration of test item in test sample (mg/L)	Determined concentration of test item in test sample (mg/L)	Percentage of nominal concentrations (%)
0	Control	ND	-
	100	93.3	93
	0.0101	0.00856	85
	0.0101	0.00958	95
48	Control	ND	-
	100	80.0	80

 

Validity criteria fulfilled:

yes

Conclusions:

No immobilisation was shown in daphnids exposed to the 100 mg/L test item concentration over the 48 hour exposure period. The 48-h EC50 value was therefore determined to be >100 mg/L (based on nominal test concentration).

Executive summary:

The acute toxicity of UB20-LDB (50%) to Daphnia magna was assessed according to OECD Guideline 202 and EU method C.2. Young daphnids (< 24 hours old) were exposed in a static test to a nominal test item concentration of 100 mg/L, a control and a solvent control (ethylene glycol) (to determine whether any adverse effects of exposure were due to the presence of the solvent) for 48-hours. The numbers of immobile organisms were recorded after 24 and 48 hours. Analysis of the 100 mg/L test preparations at 0 and 48 hours showed measured test concentrations of 93% and 80% of nominal respectively, thus results were based on the nominal test concentration. The test was considered to be valid given that none of the control daphnids showed immobilisation or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels. The solvent control group showed no effects in exposed daphnids over the 48-hour exposure period. As such it can be considered that the presence of ethylene glycol in the sample of test item had no effect on the results obtained and that any observed effects would have been as a direct result of exposure to the UB-20 ammonium salts.

 

No immobilisation was shown in daphnids exposed to the 100 mg/L test item concentration over the 48-hour exposure period. The 48-h EC50 value is therefore >100 mg/L.

Description of key information

The acute toxicity ofUB20-LDB (50%) to Daphnia magna was assessed according to OECD Guideline 202 and EU Method C.2. Young daphnids (< 24 hours old) were exposed in a static test to a nominal test item concentration of 100 mg/L, a control and a solvent control (ethylene glycol) (to determine whether any adverse effects of exposure were due to the presence of the solvent) for 48-hours. The numbers of immobile organisms were recorded after 24 and 48 hours. Analysis of the 100 mg/L test preparations at 0 and 48 hours showed measured test concentrations of 93% and 80% of nominal respectively, thus results were based on the nominal test concentration. The test was considered to be valid given that none of the control daphnids showed immobilisation or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels. The solvent control group showed no effects in exposed daphnids over the 48-hour exposure period. As such it can be considered that the presence of ethylene glycol in the sample of test item had no effect on the results obtained and that any observed effects would have been as a direct result of exposure to the UB-20 ammonium salts.

  

No immobilisation was shown in daphnids exposed to the 100 mg/L test item concentration over the 48-hour exposure period. The 48-h EC50 value is therefore >100 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 100 mg/L