Reaction mass of 4-(2-methylbutan-2-yl)phenol and 2,4-bis(2-methylbutan-2-yl)phenol

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:

22 January 2016 to 18 March 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch OP: C605E003.1
Purity/composition correction factor: No correction factor required
Test item handling: Use amber glassware or wrap container in aluminum-foil. To liquefied and homogenize: heat the test item until maximum 130°C under nitrogen.Stability at higher temperatures: Yes, maximum temperature: 130°C; maximum duration: multiple hours if stored under nitrogen. If heated only minutes
Chemical name (IUPAC), synonym or trade name: Phenol, 1,1-dimethylpropyl derivs.
CAS Number: 1639131-79-5
Molecular structure: Reaction mass so not available
Molecular formula: Reaction mass so not available
Molecular weight: Reaction mass so not available
Highly reactive to water: Not indicated by sponsor
Volatile: 20°C ≤ 1.9 Pa = ≤ 1.4 x 10-2 mm Hg; 25°C ≤ 3.1 Pa = ≤ 2.3 x 10-2 mm Hg (determined at WIL Research Europe, project 509995)

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from all WAFs and the control according to the schedule below.
Frequency: at t=0 h and t=48 h
Volume: 2.0 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis.At the end of the exposure period, the replicates were pooled at each concentration before sampling.Additionally, reserve samples of 2.0 mL were taken from all WAFs and the control for possible analysis. If not used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

yes

Details on test solutions:

The batch of Phenol, 1,1-dimethylpropyl derivs. tested was a colourless to pale yellow solid of unknown or variable composition. No correction was made for the composition of the test item.Handling of test item and preparation of test solutions was performed under dimmed light.The test item was heated in a water bath (90°C) to liquefy and homogenize. Sub stocks were weighed for several projects. These sub stocks were stored under nitrogen in the dark and re-liquefied (100°C) and homogenized before use.All test solutions were prepared separately at different loading rates applying three days of magnetic stirring to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixtures were left to stabilize for one hour where after the clear and colourless Water Accommodated Fractions (WAFs) were siphoned out and used for testing.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.
Breeding
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Medium: M7, as prescribed by Dr. Elendt-Schneider

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

The hardness: 180 mg/L expressed as CaCO3 and the pH: 7.7 ± 0.3.

Test temperature:

18-22°C, constant within 2°C.

pH:

pH: 6.0-8.5, not varying by more than 1.5 units

Dissolved oxygen:

oxygen: ≥3 mg/L at the end of the test

Salinity:

Not applicable - freshwater.

Conductivity:

Not specified in the study report

Nominal and measured concentrations:

WAFs prepared at loading rates of 0.10, 0.18, 0.32, 0.56 and 1.0 mg/L

Details on test conditions:

Combined limit/range-finding test
The study started with a combined limit/range-finding test. Twenty daphnids per concentration (four replicates, five daphnids per replicate) were exposed to a control and a WAF prepared at a loading rate of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:-Ten daphnids per concentration (in duplicate, 5 per replicate) were exposed to WAFs prepared at loading rates of 1.0 and 10 mg/L
;-Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration.

Final testT
est concentrations
Phenol, 1,1-dimethylpropyl derivs.: WAFs prepared at loading rates of 0.10, 0.18, 0.32, 0.56 and 1.0 mg/L
Control: Test medium without test item or other additives
Test procedure and conditionsTest duration: 48 hours
Test type: StaticTest vessels: 100 mL, all-glass
Medium: Adjusted ISO medium
Number of daphnids: 20 per concentration
Loading: 5 per vessel containing 80 mL of test solution
Light: No light
Feeding: No feeding
Aeration: No aeration of the test solutions
Introduction of daphnids: Within 25 minutes after preparation of the test solutions
Measurements and recordings
Immobility (including mortality): At 24 hours and at 48 hours.pH and dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control.
Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

0.53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Combined limit/range-finding test
After 48 hours of exposure ≥90% of the daphnids in all three WAFs were immobilized. No immobility was observed in the control group during the test period. The expected EC50 was below the concentration obtained in a WAF prepared at a loading rate of 1.0 mg/L.Samples taken from the WAF prepared at a loading rate of 1.0 mg/L were analysed. Quantification was based on the peak area of the major component, as described in WIL Research Project 509995.The initial concentration in the WAF prepared at 1.0 mg/L was 1.0 mg/L. This concentration remained stable during the 48-hour exposure period (97% of initial at the end of the test). All test conditions were maintained within the limits prescribed by the study plan.

Final test
Measured concentrations
All WAFs were analysed. Quantification was based on the peak area of the major component, as described in WIL Research Project 509995. The initial concentration in the WAFs prepared at 0.10, 0.18, 0.32, 0.56 and 1.0 mg/L was 0.092, 0.19, 0.33, 0.62 and 1.0 mg/L, respectively. These concentrations remained stable during the 48-hour exposure period (94-100% of initial at the end of the test). Based on these results, the EC50 was determined using the initial exposure concentrations of the major component.ImmobilityAfter 48 hours of exposure 25, 60 and 100% immobility was observed at 0.33, 0.62 and 1.0 mg/L, respectively. No immobility was observed at any of the lower test concentrations and the control. The responses recorded in this test allowed for reliable determination of an EC50.Experimental conditionsThese test conditions remained within the limits prescribed by the study plan (pH: 6.0-8.5, not varying by more than 1.5 units; oxygen: ≥3 mg/L at the end of the test).The temperature continuously measured in a temperature control vessel varied between 20 and 21°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C).

Results with reference substance (positive control):

The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of the daphnia was within the range determined with the historical data collected at WIL Research Europe.The 24h-EC50 was 0.69 mg/L with a 95% confidence interval between 0.59 and 1.2 mg/L.The 48h-EC50 was 0.38 mg/L with a 95% confidence interval between 0.33 and 0.43 mg/L.

Reported statistics and error estimates:

Not specified in the study report.

Number of introduced daphnids and incidence of immobility in the combined limit/range-finding test

Time (h)	Replicate	Phenol, 1,1-dimethylpropyl dervis. WAF prepared at the given loading rate (mg/L)
		Control	1.0	10	100
0	A B C D	5 5 5 5	5 5	5 5	5 5
	Total introduced	20	10	10	20
24	A B C D	0 0 0 0	1 0	5 5	5 5 5 5
	Total immobilised Effect %	0 0	1 10	10 100	20 100
48	A B C D	0 0 0 0	4 5	5 5	5 5 5 5
	Total immobilised Effect %	0 0	9 90	10 100	20 100

 

Number of introduced daphnids and incidence of immobility in the final test

Time (h)	Replicate	Initial concentration Phenol 1,1-dimethylpropyl dervis. (mg/L)
		Control	0.092	0.19	0.33	0.62	1.0
0	A B C D	5 5 5 5	5 5 5 5	5 5 5 5	5 5 5 5	5 5 5 5	5 5 5 5
	Total introduced	20	20	20	20	20	20
24	A B C D	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	3 2 1 1
	Total immobilised Effect %	0 0	0 0	0 0	0 0	0 0	7 35
48	A B C D	0 0 0 0	0 0 0 0	0 0 0 0	1 0 3 1	3 1 4 4	5 5 5 5
	Total immobilised Effect %	0 0	0 0	0 0	5 25	12 60	20 100

 

Effect parameters

Parameter	Major component of Phenol, 1,1-dimethylpropyl dervis. (mg/L)	95%-confidence interval (mg/L)
24h-EC50 48h-EC50	>1.0 0.53	n.d. 0.44 – 0.61

 

pH and oxygen concentrations (mg/L) during the final test

Initial conc. Phenol, 1,1-dimethylpropyl dervis. (mg/L)	Start (t=0 h)		End (t=48 h)
	pH	O2	pH	O2
Control 0.092 0.19 0.33 0.62 1.0	8.0 7.9 7.9 7.9 7.9 7.9	9.7 8.8 8.8 8.9 8.7 8.6	7.9 7.9 7.9 7.8 7.8 7.9	9.2 9.1 9.1 9.2 9.2 9.1

 

Acute immobilization of daphnia after 24 and 48 hours in the reference test with potassium dichromate

Concentration K2Cr2O7(mg/L)	Number Exposed	% immobile		Expected response (%) After 48 hours^1
		24h	48h	Minimal	Maximal
Control 0.10 0.18 0.32 0.56 1.0 1.8	20 20 20 20 20 20 20	0 0 0 0 15 100 100	0 0 0 35 95 100 100	0 0 0 0 0 40 100	10^2 10 10 30 100 100 100

¹Based on historical data of the previous years (n>60).

²A maximum response of 10% does not invalidate the results of the test.

Validity criteria fulfilled:

yes

Conclusions:

The 48h-EC50 was 0.53 mg/L (95% confidence interval between 0.44 and 0.61 mg/L).The concentrations were related to the major component of Phenol, 1,1-dimethylpropyl derivs.

Executive summary:

Acute Toxicity Study in Daphnia magna with Phenol, 1,1-dimethylpropyl derivs..

 

The study procedures described in this report were based on the OECD guideline No. 202, 2004. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.2, 2008 and the OECD series on testing and assessment number 23, 2000.

 

The batch of Phenol, 1,1-dimethylpropyl derivs. tested was a colourless to pale yellow solid of unknown or variable composition. No correction was made for the composition of the test item. Handling of test item and preparation of test solutions was performed under dimmed light.

 

The test item was heated in a water bath (90°C) to liquefy and homogenize. Sub stocks were weighed for several projects. These sub stocks were stored under nitrogen in the dark and re-liquefied (100°C) and homogenized before use.

 

All test solutions were prepared separately at different loading rates applying three days of magnetic stirring to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixtures were left to stabilize for one hour where after the clear and colourless Water Accommodated Fractions (WAFs) were siphoned out and used for testing.

 

Twenty daphnids per group (four replicates, five daphnids per replicate) were exposed to a control and to WAFs prepared at loading rates of 0.10, 0.18, 0.32, 0.56 and 1.0 mg Phenol, 1,1-dimethylpropyl derivs. per litre. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

 

All WAFs were analysed. Quantification was based on the peak area of the major component, as described in WIL Research Project 509995. The initial concentration in the WAFs prepared at 0.10, 0.18, 0.32, 0.56 and 1.0 mg/L was 0.092, 0.19, 0.33, 0.62 and 1.0 mg/L, respectively. These concentrations remained stable during the 48-hour exposure period (94-100% of initial at the end of the test). Based on these results, the EC50 was determined using the initial exposure concentrations of the major component.

 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

 

The 48h-EC50 was 0.53 mg/L (95% confidence interval between 0.44 and 0.61 mg/L).

 

The concentrations were related to the major component of Phenol, 1,1-dimethylpropyl derivs.

Description of key information

Key value determined in a GLP accredited laboratory study performed in accordance with OECD Guideline 202 and EU Method C.2.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.53 mg/L