6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol

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:

19 to 21 June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidance and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Hygroscopic: No
Volatile: No
Stability in water: Unknown
Solubility in water: Not indicated

Analytical monitoring:

yes

Details on sampling:

6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol: A 0.45 μm filtered solution prepared at a loading rate of 100 mg/l, the regulatory limit concentration.
Controls: Test medium without test substance or other additives.
Sampling for analysis of test concentrations: Singular samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency at t=0 h and t=48 h
Volume 2 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 ml were taken 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:

no

Details on test solutions:

The standard test procedures required generation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system should be prevented (e.g. film of the test substance on the water surface).
The batch of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol tested was a white powder with a purity of 99.4% and test substance was not completely soluble in test medium at the concentrations tested.
Preparation of test solutions started with a concentration of 100 mg/l applying 2 days of magnetic stirring to ensure maximum dissolution of the test substance in the test medium. The resulting dispersion was filtered through a membrane filter (Whatman, RC55) to remove the undissolved test material. The lower test concentrations were prepared by subsequent dilutions of the filtrate in test medium. The final test solutions were all clear and colourless.

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 (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

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.

Hardness:

180 mg/l expressed as CaCO3

Test temperature:

18.5 to 20.3°C

pH:

7.7 ± 0.3.

Dissolved oxygen:

8.7 - 9.3 mg/l

Salinity:

No applicable

Nominal and measured concentrations:

measured concentrations

Details on test conditions:

Test procedure and conditions
Test duration: 48 hours
Test type: Static
Test 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: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnids: Within half an hour after preparation of the test solutions.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

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

Details on results:

Analysis of the samples taken during the test showed that the measured concentration in the filtrate prepared at 100 mg/l was below 4 μg/l. A small peak was observed at the start and the end of exposure but the concentration could not be quantified. This value was identical to the value obtained in the water solubility test (NOTOX Project 499464). Hence, due to the extremely low solubility of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in water and test medium, actual concentrations could not be accurately measured. Testing was performed at the limit of solubility. Note that the small peak observed in the control sample at the study start was not observed after 48 hours. It was consequently considered that this was introduced during sample processing and that it was thus not present in the actual test solution with daphnia.
No effects on mobility of daphnia were observed during the test period in any of the concentrations tested.

Results with reference substance (positive control):

The reference test was carried out to check the sensitivity of the test system as used by NOTOX.
Daphnia were exposed for a maximum of 48 hours to K2Cr2O7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/l and to a blank control. Twenty daphnia were exposed per concentration.
The reference substance, potassium dichromate (K2Cr2O7, art. 1.04864, batch no. K34869764 607) was obtained from Merck, Darmstadt, Germany.
The actual responses in this reference test with K2Cr2O7 were just outside (below) the ranges of the expected historical responses at the different concentrations, i.e. a 48h-EC50 between 0.3 and 1.0 mg/l. Hence, the sensitivity of this batch of D. magna was slightly higher when compared to the historical data collected at NOTOX.
The 24h-EC50 was 0.49 mg/l with a 95% confidence interval between 0.45 and 0.55 mg/l.
The 48h-EC50 was 0.28 mg/l with a 95% confidence interval between 0.14 and 0.65 mg/l.
The raw data from this study are kept in the NOTOX archives. The test described above was performed under GLP with a QA-check.

Reported statistics and error estimates:

Not specified

Table 1: Acute immobilisation of daphnids after 24 and 48 hours

Test group 6,6’-di-tert-butyl-4’4’-butylidenedi-m-cresol (% of filtrate prepared at 100 mg/l)	Vessel number	Number Daphnia exposed	Response at 24h		Response at 48h
			Number	Total %	Number	Total %
Control	A	5	0	0	0	0
	B	5	0		0
	C	5	0		0
	D	5	0		0
0.1	A	5	0	0	0	0
	B	5	0		0
1.0	A	5	0	0	0	0
	B	5	0		0
10	A	5	0	0	0	0
	B	5	0		0
100 [<4μg/l]	A	5	0	0	0	0
	B	5	0		0
	C	5	0		0
	D	5	0		0

[ ] between brackets: actual measured concentration.

 

Table 2: Effect parameters

Parameters	Concentration 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol
	Loading rate (mg/l)	Measured (μg/l)
NOEC	100	<4
24&48h-EC50	>100	>4

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol did not induce acute immobilisation of Daphnia magna at a 0.45 μm filtered solution prepared at a loading rate of 100 mg/l after 48 hours of exposure (NOEC). The actual measured concentration was below 4 μg/l, being the limit of quantification of the analytical method.
The 48h-EC50 exceeded the limit of solubility in test medium that was reached by filtration of a solution prepared at a loading rate of 100 mg/l through a 0.45 μm filter. The actual measured concentration was below 4 μg/l, being the limit of quantification of the analytical method.

Executive summary:

Acute Toxicity Study in Daphnia magna with 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol.

 

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, the ISO International Standard 6341, 1996 and the OECD series on testing and assessment number 23, 2000. The study was performed in compliance with GLP.

 

The batch of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol tested was a white powder with a purity of 99.4% and test substance was not completely soluble in test medium at the concentrations tested.

 

Preparation of test solutions started with a concentration of 100 mg/l applying 2 days of magnetic stirring to ensure maximum dissolution of the test substance in the test medium. The resulting dispersion was filtered through a membrane filter to remove the undissolved test material.

 

A combined limit/range-finding test was performed exposing twenty Daphnia magna per test group (5 per replicate, 4 replicates) to a control and a 0.45 μm filtered solution prepared at a loading rate of 100 mg/l in the limit test. In the combined range-finding test ten daphnia per test group (5 per replicate, 2 replicates) were exposed to dilutions representing 0.1, 1.0 and 10% of the filtrate. The total test period was 48 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test period.

 

Analysis of the samples taken during the test showed that the measured concentration in the filtrate prepared at 100 mg/l was below 4 μg/l. A small peak was observed but the concentration could not be quantified. This value was identical to the value obtained in the water solubility test (NOTOX Project 499464). Hence, due to the extremely low solubility of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in water and test medium, actual concentrations could not be accurately measured. Testing was performed at the limit of solubility.

 

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

 

6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol did not induce acute immobilisation of Daphnia magna at a 0.45 μm filtered solution prepared at a loading rate of 100 mg/l after 48 hours of exposure (NOEC). The actual measured concentration was below 4 μg/l, being the limit of quantification of the analytical method.

 

The 48h-EC50 exceeded the limit of solubility in test medium that was reached by filtration of a solution prepared at a loading rate of 100 mg/l through a 0.45 μm filter. The actual measured concentration was below 4 μg/l, being the limit of quantification of the analytical method.

 

In conclusion; due to the extreme low solubility of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in test medium concentrations toxic for Daphnia magna could not be reached.

Description of key information

Key value determined in study utilising OECD & EU test guidance.

due to the extreme low solubility of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in test medium concentrations toxic for Daphnia magna could not be reached.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol did not induce acute immobilisation of Daphnia magna at a 0.45 μm filtered solution prepared at a loading rate of 100 mg/l after 48 hours of exposure (NOEC). The actual measured concentration was below 4 μg/l, being the limit of quantification of the analytical method.

The 48h-EC50 exceeded the limit of solubility in test medium that was reached by filtration of a solution prepared at a loading rate of 100 mg/l through a 0.45 μm filter. The actual measured concentration was below 4 μg/l, being the limit of quantification of the analytical method.

Due to the extreme low solubility of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in test medium concentrations toxic for Daphnia magna could not be reached.

Data from a supporting study are also provided, but the reliability of this data is difficult to assess. There is inadequate detail in the report to determine actual solubility of the substance since dose levels expressed are above the water solubility of the substance. The data have been collected in accordance with the principles of GLP but without analytical support to determine the concentration in solution. There are also no details available in the report to determine whether undissolved test sample was present in test flasks.

Volumes expressed are, however, significantly above the water solubility of the substance.

The loading rates from the most reliable data are therefore presented as the key value.