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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 June 2012 to 22 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 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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:

A 0.45 μm filtered solution prepared at a loading rate of 100 mg/l, the regulatory limit concentration.
Singular samples for possible analysis were taken from all test concentrations and the control.

Vehicle:

no

Details on test solutions:

The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system was prevented as much as possible (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.
After preparation, volumes of 50 ml were added to each replicate of the respective test concentration.
Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.
Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford,
Mass., USA) with the following composition:
NaNO3 - 500 mg/l
K2HPO4.3H2O - 52 mg/l
MgSO4.7H2O - 75 mg/l
Na2CO3.10H2O - 54 mg/l
C6H8O7.H2O - 6 mg/l
NH4NO3 - 330 mg/l
CaCl2.2H2O - 35 mg/l
C6H5FeO7.xH2O - 6 mg/l
H3BO3 - 2.9 mg/l
MnCl2.4H2O - 1.81 mg/l
ZnCl2 - 0.11 mg/l
CuSO4.5H2O - 0.08 mg/l
(NH4)6Mo7O24.4H2O - 0.018 mg/l
Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl - 15 mg/l
MgCl2.6H2O - 12 mg/l
CaCl2.2H2O - 18 mg/l
MgSO4.7H2O - 15 mg/l
KH2PO4 - 1.6 mg/l
FeCl3.6H2O - 64 μg/l
Na2EDTA.2H2O - 100 μg/l
H3BO3 - 185 μg/l
MnCl2.4H2O - 415 μg/l
ZnCl2 - 3 μg/l
CoCl2.6H2O - 1.5 μg/l
CuCl2.2H2O - 0.01 μg/l
Na2MoO4.2H2O - 7 μg/l
NaHCO3 - 50 mg/l
Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO3/l)
pH 8.1 ± 0.2

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period specified

Hardness:

Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO3/l)

Test temperature:

The temperature of the test medium was 22.9°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 22.1 and 22.7°C.

pH:

pH 8.1 ± 0.2

Dissolved oxygen:

Not specified

Salinity:

N/A freshwater used for study

Nominal and measured concentrations:

Measured concentrations.

Details on test conditions:

Preparation of test solutions: The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system was prevented as much as possible (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.
After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.
Test concentrations
6,6’-di-tert-butyl-4,4’-butylidenedim-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.
Replicates: 6 replicates of the control and the undiluted filtrate, 3 replicates of each filtrate dilution, 1 or 2 extra replicates of each group for sampling after 24 hours, 1 or 2 replicates of the filtrate and the dilutions without algae.
Test procedures and conditions
Test duration: 72 hours
Test type: Static
Test vessels: 100 ml, all-glass, containing 50 ml of test solution
Medium: M2
Cell density: An initial cell density of 1 x 104 cells/ml.
Illumination: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 99 to 105 μE.m-2.s-1.
Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Measured test substance concentrations: At the start of the test, the actual test concentration in a sample taken from the undiluted filtrate 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). After 24 hours of exposure, the concentration had dropped below the limit of detection. 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.
Reduction of growth rate and inhibition of yield: No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group. Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
The EC50 for both growth rate reduction (ERC50: 0-72h) and yield inhibition (EYC50: 0-72h) 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.
The NOEC for growth rate reduction and yield inhibition exceeded the concentration present in 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.

Results with reference substance (positive control):

This reference test was carried out to check the sensitivity of the test system used by NOTOX to Potassium dichromate (Merck, Art. 1.04864, Batch K34869764 607).
Algae were exposed for a period of 72 hours to K2Cr2O7 (Potassium dichromate) concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l and to a control. The initial cell density was 1.0 x 104 cells/ml.
Potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations 0.32 mg/l and higher.
The EC50 for growth rate reduction (ERC50: 0-72h) was 0.98 mg/l with a 95% confidence interval ranging from 0.74 to 1.3 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3mg/l. Hence, the ERC50: 0-72h for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (EYC50: 0-72h) was 0.44 mg/l with a 95% confidence interval ranging from 0.32 to 0.61 mg/l. The historical ranges of the 72h EC50 for yield inhibition lie between 0.43 and 1.1 mg/l. Hence, the EYC50: 0-72h for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Not specified

Table 1: Mean cell densities (x 10⁴cells/ml)

Test group 6,6’-di-tert-butyl-4-4’-nutylidenedi-m-cresol (% of filtrate prepared at 100 mg/l)	Exposure time (hours)
	0	24	48	72
Control	1.0	6.5	32.0	142.6
0.10	1.0	6.3	32.8	141.9
1.0	1.0	6.2	32.3	135.6
10	1.0	6.4	31.6	133.5
100 [<4μg/l]	1.0	6.3	33.3	146.6

[ ] between brackets: actual measured concentration.

 

Table 2: Percentage reduction of growth rate (total test period) and percentage inhibition of yield

Test group 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol (% of filtrate prepared at 100 mg/l)	Mean growth rate		Yield (0-72 h)
	μ(0-72 h)	Reduction (%)	x10^4cells/ml	Inhibition (%)
Control	0.06884		141.65
0.10	0.06880	0.1	140.95	0.5
1.0	0.06817	1.0	134.62	5.0
10	0.06795	1.3	132.52	6.4
100 [<4μg/l]	0.06925	-0.6	145.56	-2.8

[ ] between brackets: actual measured concentration.

 

Table 3: Percentage reduction of growth rate at different time intervals

Test group 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol (% of filtrate prepared at 100 mg/l)	Mean growth rate
	μ (0-24h)	Reduction (%)	μ (24-48h)	Reduction (%)	μ (48-72h)	Reduction (%)
Control	0.07775		0.06647		0.06230
0.10	0.07674	1.3	0.06863	-3.2	0.06103	2.0
1.0	0.07615	2.1	0.06859	-3.2	0.05979	4.0
10	0.07710	0.8	0.06676	-0.4	0.05999	3.7
100 [<4μg/l]	0.07667	1.4	0.06936	-4.3	0.06171	0.9

[ ] between brackets: actual measured concentration.

 

Table 4: Effect parameters

Parameter	Concentration 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol
	Loading rate (mg/l)	Measured (μg/l)
NOERC	100	4
72h-ERC10	>100	>4
72h-ERC50	>100	>4
NOEYC	100	4
72h-EYC10	>100	>4
72h-EYC50	>100	>4

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol no reduction of growth rate or inhibition of yield was recorded at any of the concentrations of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol tested.
The EC50 for both growth rate reduction (ERC50: 0-72h) and yield inhibition (EYC50: 0-72h) 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.
The NOEC for growth rate reduction and yield inhibition exceeded the concentration present in 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.
Due to the extremely low solubility of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in test medium, concentration levels that might be toxic for algae could not be reached.

Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test 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. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009, the ISO International Standard 8692, 2004 and the OECD series on testing and assessment number 23, 2000. The study was performed in accordance with the Principles of Good Laboratory Practice (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 6 replicates of exponentially growing algal cultures 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 three replicates per test group were exposed to dilutions representing 0.1, 1.0 and 10% of the filtrate. The total test period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 hours of exposure and at the end of the test period.

At the start of the test, the actual test concentration in a sample taken from the undiluted filtrate 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). After 24 hours of exposure, the concentration had dropped below the limit of detection. 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 no reduction of growth rate or inhibition of yield was recorded at any of the concentrations of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol tested.

The EC50 for both growth rate reduction (ERC50: 0-72h) and yield inhibition (EYC50: 0-72h) 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.

The NOEC for growth rate reduction and yield inhibition exceeded the concentration present in 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.

Due to the extremely low solubility of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in test medium, concentration levels that might be toxic for algae could not be reached.

Description of key information

Key value determined using OECD & EU test guidance.

Due to the extremely low solubility of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in test medium, concentration levels that might be toxic for algae could not be reached.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

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

6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol no reduction of growth rate or inhibition of yield was recorded at any of the concentrations of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol tested.

The EC50 for both growth rate reduction (ErC50: 0-72h) and yield inhibition (EyC50: 0-72h) 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.

The NOEC for growth rate reduction and yield inhibition exceeded the concentration present in 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.

Due to the extremely low solubility of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in test medium, concentration levels that might be toxic for algae could not be reached.