6,6'-di-tert-butyl-2,2'-methylenedi-p-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:

1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(1984)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material: not reported

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.63, 1.3, 2.5 and 5.0 mg/L (nominal), measured: 0.56, 1.2, 2.4 and 4.8 mg/L

Vehicle:

yes

Details on test solutions:

2,2'-methylenebis(6-tert-butyl-4-methylphenol) was mixed with DMF/Castor oil HCO-40 (3/1(w/w)) and dissolved into test medium to prepare the nominal concentrations described above (No precipitations as well as emulsions were observed in all the exposure levels). To set the
dispersant concentrations equal (i.e. 100 µL/L) between the nominal concentrations, DMF/Castor oil HCO-40 solution was also used for the preparations. Due to low water solubility of 2,2'-methylenebis(6-tert-butyl-4-methylphenol), homogenous solution could be attained only at (and less than) the nominal concentration of 5.0 mg/L with the maximum allowable dispersant concentration of 100 µL/L.

Test organisms (species):

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

Details on test organisms:

Green algae (Selenastrum capricornutum), the strain ATCC22662 obtained from American Type Culture Collection, and subcultured at the testing facility.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

no remarks

Post exposure observation period:

not reported

Hardness:

not data

Test temperature:

23.5 to 23.7 °C measured at each vessel at the beginning and the end of the test.

pH:

7.6 to 7.7 measured at each vessel at the beginning of the test.
7.4 - 7.5 measured at each vessel at the end of the test.

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

Nominal Concentrations: 0.63, 1.3, 2.5, 5.0 mg/L, solvent control and control
Measured concentrations: 0.56, 1.2, 2.4 and 4.8 mg/L at the beginning (0h) of the test.
Measured concentrations: 0.47, 0.91, 2.1 and 4.2 mg/L at the end (72h) of the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL glas Erlenmeyer flask (silicon plugs breathable)
- Test volume: 100 mL
- Initial cell density: 10000 cells/mL
-Light conditions: 4000-5000 lx, continuous
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1

GROWTH MEDIUM
- OECD medium used: yes

- Determination of cell concentrations: ISOTON II from Beckman Coulter, Inc.

- Range finding study: 0.5 and 5.0 mg/L
- Test concentrations: 0.63, 1.3, 2.5 and 5.0 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, lot No.ACQ2610 from Wako Pure Chemical Industries, Ltd

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: actual concentration measured and greater than 80% of the nominal

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: actual concentration measured and greater than 80% of the nominal

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: actual concentration measured and greater than 80% of the nominal

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: actual concentration measured and greater than 80% of the nominal

Details on results:

The determined 72 hours EC50 values were >5.0 mg/L based on the nominal concentrations. The NOEC´s for biomass and growth rate were evaluated to be 0.63 mg/L and 1.3 mg/L, respectively.

Results with reference substance (positive control):

The 72 h-EC50 value (Biomass) determined was 0.44 mg/L.

Reported statistics and error estimates:

The NOEC’s described above were defined as the maximum nominal concentrations at which biomass and growth rate were not significantly different from the control (p = 5%).

Cell density measured during the experiment:

 

NominalConcentration (mg/L)	Mean Cell Density (x10^4cells/mL) +/- SD
	0 h	24 h	48 h	72 h
Control	I.+/-0	7.67 +/-0.32	45.9 +/-0.9	278 +/-7
Solvent Control	II. +/-0	7.81 +/-0.21	44.1 +/-1.7	280 +/-26
0.63	III. +/-0	7.27 +/-0.56	42.9 +/-1.2	271 +/-3
1.3	IV. +/-0	7.32 +/-0.32	42.4 +/-0.3	242 +/-7
2.5	V. +/-0	7.52 +/-0.25	41.0 +/-1.7	226 +/-12
5.0	1.00 +/-0	7.45 +/-0.14	37.6 +/-1.5	185 +/-8

 

Growth rate curve (Area under the curve and the slope evaluated)

 

NominalConcentration (mg/L)	0 – 72 h		24 – 48 h		24 – 72 h
	Mean Area	Inhibition %	Mean Rate	Inhibition %	Mean Rate	Inhibition %
Control	45569440	0	0.0745	0	0.0748	0
Solvent Control	45426080	0.3	0.0721	3.2	0.0745	0.4
0.63	43989280	3.5	0.0740	0.7	0.0754	-0.9
1.3	40351360	11.5	0.0732	1.7	0.0729	2.5
2.5	38121920	16.3	0.0706	5.3	0.0708	5.3
5.0	32365760	29.0	0.0674	9.5	0.0669	10.6

 

Solvent Control: DMF/HCO－40＝3／1（100μL/L）

Validity criteria fulfilled:

yes

Remarks:

The test was valid since the cell density in control increased by 278 times at the termination of experiment.

Conclusions:

The determined 48 h-EC50 value was more than 5.0 mg/L (for biomass and growth rate) based on the nominal concentrations. The NOEC value for biomass (0-72 h) was evaluated to be 0.63 mg/L. The NOEC’s for growth rate were 2.5 mg/L (24-48 h) and 1.3 mg/L (24-72 h).

Executive summary:

In order to test acute toxicity to algae, Selenastrum capricornutum, of 2,2'-methylenebis(6 -tert-butyl-4 -methylphenol) was exposed to the test solution of four nominal concentrations (0.63, 1.3, 2.5 and 5.0 mg/L) of 2,2'-methylenebis(6 -tert-butyl-4 -methylphenol), solvent control and control for a period of 72 hours under static conditions. The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. The measured concentrations confirmed that deviation from the nominal concentrations was less than 20 %( measured concentrations were in the range of 70 - 84 % of the nominal concentrations.

Due to low water solubility of the substance, maximum dispersable concentration was limited to be 5.0 mg/L (nominal).

The determined 48 h-EC50 value was more than 5.0 mg/L (for biomass and growth rate) based on the nominal concentrations. The NOEC value for biomass (0-72 h) was evaluated to be 0.63 mg/L. The NOEC’s for growth rate were 2.5 mg/L (24-48 h) and 1.3 mg/L (24-72 h).

Experimental designs and results were well documented and prescribed conditions in the guideline were well satisfies.

Description of key information

The 48 hr-EC50 was > 5.0 mg/L (for biomass and growth rate) based on nominal concentrations.
The NOEC value for biomass (0-72 hr) was evaluated to be 0.63 mg/L, while the NOEC values for growth rate was 1.3 mg/L. As the reported NOECs are above the water solubility no PNEC can be derived based on these results.

 

Additional information

Two studies are available that asses the toxicity of DBMC towards algae and cyanobacteria.

 

The key study from the Environmantal Agency of Japan (1999) is assessed in the OECD SIDS Report (2001) and the PBT Report (2009) of the European Chemical Bureau (ECB). The EC50 value obtained from this study is > 5.0 mg/L and the NOEC is 0.63 mg/L. However, the relevant test was performed with a solubilizer in order to overcome the low water solubility of 0.007 mg/L. As a consequence, no effects have been observed up to the limit of the water solubility.

 

The supporting study (Zhang, 2021) addresses the toxicity of DBMC to cyanobacteria. The 7d EC50 was determined to be 7.8 mg/L and the 7d NOEC is 3.125 mg/L. Also in this case, no toxic effects have been observed up to the limit of water solubility.