2,2',6,6'-tetra-tert-butyl-4,4'-methylenediphenol

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: A guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 100 mg of test material was dissolved in dimethylformamide and the volume adjusted to 100 mL to give a final concentration of 1 mg/mL. An aliquot of 100 uL of this stock solution was dispersed in reconstituted water and the volume adjusted to 1 L to give a final concentration of 0.10 mg/L of test material.
- Controls: Blank and solvent group.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution): 100 uL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: In-house laboratory cultures
- Age at study initiation (mean and range, SD): less than 48 hours
- Method of breeding: Partthenogenesis
- Feeding during test: Did not recieve food during the test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

250 mg/L (theoretical value)

pH:

7.8 + or - 0.2

Nominal and measured concentrations:

0.10 mg/L of test material

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL glass jar filled with 200 mL of test solution
- Aeration: No
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (see table)
- Conductivity: <5 uS cm-1
- Intervals of water quality measurement: 0, 24, and 48 hours (see table)

OTHER TEST CONDITIONS
- Adjustment of pH: With NaOH and HCl
- Photoperiod: 16 hours light, 8 hours dark, 20 min dawn and dusk periods

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: No adverse reactions to exposure were observed.
- Other biological observations: No adverse reactions to exposure were observed.
- Mortality of control: 0
- Other adverse effects control: No adverse reactions to exposure were observed.
- Abnormal responses: No adverse reactions to exposure were observed.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: No

Preliminary solubility work indicated that a test concentration of 0.10 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and the limitations placed by the guidline in the amount of vehicle that could be used.

Physical-Chemical Measurements
Nominal Concentration (mg/L)		0 Hours				24 Hours	48 Hours
		pH	O2mg/L	%ASV	T°C	T°C	pH	O2mg/L	%ASV	T°C
Control	R1	8.0	8.2	92	21.0	21.0	8.0	8.0	90	21.0
	R2	8.0	8.2	92	21.0	21.0	8.0	8.0	90	21.0
Solvent Control	R1	8.0	8.2	92	21.0	21.0	8.0	8.1	91	21.0
	R2	8.0	8.2	92	21.0	21.0	8.0	8.0	90	21.0
0.10	R1	8.0	8.1	91	21.0	21.0	8.0	8.0	90	21.0
	R2	8.0	8.2	92	21.0	21.0	8.0	8.0	90	21.0
	R3	8.0	8.2	92	21.0	21.0	8.0	8.0	90	21.0
	R4	8.0	8.2	92	21.0	21.0	8.0	8.0	90	21.0

Cumulative Immobilization Data in the Definitive Study
Nominal Concentration (mg/L)		Cumulative ImmobilizationDaphnia (Initial Population: 10 Per Replicate)
		24 hours			48 hours
		No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control	R1	0	0	0	0	0	0
	R2	0			0
Solvent Control	R1	0	0	0	0	0	0
	R2	0			0
0.10	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0

Conclusions:

The 48-Hour EC50for the test material to Daphnia magna based on nominal test concentrations was greater than 0.10 mg/L and correspondingly the No Observed Effect Concentrations was >= 0.10 mg/L.

Executive summary:

A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the OECD Guidelines for Testing Chemicals (1984) No 202, “Daphniasp, Acute Immobilization Test and Reproduction Test”. Following a preliminary range-finding study, forty daphnids (4 replicates of 10 animals) were exposed to an aqueous solution of the test material at a concentration of 0.10 mg/L for 48 hours under static conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. The 48-Hour EC₅₀for the test material to Daphnia magna based on nominal test concentrations was greater than 0.10 mg/L and correspondingly the No Observed Effect Concentrations was >= 0.10 mg/L. The test concentration of 0.10 mg/L was the highest attainable test concentration due to the limited solubility of the test material in water and auxiliary solvent, and having due regard for the amount of auxiliary solvent permitted in the test under the OECD Guidelines.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

August 1987 to February 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The analytical monitoring has not been performed.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: U.S. Environmental Protection Agency (1975). Methods for acute toxicity testing with fish, macro-invertebrates and amphibians. EPA-660/3-75-009.

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES of the substance are all available in section 4 "Physical and chemical properties".

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test solution added to the reconstituted freshwater. For details on reconstituted freshwater, refer to the "details on test conditions" section.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus
- Source: Strain obtained via ICI's Brixham Laboratory from IRChA, France
- Age at study initiation (mean and range, SD): Less then 24 hours.
Procedure: Youns for use in acute toxicity tests are collected from cultures aged between 14 and 35 days. 24 h before a test is set up the culture is
sieved and the young discarded. The culture is then re-sieved 23 h later so that the D magna retained on the sieve are less than 24 h old. The
D magna are left for 1 h and then those swimming actively are used for testing. Young for testing are not taken from cultures which contain adults
containig, ephippia; these cultures are discarded. The procedures described above were followed for the production and collection of the less than 24h old D. magna used in this experiment.

ACCLIMATION
- Acclimation conditions (same as test or not): The D. magna are cultured in glass vessels (0.5 to 4 litre) containing reconstituted freshwater. New cultures are started with animals less than 24 h old, at a density of about 20 per litre. The water in the culture vessels is renewed weekly. The cultures are sieved regularly to remove young. Adults are first removed temporarily by pipette and the culture vessel contents passed through a sieve (2.5 mesh/mm). The water is returned, with the adult D magna, to the culture vessel. The young D. magna are retained on the sieve and can be used to start a new culture. Cultcure are discarded when approxtmately 5 weeks old .
- Type and amount of food: Chlorella vulgaris at a concentration of 1 x 10^6 cells/ml.
- Feeding frequency: fed daily

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

166 mg/L as CaCO3

Test temperature:

18-22°C

pH:

7.8-8.0

Dissolved oxygen:

9.0-9.2 mg/L

Nominal and measured concentrations:

Nominal concentrations: 500 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass crystallising dishes
- Material, size, headspace, fill volume: 300 mL
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): Duplicates
- No. of vessels per control (replicates): Duplicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: a reconstitued freshwater is prepared by dissolving the following amounts of Analar grade salts in glass-distilled deionised water:
* NaHCO3: 192 mg/L
* CaSO4·2H20: 120 mg/L
* MgS04. 7H20: 240 mg/L
* KCl: 8 mg/L
Before use for culturing, a soil extract is added to the reconstituted freshwater at a rate of 20 ml/L . The soil extract is prepared by autoclaving 100 g soil per litre of reconstituted freshwater for 15 minutes at 120°C. Solids are removed by filtration through Whatman GF/C paper.

OTHER TEST CONDITIONS
- Photoperiod: artificial light 16h light/8h dark cycle

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 24h and 48h the numbers of immobilized D. magna were recorded. D. magna were considered to be immobile if, when the contents of the dish were briefly stirred they did not swim during a 10 second period of observation.

TEST CONCENTRATIONS
- Test concentrations: 500 and 1000 mg/L

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Note: No immobilization recorded, with the exception of one which was trapped at the surface in one vessel at the highest concentration tested (1000 mg/L).

Results with reference substance (positive control):

No reference substance

Reported statistics and error estimates:

Not reported in the study report

Validity criteria fulfilled:

yes

Executive summary:

The acute toxicity of 4,4'-methylene-bis-2,6-di-tert-butyl phenol to the crustacean zooplankter, Daphnia magna was determined in a static water test without renewal of the test solutions. During a 48h exposure period, only 1 of 20 D. magna was immobilised at 1000

mg/L, the highest concentration tested. The 24h and 48h EC50 value were therefore both greater than 1000 mg/L.

Description of key information

Two acute studies in Daphnia magna have confirmed no that the test substance does not have any toxic effects on the tested organisms up to its solubility limit in the test media. 

Additional information

The substance is not acutely toxic to Daphnia magna up to its solubility limit in the test media. One older study was conducted with nominal saturate concetrations of 1000 mg/l, while a newer study confirmed the solubility and stabilty in the test medium at the solubility limit and confirmed no toxicity at the solubility limit. This was also confirmed in a longterm daphnia reproduction study that revealed no effects up to the highest achievable test concentration.