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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
GLP compliance:
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material: not reported
Analytical monitoring:
Details on sampling:
-Concentrations: 0.50, 1.6 and 5.0 mg/L of 2,2'-methylenebis(6-tert-butyl-4-methylphenol) were measured during 48 hours as a limit tst.
The nominal concentration of 2,2'-methylenebis(6-tert-butyl-4-methylphenol) were 0.50, 0.90, 1.6, 2.8 and 5.0 mg/L plus control and solvent control.
Exposure water sampled at the initiation and the termination (i.e. 48 h) of the exposure were extracted with hexane and analyzed for 2,2'-methylenebis(6-tert-butyl-4-methylphenol) by using GC-MS.
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 dilution water to prepare the nominal concentrations (No precipitations as well as emulsions were observed in all the exposure levels). To set the dispersant concentrations equal (i.e. 100 mg/L) between the nominal concentrations, MF/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 this maximum allowable dispersant concentration of 100 mg/L.
Test organisms (species):
Daphnia magna
Details on test organisms:
Screened female Daphnia, of sufficient size and health were macroscopically separated from restless juveniles ring.
These were transferred into prepared container and bred new teenagers.
The parent animals were housed 2-4 weeks under the following conditions.
1 week, from birth to young maturity.
The young animals were at least 2 times removed.
2-4 weeks later, females with juveniles were intracameral selected on the day before the test starts.
The 24 h old juveniles were used for the test.
Sterile, male and dormant eggs did not occur. In addition, the mortality rate was (one animal died 100 animals during rearing) 1%.

- dilution water: dechlorinated tap water of the city Takarazuka
- animals: 20 - 40 animals/L water, in adult animals less than 20 animals/L water
- Temperaure: 20 ± 1°C
- Photoperiod: 16 hlight / 8 h dark
- Feeding during test: approach concentrating Chlorella, which are commercially available; product name: raw Chlorella V12 from Chlorella Industry Co., Ltd.; before adding feed, 1% suspension = 1100 mg carbon /L
- amount of feed: As a general rule, per capita = 0.1 ~ 0.2 mg C/day

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Post exposure observation period:
not reported
55.2 mg CACO3/L = 3.1 °dH
Test temperature:
ranged from 19.7 to 20.0°C
ranged from 8.1 to 8.2
Dissolved oxygen:
ranged from 8.2 to 8.6 mgl/L (more than 60% saturation)
not reported.
Nominal and measured concentrations:
nominal concentrations:
0.50, 0,90, 1.6, 2.8, 5,0 mg/L, solvent control and control
Details on test conditions:
- Test vessel: 100 mL beaker
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): one
- No. of vessels per vehicle control (replicates): one
- Feeding: none

- Source/preparation of dilution water: Dechlorinated tap water fro the city of Takarazuka
- pH: 8.1
- Hardness: 55.2 mg CACO3/L = 3.1 °dH
- Photoperiod: 16 h light / 8 h dark

- Limit Test concentrations: 0.50, 1.6 and 5.0 mg/L
- Test concentrations: 0.50, 0.90, 1.6, 2.8 and 5.0 mg/L
- Vehicle concentration: 100 mg/L highly, used for 5 mg/L
Reference substance (positive control):
Potassium dichromate, lot No. ACQ2610, Wako Pure Chemical Industries, Ltd.
48 h
Dose descriptor:
Effect conc.:
> 4.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: immobility
48 h
Dose descriptor:
Effect conc.:
0.74 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: immobility
Details on results:
not reported
Results with reference substance (positive control):
The 48 h-EC50 value of the reference substance was determined as 0.54 mg/L.
Reported statistics and error estimates:
Since the rate of immobility observed at the maximum concentration (i.e. 5.0 mg/L nominal) was less than 50 %, no statistical method (e.g. Binominal, Moving average or Probit method) was employed for determining the EC50 value. A regression line for GC-MS analyses was derived by least square calculations. Numerical values were rounded according to the method specified in JIS.

Cumulative number of immobilized daphnia:

Measured Concentration [mg/L]

Cumulative Number of Immobilized Daphnia (Percent immobility)

24 h

48 h


0 (0)

0 (0)

Solvent Control

0 (0)

0 (0)


0 (0)

0 (0)


0 (0)

0 (0)


0 (0)

1 (5)


0 (0)

1 (5)


0 (0)

7 (35)

Validity criteria fulfilled:
(- The immobilisation and other abnormalities in the control id not exceed 10 % by the end of the test. - The dissolved oxygen concentration remained above 3 mg/L throughout the exposure period.)
The determined 48 h-EC50 value was more than 4.8 mg/L based on the geometric mean of the measured concentrations. The 48h-NOEC was evaluated to be 0.74 mg/L.
Executive summary:

In order to test acute toxicity to invertebrates (Daphnia magma) of 2,2'-methylenebis(6-tert-butyl-4-methylphenol) was exposedto the test solution of five nominal concentration of 2,2'-methylenebis(6-tert-butyl-4-methylphenol)

( 0.50, 0.90, 1.6, 2.8 and 5.0 mg/L), control and solvent control for a period of 48 hours under static conditions.Mobility and visible abnormalities were recorded at 24 and 48 h. The measured concentrations cofirmed that deviation from the nominal concentrations was less than 20 %, with the exception of 1.6 mg / L, the measured concentration was 1.1 mg / L (71%) (the other concentrations were in range of 80 - 96 % of the nominal concentrations). After 48 hours an EC50 of >4.8 mg/L was determined and a NOEC of 0.74 mg/L was calculated.

Due to low water solubility of the substance, maximum dispersable concentration was limited to be 5.0 mg/L (nominal). Experimental designs and results were well documented and prescribed conditions in the guideline were well satisfies.

Description of key information

The lowest observed 48-h-EC50 for freshwater invertebrates is > 4.8 mg/L. As the reported EC50 is above the water solubility no PNEC can be derived based on this result.

Key value for chemical safety assessment

Additional information

The above study is assessed in the OECD SIDS Report (2001) and the PBT Report (2009) of the European Chemical Bureau (ECB). This test can be regarded as reliable without restrictions.

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. No PNEC can be derived based on this result.