Reaction mass of 4-tert-butyl-2,5-xylenol and 6-tert-butyl-2,4-xylenol

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:

March 9 to 18, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Meets the requirements of GLP. There are no deviations from the recommended guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: acetone was used as solvent to prepare the test solutions of the amount needed. A stock solution containing 100 g/L in acetone was prepared. From this stock solution, additional solutions (4.6/10/22/46 g/L) were prepared through dilution with acetone.

- Concentration of vehicle in test medium (stock solution and final test solution): the concentration of solvent in the test solutions was 0.1 mL/L at the most.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Name: Daphnia magna, variety STRAUS
- Strain: Berlin
- Source: Umweltbundesamt Berlin
- Age at study initiation (mean and range, SD): between 0 and 24 hours
- Feeding during test: no
- Food type: unicellular green algae (Desmodesmus subspicatus)

ACCLIMATION
- Acclimation period: 30 minutes
- Acclimation conditions (same as test or not): in dilution water
- Health during acclimation (any mortality observed): animals which showed no apparent damage were used for the test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

The test vessels were left to stand for 48 hours. After 24 and 48 hours, the immobilised daphnia were counted.

Hardness:

250 mg CaCO3/L

Test temperature:

19.7-20.6 ºC

pH:

7.4 to 7.9

Dissolved oxygen:

Above 9.0 mg/L

Nominal and measured concentrations:

Table 8.1 d Measured Concentrations
Nominal Concentration in mg/L Measured Concentration in mg/L % Recovery after 48 h % of Nominal Concentration
0 h 48 h 0 h 48 h
control < LOQ < LOQ -- -- --
solvent control < LOQ < LOQ -- -- --
0.46 0.37 0.31 86% 80% 68%
1.0 0.85 0.69 81% 85% 69%
2.2 1.79 1.68 94% 81% 76%
4.6 4.21 3.48 83% 92% 76%
10.0 9.05 8.50 94% 91% 85%

LOQ = Limit of quantification (1 mg/L in solvent, corresponding to 0.1 mg/L in aqueous medium)

The measured concentrations at t = 0 were in a range of 80 – 91% of the nominal concentrations. The measured concentrations at t = 48h were in a range of 68 - 85 % of the nominal concentrations and 81 - 94 % of the start concentrations. Therefore, the determination of the biological results was based on the geometric mean of the measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Schott flasks, closed with blue lids
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: no headspace
- No. of organisms per vessel: 5
Main study:
- No. of vessels per concentration (replicates): 4 vessels, each containing 20 mL test solution and five daphnia
- No. of vessels per control (replicates): 4 vessels, each containing 20 mL dilution water and five daphnia
- No. of vessels per solvent control (replicates): 4 vessels, each containing 20 mL dilution water and five daphnia
Positive control:
- No. of vessels per positive control (replicates): 4 vessels, each containing 20± 5 mL test solution and five daphnia
- No. of vessels per control (replicates): 4 vessels, each containing 20± 5 mL dilution water and five daphnia

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water with an enrichment of certain minerals (as demanded in the guidelines) is used in the test. After preparation, the dilution water is aerated, and the pH is adjusted to 7.8 ± 0.2.

OTHER TEST CONDITIONS
- pH: 7.4 to 7.9
- Photoperiod: no lighting

TEST CONCENTRATIONS
- Test concentrations: 0.46 / 1.0 / 2.2 / 4.6 / 10 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval not determinable

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval not determinable

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

3.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval not determinable

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval not determinable

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

8.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval not determinable

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

8.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval not determinable

Details on results:

The values are based on the measured concentrations. If a sigmoidal fit is used, no confidence interval can be calculated. Since there were only two values between 0 % and 100 % immobility, a linear fit with a probability-y-axis could not be performed.

Results with reference substance (positive control):

- Results with reference substance valid? yes

Parameter Value 95%-confidence interval
NOEC 24 h 0.5 mg/L not determinable
24h EC50i 1.2 mg/L not determinable
24h EC100i 2.5 mg/L not determinable

Reported statistics and error estimates:

The analytical determinations of the test item showed acceptable correlation between nominal and measured concentrations and good recovery rates after 48 h (compared with the start concentrations). Therefore, the test item can be stated as stable under the test conditions. The determination of the results was based on the measured concentrations, as concentration at the beginning of the test was lower than the nominal concentrations. This is considered to be due to the volatility of the test item. Throughout the test, concentrations were stable: at the end of the test, the measured concentrations were > 80% of the start concentrations in all treatments. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test is considered valid.

Validity criteria fulfilled:

yes

Remarks:

The 24h-EC50i of K2Cr2O7 was in the recommended range; immobilisation in the controls did not exceed 10 %; the concentration of dissolved oxygen at the end of the test was higher than 3 mg/L.

Conclusions:

The following results were determined for the test item (species:Daphnia magna):
 
24h-NOEC = 1.7 mg/L
48h-NOEC = 1.7 mg/L
24h-EC50i= 3.8 mg/L
48h-EC50i= 3.5 mg/L

Executive summary:

A first experiment, conducted in beakers, could not be used for evaluation, as analytical determinations showed very bad stability of test item in the solutions. This was due to evaporation from the open vessels. As the test item is volatile, the experiment was performed in closed vessels without headspace using five concentrations ranging from 0.46 to 10 mg/L (nominal concentrations). Twenty daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted.

Two concentrations showed toxicity between 0 and 100% immobilisation. The 24h-EC50i of potassium dichromate was tested in a current reference test. The value was determined as 1.2 mg/L, lying within the demanded range of 0.6 – 1.7 mg/L. At the beginning and at the end of the test, the content of the test item in the test solutions was determined using GC-analysis. Start concentrations lay in a range of 80 to 92% of the nominal concentrations. The recovery after 48 hours ranged from 81 to 94 % of the start concentration, the correlation between nominal and measured concentration was acceptable. The determination of the biological results was based on the measured concentrations. The following results were determined for the test item (species: Daphnia magna).

24h-NOEC = 1.7 mg/L
48h-NOEC = 1.7 mg/L
24h-EC50i = 3.8 mg/L
48h-EC50i = 3.5 mg/L

Description of key information

Key study: Experimental result: Test according to OECD guideline 202 and EU method C.2. GLP study.

48h-EC50= 3.5 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

3.5 mg/L

Additional information

Key study: Experimental result: Test according to OECD guideline 202 and EU method C.2.

Daphnia were exposed to the test item for 48 hours in a static system.  

The following results were determined for the test item:
24h-NOEC = 1.7 mg/L
48h-NOEC = 1.7 mg/L
24h-EC50i= 3.8 mg/L
48h-EC50i= 3.5 mg/L