2,5-di-tert-butyl-p-benzoquinone

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:

05.10.2016-16.12.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0010436
- Expiration date of the lot/batch: 03.05.2017
- Purity test date: 23.05.2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature (20 ± 5°C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: not specififed
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

For both experiments a saturated solution was prepared. This was done by weighing the
nominal load 100 mg/L (real load 100.0 mg/L both experiments), adding the corresponding
amount of dilution water and shaking vigorously for 24 hours. Because photosensitivity of
the test item can’t be excluded, in the second experiment the flasks were shaken for 24 ±
1 hours in darkness. The resulting solution was filtrated through 0.45 μm PTFE filters.

Analytical monitoring:

yes

Details on sampling:

To 200 mL test- and control solution 10 g NaCl was added, then the solution was extracted two times with the solvent dichloromethane (2 x 4 mL), the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane after addition of 150 μL ISTD stock solution (1000 mg/L in dichloromethane) and the solution was measured via GC/FID. Twentyfold dilution was achieved during extraction.

Details on test solutions:

Deionised water with an enrichment of certain minerals (dilution water as demanded in the
guidelines) was used in the test.
Dilution water specification
Parameter Concentration in mg/L
CaCl2*2H2O 293.80
MgSO4*7H2O 123.30
NaHCO3 64.80
KCl 5.80
Resulting hardness in mmol/L: 2.502
Resulting hardness in mg CaCO3/L: 250
Deviations from the nominal weighted loads were less than 5%.
After preparation, the dilution water was aerated.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species Daphnia magna
Variety STRAUS
Strain Berlin
Sex female
Age between 0 and 24 hours
Origin Umweltbundesamt Berlin
In-house breeding since 27. September 2007
Selection of the test system was made following the proposal of the guidelines.

Daphnia magna is bred in the LAUS GmbH throughout the year. The animals are kept for
the use in toxicity tests. They multiply by parthenogenesis, thus being genetically identical.
The husbandry is performed similar to the method described in the OECD guideline, following
SOP 115 002 01 („Zucht und Hälterung von Daphnia magna STRAUS“), Version 12
from 02. Feb. 2015.
Vessels preserving glasses, nominal volume 2 L
Medium M4-Medium (recipe of ELENDT)
Food unicellular green algae (Desmodesmus subspicatus)
Medium renewal twice a week
Photo period 16 hours light/8 hours dark, using neon tubes
Temperature 20 +- 2 °C

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

not specified

Test temperature:

21 C

pH:

7.7-7.9

Dissolved oxygen:

8.6-9.2 mg/L

Salinity:

not specified

Conductivity:

not specified

Nominal and measured concentrations:

Nominal 100 mg/l
Measured 0h=0.55mg/L, 24h old=0.38 mg/L, 24h new=0.53mg/L, 48h=0.39mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Closed glass flasks, total volume 65 mL
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume:
- Aeration: yes

Selection of Daphnia
19.5 hours (first experiment) resp. 19 hours (second experiment) before the start of the
test, the adult animals were separated from the young. 0.5 hours before test start, the
adults were caught with the help of a glass tube, and the newborn Daphnia, aged between
0 and 19 hours, were sieved from the medium and immediately placed into a 250 mLbeaker
containing dilution water. After a settling-in period of 30 minutes, animals which
showed no apparent damage were used for the test.
Switching from M4-medium (husbandry) to Dilution water (test) has been shown not to
cause any detrimental effects for test Daphnia.

Study Performance
Using a glass tube, the Daphnia were caught and lifted from the beaker. They were put on
a small sieve, and the medium surrounding the animals was sucked off using absorbent
paper. Immediately after that, the animals were put into the respective test solution.
The test vessels were left to stand for 24 hours. Then, the vessels were inspected for immobilised
Daphnia, and medium renewal was performed: the Daphnia were put into new
test vessels with freshly prepared test solution. After 48 hours (first experiment) resp. 49
hours (second experiment), the immobilised Daphnia were counted again. Daphnia are
considered immobilised if they perform no movements or are only able to move their antennae
when the beaker is gently agitated.
The pH, the concentration of dissolved oxygen and the content of the test item in the test
vessels were measured at the beginning and at the end of the test and at the medium renewal
in the old and the new test solution.

Experimental Conditions
Date of performance 11. – 13. Oct. 2016 (first experiment)
18. – 20. Oct. 2016 (second experiment)
Treatments 100 mg/L (concentration)
Temperature 19.0 – 20.4 °C (first experiment)
18.5 – 20.5 °C (second experiment)
Duration 48 hours
Observation times 24 and 48 hours
Medium renewal after 24 hours
Feeding none
Lighting 16 hours diffuse lighting/ 8 hours dark
Test vessels closed glass flasks, total volume 65 mL
Replicates (Treatments) 4 vessels, each completely filled with test solution and 5
Daphnia
Replicates (Blank control) 4 vessels, each completely filled with dilution water and 5
Daphnia

Reference substance (positive control):

yes

Remarks:

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

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 0.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

not determinable

Remarks:

limit of solubility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

not determinable

Remarks:

limit of solubility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

not determinable because of methodological limitations

Remarks:

limit of solubility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 0.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

not determinable

Remarks:

limit of solubility

Results with reference substance (positive control):

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a
current reference study. The 24h-EC50 value was determined as 1.7 mg/L, lying within the
demanded range of 0.6 – 2.1 mg/L stated in the OECD guideline.

Reported statistics and error estimates:

No observations were made which might cause doubts concerning the validity of the study
outcome. All validity criteria were met.

Table 8.1-a Immobility

Nominal Concentration in mg/L	Immobility 24 hours					Immobility 48 hours
	absolute				in %	absolute				in %
Blank control	0	0	0	0	0	0	0	0	0	0
100	0	0	0	0	0	0	0	0	0	0

Table 8.2-a pH-values

Nominal Concentration in mg/L	pH
	0h	24h old	24h new	48h
Blank control	7.8	7.7	7.8	7.9
100	7.7	7.7	7.8	7.9

Table 8.2-b O2-values

Nominal Concentration in mg/L	O2 concentration in mg/L
	0h	24h old	24h new	48h
Blank control	9.2	8.8	9	8.7
100	9.2	8.7	9	8.6

Validity criteria fulfilled:

yes

Conclusions:

Two valid experiments were performed.
20 Daphnia were exposed to the test item for the treatment for 48 hours in a semi static
test system. Both experiments were performed as a limit test at the nominal concentration
100 mg/L. The concentration showed no toxicity. None of the animals were immobilised
neither in the treatment nor in the blank control.
At the beginning and at the end of the test, the content of the test item in the test solutions
was determined using GC/FID -determination. In the first experiment, the measured concentrations
were lower than during validation. This might be caused by photosensitivity of
the test item. Therefore, the experiment was repeated. For preparation of the saturated
solution, in the second experiment the flasks were shaken for 24 hours in darkness. The
measured concentrations in the second experiment were in the expected range. Therefore,
for evaluation, only the measured concentrations of the second experiment were
used.
Because of the poor solubility of the test item, the correlation between nominal and measured
concentrations was weak. Therefore, the determination of the biological results was
based on the geometric mean of the measured concentrations in the second experiment.
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a
current reference study. The 24h-EC50 value was determined as 1.7 mg/L, lying within the
demanded range of 0.6 – 2.1 mg/L stated in the OECD guideline.
No observations were made which might cause doubts concerning the validity of the study
outcome. All validity criteria were met.
The result of the test is considered valid.

Executive summary:

Two valid experiments were performed.

Both experiments were performed as a limit test at the nominal concentration 100 mg/L. 20

Daphnia were exposed to the test item for 48 hours in a semi static test system. After 24

and 48 hours, the immobilised Daphnia were counted.

The concentration showed no toxicity. None of the animals were immobilised neither in the

treatment nor in the blank control.

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a

current reference study. The 24h-EC50 value was determined as 1.7 mg/L, lying within the

demanded range of 0.6 – 2.1 mg/L stated in the OECD guideline.

At the beginning and at the end of the test, the content of the test item in the test solutions

was determined using GC/FID -determination. In the first experiment, the measured concentrations

were lower than during validation. This might be caused by photosensitivity of

the test item. Therefore, the experiment was repeated. For preparation of the saturated

solution, in the second experiment the flasks were shaken for 24 hours in darkness. The

measured concentrations in the second experiment were in the expected range. Therefore,

for evaluation, only the measured concentrations of the second experiment were

used.

Because of the poor solubility of the test item, the correlation between nominal and measured

concentrations was weak. Therefore, the determination of the biological results was

based on the geometric mean of the measured concentrations in the second experiment.

The following results were determined for the test item 2,5-Di-tert-butyl-1,4-benzoquinone

(species: Daphnia magna).

48h-NOEC ≥ 0.45 mg/L (limit of solubility)

48h-LOEC > 0.45 mg/L (limit of solubility)

24h-EC50 > 0.45 mg/L (limit of solubility)

48h-EC50 > 0.45 mg/L (limit of solubility)

Description of key information

The results of the short-term aquatic toxicity study on daphnia magna showed the 48 hour EC50 is >0.45 mg/l at the limit of solubility.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.45 mg/L