2,6-di-tert-butyl-4-nonylphenol

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

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)

Version / remarks:

2004

Deviations:

yes

Remarks:

No major (the test temperature increased by more than 1.0 °C during the test at several time points. The test is still valid, as it was shown that validity criteria are met).

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal loading rates of 6.25, 12.5, 25.0, 50.0 and 100 mg/L and a blank control
- Sampling method: Analytical samples taken at 0 hours (initial value) and 24 hours from fresh test solutions and 48 hours aged test solution were analysed from control and all test item loading rates.
- Sample storage conditions before analysis: deep frozen (≤ - 18 °C)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
The test item is poorly soluble in water. Therefore the test item is applied as water accommodated fraction (WAF). The WAF (S1) was prepared by weighing 100 mg test item on a weighing scoop and transferred to a volumetric flask. Test medium was added up to the bench mark. The solution was stirred by low energy stirring in a closed vessel at room temperature in the dark over 48 hours to dissolve a maximum amount of the test item. Subsequently the undissolved test item was allowed to float for a period of 1 h until the phases have separated. The solution showed oil lay on top. The WAF was withdrawn from the middle of the preparation vessel by suction (according to OECD Series on Testing and Assessment No. 23). Transfer of undissolved test item was avoided. The test item loading rates were prepared by dilution of adequate aliquots of the next highest application solution with test medium. The preparation procedure was repeated after 24 hours. ~50 mL of the prepared solutions were transferred to each test vessel.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone V
- Source: Federal Environment Agency in Berlin/Germany
- Feeding during test: no
- Culture conditions:
Daphnia magna was bred as single culture (1 daphnid per 100 mL) in Elendt M4 medium. The pHvalue of the aerated water was within a range of 6.0 – 9.0. The dissolved oxygen was above 60 % saturation and the total hardness 140 - 250 mg/L (as CaCO3), corresponding to 7.8 - 14°dH. The animals were fed with single cell green algae (Desmodesmus subspicatus, formerly Scenedesmus subspicatus) at least three times a week.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
A pipette was used to separate the young daphnids from the adults. Freshly hatched daphnids less than 24 hours old were used for the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

13°dH (232 mg/L CaCO3)

Test temperature:

20.1 ± 0.8 °C (Std. Dev.)

pH:

7.87 ± 0.16 (Std. Dev.)

Dissolved oxygen:

8.7 ± 0.3 mg/L (Std. Dev.)

Nominal and measured concentrations:

nominal loading rates of 6.25, 12.5, 25.0, 50.0 and 100 mg/L, measured: not detectable (control); 0.0846, 0.121, 0.266, 0.519 and 1.70 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beakers
- Material, size, headspace, fill volume: glass, 50 mL fill volume
- Renewal rate of test solution (frequency/flow rate): every 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 test medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: (0 (fresh), 24 (aged and fresh) and 48 hours (aged)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod /8 hours darkness daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 48h-EL50 and 48h-EC50

RANGE-FINDING STUDY
- Test concentrations: Following a semi-static range-finding test with nominal test item loading rates of 0.001, 0.01, 0.100, 1.00 and 10.0 mg/L and control a semi-static main test with nominal loading rates of 6.25, 12.5, 25.0, 50.0 and 100 mg/L and control was performed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.124 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

10.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality

Details on results:

- Biological observations:
After 24 hours of exposure no immobilisation was observed in the control as well as up to and including 25.0 mg/L. No immobilisation higher than the allowed control immobilisation was observed at the two highest test item loading rates 50.0 and 100 mg/L. The daphnids did partly swim at the surface of the test item loading rates 50.0 and 100 mg/L.

After 48 hours of exposure no immobilisation was observed in the control. 30 % of immobilisation was observed at 6.25 mg/L. In the test item loading rate of 12.5 mg/L, 50 % of the daphnids were immobile. 85 % of the daphnids were immobile at 25.0 mg/L and 95 % at test item loading rate 50.0 mg/L. At the highest test item loading rate 100 mg/L all daphnids were immobile. The daphnids did swim at the surface of all test item loading rates.


- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: In order to check the validity of the results, the toxicity of the reference item potassium dichromate was tested at 1.00 and 2.00 mg/L with 20 test organisms per test concentration. The results indicate an EC50 (24 h) of the reference item potassium dichromate between 1.00 and 2.00 mg/L. Since the results fall within the recommended rage of the OECD guideline 202 and the historical data generated with the reference item at the testing facility, the daphnids were suitable for the determination of the toxicological effects of the test item.

Reported statistics and error estimates:

The values for EL50/ EC50 were determined by Probit analysis using linear max. likelihood regression. The evaluation of data was performed by ToxRat Professional 3.2.1.

Validity criteria fulfilled:

yes

Conclusions:

48h-EC50: 0.124 mg/L

Executive summary:

In the Klimisch 1 GLP study from Lange (2017) the acute toxicity of 2,6-Di-tert-butyl-4-nonylphenol on Daphnia magna was determined in an 48 hour semi-static test according to OECD 202 with 24 h renewal interval. The test was performed with nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg test item/L prepared as WAF and a blank control. Four replicates with 5 daphnids each were set up.

After 48 hours 0, 30, 50, 85, 95 and 100 % immobilization was observed in the control and at nominal test concentrations of 6.25, 12.5, 25, 50 and 100 mg test item /L, respectively. The EL50 was 10.9 mg/L. Analytical measurements resulted in following geometric mean measured concentrations for the 6.25, 12.5, 25, 50 and 100 mg test item/L treatment levels: 0.0846, 0.121, 0.266, 0.519 and 1.70 mg/L, respectively. Therefore the data were evaluated based on mean measured concentrations. The EC50 is 0.124 mg /L.

The results are considered relevant and reliable for the risk assessment

Description of key information

48h-EC50: 0.124 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.124 mg/L

Additional information

In the Klimisch 1 GLP study from Lange (2017) the acute toxicity of 2,6-Di-tert-butyl-4-nonylphenol on Daphnia magna was determined in an 48 hour semi-static test according to OECD 202 with 24 h renewal interval. The test was performed with nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg test item/L prepared as WAF and a blank control. Four replicates with 5 daphnids each were set up.

After 48 hours 0, 30, 50, 85, 95 and 100 % immobilization was observed in the control and at nominal test concentrations of 6.25, 12.5, 25, 50 and 100 mg test item /L, respectively. The EL50 was 10.9 mg/L. Analytical measurements resulted in following geometric mean measured concentrations for the 6.25, 12.5, 25, 50 and 100 mg test item/L treatment levels: 0.0846, 0.121, 0.266, 0.519 and 1.70 mg/L, respectively. Therefore the data were evaluated based on mean measured concentrations. The EC50 is 0.124 mg /L.

The results are considered relevant and reliable for the risk assessment.