4-sec-butyl-2,6-di-tert-butylphenol

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26-Sep-2011 to 20-Oct-2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

OECD Guideline for the Testing of Chemicals, No. 203, Fish, Acute Toxicity Test, 1992.

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

EU Commission Directive 92/69/EEC, C.1: Acute Toxicity for Fish, 1992.

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

For determination of the actual test item concentration in this semi-static test, duplicate samples were taken from the test medium and the solvent control at the start of the first and last renewal period (Days 0 and 3). At the end of these renewal periods (Days 1 and 4), additional samples were taken for determination of the stability of the test item during the renewal periods of 24 hours.
All samples were taken from the approximate center of the aquaria without mixing of the test medium. Immediately after sampling, acetonitrile was added to each sample in the ratio 1:1 to stabilize the samples during the storage period. Thereafter, all samples were stored at about -20 °C. Based on a pre-experiment, the test item was stable under these storage conditions.
The concentration of the test item was analytically determined in all test medium samples. From the solvent control samples, only one of the duplicate samples was analyzed per sampling time.

Vehicle:

yes

Remarks:

organic solvent N,N-Dimethylformamide p.a. (DMF)

Details on test solutions:

Due to the low water solubility of the test item, the organic solvent N,N-Dimethylformamide p.a. (DMF) was used to dose the test item. DMF was chosen based on its solubilizing properties and its relative non-toxicity to fish. The preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.
At the start of the test and before each test medium renewal, a concentrated stock solution of the test item in DMF was freshly prepared by completely dissolving nominal 150 mg (range: 149.8 to 150.4 mg) of the test item in 10 mL DMF by stirring for 5 minutes at room temperature. This intensively stirred stock solution was diluted with DMF to obtain the application solution with a test item concentration of 1.5 mg/mL. A volume of 1.084 mL of this application solution was added to 10.84 L test water to obtain the test medium with the nominal concentration of 150 μg/L. The test medium was freshly prepared just before the introduction of the fish (= start of the exposure) and prior to each test medium renewal.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The study was performed with zebra fish (Brachydanio rerio). The fish were obtained from a breeding culture at Harlan Laboratories. No medication was applied during holding and acclimatization. Prior to test start, the test fish were acclimated for one week to the test water and temperature.
During holding and acclimatization until one day before the start of the test, the fish were fed with a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany). During holding and acclimatization, no fish died in the test fish batch and all fish were healthy.
From the acclimated test fish batch, 10 fish were measured at the start of the test. The mean body length of the fish was 2.7 ± 0.25 cm (Mean ± SD), the mean body wet weight was 0.14 ± 0.03 g (Mean ± SD). These measured fish were not used for the test.
The test method and test species are recommended by the international test guidelines.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

1.25 mmol/L (125 mg/L as CaCO3)

Test temperature:

The water temperature was 21-22°C during the test.

pH:

The pH values in all treatments ranged from 6.9 to 7.0.

Dissolved oxygen:

The oxygen concentration was at least 7.1 mg/L, corresponding to at least 80% oxygen saturation.

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

The only concentration tested was nominal 150 μg/L

Details on test conditions:

Material
Since the test item was determined to be volatile (according to the results of a pre-experiment without GLP) the test was performed in a closed system. One 10-liter flask was used for the single treatment.
The test vessels were labeled with the study number and all necessary additional information to ensure unique identification.

Experimental Conditions
The water temperature in the test vessels was maintained at 21-22 °C. A 16-hour light to 8-hour dark photoperiod, with a 30-minute transition period was used (light intensity during the light period was approximately in the range 180 to 480 Lux. The test duration was 96 hours and the fish were not fed during the test.

Study Design
The test was performed according to the threshold approach which means that the fish test is carried out with one concentration close to or slightly higher than the lowest EC50 value obtained with algae and daphnia. Thus, the only concentration tested was nominal 150 μg/L which corresponds to the EC50 value of the acute daphnia test with the same test item (Harlan Study No. D12548). Additionally, a control and a solvent control group were tested in parallel.
Since the test item was determined to be volatile (according to the results of a pre-experiment without GLP) the test was performed in a closed system. The flask was nearly completely filled to keep the air-space in the test flask as small as possible and was tightly sealed with a teflon stopper.
A semi-static test procedure with a daily test medium renewal was chosen to keep the concentration of 2,6-di-tert-butyl-4-sec-butylphenol in the test medium as constant as possible during the test period of 96 hours. The semi-static test procedure was also chosen to keep the oxygen concentration in the closed system sufficiently high and to provide best possible environmental conditions for the test fish.
Furthermore, due to the relatively high log Kow (octanol-water-coefficient) of the test item the test flasks were preconditioned for one hour with the test medium prior to test start and prior to each test medium renewal as a precautionary measure to minimize potential adsorbtion to the glass walls of the test vessels.
At the start of the exposure, seven fish were introduced into each test vessel in a random order.
The loading rate was 0.09 g fish wet weight per liter test medium. Thus, the requirement of a loading rate not exceeding 1 g fish/L was fulfilled.

Evaluations
Determination of the LC50, NOEC, LOEC, LC0 and LC100
The test fish were observed for mortality and visible abnormalities after approximately 3, 24, 48, 72 and 96 hours test duration.
The NOEC and LC0 were determined directly from the raw data. The LOEC, LC100 and LC50 at the observation times could not be quantified due to the absence of a toxic effect of the test item at the tested concentration.

Water Quality Criteria
The water temperature, pH values and oxygen concentrations were measured at the start and the end of each renewal period in the test medium, the control and the solvent control.
At the same times the appearance of the test medium was recorded.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 150 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 150 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 150 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The analytically measured test item concentrations in the samples from the start and the end of the test medium renewal periods showed a high variation (range of 53 to > 592 μg/L). These results were attributed to specific properties of the test item (e.g. inhomogeneous distribution in the water column), since also the measurements in the duplicate samples were hardly reproducible.
Due to the high variation between the measured concentrations a mean measured test item concentration could not be calculated. However, it was estimated to be > 280 μg/L (arithmetic mean over all measurements) and was far above the threshold concentration of 150 μg/L.
Therefore, the biological results were based on the nominal concentration of 150 μg/L.
In the control, the solvent control and the test medium with the nominal concentration of 150 μg/L all fish survived until the end of the test and no visible abnormalities were observed at the test fish. Therefore, the 96-hour NOEC and LC0 of 2,6-di-tert-butyl-4-sec-butylphenol to zebra fish were both determined to be at least 150 μg/L.
The 96-hour NOEC and LC0 might even be higher than this concentration, but concentrations exceeding 150 μg/L were not tested. The 96-hour LOEC, LC50 and LC100 were clearly higher than 150 μg/L. These values could not be quantified due to the absence of toxicity of 2,6-di-tertbutyl-4-sec-butylphenol at the tested concentration.
Summarizing the biological results, the test item 2,6-di-tert-butyl-4-sec-butylphenol had no acute toxic effects on zebra fish up to the nominal concentration of 150 μg/L under the present test conditions. According to the threshold approach this result showed, that zebra fish is not the most sensitive test organism compared to daphnia and algae.
No remarkable observations were made concerning the appearance of the test medium. It was a clear solution throughout the entire test duration.
The pH values in all treatments ranged from 6.9 to 7.0. The oxygen concentration was at least 7.1 mg/L, corresponding to at least 80% oxygen saturation. The water temperature was 21-22°C during the test.
The test was considered to be valid, since no mortality in the control and solvent control was observed and the validity criterion of at least 60% oxygen saturation was fulfilled.

Validity criteria fulfilled:

yes

Conclusions:

The biological results (based on nominal test item concentration) are as follows:
– 96-hour LC50: >150 μg/L
– 96-hour LC0: ≥150 μg/L
– 96-hour LC100: >150 μg/L
– 96-hour NOEC: ≥150 μg/L
– 96-hour LOEC: >150 μg/L

Executive summary:

The acute toxicity of the test item 2,6-di-tert-butyl-4-sec-butylphenol to zebra fish (Brachydanio rerio) was determined in a 96-hour test according to the EU Commission Directive 92/69/EEC, Part C.1, the Commission Regulation (EC) No 440/2008, Part C.1 and the OECD Guideline for Testing of Chemicals No. 203 (1992).

 

The test was performed according to the threshold approach which means that the fish test is carried out with one concentration close to or slightly higher than the lowest EC50 value obtained with algae and daphnia. Thus, the only concentration tested was nominal 150 μg/L which corresponds to the EC50 value of the acute daphnia test with the same test item (Harlan Study No. D12548). Additionally, a control and a solvent control group were tested in parallel.

 

Due to the low water solubility of the test item, the organic solvent N,N-Dimethylformamide was used to dose the test item. Due to the volatile characteristic of the test item the test was performed in a closed system. A semi-static test design with daily test medium renewal was chosen to keep the test item concentrations as stable as possible during the test period. The preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

 

The analytically measured test item concentrations in the samples from the start and the end of the test medium renewal periods showed a high variation (range of 53 to > 592 μg/L). These results were attributed to specific properties of the test item (e.g. inhomogeneous distribution in the water column), since also the measurements in the duplicate samples were hardly reproducible.

 

Due to the high variation between the measured concentrations a mean measured test item concentration could not be calculated. However, it was estimated to be > 280 μg/L (arithmetic mean over all measurements) and was far above the threshold concentration of 150 μg/L.

Therefore, the biological results were based on the nominal concentration of 150 μg/L.

 

During the test period of 96 hours, no mortality or other visible abnormalities were determined in the test fish exposed to the control, the solvent control and at the test concentration of nominal 150 μg/L. The biological results (based on nominal test item concentration) are as follows:

– 96-hour LC50: >150 μg/L

– 96-hour LC0: ≥150 μg/L

– 96-hour LC100: >150 μg/L

– 96-hour NOEC: ≥150 μg/L

– 96-hour LOEC: >150 μg/L

 

Summarizing the biological results, the test item 2,6-di-tert-butyl-4-sec-butylphenol had no acute toxic effects on zebra fish up to the nominal concentration of 150 μg/L under the present test conditions. According to the threshold approach this result showed, that zebra fish is not the most sensitive test organism compared to daphnia and algae.

Description of key information

The biological results (based on nominal test item concentration) are as follows:

– 96-hour LC50: >150 μg/L

– 96-hour LC0: ≥150 μg/L

– 96-hour LC100: >150 μg/L

– 96-hour NOEC: ≥150 μg/L

– 96-hour LOEC: >150 μg/L

Additional information

The acute toxicity of the test item 2,6-di-tert-butyl-4-sec-butylphenol to zebra fish (Brachydanio rerio) was determined in a 96-hour test.

The test was performed according to the threshold approach which means that the fish test is carried out with one concentration close to or slightly higher than the lowest EC50 value obtained with algae and daphnia. Thus, the only concentration tested was nominal 150 μg/L which corresponds to the EC50 value of the acute daphnia test with the same test item (Harlan Study No. D12548). Additionally, a control and a solvent control group were tested in parallel.

 

Due to the low water solubility of the test item, the organic solvent N,N-Dimethylformamide was used to dose the test item. Due to the volatile characteristic of the test item the test was performed in a closed system. A semi-static test design with daily test medium renewal was chosen to keep the test item concentrations as stable as possible during the test period.

Due to the high variation between the measured concentrations a mean measured test item concentration could not be calculated. However, it was estimated to be > 280 μg/L (arithmetic mean over all measurements) and was far above the threshold concentration of 150 μg/L.

Therefore, the biological results were based on the nominal concentration of 150 μg/L.

 

During the test period of 96 hours, no mortality or other visible abnormalities were determined in the test fish exposed to the control, the solvent control and at the test concentration of nominal 150 μg/L. The biological results (based on nominal test item concentration) are as follows:

– 96-hour LC50: >150 μg/L

– 96-hour LC0: ≥150 μg/L

– 96-hour LC100: >150 μg/L

– 96-hour NOEC: ≥150 μg/L

– 96-hour LOEC: >150 μg/L

 

Summarizing the biological results, the test item 2,6-di-tert-butyl-4-sec-butylphenol had no acute toxic effects on zebra fish up to the nominal concentration of 150 μg/L under the present test conditions. According to the threshold approach this result showed, that zebra fish is not the most sensitive test organism compared to daphnia and algae.