4-tert-butylpyrocatechol

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 23 Jun 2010 to 25 Sep 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-tert-butylpyrocatechol

EC Number:

202-653-9

EC Name:

4-tert-butylpyrocatechol

Cas Number:

98-29-3

Molecular formula:

C10H14O2

IUPAC Name:

4-tert-butylbenzene-1,2-diol

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
For the determination of the actual test item concentrations in this semi-static test, duplicate samples were taken from each treatment at the start and end of each test medium renewal period. However, from the highest nominal test concentrations of 1.91 and 4.0 mg/L, the last samples were taken after 24 hours, since all fish were dead at that observation date. From the nominal test concentrations of 0.44 and 0.91 mg/L, the last samples were taken after 48 hours, since all fish were dead at that observation date.
- Sampling method: All samples were taken from the approximate center of the aquaria without mixing of the test media.
- Sample storage conditions before analysis: Immediately after sampling, the pH of each medium was adjusted to 6.0. All the samples were analyzed immediately after sampling.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution with a nominal concentration of 20 mg/L was prepared daily at the start of the test and prior each test medium renewal by completely dissolving 60 mg of test item (dosed amounts: 60.1 and 60.2 mg) in 3000 mL of test water (pH 6.5) under slightly stirring for 15 minutes at room temperature. This stock solution was used in a series of dilutions to prepare the test media of all test item concentrations. The test media were freshly prepared just before introduction of the fish (= start of the exposure) and before each test medium renewal.
- Eluate: no
- Differential loading: no
- Controls: a blank (test water without test item) was tested in parrallel.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The appearance of the test media was visually recorded at the start of the test and once daily during the test in the freshly prepared and old test media.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Age at study initiation (mean and range, SD): no data
- Length at study initiation (mean, +/- SD): 2.8 +/- 0.21 cm
- Weight at study initiation (mean +/- SD): 0.17 +/- 0.04g
- Method of breeding: The fish were obtained from a breeding culture at Harlan Laboratories. No medication was applied during holding and acclimatization.
- Feeding during test: no


ACCLIMATION
- Acclimation period: Prior to test start, the test fish were acclimated for one week to the test water and temperature.
- Type and amount of food: 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).
- Health during acclimation (any mortality observed): During the last two weeks prior to the test, no fish died in the test fish batch and all fish were healthy.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Hardness:

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

Test temperature:

The water temperature was maintained at 21°C.

pH:

The pH values in all treatments ranged from 6.5 (after adjustment) to 6.7.

Dissolved oxygen:

The oxygen concentration was always 8.2 mg/L or higher.

Salinity:

Not applicable

Nominal and measured concentrations:

nominal concentrations: 0, 0.10, 0.21, 0.44, 0.91, 1.91 and 4.0 mg/L
measured concentrations: 0, 0.065, 0.16, 0.37, 0.85, 1.95, 4.3 mg/L

Details on test conditions:

TEST SYSTEM
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass test vessel with 5 liters of test medium
- Aeration: The test vessels were slightly aerated during the test period.
- Type of flow-through (e.g. peristaltic or proportional diluter): none (semi-static test)
- Renewal rate of test solution (frequency/flow rate): medium renewal after 24 hours. During this semi-static test, the test fish were daily placed into a new test vessel with freshly prepared test medium of the corresponding treatment.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.24 g fish wet weight per liter test medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water. It consisted of analytical grade salts dissolved in purified water to obtain the following nominal concentrations:
CaCl2 × 2H2O: 147 mg/L
MgSO4 × 7H2O: 61.5 mg/L
NaHCO3: 32.5 mg/L
KCl: 2.9 mg/L

- Total organic carbon: no data
- Particulate matter: no data
- Alkalinity: 0.4 mmol/L
- Ca/mg ratio: 4:1 (based on molarity)
- Na/K ratio: 10:1 (based on molarity)
- Conductivity: no data
- Culture medium different from test medium: no data
- Intervals of water quality measurement: The water temperature, pH values and oxygen concentrations were measured for each treatment with surviving fish at the start of the test and once daily during the test in the freshly prepared and old test media.
At the same dates the appearance of the test media was recorded.

OTHER TEST CONDITIONS
- Adjustment of pH: yes. The pH of the test water was adjusted to 6.5 with a 1 M hydrochloric acid solution.
- Photoperiod: a 16-hour light to 8-hour dark photoperiod, with a 30-minute transition period
- Light intensity: approximately within the range of 180 to 240 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The test fish were observed for mortality and visible abnormalities after approximately 3, 24, 48, 72 and 96 hours test duration. Dead fish were removed at least once daily and discarded. The LC50 and the 95%-confidence interval at the observation dates were calculated by Probit Analysis. The NOEC, LOEC, LC0 and LC100 were determined directly from the raw data.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.1
- Range finding study
- Test concentrations: 0, 1.0, 10 and 100 mg/L in the first pre-test (pH 7.4 - 7.5); 0, 1.0, 10 and 100 µg/L in the second pre-test (pH 7.5 - 7.6); 0, 0.10, 1.0, 10 and 100 mg/L in the third pre-test (pH 6.5 - 6.9).
- Results used to determine the conditions for the definitive study: yes, the selection of the test concentrations was based on the results of range-finding tests and of a pre-experiment to determine the solubility of the test item (non-GLP).

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence limits: 0.075 - 0.18 mg/L

Effect concentrations 2

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

0.37 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 3

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.065 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 4

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

In the control and at the mean measured test concentrations up to and including 0.065 mg/L, all fish survived until the end of the test and no visible abnormalities were observed in the test fish. At the next higher mean measured test concentration of 0.16 mg/L, all test fish showed one or several visible abnormalities and six fish died at this test concentration after 96 hours of test duration. At the two next higher mean measured test concentrations of 0.37 and 0.85 mg/L, all test fish died after 48 hours test duration. At the two highest mean measured test concentrations of 1.95 and 4.3 mg/L, all fish were dead already after 24 hours of test duration.

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions.

Any other information on results incl. tables

Sublethal observations / clinical signs:

In the analyzed test medium samples from the start of the test medium renewal periods, the measured test item concentrations ranged between 83 and 113% of the nominal values. This shows the correct dosage of the test item. During the test medium renewal periods of 24 hours, a decrease of the test item concentration in the test media was determined. At the end of the test medium renewal periods, 38 to 102% of the nominal values were found.

The biological results were based on mean measured concentrations calculated as time-weighted means of the test item concentrations measured at the start of the test medium renewals and the concentrations measured in the samples at the end of the renewal periods (see Table 2).

Table 1: Mortality and visible abnormalities observed in the test fish (number of fish tested per treatment: 7)

Abbreviations of visible abnormalities for which fish were observed during the study

AP: Apathy / OB: Fish mainly at the water surface

Nominal test item concentration (mg/L)	Mean measured concentration (mg/L)	Number of abnormal and dead fish / number of dead fish Type of visible abnormalities
		Observation time
		3 hours	24 hours	48 hours	72 hours	96 hours
Control	--	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
0.10	0.065	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
0.21	0.16	0 / 0	0 / 0	0 / 0	7 / 4 AP,OB	7 / 6 AP,OB
0.44	0.37	0 / 0	0 / 0	7 / 7	-/-	-/-
0.91	0.85	0 / 0	7 / 2 AP,OB	7 / 7	-/-	-/-
1.91	1.95	0 / 0	7 / 7	-/-	-/-	-/-
4.0	4.3	0 / 0	7 / 7	-/-	-/-	-/-
LC50*		n.d.	0.99	0.25	0.15	0.12
95% C.I. *		n.d.	0.72 – 1.4	0.18– 0.34	n.d.	0.075 – 0.18

-/-:              all fish dead

95% C.I.:    95% confidence interval of the LC₅₀

*:                based on mean measured concentration

n.d.:       could not be determined

Table 2: Mean measured test item concentration

Nominal test item concentration (mg/L)	Mean measured test item concentration
	(mg/L)
0.10	0.065
0.21	0.16
0.44	0.37
0.91	0.85
1.91	1.95
4.0	4.3

Table 3: First pre-test: Effects of 4 -TBC on Zebra fish, pH 7.4 - 7.5

Nominal test item concentration(mg/L)	Number of abnormal and dead fish / number of dead fish Type of visible abnormalities
	Observation time
	3 hours	24 hours
Control	0 / 0	0 / 0
1.0	0 / 0	3 / 3
10	3 / 3	-/-
100	3 / 3	-/-

-/-: all fish dead

Table 4: Second pre-test: Effects of 4 -TBC on Zebra fish, pH 7.5 - 7.6

Nominal test item concentration (µg/L)	Number of abnormal and dead fish / number of dead fish Type of visible abnormalities
	Observation time
	3 hours	24 hours	48 hours	72 hours	96 hours
Control	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
1.0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
10	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
100	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0

Table 5: Third pre-test: Effects of 4 -TBC on Zebra fish, pH 6.5 - 6.9

Nominal test item concentration (mg/L)	Number of abnormal and dead fish / number of dead fish Type of visible abnormalities
	Observation time
	3 hours	24 hours	48 hours	72 hours	96 hours
Control	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
0.10	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
1.0	0 / 0	0 / 0	3 / 3	-/-	-/-
10	0 / 0	3 / 3	-/-	-/-	-/-
100	3 / 3	-/-	-/-	-/-	-/-

-/-: all fish dead

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

(no mortality in the control group, oxygen saturation > 60%)

Conclusions:

Based on the results of this study, 4-tert-butylpyrocatechol would be considered as very toxic for Zebra fish.

Executive summary:

In a 96 -hour acute toxicity study (Harlan, 2010), Brachydanio rerio were exposed to 4 -tert-butylpyrocatechol (4 -TBC) under semi-static conditions according to the Commission Regulation (EC) n° 440/2008, Part C.1 and the OECD Guideline for Testing of Chemicals, n°203 (1992).

Zebra fish were exposed to control and test item at nominal concentration of 0.10, 0.21, 0.44, 0.91, 1.91 and 4 .0 mg/L.

In the analyzed test medium samples from the start of the test medium renewal periods, the measured test item concentrations ranged between 83 and 113% of the nominal values. During the test medium renewal periods of 24 hours, a decrease of the test item concentration in the test media was determined. At the end of the test medium renewal periods, 38 to 102% of the nominal values were found.

The biological results were based on mean measured concentrations calculated as time-weighted means of the test item concentrations measured at the start of the test medium renewals and the concentrations measured in the samples at the end of the renewal periods.

Nominal test item concentration (mg/L)	Mean measured test item concentration
	(mg/L)
0.10	0.065
0.21	0.16
0.44	0.37
0.91	0.85
1.91	1.95
4.0	4.3

The test fish were observed for mortality and visible abnormalities after approximately 3, 24, 48, 72 and 96 hours test duration.

In the control and at the mean measured test concentrations up to and including 0.065 mg/L, all fish survived until the end of the test and no visible abnormalities were observed in the test fish. At the next higher mean measured test concentration of 0.16 mg/L, all test fish showed one or several visible abnormalities and six fish died at this test concentration after 96 hours of test duration. At the two next higher mean measured test concentrations of 0.37and 0.85 mg/L, all test fish died after 48 hours test duration. At the two highest mean measured test concentrations of 1.95 and 4.3 mg/L, all fish were dead already after 24 hours of test duration.

Thus, the 96‑hour NOEC and LC₀of 4 -TBC to zebra fish were both determined to be 0.065 mg/L. The 96‑hour LOEC was 0.16 mg/L.

The 96‑hour LC₅₀ of 4 -TBC was calculated to be 0.12 mg/L with a 95% confidence interval of 0.075 to 0.18 mg/L. The 96‑hour LC₁₀₀ was 0.37 mg/L.

In summary, the biological test results were as follows:

- 96-hour LC50:	0.12 mg/L (95% CL: 0.075 – 0.18 mg/L)
- 96-hour NOEC and LC0:	0.065 mg/L
- 96-hour LC100:	0.37 mg/L
- 96-hour LOEC:	0.16 mg/L

Based on the results, 4 -TBC was very toxic for Zebra fish.

This study is classified as acceptable and satisfies the guideline requirements for acute fish toxicity study.