tert-butyl α,α-dimethylbenzyl peroxide

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:

August - November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from all test concentrations and the control throughout the test on fresh and old solutions as follows: at days 0, 1, 2 and 3 for fresh solutions and 1, 2, 3 and 4 for old solutions.

Vehicle:

no

Details on test solutions:

For both range-finding test and definitive tests, as the test item was suspected to be unstable in water (due to its chemical nature), saturated stock solutions were prepared using slow-stirring conditions described in the OECD 123 guideline. The method used a special temperature controlled glass-jacketed test vessel (around 8000 mL of capacity), with a sampling tap at the lower end and a magnetic stir bar at its bottom.

RANGE-FINDING TEST
A known volume of test medium was first poured into the flask then a known quantity of test item (855µL) was gently added at the surface of medium (loading rate of around 100 mg/L). The solution was stirred at 20°C with a magnetic stir bar at a speed avoiding formation of an emulsion. A speed of approximately 150 rpm was set up in order to create a vortex depth of around 0.5 cm: the aqueous phase was kept under stirring during approximately 72 hours, and then drawn off as a clear solution after a rest phase of 1h without agitation.
Tests solutions were then prepared from dilutions of this saturated solution.
The pH of the test solutions were not adjusted.
Test solutions were not renewed throughout the 96h test period (static test).
Fish were exposed under static conditions to a series of dilutions (11, 33 and 100%) of the saturated stock solution in dilution water. One tank was prepared at each test concentration and each tank contained 7 fish.

DEFINITIVE TEST
As chemical analysis conducted during the preliminary test showed complete disappearance of the test item in test solutions throughout the 96h period, this definitive was conducted with semi-static conditions: a new test item stock solution (At around 100 mg/L loading rate) was prepared every day.
The pH of the test solutions were not adjusted.
Test solutions were renewed every day throughout the 96h test period (semi-static test).
Based on the results of the range-finding test, the definitive test was conducted under semi-static conditions at the following series of dilutions: 19.8, 29.6, 44.4, 66.7 and 100%. The range of concentrations was obtained from dilutions of the saturated solution. One tank was prepared at each test concentration and the control group. Each tank contained 7 fish.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The test organisms were Danio rerio (Teleostei, Cyprinidae), obtained on the 28 July 2016 from a recognised supplier: La Grande Rivière France (69490 Saint-Forgeux). All fish were in good health and free from any apparent malformation. Mortality of the batch was less than 5% in the week preceding the start of the study. No disease treatments were administered throughout holding and testing.
Holding was maintained within the laboratory at a temperature of 21-25°C in glass tanks. A light cycle of 12 h light and 12 h dark was applied, illumination being provided by fluorescent tubes (intensity between 400 and 800 lux at the surface of the tanks). Tanks were aerated to ensure that the dissolved oxygen concentration is at least 60% of air saturation value in holding tanks and in test tanks. During holding, fish were fed twice per day with ground flake food TetraMin®.
The fish were not fed for a period of 24h prior to test commencement or throughout the duration of the test.
Fish were added to the test tanks within 30 min of the completion of preparation of the test solutions. All test fish were weighed prior to the test and a representative number of test fish batch (10 at random) was measured after the test to assess compliance with guideline criteria.

Fish were observed at least after 24h, 48h, 72h and 96h (end of the exposure period). Fish were considered dead if touching the caudal peduncle produces no reaction and no breathing movements are visible. Their number per concentration at each time interval was recorded. Dead fish were removed as soon as observed. Any visible abnormalities (e.g. loss of equilibrium, changes in swimming behaviour, respiratory function, pigmentation etc) was also recorded. All surviving fish were sacrificed at the end of the definitive test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

Definitive test: mean measured temperature: 22.8°C, min.: 22.2°C, max.: 23.7°C.

pH:

fresh solutions: 7.5 - 8.0
old solutions: 7.5 - 7.8

Dissolved oxygen:

fresh solutions: 87.0 - 100.1 % saturated solution
old solutions: 72.5 - 92.0

Salinity:

NA

Nominal and measured concentrations:

Nominal test solutions expressed as % saturated stock solution: 0, 19.8, 29.6, 44.4, 66.7 and 100.0
Measured test item concentrations are presented below.

Details on test conditions:

All testing was performed using a synthetic medium. Chemical reagents used for the preparation of this dilution water were of “analytical grade”; it is prepared according to the protocol described in standard NF EN ISO 6341:
The following stock solutions were prepared:
- CaCl2, 2H2O: 11.76 g per litre of water,
- MgSO4, 7H2O: 4.93 g per litre of water,
- NaHCO3: 2.59 g per litre of water,
- KCl: 0.23 g of per litre of water,
Dilution water was obtained by adding a concentration of 25 mL/L of each of the stock solutions and then aerating the solution until oxygen saturation.
Sum of Ca and Mg ions in dilution water is of 2.5 mmol/L and ratios of Ca/Mg et Na/K are 4 and 10 respectively.

Test tanks (filled with 2.5 L of test solution) containing fish were incubated with a photo-period of 9 hours of darkness per 24 h, at a temperature ranging from 21°C to 25°C but remaining constant within ± 2°C during the test.
All test tanks were made of clear glass and covered by plates with ventilation holes to minimise evaporation and avoid the entry of dust into the solutions.
Test solutions were prepared using the synthetic medium. Chemical reagents used for the preparation of the dilution water was of “analytical grade”.
The control treatment was maintained under the same conditions, with no test item.

pH and dissolved oxygen concentration were measured for all tanks at the beginning of the definitive test and thereafter at 24h intervals. The total hardness of a sample of the medium used to prepare the test solutions was measured and expressed as mg CaCO3/L. The temperature was continuously recorded in one tank.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

2.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear and no precipitation was observed at the end of the test.

Results with reference substance (positive control):

The sensitivity of the test system and the methodology are evaluated with every batch of fish by performing an acute toxicity test on potassium dichromate. The values of 24h-LC50 obtained with the batch used was as follows:
Batch 16/Br/01/2: 24h-LC50 = 173.6 mg/L (obtained on 11 August 2016)
For reference, the former AFNOR standard NF T 90-303 stated that the 24h-LC50 of the potassium dichromate with respect to Danio rerio must be between 200 and 400 mg/L, however, results obtained are consistent with the laboratory control quality chart.

Reported statistics and error estimates:

Statistical analysis was performed with the software ToxRatPro Version 3.1.0. A Probit analysis using linear max. likelihood regression was statistically considered to be more adapted to the raw data obtained.

Sublethal observations / clinical signs:

Test item determination - concentrations used for statistical analysis in the range-finding test

Test solution (% saturated stock solution)	Test item concentration (mg/L)					Deviation of measured conc. from 0h (%)	Retained conc. (mg/L)*
	T0	T24h	T48h	T72h	T96h
Control	< LD	< LD	< LD	< LD	< LD	NA	NA
1.0	0.92	0.15	0.05	0.02	0.01	98.9	0.06
10.0	2.41	0.34	0.14	0.08	0.02	99.4	0.17
100.0	10.08	1.43	0.66	0.40	0.19	98.1	0.93

< DL (0.010 mg/L): concentration lower than the Detection Limit of the analytical method

< QL (0.030 mg/L): concentration lower than the Quantification Limit of the analytical method

When test item measured concentration is < DL, DL value was used

When test item measured concentration is < QL, QL value was used

NA: Not Applicable

*Concentrations extrapolated as geometric mean of measured concentrations

Test item determination - concentrations used for statistical analysis in the definitive test

Test solution (% saturated stock solution)	Test item measured concentrations (mg/L)												Retained concentrations (mg/L)*
	T0/T24			T24/T48			T48/72			T72/96
	fresh	old	Deviation T0/T24	fresh	old	Deviation T24/T48	fresh	old	Deviation T48/T72	fresh	old	Deviation T72/T96
Control	<DL	<DL	NA	<DL	<DL	NA	<DL	<DL	NA	<DL	<DL	NA	NA
19.8	1.31	0.26	80.0%	0.95	0.22	76.8%	1.32	0.28	78.9%	1.23	0.35	71.6%	0.57
29.6	1.90	0.31	83.5%	1.58	0.27	82.6%	2.05	0.37	82.0%	1.64	0.09	94.3%	0.65
44.4	2.91	0.39	86.6%	2.47	0.40	83.8%	3.04	0.50	83.6%	3.01	0.73	75.8%	1.18
66.7	4.30	0.40	90.8%	3.71	0.41	88.9%	4.57	0.36	92.2%	4.41	0.40	90.9%	1.29
100.0	6.38	0.58	91.0%	5.63	0.77	86.2%	6.98	0.73	89.6%	7.25	1.90	73.7%	2.41

< DL (0.010 mg/L): concentration lower than the Detection Limit of the analytical method

< QL (0.030 mg/L): concentration lower than the Quantification Limit of the analytical method

When test item measured concentration is < DL, DL value was used

When test item measured concentration is < QL, QL value was used

NA: Not Applicable

*Concentrations extrapolated as geometric mean of measured concentrations

Cumulative lethality in the range-finding test

Test solution (% saturated stock solution)	Cumulative dead fish					Mortality at the end of the period
	T5H	T24h	T48h	T72h	T96h
Control	0	0	0	0	0	0.0%
11	0	0	0	0	0	0.0%
33	0	0	0	0	0	0.0%
100	2	4	5	5	5	71.4%

Cumulative lethality in the definitive test

Test solution (% saturated stock solution)	Cumulative dead fish					Mortality at the end of the period
	T0	T24h	T48h	T72h	T96h
Control	0	0	0	0	0	0.0%
19.8	0	0	0	0	0	0.0%
29.6	0	0	0	0	0	0.0%
44.4	0	0	0	0	0	0.0%
66.7	0	0	0	0	0	0.0%
100.0	0	1	4	5	6	85.7%

Validity criteria fulfilled:

yes

Remarks:

see details in the Conclusions

Conclusions:

The definitive test met the validity criteria of the test guideline detailed as follows:
- The mortality in the control did not exceed 10% at the end of the test;
- The dissolved oxygen in the test tanks remained above 60% of the air saturation value at the end of the test;
- The pH did not vary by more than 1 unit.

Executive summary:

The acute toxicity of the test item to Danio rerio exposed for 96h was assessed according to the OECD 203 Guideline and GLP requirements.

As the test item was suspected to be unstable in water (due to its chemical nature), test solutions were prepared from dilutions of a saturated solution (at 100 mg/L loading rate) prepared using slow-stirring conditions described in the OECD 123 guideline.

Chemical analysis of the test item throughout the test period has shown instability of the substance. Thus geometric means of measured concentrations were used to determine the effects as follows:

96h-LC10 = 1.91 mg/L and 96h-LC50 = 2.17 mg/L.

The definitive test met all the validity criteria of the test guideline and thus this experiment can be considered as valid.

Description of key information

The acute toxicity to fish of tert-butyl cumyl peroxide was assessed according to the OECD Guideline 203 and GLP requirements: LC50_96h was determined as 2.17 mg/L based on geometric means of measured concentrations.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

2.17 mg/L

Additional information

The acute toxicity of the test item to Danio rerio exposed for 96h was assessed according to the OECD 203 Guideline and GLP requirements (Chastenet, 2018).

As the test item was suspected to be unstable in water (due to its chemical nature), test solutions were prepared from dilutions of a saturated solution (at 100 mg/L loading rate) prepared using slow-stirring conditions described in the OECD 123 guideline.

Chemical analysis of the test item throughout the test period has shown instability of the substance. Thus geometric means of measured concentrations were used to determine the effects as follows:

96h-LC10 = 1.91 mg/L and 96h-LC50 = 2.17 mg/L.

The definitive test met all the validity criteria of the test guideline and thus this experiment can be considered as valid.