tert-butyl α,α-dimethylbenzyl peroxide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GlP OECD 201 guideline study without deviation

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Storage of samples: None
Samples treatment: Dilution with acetonitrile
Test item concentration analytical measurements were performed on abiotic samples.

Vehicle:

no

Details on test solutions:

Either in the range-finding test or in the definitive test, stock solution was prepared by saturating one litre of medium with 100 mg of TERT-BUTYL CUMYL PEROXIDE using slow-stirring method. The corresponding stock solution was then separated after 24 h of slow-stirring.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Pseudokirchneriella subcapitata, CCAP 278/4 stock are obtained from the Culture Centre of Algae and Protozoa (Ambleside, UK). The conditions used for culturing algae are described in Annex 2 of the OECD 201 guideline. Two flasks,each containing approximately 100 mL of axenic stock culture of algae are incubated at 23 ± 1 °C under lighting (photoperiod: 16 hours of illumination, 8 hours of darkness), slowly continuously shaken. These stock cultures are renewed every week, using two new cultures. The quality of the stock culture was verified for the absence of micro-organisms and deformed cells under microscopic observation before use. Four days before the beginning of the study two pre-cultures were prepared by inoculating each stock suspension of algae (5 mL) into sterile dilution water (500 mL). The pre-cultures were incubated under the same conditions as those used for the stock cultures. Only one of the two pre-cultures was used to inoculate the test flasks; the second one was to be used only if the first one was damaged. At the beginning of the test, the cell concentration of the pre-culture was determined. The result was used to calculate the volume to be introduced into each test flask in order to get an initial cell concentration of 10^4 cells/mL. The cell density of the pre-culture was about 4.55 x 10^6 cells/ml for the preliminary test and about 4.99 x 10^6 cells/ml for the definitive test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

23 ± 1°C

pH:

see "any other information on results incl. tables"

Dissolved oxygen:

see "any other information on results incl. tables"

Salinity:

Not relevant

Nominal and measured concentrations:

Preliminary test, nominal concentrations: 100, 50, 10, 5, 1 and 0.1 mg/L. Number of replicates: 3.
Definitive test, nominal concentrations: 100, 54.9, 30.2, 16.6, 9.1 and 5.0 mg/L. Number of replicates: 3.

Details on test conditions:

The incubation was performed in a phytoculture cabinet that allows test flasks to be incubated under precise conditions: temperature was set to 23 ± 1°C ; flasks were continuously shaken with a rotation at 20 rpm and constantly illuminated by 8 fluorescent tubes between 6,000 and 10,000 lux (Mazdafluor, white industry 33).

Determination of algae concentration was carried out using cytofluorimetry with a Cytofluor 2350 device in the range-finding test. In the definitive test, cells were numbered with a cell counter (Beckman Coulter Z2).

Flasks were stoppered with cellulose bungs and placed in a phytoculture cabinet (Strader DCS Pulsar).

An inoculated control flask (labelled T) was prepared and incubated under the same conditions, with no test item. This was used for determining algae growth. A non-inoculated blank (labelled Bl) containing only dilution water and test item was also prepared and incubated.

Dissolved O2 and pH were measured in the control and the highest concentration, in non-inoculated flask at the beginning of the test and in an inoculated flask at the end of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.58 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

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, colourless over the period of the test. No precipitation was observed at the end of the test. Microscopic observation confirmed that the algae appeared normal at the end of the test: the normal shape of P. subcapitata algae is a crescent shaped cell with an average length of 5-10 µm.

Results with reference substance (positive control):

The sensitivity of the test system and the methodology are evaluated every two months by performing an algal growth inhibition test on potassium dichromate. The nearest values of ErC50 and EbC50 obtained on 10/01/13 were respectively 0.74 mg/L and 0.38 mg/L. For information, ISO 8692 reports the following results for an inter-laboratory exercise on potassium dichromate: ErC50: 0.60 to 1.03 mg/L EbC50: 0.20 to 0.75 mg/L.

Reported statistics and error estimates:

The growth inhibition data are analyzed using an Excel program. It was designed to calculate the EC50 value and the 95% confidence interval. Probit analysis is generally used to calculate the 24, 48 and 72- hour EC50 values.

Measured pH and O2 concentrations in the different test systems

 

Concentrations (nominal) mg/L	pH		Dissolved O2 (mg/L)
	T0	T72h	T0	T72h
0 (Bl)	8.06	7.98	9.2	9.4
0 (T)	8.06	10.17	9.2	10.9
100	8.05	9.57	9.4	10.3
54.9	8.05	9.98	9.4	10.6
30.2	8.06	10.27	9.4	10.8
16.6	8.06	10.30	9.4	10.8
9.1	8.05	10.28	9.4	10.8
5	8.05	10.26	9.4	10.8

 

An increase in the pH is observed. This may be associated to consumption of the dissolved CO2 due to the growth of algae.

 

The average percentage inhibition of biomass (IAi) and cell multiplication (Iµi) for preliminary test and for definitive test are presented in tables hereafter:

 

Range finding test - Average percentage inhibition of cell growth (IAi) and growth rate (Iµi)

 

Concentration (nominal) (mg/L)	IAi   (%)	Iµi   (%)
100	75.3	27.5
50	35.1	9.6
10	6.1	0.3
5	0	0
1	3.3	0.4

 

 

Definitive test - Average percentage inhibition of cell growth (IAi) and growth rate (Iµi)

 

Concentration (nominal) (mg/L)	IAi   (%)	Iµi   (%)
100	27.3	5.34
54.9	9.8	1.1
30.2	0.6	0
16.6	0	0
9.1	0	0
5	0	0

 

 

The table hereafter presents the concentrations (mg/L) of TERT-BUTYL CUMYL PEROXIDE measured in non- inoculated test solutions (without algae) at the beginning and at the end of the test in all concentration tested.

 

Nominal and measured concentrations of the test item at the beginning and at the end of the exposure period

 

Concentrations of TERT-BUTYL CUMYL PEROXIDE
Nominal Concentration (mg/L)	Measured in non-inoculated solutions
	Initial (mg/L)	Final (mg/L)	Geometric mean*
			(mg/L)
0	<DL	<DL	-
5	0.51	<DL	0.13
9.1	0.92	<DL	0.18
16.6	1.81	<DL	0.25
30.2	3.29	<DL	0.33
54.9	5.93	<DL	0.45
100	8.22	<DL	0.53

 

< DL : concentration lower than the Detection Limit of the analytical method (0.020 mg/L).

< QL : concentration lower than the Quantification Limit of the analytical method (0.068 mg/L).

* Concentrations extrapolated as geometric mean between measured initial and final abiotic values. When value is lower than detection limit, half the value of quantification limit is used for the calculation.

Validity criteria fulfilled:

yes

Conclusions:

No toxicity was observed at the highest tested concentration i.e. 100 mg/L (nominal concentration).

Executive summary:

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata exposed to the test item TERT-BUTYL CUMYL PEROXIDE for a duration of 72 hours was assessed according to the OECD Guideline 201. Algae were exposed to 100 mg/L of TERT-BUTYL CUMYL PEROXIDE dissolved in dilution water. The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours. The concentrations of test item causing a 50% reduction in biomass (EbC50) and in growth rate (ErC50) were estimated. It was not possible to determine No Observable Effect Concentrations (NOEC).

The results were as follows:

 

Cell growth – Biomass (b): EbC50-72h > 100 mg/L based on nominal concentrations

Growth rate – Cell multiplication (r): ErC50-72h > 100 mg/L based on nominal concentrations

 

No toxicity was observed at the highest tested concentration i.e. 100 mg/L (nominal concentration).

 

The study was performed in compliance with the following quality criteria:

 

• The biomass in the control cultures has increased exponentially by a factor of 98 higher than 16 within the 72-hour test period.This corresponds to a specific growth rate of 1.529 day-1.

 

• The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures did not exceed 35%. This criterion applies to the mean value of coefficients of variation calculated for replicate control cultures.

 

• The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.

Description of key information

The acute toxicity to algae was assessed according to the OECD Guideline 201 and GLP requirements: no toxicity was observed at the highest tested concentration i.e. 100 mg/L (nominal concentration). EC10 was determined as 0.58 mg/L based on geometric means of measured concentrations.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.58 mg/L

Additional information

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata exposed to the test item TERT-BUTYL CUMYL PEROXIDE for a duration of 72 hours was assessed according to the OECD Guideline 201. Algae were exposed to 100 mg/L of TERT-BUTYL CUMYL PEROXIDE dissolved in dilution water. The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours. The concentrations of test item causing a 50% reduction in biomass (EbC50) and in growth rate (ErC50) were estimated. It was not possible to determine No Observable Effect Concentrations (NOEC), however EC10 is given.

The results were as follows:

 Cell growth – Biomass (b): EbC50-72h > 100 mg/L based on nominal concentrations

Growth rate – Cell multiplication (r): ErC50-72h > 100 mg/L based on nominal concentrations and ErC10-72h = 0.58 mg/L based on geometric means of measured concentrations.

 

The study was performed in compliance with the following quality criteria:

 

• The biomass in the control cultures has increased exponentially by a factor of 98 higher than 16 within the 72-hour test period. This corresponds to a specific growth rate of 1.529 day-1.

 

• The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures did not exceed 35%. This criterion applies to the mean value of coefficients of variation calculated for replicate control cultures.

 

• The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.