tert-pentyl 2-ethylperoxyhexanoate

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:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with minor deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

minor deviations to study plan (see 3principle of method if other than guideline"

Principles of method if other than guideline:

Two deviations was issued for this study:
- Analysis was performed only on inoculated samples as abiotic samples were not available.
- Experimentation started on 14/01/13 when study plan was signed on 17/01/13

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Storage of samples: none
Samples treatment: dilution with acetonitrile

Vehicle:

no

Details on test solutions:

For the range finding test and for the definitive test a stock parent solution at 100 mg/L of the test item was prepared before the starting test by mixing 100 mg of the test item TERT-AMYL PEROXY-2-ETHYLHEXANOATE in 1 liter of dilution water during 1h30 in a slow stirring flask. After a decantation period of one hour, the lower phase was used to prepare the convenient range of nominal concentrations.

Test organisms (species):

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

Details on test organisms:

Pseudokirchneriella subcapitata, CCAP 278/4 stock (previously named Raphidocelis subcapitata and Selenastrum capricornutum) 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.

Three 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 1.4 x 106 cells/ml for the preliminary test and about 1.14 x10^6 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:

set to 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, 0.5, 0.1 mg/L
Definitive test, nominal concentrations: 30, 11.6, 4.48, 1.72, 0.66, 0.26 and 0.10mg/L.
Measured concentrations: see "Any other information on results incl. tables"

Details on test conditions:

Preliminary test
All concentrations were prepared as duplicates. After 24, 48, and 72 h of incubation, a volume of 200 μL was sampled from each test flask, pipetted into a quartz microplate. Fluorescence was then determined using a cytofluorimeter and by comparison with a calibration range, cell density was determined, according to the measured fluorescence.

Definitive test
Test flasks and blanks were prepared in triplicate. Six replicates were used for the control.
After 24, 48 and 72 h of incubation, about 1 mL was sampled from each test flask. After 24 and 48 hours test flasks were replaced in the same position in the rotary shaker. Samples were stored in darkness until determination of algae concentration by cell counter cell.

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.

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 120 rpm and constantly illuminated by 8 fluorescent tubes between 6,000 and 10,000 lux (Mazdafluor, white industry 33).

Physico-chemical parameters were measured using a METTLER TOLEDO 345 pH meter for measurement of pH and with a WTW OXI 538 oxymeter for dissolved oxygen measurement. 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

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.023 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (0.014-0.035)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (0.19-0.44)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.004 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: (0.001-0.01)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.044 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: (0.021-0.075)

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 January 10, 2013 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 and EC10 values and the 95% confidence interval. Probit analysis is generally used to calculate the 24, 48 and 72-hour EC values.

Dissolved oxygen and pH were measured in the test solutions at the beginning and at the end of the test and are presented in Table 1. Measurements were carried out in blank non-inoculated solution at T0 and T72 and in control inoculated solution at T72.

 

Table 1: 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)	8.04	7.98	8.8	8.9
0 (T)	-	9.31	-	9.7
0.10	7.96	9.24	9.3	9.8
0.26	7.98	9.22	9.3	9.8
0.66	7.99	8.87	9.2	9.6
1.72	7.98	8.38	9.2	9.2
4.48	7.99	8.11	9.2	8.9
11.6	7.99	8.04	9.2	8.8
30	7.98	8.01	9.2	8.8

 

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 table 2 and 3 respectively:

 

Table 2: Range finding test - Average percentage inhibition of cell growth (IAi) and growth rate (Iµi)

 

Concentration (nominal) (mg/L)	IAi (%)	Iµi (%)
0	0.00	0.00
100	100.00	100.00
50	100.00	165.88
10	88.40	51.19
5	79.40	35.32
1	49.68	15.20
0.5	38.45	8.97
0.1	24.93	3.53

 

 

Table 3: Definitive test - Average percentage inhibition of cell growth (IAi) and growth rate (Iµi)

 

Concentration (nominal) (mg/L)	IAi (%)	Iµi (%)
0	0.00	0.00
30.0	96.27	77.09
11.6	89.37	60.76
4.48	81.99	44.56
1.72	65.89	24.22
0.66	29.56	7.64
0.26	15.11	3.27
0.10	18.02	3.64

 

  

The table 4 presents the concentrations (mg/L) of TERT-AMYL PEROXY-2-ETHYLHEXANOATE measured in non-inoculated test solutions (without algae) at the beginning and at the end of the test at the highest concentration tested.

 

Table 4: Nominal and measured concentrations of the test item at the beginning and at the end of the exposure period

  

Test item concentration
Nominal   (mg/L)	Measured in inoculated solutions
	Initial T0 (mg/L)	T24h (mg/L)	T48h (mg/L)	Final T 72h (mg/L)	Extrapolated* (mg/L)
30.0	2.57	1.61	1.26	0.67	1.386
11.6	0.95	0.54	0.43	0.35	0.512
4.48	0.34	0.17	0.15	0.09	0.165
1.72	0.12	0.06	0.05	< QL	0.050
0.66	0.04	< QL	< QL	< QL	0.017
0.26	< QL	< DL	< QL	< DL	0.011
0.10	< QL	< QL	< DL	< DL	0.011
0 (Bl)	< DL	< DL	< DL	< DL	-

 

< DL : Concentration lower than the Detection Limit of the analytical method (0.0087 mg/L, i.e. 8.7 µg/L).

< QL : concentration lower than the Quantification Limit of the analytical method (0.029 mg/L, i.e. 29 µg/L).

 

*Concentrations  extrapolated  using  the  geometric  means  (0-24,  24-48,  48-72)  of  measured concentrations of TERT-AMYL PEROXY-2-ETHYLHEXANOATE, according to the Annex 2 of the OECD 23 guideline.

When a value is lower than the detection limit, the value of detection limit is used for the calculation. When a value is lower than the quantification limit, half the value of quantification limit is used for the calculation.

 

Concentrations of TERT-AMYL PEROXY-2-ETHYLHEXANOATE were measured in inoculated series(with algae), as abiotic samples were not available.

Validity criteria fulfilled:

yes

Conclusions:

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata exposed to the test item TERT-AMYL PEROXY-2-ETHYLHEXANOATE for a duration of 72 hours was assessed according to the OECD Guideline 201. The results were as follows (mg/L, extrapolated concentrations using the geometric means (0-24, 24-48, 48-72) of measured concentrations, according to the Annex 2 of the OECD 23 guideline):

Growth rate – Cell multiplication (r): ErC50-72h= 0.28
Growth rate – Cell multiplication (r): ErC10-72h= 0.023

Executive summary:

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata exposed to the test item TERT-AMYL PEROXY-2-ETHYLHEXANOATE for a duration of 72 hours was assessed according to the OECD Guideline 201.

 

Algae were exposed to 30 to 0.1 mg/L (nominal concentrations) of TERT-AMYL PEROXY-2-ETHYLHEXANOATE 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. The results were as follows (mg/L, extrapolated concentrations using the geometric means (0-24, 24-48, 48-72) of measured concentrations according to the Annex 2 of the OECD 23 guideline):

 

Cell growth – Biomass (b): EbC50-72h= 0.044

Cell growth – Biomass (b): EbC10-72h= 0.004

Growth rate – Cell multiplication (r): ErC50-72h= 0.28

Growth rate – Cell multiplication (r): ErC10-72h= 0.023

 

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

 

• The biomass in the control cultures has increased exponentially by a factor of 73 higher than 16 within the 72-hour test period.This corresponds to a specific growth rate of 1.430 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

OECD 201, GLP, key study, validity 1:
Growth rate – Cell multiplication (r): ErC50-72h= 0.28 mg/L
Growth rate – Cell multiplication (r): ErC10-72h= 0.023 mg/L
(geometric means (0-24, 24-48, 48-72) of measured concentrations)

Key value for chemical safety assessment

EC50 for freshwater algae:

0.28 mg/L

EC10 or NOEC for freshwater algae:

0.023 mg/L

Additional information

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata exposed to the test item TERT-AMYL PEROXY-2-ETHYLHEXANOATE for a duration of 72 hours was assessed according to the OECD Guideline 201.

 

Algae were exposed to 30 to 0.1 mg/L (nominal concentrations) of TERT-AMYL PEROXY-2-ETHYLHEXANOATE 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. The results were as follows (mg/L, extrapolated concentrations using the geometric means (0-24, 24-48, 48-72) of measured concentrations, according to the Annex 2 of the OECD 23 guideline):

 

Cell growth – Biomass (b): EbC50-72h= 0.044

Cell growth – Biomass (b): EbC10-72h= 0.004

Growth rate – Cell multiplication (r): ErC50-72h= 0.28

Growth rate – Cell multiplication (r): ErC10-72h= 0.023

 

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

 

• The biomass in the control cultures has increased exponentially by a factor of 73 higher than 16 within the 72-hour test period.This corresponds to a specific growth rate of 1.430 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%.