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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 19 November 2004 to 09 December 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was performed according to OECD Guideline 201 and EU Method C3 with GLP statement. All validity criteria were fulfilled. Nevertheless, as no details are available on the test solution preparation method (i.e. phase separation; whether droplets of undissolved test substance may have been incorporated into the test solutions), the study has been quoted Klimish 2 and it has been decided to perform a new algae toxicity test in 2013.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The concentrations of test item were measured in non-inoculated test solutions (without algae) at the beginning and at the end of the test. In addition, measurements were performed at the end of the test in solutions inoculated with algae.
Vehicle:
no
Details on test solutions:
A stock solution of the test item in water was prepared 72 hours before the start of the beginning of the test by mixing 92 µL of test item in 1 litre of dilution water.
For the definitive test, volumetric flasks were filled with adequate volumes of the test item stock solution and dilution water in order to obtain the test solutions at concentrations varying from 30 mg/L to 0.6 mg/L (nominal). Flasks were stoppered with cellulose bungs and placed in the phytoculture cabinet.

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata, CCAP 278/4 stock were obtained from the Culture Centre of Algae and Protozoa (Ambleside, UK).

The conditions used for culturing algae are described in Annex 1 of the method C3. 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.

3 to 5 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 10e4 cells/mL.
The cell density of the pre-culture was about 2.773 x 10e6 cells/mL for the preliminary test and about 1.072 x 10e6 cells/mL for the definitive test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
none
Hardness:
Not applicable
Test temperature:
23.2 ± 1°C
pH:
see "any other information on results incl. tables"
Dissolved oxygen:
see "any other information on results incl. tables"
Salinity:
Not applicable
Nominal and measured concentrations:
Definitive test nominal concentrations: 0, 0.6, 1.3, 2.8, 6.2, 13.6 and 30 mg/L
Details on test conditions:
Physico-chemical parameters were measured using a METTLER TOLEDO 345 pHmeter for measurement of pH and with a WTW OXI 538 oxymeter for dissolved oxygen measurement.

The test was performed in a phytoculture cabinet that allows test flasks to be incubate under precise conditions. Temperature is set to 23°C; flasks were continuously shaken with a rotation of 20 rpm and constantly illuminated by 8 fluorescent tubes. The temperature was measured continuously in one test flask.

The study was performed using 120 mL glass bottles stoppered with cellulose bungs. Test flasks, controls and blanks were prepared in triplicate.

After 24, 48, and 72 h of incubation, about 1 mL was sampled from each test flask under agitation and introduced into culture tubes. After 24 and 48 hours test flasks were replaced in the same position in the rotary shaker. Culture tubes were stored in darkness until determination of algae concentration by microscopic counting. Measured concentrations were used for calculating the percentages inhibition and plotting the growth curves.
Reference substance (positive control):
yes
Remarks:
sodium dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.353 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL = 0.309-0.394 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.766 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL = 0.721-0.811 mg/L
Details on results:
As shown in the previous table, the final concentration of the test item was not within the designated limit of 80 % of the initial concentration in non-inoculated flasks. Thus, the test item concentration was not maintained throughout the duration of the test.
The lost of test substance in the test medium could be related to adsorption to the increasing algal biomass or to hydrolysis in aqueous media.
Therefore, it was decided to express the effective concentrations as initial measured concentrations. In addition, it allows to compare these results with the algae toxicity test performed in 2013.
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 sodium dichromate. The most recent values of ErC50 and EbC50 obtained on October 9 2004 were respectively 1.00 mg/L and 0.50 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:
In the test report, 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. The No-Observed-Effect Concentration (NOEC) is determined by statistic analysis using the Dunnett’s test.
A more relevant statistical analysis using ToxRat Version 2.10 has been performed and has been attached in this RSS.

Measured pH and O2 concentrations in the definitive test

 

Nominal concentrations

(mg/L)

pH

Dissolved O2 (mg/L)

T0

T72h

T0

T72h

0 (Bl)

7.91

7.91

9.0

8.8

0 (T)

7.91

9.94

9.0

11.8

0.6

7.89

10.14

9.2

12.2

1.3

7.88

9.91

9.2

12.1

2.8

7.90

8.82

9.2

10.4

6.2

7.90

8.00

9.2

9.1

13.6

7.88

7.92

9.2

9.0

30

7.86

7.91

9.2

8.9

 Measurements were carried out in non-inoculated solutions at T0 and in inoculated solutions after T72 of incubation.

Test item concentrations and percentage of inhibition

Nominal concentrations (mg/L)

Measured in non inoculated solutions

Geometric mean measured concentrations (mg/L)

Percentage inhibition of growth rate

Initial

Final *

0

0

<LD

-

0.00

0.6

0.062

<LD (0.064)

0.027

-1.2

1.3

0.172

0.053 (0.138)

0.095

2.7

2.8

0.415

0.218 (0.196)

0.301

16.1

6.2

0.991

0.551 (0.713)

0.739

65.3

13.6

1.923

1.418

1.651

95.9

30

4.225

2.820

3.452

110.0

<LD: concentration lower than the detection limit of the analytical method (23µg/L)

* within parenthesis: concentrations of the test item measured in inoculated series (with algae).

Validity criteria fulfilled:
yes
Remarks:
see details in the section below
Conclusions:
Effective concentrations have been calculated using initial measured concentrations of the test item. Concentrations were measured by gas chromatography coupled with mass spectrometry.
The 72h-ErC50 and 72h-ErC10 were evaluated at 0.766 mg/L and 0.353 mg/L, respectively.
The definitive test met the validity criteria of the test guideline detailed as follows:
- The biomass in the control cultures increased exponentially by a factor of 95.8 within the 72-hour test period (required: at least 16-fold)
- The coefficient of variation of average specific growth rates during the whole test period in the control cultures was 4% (required: < 7%)
- The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures were 13.8% (required: < 35%)
Executive summary:

The determination of the growth inhibition of the freshwater algal Pseudokirchneriella subcapitata exposed to OO-t-BUTYL-O-(2-ETHYLHEXYL)MONOPEROXYCARBONATE for a duration of 72h was performed following the OECD Guideline 201 and EU Method C3 with GLP statement.

For the definitive test, volumetric flasks were filled with adequate volumes of the test item stock solution and dilution water in order to obtain the test solutions at concentrations varying from 30 mg/L to 0.6 mg/L (nominal).

Algae were exposed to the range of concentrations of the test item.

Effective concentrations have been calculated using initial measured concentrations of the test item which were determined by gas chromatography coupled with mass spectrometry. The 72h-ErC50 and 72h-ErC10 were evaluated at 0.766 mg/L and 0.353 mg/L, respectively.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was performed according to OECD Guideline 201 and EU Method C3 with GLP statement. All validity criteria were fulfilled. The study has been quoted Klimish 2 because of disappearance of the test substance from solution, related to adsorption to increasing algal biomass or to hydrolysis in aqueous media.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Storage of samples: None
Samples treatment: Dilution with methanol

The concentrations (mg/L) of OO-t-BUTYL-O-(2-ETHYLHEXYL)MONOPEROXYCARBONATE were measured in non-inoculated test solutions (without alga) at the beginning and at the end of the test.
Vehicle:
no
Details on test solutions:
For the range finding test, a 100 mg/L stock solution of was prepared 1h30 before the beginning of the test. After decantation, it was used to prepare by dilution the convenient range of nominal concentrations: 5, 1, 0.5 and 0.1 mg/L.

For the definitive test, a 100 mg/L stock solution was prepared 1h30 before the beginning of the test. After decantation, it was used to prepare the convenient range of nominal concentrations: 5, 2.6, 1.36, 0.7, 0.36, 0.2 and 0.1 mg/L.

It should be noted that these nominal concentrations are based on the stock solution concentration (100 mg/L). Due to the known water solubility determined according to OECD 105 guideline(5.39 mg/L), real concentrations are much lower (see analytical results).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata, CCAP 278/4 stock were 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 10e4 cells/mL.
The cell density of the pre-culture was about 1.14 x 10e6 cells/ml for the preliminary test and about 3.41 x 10e6 cells/ml for the definitive test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not applicable
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 applicable
Nominal and measured concentrations:
Range finding test nominal concentrations: 5, 1, 0.5 and 0.1 mg/L.

Definitive test nominal concentrations: 5, 2.6, 1.36, 0.7, 0.36, 0.2 and 0.1 mg/L.

It should be noted that these nominal concentrations are based on the stock solution concentration (100 mg/L). Due to the known water solubility (5.39 mg/L), real concentrations are much lower (see analytical results)
Details on test conditions:
In the preliminary test, algae were exposed under static conditions to a series of 4 nominal concentrations (5, 1, 0.5 and 0.1 mg/L). 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.
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.


In the definitive test, 7 nominal concentrations tested were: 5, 2.6, 1.36, 0.7, 0.36, 0.2, and 0.1 mg/L (factor 1.92). All concentrations were prepared as triplicates. A non-inoculated blank (labelled Bl) containing only dilution water and test item was also prepared and incubated (for analytical monitoring)

After 24, 48 and 72h 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.
The pH and dissolved O2 were measured for all concentrations, at the beginning and the end of the test.

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).
The study was performed using 120 mL glass bottles stoppered with cellulose bungs.
Filling volume: 50 mL.

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.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.109 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 0.090-0.125
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.214 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 0.199-0.230
Details on results:
As shown in the previous table, the final concentration of the test item was not within the designated limit of 80 % of the initial concentration in non-inoculated flasks. Thus, the test item concentration was not maintained throughout the duration of the test.
The loss of test substance in the test medium could be related to adsorption to the increasing algal biomass or to hydrolysis in aqueous media.
Therefore, it was decided to express the effective concentrations as initial measured concentrations. For the lowest initial concentrations, where < QL is mentioned, half the quantification limit (QL) was used, i.e. 0.013 mg/L.
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 October 18 2012 were respectively 1.1 mg/L and 0.64 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:
In the test report, 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. The No-Observed-Effect Concentration (NOEC) is determined by statistic analysis using the Dunnett’s test.
A more relevant statistical analysis using ToxRat Version 2.10 has been performed and has been attached in this RSS.

Measured pH and O2 concentrations in the definitive test

 

Nominal concentrations

(mg/L)

pH

Dissolved O2 (mg/L)

T0

T72h

T0

T72h

0 (Bl)

7.94

7.98

9.1

8.6

0 (T)

7.94

9.94

9.1

10.1

0.10

7.96

9.93

9.1

10.1

0.20

7.96

9.82

9.1

10.0

0.36

7.98

9.71

9.0

9.9

0.70

7.97

9.55

9.1

9.8

1.36

7.98

8.65

9.1

9.4

2.6

7.98

8.15

9.1

8.8

5

8.00

7.93

9.1

8.6

 

Measurements were carried out in blank non-inoculated solution at T0 and T72 and in control inoculated solution at T72.

 

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

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

 

Nominal concentrations

(mg/L)

Measured in non inoculated solutions

Initial

(mg/L)

Final

(mg/L)

0

< DL

< DL

0.1

< QL

< DL

0.2

< QL

< DL

0.36

0.03

< DL

0.70

0.06

< DL

1.36

0.11

< DL

2.6

0.23

< QL

5

0.47

0.03

 

< DL : concentration lower than the Detection Limit of the analytical method (0.008 mg/L).

< QL : concentration lower than the Quantification Limit of the analytical method (0.026 mg/L).

Concentrations measured in non-inoculated test solutions (without algae) at the beginning and at the end of the test

 

It should be noted that these nominal concentrations are based on the stock solution concentration (100 mg/L). Due to the known water solubility (5.39 mg/L), real concentrations are much lower.

Nominal concentrations (mg/L) Inhibition of cell growth (%) Inhibition of growth rate (%)
0 0.00 0.00
5 100.00 111.08
2.6 93.01 51.33
1.36 56.29 13.71
0.70 32.75 5.96
0.36 21.54 4.58
0.20 15.40 3.79
0.11 8.06 2.67
Validity criteria fulfilled:
yes
Remarks:
see details in the section below
Conclusions:
Effective concentrations have been calculated using initial measured concentrations of the test item. Concentrations were determined by HPLC/MS/MS. The 72h-ErC50 and 72h-ErC10 were evaluated at 0.214 mg/L and 0.109 mg/L.
The definitive test met the validity criteria of the test guideline detailed as follows:
- The biomass in the control cultures increased exponentially by a factor of 103.0 within the 72-hour test period (required: at least 16-fold)
- The coefficient of variation of average specific growth rates during the whole test period in the control cultures was 2.3% (required: < 7%)
- The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures were max 21.5% (required: < 35%)
Executive summary:

The determination of the growth inhibition of the freshwater algal Pseudokirchneriella subcapitata exposed to OO-t-BUTYL-O-(2-ETHYLHEXYL)MONOPEROXYCARBONATE for a duration of 72h was performed following the OECD Guideline 201 with GLP statement. The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours. Algae were exposed to 0.1 to 5 mg/L (nominal concentrations) of test substance.

Effective concentrations have been calculated using initial measured concentrations of the test item which were determined by HPLC/MS/MS. The 72h-ErC50 and 72h-ErC10 based on growth rate were evaluated at 0.214 mg/L and 0.109 mg/L respectively. All the validity criteria of the test guideline have been fulfilled.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
2013-09-17 to 2013-09-20
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was performed according to OECD Guideline 201 with GLP statement with some modifications. We believe that these modifications caused the high variability observed within the results. Thus, this study did not meet the coefficient of variation criteria required by the OECD 201 guideline. We classified this study as Klimisch 3.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Storage of samples: None
In a preceding study, OO-t-butyl-O-(2-ethylhexyl)monoperoxycarbonate was highly suspected to adsorb to algae and it was demonstrated that its half-life is short (43 hours). For these reasons, it was decided to monitore OO-t-butyl-O-(2-ethylhexyl)monoperoxycarbonate in non-inoculated solutions throughout the study.
Vehicle:
no
Details on test solutions:
As the test item has a low solubility, the WAF method was applied to the 3 highest concentrations (10, 5.21 and 2.71 mg/L); measured amounts of test item are dropped directly in Slow Stir flasks filed with test medium (no head space). The mixtures were strongly stirred during about 20 hours; then, after 30 minutes of decantation, the aqueous phase was taken out for the test. The 4 lowest concentrations (1.41, 0.735, 0.38 and 0.2 mg/L) were obtained by dilution of the 10 mg/L solution.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata, CCAP 278/4 stock (previously named Raphidocelis subcapitata and Selenastrum capricornutum) were obtained from the Culture Centre of Algae and Protozoa (Ambleside, UK).
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 applicable
Nominal and measured concentrations:
Nominal concentrations: 10, 5.208, 2.712, 1.412, 0.735, 0.383, 0.199 mg/L.

Initial concentrations measured: 2.55, 1.06, 0.76, 0.46, 0.24, 0.10, 0.052 mg/L.
Final concentrations measured: 1.36, 0.91, 0.71, 0.34, 0.18, 0.073, 0.045 mg/L

Corresponding geometrical means: 1.86, 0.99, 0.73, 0.39, 0.21, 0.085, 0.048 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL-flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 100 mL, no headspace
- Initial cells density: 5000 cells/mL
- Control end cells density: 51500 cells/mL
- No. of organisms per vessel: 500000
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: none
- Photoperiod: continous lighting
- Light intensity and quality: 6000-10000 lux (Mazdafluor, white industry)
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell density was estimated in each flask every 24 hours by flow-cytometry

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.92
- Range finding study: previous OECD 201 guideline studies allowed us to determine concentrations to be tested
- Test concentrations: 10, 5.208, 2.712, 1.412, 0.735, 0.383, 0.199 mg/L (nominal)
- Results used to determine the conditions for the definitive study: geometrical means of initial and final concentrations (both measured)
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: low half life of the substance (close to 20 hours)
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 may 2013 in closed bottle (HEPES Buffer) were respectively 0.63 mg/L and 0.29 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 EC50 and EC10 values. The No-Observed-Effect Concentration (NOEC) is determined by statistic analysis using the Dunnett’s test.

Table 3: Average percentage inhibition of cell growth (IAi) and growth rate (Iµi) after 72h.

Concentration (nominal) (mg/L)

IAi %

Iµi %

T

0

0

10

96.95

88.94

5.208

98.05

91.36

2.712

99.38

95.38

1.412

98.90

94.95

0.735

100

99.53

0.383

91.16

56.23

0.199

56.27

18.24

Validity criteria fulfilled:
no
Remarks:
As explained above, for reasons inherent to the experimental design, the present study did not meet the variability criteria.
Conclusions:
The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata exposed to the test item OO-t-BUTYL-O-(2-ETHYLHEXYL)MONOPEROXYCARBONATE for a duration of 72 hours was assessed according to the OECD Guideline 201. But as validity criteria were not fulfilled in this study, it was not possible to use these data.
Executive summary:

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata exposed to the test item OO-t-BUTYL-O-(2 -ETHYLHEXYL)MONOPEROXYCARBONATE for a duration of 72 hours was assessed according to the OECD Guideline 201. Algae were exposed to test item dissolved in dilution water. The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours.

Nevetheless, validity criteria according to the OECD guideline were not fulfilled and thus it was not possible to consider the results of this study to assess the toxicity of OO-t-BUTYL-O-(2-ETHYLHEXYL)MONOPEROXYCARBONATE to algae.

Description of key information

GLP study performed according to OECD 201 guideline: 72h-ErC50 of 0.214 mg/L and 72h-ErC10 of 0.109 mg/L (initial measured concentrations, growth rate inhibition)

Key value for chemical safety assessment

EC50 for freshwater algae:
0.214 mg/L
EC10 or NOEC for freshwater algae:
0.109 mg/L

Additional information

Two studies were performed according to OECD Guideline 201 with GLP statement. The growth inhibition of the freshwater algal Pseudokirchneriella subcapitata exposed to OO-t-BUTYL-O-(2-ETHYLHEXYL)MONOPEROXYCARBONATE was determined for a duration of 72h.

In the first study (Gancet, 2005), algae were exposed to concentrations varying from 0.6 to 30 mg/L (nominal).

Effective concentrations have been calculated using initial measured concentrations of the test item which were determined by gas chromatography coupled with mass spectrometry. The 72h-ErC50 and 72h-ErC10 were evaluated at 0.766 mg/L and 0.353 mg/L, respectively. All the validity criteria of the test guideline have been fulfilled.

Nevertheless, as no details are available on the test solution preparation method (i.e. phase separation; whether droplets of undissolved test substance may have been incorporated into the test solutions), the study has been quoted Klimish 2 and it has been decided to perform a new algae toxicity test in 2013 in order to confirm or not these results.

In this second study (Gancet, 2013), algae were exposed to 0.1 to 5 mg/L (nominal concentrations) of test substance. The test item concentration was not maintained throughout the duration of these algae tests. The loss of test substance in the test medium could be related to adsorption to the increasing algal biomass or to hydrolysis in aqueous media. Therefore, it was decided to express the effective concentrations as initial measured concentrations which were determined by HPLC/MS/MS. The 72h-ErC50 and 72h-ErC10 based on growth rate were evaluated at 0.214 mg/L and 0.109 mg/L respectively. All the validity criteria of the test guideline have been fulfilled.

Both algae toxicity tests showed ErC50 and ErC10 < 1 mg/L. It was decided to use the results of Gancet, 2013 study which were more detailed in the study report and gave the lowest effective concentrations.