Cyclohexylidenebis[tert-butyl] 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:

2002-07-09 to 2003-07-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP; OECD guideline study with restictions "The coefficient of analysis of the daily growth rate in the control replicates and coefficient of analysis of the average specific growth rates in the control replicates were not reported." according to the current guideline (adopted 2006)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 7th June 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

31st July 1992

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
First test: At t0 hour, a sample was taken from the limit test solution container (loading rate of 1000 mg/L). At T24, T48 and T72 hours, samples were taken from each limit test solution replicate, pooled (5 mL in total). Further samples were also taken at T24, T48 and T72 hours from the limit test solution which contained no algae and had been run alongside to determine the influence of adsorption and/or bioaccumulation on the possible decrease in test item concentration throughout the test.
Second test: At T0 hour, samples were taken from all test solution container, except for the control. At T24, T48 and T72 hours, samples were taken from all test replicate groups at each loading rate, pooled by concentration (5 mL in total). Further samples were also taken at T24, T48 and T72 hours from the solutions which contained no algae.
Third test: At T0 hour, samples were taken from each test solution container, except for the control. At T24, T48 and T72 hours, samples were taken from all test replicate groups at each loading rate, pooled by concentration (5 mL in total). Further samples were also taken at T24, T48 and T72 hours from the solutions which contained no algae.
- Sample storage conditions before analysis: storage at - 20 °C

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Each test solution was prepared by stirring the test item in LC reconstituted water at the appropriate loading rate between 20 and 24 hours after a filtration through a filter of porosity 0.45 µm, the WSF was collected and used as the test solution.
- Eluate: water
- Differential loading: preculture loading: new cultures are loaded at a concentration of 1 x 10000 cells/mL.
- Controls: yes (water)

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source: Culture Collection of algae and protozoa, Institute of the freshwater ecology, Far Sawrey, Ambleside, Cumbria, LA22 OLP, UK
- Method of cultivation: the algae are cultured under sterile conditions and maintained at exponential growth rate

ACCLIMATION
- Acclimation period: 7 days
- Culturing media and conditions: the same used in the test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

34 ± 17 mg/L as CaCO3

Test temperature:

First test: 23.5 °C and 24.6 °C
Second test: 23.1 °C and 23.5 °C
Third test: 22.7 °C and 23.9 °C

pH:

First test: 7.42 and 7.98
Second test: 7.79 and 8.87
Third test: 7.68 and 9.19

Nominal and measured concentrations:

-nominal concentration:
First test 1, 10, 100, 1000 mg/L;
Second test: 0.100, 0.215, 0.464, 1.00, 2.15, 4.64, 10.0 mg/L;
Third test: 0.100, 0.215, 0.464, 1.00, 2.15, 4.64, 10.0 mg/L
- The initial concentration in the limit test solution was slightly lower than the limit of quantification (0.0394 mg/L) but was estimated as 0.0283 mg/L.
The initial concentration in the second test of highest concentration was 0.513 mg/L, while the highest initial concentration measured in solution of the third test was 0.484 mg/L. Measured concentrations in solutions were systematically lower than the limit of quantification at the end of these tests (72 h).

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Aeration: was not used during the test but the cultures were constantly agitated
- Initial cells density: 10000 cells/mL
- Control end cells density: 389800 cells/mL
- No. of vessels per concentration (replicates): 3 (in the range-finding test only 2 replicates)
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water
- Conductivity: < 10 µS/cm
- Culture medium different from test medium: yes
- Intervals of water quality measurement: Temperature: controlled daily in a flask containing test water but no algae; pH: the pH values of the control and of all the test loading rates were measured at the beginning and the end of the tests.

OTHER TEST CONDITIONS
- Light intensity and quality: approximately 7000 lux

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: The number of the cells in solutions was calculated at T0 as 1 x 10000 cells/mL. For the remaining observation times (T24, T48 and T72 hours), cell numbers were determined using a Malassez cell counter.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: Growth rate inhibitions at 1, 10 and 100 mg/L nominal were 48 %, 57 % and 60 %, respectively, relative to the control, at T72 hours. Growth inhibitions at 1, 10 and 100 mg/L nominal were 77 %, 87 % and 86 %, respectively, relative to the control, at T72 hours.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: expressed as initial loading rate EL50, corresponding to approx. 0.5 mg/L expressed as actual (measured) concentration

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: expressed as initial loading rate EL50, corresponding to approx. 0.5 mg/L expressed as actual (measured) concentration

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: expressed as initial loading rate EL50, corresponding to approx. 0.5 mg/L expressed as actual (measured) concentration

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: expressed as initial loading rate EL50, corresponding to approx. 0.5 mg/L expressed as actual (measured) concentration

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: expressed as initial loading rate EL50, corresponding to approx. 0.5 mg/L expressed as actual (measured) concentration

Details on results:

- First test: Inhibitions of the growth rate and growth at 1000 mg/L nominal were 63 % and 87 %, respectively, relative to the control, at T72 hours (Limit test).
- Second test: Based on nominal concentrations, the test item was found to be less toxic in this second test than in first one.
The 24, 48, 72-hour ErL50s and the 72-hour EbL50 could not be calculated since the inhibition percentage of the growth at highest loading rate (1000 mg/L) was markedly lower than 50 % at the corresponding time (8 %, 8 %, 1 % and 13 %, respectively).
-Third test: Based on nominal concentrations, the toxicity results of this third test were in accordance with those of second one. As for the second test, the 24, 48, 72-hour ErL50s and the 72-hour EbL50 could not be calculated since the inhibition percentage of the growth rate or growth rate or growth at the highest loading rate (1000 mg/L) was markedly lower than 50 % at the corresponding time (27 %, 6 %, 2 % and 13 %, respectively).
Taking into account the results of these three tests, chemical analysis was carried out to determine the actual concentration of the test item in:
1) the limit test solution (nominal loading rate of 1000 mg/L) throughout the first test (0, 24, 48 and 72 hours),
2) the second test solution of the highest concentration (nominal loading rate of 10.0 mg/L) at the beginning and the end of the test (0 and 72 hours),
3) all solutions of the third test (nominal loading rates from 0.100 to 10.0 mg/L) at the beginning and the end of the test (0 and 72 hours).

Toxicity parameters:
Based on the results of second and third tests, there was no significant effect on algal growth up to approximately 0.5 mg/L, considered as the limit of solubility of the test item under our experimental conditions. The study was therefore stopped.
Hence, the ErC50 at each of the measured growth intervals and the 72-hour EbC50 were > 10.0 mg/L expressed as initial loading rate (EL50), corresponding to approximately 0.5 mg/L expressed as actual concentration (measured concentration at the beginning of the test in the saturated solution obtained from this loading rate of 10.0 mg/L).
The NOEL at 72 hours was calculated as >= 10.0 mg/L expressed as initial loading rate (EL50), corresponding to approximately 0.5 mg/L expressed as actual concentration (p = 0.5), based on the specific growth rate and biomass data.

Reported statistics and error estimates:

- Determination of the ErL50 and the EbL50: Specific growth rate can be defined as the rate of increase in the natural log of the cell number over specific intervals. The loading rate causing a reduction of growth rate to 50 % that of the control (ErL50) is obtained for each observation time by calculation from the average specific growth rate at each loading rate.
Biomass, in this case may be defined as the number of cells per mL of solution x time in hours and therefore represents an area. The biomass at 72 hours is determined using the trapezoidal method from number of cells observed at T0, T24, T48 and T72 hours. The loading rate causing a reduction of biomass at 72 hours to 50% that of the control is then calculated.
When for at least two loading rates, inhibition is > 0 % and < 100 %, the EL50 is calculated according to Pobit analysis. The confidence interval limits are calculated statistically according to Fieller`s method.
When at only one loading rate, inhibition is > 0 % and < 100 %, the EL50 is also calculated by Probit analysis. In this case, the highest loading rate causing no inhibition and the lowest loading rate producing 100 % inhibition are used as confidence limits.
If at all loading rates, inhibition is 0 % or 100 %, the EL50 corresponds to the geometric mean of the highest loading rate causing no inhibition and the lowest loading rate producing 100 % inhibition.
- Determination of the NOEL
After checking of normality of the data with Chi-square and Shapiro-Wilks tests as well as variance homogeneity (Bartlett test), the NOEL is determined by ANOVA (the Bonferroni T-test) using the individual replicates of the area under the curve and the specific growth rate.

Validity criteria fulfilled:

yes

Conclusions:

The 72 hour ErC50 and the EbC50 of cyclohexylidenebis[tert-butyl] peroxide in a static test system are > 10.0 mg/L expressed as initial loading rate (EL50), corresponding to approximately 0.5 mg/L expressed as actual concentration (the assumed limit of solubility), for Pseudokirchneriella subcapitata.
The NOEC at 72 hours is >= 10.0 mg/L expressed as initial loading rate (NOEL), corresponding to approximately 0.5 mg/L expressed as actual concentration, based on the specific growth rat and biomass data.

Executive summary:

The acute toxicity of cyclohexylidenebis[tert-butyl] peroxide was evaluated in the algal strain Pseudokirchneriella subcapitata using a 72 hour static test according to OECD guideline no. 201 and EU method C.3. As the equilibrium between the test item and the aqueous phase depends on the ratio of the test item to liquid a WSF (water-soluble fraction) was prepared for each test solution separately. Test solutions were stirred between 20 and 24 hours and WSFs were obtained by filtration through filters of porosity 0.45 µm.

The 72 hour ErC50 and the EbC50 of cyclohexylidenebis[tert-butyl] peroxide in a static test system are > 10.0 mg/L expressed as initial loading rate (EL50), corresponding to approximately 0.5 mg/L expressed as acutual concentration (the assumed limit of solubility), for Pseudokirchneriella subcapitata. The NOEC at 72 hours is >= 10.0 mg/L expressed as initial loading rate (NOEL), corresponding to approximately 0.5 mg/L expressed as actual concentration, based on the specific growth rat and biomass data.

Description of key information

The 72 hour ErC50 and the EbC50 of cyclohexylidenebis[tert-butyl] peroxide in a static test system are > 10.0 mg/L expressed as initial loading rate (EL50), corresponding to approximately 0.5 mg/L expressed as actual concentration (the assumed limit of solubility), for Pseudokirchneriella subcapitata.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.5 mg/L

Additional information

The acute toxicity of cyclohexylidenebis[tert-butyl] peroxide was evaluated in the algal strain Pseudokirchneriella subcapitata using a 72 hour static test according to OECD guideline no. 201 and EU method C.3. As the equilibrium between the test item and the aqueous phase depends on the ratio of the test item to liquid a WSF (water-soluble fraction) was prepared for each test solution separately. Test solutions were stirred between 20 and 24 hours and WSFs were obtained by filtration through filters of porosity 0.45 µm. The 72 hour ErC50 and the EbC50 of cyclohexylidenebis[tert-butyl] peroxide in a static test system are > 10.0 mg/L expressed as initial loading rate (EL50), corresponding to approximately 0.5 mg/L expressed as acutual concentration (the assumed limit of solubility), for Pseudokirchneriella subcapitata. The NOEC at 72 hours is >= 10.0 mg/L expressed as initial loading rate (NOEL), corresponding to approximately 0.5 mg/L expressed as actual concentration, based on the specific growth rat and biomass data.