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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2008-08-19 to 2008-09-11
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented study performed under standard guidelines (OECD 202 and EC C2) and GLP. However the measured concentrations were 69-76% and 78-79% of the nominals at the beginning and the end of the test, respectively. The results were based on measured concentrations.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
Commission Directive 92/69/EEC
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
2008-11-12
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 2.2, 4.6, 10, 22 and 46 mg/L for nominal concentrations.
- Sampling method: For the determination of the actual test item concentrations, duplicate samples were taken from each treatment before the start of the test and at the end of the test after 48 hours. The concentrations of the test item tert-Butyl-peroxybenzoate were analyzed in the duplicate test media samples from the nominal test concentrations of 10 and 22 mg/L from both sampling times (0 and 48 hours). The samples of the nominal test concentrations of 2.2 and 4.6mg/L were not analyzed since the concentrations were below the 48 hour NOEC determined in this test. The samples of the nominal test concentrations of nominal 46 mg/L were not analyzed since the same high toxic effect (EC100) was determined at the lower nominal concentration of 22 mg/L. The test concentration of 46 mg/L was therefore not considered to belong to the relevant part of the concentration-effect curve. From the control, only one of the duplicate samples was analyzed from each of the sampling times.
- Sample storage conditions before analysis: Samples were stored at about -20°C and protected from light immediately after sampling (protected from light until analysis). Based on pre-experiments for investigation of the storage stability (non-GLP), the test item is sufficiently stable in the test water under the storage conditions.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest nominal concentration of 46mg/L was prepared by dissolving 109 mg of test item completely in 2355 mL of test water under intense stirring for 3 hours at room temperature in the dark in a completely filled and tightly closed stirring vessel. This intensively mixed highest test concentration was used to prepare the test media of all lower test concentrations. The test media were prepared just before introduction of the daphnids (i.e., start of the test).
- Eluate: no data
- Differential loading: no data
- Controls: test water without test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no data
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water fleas
- Strain: Strauss, clone 5
- Source: University of Sheffield / UK in 1992.
- Age at study initiation (mean and range, SD): 6-24 hours old.
- Weight at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): no data
- Valve height at study initiation, for shell deposition study (mean and range, SD): no data
- Peripheral shell growth removed prior to test initiation: no data
- Method of breeding: The lignt conditions during the study was 16 hours light and 8 hours darkness with a 30 minute transition period.
- Feeding during test: The daphnids were not fed during the test
- Food type: not applicable
- Amount: 20 daphnids were divided into four replicates of five daphnids each.
- Frequency: no data

ACCLIMATION
- Acclimation period: no data
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: see below
- Feeding frequency: no data
- Health during acclimation (any mortality observed): no data

QUARANTINE (wild caught)
- Duration: not applicable
- Health/mortality: not applicable
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
no remarks
Post exposure observation period:
none
Hardness:
Water hardness = 2.5 mmol/L (= 250 mg/L as CaCO3)
Test temperature:
The test was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water temperature was maintained at 21°C. See table 1 in "any other information on materials and methods incl. tables" field.
pH:
The pH was measured at the beginning and the end of the test. See table 1 in "any other information on materials and methods incl. tables" field.
Dissolved oxygen:
The dissolved oxygen concentration was measured at the beginning and the end of the test. See table 1 in "any other information on materials and methods incl. tables" field.
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentrations : 2.2, 4.6, 10, 22, 46 mg/L.
The mean measured concentrations (calculated as the average over all measurements per test concentration) in the two critical test concentrations where 0 and 100% effects were observed, were 7.7 (nominal 10mg/L) and 16mg/L (nominal 22mg/L), corresponding to 77 and 73% of nominal.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass tubes
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 50 mL
- Aeration: The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, the test water was not aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: no data

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: in-house prepared by a water purification system (Millipore)
- Total organic carbon: no data
- Particulate matter: no data
- Metals: no data
- Pesticides: no data
- Chlorine: no data
- Alkalinity: no data
- Ca/mg ratio: no data
- Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data
- Other: Appearance of the test media during the test period = no remarkable observations, clear test medium.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness with a 30 minute transition period
- Light intensity: 200 to 1200 Lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The immobility of the daphnids was determined by visual inspection after 24 and 48 hours of exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: About 2
- Justification for using less concentrations than requested by guideline: none
- Range finding study: yes
- Test concentrations of the range finding study : 0, 9.81 and 24.5 mg/L
- Results used to determine the conditions for the definitive study: not specified but the selection of the test concentrations was based on the results of a range-finding test (without GLP).
Reference substance (positive control):
no
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
11 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CI = 7.7-16 mg/L
Key result
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
16 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
11 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CI = 7.7-16 mg/L
Key result
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
16 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
7.7 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: no data
- Observations on body length and weight: no data
- Other biological observations: no data
- Mortality of control: During the test, no immobilized test organisms were determined in the control.
- Other adverse effects control: no data
- Abnormal responses: no data
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no data
- Effect concentrations exceeding solubility of substance in test medium: no data
Results with reference substance (positive control):
For evaluation of the quality of the daphnia clone and the experimental conditions, potassium bichromate is tested as a positive control twice a year. The result of the latest positive control test in March 2008 (48-hour EC50: 0.73 mg/L, RCC Study No. B83744) indicated that the sensitivity of the test organisms was within the historical range of the Harlan Laboratories (48-hour EC50 from 1996 to 2008: 0.53-1.1 mg/L).
Reported statistics and error estimates:
The 24 and 48 hour EC50 could not be calculated by Probit Analysis or Moving Average Interpolation due to the steep concentration-effect relationship. Instead, the 48 hour EC50 was determined as the geometric mean value of the two consecutive test concentrations with 0 and 100% immobility, and the confidence limits for the EC50 as the test concentrations with 0 and 100% immobility.

Table 2: Effect of tert-Butyl peroxybenzoate on the mobility of Daphnia magna

Nominal test item concentration (mg/L)

No. of daphnids tested

Immobilized daphnids after 24 hours

Immobilized daphnids after 48 hours

No.

%

No.

%

Control

2.2

4.6

10 (7.7*)

22 (16*)

46

20

20

20

20

20

20

0

0

0

0

20

20

0

0

0

0

100

100

0

0

0

0

20

20

0

0

0

0

100

100

* mean measured test item concentration

Validity criteria fulfilled:
yes
Remarks:
During the test, no immobilized test organisms were determined in the control; the dissolved oxygen concentration at the end of the test was > 3 mg/L in control and test vessels.
Conclusions:
A study was realised in agreement with OECD Guideline 202 and EU Commission Directive 92/69/33C, Part C.2. The validity criteria were all fulfilled. The EC50 (48h) = 11 mg/L.
Executive summary:

The acute toxicity of the test item tert-Butyl peroxybenzoate (CAS 614 -45 -9) to Daphnia magna (6 -24 hours old) was determined in a 48-hours static test according to the EU Commission Directive 92/69/EEC, Part C.2 and the OECD Guideline for Testing of Chemicals, No. 202 (2004). A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield / UK in 1992. About test conditions, the pH values; the dissolved oxygen concentrations; and the water temperature were determined at each test concentration and in the control at the beginning and the end of the test. Also, the test was performed with a photoperiod of 16 hours light and 8 hours darkness with a 30 minutes transition period (200 to 1200 Lux). Test vessel used was glass tube (50mL) with a closed system because the test item was determined to be volatile. The nominal test item concentrations tested were 2.2, 4.6, 10, 22, and 46 mg/L. The selection of this test concentrations was based on the results of a range-finding test (without GLP), no detail in this report. Additionally, a control group was tested in parallel. The number of vessels per concentration and control was 4, and for each treatment, 20 daphnids were divided into 4 replicates of 5 daphnids each. No remarkable observations were found in the test media during the test period.

The mean measured concentrations (calculated as the average over all measurements per test concentration) were 7.7mg/L (nominal 10mg/L) and 16mg/L (nominal 22mg/L), corresponding to 77 and 73% of nominal.

The reported biological results were based on the mean measured test item concentrations. During the test, no immobilized test organisms were determined in the control. The 24- and 48- hours EC50 of the test item was calculated to be 11 mg/L with 95% Confidence limits of 7.7 and 16 mg/L. The 24- and 48-hour NOEC was 7.7 mg/L. The 24- and 48-hour EC100 was 16 mg/L.

Description of key information

48-hours static test according to the EU Commission Directive 92/69/EEC, Part C.2 and the OECD Guideline for Testing of Chemicals, No. 202. The 24- and 48- hour EC50s of the test item was calculated to be 11 mg/L (based on measured concentrations).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
11 mg/L

Additional information

The acute toxicity of the test item tert-Butyl peroxybenzoate (CAS 614 -45 -9) to Daphnia magna (6 -24 hours old) was determined in a 48-hours static test according to the EU Commission Directive 92/69/EEC, Part C.2 and the OECD Guideline for Testing of Chemicals, No. 202 (2004). A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield / UK in 1992. About test conditions, the pH values; the dissolved oxygen concentrations; and the water temperature were determined at each test concentration and in the control at the beginning and the end of the test. Also, the test was performed with a photoperiod of 16 hours light and 8 hours darkness with a 30 minutes transition period (200 to 1200 Lux). Test vessel used was glass tube (50mL) with a closed system because the test item was determined to be volatile. The nominal test item concentrations tested were 2.2, 4.6, 10, 22, and 46 mg/L. The selection of this test concentrations was based on the results of a range-finding test (without GLP), no detail in this report. Additionally, a control group was tested in parallel. The number of vessels per concentration and control was 4, and for each treatment, 20 daphnids were divided into 4 replicates of 5 daphnids each. No remarkable observations were found in the test media during the test period.

The mean measured concentrations (calculated as the average over all measurements per test concentration) were 7.7mg/L (nominal 10mg/L) and 16mg/L (nominal 22mg/L), corresponding to 77 and 73% of nominal.

The reported biological results were based on the mean measured test item concentrations. During the test, no immobilized test organisms were determined in the control. The 24- and 48- hour EC50s of the test item was calculated to be 11 mg/L with 95% Confidence limits of 7.7 and 16 mg/L. The 24- and 48-hour NOEC was 7.7 mg/L. The 24- and 48-hour EC100 was 16 mg/L.