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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 2008-06-02 to 2008-07-11
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed following OECD Guideline 202, under GLP.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. certificate)
Remarks:
2008-11-12
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Vapour pressure: 26 hPa (at 20°C)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, and 150 mg/L
- Sampling method: The test item (52.59 mg) was dissolved in acetonitrile and made up to the mark in a 50 mL volumetric flask to prepare a stock solution of 1052 mg/L. Defined volumes of this stock solution were diluted with acetonitrile/water (v/v; 1/1) to obtain calibration solutions of the test item in the range of 2.63 to 168 mg/L. A second series of calibration solutions in the range of 1.09 to 87.0 mg test item /L, based on a stock solution of 1087 mg/L was prepared. The total calibration range used was 1.09 to 168 mg/L. These solutions were used to calibrate the HPLC-system.
- Sample storage conditions before analysis: no storage period.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:For determination of the actual concentrations of the test item Di-t-butyl peroxide, quadriplicate samples were taken from each treatment before the test start and at the end of the test after 48 hours. For the 48-hour stability samples, the contents of the respective replicates were combined prior to sampling. Immediately after sampling, acetonitrile in the ratio of 1:1 was added to all samples of the single test concentration from both sampling times to stabilize these samples. Thereafter, duplicate samples of the single test concentration were immediately analyzed. From the control, only one of the duplicate samples from both sampling times was analyzed immediately after sampling. The other duplicate samples of each treatment were stored deep-frozen (at about -20 °C). These samples were not analyzed.
- Eluate: distilled
- Differential loading: cf. Method description above
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnid
- Strain: 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
- Method of breeding: The clone has been bred at Harlan Laboratories in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.
- Feeding during test: No
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
20°C
pH:
7.6-7.8
Dissolved oxygen:
no data
Salinity:
not applicable
Nominal and measured concentrations:
nominal conc.: 0, and 152 mg/L
measured conc.: 0, and 73.1 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL glass tubes
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 50 mL of test medium
- Aeration: no data
- Type of flow-through (e.g. peristaltic or proportional diluter): no data
- 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): 5
- Biomass loading rate: no data

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no data
- Photoperiod: 16:8
- Light intensity and quality: from 500 to 630 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter: Coulter Conter, Model ZM
- Chlorophyll measurement: none
- Other: none

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 150
- Test concentrations: 0, and 150 mg/L
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 73.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
>= 73.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 73.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 73.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
no data
Results with reference substance (positive control):
For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate 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, study B83744) indicated that the sensitivity of the test organisms was within the internal historical range (48-hour EC50 from 1996 to 2008: 0.53 1.1 mg/L).
Reported statistics and error estimates:
no data

Table: Effect of di-t-butyl peroxide on the mobility of Daphnia magna.

Nominal
test item concentration

No. of
daphnids
tested

Immobilized
daphnids after
24 hours

Immobilized
daphnids after
48 hours

(mg/L)

 

No.

%

No.

%

Control

20

0

0

0

0

150
(73.1)#

20

0

0

0

0

 

#:   mean measured concentration

Validity criteria fulfilled:
yes
Conclusions:
Di-t-butyl peroxide to Daphnia magna was determined in a 48‑hour static test, under GLP and according to OECD guideline. The mean measured test concentration was 48.9% of the nominal value (i.e. 73.1 mg/L). In the control and at the mean measured test item concentration of 73.1 mg/L (nominal 150 mg/L), no immobilized test organisms were observed during the test period of 48 hours. Thus, EC50(72h) >73.1 mg/L and the No effect concentration >= 73.1 mg/L.
Executive summary:

The acute toxicity of the test item di-t-butyl peroxide to Daphnia magna was determined in a 48‑hour static test according to the EU Commission Directive 92/69/, Part C.2 and the OECD Guideline for Testing of Chemicals, No. 202 (2004), under GLP. A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test organisms up to and including a nominal concentration of 150 mg/L. Thus, the only nominal concentration tested was 150 mg/L. Additionally, a control group was tested in parallel. The analytically determined test item concentration in the analyzed test medium was 126% of the nominal value (189 mg/L) at the start of the test. During the test period of 48 hours, a decrease of the test item concentration in the test medium was determined. At the end of the test, 19% of the nominal value (28 mg/L) was found, presumably due to loss to the atmosphere despite the use of closed vessels.

The mean measured test concentration was 48.9% of the nominal value. Therefore, the reported biological results are related to the mean measured test item concentration of 73.1 mg/L. In the control and at the mean measured test item concentration of 73.1 mg/L (nominal 150 mg/L), no immobilized test organisms were observed during the test period of 48 hours.

Description of key information

The geometric mean was used in place of nominal concentrations. Finally a 48h-EC50 > 73.1 mg/L was calculated. This was based on no observed effect at the highest concentration tested.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
73.1 mg/L

Additional information

A reliable GLP study on Daphnia magna following OECD 202 is available. No immobilization was observed in a limit test at 150 mg/L. However, analytical measurements showed concentration of the substance decreased in the medium from 126% at test initiation to 19% at test termination, presumably due to loss to the atmosphere despite the use of closed vessels. The geometric mean was used in place of nominal concentrations. Finally a 48h-EC50 > 73.1 mg/L was calculated. This is considered acceptable as test substance concentrations were still measurable at the end of the study.