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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to protocol.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
WAF was tested, endpoints expressed as loading rates.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Aqueous samples were taken from the test vessel directly in 10 mL glass HPLC vials. The HPLC vials were placed in the auto sampler, without further dilutions. Samples were analyzed using the HPLC system.
Vehicle:
no
Details on test solutions:
Preparation of solutions
The test chemical is poorly soluble and potentially prone to hydrolysis and thermal degradation. For this reason a preparation method that allows the test chemical and potentially any degradation products the possibility to dissolve up to their maximum solubility limit in the test media was chosen. A WAF approach was therefore used for this test. A traditional stock solution and subsequent dilutions were not made. Use of a solvent was also not chosen as this would encourage testing at above the water solubility irrelevant for aquatic hazard determination. WAF vessels were not diluted and were each prepared separately.
A WAF solution was prepared for the only test concentration by adding of an accurate amount of the test substance to the test media and allowing it
equilibrate under slow agitation over 24 hours in a sealed vessel. It was then considered loaded with the test substance and / or breakdown products at their corresponding water solubility limits.
The test solution was carefully removed from each vessel avoiding the transfer of un-dissolved test material. The resulting solution was then split equally. One was used directly for testing the other was centrifuged at 8000 rpm for 10 minutes at 20 ºC and then transferred to test vessels for testing leaving a few milliliters at the bottom of the centrifuge tube. A centrifuged and non centrifuged replicate of 100 mg/L loading were therefore tested.
Due to the expected degradation of the dissolved fraction test vessels were replenished after 24 hours with a freshly prepared WAF to ensure a more stable exposure concentration. Due to the relatively low toxicity of the expected degradation products these were not quantified at this stage.

Test concentrations
100 mg/L loading centrifuged and non centrifuged and a control without test substance were used for testing as previously described (avoiding transfer of visible test chemical from the WAF). At the start of the test a new WAF was prepared as previously described. After 24 hours it was used in the same fashion to replenish the test solutions.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test animals were taken from a Daphnia magna stock, cultured in conformity with the relevant Standard Operation Procedure. The animals used in the test were less than 24 hours old were obtained from parent animals aged between 2-4 weeks. Daphnia were originally obtained from NOTOX B.V ‘s-Hertogenbosch, the Netherlands.

The sensitivity of the Daphnia was checked by performing an inhibition test with a reference compound (potassium dichromate) twice a year. The sensitivity was tested for compliance with the guidelines
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
12.2 ºdH which is equivalent to 210 mg/L as CaCO3.
Test temperature:
20.8 to 21.2 °C
pH:
7.8 to 8.3
Dissolved oxygen:
8.3 to 9.1 mg O2/L
Nominal and measured concentrations:
Nominal: 100 mg/L WAF
Measured: see attached table
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL glass beakers
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
The test medium was Dutch Standard Water (DSW), having a pH of approximately 8.2, and a conductivity between 550 and 650 µS/cm containing per liter of deionized water: 200 mg of CaCl2·2H2O, 180 mg of MgSO4·7H2O, 100 mg of NaHCO3 and 20 mg of KHCO3. The guideline criteria requires the CaCO3 content to be between 10 and 250 mg/L (approximately 1-14 ºdH). Hardness in the test water was measured once at the start of the study
using the appropriate Dr Lange test kit. This was validated by analyzing a CaCl2 Standard. The dilution was saturated with oxygen before the start of the test. This is done by thorough aeration for at least 30 minutes before use.
- Culture medium different from test medium: yes, Elendt M4 medium is used as culture medium

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light
- Light intensity: ambient light provided by fluorescent tubes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mobility

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: limit test was performed
- Range finding study
- Results used to determine the conditions for the definitive study: Two range finding tests in sealed and open conditions showed no effect in
preliminary studies. The data was sufficient to proceed in this manner with the definitive study.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: endpoint based on loading rate
Details on results:
Due to the speed the test chemical reaches saturation in water, preliminary information on half-life and preliminary studies, a one day stirring period for the WAF vessels was chosen. This means that a mixture of the parent peroxide, degradation products (including temporary intermediate breakdown products) and any impurities were essentially tested for their effects on the test organism at their maximum achievable concentrations. The predicted main breakdown components being 2-ethylhexan-ol and 2-metylbutan2-ol (Ref 6). The presence of these components was not confirmed analytically in the scope of this study.
All critical test validity criteria were met. The analytical measurements at T=0 did not meet the required quality criteria and are therefore not reported. There were however sufficient measurement of old and new solutions with an alternative method in order to demonstrate the presence of and subsequent rapid disappearance of the parent material between solution refreshments. The parent material was shown to rapidly degrade in less than 24 hours.
An exact EL50 determination was not possible as the test material displayed no toxicity at its maximum achievable solubility in test medium with test solution replenishment after 24 hours.
No effects were observed and the test material and subsequent degradation products generated from a loading of 100 mg/L can be concluded as not acutely toxic to Daphnia magna.
Results with reference substance (positive control):
The EC50 value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L.
Reported statistics and error estimates:
No statistics performed as no animals died during the test
Validity criteria fulfilled:
yes
Conclusions:
This study is reliable. It is performed according to OECD guideline 202 under GLP and all validity criteria were fulfilled.
The test substance has a very low water solubiltiy and was stable during the test. Water Accommodated Fractions were created and endpoints are based on loading rates. The calculated endpoints are adequate for C&L and risk assessment purposes.
Executive summary:

In order to predict the effects of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate in the aquatic environment, an acute toxicity test to Daphnia magna was conducted in accordance with OECD test guidelines and the OECD Principles of Good Laboratory Practice. Minor modifications to the guideline were applied based on the preliminary and existing data on the test substance. The main adaption being the preparing of the test chemical as a water accommodated fraction (WAF).

The toxicity of the test chemical to Daphnia Magna prepared as a Water Accommodated Fraction (WAF) was determined in a semi static system over an exposure period of 48 hours at a limit concentration of 100 mg/L loading of the test material.

No significant effect in comparison to the control was observed at the only concentration tested.

The NOELR (no observed effect loading rate) can be expressed as 100.0 mg/L (Loading)

The EL50 (48h) could not be determined and may only be expressed as > 100 mg/L (Loading).

Alternatively the effects can be expressed as the parent material having no toxicity at the maximum achievable solubility in test medium. A solution refreshment was applied to ensure a more stable exposure.

The following quality criteria have been met in the present study:

- Immobilization in the control did not exceed 10%.

- Oxygen concentration did not fall below 3 mg/L at any point during the study.

- The EC50 value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L (Documented as part of GLP laboratory maintenance).

- No daphnids were trapped on the surface for any length of time.

- The quality criteria for the analytical method were met

The following quality criterion could not be analytically determined:

- Test substance stability could not be determined due to rapid degradation of the parent material. Replenishment of the test solution after 24 hours was therefore carried out to ensure a more stable exposure and a realistic worst case scenario.

Chemical analysis demonstrated that the parent test chemical was present at its solubility limit in test medium in fresh solutions and that it was no longer detectable after 24 hours. It is well known that this group of chemicals are hydrolytically and thermically instable and the soluble fraction of the parent component was therefore shown to rapidly degrade. Non centrifuged test solutions were also tested as a dispersion was suspected. A slightly higher concentration was indeed measured. No effects were observed on the test organisms.

Description of key information

One GLP OECD 202 test is availble. The test was prepared as a Water accomodated Fraction (WAF): The EL50 (48h) could not be determined and may only be expressed as > 100 mg/L (Loading). Alternatively the effects can be expressed as the parent material having no toxicity at the maximum achievable solubility in test medium

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

In order to predict the effects of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate in the aquatic environment, an acute toxicity test to Daphnia magna was conducted in accordance with OECD test guidelines and the OECD Principles of Good Laboratory Practice. Minor modifications to the guideline were applied based on the preliminary and existing data on the test substance. The main adaption being the preparing of the test chemical as a water accommodated fraction (WAF).

The toxicity of the test chemical to Daphnia Magna prepared as a Water Accommodated Fraction (WAF) was determined in a semi static system over an exposure period of 48 hours at a limit concentration of 100 mg/L loading of the test material.

No significant effect in comparison to the control was observed at the only concentration tested.

The NOELR (no observed effect loading rate) can be expressed as 100.0 mg/L (Loading)

The EL50 (48h) could not be determined and may only be expressed as > 100 mg/L (Loading).

Alternatively the effects can be expressed as the parent material having no toxicity at the maximum achievable solubility in test medium. A solution refreshment was applied to ensure a more stable exposure.

The following quality criteria have been met in the present study:

- Immobilization in the control did not exceed 10%.

- Oxygen concentration did not fall below 3 mg/L at any point during the study.

- The EC50 value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L (Documented as part of GLP laboratory maintenance).

- No daphnids were trapped on the surface for any length of time.

- The quality criteria for the analytical method were met

The following quality criterion could not be analytically determined:

- Test substance stability could not be determined due to rapid degradation of the parent material. Replenishment of the test solution after 24 hours was therefore carried out to ensure a more stable exposure and a realistic worst case scenario.

Chemical analysis demonstrated that the parent test chemical was present at its solubility limit in test medium in fresh solutions and that it was no longer detectable after 24 hours. It is well known that this group of chemicals are hydrolytically and thermically instable and the soluble fraction of the parent component was therefore shown to rapidly degrade. Non centrifuged test solutions were also tested as a dispersion was suspected. A slightly higher concentration was indeed measured. No effects were observed on the test organisms.