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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:
no chemical analyses have been performed; WAF method used.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test chemical is extremely poorly soluble and potentially prone to hydrolysis. 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 therefore not made during this test. Use of a solvent was also not chosen as this would encourage testing at above the water solubility irrelevant for aquatic hazard determination.
A WAF was prepared at 100 mg/L loading 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.
Each WAF was then considered loaded with the test substance and / or breakdown products at their corresponding water solubility limits and was subsequently transferred to test vessels avoiding transfer of any undissolved material.
Hydrolysis data is not available and efforts to measure the test chemical analytically failed as water solubility is below the detection limit of the available analytical method. Therefore to ensure that 24 hours was sufficient to allow equilibration (in the absence of analytics) a series of non GLP preliminary tests using WAF preparations ranging from 24 hours to 2 weeks were conducted.

The 100 mg/L WAF and corresponding control without test substance were used directly (avoiding transfer of visible test chemical) for testing after a settling period. After settling and transfer of the solution for testing, the agitation of the WAF vessels was restarted. The same procedure was then repeated after 24 hour for solution refreshment. Due to the large excess of test chemical and extremely low solubility and good stability in the solid form preparation of a new WAF solution was not required.
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.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
Conductivity was measured in the dilution water as 609 μs/cm and total hardness was measured as ºdH of 9.0, which is equivalent to 160 mg/L as CaCO3.
Test temperature:
21.45 - 22.25 °C
pH:
8.1 - 8.2
Dissolved oxygen:
8.7 - 8.8 mg O2/L
Nominal and measured concentrations:
Nominal loading rate: 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL glass beakers
- Type (delete if not applicable): open
- Aeration: no
- Renewal rate of test solution: after 24h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test medium was Dutch Standard Water (DSW), having a pH of approximately 8.2, and a conductivity between 550 and 650 s/cm, containing per liter of de-ionized 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.

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

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

TEST CONCENTRATIONS
- A limit test was performed at 100 mg/L loading as no effects were seen up to this level in the range finding studies.
- Range finding study
- Results used to determine the conditions for the definitive study:
All preliminary tests conducted showed no significant effects on the test organism at 100 mg/L loading, regardless of there being a short (24 hour) or longer (2 week) stirring period. A 24 hour period was therefore used for the definitive test as no toxic effects were observed during preliminary testing.
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: loading rate
Details on results:
One animal died in the test concentration during the test. This effect did not give a significant difference from the control.
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:
The limit test concentration and control were compared to one another for significant difference using the Wilcoxon two sample test.
Validity criteria fulfilled:
no
Remarks:
Not possible to determine test chemical stability. A large excess of the test chemical was loaded to the test media in the WAF to ensure stabilization at the water solubility limit even if degradation of the test chemical was to occur.
Conclusions:
This study is reliable without restrictions. It is performed according to OECD guideline 202 under GLP and all critical validity criteria were fulfilled.
The test substance concentration was not monitored during the test as chemical analysis was technically not feasible. The calculated endpoints are adequate for C&L and risk assessment purposes.
Executive summary:

In order to predict the effects of Dihexadecyl peroxodicarbonate in an aquatic environment, an acute toxicity test to Daphnia magna was conducted in accordance with OECD test guidelines and with the OECD Principles of Good Laboratory Practice. Some modifications to the guideline were applied due to inherent properties of the test chemical.

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 loading concentration of 100 mg/L.

For the tested concentration no statistically significant effect in comparison to the control was observed.

The NOELR (no observed effect loading rate) can be expressed as 100 mg/L (Loading)

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

The test substance was concluded as not acutely toxic to Daphnia at its solubility limit in the test media.

Chemical analysis was not possible due to the solubility limit of the test chemical being below the detection limit of the available analytical method. Efforts were made to maximize exposure to ensure a worst case scenario therefore.

The following quality criteria have been met in this 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 following quality criterion was not met:

- Not possible to determine test chemical stability. Test solutions were therefore replaced after 24 hours to maximize exposure. A large excess of the test chemical was loaded to the test media in the WAF to ensure stabilization at the water solubility limit even if degradation of the test chemical was to occur.

Description of key information

The NOELR (no observed effect loading rate) can be expressed as 100 mg/L (Loading)

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

The test substance was concluded as not acutely toxic to Daphnia at its solubility limit in the test media.

Key value for chemical safety assessment

Additional information