Dioctanoyl peroxide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date 4-11-2015 Experimental completion date 25-11-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

The solubility limit of the test substance is below the quantification limit of the HPLC analytical method. Analytical measurement was therefore not possible.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Solubility: Not determined; technically not feasible.
Storage: -20°C
Purity: 99.2%
Lot/Batch: 1204350050

Analytical monitoring:

no

Details on sampling:

Chemical analysis was not possible due to extremely low water solubility. No samples were therefore taken.

Vehicle:

no

Details on test solutions:

De-ionised water
The de-ionised water used in the study contained less than 10 μg/L of copper (not measured under GLP), with a conductivity of less than 5 μS/m and less than 2.0 mg/L NPOC-content.

Test vessels
As test vessels 50 ml glass beakers were used, containing 50 ml of test solution. All vessels were covered by glass plates during the test.

Test solution
The test material has extremely low solubility. Solubility is also below the capabilities of the analytical HPLC method. Chemical analysis can therefore not be conducted. Due to the low solubility preparation of the test solutions in a standard manner via a stock solution and further dilution is not possible. For this reason WAF's were prepared for each test concentration separately. This was achieved by accurate weighing of the test substance with an analytical balance and loading of the test material to 2 liter of test medium. The resulting solutions were stirred slowly for 7 days. This time span was shown in preliminary studies to cause some slight effects in algae species and was therefore in the absence of analysis presumed to be the worst case, and was applied to both definitive algae and Daphnia testing. After 7 days of slow stirring the solutions were stopped and allowed to rest for one hour. After which liquid from each of the WAF vessels was tapped off directly into the test vessels. The tap was positioned so as to remove liquid from the water phase of the vessel avoiding transfer of undissolved material on the surface or bottom of the vessels. The test solutions were then ready for addition of test organisms or for physical chemical measurements. WAF's were then restarted again for solution refreshment after 24 hours using the same procedures.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test animals were taken from a Daphnia magna stock. 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 Wil Research (Formerly NOTOX B.V.) 's-Hertogenbosch. The sensitivity of the Daphnia was checked by performing an immobilisation test with a reference compound (potassium dichromate) twice a year. The sensitivity was tested for compliance with the relevant study guideline.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

140 and 250 mg/l CaCO3 content (approximately 7.8-14 °dH).

Test temperature:

The test temperature was set at 20°C and the actual temperature should remain constant within ± 2°C.

pH:

ca. 8.2

Conductivity:

550-650 μS/cm

Nominal and measured concentrations:

Direct addition of the test material to 2 L of test medium was carried out to achieve the following test substance loadings: 100 and 1000 mg/L and a control in test medium.

Details on test conditions:

Test medium
The test medium was Dutch Standard Water (DSW), having a pH of approximately 8.2, and a typical conductivity of between 550 and 650 μS/m, 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 recommends the CaCO3 content be between 140 and 250 mg/L (approximately 7.8-14 °dH). Water hardness parameters are measured monthly in the DSW system using the appropriate Hach
Lange test kit. These are validated by analyzing a CaCl2 Standard. The dilution water was saturated with oxygen before the start of the test.

Test conditions
The test was carried out in a temperature-controlled room. The test temperature was set at 20°C and the actual temperature should remain constant within ± 2°C. The light regime was 16 h of ambient light per day, provided by fluorescent tubes.

Other apparatus
Apparatus was used according to the relevant Standard Operation Procedure as detailed in the study plan. The dissolved oxygen concentrations and conductivity were determined electrochemically using an oxygen electrode/conductivity electrode and meter. The pH was determined with a pH meter. The temperature was measured with a temperature sensor and recorder. The total hardness was measured using Hach Lange test kits and checked for accuracy with appropriate standard solutions

Test procedures
The test was performed as a semi-static limit test for 48 hours. 20 animals divided into 4 batches of 5 animals will be tested at each test concentration and in the control. Those animals which were not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobile and were recorded. The number of animals being trapped at the surface was also determined if applicable. These animals were not regarded as immobile. The daphnids were randomly placed in the test fluids and the test vessels will be placed in a random manner within each group. The test vessels were not aerated during the test and the animals were not fed. The test will at least be inspected at 0,24 and 48 hours and refreshed after 24 hours using identical methods to those used at the start of the test. Animals will be transferred carefully to newly filled beakers using a glass Pasteur pipette.

Evaluation of data
In this study no effects were observed with the highest test material loading. No statistical calculations were deemed necessary. A single immobilized animal was observed in the control.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: concentration based on loading rate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: concentration based on loading rate

Details on results:

Water quality
The temperature varied from 19.7 to 21.4 °C during the test and therefore exceeded the study plan requirements by 0.7 °C.
Conductivity was between 673 and 688 μS/m and hardness was 12.3-13.2 ° dH during the periodic checks of the DSW system in which the test fell. Water quality parameters from the guideline were therefore also met. The normal expected range of the conductivity (550-650 μS/cm) was exceeded slightly but did not exceed the acceptable range as indicated in the standard operating procedure for Dutch Standard Water (Maximum 700 μS/m) and was therefore considered acceptable for testing.

Statistical evaluation of effects
Statistical calculations were not required as no mobility inhibition was observed. The highest concentration was therefore representative of the nominal NOELR for this substance.
The NOELR of the test material was determined as 1000 mg/L a LOELR was not determined as no effects were observed. The EL50 may therefore also be expressed as > 1000 mg/L. Alternatively the test substance may be concluded to have no acute effects at its solubility limit.

Quality criteria
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.

Biological observations

Nominal concentration mg/L		0 hours	24 hours	48 hours
Control	I	5	5	4
	II	5	5	5
	III	5	5	5
	IV	5	5	5
Total		20	20	19
100	I	5	5	5
	II	5	5	5
	III	5	5	5
	IV	5	5	5
Total		20	20	20
1000	I	5	5	5
	II	5	5	5
	III	5	5	5
	IV	5	5	5
Total		20	20	20

pH Measurements

Nominal test concentration (mg/L)	pH
	0h	24h fresh	24h old	48h
Control	8.1	7.8	7.8	7.6
100	8.1	7.8	7.9	7.9
1000	8.0	7.8	7.9	8.0

Oxygen Measurements

Nominal test concentration (mg/L)	O2mg/L
	0h	24h fresh	24h old	48h
Control	8.5	8.7	8.5	8.6
100	8.4	8.7	8.6	8.4
1000	8.6	8.9	8.4	8.6

Validity criteria fulfilled:

yes

Conclusions:

All of the biological quality criteria were met and the study data can be considered to be an accurate representation of the effects of the soluble components of the test material to Daphnia magna in an acute semi-static test.
In the absence of analytics the influence stirring time was investigated thoroughly prior to the study and the worst case approach chosen for definitive testing. The test regime chosen was semi-static to improve exposure and the loading rate was elevated to the maximum of 1000 mg/L.
The loading rate is considered the most appropriate way of expressing the toxicity of this substance.
The loading rate considers all soluble components, impurities, and degradation products that result from the initial loading of the test substance.

Executive summary:

In order to predict the effects of the test substance in an aquatic environment, an acute immobilization test to Daphnia magna was conducted in accordance with OECD test guideline and with the OECD Principles of Good Laboratory Practice.

The toxicity of the test chemical to juvenile daphnids was determined in a semi-static system over an exposure period of 48 hours. Nominal loadings of 100 and 1000 mg/L were tested including the required control group.

The NOELR of the test material was determined as 1000 mg/L a LOELR was not determined as no effects were observed. The EL₅₀ may therefore also be expressed as > 1000 mg/L. Alternatively the test substance may be concluded to have no acute effects at its solubility limit.

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

• No daphnids were irreversibly trapped on the surface for any length of time

Analytical measurement was not possible due to the extremely low solubility of the test substance. Additional measures were therefore taken to ensure that the test represented the worst case in the absence of analytics. An extended stir of 7 days was used to ensure that solubility had been reached, extended preliminary tests were carried out and semi-static replacement was used to maximize exposure. The study is therefore considered to represent a realistic worst case for acute toxicity to Daphnia magna for the test substance.

Description of key information

In order to predict the effects of the test substance in an aquatic environment, an acute immobilization test to Daphnia magna was conducted in accordance with OECD test guideline and with the OECD Principles of Good Laboratory Practice.

The toxicity of the test chemical to juvenile daphnids was determined in a semi-static system over an exposure period of 48 hours. Nominal loadings of 100 and 1000 mg/L were tested including the required control group.

The NOELR of the test material was determined as 1000 mg/L a LOELR was not determined as no effects were observed. The EL₅₀may therefore also be expressed as > 1000 mg/L. Alternatively the test substance may be concluded to have no acute effects at its solubility limit.

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 EC₅₀value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L

• No daphnids were irreversibly trapped on the surface for any length of time

Analytical measurement was not possible due to the extremely low solubility of the test substance. Additional measures were therefore taken to ensure that the test represented the worst case in the absence of analytics. An extended stir of 7 days was used to ensure that solubility had been reached, extended preliminary tests were carried out and semi-static replacement was used to maximize exposure. The study is therefore considered to represent a realistic worst case for acute toxicity to Daphnia magna for the test substance.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1 000 mg/L