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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:
May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD 202 GLP study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
The test chemical was therefore prepared using the water accommodated fraction method (WAF)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
Analytical monitoring was attempted prior to the start of this study as part of a water solubility test. Due to the extremely low water solubility chemical analysis was concluded to be technically not feasible at the time this study was conducted and due to the lack of effects found in preliminary tests this was not considered a critical limitation.
Vehicle:
no
Details on test solutions:
De-ionized water
The de-ionized water used contained not more than 0.01 mg/L of copper, had a TOC-content of not more than 2.0 mg/L and a conductivity of less than 5 S/cm. This water was produced from tap water in a water purification system according to the relevant Standard Operation Procedure.

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 (Standard Operation Procedure O1) . 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.


Preparation of 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 due to analytical detection limitations and efforts to measure the test chemical failed in the water solubility study as the water solubility is below the detection limit of the available analytical method. No confirmation of maximum saturation could therefore be given. Previous experience has shown that poorly soluble peroxide substances tend to reach their maximum solubility within 24 hours.
Test organisms (species):
Daphnia magna
Details on test organisms:
Source and maintenance of the 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. Hambakenwetering 7 5231 DD ‘s-Hertogenbosch P.O. Box 3476 5203 DL ‘s-Hertogenbosch the Netherlands.

Quality control of the test organisms
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 concentrations
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 type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
media refreshment at 24 hour intervals
Hardness:
12.2 ºdH of which is equivalent to 217.8 mg/L as CaCO3
Test temperature:
20.85 to 21.15 °C
pH:
7.9 to 8.4
Dissolved oxygen:
8.4 to 8.6 mg/L
Nominal and measured concentrations:
100 mg/L loading
Details on test conditions:
Test medium
Dilution water was used directly as the test medium.

Test flasks
As test vessels 50 mL beakers were used containing approximately 50 mL of test solution. .

Test room, temperature control and light regime The test was carried out in a temperature-controlled room. The test temperature was set between 18 and 22°C and the actual temperature should remain constant within ± 1°C. The light regime was 16 h of ambient light per day, provided by fluorescent tubes.

General test principles and procedures
The test was performed semi static test for 48 hours. 20 animals divided into 4 batches of 5 animals were tested in the control and at the limit test concentration. Those animals which are not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobile. The number of animals trapped at the surface was also considered and recorded if observed. These animals were not regarded as immobile and were made to re-submerge. In summary the daphnids were randomly placed in the test fluids and the test vessels were 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 was inspected at 0, 24 and 48 hours. The solutions were replaced after 24 hours with the original WAF preparations that were allowed to turn for an additional 24 hours. Due to the visual presence of a large excess of the test chemical a new WAF was not considered necessary. Animals were carefully transferred using a glass pipette
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
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

Preliminary test

All preliminary tests conducted showed no significant effects on the test organism at 100 mg/L loading after 24 hour stirring period. A 24 hour period was therefore used for the definitive test as no toxic effects were observed during preliminary testing. All preliminary testing was not conducted under GLP

Definitive test

The NOELR was determined as 100 mg/L. An EL50 (48h) could not be reached and may therefore be expressed as >100 mg/L. No difference was observed between the control and treatment.

pH, temperature ,oxygen, conductivity and hardness

The pH measurements show that the pH remained stable in the test in all concentrations. The temperature varied from 20.85 to 21.15 °C during the test. The oxygen concentration did not fall below 3 mg/L at any point during the test. Conductivity was measured in the dilution water as 638 µs/cm and total hardness was measured as ºdH of 12.2(measured in first definitive test), which is equivalent to 217.8 mg/L as CaCO3 (Ref 8). All measurements were in accordance with the required conditions described in the study plan and / or the guideline. See tables 1 & 2 for additional measurements. During the second definitive study the conductivity was not measured again but was taken from the corresponding log book for the DSW system. The conductivity was recorded as 612 µs/cm. The test water was therefore considered acceptable. Test medium was automatically made by a pumping system to a standard composition the hardness and conductivity were therefore not measured again fully during the second definitive test. The corresponding deviation from the study plan is reported under deviations.

Validity criteria fulfilled:
yes
Remarks:
Immobilization in the control did not exceed 10%; O2 concentration did not fall below 3 mg/L at any point during the study; No daphnids were trapped on the surface for any length of time.
Conclusions:
Under the conditions of the test An EL50 (48h) could not be reached and may therefore be expressed as >100 mg/L.
Executive summary:

In order to predict the effects of Bis (2,4-dichlorobenzoyl) peroxide 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 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 by loading of a large excess of the test chemical and by refreshment of the test solution after 24 hours.

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: It was 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 during preparation.

Description of key information

The results of an OECD 202 test indicate that under the conditions of the test the EL50 (48h) could not be reached and may only be expressed as > 100 mg/L. 

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

One OECD 202 GLP study is available.

In order to predict the effects of Bis (2,4-dichlorobenzoyl) peroxide 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 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 by loading of a large excess of the test chemical and by refreshment of the test solution after 24 hours.

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: It was 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 during preparation.