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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
no chemical analyses performed.
GLP compliance:
no
Remarks:
As the tests have not fulfilled all required endpoints and as no analytical chemistry was conducted during the tests, the study was considered not fit for regulatory submission, therefore the study is reported with no claim of compliance with GLP.
Specific details on test material used for the study:
Identification: [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide CAS 2212-81-9
Batch: 1403454131
Purity: 91.1%
Physical State/Appearance: White flakes
Expiry Date: 01 January 2024
Storage Conditions: Room temperature in the dark
Analytical monitoring:
no
Details on sampling:
Duplicate water samples were taken from the control and 100% v/v saturated solution test group for quantitative analysis. Samples of the fresh test preparations were taken from the bulk test preparations on Days 0, 6, 13 and 20 and of the expired test preparations (replicates pooled) on Days 1, 7, 14 and 21. All samples were stored frozen for analysis at the end of the test; however, in error the samples were disposed of before any analysis could be conducted and therefore no measured concentrations have been determined.
Vehicle:
no
Details on test solutions:
Information provided by the Sponsor indicated that the test item had a low water solubility and high log Kow. The quoted water solubility for the test item was 0.04 mg/L.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (OECD 2019) and therefore the test solutions were prepared using a slow-stir saturated solution method of preparation.

A nominal amount of test item (110 mg) was added to the surface of 11 liters of test water in duplicate and stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour before the aqueous was removed by siphoning to produce the 100% v/v saturated solution.

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnia were maintained in 150 mL glass vessels containing Elendt M7 medium (see Annex 2) in a temperature controlled room at approximately 20 C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and tetramin fish food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
242 to 294 mg/L as CaCO3
Test temperature:
19 to 22 °C
pH:
8.6 to 9.7
Dissolved oxygen:
≥8.6 mg O2/L
Nominal and measured concentrations:
Based on information provided by the Sponsor, a "limit test" at a concentration of 100% v/v saturated solution was selected for the definitive test.
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass vessels which were then covered with a plastic lid to reduce evaporation
- Type (delete if not applicable): open
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M7
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h
- Light intensity: 624 - 1183 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Parental mortality, -length, number of offspring

TEST CONCENTRATIONS
- Spacing factor for test concentrations: N.A.: Limit test
- Justification for using less concentrations than requested by guideline: Limit test
Reference substance (positive control):
not specified
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
no. of offspring per parent alive at the end of the test
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
parent
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: parental body length
Details on results:
On Days 16, 18, 19, 20 and 21 of the definitive test, no record of the adult condition, number of neonates produced or condition of neonates was recorded in replicate 10 of the 100% v/v saturated solution test group. Inspection of the data suggests neonates would have been produced on one of the days with missing data, therefore for the purpose of analysis of reproduction, data from this replicate was excluded.

Parent mortality:
Mortalities (immobilization) of the parental generation Daphnia were recorded in both the control and 100% v/v saturated solution test groups resulting in 20% mortality in the control and 10% mortality at the 100% v/v saturated solution by the end of the test. However, statistical analysis of the mortality data using the Student’s t- test showed that the observed mortalities in the 100% v/v saturated solution test group were not significantly different (P≥0.05) when compared to the control group.

Parental length:
After 21 days the length of each surviving adult was determined. A reduction in body length in the 100% v/v saturated solution test group of 3.6% was observed when compared to the control. Statistical analysis of this data showed a statistically significant effect; however, as the effect was less than a 10% reduction this result was considered not to be biologically significant.

Reproduction:
A reduction in neonate numbers produced in the 100% v/v saturated solution test group of 3.4% was observed when compared to the control. Analysis of the data obtained on Day 21 showed that the numbers of live young produced per adult by the control group were not significantly different (P≥0.05) from the 100% v/v saturated solution test group.

This study showed that there were no toxic effects at saturation.
Reported statistics and error estimates:
For the determination of the "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) the number of parental generation Daphnia mortalities for the control and each test group were estimated by inspection of the mortality data. For the estimation of the LOEC and the NOEC the numbers of live young produced per adult over the duration of the test (excluding any parents which died accidentally or inadvertently) for the control and each test group were compared using the Student-t test incorporating Shapiro Wilk's test on normal distribution and Levene’s test on variance homogeneity. Results from the control and each test group’s Daphnia length data, determined for the surviving daphnids on termination of the test, were compared using Student-t test incorporating Shapiro Wilk's test on normal distribution and Levene’s test on variance homogeneity. All statistical analyses with the exception of the values for parental generation mortalities were performed using the ToxRatPro software package (TOXRAT).
Validity criteria fulfilled:
yes
Remarks:
only chemical analyses were not performed, otherwise all validity criteria were met.
Conclusions:
Even though no chemical analyses were performed on the test concentrations, this study can be used as supporting evidence that the test substance does not cause a long-term effect on aquatic invertebrates.
Executive summary:

Introduction:

A study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, "Daphniamagna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008

Method:

Information provided by the Sponsor indicated that the test item had a low water solubility and high log Kow. The quoted water solubility for the test item was 0.04 mg/L, therefore the test solutions were prepared using a slow-stir saturated solution method of preparation.

Based on information provided by the Sponsor, a "limit test" at a concentration of 100% v/v saturated solution was selected for the definitive test. Daphnia magna were exposed (10 replicates of a single daphnid per group) to a test solution of the test item at a nominal concentration of 100% v/v saturated solution for a period of 21 days at a temperature of 19 to 22 °C under semi‑static test conditions. The test solutions were renewed daily throughout the test. The test item solutions were prepared by stirring an excess (10 mg/L) of test item in test water using a magnetic stirrer at a rate sufficient to produce a dimple at the water surface for 23 hours. After the stirring period the preparation wasallowed to stand for 1 hour before the aqueous was removed avoiding any undissolved test itemto produce the 100% v/v saturated solution of the test item.

The numbers of live and dead adult daphnia and young daphnids (live and dead) were determined daily. The daphnia were fed daily with a mixture of algal suspension andtetraminfish food suspension.

Results:

Exposure of Daphnia magna to the test item gave the following results based on the loading rates:

No effects on parent mortality, parental body length and number of offsrping: NOEC ≥ 10 mg/L loading or 100% saturated solution.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
In the WAF solutions iron precipitation was observed. Therefore, Iron was re-dosed to the test solutions before adding them to the test vessels. Extractions of the samples were made to recover the parent material from algae added to the test as food.
GLP compliance:
no
Remarks:
Performed in a lab that works according to standard operating procedures and own quality system.
Specific details on test material used for the study:
Batch/Lot Tested: 1403454131
Stability: Sample stable in refrigerator and freezer. Strongly adsorbing under test conditions
Solubility: 20-40 ug/L
Safe Storage: Material was stored in fridge or freezer in sealed container despite having good stability at room temperature.
Analytical monitoring:
yes
Details on sampling:
New and old samples (24h&72h) were taken throughout the study. Test material was extracted directly from the sample vessels to minimise test material loss. Pipetting of the test material was also avoided where possible and if needed was only conducted using primed pipettes previously rinsed with the relevant test solution.
Vehicle:
no
Details on test solutions:
Preparation of the stock solutions:
Due to the very poor solubility of the test material and the desire to avoid high energy preparation techniques and the use of solvents a traditional stock solution was not made. Solvents can preferentially dissolve other components of the test material such as impurities creating effects not relevant when the test material is dissolved in water.

Preparation of the test solutions:
3 x 10L (weeks 1&2) and 3 x 2L (week 3) Duran flasks equipped with a Teflon drain taps were filled with the test medium, using volumetric flasks. A control replicate without test material was prepared as well as 1 mg/L and a 10 mg/L water accommodated fractions, achieved by direct weighing of the test material with an analytical balance. The solutions were allowed to stir slowly but continually for at least 72 hours before the first test solution was removed. 1 hour prior to the removal of the test solution the
stirring was stopped to minimize the chance of undissolved test material transfer. At each solution replenishment 500 mL of test solution was removed from the WAF. To this the appropriate amount of the standard vitamin component was added and the iron component was re-dosed. The test solution was then used directly and was transferred to the test vessels. At this point the new test solution samples were also taken for chemical analysis.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test animals were taken from a Daphnia magna clone 5 stock, (Origin: Charles River Laboratories, The Netherlands) cultured in conformity with the relevant SOP. The parent animals were cultured in test medium from the day they were born. The animals used in the test were less than 24 hours old and were obtained from parent animals reproducing parthenogenically and having an age of 2-4 weeks (having previously produced at least one brood before use). The culture is checked half-yearly for sensitivity by a reference test with an appropriate reference toxicant. The most recent reference test results were within the range given in the test guideline. Furthermore there was no abnormal mortality or winter egg formation. The culture was therefore deemed suitable for use.

Feeding:
Test animals were fed a diet of 0.1 to 0.2 mg carbon per daphnid per week day (in 50 mL) or 0.2 to 0.4 mg (in 100 mL) (Friday to Sunday) during weekends, in the form of the algal strain Chlorella vulgaris.
The strain is cultured in the Environmental Chemistry laboratory and total organic carbon content has previously been measured. Algae was added prior to test solution to promote thorough mixing and less association with glass surfaces.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Test temperature:
20.4 - 21.7 °C
pH:
7.7 - 8.6
Dissolved oxygen:
8.0 - 9.1 mg O2/L
Nominal and measured concentrations:
The following nominal test concentrations were prepared: 1 mg/L & 10 mg/L including a control not containing the test material.
Time weighted mean measured concentrations: 21.6 ug/L in the 1mg/L replicate and 60.3 ug/L in the 10 mg/L replicate.
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL (nominal) glass beakers were used during weekdays, containing approximately 50 mL of test solution and covered by glass plates during the test. For the weekend 100 mL beakers were used containing approximately 100 mL of test medium so as to ensure sufficient food for the weekend period.
- Type: open
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): 5 times per week (not during weekends)
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h light, provided by “day light” fluorescent tubes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : reproduction, parental lenght, -weight and -mortality.
Reference substance (positive control):
yes
Remarks:
no further details
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate
Basis for effect:
other: parental length
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate
Basis for effect:
other: parental weight
Details on results:
Reproduction:
No significant effects on reproduction were detected in the highest tested concentration.
Due to the mean measured exposure (60.3 ug/L) exceeding the reported solubility limit of the test substance, the test substance should be considered to have no chronic effects on Daphnia reproduction up to its solubility limit.

Length:
No effects on length were detected in the highest tested concentration.

Weight:
Three were not sufficient replicates to allow a statistical analysis of the dry weight endpoint. However there was clear overlap between the dry weight of the animals at the highest concentration and in the controls. Indicating, the same conclusion as drawn for the reproduction and length endpoints.

Parental mortality:
An EC50 based on survival of parent animals could not be determined as no significant adult mortality occurred in the highest test concentration. The EC50 for adult mortality can therefore only be expressed as > 10 mg/L based on nominal concentrations or EC50 > the solubility limit based on time weighted mean measurements.
Results with reference substance (positive control):
The culture is checked half-yearly for sensitivity by a reference test with an appropriate reference toxicant. The most recent reference test results were within the range given in the test guideline.

It is not uncommon for the first brood if laid early (before 8 days) to be week and/or non-mobile. This was also observed during this study for the first juveniles born on days 6/7. These juveniles were present at

all concentrations including the control and were excluded from statistical analysis and scored as dead/immobile.

Validity criteria fulfilled:
yes
Conclusions:
The study met the biological and analytical validity criteria for a valid test and is expected to be a valid representation of the effects of the test material up to its solubility limit. Analysis demonstrated the level of the exposure and could be recovered from the test replicates via extraction.
The adsorbing properties of the test material are inherent to it and cannot be avoided and resulted in variation in the old solution determinations. However the material did remain in the test system and was available to the test organism via grazing of the algae. This is considered a relevant route of expose for
this material should it enter the environment. However in reality the test material is unlikely to be able to pass the water treatment plant.
Executive summary:

The purpose of this study was to assess the toxicity of the test substance loaded by water accommodated fractions to the test medium and the subsequent testing of the dissolved fresh water fractions, on the reproductive efficacy of Daphnia magna STRAUS - clone 5, in a semi-static test complying with the OECD Guideline No. 211 (OECD, 2012).

The primary test criterion used to indicate the toxicity of the test substance was reproductive capacity expressed as the total number of neonates per surviving parent animal at the end of the study due to there being no test substance related parent mortality. Data on parental length and dry-weight was also generated for use in determining secondary endpoints as required.

The nominal concentrations used in the study were as follows:

1 and 10 mg/L water accommodated fractions (WAF) including a test medium only control was tested.

All concentrations given refer to the technical product as supplied by the sponsor.

The following quality criteria were met:

- Cultures were in good health (i.e. disease free, no ephippia or males, no discolored animals valid reference test).

- No animals died in the control group over the test period.

- The average number ofjuveniles per parent animal alive at the end of the test in the control was 188 after 21 days (minimum acceptable = 60)

Reproduction (Primary endpoint)

A NOEC for reproduction and length for the substance as time weighted average test material was determined as the limit of solubility of the test material (63 µg/L), this is equal to a NOELR of 10 mg/L.

The test substance may therefore be concluded as non-toxic to the reproduction of Daphnia Magna up to its solubility limit. Hence no chronic effects to daphnia would be expected. With additional analysis it was demonstrated that sufficient test material remained in the test system and was available to the test organisms via consumption of the algae.

Description of key information

The analgous test substance CAS 25155-25-3 [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide did not have any effects to daphnids at its maximum achievable solubility in test medium (generated from a 10 mg/L water accommodated fraction).

This is confirmed by a non-GLP study according to OECD guideline 211 with the registered substance (CAS 2212 -81 -9). A NOEC for reproduction and length, for the substance as time weighted average test material was determined as the limit of solubility of the test material, this is equal to a NOELR of 10 mg/L.

Another non-GLP study according to OECD guideline 211 with the registered substance (CAS 2212 -81 -9) without chemical analyses of the test concentrations confirms this result as well.

No effects were found at the water solubility limit in any of the studies. The test substance may therefore be concluded as non-toxic to the reproduction of Daphnia Magna up to its solubility limit. Hence no chronic effects to daphnia would be expected.

Key value for chemical safety assessment

Additional information

The long-term toxicity to Daphnia magna of analogous CAS 25155 -25 -3 [1,3(or 1,4)-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide was evaluated. The study was performed in accordance with OECD testing guideline 211 and GLP requirements.

The test substance at its maximum achievable solubility in test medium (generated from a 10 mg/L water accommodated fraction) did not have any detectable effects on the reproduction or length of Daphnia magna in a 21 day chronic study under semi static conditions with daily refreshment. The NOELR (No observed effect loading rate) can therefore be expressed as 10 mg/L. 

This is confirmed by a non-GLP study according to OECD guideline 211 with the registered substance (CAS 2212 -81 -9).

The test substance was loaded by water accommodated fractions to the test medium and subsequently the effect of the dissolved fresh water fractions, on the reproductive efficacy of Daphnia magna STRAUS - clone 5, in a semi-static setup was tested.

The primary test criterion used to indicate the toxicity of the test substance was reproductive capacity expressed as the total number of neonates per surviving parent animal at the end of the study due to there being no test substance related parent mortality. Data on parental length and dry-weight was also generated for use in determining secondary endpoints as required.

The nominal concentrations used in the study were as follows:

1 and 10 mg/L water accommodated fractions (WAF) including a test medium only control was tested.

All concentrations given refer to the technical product as supplied by the sponsor.

A NOEC for reproduction and length for the substance as time weighted average test material was determined as the limit of solubility of the test material (63 µg/L), this is equal to a NOELR of 10 mg/L.

The test substance may therefore be concluded as non-toxic to the reproduction of Daphnia Magna up to its solubility limit. Hence no chronic effects to daphnia would be expected. With additional analysis it was demonstrated that sufficient test material remained in the test system and was available to the test organisms via consumption of the algae.

A second non-GLP study confirms these results as well. A "limit test" at a concentration of 100% v/v saturated solution was selected for the definitive test. Daphnia magna were exposed (10 replicates of a single daphnid per group) to a test solution of the test item at a nominal concentration of 100% v/v saturated solution for a period of 21 days at a temperature of 19 to 22 °C under semi‑static test conditions. The test solutions were renewed daily throughout the test. The test item solutions were prepared by stirring an excess (10 mg/L) of test item in test water using a magnetic stirrer at a rate sufficient to produce a dimple at the water surface for 23 hours. After the stirring period the preparation was allowed to stand for 1 hour before the aqueous was removed avoiding any undissolved test item to produce the 100% v/v saturated solution of the test item.

The numbers of live and dead adult daphnia and young daphnids (live and dead) were determined daily. The daphnia were fed daily with a mixture of algal suspension andtetraminfish food suspension.

No effects were seen on any of the effect parameters measured and the NOELR is therefore equal to 10 mg/L or equal to the water solubiltiy limit of the test substance.