1-Butanone, 3-(dodecylthio)-1-[(1R,2S)-2,6,6-trimethyl-3-cyclohexen-1-yl]-, rel-

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

Run on 2013-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification

Remarks:

(Q)SAR method with established scientific validity. The substance falls within the applicability domain of the model.

Justification for type of information:

1. SOFTWARE
ECOSAR

2. MODEL (incl. version number)
ECOSAR v1.11

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
C/C=C/C(C1C(C)C=CCC1(C)C)=O
log Kow : 3.6 (estimated result based on differents QSARs)

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See "Any other information on results incl. tables"

5. APPLICABILITY DOMAIN
See "Any other information on results incl. tables"

6. ADEQUACY OF THE RESULT
See "Any other information on results incl. tables"

Qualifier:

according to guideline

Guideline:

other: REACH guidance on QSARs R.6, May 2008

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Remarks:

(not relevant)

Test organisms (species):

other: no data

Test type:

not specified

Water media type:

not specified

Total exposure duration:

48 h

Remarks on exposure duration:

none

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

3.802 mg/L

Remarks on result:

other: ECOSAR Class: Neutral Organics SAR ( Baseline toxicity)

Validity of model:

1. Defined Endpoint: short term toxicity to aquatic invertebrates (Daphnid).

2. Unambigous algorithm: The ECOSAR class determined was Vinyl/Allyl Ketones. However, based on observed effects for daphnids, this chemical class is not expressing excess toxicity compared to neutral organics. The class-specific SAR equation is yielding lower toxicity values than estimated from baseline toxicity (neutral organics), therefore the neutral organics SAR is used to estimate the toxicity of the substance for daphnids:

Linear regression QSAR; Log 48h-LC50 (mmol/L) = -0.858*log Kow + 1.3848.

To convert the LC50 from mmol/L to mg/L, multiply by the molecular weight.

3. Applicability domain: applicable to chemicals with log Kow less than 5.0 and molecular weight less than 1000 g/mol. The model can be suitable for chemicals class of neutral organic.

4. Statistical characteristics: N = 98 + 31 and the coefficient of determination R² = 0.7704.

5. Mechanistic interpretation: related to the partitioning of the substance from water into the organisms lipid phase.

Adequacy of prediction: the substance falls within the applicability domain described above and therefore the predicted value can be considered reliable.

Validity criteria fulfilled:

yes

Conclusions:

delta-Damascone was predicted to have an 48h-LC50 of 3.802 mg/L to daphnid after 48h of exposure. The substance falls within the applicability domain and therefore the predicted value can be considered reliable.

Executive summary:

The short term toxicity to daphnid of delta-Damascone was estimated using the ECOSAR v1.11 QSAR model available from the U.S. EPA.

The estimation 48h-LC50 was 3.802 mg/L (neutral organics class), calculated from an estimated log Kow value at 3.6 (mean of QSAR results) and a linear regression:

Log 48h-LC50 (mmol/L) = -0.858*log Kow + 1.3848

The substance falls within the applicability domain and therefore the predicted value can be considered reliable.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

Run on 2013-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification

Remarks:

Q)SAR method with established scientific validity. This substance is not related to an existing ECOSAR class. Estimation provided use the Neutral Organics QSAR equations which represent baseline toxicity potential (minimum toxicity) assuming a simple non-polar narcosis model. Without empirical data on structurally similar chemicals, it is uncertain if this substance will present significantly higher toxicity above baseline estimates. The substance falls within the applicability domain of the model.

Justification for type of information:

1. SOFTWARE
ECOSAR

2. MODEL (incl. version number)
ECOSAR v1.11

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC1(C)CC=CC(C)C1C(CC(SCC(O)=O)C)=O
log Kow : 3.1 (estimated result based on differents QSARs)

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See "Any other information on results incl. tables"

5. APPLICABILITY DOMAIN
See "Any other information on results incl. tables"

6. ADEQUACY OF THE RESULT
See "Any other information on results incl. tables"

Qualifier:

according to guideline

Guideline:

other: REACH guidance on QSARs R.6, May 2008

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Remarks:

(not relevant)

Test organisms (species):

other: no data

Test type:

not specified

Water media type:

not specified

Total exposure duration:

48 h

Remarks on exposure duration:

none

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

150.992 mg/L

Remarks on result:

other: > limit of solubility. ECOSAR Class: Neutral Organics-acid (acid moeity found: predicted values multiplied by 10).

Validity of model:

1. Defined Endpoint: short term toxicity to aquatic invertebrates (Daphnid).

2. Unambigous algorithm: This substance is not related to an existing ECOSAR class. Estimation provided use the Neutral Organics QSAR equations which represent baseline toxicity potential (minimum toxicity) assuming a simple non-polar narcosis model. Without empirical data on structurally similar chemicals, it is uncertain if this substance will present significantly higher toxicity above baseline estimates. Acid moeity was found, therefore predicted values were multiplied by 10:

Linear regression QSAR; Log 48h-LC50 (mmol/L) = -0.858*log Kow + 1.3848.

To convert the LC50 from mmol/L to mg/L, multiply by the molecular weight.

3. Applicability domain: applicable to chemicals with log Kow less than 5.0 and molecular weight less than 1000 g/mol. The model can be suitable for chemicals class of neutral organic.

4. Statistical characteristics: N = 98 + 31 and the coefficient of determination R² = 0.7704.

5. Mechanistic interpretation: related to the partitioning of the substance from water into the organisms lipid phase.

Adequacy of prediction: the substance falls within the applicability domain described above and therefore the predicted value can be considered reliable.

Validity criteria fulfilled:

yes

Conclusions:

delta-Damascone mercapto acetic acid was predicted to have an 48h-LC50 > solubility limit, estimated at 84.53 mg/L to daphnid after 48h of exposure. The substance falls within the applicability domain and therefore the predicted value can be considered reliable.

Executive summary:

The short term toxicity to daphnid of delta-Damascone mercapto acetic acid was estimated using the ECOSAR v1.11 QSAR model available from the U.S. EPA. The estimation 48h-LC50 was greater than the solubility limit, estimated at 84.53 mg/L (neutral organics-acid), calculated from an estimated log Kow value at 3.1 (mean of QSAR results) and a linear regression (acidmoeity was found, therefore predicted values were multiplied by 10):

Log 48h-LC50 (mmol/L) = -0.858*log Kow + 1.3848

The substance falls within the applicability domain and therefore the predicted value can be considered reliable.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

Run on 2013-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification

Remarks:

Q)SAR method with established scientific validity. This substance is not related to an existing ECOSAR class. Estimation provided use the Neutral Organics QSAR equations which represent baseline toxicity potential (minimum toxicity) assuming a simple non-polar narcosis model. Without empirical data on structurally similar chemicals, it is uncertain if this substance will present significantly higher toxicity above baseline estimates. The substance falls within the applicability domain of the model.

Justification for type of information:

1. SOFTWARE
ECOSAR

2. MODEL (incl. version number)
ECOSAR v1.11

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC1(C)CC=CC(C)C1C(CC(SCCC(O)=O)C)=O
log Kow : 3.3 (estimated result based on differents QSARs)

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See "Any other information on results incl. tables"

5. APPLICABILITY DOMAIN
See "Any other information on results incl. tables"

6. ADEQUACY OF THE RESULT
See "Any other information on results incl. tables"

Qualifier:

according to guideline

Guideline:

other: REACH guidance on QSARs R.6, May 2008

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Remarks:

(not relevant)

Test organisms (species):

other: no data

Test type:

not specified

Water media type:

not specified

Total exposure duration:

48 h

Remarks on exposure duration:

none

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

106.724 mg/L

Remarks on result:

other: > limit of solubility. ECOSAR Class: Neutral Organics-acid (acid moeity found: predicted values multiplied by 10).

Validity of model:

1. Defined Endpoint: short term toxicity to aquatic invertebrates (Daphnid).

2. Unambigous algorithm: This substance is not related to an existing ECOSAR class. Estimation provided use the Neutral Organics QSAR equations which represent baseline toxicity potential (minimum toxicity) assuming a simple non-polar narcosis model. Without empirical data on structurally similar chemicals, it is uncertain if this substance will present significantly higher toxicity above baseline estimates. Acid moeity was found, therefore predicted values were multiplied by 10:

Linear regression QSAR; Log 48h-LC50 (mmol/L) = -0.858*log Kow + 1.3848.

To convert the LC50 from mmol/L to mg/L, multiply by the molecular weight.

3. Applicability domain: applicable to chemicals with log Kow less than 5.0 and molecular weight less than 1000 g/mol. The model can be suitable for chemicals class of neutral organic.

4. Statistical characteristics: N = 98 + 31 and the coefficient of determination R² = 0.7704.

5. Mechanistic interpretation: related to the partitioning of the substance from water into the organisms lipid phase.

Adequacy of prediction: the substance falls within the applicability domain described above and therefore the predicted value can be considered reliable.

Validity criteria fulfilled:

yes

Conclusions:

delta-Damascone mercapto propionic acid was predicted to have an 48h-LC50 > solubility limit, estimated at 47.31 mg/L to daphnid after 48h of exposure. The substance falls within the applicability domain and therefore the predicted value can be considered reliable.

Executive summary:

The short term toxicity to daphnid of delta-Damascone mercapto propionic acid was estimated using the ECOSAR v1.11 QSAR model available from the U.S. EPA. The estimation 48h-LC50 was greater than the solubility limit, estimated at 47.31 mg/L (neutral organics-acid), calculated from an estimated log Kow value at 3.3 (mean of QSAR results) and a linear regression (acid moeity was found, therefore predicted values were multiplied by 10):

Log 48h-LC50 (mmol/L) = -0.858*log Kow + 1.3848

The substance falls within the applicability domain and therefore the predicted value can be considered reliable.

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 09 January to 23 February 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study was performed according to OECD Guideline 202 and EU Method C.2 with GLP statement. Based on the very low water solubility of the test substance, it's technically not feasible to obtain a relevant endpoint value.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

30th August 2005

Specific details on test material used for the study:

- Storage condition of test material: Approximately 4 °C under nitrogen in the dark

Analytical monitoring:

yes

Details on sampling:

See details on analytical methods

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: the test material was prepared using a preliminary solution in dimethylformamide (DMF). Stock solution: 1mg/ml test material in DMF. An aliquot (500ul) of the stock solution was dispersed in reconstitued water and the volume adjusted to 5 liters to give the 0.1 mg/l test concentration. Each solvent stock solution was inverted several time to ensure adequate mixing and homogeneity. The concentration and stability of the test material in the centrifuged and untreated samples were verified by chemical anylysis at 0 and 48 hours (See "Background attached material" fore more details)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution): 0.01%
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: no data
- Source: derived from in-house laboratory cultures.
- Age at study initiation (mean and range, SD): less than 24 hours old
- Weight at study initiation (mean and range, SD): Not Applicable/Not required in OECD 202 Guideline
- Length at study initiation (length definition, mean, range and SD): Not Applicable/Not required in OECD 202 Guideline
- Valve height at study initiation, for shell deposition study (mean and range, SD): Not Applicable/Not required in OECD 202 Guideline
- Peripheral shell growth removed prior to test initiation: Not Applicable/Not required in OECD 202 Guideline
- Method of breeding: polypropylene vessels
- Feeding during test
- Food type: None
- Amount: None
- Frequency: Never


ACCLIMATION
- Acclimation period: Not Applicable/Not required in OECD 202 Guideline
- Type and amount of food: algae (Chlorella sp.)
- Feeding frequency: daily
- Health during acclimation (any mortality observed):

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

none

Post exposure observation period:

Not Applicable

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaC03.

Test temperature:

20 ± 1 °C (See Table 6.1.3/1 in "Any other information on material & Methods incl Tables")

pH:

See Table 6.1.3/1 in "Any other information on material & Methods incl Tables"
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCI. pH and dissolved oxygen were recorded at the start and termination of the test using a WTW pH/Oxi 3401 pH and dissolved oxygen meter. There were no treatment related differences for oxygen concentration or pH.

Dissolved oxygen:

See Table 6.1.3/1 in "Any other information on material & Methods incl Tables"
Reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
pH and dissolved oxygen were recorded at the start and termination of the test using a WTW pH/Oxi 3401 pH and dissolved oxygen meter. There were no treatment related differences for oxygen concentration or pH.

Salinity:

Not applicable

Nominal and measured concentrations:

See Table 6.1.3/2: Nominal and measured concentrations in "Any other information on Material & Methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass jars containing ca. 200 ml of test preparation
- Type (delete if not applicable): covered to reduce evaporation, not aerated
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): no flow-through system used, static system
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: Not Applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water. (Stock solutions: a.) CaCl2.2H2O 11.76 g/l, b.) MgS04.7H2O 4.93 g/l, c.) NaHC03 2.59 g/ld.) KCI 0.23 g/l. Preparation: An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS/cm. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCI and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
- Total organic carbon: Not Applicable (for natural water only)
- Particulate matter: Not Applicable (for natural water only)
- Metals: Not Applicable (for natural water only)
- Pesticides: Not Applicable (for natural water only)
- Chlorine: Not Applicable (for dechlorinated tap water only)
- Alkalinity: Not Applicable
- Ca/mg ratio: Not Applicable/Not required in OECD Guideline 202
- Conductivity: <5 µS/cm (normally required for natural water only)
- Culture medium different from test medium: no
- Intervals of water quality measurement: Not Applicable (for natural water only)

OTHER TEST CONDITIONS
- Adjustment of pH: Tests carried out without adjustment of pH
- Photoperiod: 16 hours light and 8 hours darkness with 20 min dawn and dusk transition periods
- Light intensity: No data/ Not required in OECD Guideline 202

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (one concentration tested: 0.1 mg/l)
- Justification for using less concentrations than requested by guideline: insolubility of the test material (highest solubility: 0.1 mg/l in the test conditions)
- Range finding study
- Test concentrations: 0.010, 0.10 mg/l
- Results used to determine the conditions for the definitive study: yes (No immobilisation was observed at the test concentrations of 0.010, 0.10 mg/l. )

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

(immobilisation)

Remarks on result:

other: 95% CL: -

Duration:

24 h

Dose descriptor:

other: Maximum concentration tested giving no observed effect

Effect conc.:

100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

(immobilisation)

Remarks on result:

other: 95% CL: -

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

(immobilisation)

Remarks on result:

other: 95% CL: -

Duration:

48 h

Dose descriptor:

other: Maximum concentration tested giving no observed effect

Effect conc.:

100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

(immobilisation)

Remarks on result:

other: 95% CL: -

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 72 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

(Immobilisation)

Remarks on result:

other: 95% CL: -

Duration:

24 h

Dose descriptor:

other: Maximum concentration tested giving no observed effect

Effect conc.:

72 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

(immobilisation)

Remarks on result:

other: 95% CL: -

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 72 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

(immobilization)

Remarks on result:

other: 95% CL: -

Duration:

48 h

Dose descriptor:

other: Maximum concentration tested giving no observed effect

Effect conc.:

72 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

(immobilisation)

Remarks on result:

other: 95% CL: -

Details on results:

- Behavioural abnormalities: NONE
- Observations on body length and weight: Not Applicable
- Other biological observations: NONE
- Mortality of control: Not Applicable
- Other adverse effects control: NONE
- Abnormal responses: NONE
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: NONE
- Effect concentrations exceeding solubility of substance in test medium: Not Applicable

Results with reference substance (positive control):

- Results with reference substance valid? YES
- Mortality: no immobilisation < 1.0 mg/l (control, 0.32, 0.56 mg/l). Immobilization observed from 1 mg/l (1.0, 1.8 & 3.2 mg/l)
- EC50/LC50: 48-hours LC50= 0.97 mg/l with 95% confidence limits of 0.85 - 1.1 mg/l.

Reported statistics and error estimates:

The EC50 value and associated confidence limits at 24 hours and the slope of the response curve and its standard error were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).
The EC50 value and associated confidence limits at 48 hours were calculated using the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used where two or more partial responses to exposure are shown.
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

Immobilization data

There was no immobilization in 20 daphnids exposed to a test concentration of 0.10 mg/l for a period of 48 hours. Inspection of the immobilization data gave the following results:

 

Time (hours)	EC50 (mg/l)	95% Confidence limit (mg/l)
24 48	> 0.10 > 0.10	- -

 

The maximum concentration tested giving no observed effect after 24 and 48 hours exposure was 0.10 mg/L. The maximum concentration tested giving no observed effect is based upon zero immobilization at this concentration.

The test concentration of 0.10 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guidelines. Other various recognized auxiliary solvents were used during preliminary solubility work, however, DMF was found to give the best testable dispersion of the test material in water. At higher test concentrations there was a marked precipitation of the test material on addition of the solvent stock solution to water.

 

Verification of test concentrations

(For more details, see Verification of Concentration in “attached background material”)

 

A study to determine the generalphysico-chemical properties of the test material (SafepharmLaboratories Project Number 161/439) determined the water solubility of the test material to be 0.090 mg/l using the shake flask method. Preliminary solubility work showed that the highest attainable test concentration (by visual inspection of the test media) that could be prepared was 0.10 mg/l using a preliminary solution in DMF.

Analysis of the untreated samples at 0 hours showed the test concentrations to range from 118% to 129% of nominal and analysis of the centrifuged samples at 0 hours showed the test concentrations to range from 87% to 91% of nominal value.

One of the untreated sample results at 0 hours was higher than the allowable limits of 80% to 120% of nominal concentration. This was considered to be due to analytical variation and considered not to have affected the outcome of the test as all other untreated sample results throughout the duration of the test were within the allowable limits indicating the test system was dosed correctly.

The results of the untreated test samples were similar to those obtained from the media preparation trials conducted (110% of nominal). The results of the centrifuged test samples were significantly higher than those obtained from the media preparation trials conducted (55% of nominal after centrifugation at 40000 g for 30 minutes). It was considered unnecessary to stir the solvent spike preparation for a prolonged period in order to maximize the dissolved test material concentration as the preliminary media preparation trials conducted indicated that overall a decline in measured concentration was seen with increased preparation time.

Analysis of the untreated samples at 48 hours showed test concentrations to range from 99% to 100% of nominal value. Analysis of the centrifuged samples at 48 hours showed test concentrations to range from 57% to 58% of nominal value.

The results obtained from the centrifuged test samples show a decline in measured test concentration over the test period and wasinlinewith the results obtained from the media preparation trials conducted.

Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations of the centrifuged test media in order to give a "worst case" analysis of the data. The 48-Hour EC50 based on the time-weighted mean measured test concentrations of the centrifuged test media was greater than 0.072 mg/L and the maximum concentration tested giving no observed effect was 0.072 mg/L.

 

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than 0.10 mg/L.
Based on the time-weighted mean measured test concentrations of the centrifuged test media the 48-Hour EC50 was estimated to be greater than 0.072 mg/L and the maximum concentration tested giving no observed effect was 0.072 mg/L.

Executive summary:

Introduction.A study was performed to assess the acute toxicity of the test material toDaphnia magna.The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202,"Daphnia sp, Acute Immobilization Test" referenced as Method C.2 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

 

Methods.A Determination of the General Physico-chemical Properties study conducted on the test material showed the water solubility value of the test material to be 0.090 mg/l using the shake flask method of preparation.

 

Preliminary solubility work showed that the highest attainable test concentration (by visual inspection of the test media) was 0.10 mg/l using a preliminary solution prepared in dimethylformamide (DMF). Media preparation trials were conducted in order to determine the amount of dissolved test material present and the most appropriate method of test media preparation. The results of these trials indicated that using a solvent spike method of preparation was the most suitable for this test material and that after centrifugation at 40000gfor 30 minutes a dissolved concentration of approximately 0.055 mg/l was obtained. Given the range of tests to be conducted on this material in order to maintain consistency it was considered inappropriate to centrifuge the test preparations prior to exposure. Instead samples were taken for analysis following centrifugation, the results of which gave an indication of the dissolved and hencebioavailabletest concentration.

 

Following a preliminary range-finding test, twentydaphnids(4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a concentration of 0.10 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

A positive control conducted approximately every six months used potassium dichromate as the reference material.Daphnia magnawas exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

 

Results.The 48-Hour EC50 for the test material to Daphnia magna based on nominal test concentrations was greater than 0.10 mg/L. The test concentration of 0.10 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent, and having due regard to the amount of auxiliary solvent permitted in the test under the OECD Guidelines.

 

Analysis of the untreated samples at 0 hours showed the test concentrations to range from 118% to 129% of nominal and analysis of the centrifuged samples at 0 hours showed the test concentrations to range from 87% to 91% of nominal value.

 

One of the untreated sample results at 0 hours was slightly higher than the allowable limits of 80% to 120% of nominal concentration. This was considered to be due to analytical variation and considered not to have affected the outcome of the test as all other untreated sample results throughout the duration of the test were within the allowable limits indicating the test system was dosed correctly.

 

The results of the untreated test samples were similar to those obtained from the media preparation trials conducted (110% of nominal). The results of the centrifuged test samples were significantly higher than those obtained from the media preparation trials conducted (55% of nominal after centrifugation at 40000gfor 30 minutes).Itwas considered unnecessary to stir the solvent spike preparation for a prolonged period in order to maximize the dissolved test material concentration as the preliminary media preparation trials indicated that overall a decline in measured concentration was seen with increased preparation time.

 

Analysis of the untreated samples at 48 hours showed test concentrations to range from 99% to 100% of nominal value. Analysis of the centrifuged samples at 48 hours showed test concentrations to range from 57% to 58% of nominal value.

 

The results obtained from the centrifuged test samples showed a decline in measured test concentration over the test period and wasinlinewith the results obtained from the media preparation trials conducted.

 

Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations of the centrifuged test media in order to give a "worst case" analysis of the data. The 48-Hour EC50 based on the time-weighted mean measured test concentrations of the centrifuged test media was greater than 0.072 mg/l and the maximum concentration tested giving no observed effect was 0.072 mg/l.

 

The 48-Hour EC50 for the reference material to Daphnia magna based on nominal concentrations was 0.97 mg/l with 95% confidence limits of 0.85 - 1.1 mg/l.

Reference 5

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 08 August 2012 to 06 November 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study was performed according to international guidelines without GLP statement. The lack of consistency between the replicats and between the concentration levels leads to conclude that aqueous studies on this substance will not be reproducible and therefore are not considered technically possible.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidance document No. 23

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Schäfers et al., 2009. Environmental properties of long-chain alcohols, part 2: Structure-activity relationship for chronic aquatic toxicity of long-chain alcohols. Ecotoxicology and Environmental Safety, 72 (4), pp 996-1005.

Deviations:

not specified

Principles of method if other than guideline:

The primary objective was to determine the method of preparation for a water accommodated fraction (WAF) of the test media suitable for executing a Daphnia magna reproduction test according to OECD211. Two different WAF preparation approaches were tested. The different WAF preparations obtained were applied in two Daphnia magna acute immobilisation tests carried out under experimental conditions similar to the conditions applied for chronic tests on Daphnia magna. Therefore, a concentrated algal suspension was added to the test vessels to feed the experimental animals. Daphnia magna were exposed to the different WAF preparations with loadings of 100, 10 and 1 mg/L under static conditions for 48 hours. The number of immobile daphnids was determined after 24 h and 48 h. The tests were performed according to the OECD guideline 202 and the EC method C2. The results provide important information for the selection of a correct media preparation regime that controls the exposure concentration. Such testing is a recommendation of the OECD guidance document on aquatic toxicity testing of difficult substances and mixtures, number 23.

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

Samples of the fresh and aged solutions for each preparation were analysed.

Vehicle:

no

Details on test solutions:

The test solutions were prepared by two approaches (WAF prepared by direct suspension or bottle-coating method) under two sets of conditions (46 h stirring/2 h sedimentation or 24 h stirring/24 h sedimentation). Each loading (100, 10 and 1 mg test substance/L) was prepared individually under sterile conditions and used within 1-2 hours of preparation.

WAF PREPARED BY DIRECT SUSPENSION:
Solutions for each loading were prepared by weighing 200, 20, and 2 mg of the test item onto Teflon plates which were then placed into sterile 2 L bottles with 2 L of dilution water. This was performed under sterile conditions. The solutions were then stirred at 300 rpm at room temperature (21 ± 2°C) for:
a) 46 h followed by a 2 h sedimentation period to separate the insoluble parts of the solution from the aqueous phase before use
b) 24 h followed by a 24 h sedimentation period to separate the insoluble parts of the solution from the aqueous phase before use

WAF PREPARED BY BOTTLE-COATING METHOD:
A solution (noted 1) was prepared by dissolving 1000 mg of the test item in 5 mL of acetone solvent (used for the 100 mg/L loading). For the 10 mg/L loading (noted 2), 500 µL of solution 1 was diluted with 4.5 mL of acetone. The 1 mg/L loading (noted 3) was prepared by diluting 500 µL of solution 2 with 4.5 mL acetone. A 1 mL aliquot of the appropriate acetone solution was pipetted into a sterile 2-L WAF bottle that had been warmed to 60°C. The bottle was slowly turned to cover a maximum area of the glass walls with the acetone solution and continued until the solvent was no longer visible. Two litres of dilution water were added to each vessel and the solutions stirred at 300 rpm at room temperature (21 ± 2°C) for:
a) 46 h followed by a 2 h sedimentation period to separate the insoluble parts of the solution from the aqueous phase before use
b) 24 h followed by a 24 h sedimentation period to separate the insoluble parts of the solution from the aqueous phase before use

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: no data
- Source: German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene (Berlin).
- Age at study initiation (mean and range, SD): newborn Daphnia between 4 and 24h old.
- Weight at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): no data
- Valve height at study initiation, for shell deposition study (mean and range, SD): no data
- Peripheral shell growth removed prior to test initiation: no data
- Method of breeding: no data
- Feeding during test: no

ACCLIMATION
To maintain the stock culture, batches of 30-50 adult daphnids were held at room temperature in ca. 1.8L of dilution water for one week. During this week, the daphnids were fed daily with an algal suspension (Desmodesmus subspicatus containing Artemio Fluid JBL). Algae growing in the log-phase were centrifuged and the pellet was re-suspended in a few mL of medium. Thirty millilitres of this suspension was given to 1L of medium. The water was changed once per week.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

none

Post exposure observation period:

None

Hardness:

1.1 mmol/L

Test temperature:

The temperature of the incubator was between 19.9ºC - 20.3ºC from test start to end for all tests.

pH:

The pH of all treatments ranged from 8.02 – 9.42.

Dissolved oxygen:

The oxygen saturation in all test vessels ranged between 6.4 – 8.2 mg O2/L.

Salinity:

Not applicable

Nominal and measured concentrations:

See tables 6.1.3/1 to 6.1.3/5 in "Any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass flasks stoppered with silicone/foam lids
- Aeration: the test vessels were not aerated during the test
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable

TEST MEDIUM / WATER PARAMETERS
Dechlorinated local tap water. The tap water is sourced from the Schmallenberg district water production plants, mostly fed by small springs and percolation. The purification process occurs on site at Fraunhofer IME and includes filtration with activated charcoal, passage through a lime-stone column, and aeration to the point of oxygen saturation. To avoid copper contamination, plastic water pipes are used in the test facilities.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h light/8h dark cycle
- Light intensity: 529-569 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The number of immobile daphnids in each treatment was visually determined daily and the immobile daphnids were removed. Any abnormalities in appearance and behaviour were also recorded. Daphnids were classed as immobile when they did not swim 15 seconds after gentle perturbation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: factor 10
- Range finding study: no

Reference substance (positive control):

no

Details on results:

See table 6.1.3/6 in "Any other information on results incl. tables".

The WAF preparation with the bottle coating method is capable of producing higher test item concentrations in water compared to that of the direct dissolving method. However, the bottle coating method was leading to the adherence of animals to the media surface leading to high mortality and thus preventing the determination of the specific effects defined in the guideline.
Based on the outcome from the preliminary investigations on WAF preparations (total lack of reproducibility within the various replicates and concentration levels) and with respect to the results of the acute immobilisation tests on Daphnia magna, it was concluded that it is not technically feasible to get the substance in solution and to obtain reliable concentrations and that as a consequence it is not possible to conduct a chronic Daphnia test and that aquatic toxicity tests with this substance are likely not to be technically feasible.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

No statistical evaluation of test organism immobility was performed

Chemical analysis of WAF solutions - 46 h stirring/ 2 h sedimentation:

Analysis of the fresh and aged direct suspension and acetone WAF loadings, stirred for 46 h followed by sedimentation for 2 h, was carried out. In the fresh loadings, only traces (0.49-0.81ng/mL) of the test substance found in the highest concentration tested (100 mg/L loading) after direct suspension of the test item. In contrast, the acetone WAF lead to decent concentrations (>50 ng/mL) for the highest treatment. The use of lower loadings (10 mg/L and 1 mg/L) was reflected in the measured concentrations. Noticeable was the higher amount of test substance in the samples with the lowest nominal loading (“1 acetone”) compared with the samples having a nominal loading of 10 mg/L (“10 acetone”). In addition to that a high deviation in the lowest treatment was observed indicating that the test item was not homogeneously dissolved. The control sample showed no contamination with the test item. (see table 6.1.3/1 below)

Table 6.1.3/1: Test item concentrations and recoveries of the WAF loadings without algae (fresh)

Conc.	Prep. method	Separated volume of extract	Complete volume of extract	Test water sample volume	Conc.  in LC-MS sample	Conc. in                   test water sample
[mg/L]	-	[mL]	[mL]	[mL]	[ng/mL]	[ng/mL]
100 #1	Direct	1.4	74	51	0.475	0.49
100 #2	Direct	1.4	72	49	0.768	0.81
10 #1	Direct	1.4	77	50	0	0.00
10 #2	Direct	1.4	70	49	0	0.00
1 #1	Direct	1.4	74	49	0	0.00
1#2	Direct	1.4	74	51	0	0.00
100 #1	Acetone	1.4	76	52	66.654	69.58
100 #2	Acetone	1.4	76	49	48.349	53.56
10 #1	Acetone	1.4	76	49	7.638	8.46
10 #2	Acetone	1.4	75	51	8.65	9.09
1 #1	Acetone	1.4	73	51	15.451	15.80
1#2	Acetone	1.4	76	46	52.609	62.09
Control #1	-	1.4	73	51	0	0.00
Control #2	-	1.4	78	45	0	0.00

For the aged suspensions, again, no test item was found in the water samples previously prepared by direct suspension. The concentration in the other samples (100-1 acetone) was generally decreasing. The concentrations measured reflected the differences observed for the fresh material. Again, the control samples showed no contamination with the test item (see table 6.1.3/2 below).

Table 6.1.3/2: Test item concentrations and recoveries of the WAF loadings with algae (aged)

Conc.	Prep. method	Separated volume of extract	Complete volume of extract	Test water sample volume	Conc.  in LC-MS sample	Conc. in                   test water sample
[mg/L]	-	[mL]	[mL]	[mL]	[ng/mL]	[ng/mL]
100	Direct	2.8	71	52	0	0.00
10	Direct	2.8	72	51	0	0.00
1	Direct	2.8	74	50	0	0.00
100	Acetone	2.8	74	50	32.574	17.22
10	Acetone	2.8	70	50	1.822	0.91
1	Acetone	2.8	72	49	7.45	3.91
Control	-	2.8	72	50	0	0.00

Chemical analysis of WAF solutions - 24 h stirring/ 24 h sedimentation:

Analysis of the freshly prepared direct suspension and acetone WAFs, stirred for 24 h followed by sedimentation for a further 24 h, was carried out. Gentle stirring of the WAF solution was stopped after 24 h followed by an extended “sedimentation period” lasting 24 h to improve the sample material. Fresh and aged (after 24 h incubation with algae) solutions were analysed. 

Generally, the quality of the results was increased by the optimised WAF preparation procedure. A lower deviation between the replicates was observed. With respect to the analysis of WAF samples prepared by direct suspension, the result of the first trial with shortened sedimentation time was mostly confirmed. However, lower concentrations were found for samples prepared by the acetone WAF procedure with around 30 ng/mL for 100 mg/L and 10 mg/L. The 100 times dilution of the WAF (1 mg/L) lead to a significantly lower concentration of around 1.5 ng/mL.

Aged samples with algae contained approximately a third of the concentration measured before for the fresh samples. However, the concentration measured in the treatment with the highest dilution was below LOD.

The preparation and analysis of the optimised WAFs was repeated to prove the repeatability of the results obtained. Slightly higher concentrations compared to the first run were measured in samples prepared by direct suspension. The acetone WAF lead to fairly comparable results for the two highest treatments, however, a higher deviation between the replicated measurements was observed. Concentrations found in the lowest treatment were 10 times higher compared to the solutions prepared with the shorter sedimentation time.

 

Table 6.1.3/3: Test item concentrations and recoveries of the optimised WAF loadings without algae (fresh)

Conc.	Prep. method	Separated volume of extract	Complete volume of extract	Test water sample volume	Conc.  in LC-MS sample	Conc. in                   test water sample
[mg/L]	-	[mL]	[mL]	[mL]	[ng/mL]	[ng/mL]
100 #1	Direct	5.0	72	50	1.491	0.43
100 #2	Direct	5.0	74	50	0	0.00
10 #1	Direct	5.0	76	50	0	0.00
10 #2	Direct	5.0	73	49	0	0.00
1 #1	Direct	5.0	76	50	0	0.00
1#2	Direct	5.0	78	50	0	0.00
100 #1	Acetone	5.0	73	49	57.075	24.29
100 #2	Acetone	5.0	76	51	68.095	28.99
10 #1	Acetone	5.0	75	49	109.075	33.39
10 #2	Acetone	5.0	75	51	123.047	36.19
1 #1	Acetone	5.0	78	50	4.774	1.49
1#2	Acetone	5.0	76	49	5.264	1.63
Control #1	-	5.0	79	51	0	0.00
Control #2	-	5.0	73	50	0	0.00

 

Table 6.1.3/4: Test item concentrations and recoveries of the optimised WAF loadings with algae (aged)

Conc.	Prep. method	Separated volume of extract	Complete volume of extract	Test water sample volume	Conc.  in LC-MS sample	Conc. in                   test water sample
[mg/L]	-	[mL]	[mL]	[mL]	[ng/mL]	[ng/mL]
100 #1	Direct	75.0	75	51	14.461	0.28
100 #2	Direct	75.0	75	51	11.146	0.22
10 #1	Direct	75.0	75	49	0	0.00
10 #2	Direct	75.0	75	51	0.666	0.01
1 #1	Direct	75.0	75	50	0	0.00
1#2	Direct	75.0	75	52	0	0.00
100 #1	Acetone	5.0	76	52	31.186	9.12
100 #2	Acetone	5.0	73	51	34.632	9.91
10 #1	Acetone	5.0	76	51	28.977	8.64
10 #2	Acetone	5.0	83	50	30.959	10.28
1 #1	Acetone	75.0	75	49	4.851	0.10
1#2	Acetone	75.0	75	51	3.977	0.08
Control #1	-	75.0	75	50	0	0.00
Control #2	-	75.0	75	50	0	0.00

Table 6.1.3/5: Test item concentrations and recoveries of the repeated optimised WAF loadings without algae (fresh)

Conc.	Prep. method	Separated volume of extract	Complete volume of extract	Test water sample volume	Conc.  in LC-MS sample	Conc. in                   test water sample
[mg/L]	-	[mL]	[mL]	[mL]	[ng/mL]	[ng/mL]
100 #1	Direct	75.0	75	51	250.196	4.91
100 #2	Direct	75.0	75	51	89.341	1.75
10 #1	Direct	75.0	75	50	79.407	1.59
10 #2	Direct	75.0	75	50	93.244	1.86
1 #1	Direct	75.0	75	50	2.212	0.04
1#2	Direct	75.0	75	51	1.594	0.03
100 #1	Acetone	5.0	76	51	130.302	38.84
100 #2	Acetone	5.0	76	51	177.042	52.77
10 #1	Acetone	5.0	74	50	58.248	17.24
10 #2	Acetone	5.0	73	51	81.352	23.29
1 #1	Acetone	25.0	75	50	177.125	10.63
1#2	Acetone	25.0	75	51	184.242	10.84
Control #1	-	75.0	75	50	0	0.00
Control #2	-	75.0	75	52	0	0.00

Immobilisation effects:

No signs of disease or stress, such as discolouration or abnormal behavior, were observed in any replicate due to the test concentration. However, in the acetone WAFs (both 2 and 24 h sedimentation preparations) the test organisms were observed ‘sticking’ to the surface of the test media. 

Table 6.1.3/6: Immobilisation of daphnids after 48 h exposure toThiadamascone via different WAF preparations

Nominal concentration	Direct dissolving (46 h /2 h)		Acetone (46 h /2 h)		Direct dissolving (24 h /24 h)		Acetone (24 h /24 h)
[mg/L]	24 h [%]	48 h [%]	24 h [%]	48 h [%]	24 h [%]	48 h [%]	24 h [%]	48 h [%]
Control	0	0	0	0	0	5	0	5
1	15	15	0	20	0	5	0	0
10	25	35	5	25	20	25	5	20
100	0	20	0	15	5	10	0	0

Validity criteria fulfilled:

yes

Conclusions:

Based on the outcome from the preliminary investigations on WAF preparations (total lack of reproducibility within the various replicates and concentration levels) and with respect to the results of the acute immobilisation tests on Daphnia magna, it was concluded that it is not technically feasible to get the substance in solution and to obtain reliable concentrations and that as a consequence it is not possible to conduct a chronic Daphnia test and that aquatic toxicity tests with this substance are likely not to be technically feasible.

Executive summary:

This study was performed to assess the influence of different water accommodated fractions (WAF) preparations on the recovery of the test substance. WAF preparations were applied in a Daphnia magna acute immobilisation test (according to OECD Guideline 202 and EU Method C.2 without GLP statement) which were carried out as preliminary tests for a chronic test on Daphnia magna according to OECD Guideline 211.

Since the substance was known to have a difficult testing profile, extensive investigations were carried out to correctly prepare the test solutions. These were based on two approaches. The direct suspension of the test item in water (direct suspension method) and the coating of the inner surface of the WAF bottle with the test item applied in a small aliquot of acetone prior to mixing with water (bottle coating method) were compared.

Three different loadings of 100, 10 and 1 mg/L were prepared. Both approaches were tested under two sets of conditions (46 h stirring/2 h sedimentation or 24 h stirring/24 h sedimentation), in accordance with the OECD guidance document No. 23 and the Schäfers method. Each loading was prepared individually under sterile conditions and used within 1-2 hours of preparation. Samples of the fresh and aged (after 48 h incubation with algae) solutions for each preparation were analysed by LC/MS-MS after liquid

extraction.

Only traces of the test item were recovered in both fresh and aged samples of WAF prepared by the direct suspension method followed by a 2 hours sedimentation period.. Extending the sedimentation period to 24 hours did not lead to an optimized recovery. WAFs prepared by the bottle coating method contained higher concentrations of the test item. Following the 2 hours and 24 hours sedimentation period, 62% and 27% of the highest

loading (100 mg/L) were recovered, respectively, However, the concentrations decreased to 27% and 9% of the nominal loading after 48 hours, respectively.

Daphnids were placed into the test medium prepared with the nominal loadings of 100, 10, and 1 mg test substance/L. A control of dilution water only was run in parallel. The test was conducted under static conditions for 48 h. Effects on immobilisation were determined after 24 and 48 hours. The bottle coating method is capable of producing higher test item concentrations in water compared to that of the direct dissolving method. However,

this method was leading to the adherence of animals to the media surface leading to high mortality and thus preventing the determination of effects defined in the guideline.

Based on the outcome from the preliminary investigations on WAF preparations (total lack of reproducibility within the various replicates and concentration levels) and with respect to the results of the acute immobilisation tests on Daphnia magna, it was concluded that it is not technically feasible to get the substance in solution and to obtain reliable concentrations and that as a consequence it is not possible to conduct a chronic Daphnia test and that aquatic toxicity tests with this substance are likely not to be technically feasible.

Description of key information

Based on the very low water solubility of the registered substance, it's technically not feasible to obtain a relevant endpoint value. The substance had no acute toxic effects on daphnids up to and significantly higher than its solubility limit in test water.

Additional information

Two studies are available to asses the acute toxicity of the registered substance to aquatic invertebrates (Daphnia magna) in a 48h static test.

The first study (Safepharm, 2006), evaluated as the key study, was performed according to OECD Guideline No 202, EU Method C.2 with GLP statement and the second study (Fraunhofer, 2013), evaluated as the supporting study, was performed according to OECD Guideline No 202, EU Method C.2 without GLP statement.

Based on the very low water solubility of the substance, it's technically not feasible to obtain a relevant endpoint value. According to the key study, the substance had no acute toxic effects on fish up to and significantly higher than its solubility limit in test water.

In addition, based on the outcome of the supporting study, the preliminary investigations on WAF preparations using two differents methods of exposure (direct suspension of the test item in water and coating the inner surface of the WAF bottle with the test item), resulting in total lack of reproducibility within the various replicates and concentration levels. With respect to the results of the acute immobilisation tests on Daphnia magna, it was concluded that it is not technically feasible to get the substance in solution and to obtain reliable concentrations and results and that as a consequence it is not possible to conduct a chronic Daphnia test and that aquatic toxicity tests with this substance are considered not technically feasible.

Moreover, as a strong and consistent evidence of mineralisation of the registered substance was provided, the acute toxicities of the degradation products, which are the relevant species for the environmental risk assessment, were determined using the ECOSAR v1.11 QSAR model available from the U.S. EPA. The assessed degradation products were Delta-Damascone mercapto acetic acid (DD-ma) (which is the main degradation product) and two other larget metabolites (Delta-Damascone mercapto proprionic acid (DD-mp) and Delta-Damascone (DD)). The estimated 48h-LC50 values were > 84.53 mg/L for DD-ma, > 47.31 mg/L for DD-mp and 3.80 mg/L for DD. According to these data, the degradation products are not expected to be toxic (T) in a PBT assessment context.