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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

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: Green algae

Test type:

not specified

Water media type:

not specified

Total exposure duration:

96 h

Remarks on exposure duration:

none

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

5.216 mg/L

Remarks on result:

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

Validity of model:

1. Defined Endpoint: Toxicity to aquatic algae.

2. Unambigous algorithm: The ECOSAR class determined was Vinyl/Allyl Ketones. However, based on observed effects for green algae, 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 green algae:

Linear regression QSAR; Log 96h-EC50 (mmol/L) = -0.6922*log Kow + 0.9253. To convert the EC50 from mmol/L to mg/L, multiply by the molecular weight.

3. Applicability domain: applicable to chemicals with log Kow less than 6.4 and molecular weight less than 1000 g/mol. The model can be suitable for chemicals class of neutral organic.

4. Statistical characteristics: N = 41+9 and the coefficient of determination R² = 0.6782.

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 96h-EC50 of 5.216 mg/L to green algae after 96h of exposure. The substance falls within the applicability domain and therefore the predicted value can be considered reliable.

Executive summary:

Toxicity to green algae of delta-Damascone was estimated using the ECOSAR v1.11 QSAR model available from the U.S. EPA. The estimation 96h-EC50 was 5.216 mg/L (neutral organics class), calculated from an estimated log Kow value at 3.6 (mean of QSAR results) and a linear regression:

Log 96h-EC50 (mmol/L) = -0.6922*log Kow + 0.9253

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

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

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: Green algae

Test type:

not specified

Water media type:

not specified

Total exposure duration:

96 h

Remarks on exposure duration:

none

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

171.173 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: Toxicity to aquatic algae.

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 96h-EC50 (mmol/L) = -0.6922*log Kow + 0.9253. To convert the EC50 from mmol/L to mg/L, multiply by the molecular weight.

3. Applicability domain: applicable to chemicals with log Kow less than 6.4 and molecular weight less than 1000 g/mol. The model can be suitable for chemicals class of neutral organic.

4. Statistical characteristics: N = 41+9 and the coefficient of determination R² = 0.6782.

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 96h-EC50 > solubility limit, estimated at 84.53 mg/L to green algae after 96h of exposure. The substance falls within the applicability domain and therefore the predicted value can be considered reliable.

Executive summary:

Toxicity to green algae of delta-Damascone mercapto acetic acid was estimated using the ECOSAR v1.11 QSAR model available from the U.S. EPA. The estimation 96h-EC50 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 (acid moeity was found, therefore predicted values were multiplied by 10):

Log 96h-EC50 (mmol/L) = -0.6922*log Kow + 0.9253

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

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

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: Green algae

Test type:

not specified

Water media type:

not specified

Total exposure duration:

96 h

Remarks on exposure duration:

none

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

130.588 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: Toxicity to aquatic algae.

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 96h-EC50 (mmol/L) = -0.6922*log Kow + 0.9253. To convert the EC50 from mmol/L to mg/L, multiply by the molecular weight.

3. Applicability domain: applicable to chemicals with log Kow less than 6.4 and molecular weight less than 1000 g/mol. The model can be suitable for chemicals class of neutral organic.

4. Statistical characteristics: N = 41+9 and the coefficient of determination R² = 0.6782.

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 96h-EC50 > solubility limit, estimated at 47.31 mg/L to green algae after 96h of exposure. The substance falls within the applicability domain and therefore the predicted value can be considered reliable.

Executive summary:

Toxicity to green algae of delta-Damascone mercapto propionic acid was estimated using the ECOSAR v1.11 QSAR model available from the U.S. EPA. The estimation 96h-EC50 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 96h-EC50 (mmol/L) = -0.6922*log Kow + 0.9253

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

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 09 January to 03 March 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 201 and EU Method C.3 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 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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:

- Concentrations: 0 (solvent control): two samples of the solvent control (replicates R1 - R4 pooled) and the 0.10 mg/l test group (replicates R1 - R3, R4 - R6 and R7 - R9 pooled) were taken at each occasion. One sample was analysed untreated and one sample after centrifugation (40000 g for approximately 30 minutes).
- Sampling method: Samples were taken from the control and each test group at 0 and 72 hours for quantitative analysis.
- Sample storage conditions before analysis: Further samples (in duplicate) were taken at 0 hours and stored at approximately -20°C for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.

The method of analysis, stability, recovery and test preparation analyses are described in "Details on analytical treatment"

Vehicle:

yes

Details on test solutions:

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 study under the OECD Guideline. Other various recognized auxiliary solvents were used during preliminary solubility work, however, dimethylformamide 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.

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1mg/ml in dimethylformamide (DMF) was made by serial dilutions in DMF. An aliquot (400ul) of this stock solution was dispersed in 4 liters of algal suspension to give the required test concentration of 0.10 mg/l. Culture media used: AAP-medium (US EPA)
Each of the solvent stock solutions were inverted several times to ensure adequate mixing and homogeneity.
- Controls: The concentration and stability of the test material in the test centrifuged and and untreated samples were verified by chemical analysis at 0 (fresh media) and 72 hours
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Stock solutions: 100 %. Final test solutions: 0.01 %
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: Scenedesmus subspicatus CCAP 276/20
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): not mentionned in study report
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (AAP medium). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 24 ± 1°C.


ACCLIMATION
- Acclimation period: Not Applicable
- Culturing media and conditions (same as test or not): same as test medium (without sodium carbonate)
- Any deformed or abnormal cells observed: none

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

none

Post exposure observation period:

Not Applicable

Hardness:

Not required in OECD 201 Guideline/Not Applicable

Test temperature:

The flasks were incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1°C. The temperature within the incubator was recorded daily.

pH:

The pH values of the control cultures were observed to increase from pH 7.4 ± 0 at 0 hours to pH 7.9 ± 0 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Dissolved oxygen:

Not required in OECD 201 Guideline/Not Applicable

Salinity:

Not applicable

Nominal and measured concentrations:

See Table 6.1.5/2 in "Remaks on results including tables and figures"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flasks (closed) filled to 100 ml
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: The flasks were plugged with polyutethane foam bungs and incubated (INFORS Multitron® Version 2
incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 150 rpm for 72 hours.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not Applicable
- Renewal rate of test solution (frequency/flow rate): Not Applicable
- Initial cells density: 12700 cells/ml
- Control end cells density: Yes, determined using a Coulter Multisizer Particle Counter
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3


GROWTH MEDIUM
- Standard medium used: yes (AAP medium)
- Detailed composition if non-standard medium was used: Not Applicable


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP)
- Total organic carbon: none (not required in OECD Guideline 201/Not Applicable)
- Particulate matter: not required in OECD Guideline 201/Not Applicable
- Metals: not required in OECD Guideline 201/Not Applicable
- Pesticides: not required in OECD Guideline 201/Not Applicable
- Chlorine: not required in OECD Guideline 201/Not Applicable
- Alkalinity: not required in OECD Guideline 201/Not Applicable
- Ca/mg ratio: not required in OECD Guideline 201/Not Applicable
- Conductivity: not required in OECD Guideline 201/Not Applicable
- Culture medium different from test medium: no
- Intervals of water quality measurement: not required in OECD Guideline 201/Not Applicable


OTHER TEST CONDITIONS
- Sterile test conditions: no data
- Adjustment of pH: no
- Photoperiod: continuous illumination for 72 hours.
- Light intensity and quality: intensity ca. 7000 lux
- Salinity (for marine algae): Not Applicable


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter
- Chlorophyll measurement: no
- Other: none


TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable, only one concentration could be tested due to the limit of solubility (0.1 mg/ml) that was achieved using the maximum of solvent (DMF) recommended by the OECD guideline
- Range finding study
- Test concentrations: 0.1, 0.01 mg/l for 72 hours
- Results used to determine the conditions for the definitive study: no significant inhibition of growth rate were recorded at the test concentrations of 0.10 and 0.01 mg/I.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: -

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: -

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: -

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 21 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: -

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 21 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: -

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

21 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: -

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.
- Any stimulation of growth found in any treatment: none

Results with reference substance (positive control):

None

Reported statistics and error estimates:

A Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981 ) was carried out on the area under the growth curve data at 72 hours for the solvent control and the 0.10 mg/l test concentration to determine any statistically significant differences between the test
and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Growth data

From the Table of Cell densities, it is clear that neither the growth (r) nor the biomass (b) of Scenedesmus subspicatus (CCAP 276/20) were affected by the presence of the test material over the 72-Hour exposure period. The data show that the cell concentration of the control cultures increased by a factor of 71 after 72 hours and the cell concentration of the solvent control cultures increased by a factor of 93 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

 

The Graph representing the mean cell densities versus time is joined in “Attached background material”.

 

Table 6.1.5/1: Cell densities

Nominal Concentration (mg/l)		Cell Densities (cells/ml x 100)
		0h	24h	48h	72h
Control	R1	103	552	1730	7770
	R2	115	599	1580	7870
	R3	109	659	1590	7550
	Mean	109	603	1630	7730
Solvent Control	R1	122	591	2260	11200
	R2	105	481	2110	10100
	R3	113	635	2240	10400
	Mean	114	569	2200	10600
0.10	R1	114	479	2130	10400
	R2	114	459	1690	10300
	R3	113	583	2360	11400
	R4	117	659	2090	10000
	R5	117	608	2450	10500
	R6	125	531	1940	11800
	Mean	117	553	2110	10700

 

Verifications of concentrations

 

Table 6.1.5/2: Verification of test concentration

Sample	Nominal Concentration (mg/l)	Concentration Found (mg/l)	% of nominal concentration
0 hours	Solvent Control U 0.10 R1-R3 U 0.10 R4-R6 U Solvent control C 0.10 R1-R3 C 0.10 R4-R6 C	< LOQ 0.152 0.164 0.0339/<LOQ 0.100 0.0983	- 152 164 -/- ** 100 98
72 hours	Solvent Control U 0.10 R1-R3 U 0.10 R4-R6 U Solvent control C 0.10 R1-R3 C 0.10 R4-R6 C	< LOQ 0.0344 0.0219 <LOQ 0.0464 0.00406	- 34 22 - 5 4
U = Untreated C = centrifuged at 40’000 g for 30 min at 21°C in polycarbonate tubes LOQ = Limit of detection R1-R6 = Replicates ** duplicate samples, stored frozen prior to analysis

 

Analysis of the test preparations at 0 hours (see Appendix 1) showed measured test concentrations of the untreated test samples to range from 152% to 164% of nominal whilst measuredconcentrations in the range of 98% to 100% of nominal were obtained from the centrifuged test samples. The measured concentrations of the untreated test samples were higher than the allowable limits of 80% to 120% of nominal. This was considered to be possibly due to the presence of small, dispersed globules of undissolved test material that may not have been detected by visual examination. However, given that the results are based on centrifuged analysis only this was considered to have had no significant impact on the outcome of the test.

Analysis of the test preparations 72 hours showed measured test concentrations of the untreated test samples to range from 22% to 34% of nominal whilst measured test concentrations in the range of 4% to 5% of nominal were obtained from the centrifuged test samples.

Given that the preliminary stability analyses conducted (see “Verification of test concentration” in attached background material) indicated that the test material was stable in culture medium over the test period this decline in both the untreated and centrifuged test samples was considered to be due to adsorption of the test material to the algal cells present. Adsorption was not a factor in the preliminary stability analyses conducted since no algal cells were present.

Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations of the centrifuged test media in order to give a "worst case" analysis of the data.

The geometric mean measured test concentrations were determined to be:

Nominal Test Concentration (mg/l)	Geometric Mean Measured Test Concentration (mg/l)	Expressed as a % of the nominal Test Concentration
0.10 (R1-R3)	0.022	22
0.10 (R4-R6)	0.020	20

 

The following results were determined from the data based on the geometric mean measured test concentrations of the centrifuged test media.

 

EbC50 (72 h) :> 0.021 mg/l

ErC50(0-72 h) :> 0.021 mg/l

No Observed Effect Concentration (NOEC)=0.021 mg/l.

Validity criteria fulfilled:

yes

Remarks:

The three criteria of OECD Guideline 201 were fullfilled (Biomass increasing Factor > 16, mean coefficient variation <35% and variation of average specific growth rate of control < 7%

Conclusions:

The effect of the test material on the growth of Scenedesmus subspicatus has been investigated and gave EC50 value of greater than 0.10 mg/L. Correspondingly the No Observed Effect Concentration was 0.10 mg/L.
Based on the geometric mean measured test concentrations of the centrifuged test media the EC50 value was estimated to be greater than 0.021 mg/L. Correspondingly the No Observed Effect Concentration was 0.021 mg/L.

Executive summary:

Introduction.A study wasperfonnedto assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus.The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201, "Alga,GrowthInhibition Test" referenced as Method C.3 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 (Safepharm Laboratories Limited Project Number: 161/439) showed that the water solubility value of the test material was 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 the media preparation trials indicated that using a solvent spike method of preparation was the most suitable for this test material and indicated that after centrifugation at 40000 g for 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 not considered appropriate 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,Scenedesmus subspicatus was exposed to an aqueous solution of the test material at a concentration of 0.10 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®MultisizerParticle Counter.

 

Results.Exposure of Scenedesmus subspicatus to the test material gave EC50 values of greater than 0.10 mg/l and correspondingly the No Observed Effect Concentration was 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 test preparations at 0 hours showed measured test concentrations of the untreated test samples to range from 152% to 164% of nominal whilst measured concentrations in the range of 98% to 100% of nominal were obtained from the centrifuged test samples. The measured concentrations of the untreated test samples were higher than the acceptable limits of 80% to 120% of nominal. This was considered to be possibly due to the presence of small, dispersed globules ofundissolvedtest material that may not have been detected by visual examination.

However, given that the results are based on centrifuged analysis only this was considered to have had no significant impact on the outcome of the test.

Analysis of the test preparations at 72 hours showed measured test concentrations of the untreated test samples to range from 22% to 34% of nominal whilst measured test concentrations in the range of 4% to 5% of nominal were obtained from the centrifuged test samples.

Given that the preliminary stability analyses conducted indicated that the test material was stable in culture medium over the test period this decline in both the untreated and centrifuged test samples was considered to be due to adsorption of the test material to the algal cells present. Adsorption was not a factor in the preliminary stability analyses conducted since no algal cells were present.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations of the centrifuged test media in order to give a "worst case" analysis of the data. The EC50 value based on the geometric mean measured test concentrations of the centrifuged test media was greater than 0.021 mg/l and correspondingly the No Observed Effect Concentration was 0.021 mg/l.

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 toxic effects on algae up to and significantly higher than its solubility limit in test water.

Additional information

One key study, performed according to OECD Guideline No 201, EU Method C.3 with GLP statement, is available to assess the effect of the registered substance on the growth of the green alga Scenedesmus subspicatus.

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

Moreover, as 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 96h-EC50 values were > 84.53 mg/L for DD-ma, > 47.31 mg/L for DD-mp and 5.22 mg/L for DD. According to these data, the degradation products are not expected to be toxic (T) in a PBT assessment context.