Androsta-1,4-diene-17-carbothioic acid, 6,9-difluoro-11-hydroxy-16-methyl-3-oxo-17-(1-oxopropoxy)-, (6.alpha.,11.beta.,16.alpha.,17.alpha.)-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18th November 2015 - 2nd March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Thio Acid Propionate
Physical state/Appearance: Off white solid
Batch: 0000135240
Purity: 97.76%
Expiry Date: 26 July 2017
Storage Conditions: approximately 4 ºC in the dark over silica gel

Analytical monitoring:

yes

Remarks:

HPLC/UV

Details on sampling:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of
Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic
replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104 - 105 cells/mL.

A positive control (Envigo Study Number MG09PL) used potassium dichromate as the reference item. The positive control was conducted between 07 December 2015 and 10 December 2015.

The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Details on test solutions:

Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.

Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of
Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic
replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of
approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104 - 105 cells/mL.

A positive control (Envigo Study Number MG09PL) used potassium dichromate as the reference item. Details of the positive control are given in Annex 2. The positive control
was conducted between 07 December 2015 and 10 December 2015.

The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Post exposure observation period:

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.56 mg/L. Cell debris was observed in the 1.9, 6.1 and 20 mg/L test cultures whilst no intact cells were observed to be present in the 65 mg/L test cultures.

Test temperature:

24 ± 1 °C

pH:

7.5

Nominal and measured concentrations:

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Details on test conditions:

The test item solutions were prepared by dissolving 1100 mg of test item in 11 litres of culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period, as a precautionary measure, any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100 mg/L solution of the test item. This solution was then further diluted as necessary, to provide the remaining test groups.

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

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 50 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

Range-finding test:
The results showed no effect on growth rate at the test concentrations of 0.10, 1.0 and 10mg/L. However, growth was obsered to be reduced at 100mg/L. Based on this information, test concentrations of 1.0. 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 89% to 91% of nominal. A decline in measured concentrations
was observed at 72 hours in the range of 37% to 50% of nominal. This decline in measured concentrations was considered to be due to precipitation of the test item. Prior to analysis the test samples were centrifuged, removing not only the algal cells present but also any precipitated test item. The measured concentrations obtained after centrifugation are representative of the dissolved and hence bioavailable test concentrations.

Definitive Test:
Verification of Test Concentrations:
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 84% to 89% of nominal. A decline in measured test concentrations was observed at 72 hours in the range of 38% to 47% of nominal. This decline in measured concentrations was considered to be due to precipitation of the test item. Prior to analysis the test samples were centrifuged, removing not only the algal cells present but also any precipitated test item. Given that the measured concentrations obtained after centrifugation are representative of the dissolved and hence bioavailable test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations.

Inhibition of growth rate:
ErC10 (0 - 72 h): 7.7 mg/L
ErC20 (0 - 72 h): 15 mg/L
ErC50 (0 - 72 h): 50 mg/L

Where ErCx is the test concentration that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control and 0.56 mg/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.56 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 1.9 mg/L.

Inhibition of Yield:
EyC10 (0 - 72 h): 0.55 mg/L
EyC20 (0 - 72 h): 0.70 mg/L
EyC50 (0 - 72 h): 2.0 mg/L

Where: EyCx is the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as in Section 6.2.3. There were no statistically significant differences between the control and 0.56 mg/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.56 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 1.9 mg/L.

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
1.0	0.56	67
3.2	1.9	67
10	6.1	69
32	20	72
100	65	73

Validation Criteria:

The following data show that the cell concentration of the control cultures increased by a factor of 124 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.

Mean cell density of control at 0 hours : 6.42 x 10³ cells per mL

Mean cell density of control at 72 hours : 7.94 x 10⁵ cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 10% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures:

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.56 mg/L. Cell debris was observed in the 1.9, 6.1 and 20 mg/L test cultures whilst no intact cells were observed to be present in the 65 mg/L test cultures.

Water Quality Criteria:

Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures was observed to range from pH 7.1 at 0 hours to pH 6.7 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.

A concentration dependent decline on pH was observed in the test preparations at 0 hours in the range of pH 7.4 at 0.56 mg/L though to pH 4.8 at 65 mg/L. This was considered to be due to an intrinsic property of the test item and as such no adjustments were made to the pH of the test preparations prior to inoculation with algae.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results:

Growth Rate: EC50 (mg/L)=50; NOEC (mg/L)=0.56; LOEC (mg/L)=1.9
Yield: EC50=2.0; NOEC=0.56; LOEC=1.9

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods:

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that the test item was readily soluble in culture medium using a prolonged stir method of preparation.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by dissolving 1100 mg of test item in 11 litres of culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period, as a precautionary measure, any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100 mg/L solution of the test item. This solution was then further diluted as necessary, to provide the remaining test groups.

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

Results:

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 84% to 89% of nominal. A decline in measured test concentrations was observed at 72 hours in the range of 38% to 47% of nominal. This decline in measured concentrations was considered to be due to precipitation of the test item. Prior to analysis the test samples were centrifuged, removing not only the algal cells present but also any precipitated test item. Given that the measured concentrations obtained after centrifugation are representative of the dissolved and hence bioavailable test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	50	0.56	1.9
Yield	2.0	0.56	1.9

Description of key information

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods:

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that the test item was readily soluble in culture medium using a prolonged stir method of preparation.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by dissolving 1100 mg of test item in 11 litres of culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period, as a precautionary measure, any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100 mg/L solution of the test item. This solution was then further diluted as necessary, to provide the remaining test groups.

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

Results:

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 84% to 89% of nominal. A decline in measured test concentrations was observed at 72 hours in the range of 38% to 47% of nominal. This decline in measured concentrations was considered to be due to precipitation of the test item. Prior to analysis the test samples were centrifuged, removing not only the algal cells present but also any precipitated test item. Given that the measured concentrations obtained after centrifugation are representative of the dissolved and hence bioavailable test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	50	0.56	1.9
Yield	2.0	0.56	1.9

Key value for chemical safety assessment

EC50 for freshwater algae:

50 mg/L

Additional information

Validation Criteria:

The results of the test are considered valid if the following performance criteria are met:

• The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.

• The mean of the coefficients of variation of the section by section daily growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3) must not exceed 35%.

• The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%

Deviations from Study Plan:

There were no deviations from study plan.