4,6-dimethyl-2-(1-phenylethyl)-3,6-dihydro-2H-pyran

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:

Experimental start date: 27 July 2017 Experimental completion date: 04 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 12-0492
Chemical (IUPAC) Name: 4,6-dimethyl-2-(1-phenylethyl)-3,6-dihydro-2H-pyran
Physical state/Appearance: Very pale yellow liquid
Storage Conditions: Approximately 4 °C in darkness

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours, from samples run alongside the test at 24 and 48 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Preliminary Media Preparation Trial
Information provided by the Sponsor indicated the water solubility of the test item to be 18 mg/L.
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 .

Range-Finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 17 mg/L could be obtained using a saturated solution method of preparation.
A nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 0.10, 1.0 and 10% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (5.1 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
Each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Initial Experiment
A nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 liter discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 18, 32 and 56% v/v saturated solution. An aliquot (1500 mL) of each of the stock solutions was separately inoculated with algal suspension (12 mL) to give the required test concentrations of 10, 18, 32, 56 and 100% v/v saturated solution.
Each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
A nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a
0.2 µm Sartorious Sartopore filter (first approximate 1 liter discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.
A series of dilutions was made from this saturated solution to give stock solutions of 0.32, 1.0, 3.2, 10 and 32% v/v saturated solution. An aliquot (1500 mL) of each of the stock solutions was separately inoculated with 18.1 mL of algal suspension to give the required test concentrations of 0.32, 1.0, 3.2, 10 and 32% v/v saturated solution.
Each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

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. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 2 ºC.
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 10^3 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 ºC until the algal cell density was approximately 10^4 - 10^5 cells/mL.
A positive control (Envigo Study Number FP48BQ) used potassium dichromate as the reference item. The positive control was conducted between 28 November 2016 and 01 December 2016.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 24 ± 1 °C throughout the test.

pH:

The pH value of the control cultures was observed to increase from pH 8.1 at 0 hours to pH 9.5 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.

Nominal and measured concentrations:

Range-Finding Test
nominal - 0.10, 1.0, 10 and 100% v/v

Initial Experiment
nominal - 10, 18, 32, 56 and 100% v/v

Definitive Test
nominal - 0.32, 1.0, 3.2, 10 and 32% v/v

Verification of Test Concentrations
Analysis of the 3.2, 10 and 32% v/v saturated solution test preparations at 0 hours showed measured test concentrations to range from 0.43 to 4.9 mg/L. There was no significant change in the measured concentrations at 24, 48 and 72 hours, with the exception of the 3.2% v/v saturated solution sample at 72 hours where a measured concentration of 0.63 mg/L was obtained. It was considered appropriate to base the results on the 0-Hour measured test concentrations only.

Details on test conditions:

Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture. The media used in the initial experiment and the definitive test was prepared with the addition of 250 mg/L of sodium bicarbonate to prevent inhibition of growth due to the restriction in gaseous exchange associated with testing in an enclosed system (Herman et al 1990).

Range-Finding Test
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Initial Experiment
The test was conducted in 250 mL glass stoppered conical flasks. All flasks were completely filled and sealed with a ground glass stopper as an additional precaution at the request of the Sponsor. Six flasks were used for the control and three flasks were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
The flasks were incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Samples were taken at 22, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.

Definitive Test
In the definitive test, 250 mL glass stoppered conical flasks were used. Six flasks each were used for the control and three flasks were used for each treatment group. All flasks were completely filled and sealed with a ground glass stopper as an additional precaution at the request of the Sponsor.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 4.15 x 10^5 cells per mL. Inoculation of 1500 mL of test medium with 18.1 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.43 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range-finding Test
The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10% v/v saturated solution. However, growth was observed to be reduced at 100% v/v saturated solution.
Based on this information test concentrations of 10, 18, 32, 56 and 100% v/v saturated solution were selected for the initial experiment.
Chemical analysis of the 10 and 100% v/v saturated solution test preparations at 0 hours showed that measured concentration of 1.8 and 14 mg/L were obtained respectively. Analysis at 72 hours showed that measured concentrations of 0.36 and
5.5 mg/L were obtained respectively, indicating that the test item was unstable under test conditions.

Initial experiment
The results showed a significant effect on growth at every level.
Based on this information test concentrations of 0.32, 1.0, 3.2, 10 and 32% v/v saturated solution were selected for the definitive test.

Definitive test

Inhibition of Growth Rate
ErC10 (0 - 72 h): 1.5 mg/L
ErC20 (0 - 72 h): 2.0 mg/L
ErC50 (0 - 72 h): 3.5 mg/L; 95% confidence limits 3.1 – 4.0 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 and 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 the 0.32, 1.0 and 3.2% v/v saturated solution test concentrations (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 0.43 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 1.6 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h): 1.2 mg/L
EyC20 (0 - 72 h): 1.3 mg/L
EyC50 (0 - 72 h): 1.7 mg/L; 95% confidence limits 1.6 – 1.8 mg/L

Where:
EyCx is the test concentration that reduced yield by x%.
There were no statistically significant differences between the control and the 0.32, 1.0 and 3.2% v/v saturated solution test concentrations (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 0.43 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 1.6 mg/L.

Results with reference substance (positive control):

A positive control (Envigo Study Number FP48BQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h): 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h): 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Response Variable	EC50(mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	3.5	3.1	-	4.0	0.43	1.6
Yield	1.7	1.6	-	1.8	0.43	1.6

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 108 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.

Nominal cell density of control at 0 hours:          5.00 x 10³cells per mL

Mean cell density of control at 72 hours:          5.40 x 10⁵cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 11% 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%.

Observationson 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 0.32, 1.0, 3.2 and 10% v/v saturated solution, however no intact cells were observed to be present in the test cultures at
32% v/v saturated solution.

Water QualityCriteria

The pH values of the control and each test preparation are given in Table 3. Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures was observed to increase from pH 8.1 at 0 hours to pH 9.5 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.

Observations on Test Item Solubility

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.32, 1.0 and 3.2% v/v saturated solution test cultures were observed to be green dispersions, the 10% v/v saturated solution test cultures were observed to be pale green dispersions and the 32% v/v saturated solution were observed to be extremely pale green dispersions.

 

Validity criteria fulfilled:

yes

Conclusions:

The ErC50, ErC10 and NOEC were 3.5, 1.5 and 0.43 mg/l

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. Following a preliminary range-finding test and an initial experiment, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 0.32, 1.0, 3.2, 10 and 32% v/v saturated solution (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 stirring an excess (100 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius filter, first approximate
1 liter discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

Analysis of the 3.2, 10 and 32% v/v saturated solution test preparations at 0 hours showed measured test concentrations to range from 0.43 to 4.9 mg/L. There was no significant change in the measured concentrations at 24, 48 and 72 hours, with the exception of the
3.2% v/v saturated solution sample at 72 hours where a measured concentration of 0.63 mg/L was obtained. It was considered appropriate to base the results on the 0-Hour measured test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the 0-Hour measured test concentrations:

Response Variable	EC50(mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	3.5	3.1	-	4.0	0.43	1.6
Yield	1.7	1.6	-	1.8	0.43	1.6

Description of key information

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. Following a preliminary range-finding test and an initial experiment,Pseudokirchneriella subcapitatawas exposed to solutions of the test item at nominal concentrations of 0.32, 1.0, 3.2, 10 and 32% v/v saturated solution (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 stirring an excess (100 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius filter, first approximate 1 liter discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

Analysis of the 3.2, 10 and 32% v/v saturated solution test preparations at 0 hours showed measured test concentrations to range from 0.43 to 4.9 mg/L. There was no significant change in the measured concentrations at 24, 48 and 72 hours, with the exception of the 3.2% v/v saturated solution sample at 72 hours where a measured concentration of 0.63 mg/L was obtained. It was considered appropriate to base the results on the 0-Hour measured test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the 0-Hour measured test concentrations:

Response Variable	EC50(mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	3.5	3.1	-	4.0	0.43	1.6
Yield	1.7	1.6	-	1.8	0.43	1.6

Key value for chemical safety assessment

EC50 for freshwater algae:

3.5 mg/L

EC10 or NOEC for freshwater algae:

0.43 mg/L