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Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 10 July 2014; Experimental Completion Date: 22 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US EPA Ecological Effects Test Guideline OPPTS 850.1000 “Special Considerations for Conducting Aquatic Laboratory Studies”
Deviations:
no
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC, 1996 and OECD, 2000), is to expose organisms to a saturated solution of the test item in cases where the test item is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (100 mg/L) of test item in culture medium for a period of 24 hours prior to removing any undissolved test item present by filtration to give a saturated solution of the test item.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of (3R)-3-methyl-5-[(1R,3R)-2,2,3-trimethylcyclopentyl]pentan-2-one and (3S)-3-methyl-5-[(1R,3R)-2,2,3-trimethylcyclopentyl]pentan-2-one
EC Number:
942-532-4
Molecular formula:
C14H26O
IUPAC Name:
Reaction mass of (3R)-3-methyl-5-[(1R,3R)-2,2,3-trimethylcyclopentyl]pentan-2-one and (3S)-3-methyl-5-[(1R,3R)-2,2,3-trimethylcyclopentyl]pentan-2-one
Test material form:
other: liquid
Details on test material:
Identification: NACET00502
Chemical name: 3-Methyl-5-((1R)-2,2,3-trimethylcyclopenyl)petan-2-one
CAS number: 119464-63-0 (planar)
Physical state/Appearance: Clear colorless liquid
Batch: S-13-11-1
Purity: 98.5%
Expiry date: 24 December 2015
Storage conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100% v/v saturated solution (definitive test)

- Sampling method: Samples were taken for quantitative analysis from the control and the 100% v/v saturated solution test group from the bulk test preparation at 0 hours and from the pooled replicates at 96 hours.

Additional samples of the 100% v/v saturated solution were prepared at the start of the test and incubated alongside the test to provide samples for analysis at 24, 48 and 72 hours.

- Sample storage conditions before analysis: All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 96 hours and stored frozen for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
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.
Standard culture medium as defined in the test guidelines was used.

PROCEDURE:
PRELIMINARY MEDIA PREPARATION TRIAL:
Information provided indicated the test item was insoluble in water. 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.

Based on the results of the trial the test item was prepared using a saturated solution method of preparation.

RANGE-FINDING TEST:
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 96 hours.

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.

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 Gelman Acrocap filter (first approximate 500 mL 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, 1.0 and 0.10% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (6.1 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

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 96 hours.

After 96 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

A sample of each test concentration was taken for chemical analysis at 0 and 96 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.

DEFINITIVE TEST:
Based on the result of the range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable concentration, no effect on algal growth was observed.

Experimental 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. An aliquot (2 liters) of each of the 100% v/v saturated solution was inoculated with algal suspension (32.7 mL) to give the required test concentration of 100% v/v saturated solution.

The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.











Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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), Dunstaffnage Marine Laboratory, 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 ± 1ºC.

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 10E3 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 ± 1ºC until the algal cell density was approximately 10E4 - 10E5 cells/mL.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h

Test conditions

Test temperature:
Temperature was maintained at 24 ± 1 ºC throughout the test.
pH:
See water quality criteria results.
Nominal and measured concentrations:
Definitive Test:
Nominal test concentration (% v/v Saturated Solution): 100
Time Weighted Mean Measured Test Concentration (mg/L): 2.2
Expressed as a % of the 0-Hour Meased Test Concentration: 53
Details on test conditions:
DEFINITIVE TEST:
Exposure conditions:
250 mL glass conical flasks were used. Four flasks each containing 100 mL of test preparation were used for the control and 100% v/v saturated solution treatment group.

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 6.11 x 10E5 cells per mL. Inoculation of 2 liter of test medium with 32.7 mL of this algal suspension gave an initial nominal cell density of 1 x 10E4 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 96 hours.
EVALUATIONS:
Test Organism Observations:
Samples were taken at 0, 24, 48, 72 and 96 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Water Quality Criteria:
The pH of the control and 100% v/v saturated solution test preparation was determined at initiation of the test and after 96 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded hourly using the incubators’ internal pt-100 temperature probe and Multicom Software Package.













Reference substance (positive control):
yes
Remarks:
zinc chloride

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 2.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
(Time-Weighted Mean Measured Test Concentration)
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 2.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
(Time-Weighted Mean Measured Test Concentration)
Basis for effect:
other: yield
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 2.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
(Time-Weighted Mean Measured Test Concentration)
Basis for effect:
biomass
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
2.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
(Time-Weighted Mean Measured Test Concentration)
Basis for effect:
other: growth rate, yield and biomass
Details on results:
RANGE-FINDING TEST:
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

Based on this information a single test concentration of four replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration, no effect on growth was observed.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.070 to 11 mg/L. A decline in measured test concentrations was observed at 96 hours to less than the limit of quantification (LOQ) of the analytical method employed which was determined to be 0.0051 mg/L indicating that the test item was unstable over the test period.

DEFINITIVE TEST:
Verification of Test Concentrations:
Analysis of the 100% v/v saturated solution test preparations at 0, 24, 48, 72 and 96 hours showed measured test concentrations of 4.1, 2.9, 2.5, 2.0 mg/L and less than the limit of quantification (LOQ), determined to be 0.0051 mg/L respectively were obtained.

Given this decline in measured test concentrations, it was considered justifiable to base the results on the time-weighted mean measured test concentrations in order to give a “worst case” analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.0026 mg/L) was used to enable calculation of the time-weighted mean measured concentration. The time-weighted mean measured test concentration was determined to be:

Nominal test concentration (% v/v Saturated Solution): 100
Time Weighted Mean Measured Test Concentration (mg/L): 2.2
Expressed as a % of the 0-Hour Meased Test Concentration: 53

GROWTH DATA:
From the results, it is clear that the growth rate (r), yield (y) and biomass integral (b) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item at a time-weighted mean measured test concentration of 2.2 mg/L over the 96-Hour exposure period.

Accordingly the following results were determined from the data:
Inhibition of Growth Rate:
ErC05 (0 - 96 h) : >2.2 mg/L
ErC50 (0 - 96 h) : >2.2 mg/L

Statistical analysis of the growth rate data at 96 hours was carried out for the control and 2.2 mg/L test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant differences, between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100% v/v saturated solution.
Inhibition of Yield:
EyC05 (0 - 96 h) : >2.2 mg/L
EyC50 (0 - 96 h) : >2.2 mg/L

There were no statistically significant differences between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100% v/v saturated solution.

Inhibition of Biomass Integral:
EbC05 (0 - 96 h) : >2.2 mg/L
EbC50 (0 - 96 h) : >2.2 mg/L

There were no statistically significant differences between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on biomass integral was 100% v/v saturated solution.

Observations on Cultures:
All test and control cultures were inspected microscopically at 96 hours. There were no abnormalities detected in any of the control or test cultures at 96 hours.

Water Quality Criteria:
Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures was observed to increase from pH 7.3 at 0 hours to pH 8.3 - 8.4 at 96 hours. The pH deviation in the control cultures was less than 1.5 pH units after 96 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 96-Hour test period all control and test cultures were observed to be very bright green dispersions.

















Results with reference substance (positive control):
A positive control used zinc chloride as the reference item at concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 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:

96 h EC50 (growth rate): 0.43 mg/L
96 h EC50 (yield): 0.20 mg/L
96 h EC50 (biomass): 0.18 mg/L
NOEC (for all response variables): 0.10 mg/L
LOEC (for all response variables): 0.32 mg/L

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




Any other information on results incl. tables

Validity Criteria:

The following data show that the cell concentration of the control cultures increased by a factor of 234 after 96 hours. This increase was in line with the Guideline that states the enhancement must be at least by a factor of of 100 after 96 hours.

Mean cell density of control at 0 hours: 1.15 x 104cells per mL

Mean cell density of control at 96 hours: 2.70 x 106cells per mL

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 - 96 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 15%.

 

The coefficient of variation of the mean control yield was 3% after 96 hours and hence satisfied the validation criterion which states that this must not exceed 15%.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and based on the time-weighted mean measured test concentrations gave EC50 values of greater than 2.2 mg/L. The No Observed Effect Concentration was 2.2 mg/L.

This study showed that there were no toxic effects at saturation.
Executive summary:

Intoduction:

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 US EPA Ecological Effects Test Guideline OCSPP 850.5400 “Algal Toxicity” and the US EPA Ecological Effects Test Guideline OPPTS 850.1000 “Special Considerations for Conducting Aquatic Laboratory Studies”.

Methods:

Information provided by the Sponsor indicated the test item was insoluble in water. 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 a dissolved test item concentration of approximately 4.5 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a solution of the test item at a nominal concentration of 100% v/v saturated solution (six replicate flasks) for 96 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test item solution was 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 Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item.

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 100% v/v saturated solution test preparations at 0, 24, 48, 72 and 96 hours showed measured test concentrations of 4.1, 2.9, 2.5, 2.0 and less than the limit of quantification (LOQ), determined to be 0.0051 mg/L respectively were obtained.

Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations in order to give a "worst case" analysis of the data.

 

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values based on the time-weighted mean measured test concentrations of greater than 2.2 mg/L. The No Observed Effect Concentration was 2.2 mg/L.

 

This study showed that there were no toxic effects at saturation.