(1'R,2R)-2-Methyl-4-(2',2',3'-tri-nal and methylcyclopent-3'-en-1'-yl)-4-pent-enal

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:

11-08-2000 to 05-10-2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

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

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: February 2000 ; signature: April 2000

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Preliminary range finding test : 0 (control), 0.12, 1.2 and 12 mg/L nominal test item concentration
Definitive Test: 0 (control), 0.538, 1.18, 2.56, 5.55 and 11.7 mg/L analytically confirmed initial concentration
Time Weighted Mean Measured equivalent concentrations: 0 (control), of 0.255, 0.430, 0.747, 1.34 and 2.41 mg/L
Samples from control replicates and test group replicates of equal nominal concentration were pooled for analysis. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.
- Sampling method: Water samples were taken from the control and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for centrifuged and uncentrifuged quantitative analysis. Samples were taken at 0 h and 72 h for quantitative analysis. Duplicate samples were taken at 0 hours and stored frozen (approximately -20°C) for further analysis if necessary.
- Sample storage conditions before analysis: See above.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct dissolution of test item in culture medium to prepare stock; serial dilution of stock to prepare test media. The test concentrations used in the definitive study were prepared by adding an amount of test item to the vortex created in a volume of water to give an initial dispersion at a concentration of 1000 mg/L. Stirring periods of 23 hours and 95 hours were employed, both with I hour standing periods. Vortex depths of 5% and 25% were used for both stirring periods and samples taken by mid-depth siphon for chemical analysis. This work showed that increasing the stirring period and/or the vortex depth did not significantly increase the measured test concentration of the saturated solution. Therefore, a stirring period of 23 hours with a 5% vortex depth was used for all further stirring work. To ensure that saturated solutions prepared by this method contained only dissolved test item, samples were removed by mid-depth siphon and analysed with and without filtration (0.2 micron filters). Based on the results of the range-finding study test item solutions for the definitive study were prepared by stirring an excess of test item (equivalent to 1000 mg/L) in culture medium for a period of time and then removing the aqueous phase by siphon to produce a saturated solution with a measured concentration of 11.7 mg/L. The saturated solution was then further diluted, as necessary, to produce the remaining measured test concentrations of 0.538, 1.18, 2.56 and 5.55 mg/L.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No precipitate reported.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus subspicatus (or Desmodesmus subspicatus)
- Strain: CCAP 276/20.
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Scotland
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium under constant aeration and illumination (light intensity: 7000 lux) at 21 ± 1 ºC temperature.

ACCLIMATION
- Acclimation period: No. However, prior to the start of the test exposure sufficient master culture was added to approximately 100 mL culture medium in flasks at 10^4 cells/mL and stirring at 100 – 150 rpm under constant conditions until algal density 10^4 – 10^5 cells/mL.
- Culturing media and conditions (same as test or not): Yes.
Culture medium: (mg/L) - ASTM medium, according to OECD 201, Annex 3
NaNO3 25.5, MgCI2.6H2O 12.164, CaCI2.2H2O 4.41, MgSO4.7H20 14.7, K2HPO4 1.044, NaHC03 15.0, H3BO3 0.1855, MnCI2.4H2O 0.415, ZnCI2 0.00327, CoCI2.6H2O 0.00143, CuCI2.2H2O 0.000012, NaMoO4.2H2O 0.00726, FeCl3.6H2O 0.159, Na2-EDTA 0.3, . pH 7.5 +/- 0.1.
- Any deformed or abnormal cells observed: None reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ± 1ºC

pH:

0-hours pH: 7.4 – 7.5
72-hours pH: 8.2 - 9.8.
This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the gaseous CO2 transfer rate. This effect was considered exaggerated by the requirement to conduct the test in conical flasks sealed with ground glass stoppers due to the possible volatile nature of the test item, this further reduced gaseous exchange between the test vessels and the atmosphere. This increase in the control cultures was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion (increased by factor 132).

Nominal and measured concentrations:

Preliminary range finding test : 0 (control), 0.12, 1.2 and 12 mg/L nominal test item concentration
Definitive Test: 0 (control), 0.538, 1.18, 2.56, 5.55 and 11.7 mg/L analytically confirmed initial concentration
Time Weighted Mean Measured equivalent concentrations: 0 (control), of 0.255, 0.430, 0.747, 1.34 and 2.41 mg/L
Samples from control replicates and test group replicates of equal nominal concentration were pooled for analysis. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): Closed – Static. Vessels glass stoppered.
- Headspace, fill volume: 100 mL fill volume, approximately 150 mL headspace
- Aeration: Vessel shaken continuously.
- Renewal rate of test solution (frequency/flow rate): Not applicable.
- Initial cells density: nominal: ca. 1x10^4 cells/mL
- Control end cells density: Definitive test: mean: ca. 1.30 x10^6 cells/mL
- No. of vessels per concentration (replicates): 3 replicates of each test concentration
- No. of vessels per control (replicates): 3 replicates of the control
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: Yes. ASTM medium, according to OECD 201, Annex 3
NaNO3 25.5, MgCI2.6H2O 12.164, CaCI2.2H2O 4.41, MgSO4.7H20 14.7, K2HPO4 1.044, NaHC03 15.0, H3BO3 0.1855, MnCI2.4H2O 0.415, ZnCI2 0.00327, CoCI2.6H2O 0.00143, CuCI2.2H2O 0.000012, NaMoO4.2H2O 0.00726, FeCl3.6H2O 0.159, Na2-EDTA 0.3, . pH 7.5 +/- 0.1.

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No.
- Intervals of water quality measurement: Start and end of test.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: Intensity approximately 7000 lux
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter Multisizer II particle counter
- Other: Microscopic evaluation of the cells was carried out a0.5t the end of the exposure for abnormalities.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable. Concentrations justified from the results of the range finding study.
- Range finding study: Yes.
- Test concentrations: Two replicates per concentration were exposed to 0 (control), 0.12, 1.2 and 12 mg/L (nominal ; based on the results of the pre-study media preparation trial)
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: - mg/L ; time weighted mean measured concentration

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.34 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: time weighted mean measured concentration

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits: - mg/L ; time weighted mean measured concentration

Details on results:

- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the end of the exposure period revealed no abnormalities in the test item concentrations or controls.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: Not reported. It was speculated in that adsorption to algal cells may also have been a significant factor in the decrease in measured test concentrations observed over the test period
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: Low dose stimulation was observed in the range finder and in the definitive limit test.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Differences in nominal versus measured between with and without algae speculated to be due to test item adsorption to algal cells.
- Effect concentrations exceeding solubility of substance in test medium: No.

Reported statistics and error estimates:

Statistical analysis of the area under the growth curve 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 and Dunnett's multiple comparison procedure for comparing several treatments with a control. There were no statistically significant differences between the control, 0.255, 0.430, 0.747 and 1.34 mg/L test concentrations (P >:0.05), however all other test concentrations were significantly different (P

Table 1. Percentage reduction in growth rate and inhibition of yield in the definitive test

Measured initial test item concentration	Time weighted mean measured concentration		Growth Rate	Inhibition of Growth Rate	Biomass (area under curve at 72h)		Inhibition
[mg/L]	[mg/L]		[0 – 72 h]	[%]	[1x10^7]		[%]
Control		Mean	0.068	-	2.14		-
0.538		Mean	0.068	0	2.12	(+)	1
1.18		Mean	0.068	0	2.27	(+)	6
2.56		Mean	0.068	0	2.36	(-)	11
5.55		Mean	0.068	0	2.11	(-)	1
11.7		Mean	0.051	25	0.715	(-)	67

(+) : % increase in growth as compared to controls

(-) : % decrease or inhibition of growth as compared to controls

Validity criteria fulfilled:

yes

Conclusions:

The test item 48h-EC50 was > 2.4 mg/L (C.I: – mg/L) based on time weighted mean measured concentrations. The corresponding NOEC was 1.34 mg/L.

Executive summary:

The algal growth inhibition to Scenedesmus subspicatus was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth over a period of 72 hours. Following preliminary range-finding tests (conducted over a range of nominal test concentrations of 0 (control), 0.12, 1.2 and 12 mg/L), algae was exposed to an aqueous solution of the test item at analytically confirmed initial concentrations of: 0 (control), 0.538, 1.18, 2.56, 5.55 and 11.7 mg/L mg/L (three replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1°C under static conditions. The equivalent Time Weighted Mean Measured concentrations were : 0 (control), of 0.255, 0.430, 0.747, 1.34 and 2.41 mg/L. the test item solutions were prepared by stirring an excess of test item (equivalent to 1000 mg/L) in culture medium for a period of time prior to removing the aqueous phase by mid-depth siphon to produce a saturated solution. This saturated solution was then diluted as necessary, to produce the remaining test groups. Samples of centrifuged and uncentrifuged test media were analysed to determine the amount of dissolved test item present within the test system. Temperature was acceptably maintained during the test. The pH values of the control cultures were observed to increase from pH 7.0 at 0 hours to pH 8.2-9.8 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the gaseous CO2 transfer rate. This effect was considered exaggerated by the requirement to conduct the test in conical flasks sealed with ground glass stoppers due to the possible volatile nature of the test item, this further reduced gaseous exchange between the test vessels and the atmosphere. This increase in the control cultures was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion (increased by factor 132). The validity criteria of the test guideline were considered fulfilled. Chemical analysis of the test solutions at 0 hours showed measured concentrations ranging from 0.645 to 12.0 mg/L for the uncentrifuged samples and from 0.538 to 11.7 mg/L for the centrifuged samples. A marked decline was shown in the measured concentrations at 72 hours for both the uncentrifuged and centrifuged samples to below the limit of quantitation (LOQ) of the analytical method employed. This decline was considered to be due to adsorption of the test item to algal cells. On this basis effect levels were based on mean measured concentrations to give a worst case analysis of the data. The EC50 for growth rate reduction (72h-ErC50) was > 2.4 mg/L and the corresponding NOEC was 1.34 mg/L based on time weighted mean measured concentrations.

Description of key information

ErC50 (algae; growth rate) = > 2.4 (C.I. - ) mg/L based on time weighted mean measured concentrations, 72hour-freshwater, OECD TG 201, 2000

NOEC (algae) = 1.34 mg/L based on time weighted mean measured concentrations, 72hour-freshwater, OECD TG 201, 2000

Key value for chemical safety assessment

EC50 for freshwater algae:

2.4 mg/L

EC10 or NOEC for freshwater algae:

1.34 mg/L

Additional information

Key study : OECD TG 201, 2000 : The algal growth inhibition to Scenedesmus subspicatus was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth over a period of 72 hours. Following preliminary range-finding tests (conducted over a range of nominal test concentrations of 0 (control), 0.12, 1.2 and 12 mg/L), algae was exposed to an aqueous solution of the test item at analytically confirmed initial concentrations of: 0 (control), 0.538, 1.18, 2.56, 5.55 and 11.7 mg/L mg/L (three replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1°C under static conditions. The equivalent Time Weighted Mean Measured concentrations were : 0 (control), of 0.255, 0.430, 0.747, 1.34 and 2.41 mg/L. the test item solutions were prepared by stirring an excess of test item (equivalent to 1000 mg/L) in culture medium for a period of time prior to removing the aqueous phase by mid-depth siphon to produce a saturated solution. This saturated solution was then diluted as necessary, to produce the remaining test groups. Samples of centrifuged and uncentrifuged test media were analysed to determine the amount of dissolved test item present within the test system. Temperature was acceptably maintained during the test. The pH values of the control cultures were observed to increase from pH 7.0 at 0 hours to pH 8.2-9.8 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the gaseous CO2 transfer rate. This effect was considered exaggerated by the requirement to conduct the test in conical flasks sealed with ground glass stoppers due to the possible volatile nature of the test item, this further reduced gaseous exchange between the test vessels and the atmosphere. This increase in the control cultures was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion (increased by factor 132). The validity criteria of the test guideline were considered fulfilled. Chemical analysis of the test solutions at 0 hours showed measured concentrations ranging from 0.645 to 12.0 mg/L for the uncentrifuged samples and from 0.538 to 11.7 mg/L for the centrifuged samples. A marked decline was shown in the measured concentrations at 72 hours for both the uncentrifuged and centrifuged samples to below the limit of quantitation (LOQ) of the analytical method employed. This decline was considered to be due to adsorption of the test item to algal cells. On this basis effect levels were based on mean measured concentrations to give a worst case analysis of the data. The EC50 for growth rate reduction (72h-ErC50) was > 2.4 mg/L and the corresponding NOEC was 1.34 mg/L based on time weighted mean measured concentrations.