Cyclopropanecarboxylic acid, 2-[1-(3,3-dimethylcyclohexyl)ethoxy]-2-methylpropyl ester

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:

24-05-2004 to 24-06-2004

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

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23 (2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: May 2002 ; signature: September 2002

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Singular samples for possible analysis were taken from all test concentrations and the control; Frequency at t=0 h, t=24 h and t=72 h
- Sampling method: Singular samples for possible analysis were taken from all test concentrations and the control according to the schedule:
1. At the start of the test 0-hours from the freshly prepared solutions.
2. The end of the test from the 72-hour old solutions, volume: 4.5 mL for controls, 0.5, 0.8 and 1.3 mg/L test item groups; 1.5 mL for 2.0 and 3.2 mg/L test item groups ; from the approximate centre of the test vessels.
- Sample storage conditions before analysis: Samples were used on day of sampling; reserve samples were stored in a freezer until analysis (≤ -15°C). Reserve samples of 1.5 mL or 4.5 mL were taken for possible analysis if needed.

Vehicle:

yes

Remarks:

acetone

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): 3 days (in pre-culture under the same conditions as the test).
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: 3 days pre-culture.
- Culturing media and conditions (same as test or not): No.
Stock Culture Medium M1 ; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis) with the following composition: NaNO3 500 mg/L; K2HPO4.3H2O 52 mg/L; MgSO4.7H2O 75 mg/L; Na2CO3.10H2O 54 mg/L; C6H8O7.H2O 6 mg/L; NH4NO3 330 mg/L; CaCl2.2H2O 35 mg/L; C6H5FeO7.xH2O 6 mg/L; H3BO3 2.9 mg/L; MnCl2.4H2O 1.81 mg/L; ZnCl2 0.11 mg/L; CuSO4.5H2O 0.08 mg/L; (NH4)6Mo7O24.4H2O 0.018 mg/L
Pre-Culture and definitive test adjusted-Medium M2 : NH4Cl 15 mg/L; MgCl2.6H2O 12 mg/L; CaCl2.2H2O 18 mg/L; MgSO4.7H2O 15 mg/L; KH2PO 1.6 mg/L; FeCl3.6H2O 64 µg/L; Na2EDTA.2H2O 100 µg/L; H3BO3 185 µg/L; MnCl2.4H2O 415 µg/L; ZnCl2 3 µg/L; CoCl2.6HO 1.5 µg/L; CuCl2.2H2O 0.01 µg/L; Na2MoO4.2H2O 7 µg/L; NaHCO3 7 mg/L; Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/l); pH 8.1 ± 0.2
- Any deformed or abnormal cells observed: None reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

In accordance with the OECD TG 201 guidelines.

Hardness:

Ca+Mg: 0.24 mmol/l (24 mg CaCO3/l)

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 23.8 and 24.2 °C. Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).

pH:

0 hours: pH 8.2 ± 0.1 ; 72 hours: pH 8.2 (definitive test concentrations) and pH 8.2 (controls). pH did not vary more than 1.5 units.

Nominal and measured concentrations:

Range-finder test: 0.05, 0.5 and 5.0 mg/L nominal concentrations with solvent control (acetone no test item) and blank control (no test item no solvent)
Final test: 0.5, 0.8, 1.3, 2.0 and 3.2 mg/L with solvent control (acetone no test item) and blank control (no test item no solvent)
The 3.2 mg/L nominal had an equivalent 1.9 mg/L geometric mean measured average concentration

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass.
- Type (delete if not applicable): Closed - Static
- Material, size, headspace, fill volume: Glass, 100 mL, containing 50 mL test solution ; closed airtight (capped)
- Aeration: Vessel shaken continuously.
- Initial cells density: 1 x 10^4 cells/mL.
- Control end cells density: Mean (of replicates after 72 hours) 32.3 x10^4 cells/mL (solvent control) and 32.4 x10^4 cells/mL (blank control)
- No. of vessels per concentration (replicates): 3 replicates of each test concentration
- No. of vessels per control (replicates): 6 replicates of the control
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: Yes. adjusted-Medium M2.
- Detailed composition if non-standard medium was used: adjusted M2 according to OECD 201 using RO-water: NH4Cl: 15 mg/L; MgCl2.6H2O: 12 mg/L; CaCl2.2H2O: 18 mg/L; MgSO4.7H2O: 15 mg/L; KH2PO4: 1.6 mg/L; FeCl3.6H2O: 64 µg/L; Na2EDTA.2H2O: 100 µg/L; H3BO3: 185 µg/L; MnCl2.4H2O: 415 µg/L; ZnCl2: 3 µg/L; CoCl2.6H2O: 1.5 µg/L; CuCl2.2H2O: 0.01 µg/L; Na2MoO4.2H2O: 7 µg/L; NaHCO3: 7 mg/L; Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L); HEPES buffer 6 mmol/L; pH 8.1 ± 0.2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis)
- Culture medium different from test medium: Yes. Medium M1 used for stock culture medium. adjusted-Medium M2 used for pre-culture medium and test medium. See above for more information.
- Intervals of water quality measurement: Not reported.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 81 to 109 µE/m2/s.
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Spectrophotometer at 720nm with immersion probe (pathlength = 20mm). Algal medium used as blank.
- Other: Initial cell density: Cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: In definitive test justified from the results of the range finding study.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes.
- Test concentrations: 0.05, 0.5 and 5.0 mg/L nominal concentrations with solvent control (acetone no test item) and blank control (no test item no solvent) in the range-finding test. In the definitive test 0.5, 0.8, 1.3, 2.0 and 3.2 mg/L with solvent control (acetone no test item) and blank control (no test item no solvent)
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: - mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: - mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 1.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): No abnormalities reported.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: Not reported.
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: Yes. At low concentrations 0.5 mg/L nominal yield inhibition was negative between 24-72h. This was low dose stimulation and was limited to < 1% and was taken into account in the data analysis and effect level calculations.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Final test: Analysis of the samples taken from nominally 3.2 mg/L showed that measured concentrations decreased from 2.3 mg/L at the start to 1.8 mg/L after 24 hours of exposure. At the end of the 72- hour test period the measured concentration was 2.0 mg/L. Hence, analytical results obtained between 24 and 72 hours were in agreement with the results obtained during the range-finding test. This confirmed the thesis that the maximum solubility in algal growth medium (M2 medium) was approximately 2.0 mg/L and excluded possible adsorption.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate inhibition (72h-ERC50) was 1.0 mg/L with a 95% confidence interval ranging from 0.58 to 1.8 mg/L. The historical ranges for growth rate inhibition using standard ‘open’ systems range between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the present test in closed systems is within the historical range. The EC50 for cell growth inhibition (72h-EBC50) was 0.49 mg/L with a 95% confidence interval ranging from 0.28 to 0.85 mg/L. The historical ranges for yield inhibition using standard ‘open’ systems lie between 0.49 and 1.41 mg/L. Hence, the 72h-EBC50 for the present test for open or closed systems was within range.
- Other: The sensitivity of the test system was in agreement with the historical data.

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. Statistical analysis of the data was not needed as the effects recorded were not significant (<10%). No EC50-values could be calculated because the test substance proved to be non-toxic (EC50 > maximum soluble concentration).

Full details are reported in the full study report.

Table 1. Mean cell densities (x10^4 cells/mL) during the range-finding test

Time (h)	Test item, nominal concentration (mg/L)
	Solvent Control	0.05	0.5	5.0	Blank Control
0	1.0	1.0	1.0	1.0	1.0
24	7.4	7.4	7.9	4.3	7.4
48	27.6	29.1	26.3	4.4	27.7
72	52.5	49.8	49.1	6.2	47.4

 

Table 2. Percentage reduction of growth rate (total test period) and percentage inhibition of cell growth during the final test

Test item, nominal concentration (mg/L)	Growth rate (0 – 72h)		Cell Growth (0 – 72h)
	mean	% reduction	mean	% inhibition
0 (Solvent Control)	0.04824		776.88
0.5	0.04871	-1.0	773.28	0.5
0.8	0.04896	-1.5	781.40	-0.6
1.3	0.04884	-1.2	787.64	-1.4
2.0	0.04888	-1.3	815.08	-4.9
3.2 (1.9)	0.05307	-10.0	952.68	-22.6
Blank control	0.04828	-0.1	791.54	-1.9

( ) : average exposure concentration

 

Table 3. Mean cell densities (x10^4 cells/mL) during the final test

Time (h)	Test item, nominal concentration (mg/L)
	Solvent Control	0.5	0.8	1.3	2.0	3.2 (1.9)	Blank Control
0	1.0	1.0	1.0	1.0	1.0	1.0	1.0
24	3.8	4.0	3.7	4.2	5.3	4.5	4.1
48	15.0	14.0	14.4	14.3	14.3	14.8	15.2
72	32.3	33.4	34.0	33.7	33.8	45.7	32.4

( ) : average exposure concentration

Validity criteria fulfilled:

yes

Conclusions:

The test item 72h-ErC50 for growth rate reduction was > 1.9 (C.I. – ) mg/L based on geometric mean measured average concentrations. The corresponding NOEC was > 1.9 mg/L. Both effect parameters exceeded the maximum water solubility in algal test medium.

Executive summary:

The algal growth inhibition to Pseudokirchneriella subcapitata, 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 rate and cell growth over a period of 72 hours. Following a range finding study at 0.05, 0.5 and 5.0 mg/L nominal concentrations with solvent control (acetone no test item) and blank control (no test item no solvent), a definitive study was conducted under static conditions with an initial cell density of 1.0x10^4 cells/mL. Five test item solutions were employed with a concentrations of 0.5, 0.8, 1.3, 2.0 and 3.2 mg/L nominal with solvent control (acetone, no test item) and blank control (no test item, no solvent). Three replicates were tested for each test item concentration and six replicates for the control under constant illumination and shaking at a temperature of 22 ± 2 °C. Environmental conditions were within the acceptable limits. The concentrations of the test item were analytically verified via GC-FID at 0 hours (test start), 24 hours and at 72 hours (test end) of the exposure. Analysis of the samples taken from nominally 3.2 mg/L showed that measured concentrations decreased from 2.3 mg/L at the start to 1 .8 mg/L after 24 hours of exposure. At the end of the 72-hour test period the measured concentration was 2.0 mg/L. Hence, analytical results obtained between 24 and 72 hours were in agreement with the results obtained during the range-finding test. This confirmed the thesis that the maximum solubility in algal growth medium (M2 medium) was approximately 2.0 mg/L. The study met the acceptability criteria prescribed by the protocol and was considered valid. No inhibition of cell growth or reduction of growth rate was recorded at any of the concentrations of test item tested including a concentration (nominal 3.2 mg/L) exceeding the water solubility limit. In conclusion, the 72h-EC50 for both growth inhibition and growth rate reduction exceeded the maximum solubility in algal test medium, corresponding to an average exposure concentration of 1.9 mg/L.

Description of key information

EC50 (aquatic algae; growth rate) : > 1.9 mg/L (C.I. – ) based on geometric mean measured average concentrations, 72 hour, freshwater, OECD TG 201, 2004

NOEC (aquatic algae; growth rate) : > 1.9 mg/L based on geometric mean measured average concentrations, 72 hour, freshwater, OECD TG 201, 2004

Key value for chemical safety assessment

EC50 for freshwater algae:

1.9 mg/L

EC10 or NOEC for freshwater algae:

1.9 mg/L

Additional information

Key study : OECD TG 201, 2004 : The algal growth inhibition to Pseudokirchneriella subcapitata, 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 rate and cell growth over a period of 72 hours. Following a range finding study at 0.05, 0.5 and 5.0 mg/L nominal concentrations with solvent control (acetone no test item) and blank control (no test item no solvent), a definitive study was conducted under static conditions with an initial cell density of 1.0x10^4 cells/mL. Five test item solutions were employed with a concentrations of 0.5, 0.8, 1.3, 2.0 and 3.2 mg/L nominal with solvent control (acetone, no test item) and blank control (no test item, no solvent). Three replicates were tested for each test item concentration and six replicates for the control under constant illumination and shaking at a temperature of 22 ± 2 °C. Environmental conditions were within the acceptable limits. The concentrations of the test item were analytically verified via GC-FID at 0 hours (test start), 24 hours and at 72 hours (test end) of the exposure. Analysis of the samples taken from nominally 3.2 mg/L showed that measured concentrations decreased from 2.3 mg/L at the start to 1.8 mg/L after 24 hours of exposure. At the end of the 72-hour test period the measured concentration was 2.0 mg/L. Hence, analytical results obtained between 24 and 72 hours were in agreement with the results obtained during the range-finding test. This confirmed the thesis that the maximum solubility in algal growth medium (M2 medium) was approximately 2.0 mg/L. The study met the acceptability criteria prescribed by the protocol and was considered valid. No inhibition of cell growth or reduction of growth rate was recorded at any of the concentrations of test item tested including a concentration (nominal 3.2 mg/L) exceeding the water solubility limit. In conclusion, the 72h-EC50 for both growth inhibition and growth rate reduction exceeded the maximum solubility in algal test medium, corresponding to an average exposure concentration of 1.9 mg/L.