1-cyclohexylethyl butyrate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22/02/2002 - 21/03/2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study according to international guideline (OECD guideline 201) under GLP. Validity criteria are just met. The growth rate in the controls was fairly low and only one concentration gave a growth rate reduction between 20 and 80% (other concentrations have either 0% or 100% reduction).

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.81, 1.63, 3.25, 6.5 and 12.9 mg/l (nominal)
- Sampling method: Water samples (duplicates) were collected after 0 and 72 hours.
- Sample storage conditions before analysis: samples were stored at -20ºc until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An amount of test material (1.10 g) was dispersed in 11 litres of reconstituted water. This was then stirred at a temperature of 25ºC using a propeller stirrer at approximately 2000 rpm for approximately 48 hours to give an initial test material dispersion of 100 mg/l. After stirring for approximately 48 hours this dispersio undisslved test material was removed by filtration through a filter (0.2 um) to give a saturated solution of the test material (mean measured concentration of 12.9 mg/l at the end of the test).
Serial dlutions of the saturated solution were made to prepare stock solutions with nominal concentrations of 0.81, 1.63, 3.25 and 6.5 mg/l. An aliquot (1 litre) of each of these stock solutions was inoculated with algal suspension (5.0 ml) to give the required nominal test concentrations of 0.81, 1.63, 3.25, 6.5 and 12,9 mg/l). Each prepared test concentration was inverted several times to ensure adequate mixng and homogeneity.
- Controls: Yes

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus subspicatus
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Collection of algae and protozoa (CCAP), institute of Freshwater Ecology, The Ferry House, Far Sawrey, Ambleside, Cumbria.
- Method of cultivation: The culture was maintained in the laboratory at a temperature of 21 +/-1 ºC under continuous illuination of approximately 7000 lux and constant aeration.
- Initial cell density: 10^4 cells/ml

ACCLIMATION
- Culturing media and conditions (same as test or not): same

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 +/- 1ºC

pH:

7.2 - 9.4

Nominal and measured concentrations:

Nominal: 0.81, 1.63, 3.25, 6.5 and 12.9 mg/l
Time-Weighted mean measured concentrations: 0.10, 0.17, 0.34, 0.77 and 1.2 mg/l (for explanation of values used for determination of NOEC and EC50 see information on results below).

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml conical flask plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: glass, 250 ml filled with 100 ml medium
- Aeration: None
- Initial cell density: 1.03 x 10^4 cells/ml
- Control end cells density: 1.02 x 10^6 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition:
NaNO3: 25.5 mg/l
MgCl2.6H2O: 12.164 mg/l
CaCl2.2H2O: 4.41 mg/l
MgSO4.7H2O: 14.7 mg/l
K2HPO4: 1.044 mg/l
NaHCO3: 15.0 mg/l
H3BO3: 0.1855 mg/l
MnCl2.4H2O: 0.415 mg/l
ZnCl2: 0.00327 mg/l
FeCl3.6H2O: 0.159 mg/l
CoCl2.6H2O: 0.00143 mg/l
Na2.MoO4.2H2O: 0.00726 mg/l
CuCl2.2H2O: 0.000012 mg/l
Na2EDTA.2H2O: 0.30 mg/l
Na2SeO3.5H2O: 0.000010 mg/l

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionised water
- Culture medium different from test medium: No
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes
- Photoperiod: continuous illumination
- Light intensity and quality: 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0.0129, 0.129, 1.29 and 12.9 mg/l
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.95 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Remarks:

Concentrations as measured in the stability tests were used for determination of the EC50, see information on results below for more information.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.163 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Remarks:

Concentrations as measured in the stability tests were used for determination of the EC50, see information on results below for more information.

Basis for effect:

growth rate

Details on results:

For explanation of values used for determination of NOEC and EC50 see information on results below.
- Exponential growth in the control (for algal test): yes
- No abnormalities in cells detected after microscopic investigation after 0 and 72 hours

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeniety of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the area under the growth curve data at 72 hours for the control and all test concentratins to determine any statistically significant differences between the test adn control groups. All statistical analyses were performed using SAS.

Inhibition of growth rate and biomass:

Time-weighed mean measured test concentration (mg/L)	Area under curve at 72 h	% Inhibition	Growth rate (0-72h)	% Inhibition
Control	1.54E+07	-	0.064	-
0.10	1.95E+07	[27]	0.065	[2]
0.17	1.76E+07	[15]	0.062	3
0.34	2.75E+06	82	0.037	42
0.77	-2.75E+05	102	-0.018	128
1.2	-4.70E+05	103	-0.025	139

Cell densities an pH values in the definitive test:

Time-weighed mean measured test concentration (mg/L)		pH	Cell densities (cells per mL)				pH
		0h	0h	24h	48h	72h	72h
Control	R1 R2 R3 Mean	7.2 7.2 7.2	9.87E+03 9.61E+03 1.15E+04 1.03E+04	6.13E+04 7.18E+04 7.75E+04 7.02E+04	8.53E+04 8.60E+04 8.63E+04 8.59E+04	9.96E+05 1.00E+06 1.06E+06 1.02E+06	9.3 9.3 9.3
0.10	R1 R2 R3 Mean	7.1 7.1 7.1	1.08E+04 1.01E+04 1.25E+04 1.12E+04	8.69E+04 9.56E+04 8.45E+04 8.90E+04	1.56E+05 1.57E+05 1.50E+05 1.54E+05	1.17E+06 1.24E+06 1.19E+06 1.20E+06	9.3 9.4 9.3
0.17	R1 R2 R3 Mean	6.9 6.9 6.9	1.34E+04 1.34E+04 1.18E+04 1.29E+04	8.71E+04 7.87E+04 7.75E+04 8.11E+04	1.34E+05 1.32E+05 1.31E+05 1.32E+05	1.13E+06 1.06E+06 1.41E+06 1.11E+06	8.8 8.8 8.8
0.34	R1 R2 R3 Mean	6.8 6.8 6.8	1.34E+04 1.32E+04 1.34E+04 1.33E+04	2.70E+04 2.49E+04 2.44E+04 2.54E+04	2.56E+04 2.47E+04 2.41E+04 2.48E+04	1.97E+05 1.89E+05 2.01E+05 1.96E+05	7.7 7.7 7.7
0.77	R1 R2 R3 Mean	6.8 6.8 6.8	1.27E+04 1.15E+04 1.15E+04 1.19E+04	1.18E+04 6.92E+03 1.18E+04 1.02E+04	8.06E+03 5.92E+03 5.52E+03 6.50E+03	4.86E+03 2.13E+03 2.74E+03 3.24E+03	7.5 7.4 7.4
1.2	R1 R2 R3 Mean	6.7 6.7 6.7	1.29E+04 1.31E+04 1.29E+04 1.30E+04	9.22E+03 8.11E+03 1.02E+04 9.19E+03	2.58E+03 2.77E+03 2.51E+03 2.62E+03	2.09E+03 1.55E+03 3.03E+03 2.22E+03	7.2 7.2 7.2

Graphs representing mean cell densities vs. time and inhibition of growth rate and biomass based on time-weighted mean measured test concentrations are included as illustrations below.

The measured values are in line with the nominal values and all above 80%. Several stability tests have been done without algae showing that at the end of exposure the loss in concentration was maximally 40% except at the low concentration where it is reduced to 10% of nominal.

At the end of the algae test the measured values are below the Limit of Quantification (LoQ). The disappearance of the test may be due to volatilization despite precautions taken, or adsorption to algae, though not seen in the stability tests. A TWA or geometric mean between initial concentrations and LoQ is not justified because the LoQ would drive the toxicity values. In addition, it can be seen that the toxicity is highest at 24h and increasing at the highest two concentration being indicative for significant exposure to the test substance. In the stability tests (also 72h) it is also shown that significant exposure can be expected. In view of the highest toxicity seen at 24 hours, the t=0 concentration will be used to derive the EC50 and NOEC values.

Other than issues with stability of the substance also a true EC50 is difficult to determine statistically, given that there is only one valid value giving a growth rate reduction between 20 and 80%. The NOEC is nominal 1.63 mg/L and measured 1.32 mg/L (> 80%, thus no correction is needed here). The EC50 is between 1.63 mg/L and 6.5 mg/L nominal close to 3.25 mg/L (42% reduction of growth rate). The EC42 is considered to be an EC50 (because the statistically derived value is not correct).

Validity criteria fulfilled:

yes

Remarks:

>16-fold increase in cell density in controls, variation coefficients within limits.

Conclusions:

The 72h-ErC50 of Cyclobutanate towards Scenedesmus subspicatus is 3.25 and the NOEC is 1.63 mg/l.

Executive summary:

The acute toxicity of Cyclobutanate towards Scenedesmus subspicatus was investigated according to OECD guideline 201 under GLP. Algal cells were exposed to nominal concentrations of 0.81, 1.63, 3.25, 6.5 and 12.9 mg/l and were observed for 72 hours. In a preliminary study, the measured concentration at t=0 were in line with the nominal ones (80-120%). After 72h the test concentrated diminished with 40%, during storage in light or dark or with or without algae. In the main test at 72h the test concentrations were all below the LoQ. The result show that the EC50 at t=24 h is the same as at 72h and no recovery takes place after disappearance of the parent substance. This leads to the conclusion that Cyclobutanate has a direct effect on algae and therefore the toxicity values based on the geometric mean and the LoQ was not considered justified. The toxicity values have been based on the concentrations measured in the preliminary stability tests. This means that the NOEC results in a value of 0.163 (1.63 x 0.1, being worst case because at 1 mg/l the loss was 90% but at 3.25 only 40%). The EC50 results in a value 1.95 mg/l (Taking the EC42 as the EC50 and 40% loss on the nominal concentration: 3.25 x 0.6)

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See 'Attached justification'

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.52 mg/L

Remarks on result:

other: as determined from read-across to cyclobutanate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.13 mg/L

Remarks on result:

other: as determined from read-across to cyclobutanate

Description of key information

 The toxicity to aquatic algae is assessed based on read-across from the close structural analogue Cyclobutanate (CAS# 113889-23-9) and the ErC50 value and NOErC value determined at 1.52 mg/L and 0.13 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.52 mg/L

EC10 or NOEC for freshwater algae:

0.13 mg/L

Additional information

The acute toxicity of Cyclobutanate towards Scenedesmus subspicatus was investigated according to OECD guideline 201 under GLP. Algal cells were exposed to nominal concentrations of 0.81, 1.63, 3.25, 6.5 and 12.9 mg/l and were observed for 72 hours. In a preliminary study, the measured concentration at t=0 were in line with the nominal ones (80-120%). After 72h the test concentrated diminished with 40%, during storage in light or dark or with or without algae. In the main test at 72h the test concentrations were all below the LoQ. The result show that the EC50 at t=24 h is the same as at 72h and no recovery takes place after disappearance of the parent substance. This leads to the conclusion that Cyclobutanate has a direct effect on algae and therefore the toxicity values based on the geometric mean and the LoQ was not considered justified. The toxicity values have been based on the concentrations measured in the preliminary stability tests. This means that the NOEC results in a value of 0.163 (1.63 x 0.1, being worst case because at 1 mg/l the loss was 90% but at 3.25 only 40%). The EC50 results in a value 1.95 mg/l (Taking the EC42 as the EC50 and 40% loss on the nominal concentration: 3.25 x 0.6).