3,7-dimethyloct-1-en-3-yl acetate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-10-14 to 2014-12-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

adopted 2006, corrected 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Chemical name: 3,7-dimethyloct-1-en-3-ol
- CAS Number: 18479-49-7

Analytical monitoring:

yes

Details on sampling:

- Sampling method: duplicate samples taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae); simultaneously duplicate samples also taken from negative control; for sampling at end of the test, test medium of treatment replicates pooled
- Sample storage conditions before analysis: stored deep-frozen (at about -20 °C) immediately after sampling

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the highest nominal concentration of 100 mg/L was prepared in a closed system by dissolving 57.94 mg of the test item completely in 570 mL of test water using intense stirring for three hours at room temperature in the dark. The test medium of the highest test concentration was diluted with test water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test.
- Controls: negative control (test medium only), positive control (in seoarate experiment)
- Test concentration separation factor: In the definitive test, the enlarged spacing factor of 3.2 between the test concentrations was chosen based on the results of the range-finding test that documented a rather flat concentrationeffect relationship; therefore, a large concentration range had to be tested.
- Evidence of undissolved material: no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
Raphidocelis subcapitata (KORSIKOV) nov. comb.
Syn.: Kirchneriella subcapitata KORSIKOV
Syn.: Selenastrum capricornutum PRINTZ
Syn.: NIVA-CHL 1
- Strain: 61.81 SAG
- Source (laboratory, culture collection): laboratory culture, originally supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen, Germany
- Method of cultivation: The algae were cultivated at the laboratory under standardized conditions according to the test guidelines. An inoculum culture was set up four days before the start of the exposure. The algae were cultivated under the test conditions. The inoculum culture was diluted three-fold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.

ACCLIMATION
- Acclimation period: none
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Post exposure observation not performed

Hardness:

0.15 mmol/L (= 15 mg/L as CaCO3)

pH:

7.5 - 7.9

Dissolved oxygen:

not specified

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

nominal: 0 (control), 0.32, 1, 3.2, 10, 32, 100; measured: < LOQ, 0.333, 1.01, 3.21, 9.66, 30.6, 95.5 mg/L (fresh medium, 0 d); < LOQ, 0.306, 0.960, 3.13, 9.36, 30.1, 91.6 mg/L (aged medium, 3 d) (values represent rounded results obtained by calculation using exact raw data); corresponding to 92 - 104 % of nominal concentration

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed, tightly sealed with glass stoppers
- Material, size, headspace, fill volume: glass, 50 mL nominal volume, no headspace, 60 mL fill volume
- Aeration: no
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes (AAP Medium)


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: approximately 6500 Lux (range: 5700 to 7050 Lux); fluorescent tubes (Philips TLD 36W-1/840)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence measurement (BIOTEK Multi-Detection Microplate Reader, Model FLx800, wavelength: excitation 440 nm, emission 680 nm).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2, chosen based on the results of the range-finding test that documented a rather flat concentration effect relationship
- Range finding study: yes
- Test concentrations: 0 (control), 0.1, 1.0, 10, 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: yes

CULTURING APPARATUS
-Details on culturing apparatus used: incubation in a temperature-controlled water bath at a temperature of 24 °C and illuminated by fluorescent tubes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 61-66 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

28 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% confidence interval: 23-33 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

27 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 24-30 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% confidence interval: 2.6-7.3 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

27 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 35-41 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

9.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% confidence interval: 6.3-13

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 32 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

Results with reference substance (positive control):

- EC50: 72-hour EC50 for the growth rate was determined to be 1.1 mg/L

Reported statistics and error estimates:

The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and
their 95% confidence intervals were calculated by Weibull Analysis. For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Dunnett t-test or Williams t-test, where appropriate.

Table 1: Average Growth Rates (μ)

Nominal test item concentratiion [mg/L]
	0-24 h		0-48 h		0-72 h
	µ	Ir [%]	µ	Ir [%]	µ	Ir [%]
Control	1.501	0.0	1.790	0.0	1.825	0.0
0.32	1.386	7.6	1.783	0.4	1.805	1.1
1.0	1.406	6.3	1.780	0.6	1.804	1.2
3.2	1.468	2.2	1.761^*	1.6	1.766^*	3.2
10	1.405	6.4	1.719^*	4.0	1.765^*	3.3
32	1.251^*	16.6	1.480^*	17.3	1-572^*	13.9
100	0.424	71.8	0.416^*	76.8	0.286^*	84.3

*: mean value statistically significantly lower than in the control (according to Williams t-test one-sided smaller, α = 0.05)

 

Table 2: Yield Y

Nominal test item concentratiion [mg/L]
	0-24 h		0-48 h		0-72 h
	Y	Iy [%]	Y	Iy [%]	Y	Iy [%]
Control	2.3	0.0	22.9	0.0	155.7	0.0
0.32	2.0	13.4	22.5	1.4	146.4	5.9
1.0	2.0	11.4	22.4	2.0	146.4	6.0
3.2	2.2	4.3	21.5	5.8	130.4^#	16.2
10	2.0	11.7	19.7^#	13.8	129.9^#	16.5
32	1.6^#	28.2	12.0^#	47.6	72.5^#	53.4
100	0.3^#	84.8	0.8^#	96.3	0.9^#	99.4

#: mean value statistically significantly lower than in the control
(according to Dunnett t-test, one-sided smaller, α = 0.05)

 

Table 3: Analytical results

Timepoint [day]	Nominal concentration of test item [mg/L]	Measured concentration of test item x [mg/L]	Sample preparation factor F	Determined concentration of test item c [mg/L]	% of nominal concentration [%]
0 (fresh)	Control	*	0.5	< LOQ	n.a.
	0.32	0.666	0.5	0.333	104
	1	2.02	0.5	1.01	101
	3.2	6.42	0.5	3.21	100
	10	1.93	5	9.66	97
	32	6.12	5	30.6	96
	100	1.91	50	95.5	96
3 (old)	Control	*	0.5	< LOQ	n.a.
	0.32	0.612	0.5	0.306	96
	1	1.92	0.5	0.960	96
	3.2	6.26	0.5	3.13	98
	10	1.87	5	9.36	94
	32	6.02	5	30.1	94
	100	1.83	50	91.6	92

*the measured concentration was below the lowest calibration concentration
LOQ = 0.114 mg test item/L
n.a. = not applicable
The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

 

 

CONSIDERATIONS ON VALIDITY OF STUDY

In the control, the biomass increased by a factor of 239 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 16 %. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35 %. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 0.6 %. According to the OECD test guideline, the coefficient of variation must not be higher than 7 %. Thus, the validity criterion was fulfilled. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period. The pH rose in the control from 7.7 at test start to 7.9 at test end fulfilling the requirement of the OECD guideline that the pH of the control medium should not increase by more than 1.5 units during the test. The pH of the test media was in the range of 7.5 to 7.9 during the test period. The water temperature during the test was maintained at 24 °C.

Validity criteria fulfilled:

yes

Conclusions:

In a 72-hour static test according to the OECD Guideline 201 (adopted 2006, corrected 2011) and the Commission Regulation (EC) No 761/2009, C.3 with the freshwater green algal species Pseudokirchneriella subcapitata the 72-hour EC50 and EC10 for the growth rate were determined to be 64 mg/L and 27 mg/L, respectively. The 72-hour EC50 and EC10 based on yield was found to be 28 mg/L and 4.9 mg/L, respectively. The 72-hour NOEC both for the growth rate and yield was 1 mg/L.

Executive summary:

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (adopted 2006, corrected 2011) and the Commission Regulation (EC) No 761/2009, C.3. The nominal test item concentrations tested were 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (3 replicates per concentration). Additionally, a control group was tested in parallel (6 replicates). As the test item is a volatile substance, the test was performed using glass vessels completely filled with test medium, minimizing the headspace, that were tightly sealed with glass stoppers to avoid losses of test item. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. The measured concentrations of the test item in the test media of all test concentrations were between 96 and 104 % of the nominal values at the start of the test and between 92 and 98% at the end of the test. Thus, the correct dosing of the test item was confirmed. The test item was stable in the test media over the test period of 72 hours. The biological results were hence related to the nominal concentrations of the test item. The test item had a significant inhibitory effect on the growth rate μ of the algae after the test period of 72 hours at the concentration of 3.2 mg/L and at all higher test concentrations. The yield Y of the algae was statistically significantly reduced at the test concentration of 3.2 mg/L. The 72-hour NOEC based on the growth rate μ was determined to be 1.0 mg/L, since up to and including this test concentration the growth rate of the algae after 72 hours was not significantly lower than in the control. The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 32 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration. In the control, the biomass increased by a factor of 239 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 16 %. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35 %. Thus, the validity criterion was fulfilled.  The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 0.6%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period. The pH rose in the control from 7.7 at test start to 7.9 at test end fulfilling the requirement of the OECD guideline that the pH of the control medium should not increase by more than 1.5 units during the test. The pH of the test media was in the range of 7.5 to 7.9 during the test period. The water temperature during the test was maintained at 24 °C. As a result, the 72-hour EC50 and EC10 for the growth rate were determined to be 64 mg/L and 27 mg/L, respectively. The 72-hour EC50 and EC10 based on yield was found to be 28 mg/L and 4.9 mg/L, respectively. The 72-hour NOEC both for the growth rate and yield was 1 mg/L.

Description of key information

In a 72-hour static test according to the OECD Guideline 201 (adopted 2006, corrected 2011) and the Commission Regulation (EC) No 761/2009, C.3 with the read-across source substance, using the freshwater green algal species Pseudokirchneriella subcapitata, the 72-hour EC50 and EC10 based on growth rate were determined to be 64 mg/L and 27 mg/L, respectively. The 72-hour EC50 and EC10 based on yield was found to be 28 mg/L and 4.9 mg/L, respectively. The 72-hour NOEC both for the growth rate and yield was 1 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

64 mg/L

EC10 or NOEC for freshwater algae:

27 mg/L

Additional information

No experimental data on the toxicity to aquatic algae is available for the registration substance itself, but for the read-across source substance 3,7-dimethyloct-1-en-3-ol (CAS no. 18479-49-7).

 

Key information

The impact of the read across source substance on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (adopted 2006, corrected 2011) and the Commission Regulation (EC) No 761/2009, C.3. The nominal test item concentrations tested were 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (3 replicates per concentration). Additionally, a control group was tested in parallel (6 replicates). As the test item is a volatile substance, the test was performed using glass vessels completely filled with test medium, minimizing the headspace, that were tightly sealed with glass stoppers to avoid losses of test item. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. The measured concentrations of the test item in the test media of all test concentrations were between 96 and 104 % of the nominal values at the start of the test and between 92 and 98% at the end of the test. Thus, the correct dosing of the test item was confirmed. The test item was stable in the test media over the test period of 72 hours. The biological results were hence related to the nominal concentrations of the test item. The test item had a significant inhibitory effect on the growth rate μ of the algae after the test period of 72 hours at the concentration of 3.2 mg/L and at all higher test concentrations. The yield Y of the algae was statistically significantly reduced at the test concentration of 3.2 mg/L. The 72-hour NOEC based on the growth rate μ was determined to be 1.0 mg/L, since up to and including this test concentration the growth rate of the algae after 72 hours was not significantly lower than in the control. The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 32 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration. In the control, the biomass increased by a factor of 239 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 16 %. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35 %. Thus, the validity criterion was fulfilled.  The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 0.6%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period. The pH rose in the control from 7.7 at test start to 7.9 at test end fulfilling the requirement of the OECD guideline that the pH of the control medium should not increase by more than 1.5 units during the test. The pH of the test media was in the range of 7.5 to 7.9 during the test period. The water temperature during the test was maintained at 24 °C. As a result, the 72-hour EC50 and EC10 for the growth rate were determined to be 64 mg/L and 27 mg/L, respectively. The 72-hour EC50 and EC10 based on yield was found to be 28 mg/L and 4.9 mg/L, respectively. The 72-hour NOEC both for the growth rate and yield was 1 mg/L.

 

Conclusion

Based on reliable information for the read-across source substance, the 72-hour EC50 and EC10 (growth rate) for the read-across target substance are considered to be 64 mg/L and 27 mg/L, respectively. The 72-hour EC50 and EC10 based on yield are considered to be 28 mg/L and 4.9 mg/L, respectively. The 72-hour NOEC both for the growth rate and yield is determined to be 1 mg/L.