3,7-dimethyl-6-octenyl 2-methylcrotonate

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:

29 Aug - 20 Oct 2016

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:

2006, corrected 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

No. 2016/266 C3, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss federal office of public health; consumer protection directorate; notification authority for chemicals; CH-3003 Bern

Analytical monitoring:

yes

Details on sampling:

- Sampling method: additional replicates (with algae) were performed for analytical sampling under the same conditions; samples were taken after 24, 48 and 72 h; 9 mL samples were added to 6 mL acetonitril
- Sample storage conditions before analysis: all samples were deep-frozen (at about -20 °C)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: based on the results of a pre-experiment, loading rate of 100 mg/L (considering the relative density of 0.90) was mixed with test water, stirred for 3 h and filtered through a 0.45 μm membrane filter (Whatman, NC45). The filter was saturated by at least 200 mL test medium and the negative pressure of the filtration unit was reduced to a minimum to avoid losses of the volatile test item.
- Differential loading: undiluted filterate and the dilutions 1:2.2 and 1:4.6
- Controls: yes

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Strain No. 61.81 SAG
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: cultivated under standard conditions according to the test guidelines

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: yes

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

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

Test temperature:

temperature maintained at 23 °C

pH:

8.0 to 8.2 (control)
7.9 to 8.2 (test concentrations)

Nominal and measured concentrations:

control; 1:46; 1:22; 1:10; 1:4.6; 1:2.2 and undiluted filtrate (100 mg/L) (nominal)
control; n.a.; n.a.; n.a.; 0.018; 0.041 and 0.085 mg/L (geometric mean measured concentration)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: closed (volatile substance)
- Material, size, headspace, fill volume: Erlenmeyer flasks; completly filled (60 mL), without headapce and sealed with glass stoppers
- Initial cells density: 5000 cells/mL corresponding to 1.28 x 104 relative fluorescence units (x 104)
- Control end cells density: 145.8 relative fluorescence units
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition of medium: AAP-medium modified according to the International Standard ISO 14442 as a closed test system was applied (NaHCO3 concentration was increased to 250 mg/L)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured and recorded in each treatment at the start and end of the test. The temperature in the incubator was monitored and recorded continuously. The appearance of the test media was also visually controlled and recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: test water was adjusted to 7.5 with a 1 M sodium hydroxide solution
- Photoperiod: continuously
- Light intensity and quality: LED light 67 μE s-1m-2 (range: 66 to 68 μE s-1m-2)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentration: for initial cell concentration: electronic particle counter (Cell Counter CASY TT, OLS, Bremen/Germany); algal biomass in the samples was determined by fluorescence measurement (SpectraMax I3x, Molecular Devices Ltd, Wokingham Berkshire/UK).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: theoretical spacing factor of 2.2
Range finding study:
- Test concentrations: control, 1:20; 1:5 and undiluted filtrate (100 mg/L)
- Results used to determine the conditions for the definitive study: The concentration of the main test were based on ther esults of the range-finding test

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.054 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence limits

Remarks:

0.046 - 0.059 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.085 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.018 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.034 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % confidence limits

Remarks:

0.029 - 0.039 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.062 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % confidence limits

Remarks:

0.057 - 0.066 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.018 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no difference between cells of the undiluted filtrate (0.085 mg/L) and the cells of the control
- Unusual cell shape: shape and size of the algal cells were obviously not affected

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 0.9 mg/L (Potassium dichromate; October 2016)

Reported statistics and error estimates:

The 72-hour EC10 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression. 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 the Williams t-test or Welch t-test where appropriate. Statistical analysis was performed using ToxRat Professional®.

Validity criteria:

In the control, the biomass increased by a factor of 114 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 19%. 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 1.1%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%.Thus, the validity criterion was fulfilled.

Table1: Analytical Results:

Treatment /Dilution	Analytical Measured Concentration of the Test Item [mg/L]				Mean Measured Concentration (Geometric Mean)*
	0 hours	24 hours	48 hours	72 hours	[mg/L]
Dilution1:46	n.a.	n.a.	n.a.	n.a.	n.a.
Dilution1:22	n.a.	n.a.	n.a.	n.a.	n.a.
Dilution1:10	n.a.	n.a.	n.a.	n.a.	n.a.
Dilution1:4.6	0.055	0.029	<LOQ	<LOQ	0.018
Dilution1:2.2	0.12	0.081	0.036	<LOQ	0.041
UndilutedFiltrate°	0.26	0.19	0.12	<LOQ	0.085

°: Undiluted filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L

LOQ: Limit of quantification (LOQ = 0.017 mg test item/L)

*: If the measured concentration was below LOQ, half of the LOQ (½ LOQ = 0.0085 mg/L) was used to calculate the mean measured concentration

n.a.: Not analyzed since below NOEC determined in this test

Table 2: Average Growth Rates (μ)

Treatment /Dilution	Mean Measured Concentration	Average Growth Rate μ (day-1) and Inhibition of μ (Ir)
		0-24 h		0-48 h		0-72 h
	[mg/L]	μ$	Ir [%]	μ	Ir [%	μ	Ir [%]
Control	---	1.326	0.0	1.616	0.0	1.577	0.0
1:46	n.a.	1.337	-0.8	1.632	-1.0	1.601	-1.5
1:22	n.a.	1.274	3.9	1.645	-1.8	1.597	-1.2
1:10	n.a.	1.246	6.0	1.656	-2.5	1.583	-0.4
1:4.6	0.018	1.378	-4.0	1.665	-3.0	1.611	-2.1
1:2.2	0.041	1.252	5.6	1.535*	5.0	1.509*	4.3
Undiluted Filtrate°	0.085	1.005	24.2	1.058*	34.5	1.111*	29.6

°: Undiluted filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L.

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

$: No statistically significant effect at all test concentrations compared to control (according to Welch t-test, one-sided smaller, α = 0.05).

Table 3: Yield (Y)

Treatment /Dilution	Mean Measured Concentration	Yield Y (x 104) and Inhibition of Y (Iy)
		0-24 h		0-48 h		0-72 h
	[mg/L]	Y	Iy [%]	Y	Iy [%]	Y	Iy [%]
Control	---	3.6	0.0	31.2	0.0	144.5	0.0
1:46	n.a.	3.6	-1.8	32.2	-3.4	154.8	-7.2
1:22	n.a.	3.3	6.6	33.3	-6.8	153.0	-5.9
1:10	n.a.	3.2	10.7	33.9	-8.7	146.8	-1.6
1:4.6	0.018	3.8	-7.0	34.6	-11.0	159.6	-10.4
1:2.2	0.041	3.2	10.1	26.5*	15.1	117.8*	18.5
UndilutedFiltrate°	0.085	2.3*	35.9	9.4*	69.9	34.8*	75.9

°: Undiluted filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L.

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

Validity criteria fulfilled:

yes

Conclusions:

The EC50 (72h) for growth rate was > 0.085 mg/L (based on geometric mean measured concentrations).

Description of key information

ErC50 (72 h) > 0.085 mg/L (geometric mean measured concentration, OECD 201, Pseudokirchneriella subcapitata)

ErC10 (72 h) = 0.054 mg/L (geometric mean measured concentration, OECD 201, Pseudokirchneriella subcapitata)

Additional information

There is one GLP guideline study available, which determined the effects of the substance on the growth of unicellular freshwater green algae according to the principles of the OECD guideline 201. In a static test consisting five concentrations in a geometrical series with a dilution factor of 2.2 Pseudokirchneriella subcapitata was exposed to the test substance for 72 h. Due to the low water solubility of the test item, the test media were prepared following a filtration method. 100 mg/L test item was mixed in test water, and was intensively stirred for 3 hours. After stirring the emulsion was filtered through a 0.45 μm membrane filter. Undiluted filtrate (with a loading rate of 100 mg/L) and the dilutions 1:2.2, 1:4.6, 1:10, 1:22 and 1:46 of the filtrate were used as test media. The concentrations of the test item were analytically verified by HPLC-UV analysis. The analytical measurements from test media at the start of the test show the correct preparation of the test media. During the test period of 72 hours the test item concentrations in the test media decreased below the limit of quantification. Due to the decrease of the test item concentrations during the test period of 72 hours, the biological results were related to the mean measured concentrations. The mean measured concentrations were calculated as the geometric mean of the concentrations measured at all sampling dates (0, 24, 48 and 72 hours). The ErC50 (72 h) value was > 0.085 mg/L (based on the mean measured concentrations) and the ErC10 (72 h) was 0.054 mg/L.