Hex-3-en-1-yl acetate

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:

17 - 20 Jan 2017

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:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt, Hildesheim, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations and control
- Sampling method: at test start and test end; seperate replicates were prepared for analytical sampling
- Sample storage conditions before analysis: at 6 ± 2 °C until sample preparation and at room temperature until the start of the analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: preparation of seven test solutions with dilution water by adding an appropriate amount of the test item and stirred for 30 minutes at room temperature
- Controls: yes

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: SAG 61.81
- Age of inoculum (at test initiation): a 4 days old pre-culture, prepared in dilution water, was used as inoculum
- Method of cultivation: stocks prepared on nutrient medium Z-Agar according to Lüttge et al., 1994 [Lüttge, U, Schnepf, E., Läuchli, A., Nagata, T. (editors, 1994): Botanica Acta, Journal of the German Botanical Society, No. 3 Volume 107 page 111-186 (June 1994), Thieme- Verlag]. Light intensity was 35-70 µE x m^-2 x s^-1 for 24 hours per day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.0 - 23.0 °C (mean 22.5 °C)

pH:

7.59 - 8.84 (Control)
7.47 - 8.65

Nominal and measured concentrations:

0.100, 0.316, 1.00, 3.16, 10.0, 31.6 and 100 mg/L.
0.0262, 0.245, 0.701, 1.63, 7.37, 18.5 and 73.6 mg/L (based on geometric mean measured concentrations)

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile headspace flasks, with aluminium tops with PTFE seals
- Type: closed
- Headspace, fill volume: no headspace, 59 mL volume
- Initial cells density: nominal: approx. 5 x 10E+03 - 10E+04 cells/mL, current: 6305 cells/mL
- Control end cells density: yes, 399535 cells/mL
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates

GROWTH MEDIUM
- Standard medium used: yes, according to guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: yes, nutrient medium Z according to Lüttge et al. (1994)

OTHER TEST CONDITIONS
- Photoperiod: 24 h/day light
- Light intensity and quality: 4728 to 6070 lux, fluorescent tubes, cool daylight, homogeneity within ± 15% over incubation area

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: fluorometer, microscope
- Chlorophyll measurement: daily Chlorophyll a fluorescence measurement (excitation at 436 nm, emission at 685 nm)
- Other: microscopic evaluation of the cells at the start and the end of the exposure time (shape, colour, chloroplast morphology, flocculation, agglutination)

TEST CONCENTRATIONS
- Range finding study: non-GLP, closed system without headspace
- Test concentrations: nominal: 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: % growth rate inhibition 9, 19, 100 respective to the nominal conentrations 1.0, 10.0 and 100 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

21.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % Confidence intervals

Remarks:

18.6 - 44.9 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

16.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % Confidence intervals

Remarks:

8.37 - 18.5 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

7.37 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

18.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % Confidence intervals

Remarks:

7.70 - 43.0 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

16.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % Confidence intervals

Remarks:

<0.0262 - 42.1 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

7.37 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, >= 16-fold corresponding to a specific growth rate of 1.38 per day
- Observation of abnormalities: no
- Unusual cell shape: no

Results with reference substance (positive control):

- Results with reference substance valid: yes
- ErC50: with headspace 0.435 (95% confidence interval 0.414 - 0.458), without headspace 0.811 (95% confidence interval 0.763 - 0.883)

Reported statistics and error estimates:

EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield.
As a standard, One Way Analysis of Variance (ANOVA) and Dunnett’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

Analtiyical Results:

Table 1: Measured concentrations and percentage of the nominal concentration and geometric mean of the test item in fresh medium (0 hours) and old medium (72 hours) with algae

Sampling date	2017-01-17 0 h fresh medium		2017-01-20 72 h old medium		Geometric Mean
Start of analysis	2017-01-17		2017-01-20
Nominal test item concentration [mg/L]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]	[mg/L]
100	92.5	92	58.6	59	73.6
31.6	29.3	93	11.7	37	18.5
10.0	9.42	94	5.77	58	7.37
3.16	3.01	95	0.879	28	1.63
1.00	0.939	94	0.524	52	0.701
0.316	0.302	96	0.199	63	0.245
0.100	0.0927 2)	93	0.00740 1)	7	0.0262
Control		< LOQ		< LOQ	< LOQ

Meas. Conc. = Measured concentration of the test item (enrichment and dilution factor taken into account)

% = Percentage of the nominal concentration of the test item

LOQ = Limit of quantification (10 µg test item /L)

1) = value <LOQ but > LOD, therefore taken into account for geometric mean

2) = Reanalysed on 2017-01-19

Biological Results:

Table 2: Evaluation after 72 hours

Geometric mean measured test item concentration [mg/L]	Replicate No	Growth rate [d-1]	Inhibition of growth rate [%]
Control	1	1.41
	2	1.37
	3	1.36
	4	1.39
	5	1.40
	6	1.37
	Mean	1.38
0.0262	1	1.38	0
	2	1.39	-1
	3	1.25	10
	mean	1.34	3
0.245	1	1.37	1
	2	1.37	1
	3	1.36	2
	mean	1.37	1
0.701	1	1.35	3
	2	1.38	1
	3	1.31	5
	mean	1.34	3
1.63	1	1.36	2
	2	1.38	0
	3	1.36	2
	mean	1.37	1
7.37	1	1.40	-2
	2	1.36	2
	3	1.36	1
	mean	1.38	1
18.5	1	1.08	22
	2	1.06	24
	3	1.01	27
	mean	1.05	24
73.6	1	n.a.	100
	2	n.a.	100
	3	n.a.	100
	mean	n.a.	100

  n.a. = not applicable

Validity criteria fulfilled:

yes

Conclusions:

The substance was found to inhibit the growth rate of the freshwater green alga Pseudokirchneriella subcapitata after 72 h of exposure. The test was conducted according to the OECD guideline 201 (GLP). ErC10- and ErC50-value were found to be 16.1 mg/L and 21.3 mg/L, respectively.

Description of key information

ErC50 (72h) = 21.3 mg/L (geometric mean measured, OECD 201, Pseudokirchneriella subcapitata

ErC10 (72h) = 16.1 mg/L (geometric mean measured, OECD 201, Pseudokirchneriella subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:

21.3 mg/L

EC10 or NOEC for freshwater algae:

16.1 mg/L

Additional information

One study is available, in which the toxicity to freshwater algae was investigated according to OECD guideline 201 and GLP. In a static test system, the test organism Pseudokirchneriella subcapitata was exposed to five nominal concentration of 0.100, 0.316, 1.0, 3.16, 10.0, 31.6 and 100 mg/L corresponding to geometric mean measured concentrations of 0.0262, 0.245, 0.701, 1.63, 7.37, 18.5 and 73.6 mg/L. The concentrations of the test item in all test concentrations and the control were analytically verified. The measured concentrations in the fresh media (0 h) were in the range of 92 to 96% of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 7 to 63% of the nominal values of all concentrations. Therefore, all effect values given are based on the geometric mean measured test item concentrations. The EC50 values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 21.3 (18.6 - 44.9) mg/L and 18.1 (7.70 - 43.0) mg/L, respectively. The EC10 values were 16.1 (8.37 - 18.5) mg/L for growth rate and 16.4 (<0.0262 - 42.1 mg/L) mg/L for yield.