5-hexyldihydro-5-methylfuran-2(3H)-one

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:

2016-08-02 to 2016-08-05

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:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L
- Sampling method: The concentrations of the test item in all test samples and the control were analytically verified by GC-MS at the start, after 24 hours, 48 hours and the end of exposure.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until sample preparation and at room temperature until the start of the analysis

Vehicle:

yes

Remarks:

dilution water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Five test solutions in a geometrical series with a factor of 3.16: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L were freshly prepared with dilution water by adding an appropriate amount of the test item. For preparation the density of the test item was taken into account. The test solutions were stirred for 15 minutes at 1100 rpm and room temperature until the test item is completely dissolved. The concentrations are based on the results of a preliminary range finding test. Additionally a stock solution of 10.0 mg/L was prepared with dilution water (without the compounds MES and NaHCOs) for analysis of total organic carbon (TOC, according to DIN EN 1484) at the start and at the end of the exposure.
- Controls: Six replicates (without test item) were exposed under the same conditions as the test concentrations.
- Chemical name of vehicle: dilution water
- Evidence of undissolved material: no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus
- Strain: HINDÄK, SAG 61.81
- Age of inoculum: 4 d
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux corresponding to 35-70 µE * m*exp-2 * s*exp-1 for 24 hours per day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

Nominal range: 21 - 24°C, controlled at ± 2°C

pH:

8.1 ± 0.2

Salinity:

Freshwater was used.

Conductivity:

No data was provided.

Nominal and measured concentrations:

Nominal: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L
Measured: 0.825 - 1.94 - 5.93 - 26.5 - 77.1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile headspace flasks, volume: 59 mL, with aluminium tops with PTFE seals.
- Type: closed
- Fill volume: 59 mL
- Aeration: A static test will be carried out. With regard to the volatility of the test item glass flasks without headspace will be used to reduce losses of test item.
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: Nominal: approximately 5 x 103 - 104 cells/mL Current: 6305 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to guideline

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE * m*exp-2 * s*exp-1

EFFECT PARAMETERS MEASURED:
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test.
- Microscopic evaluation: Microscopic evaluation of the cells at the start and the end of the incubation period was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: yes
- Test concentrations: 0.825 - 1.94 - 5.93 - 26.5 - 77.1 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.825 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.94 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.38 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 3.62 - 5.27 95 % Confidence interval

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

9.37 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 8.22- 10.5 95 % Confidence interval

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

29.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 27.7-31.5 95 % Confidence interval

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.825 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield Inhibition

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.94 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield Inhibition

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield Inhibition

Remarks on result:

other: 0.896-1.76 95 % Confidence interval

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.02 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield Inhibition

Remarks on result:

other: 1.55-2.67 95 % Confidence intervals

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield Inhibition

Remarks on result:

other: 4.62 - 7.46 95 % Confidence interval

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities.
- Any observations that might cause a difference between measured and nominal values: No. The measured concentrations of the test item at the start of exposure were in the range of 58 - 88% of the nominal values. The concentration range at 24 and 48 h was 69 - 85% and 56 - 84%. At the end of exposure the measured concentrations with algae were in the range of 46 - 81% and without algae 65 - 85% of the nominal values. The results show that the test item was stable under the test conditions.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid: yes
- EC50: Growth Rate Inhibition: 1.15, Yield Inhibition: 0.509

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 alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha=0.05.

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Time weighted average mean measured test item concentration	Replicate	Growth rate	Inhibition of growth rate	Yield	Inhibition of yield
[mg/L]	No.	[d*exp-1]	[%]	[cells/mL]	[%]
77.1	1	n.a.	100	n.a.	100
	2	-0.272	100		100
	3	n.a.	100	n.a.	100
	Mean	(+) n.a.	100	(+) n.a.	100
26.5	1	0.878	46	81431	90
	2	0.89	46	84688	90
	3	0.878	46	81593	90
	Mean	(+) 0.882	46	(+) 82604	90
5.93	1	1.46	11	493359	42
	2	1.43	13	452455	47
	3	1.4	15	415977	52
	Mean	(+) 1.43	13	(+) 453930	47
1.94	1	1.58	4	708505	18
	2	1.5	9	557576	35
	3	1.59	3	731311	15
	Mean	(+) 1.55	5	(+) 665797	22
0.825	1	1.65	-1	886271	-3
	2	1.65	-1	894954	-4
	3	1.62	1	796796	7
	Mean	(+) 1.64	0	(-) 859340	0
Control	1	1.62		810243
	2	1.64		847482
	3	1.67		932982
	4	1.66		901916
	5	1.62		809820
	6	1.63		843197
	Mean	1.64		857607

Negative inhibition = growth stimulation n.a. = data not determinable

 

 

Measured Concentrations and Percentage of the Nominal Concentration of the test substance

Sampling date	2016-08-02 0 h with algae		2016-08-03 24 h with algae		2016-08-04 48 h with algae		2016-08-05 72 h with algae		2016-08-05 72 h without algae
Nominal test item concentration [mg/L]	Test substance
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Time weighted average mean measured test item conc. [mg/L]
100	70.4*	70	81.6	82	81.8	82	66.9	67	84.8	85	77.1
31.6	27.8*	88	26.3	83	26.5	84	25.7	81	26.4	84	26.5
10 a)	10.2	102	n.m.	n.m.	n.m.	n.m.	n.m.	n.m.	8.97	90	n.m.
10	6.28*	63	6.94	69	5.63	56	4.55	46	6.52	65	5.93
3.16	1.84*	58	2.44	77	1.69	53	1.72	54	2.36	75	1.94
1	0.88*	88	0.848	85	0.825	82	0.731	73	0.832	83	0.825
Control	< LOQ		< LOQ		< 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 (0.02 mg test item /L)

* = Reanalyses on 2016-08-03 with two replicates, mean value of three replicates

1) = Samples without MES and without algae, only on 2016-08-02 and 2016-08-05

n.m. = not measured

Validity criteria fulfilled:

yes

Conclusions:

The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 29.5 (27.7-31.5) mg/L and 5.84 (4.62 - 7.46) mg/L, respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 4.36 (3.62 - 5.27) mg/L and 1.31 (0.896 - 1.76) mg/L, respectively.

Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No 761/2009 C.3.The aim of the study was the determination of NOEC, LOEC, EC10, EC20- and EC50-values of growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6305 cells/mL. Five solutions with the nominal test item concentrations 1.00 -3.16 - 10.0 - 31.6 -100 mg/L, corresponding to the time weighted average mean measured test item concentrations 0.825 - 1.94 - 5.93 - 26.5 - 77.1 mg/L, were freshly prepared with dilution water and stirred for 15 minutes (1100 rpm, room temperature) until the solutions were visually clear.Three replicates were tested for each test item concentration and six replicates for the control.The environmental conditions were within the acceptable limits. The test media were clear throughout the test period. The concentrations of the test item in all test concentrations and the control were analytically verified by GC-MS at the start, after 24 hours, 48 hours and at the end of exposure. The measured concentrations of the test substance in the fresh media (0 h) with algae were between 58 and 88% of the nominal values.The measured concentrations in the old media (72 h) containing algae were in the range of 46 to 81% of the nominal values.Therefore all effect values given are based on the time weighted average mean measured test item concentrations with algae. The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 29.5 (27.7-31.5) mg/L and 5.84 (4.62 - 7.46) mg/L, respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 4.36 (3.62 - 5.27) mg/L and 1.31 (0.896 - 1.76) mg/L, respectively. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 0.825 mg/L, respectively.

Description of key information

ErC50 (72 h) = 29.5 mg/L (TWA, Pseudokirchneriella subcapitata)

ErC10 (72 h) = 4.36 mg/L (TWA, Pseudokirchneriella subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:

29.5 mg/L

EC10 or NOEC for freshwater algae:

4.36 mg/L

Additional information

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No 761/2009 C.3. The study was conducted under static conditions with an initial cell density of 6305 cells/mL. Five solutions with the nominal test item concentrations 1.00 -3.16 - 10.0 - 31.6 -100 mg/L, corresponding to the time weighted average mean measured test item concentrations 0.825 - 1.94 - 5.93 - 26.5 - 77.1 mg/L, were tested. Three replicates were tested for each test item concentration and six replicates for the control. The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 29.5 (27.7-31.5) mg/L and 5.84 (4.62 - 7.46) mg/L, respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 4.36 (3.62 - 5.27) mg/L and 1.31 (0.896 - 1.76) mg/L, respectively. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 0.825 mg/L, respectively.