Oct-7-en-1-ol

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:

19 April 2022 - 22 April 2022

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)

Deviations:

no

Principles of method if other than guideline:

Deviations from the Study Plan:
• The pre-culture was incubated for 5 days instead of 4 days. As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.
• The pH of the blank control shouldn’t change by more than 1.5 units. The change was 1.7 units, this was caused by the test design as a closed system. The pH increase was caused by CO2 shortage in the closed system. As normal growth was detected in the blank controls and all validity criteria were fulfilled, this was stated as uncritical.
• For the extra replicates without algae for analysis, 100 ml glass bottles were used in-stead of 250 ml glass bottles. As the size of the bottle has no influence on the outcome of the study, this was stated as uncritical.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sample Preparation
No Enrichment
2 g NaCl were added to 10 mL of the test item solution from the concentration 22 mg/L, 46 mg/L and 100 mg/L; then, the clear solution was extracted two times with the solvent di-chloromethane (2 x 4 mL), the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane after addition of 500 µL ISTD (internal standard) stock solution (1000 mg/L in dichloromethane) and the solution was measured via GC/MS.

Tenfold Enrichment
6 g NaCl were added to 100 mL of the test item solution from the concentrations 4.6 mg/L and 10 mg/L as well as the blank control; then, the clear solution was extracted two times with the solvent dichloromethane (2 x 4 mL), the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane after addition of 500 µL ISTD (internal standard) stock solution (1000 mg/L in dichloromethane) and the solution was measured via GC/MS.

The method is fully described in validation report VB21102201G926. This document and the corresponding raw data will be archived following GLP regulations.
A GC/MS-Method for the determination of Oct-7-en-1-ol was validated. For the calibration, a dilution series in dichloromethane was prepared, using a stock solution. The GC/MS-method was validated for the parameters specificity, linearity, matrix effects, instrument repeatability and carryover, accuracy and precision. Stability of the test item was tested in the stock and standard solutions, in M4 medium and in buffered algal medium.

Vehicle:

no

Details on test solutions:

A stock solution containing 99.7 mg/L test item in buffered OECD algal medium (demineralised water enriched with minerals but without algae) was prepared. For this the amount of 118 µL/L (based on a density of 0.84453 g/cm³ at 20 °C, provided by the sponsor) was pipetted in a decanting bottle, filled with the respective amount of buffered algal medium. This solution was stirred on a magnetic stirrer under closed conditions for 1 hour and 5 minutes, to guarantee complete dissolution. The lower treatments (46 / 22 / 10 / 4.6 mg/L nominal) were prepared by dilution of this stock solution with buffered algal medium.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Unicellular freshwater green alga.
Genus: Desmodesmus
Species: subspicatus
SAG Strain Number: 86.81
Taxonomic position: Chlorophyta - Chlorophyceae
Origin: The culture of Desmodesmus subspicatus was obtained in May 2020 by MBM Sciencebridge GmbH (Albrecht-von-Haller-Institut für Pflanzenwissenschaften Experimentelle Phykologie und Sammlung von Algenkulturen (SAG)).

Test type:

static

Water media type:

other: deionised water - buffered algal medium (due to closed system)

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Closed system

Test temperature:

21.8 – 22.7 °C

pH:

Controls: 7.7 - 9-4
Treatment groups: 7.8 - 9.5

Nominal and measured concentrations:

4.6 / 10 / 22 / 46 / 100 mg/L nominal concentration
2.3 / 8.2 / 19.8 / 33.8 / 86.5 mg/L mean measured concentration

Details on test conditions:

Treatments:
3 vessels, each completely filled with test solution and algae
1 glass bottle 100 mL volume, completely filled with test item solution without algae for analytical determination

Blank Control:
6 vessels, each completely filled with algal medium and algae
1 glass bottle 100 mL volume, completely filled with algal medium without algae for analytical determination

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

17.78 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

17.83 mg/L

95% CI:

13.69 - 21.73

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

16.57 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

8.25 mg/L

95% CI:

3.51 - 11.46

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

19.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

19.8 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.:

8.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: yes, because one replicate of the test concentration 4.6 mg/L was flocculated at the end of the test, this replicate was not used for the evaluation.
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other:
- Any stimulation of growth found in any treatment: yes, at 10 mg/L nominal concentration
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 0.69 mg/L (0.61 – 0.79 mg/L)

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.3.0. The details of calculation are stated in Annex 5: Statistical calculation using ToxRat® Professional 3.3.0.
Because one replicate of the test concentration 4.6 mg/L was flocculated at the end of the test, this replicate was not used for the evaluation.

Findings

Cell Numbers

The cell numbers were determined with an electronic particle counter. Because one replicate of the test concentration 4.6 mg/L was flocculated at the end of the test, this replicate was not used for the evaluation.

The means and standard deviations of the cell numbers of the blank control and the treatments are presented in the following table:

Cell Number/mL

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	2560	11717	66603	434710
	SD	0	4658	15201	87518
4.6	Mean	2560	8440	60700	404040
	SD	0	1414	22203	89491
10	Mean	2560	9927	83153	549607
	SD	0	446	7430	62031
22	Mean	2560	6580	8527	13947
	SD	0	1542	1168	4939
46	Mean	2560	7347	7227	10853
	SD	0	742	3009	6351
100	Mean	2560	7767	4080	4393
	SD	0	361	367	331

SD = Standard Deviation

Light Intensity

The light intensity is checked monthly and recorded in a control chart. The lighting corresponded to the specified range of 4440 - 8880 lux.

pH values

In the following table, the pH values measured at the start and the end of the test are stated.

pH values

Nominal Concentration in mg/L	0 h	72 h
Blank control	7.7	9.4
4.6	7.8	9.4
10	7.8	9.5
22	7.8	8.4
46	7.8	8.4
100	7.8	8.5

Microscopical Observations

In the following table, the appearance of the algae at the end of the test is stated.

Microscopical Observations

Nominal Concentration in mg/L	Normal and Healthy Appearance of the Algae
Blank control	Yes
4.6	Yes
10	Yes
22	No cells visible
46	No cells visible
100	No cells visible

 

Analytical Determination

At the beginning and at the end of the test, the content of the test item in the test solutions was determined using GC/MS-determination. The measured concentrations lay between 81 % and 95 % of the nominal concentrations at the beginning of the test and between 3 % and 92 % of the nominal concentrations at the end of the test. Therefore, the determination of the results was based on the geometric mean of the measured concentrations (see Guideline No. 201 (§39)).

Because the measured concentration of the lowest tested concentration 4.6 mg/L lay below the LOQ at both measurement days, ½ LOQ (2.3 mg/L) was used for both measurement days to calculate the geometric mean for this concentration (see OECD guidance document No. 23 § 165).

The measured concentrations for treatment and controls are given in the following table:

% of Nominal Concentration and Geometric Mean

Nominal Concentration	% of Nominal Concentration		Geometric Mean of measured concentrations
mg/L	0 h	72 h	mg/L
Blank control	--	--	--
4.6	81	3	2.3 *
10	86	78	8.2
22	88	92	19.8
46	95	57	33.8
100	89	84	86.5

* ½ LOQ (2.3 mg/L) was used for both measurement days to calculate the geometric mean for this concentration.

Growth Rate, Yield

From the cell numbers, the Growth Rate µ and the Yield were calculated. Because one replicate of the test concentration 4.6 mg/L was flocculated at the end of the test, this replicate was not used for the evaluation.

The means and standard deviations at the end of the test are given in the following table.

Growth Rate µ, Yield

Nominal Concentration in mg/L	Para- meter	Growth Rate (0-72 h) [day^-1]	Yield (0-72 h) [Cell Concentration/mL]
Blank control	Mean	1.71	432150
	SD	0.07	87518
4.6	Mean	1.68	401480
	SD	0.07	89491
10	Mean	1.79	547047
	SD	0.04	62031
22	Mean	0.55	11387
	SD	0.13	4939
46	Mean	0.43	8293
	SD	0.25	6351
100	Mean	0.18	1833
	SD	0.03	331

SD = Standard Deviation

Inhibition

Because one replicate of the test concentration 4.6 mg/L was flocculated at the end of the test, this replicate was not used for the evaluation.

The following mean inhibition values were calculated for the Growth Rate µ and the Yield.

Inhibition Values

Nominal concentration in mg/L	% Inhibition
	Growth Rate (0-72 h)	Yield (0-72 h)
Blank control	0	0
4.6	1.3	7.1
10	-4.8	-26.6
22	67.8	97.4
46	74.9	98.1
100	89.5	99.6

Negative inhibition values indicate a stimulation of algal growth compared to the blank control.

Validity

Increase Factor

The cell concentration in the blank control should increase by a factor of at least 16 within 72h.

Daily Growth Rates

Mean coefficient of variation of daily growth rates in the blank controls should be 35 % at the most.

Coefficient of variation of average growth rate during the whole test period should be 7 % at the most.

Values and assessment

The daily growth rates of the blank controls were calculated. Means, standard deviations and coefficients of variation were determined. Values and assessment can be found in the following tables.

Daily Growth Rates of the blank controls

Growth rates	days 0 – 1	days 1 – 2	days 2 – 3	CV of sectional daily growth rates	days 0 – 3
Replicate 1	0.95	2.04	2.14	39 %	1.71
Replicate 2	1.69	1.75	1.92	7 %	1.79
Replicate 3	1.38	1.70	1.77	13 %	1.62
Replicate 4	2.05	1.09	1.84	30 %	1.66
Replicate 5	1.37	1.84	1.83	16 %	1.68
Replicate 6	1.32	2.25	1.77	26 %	1.78
Mean	1.46	1.78	1.88	22 %	1.71
Standard deviation	0.37	0.39	0.14		0.07
CV	26 %	22 %	7 %		4 %

CV = Coefficient of Variation

 

Assessment

Parameter	Validity criteria	Observed value	Assessment
Increase factor biomass	factor 16 in 72 h	170	Valid
Mean coefficient of variation of daily growth rates	max. 35 %	22 %	Valid
Coefficient of variation of average growth rate during the whole test period	max. 7 %	4 %	Valid

 

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were met. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Conclusions:

One valid experiment was performed.
The study was performed using 5 concentrations ranging from 4.6 to 100 mg/L (nominal). Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concen-tration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times.
Significant inhibition of algal growth was observed at the following concentrations: 22 / 46 / 100 mg/L nominal for both endpoints.

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were deter-mined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.64 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

Because one replicate of the test concentration 4.6 mg/L was flocculated at the end of the test, this replicate was not used for the evaluation.

At the beginning and at the end of the test, the content of the test item in the test solutions was determined using GC/MS-determination. The measured concentrations were between 81 % and 95 % of the nominal concentrations at the beginning of the test and between 3 % and 92 % of the nominal concentrations at the end of the test. Therefore, the determination of the results was based on the geometric mean of the measured concentrations (see Guideline No. 201 (§39))
Because the measured concentration of the lowest tested concentration 4.6 mg/L were below the LOQ at both measurement days, ½ LOQ (2.3 mg/L) was used for both measurement days to calculate the geometric mean for this concentration (according to OECD guidance document No. 23 § 165).

The pH of the blank control should not fluctuate by more than 1.5 units. The change was 1.7 units in the blank control.

All validity criteria were met.
No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Description of key information

The toxicity of OEA was examined in an algae test according to OECD 201 and under GLP conditions. The exposure of the algae was confirmed analytically by GC/MS-determination. 

Key value for chemical safety assessment

EC50 for freshwater algae:

17.83 mg/L

EC10 or NOEC for freshwater algae:

8.25 mg/L

Additional information

The results of this study are supported by the outcome of a second algae test (also performed according to OECD 201 and under GLP conditions). The ErC50 (72 -h) and ErC10 (72 -h) were calculated to be 36.26 mg/L and 9.09 mg/L, respectively, based on nominal concentration.