2-ethylhexyl nitrate

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:

October 2021

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:

yes

Remarks:

The pre-culture was prepared 5 days before the test start. pH change in control was >1.5 units. Due to the test design as a closed system. All validity criteria were met & exponential growth was observed in control, therefore deviations were uncritical.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: 123L21
- Purity: > 99.5 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Closed vessel at room temperature (20 ± 5 °C), dry, in the dark
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Homogeneous and stable under storage conditions

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

Test solution was prepared in accordance with OECD Guidance Document No. 23, Second Edition, Guidance Document on Aqueous-phase Toxicity Testing of Difficult Test Chemicals, adopted 08. Feb. 2019. A saturated solution was prepared for the test. This was done by pipetting the nominal load of 100 mg/L corresponding to a volume of 104 µL/L test item (based on a density of 0.962 g/mL given by the sponsor), onto the surface of the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and stirring vigorously for 23 hours. After a settling time of 1 hour, the first 50 mL of the lower phase were discarded. The solution was used as highest test concentration and also for preparation of the lower concentrated treatments (2.2 / 4.6 / 10 / 22 and 46 mg/L), by dilution with algal medium.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: 86.81
- Source (laboratory, culture collection): The culture of Desmodesmus subspicatus was obtained in May 2020 by MBM Science-bridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

21.2 - 22.5 °C

pH:

See Table 1

Dissolved oxygen:

Not reported

Salinity:

N/A

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal concentrations of 2.2 / 4.6 / 10 / 22 / 46 and 100 mg/L. Concentrations were determined based on the result of a non-GLP pre-test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Closed glass flasks total volume 65 mL
- Type (delete if not applicable): Closed
- Material, size, headspace, fill volume: Glass flask, nominal volume 65 mL, completely filled with test solution
- Initial cells density: 2980 cell number/mL
- Control end cells density: 479420 cells/mL
- No. of vessels per concentration (replicates): 3 vessels, each filled with test solution and algae, 1 vessel filled with test item solution without algae for analytical determination
- No. of vessels per control (replicates): 6 vessels, each filled with algal medium and algae, 1 vessel filled with algal medium without algae for analyt-ical determination

GROWTH MEDIUM
- Standard medium used: Yes, see Table 2

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: Continuous
- Light intensity and quality: 4440-8880 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Electronic particle counter

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (CAS No. 7778-50-9)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on the measured concentrations in the solutions without algae

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on the measured concentrations in the solutions without algae

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on the measured concentrations in the solutions without algae

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on the measured concentrations in the solutions without algae

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: based on the measured concentrations in the solutions without algae

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: based on the measured concentrations in the solutions without algae

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.93 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: based on the measured concentrations in the solutions without algae

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.25 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: based on the measured concentrations in the solutions without algae

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.46 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on the measured concentrations in the solutions with algae

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on the measured concentrations in the solutions with algae

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on the measured concentrations in the solutions with algae

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on the measured concentrations in the solutions with algae

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.46 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: based on the measured concentrations in the solutions with algae

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: based on the measured concentrations in the solutions with algae

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: based on the measured concentrations in the solutions with algae

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: based on the measured concentrations in the solutions with algae

Details on results:

One replicate of the blank control showed coagulated algal cells and turbidity. This replicate was therefore excluded from evaluation of results.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: Growth rate: 0.64 mg/L (95% CI: 0.58-0.71 mg/L), Yield: 0.32 mg/L (95% CI: 0.31-0.33 mg/L)

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Ex-cel®). The estimation of the biological data was accomplished using the software Tox-Rat® Professional, version 3.3.0. Due to the very steep dose-response relationship, only the three highest concentrations were used for statistical evaluation to determine the most accurate EC10 and EC50 values possible using regression analysis. It was not possible to determine 95% confidence intervals. The highest concentration showed a significant inhibition of algal growth, the second highest concentration showed a slight stimulation of algal growth, and the third highest concentration showed a slight, non-significant inhibition of algal growth. Based on the available data, an exact determination of the EC value with a confidence interval is not possible due to mathmatical reasons, regardless of whether a nonlinear or linear regression is used. However, the EC values can be estimated as values between the highest and the second highest concentration. Despite the mathematical inaccuracy, the values for the EC10 and EC50 lay within a plausible range.

Table 3: Cell Numbers

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	2980	16768	133164	479420
	SD	0	797	9873	32929
2.2	Mean	2980	19307	73580[RL1] [MM2]	341060
	SD	0	5356	20747	39822
4.6	Mean	2980	17393	126480	472780
	SD	0	1507	17340	11719
10	Mean	2980	16580	117973	450580
	SD	0	1101	5752	19995
22	Mean	2980	16267	106447	453220
	SD	0	1222	21180	63159
46	Mean	2980	17167	138247	516680
	SD	0	387	17348	30121
100	Mean	2980	10647	20240	84573
	SD	0	3285	5960	16703

Table 4: Geometric mean of measured concentrations

Nominal Concentration	Geometric Mean
mg/L	mg/L
2.2	< LOQ
4.6	0.08
10	0.15
22	0.57
46	0.84
100	2.53

Table 5: Growth Rate and Yield

Nominal Concentration in mg/L	Parameter	Growth Rate (0-72 h) [day^-1]	Yield (0-72 h) [Cell Concentration/mL]
Blank control	Mean	1.69	476440
	SD	0.02	32929
2.2	Mean	1.58	338080
	SD	0.04	39822
4.6	Mean	1.69	469800
	SD	0.01	11719
10	Mean	1.67	447600
	SD	0.01	19995
22	Mean	1.67	450240
	SD	0.05	63159
46	Mean	1.72	513700
	SD	0.02	30121
100	Mean	1.11	81593
	SD	0.06	16703

Table 6: Inhibition values

Nominal concentration in mg/L	% Inhibition
	Growth Rate (0-72 h)	Yield (0-72 h)
Blank control	0	0
2.2	6.8*	29.0*
4.6	0.2	1.4
10	1.2	6.1
22	1.2	5.5
46	-1.5**	-7.8**
100	34.4	82.9

* inhibition of algal growth at the lowest tested concentration 2.2 mg/L was not caused by toxicity of the test item because at the higher tested concentrations nearly no inhibition of algal growth was observed, and the second highest concentration 46 mg/L showed stimulation of algal growth

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

Validity criteria fulfilled:

yes

Conclusions:

In a OECD 201 freshwater algae growth inhibition study, a 72-hour NOEC (growth rate), ECr10 and ECr50 of 0.84, 2.22 and >2.23 mg/L, respectively were determined in Desmodesmus subspicatus. All validity criteria were met and the study was considered Klimisch Code 1 (reliable without restriction).

Executive summary:

A study was conducted in Desmodesmus subspicatus was conducted according to OECD guideline 201. Test solution was prepared in accordance with OECD Guidance Document No. 23, Second Edition, Guidance Document on Aqueous-phase Toxicity Testing of Difficult Test Chemicals, adopted 08. Feb. 2019. A saturated solution was prepared for the test. This was done by pipetting the nominal load of 100 mg/L corresponding to a volume of 104 µL/L test item (based on a density of 0.962 g/mL given by the sponsor), onto the surface of the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and stirring vigorously for 23 hours. After a settling time of 1 hour, the first 50 mL of the lower phase were discarded. The solution was used as highest test concentration and also for preparation of the lower concentrated treatments (2.2 / 4.6 / 10 / 22 and 46 mg/L), by dilution with algal medium. Concentrations were determined based on the result of a non-GLP pre-test.

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. At the end of the test, the measured concentrations were much lower than at the beginning. Although the test was performed in completely filled and closed test vessels,it is highly probable that some of the test substance was removed by volatilisation during handling. Because the measured concentrations were not within the range of 20 % of the nominal concentration, the determination of the results was based on the geometric mean of the measured concentrations.

A 72-hour NOEC (growth rate), ECr10 and ECr50 of 0.84, 2.22 and >2.23 mg/L, respectively were determined in Desmodesmus subspicatus. All validity criteria were met and the study was considered Klimisch Code 1 (reliable without restriction).

Description of key information

A study was conducted in Desmodesmus subspicatus was conducted according to OECD guideline 201. Test solution was prepared in accordance with OECD Guidance Document No. 23, Second Edition, Guidance Document on Aqueous-phase Toxicity Testing of Difficult Test Chemicals, adopted 08. Feb. 2019. A saturated solution was prepared for the test. This was done by pipetting the nominal load of 100 mg/L corresponding to a volume of 104 µL/L test item (based on a density of 0.962 g/mL given by the sponsor), onto the surface of the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and stirring vigorously for 23 hours. After a settling time of 1 hour, the first 50 mL of the lower phase were discarded. The solution was used as highest test concentration and also for preparation of the lower concentrated treatments (2.2 / 4.6 / 10 / 22 and 46 mg/L), by dilution with algal medium. Concentrations were determined based on the result of a non-GLP pre-test.

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. At the end of the test, the measured concentrations were much lower than at the beginning. Although the test was performed in completely filled and closed test vessels,it is highly probable that some of the test substance was removed by volatilisation during handling. Because the measured concentrations were not within the range of 20 % of the nominal concentration, the determination of the results was based on the geometric mean of the measured concentrations.

A 72-hour NOEC (growth rate), ECr10 and ECr50 of 0.84, 2.22 and >2.23 mg/L, respectively were determined in Desmodesmus subspicatus. All validity criteria were met and the study was considered Klimisch Code 1 (reliable without restriction).

Key value for chemical safety assessment

EC50 for freshwater algae:

2.53 mg/L

EC10 or NOEC for freshwater algae:

2.22 mg/L

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