2-(ethylnitroamino)ethyl nitrate

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:

08 June 2020 – 12 June 2020

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:

Stock solutions were autoclaved for 20 min instead of 15 min (considered uncritical as the goal of sterility was achieved). The pre-culture was prepared 5 days before start instead of 2-4 days (considered uncritical as validity critiria were met).

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 4.6, 10, 22, 46 and 100 mg/L.
- Sampling method: At the beginning and at the end of the test, the content of the test item in the test solutions was determined using HPLC-UV-determination. Samples were diluted with an equal amount of acetonitrile (dilution factor 2). Further dilutions to reach the calibrated range were performed using acetonitrile/medium 50%/50% (v/v).
- Sample storage conditions before analysis: samples were kept in the fridge.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution containing 102 mg/L test item in algal medium (without algae) was prepared. For complete dissolution, the nominal load (102 mg/L) was mixed with the corresponding amount of algal medium and shaking vigorously for 23 hours and 15 minutes. The lower test concentrations were prepared by dilution of the stock solution in algal medium.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green alga Desmodesmus subspicatus
- Strain: 86.81
- Source (laboratory, culture collection): Culture was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysi-ologie of Universität Göttingen).
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 ºC. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

ACCLIMATION
- Acclimation period: 4 days (incubation) + 1 day (stored in fridge before starting the test)
- Culturing media and conditions (same as test or not): same as test (Lighting within range of 4440 – 8880 lux; 21.1 – 22.1 ºC)
- Any deformed or abnormal cells observed: Not reported.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test temperature:

21.5 – 22.2 ºC

pH:

7.4 - 7.8

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 4.6, 10, 22, 46 and 100 mg/L.
Measured concentrations for 0, 4.6, 10, 22, 46 and 100 mg/L (initial time, T0h): n.d (control), 4.89, 10.87, 23.08, 48.63 and 103.14 mg/L.
Measured concentrations for 0, 4.6, 10, 22, 46 and 100 mg/L (final time, T72h): n.d (control), 5.12, 11.60, 25.71, 57.11 and 125.41 mg/L.
Geometric mean of measured concentrations: 5, 11, 24, 53 and 114 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: flask
- Type (delete if not applicable): open (covered with perforated plastic foil acting as a stopper)
- Material, size, headspace, fill volume: glass, 65 mL capacity, 45 mL fill volume.
- Aeration: Not specified.
- Initial cells density: 2.3E+03 cells/mL
- Control end cells density: 612480 cells/mL (average)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes (OECD TG 201 medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the OECD medium was prepared as per the OECD guideline 201.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of the controls and the test solutions was checked at the beginning and at the end of the test. The temperature was continuously measured by a temperature data logger.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Constant illumination
- Light intensity and quality: 4440 – 8880 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: Before the start of incubation and every 24 hours, the cell number was determined with an electronic particle counter (Z2, Beckman Coulter).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometric factor of 2.2
- Justification for using less concentrations than requested by guideline: Not applicable; 5 concentrations used for the main test.

- Range finding study
- Test concentrations: 1, 10 and 100 mg test item/L along with negative control.
- Results used to determine the conditions for the definitive study: slight but not statistically significant inhibition was observed at the concentration 100 mg/L. Nevertheless, for precautionary reasons, a full test with concentrations of 4.6, 10, 22, 46 and 100 mg/L was performed.

Reference substance (positive control):

yes

Remarks:

(potassium dichromate)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 114 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: No significant inhibition of algal growth observed at the highest concentration tested.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 114 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: No significant inhibition of algal growth observed at the highest concentration tested.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 114 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: No significant inhibition of algal growth observed at the highest concentration tested.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 114 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: No significant inhibition of algal growth observed at the highest concentration tested.

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 114 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: No significant inhibition of algal growth observed at the highest concentration tested.

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 114 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: No significant inhibition of algal growth observed at the highest concentration tested.

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Normal and healthy appearance of the algae in all tested concentrations.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not reported.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the last study with potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was performed under GLP conditions in January/February 2020.
- ErC50 (0-72h) = 0.77 mg/L (95% CL: 0.77 -0.78 mg/L)
- EyC50 (0-72h) = 0.42 mg/L (95% CL: 0.38 -0.47 mg/L)

Reported statistics and error estimates:

The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.3.0. Statistical evaluation was performed with the nominal concentration whereas the biological results were based on the measured concentrations.

Table 1: cells number / mL

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	2320	11613	97930	612480
	SD	0	1744	10594	121531
4.6	Mean	2320	15913	142647	718293
	SD	0	5704	762	52845
10	Mean	2320	16353	109353	594140
	SD	0	7798	13648	121145
22	Mean	2320	32547	107647	651347
	SD	0	25795	23139	103575
46	Mean	2320	15253	103587	548967
	SD	0	607	7702	78677
100	Mean	2320	18940	113347	597260
	SD	0	6539	18436	89232

Table 2. pH values

Nominal Concentrationin mg/L	0 h	72 h
Blank control	7.5	7.8
4.6	7.5	7.8
10	7.5	7.8
22	7.5	7.7
46	7.4	7.7
100	7.4	7.7

Table 3. Analytical determination. Raw Data and Measured Concentrations, t = 0 h

Nominal Concentration Test Item	Area  t = 0 h	Recovery QC	Measured Conentration  t = 0 h
mg/L	mAU*min	%	mg/L
QC 10 mg/L	3.6020	103.1%	--
QC 10 mg/L	3.3785	96.7%	--
Blank control	--	--	n. d.
Blank control	--	--	n. d.
4.6	0.8481	--	4.85
4.6	0.8627	--	4.93
10	1.9491	--	11.15
10	1.8521	--	10.59
22	3.9974	--	22.88
22	4.0685	--	23.29
46	4.2434	--	48.58
46	4.2530	--	48.69
100	4.6472	--	106.40
100	4.3619	--	99.87
QC 10 mg/L	3.5276	100.9%	--
QC 10 mg/L	3.4587	99.0%	--

Table 4. Analytical determination. Raw Data and Measured Concentrations, t = 72 h

Nominal Concentration Test Item	Area  t = 0 h	Recovery QC	Measured Conentration  t = 0 h
mg/L	mAU*min	%	mg/L
QC 10 mg/L	2.981	85.3%	--
QC 10 mg/L	3.0271	86.6%	--
Blank control	--	87.0%	n. d.
Blank control	--	87.0%	n. d.
4.6	0.8069	87.0%	5.30
4.6	0.7534	87.0%	4.95
10	1.7893	87.0%	11.76
10	1.7411	87.0%	11.44
22	3.8508	87.0%	25.33
22	3.9685	87.0%	26.10
46	4.408	87.0%	57.99
46	4.2744	87.0%	56.23
100	4.7483	87.0%	124.93
100	4.7849	87.0%	125.89
QC 10 mg/L	3.0984	88.7%	--
QC 10 mg/L	3.0586	87.5%	--

Because the deviation of the QC sample was higher than ± 3 %, the sensitivity correction factor from the QC samples (mean 87 %) was taken into account at the end of the test after 72 hours, using the following equation:

Cmeasured (mg/L) = ((measured value- a) / b) x (100 / QC Recovery Rate (%)) x factor

with: a = intercept, b = slope and factor = dilution or enrichement factor

Table 5. Mean Measured Concentration and % of Nominal

Nominal Concentration Test Item	Mean Measured Concentration  t = 0 h	% of Nominal	Mean Measured Conentration  t = 72 h	% of Nominal	Geometric Mean
mg/L	mg/L	%	mg/L	%	mg/L
4,6	4.89	106%	5.12	111%	5
10	10.87	109%	11.60	116%	11
22	23.08	105%	25.71	117%	24
46	48.63	106%	57.11	124%	53
100	103.14	103%	125.41	125%	114

Table 6: 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	1.85	610160
	SD	0.07	121531
4.6	Mean	1.91	715973
	SD	0.02	52845
10	Mean	1.84	591820
	SD	0.07	121145
22	Mean	1.88	649027
	SD	0.06	103575
46	Mean	1.82	546647
	SD	0.05	78677
100	Mean	1.85	594940
	SD	0.05	89232

Table 7: Inhibition values

Nominal concentration in mg/L	% Inhibition
	Growth Rate (0-72 h)	Yield (0-72 h)
Blank control	0	0
4.6	-3.2	-17.3
10	0.5	3.0
22	-1.3	-6.4
46	1.8	10.4
100	0.3	2.5

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

Table 8: 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	1.589	2.056	1.565	16 %	1.737
Replicate 2	1.487	2.215	1.710	21 %	1.804
Replicate 3	1.830	1.807	2.060	7 %	1.899
Replicate 4	1.720	2.197	1.834	13 %	1.917
Replicate 5	1.477	2.271	1.977	21 %	1.908
Replicate 6	1.508	2.272	1.772	21 %	1.850
Mean	1.602	2.136	1.820	17 %	1.853
Standard deviation	0.144	0.180	0.180		0.071
CV	9 %	8 %	10 %		4 %

CV: coefficient of variation

Table 9: Validity assessment

Parameter	Validity criteria	Observed value	Assessment
Increase factor biomass	factor 16 in 72 h	264	Valid
Mean coefficient of variation of daily growth rates	max. 35 %	17 %	Valid
Coefficient of variation of average growth rate during the whole test period	max. 7 %	4 %	Valid

Table 10: Cell numbers measured (after 1:20 dilution of test cultures)

Nominal Concentration in mg/L	Cell Number/mL
	0 h	24 h	48 h	72 h
Blank control	116	568	4438	21234
	116	513	4701	25998
	116	723	4404	34566
	116	648	5833	36521
	116	508	4921	35542
	116	524	5082	29883
4.6	116	1125	7129	35432
	116	629	7172	33547
	116	633	7096	38765
10	116	629	6217	35678
	116	1266	5304	29876
	116	558	4882	23567
22	116	1083	5437	36787
	116	3100	6511	34127
	116	699	4199	26788
46	116	729	4897	26768
	116	771	5618	23899
	116	788	5023	31678
100	116	776	6211	34561
	116	1324	6188	29345
	116	741	4603	25683

Table 11: Cell numbers calculated (dilution factor 20)

Nominal Conc. in mg/L	Replicate	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	1	2320	11360	88760	424680
	2	2320	10260	94020	519960
	3	2320	14460	88080	691320
	4	2320	12960	116660	730420
	5	2320	10160	98420	710840
	6	2320	10480	101640	597660
	Mean	2320	11613	97930	612480
	SD	0	1744	10594	121531
4.6	1	2320	22500	142580	708640
	2	2320	12580	143440	670940
	3	2320	12660	141920	775300
	Mean	2320	15913	142647	718293
	SD	0	5704	762	52845
10	1	2320	12580	124340	713560
	2	2320	25320	106080	597520
	3	2320	11160	97640	471340
	Mean	2320	16353	109353	594140
	SD	0	7798	13648	121145
22	1	2320	21660	108740	735740
	2	2320	62000	130220	682540
	3	2320	13980	83980	535760
	Mean	2320	32547	107647	651347
	SD	0	25795	23139	103575
46	1	2320	14580	97940	535360
	2	2320	15420	112360	477980
	3	2320	15760	100460	633560
	Mean	2320	15253	103587	548967
	SD	0	607	7702	78677
100	1	2320	15520	124220	691220
	2	2320	26480	123760	586900
	3	2320	14820	92060	513660
	Mean	2320	18940	113347	597260
	SD	0	6539	18436	89232

Table 12: Growth rate over periods of test duration

Nominal Conc. in mg/L	Replicate	Growth Rate µ [day^-1]
		24 h	48 h	72 h
Blank control	1	1.59	1.82	1.74
	2	1.49	1.85	1.80
	3	1.83	1.82	1.90
	4	1.72	1.96	1.92
	5	1.48	1.87	1.91
	6	1.51	1.89	1.85
	Mean	1.60	1.87	1.85
	SD	0.14	0.05	0.07
4.6	1	2.27	2.06	1.91
	2	1.69	2.06	1.89
	3	1.70	2.06	1.94
	Mean	1.89	2.06	1.91
	SD	0.33	0.00	0.02
10	1	1.69	1.99	1.91
	2	2.39	1.91	1.85
	3	1.57	1.87	1.77
	Mean	1.88	1.92	1.84
	SD	0.44	0.06	0.07
22	1	2.23	1.92	1.92
	2	3.29	2.01	1.89
	3	1.80	1.79	1.81
	Mean	2.44	1.91	1.88
	SD	0.77	0.11	0.06
46	1	1.84	1.87	1.81
	2	1.89	1.94	1.78
	3	1.92	1.88	1.87
	Mean	1.88	1.90	1.82
	SD	0.04	0.04	0.05
100	1	1.90	1.99	1.90
	2	2.43	1.99	1.84
	3	1.85	1.84	1.80
	Mean	2.06	1.94	1.85
	SD	0.32	0.09	0.05

Table 13: Yield

Nominal Conc. in mg/L	Replicate	Yield [Cell Concentration/mL]
		72 h
Blank control	1	422360
	2	517640
	3	689000
	4	728100
	5	708520
	6	595340
	Mean	610160
	SD	121531
4.6	1	706320
	2	668620
	3	772980
	Mean	715973
	SD	52845
10	1	711240
	2	595200
	3	469020
	Mean	591820
	SD	121145
22	1	733420
	2	680220
	3	533440
	Mean	649027
	SD	103575
46	1	533040
	2	475660
	3	631240
	Mean	546647
	SD	78677
100	1	688900
	2	584580
	3	511340
	Mean	594940
	SD	89232

Table 14: % Inhibition at the end of the test (72 hours)

Nominal Conc. in mg/L	Replicate	% Inhibition Growth Rate µ 72 hours	% Inhibition Yield 72 hours
Blank control	1	6.26	30.78
	2	2.62	15.16
	3	-2.50	-12.92
	4	-3.49	-19.33
	5	-3.00	-16.12
	6	0.12	2.43
	Mean	0	0
	SD	3.84	19.92
4.6	1	-2.95	-15.76
	2	-1.97	-9.58
	3	-4.57	-26.68
	Mean	-3.16	-17.34
	SD	1.31	8.66
10	1	-3.07	-16.57
	2	0.12	2.45
	3	4.39	23.13
	Mean	0.48	3.01
	SD	3.74	19.85
22	1	-3.62	-20.20
	2	-2.27	-11.48
	3	2.08	12.57
	Mean	-1.27	-6.37
	SD	2.98	16.98
46	1	2.10	12.64
	2	4.14	22.04
	3	-0.93	-3.45
	Mean	1.77	10.41
	SD	2.55	12.89
100	1	-2.50	-12.90
	2	0.44	4.19
	3	2.84	16.20
	Mean	0.26	2.49
	SD	2.68	14.62

Note: Individual inhibition values of each blank control replicate were compared to the mean inhibition value of the control groups. The sum of the deviations (mean value) of the controls results in 0% inhibition. Positive and negative values denote relative inhibition and relative stimulation, respectively.  

Validity criteria fulfilled:

yes

Remarks:

(increase of biomass in control during 72h>16 fold; coefficient of variation of the mean specific growth rate among replicates in control (t0-t72)<7%; the mean of the replicate coefficients of variation in section-by-section growth rate in control < 35%)

Conclusions:

In an Algae Growth Inhibition Test with Desmodesmus subspicatus green algae, the growth rate inhibition effect concentrations 72 h-ErC50 and 72 h-ErC10 were determined to be > 114 mg/L.

Executive summary:

An Algae Growth Inhibition Test was performed with Desmodesmus subspicatus on the test substance over a period of 72 h in static conditions according to OECD Guideline 201, following GLP. OECD medium was used for algae culturing. In the previous solubility study the test item was found soluble in OECD medium and thus no vehicle was needed. In a preliminary range finding study with concentrations of 1, 10 and 100 mg/L, slight but not statistically significant inhibition was observed at the concentration 100 mg/L. Nevertheless, for precautionary reasons, a full test with concentrations of 4.6, 10, 22, 46 and 100 mg/L was performed. Negative control consisted of test medium alone and potassium dichromate was used as reference substance.Three replicates were performed for each concentration of the test item, along with six replicates for the negative control group. A validated analytical method based on HPLC/UV was used to monitor the concentration and stability of the active ingredient in the test solution. The measured concentrations lay between 103 % and 109 % of the nominal concentrations at the beginning of the test and between 111 % and 125 % 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. All validity criteria were fulfilled. No significant inhibition of algal growth was observed at any tested concentration. Based on these results, the growth rate inhibition effect concentrations were determined to be 72 h-ErC10 > 114 mg/L, 72 h-ErC50 > 114 mg/L and 72h-NOEC ≥ 114 mg/L, and the yield inhibition effect concentrations were 72h-EyC10 > 114 mg/L, 72h-EyC50 > 114 mg/L and 72h-NOEC ≥ 114 mg/L.

Description of key information

Key study. Test method according to OECD 201, GLP study. In an Algae Growth Inhibition Test with Desmodesmus subspicatus green algae, the growth rate inhibition effect concentrations 72 h-ErC50 and 72 h-ErC10 were determined to be > 114 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

114 mg/L

EC10 or NOEC for freshwater algae:

114 mg/L