2-(methylnitroamino)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:

06 July 2020 – 10 July 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)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: As the test item was phlegmatized in 15 % water for safety reasons during transportation, before the test item preparation for administration, the test item was dried under vacuum in the desiccator and weighed. The procedure of drying in the desiccator under vacuum and weighing was repeated until a constant weight was obtained.

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 106.0 mg/L test item in algal medium (without algae) was prepared. For complete dissolution, the nominal load was mixed with the corresponding amount of algal medium and shaking vigorously for 23 hours. 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 May 2020 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; 21.3 – 22.6 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:

21.2 – 22.2 ºC

pH:

7.0 - 7.6

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), 5.30, 11.02, 23.81, 49.59 and 110.83 mg/L.
Measured concentrations for 0, 4.6, 10, 22, 46 and 100 mg/L (final time, T72h): n.d (control), 5.45, 11.32, 24.74, 50.53 and 128.87 mg/L.
Geometric mean of measured concentrations: 5, 11, 24, 50 and 120 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: 3.8E+03 cells/mL
- Control end cells density: 237084 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: the treatments showed no toxicity and even a stimulation of algal growth was observed in each concentration compared to the blank control. 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.:

> 120 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

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

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 120 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

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

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 120 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

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

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 120 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

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

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 120 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

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

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 120 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: No 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 stimulation of growth found in any treatment: yes, stimulation of algal growth was showed at all concentrations compared to the blank control.
- 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 May 2020.
- ErC50 (0-72h) = 1.00 mg/L (95% CL: 0.97-1.03 mg/L)
- EyC50 (0-72h) = 0.35 mg/L (95% CL: 0.32-0.39 mg/L)

Reported statistics and error estimates:

As algal growth in the treatments was higher than in the blank control, no statistical determination of the EC values, NOEC and LOEC were performed.

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	3820	14748	49168	237084
	SD	0	2870	5978	42564
4.6	Mean	3820	14587	126887	636507
	SD	0	615	9087	18201
10	Mean	3820	17327	136460	705160
	SD	0	1489	1311	152639
22	Mean	3820	15193	130860	632433
	SD	0	518	10997	16565
46	Mean	3820	15840	136507	617573
	SD	0	1709	8046	20244
100	Mean	3820	13460	104380	481307
	SD	0	322	18193	33984

Table 2. pH values

Nominal Concentrationin mg/L	0 h	72 h
Blank control	7.5	7.5
4.6	7.5	7.2
10	7.5	7.2
22	7.5	7.3
46	7.6	7.2
100	7.6	7.0

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	3.5795	101.7 %	--
QC 10 mg/L	3.5758	101.6 %	--
Blank control	n.a.	--	n. d.
Blank control	n.a.	--	n. d.
4.6	0.8686 *	--	5.2
4.6	0.8983 *	--	5.4
10	1.8832 *	--	10.9
10	1.9332 *	--	11.2
22	4.1230 *	--	23.4
22	4.2801 *	--	24.2
46	8.8209 *	--	49.6
46	8.8303 *	--	49.62
100	4.8948 **	--	110.68
100	4.9076 **	--	110.97
QC 10 mg/L	3.5829	101.8%	--
QC 10 mg/L	3.5884	101.9%	--

* 2-fold diluted before measurement

** 8-fold diluted before measurement

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

Nominal Concentration Test Item	Area  t = 72 h	Recovery QC	Measured Conentration  t = 72 h
mg/L	mAU*min	%	mg/L
QC 10 mg/L	3.5564	101.0 %	--
QC 10 mg/L	3.5252	100.2 %	--
Blank control	--	--	--
Blank control	--	--	--
4.6	0.9165 *	--	5.49
4.6	0.9035 *	--	5.41
10	1.9639 *	--	11.33
10	1.9630 *	--	11.32
22	4.3695 *	--	24.74
22	4.3705 *	--	24.75
46	8.9737 *	--	50.42
46	9.0125 *	--	50.63
100	5.7025 **	--	128.70
100	5.7173 **	--	129.03
QC 10 mg/L	3.5487	100.8 %	--
QC 10 mg/L	3.5492	100.8 %	--

* 2-fold diluted before measurement

** 8-fold diluted before measurement

As the deviation from the nominal concentration of the QC samples was lower than ± 3 %, the sensitivity correction factor from the QC samples was not taken into account.

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	5.30	115 %	5.45	118%	5
10	11.02	110 %	11.32	113%	11
22	23.81	108 %	24.74	112%	24
46	49.59	108 %	50.53	110%	50
100	110.83	111 %	128.87	129%	120

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	1.37	233264
	SD	0.06	42564
4.6	Mean	1.71	632687
	SD	0.01	18201
10	Mean	1.73	701340
	SD	0.07	152639
22	Mean	1.70	628613
	SD	0.01	16565
46	Mean	1.70	613753
	SD	0.01	20244
100	Mean	1.61	477487
	SD	0.02	33984

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	-24.31	-171.23
10	-26.45	-200.66
22	-24.16	-169.49
46	-23.58	-163.12
100	-17.49	-104.70

* 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 *	--	--	--	--	--
Replicate 2	1.220	1.432	1.665	15%	1.439
Replicate 3	1.212	1.263	1.388	7%	1.288
Replicate 4	1.622	0.768	1.663	37%	1.351
Replicate 5	1.209	1.333	1.529	12%	1.357
Replicate 6	1.421	1.265	1.585	11%	1.424
Mean	1.337	1.212	1.566	17%	1.372
Standard deviation	0.183	0.258	0.115		0.061
CV	14%	21%	7%		4%

CV: coefficient of variation

* In replicate 1 of the blank control the algal cells were coagulated. Therefore, the determination of the cell numbers was falsified. Hence, this replicate was excluded from the evaluation.

Table 9: Validity assessment

Parameter	Validity criteria	Observed value	Assessment
Increase factor biomass	factor 16 in 72 h	62	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	191 *	589 *	930 *	3636 *
	191	647	2708	14309
	191	642	2271	9098
	191	967	2084	10996
	191	640	2426	11192
	191	791	2803	13676
4.6	191	744	5822	31606
	191	694	6563	32825
	191	750	6648	31045
10	191	945	6887	44036
	191	797	6826	31545
	191	857	6756	30193
22	191	740	6255	31599
	191	789	6197	30805
	191	750	7177	32461
46	191	867	7279	30600
	191	810	6685	32001
	191	699	6512	30035
100	191	671	4452	22404
	191	690	6224	23992
	191	658	4981	25800

* In replicate 1 of the blank control the algal cells were coagulated. Therefore, the determination of the cell numbers was falsified. Hence, this replicate was excluded from the evaluation.

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	3820 *	11780 *	18600 *	72720 *
	2	3820	12940	54160	286180
	3	3820	12840	45420	181960
	4	3820	19340	41680	219920
	5	3820	12800	48520	223840
	6	3820	15820	56060	273520
	Mean	3820	14748	49168	237084
	SD	0	2870	5978	42564
4.6	1	3820	14880	116440	632120
	2	3820	13880	131260	656500
	3	3820	15000	132960	620900
	Mean	3820	14587	126887	636507
	SD	0	615	9087	18201
10	1	3820	18900	137740	880720
	2	3820	15940	136520	630900
	3	3820	17140	135120	603860
	Mean	3820	17327	136460	705160
	SD	0	1489	1311	152639
22	1	3820	14800	125100	631980
	2	3820	15780	123940	616100
	3	3820	15000	143540	649220
	Mean	3820	15193	130860	632433
	SD	0	518	10997	16565
46	1	3820	17340	145580	612000
	2	3820	16200	133700	640020
	3	3820	13980	130240	600700
	Mean	3820	15840	136507	617573
	SD	0	1709	8046	20244
100	1	3820	13420	89040	448080
	2	3820	13800	124480	479840
	3	3820	13160	99620	516000
	Mean	3820	13460	104380	481307
	SD	0	322	18193	33984

* In replicate 1 of the blank control the algal cells were coagulated. Therefore, the determination of the cell numbers was falsified. Hence, this replicate was excluded from the evaluation.

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	1.13 *	0.79 *	0.98 *
	2	1.22	1.33	1.44
	3	1.21	1.24	1.29
	4	1.62	1.19	1.35
	5	1.21	1.27	1.36
	6	1.42	1.34	1.42
	Mean	1.34	1.27	1.37
	SD	0.18	0.06	0.06
4.6	1	1.36	1.71	1.70
	2	1.29	1.77	1.72
	3	1.37	1.77	1.70
	Mean	1.34	1.75	1.71
	SD	0.04	0.04	0.01
10	1	1.60	1.79	1.81
	2	1.43	1.79	1.70
	3	1.50	1.78	1.69
	Mean	1.51	1.79	1.73
	SD	0.09	0.00	0.07
22	1	1.35	1.74	1.70
	2	1.42	1.74	1.69
	3	1.37	1.81	1.71
	Mean	1.38	1.77	1.70
	SD	0.03	0.04	0.01
46	1	1.51	1.82	1.69
	2	1.44	1.78	1.71
	3	1.30	1.76	1.69
	Mean	1.42	1.79	1.70
	SD	0.11	0.03	0.01
100	1	1.26	1.57	1.59
	2	1.28	1.74	1.61
	3	1.24	1.63	1.64
	Mean	1.26	1.65	1.61
	SD	0.02	0.09	0.02

* In replicate 1 of the blank control the algal cells were coagulated. Therefore, the determination of the cell numbers was falsified. Hence, this replicate was excluded from the evaluation.

Table 13: Yield

Nominal Conc. in mg/L	Replicate	Yield [Cell Concentration/mL]
		72 h
Blank control	1	68900 *
	2	282360
	3	178140
	4	216100
	5	220020
	6	269700
	Mean	233264
	SD	42564
4.6	1	628300
	2	652680
	3	617080
	Mean	632687
	SD	18201
10	1	876900
	2	627080
	3	600040
	Mean	701340
	SD	152639
22	1	628160
	2	612280
	3	645400
	Mean	628613
	SD	16565
46	1	608180
	2	636200
	3	596880
	Mean	613753
	SD	20244
100	1	444260
	2	476020
	3	512180
	Mean	477487
	SD	33984

* In replicate 1 of the blank control the algal cells were coagulated. Therefore, the determination of the cell numbers was falsified. Hence, this replicate was excluded from the evaluation.

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	24.84 *	66.53 *
	2	-4.89	-21.05
	3	6.11	23.63
	4	1.51	7.36
	5	1.08	5.68
	6	-3.80	-15.62
	Mean	0!	0!
	SD	4.45	18.25
4.6	1	-24.15	-169.35
	2	-25.07	-179.80
	3	-23.72	-164.54
	Mean	-24.31	-171.23
	SD	0.69	7.80
10	1	-32.21	-275.93
	2	-24.11	-168.83
	3	-23.04	-157.24
	Mean	-26.45	-200.66
	SD	5.02	65.44
22	1	-24.15	-169.29
	2	-23.53	-162.48
	3	-24.80	-176.68
	Mean	-24.16	-169.49
	SD	0.64	7.10
46	1	-23.37	-160.73
	2	-24.45	-172.74
	3	-22.91	-155.88
	Mean	-23.58	-163.12
	SD	0.79	8.68
100	1	-15.79	-90.45
	2	-17.45	-104.07
	3	-19.22	-119.57
	Mean	-17.49	-104.70
	SD	1.72	14.57

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.  

* In replicate 1 of the blank control the algal cells were coagulated. Therefore, the determination of the cell numbers was falsified. Hence, this replicate was excluded from the evaluation.

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 > 120 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, no significant inhibition was observed up to the concentration of100 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 108 % and 115 % of the nominal concentrations at the beginning of the test and between 110 % and 129 % 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 inhibition of algal growth was observed at any tested concentration. Furthermore, stimulation of algal growth was showed at all concentrations compared to the blank control. Therefore, no statistical evaluation was performed. Based on these results, the growth rate inhibition effect concentrations were determined to be 72 h-ErC10 > 120 mg/L, 72 h-ErC50 > 120 mg/L and 72h-NOEC ≥ 120 mg/L, and the yield inhibition effect concentrations were 72h-EyC10 > 120 mg/L, 72h-EyC50 > 120 mg/L and 72h-NOEC ≥ 120 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 > 120 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

120 mg/L

EC10 or NOEC for freshwater algae:

120 mg/L