2-(ethylnitroamino)ethyl nitrate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 June 2020 – 12 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

The temperature in the experiment was in the range 15.8 – 24.5ºC, and therefore not in the desired range (18 - 22ºC). As no immobility was observed in the blank control and the treatment, this was stated as uncritical.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 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 test solution containing 101 mg/L test item in dilution water was prepared. For complete dissolution the loading rate 101 mg/L was mixed with the corresponding amount of dilution water and shaked vigorously for 24 hours.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus, Berlin.
- Age at study initiation (mean and range, SD): between 0 and 24 hours
- Method of breeding: Daphnia magna is bred in the lab facility throughout the year. They multiply by parthenogenesis, thus being genetically identical. The keeping is performed similar to the method described in the OECD guideline.
Vessels: preserving glasses, nominal volume 2 L
Medium: M4-Medium (recipe of ELENDT)
Food: green algae (Desmodesmus subspicatus)
Medium renewal: twice a week
Photoperiod: 16/8 hours, using neon tubes
Temperature: 20 ± 2ºC
- Source: Umweltbundesamt Berlin
- Feeding during test: No
- Food type: green algae (Desmodesmus subspicatus)

ACCLIMATION
- Acclimation period: yes, not specified.
- Acclimation conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

None

Test temperature:

15.8 – 24.5ºC

pH:

7.2 (T0 and T48)

Dissolved oxygen:

8.5 to 8.7 mg O2/L

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 100 mg/L.
Measured concentrations for 0 and 100 mg/L (initial time, T0h): n.d. (control) and 97.55 mg/L.
Measured concentrations for 0 and 100 mg/L (final time, T48h): n.d. (control) and 109.75 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Material, size, headspace, fill volume: glass beakers, nominal volume 50 mL, tall shape.
- Volume of solution: 20 ± 5 mL
- Aeration: not specified.
- Renewal rate of test solution (frequency/flow rate): Not applicable (static test)
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: 4 mL of test media per daphnia.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Medium M4 ELENDT was prepared from concentrated stock solutions in deionized water as described in the OECD 202 guideline.
- Intervals of water quality measurement: pH and dissolved oxygen were measured at the start and at the end of the test (including Controls).

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16/8 hours, using neon tubes.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : the immobilised daphnia were counted after 24 and 48 h. Daphnia were considered immobilised when they did not perform any movements or were only able to move their antennae when the beaker was gently agitated. Daphnia which were trapped at the surface of the test solution were also considered immobilised.

VEHICLE CONTROL PERFORMED: Not applicable

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: No significant toxicity against daphnia was observed at any concentration in the range finding test. Based on these results, a limit test at 100 mg/L was conducted for the main study.

Reference substance (positive control):

yes

Remarks:

(potassium dichromate)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: No effects observed at the highest tested concentration.

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: No effects observed at the highest tested concentration.

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: No effects observed at the highest tested concentration.

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: No effects observed at the highest tested concentration.

Details on results:

- Behavioural abnormalities:
- Mortality of control: No mortality was observed over a period of 48 h in the control group.
- Immobilisation of control: No immobilisation was observed over a period of 48 h in the control group.
- Abnormal responses: no signs of stress or other abnormal behaviour was reported.
- 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, potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study. The 24h-EC50 value was within the requested range for the 24h-EC50 between 0.6 - 2.1 mg/L of potassium dichromate K2Cr2O7 as specified in the OECD guideline.
- EC50-24 h: 2.01 mg/L.

Table 1: Immobility

Nominal Concentration in mg/L	Immobility 24 hours					Immobility 48 hours
	absolute				in %	absolute				in %
Blank control	0	0	0	0	0	0	0	0	0	0
100	0	0	0	0	0	0	0	0	0	0

Table 2: pH and O2

Nominal Concentration in mg/L	pH		O2-Concentration in mg/L
	0 h	48 h	0 h	48 h
Blank control	7.3	7.2	8.8	8.6
100	7.2	7.2	8.7	8.5

Table 3: 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	3.7949	108.6%	--
QC 10 mg/L	3.6250	103.7%	--
Blank control	--	--	n. d.
Blank control	--	--	n. d.
100	4.5422	--	104.00
100	3.9790	--	91.10
QC 10 mg/L	3.8248	109.5%	--
QC 10 mg/L	2.9516	84.5%	--

Table 4: Raw Data and Measured Concentrations, t = 48 h

Nominal Concentration Test Item	Area  t = 48 h	Recovery QC	Measured Conentration  t = 48 h
mg/L	mAU*min	%	mg/L
QC 10 mg/L	3.0821	88.2%	--
QC 10 mg/L	3.1052	88.9%	--
Blank control	--	86.8%	n. d.
Blank control	--	86.8%	n. d.
100	4.1828	86.8%	110.27
100	4.1297	86.8%	108.87
QC 10 mg/L	2.9487	84.4%	--
QC 10 mg/L	3.0044	86.0%	--

Because the deviation of the QC sample was higher than ± 3 %, the sensitivity correction factor from the QC samples (mean 86.8 %) was taken into account at the end of the test after 48 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 = 48 h	% of Nominal
100 mg/L	97.55	98 %	109.75	110 %

Validity criteria fulfilled:

yes

Remarks:

(mortality in control < 10% (no immobilisation observed); dissolved oxygen concentration > 3 mg O2/L)

Conclusions:

In a short-term toxicity test to daphnia magna, the 48h-EC50 of the test substance was found to be higher than 100 mg/L.

Executive summary:

An acute aquatic toxicity study with Daphnia magna was conducted on test item according to OECD guideline 202, following GLP. In a preliminary range finding study no significant toxicity against daphnia was observed at the tested concentrations of 0, 1, 10 and 100 mg/L. Based on these results, the main study was performed as a limit test using the concentration of 100 mg/L. 20 Daphnia were exposed to the test item for the treatment for 48 hours in a static test system in 4 replicates of 5 daphnids per replicate. Elendt M4 medium was used as test medium. No vehicle was used since the test item was found soluble in test medium at the highest concentration tested. Negative control consisted of test medium alone and a solution of potassium dichromate was used as reference substance. 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 were 98 % of the nominal concentration at the beginning and 110 % at the end of the test. Therefore, the determination of the biological results was based on the nominal concentration. After 24 and 48 hours, the immobilised daphnia were counted. The concentration showed no toxicity. None of the animals was immobilised in the treatment and the negative control. Dissolved oxygen was higher than 3 mg/L in control and test vessels at the end of the test. Based on these results, the following conclusions can be established: 24h-EC50 > 100 mg/L, 48h-EC50 > 100 mg/L, 48h-LOEC > 100 mg/L and 48 h-NOEC ≥ 100 mg/L.

Description of key information

Key study. Test method according to OECD 202, GLP study. The 48h-EC50 of the test substance was found to be higher than 100 mg/L in Daphnia magna.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L