nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 October 2016 - 12 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide

EC Number:

946-436-3

Molecular formula:

UVCB

IUPAC Name:

nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide

impurity 1

Reference substance name:

(p-chlorophenoxy)isobutyroyl chloride

EC Number:

255-286-1

EC Name:

(p-chlorophenoxy)isobutyroyl chloride

Cas Number:

41267-93-0

Molecular formula:

C10H10Cl2O2

IUPAC Name:

2-(4-chlorophenoxy)-2-methylpropanoyl chloride

impurity 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

Test material form:

solid

Details on test material:

Identification: Nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide
Appearance: Light yellow lumps
Test item storage: At room temperature; Store in closed container

Specific details on test material used for the study:

- Irritant/corrosive: yes
- Solubility in water: 5-10%
- Stability in water: stable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 and t=48 h
Volume: 2.0 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer (≤ -15°C) until analysis. Silanised vials were used to store the samples.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: Direct application to test medium.
* For the range-finding test the test solution was prepared at a loading rate of 100 mg/L and stirred for three days. The dispersion was filtered through a 0.45 μm membrane filter (Whatman, RC55) to remove any undissolved material. The resulting saturated solution was used as the highest test concentration and lower test concentrations were prepared by dilution of this saturated solution in test medium. All final test solutions were clear and colourless. A slight layer of foam was observed in the two highest concentrations.
* For the final test the test solution was prepared at 1.0 mg/L applying 15 minutes of stirring, which resulted in a clear and colourless solution that was used as the highest test concentration. Lower test concentrations were prepared by dilution of the highest test concentration in test medium. The glassware used in the final test was incubated with test concentrations 24 hours prior to the start of the test to improve the stability of the exposure concentrations, as the test item tends to adsorb to glass. The solutions used for incubation were discharged before the start of the test and the daphnids were exposed to freshly prepared solutions.
- Controls: test medium without test item or other additives (blank-control)

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus, 1820
- Source: in-house laboratory culture with a known history. At least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Age of parental stock: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Age at study initiation: < 24 hours, from parental daphnids of more than two weeks old.
- Feeding during test: no

ACCLIMATION
- Acclimation period: no

BREEDING:
- Method: each batch was started with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium (M7) in an all-glass culture vessel. After 7 days of cultivation half of the medium was renewed twice a week.
- Feeding: daily, a suspension of fresh water algae.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg CaCO3/L

Test temperature:

19-20 °C

pH:

7.8-8.0

Dissolved oxygen:

8.9-9.0 mg O2/L

Nominal and measured concentrations:

Nominal test concentrations: 0.022, 0.046, 0.1, 0.22, 0.46 and 1.0 mg/L
Average exposure concentrations (t=0-48 h): 0.0083, 0.018, 0.053, 0.13, 0.28 and 0.66 mg/L, respectively corresponding to nominal concentrations above.
Measured concentrations were 72-80% of nominal at the start of the test and had decreased to 23-69% of initial at the end of the test, therefore final results were based on the geometric mean measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all-glass
- Fill volume: 80 mL, pre-incubated with test concentrations for 24 hours
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Standard medium used: yes, adjusted ISO medium
- Source/preparation of dilution water: tap water purified by reverse osmosis
- Culture medium different from test medium: yes, M7 was used as culture medium

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours, daily

EFFECT PARAMETERS MEASURED: mobility at 24 and 48 hours.
- Additional measurements: pH and dissolved oxygen at the beginning, after 24 hours of exposure and at the end of the test, for all concentrations and the control. Temperature of the medium: continuously in a temperature control vessel.

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100% of a saturated solution prepared at 100 mg/L
- Results used to determine the conditions for the definitive study: yes, the EC50 was expected to be between 0.1% and 10% of a saturated solution prepared at 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (November 2016)

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.115 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI 0.098-0.133 mg/L

Details on results:

- No biological, behavioural or other abnormalities were observed in the control group or any of the test concentrations.
- One daphnia was observed trapped at the surface after 24 hours at the average exposure concentration of 0.28 mg/L (this daphnia was reimmersed into the respective solutions before recording of mobility).
- Because the test solutions did not remain stable over the 48 h period (23-69% of initial measured concentrations), the final results were based on geometric mean measured concentrations (t=0-48 h).
- Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 48h-EC50: 0.58 mg/L, 95%-confidence interval: 0.49-0.67 mg/L
- Other: results fell within the historical range.

Reported statistics and error estimates:

The EC50-values were calculated from the weibits of the percentages affected daphnids and the logarithms of the corresponding test item geometric mean measured concentrations using the maximum likelihood estimation method.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

In the range-finding test, no daphnids became immobilized in the control or in the lowest concentration tested throughout the test duration. In the higher test concentrations, effects were complete after 24 hours of exposure. Microscopic observations of daphnids exposed to 1.0% of the saturated solution at prepared at 100 mg/L revealed no test item attached to their bodies at 24 hours of exposure.

Table 1 Average exposure concentration versus nominal concentration

Test item^1 Nominal concentration (mg/L)	Measured concentrations (mg/L)		Average exposure (mg/L)
	t=0h	t=48h
0.022	0.017	0.0040	0.0083
0.046	0.033	0.0097	0.018
0.1	0.077	0.036	0.053
0.22	0.17	0.094	0.13
0.46	0.35	0.23	0.28
1	0.80	0.55	0.66

¹ Nitrate amine salt of N-[3-dimethylaminopropyl]- C₁₄-C₂₀ amides, saturated, reaction products with ethylene oxide.

Table 2 Number of introduced daphnids and incidence of immobility in the final test

Time (h)	Replicate		Test item^1, Average exposure concentration (mg/L)
		Control	0.0083	0.018	0.053	0.13	0.28	0.66
0	A	5	5	5	5	5	5	5
	B	5	5	5	5	5	5	5
	C	5	5	5	5	5	5	5
	D	5	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20	20
24	A	0	0	0	0	1	5	5
	B	0	0	0	0	0	5	5
	C	0	0	0	0	3	5(1)	5
	D	0	0	0	0	2	4	5
	Total immobilized	0	0	0	0	6	19	20
	Effect %	0	0	0	0	30	95	100
48	A	0	0	0	0	3	5	5
	B	0	0	0	0	3	5	5
	C	0	0	0	0	4	5	5
	D	0	0	0	0	4	5	5
	Total immobilized	0	0	0	0	14	20	20
	Effect %	0	0	0	0	70	100	100

¹ Nitrate amine salt of N-[3-dimethylaminopropyl]- C₁₄-C₂₀ amides, saturated, reaction products with ethylene oxide

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

1). In the control, no daphnids became immobilised or showed other signs of disease or stress. 2). The oxygen concentration at the end of the test was ≥ 3 mg/L in control and test vessels.

Conclusions:

The 48h-EC50 of Nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide, towards Daphnia magna, was 0.115 mg/L based on geometric mean measured concentrations.

Executive summary:

In a 48 h acute toxicity study conducted according to OECD guideline 202 and GLP principles, water fleas (Daphnia magna) were exposed to the test substance under static conditions at the following nominal concentrations: 0 (blank-control), 0.022, 0.046, 0.1, 0.22, 0.46 and 1.0 mg/L. Exposure concentrations were varified by a validated analytical method to be 0, 0.0083, 0.018, 0.053, 0.13, 0.28 and 0.66 mg/L. Measured concentrations were 72 -80% of nominal at the start of the test and decreased to 23 -69% of initial at the end of the test. The incidence of immobilisation was recorded at 24 and 48 hours.

The 48h-EC50 was 0.115 mg/L based on geometric mean measured concentrations. The acceptability criteria were met and the study is considered to be reliable with no restrictions.