N,N-bis(2-ethylhexyl)formamide

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 - 23 Apr 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: "Daphnia sp., Acute Immobilization Test" stipulated in the "Testing Methods for New Chemical Substances" of Japanese Chemical Substances Control Law

Version / remarks:

March 31, 2011, Yakushokuhatsu 0331 No. 7

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document, No. 23 (Second Edition), "Guidance Document onAqueous-phaseAquatic Toxicity Testing of Difficult Test Chemicals"

Version / remarks:

February 2019

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N-bis(2-ethylhexyl)formamide

EC Number:

860-695-9

Cas Number:

88247-41-0

Molecular formula:

C17H35NO

IUPAC Name:

N,N-bis(2-ethylhexyl)formamide

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 4.76, 8.57, 15.4, 27.8, 50% of stock solution content
- Sampling method: Approximately 10-50 mL were sampled at the start and end of the exposure. Another solution sampled separately from the preparation container (at the start of exposure). One test vessel in each test level (at the end of exposure).

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test sample (0.110 g) and dilution water (1100 mL) were mixed to prepare the nominal concentration of 100 mg/L. Then they were sealed and gently stirred with a magnetic stirrer for 48 hours at about 20°C. After stirring, they were settled for 1 hour as it was, and the middle layer was collected as the stock solution. Required volumes of the stock solution and dilution water processing similar to the control without the test item were mixed and stirred in a preparation container to prepare the test solution which was divided into each test vessel.
- Controls: Dilution water without the test item, whi~h was treated in the same manner as the test solution except for collecting the middle layer, was used for the control.
- Test concentration separation factor: a geometric series with a factor of 1.8
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test solutions in the exposure levels and the control were colorless and clear at the start and end of exposure.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna Clone A
- Age at study initiation (mean and range, SD): less than 24-hours old
- Stage and instar at study initiation: juveniles
- Source: The University of Sheffield, UK, supplied on July 9, 1990
- Age of parental stock (mean and range, SD): The parent animals used for the test were same lot and their age was 21-day old
- Feeding during test : No

ACCLIMATION
- Acclimation period: Young daphnids produced by parents that were cultured in this test facility were used
- Acclimation conditions (same as test or not): The parents to obtain young daphnids were bred in the same quality of water (dechlorinated tap water), water temperature (20±1 °C) and photoperiod (16-hour light/8-hour dark) as used in the study.
- Type and amount of food: Chlorella vulgaris of 0.1-0.2 mgC (Organic carbon content)/day per Daphnia was fed to the parents
- Feeding frequency: once a day
- Health during acclimation (any mortality observed): 100% survival

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

40 mg/L CaCO3

Test temperature:

20.0 - 20.1 °C

pH:

7.7 - 7.9

Dissolved oxygen:

7.7 - 8.2 mg/L

Nominal and measured concentrations:

- Nominal: control, 4.76, 8.57, 15.4, 27.8, 50% of stock solution content
- Measured (at start): < LOQ, 0.0666, 0.135, 0.290, 0.513, 0.801 mg/L
- Measured (at the end): < LOQ, 0.0631, 0.120, 0.252, 0.494, 0.804 mg/L
- Measured (time-weighted mean): 0.0648, 0.128, 0.270, 0.503, 0.802 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL stoppered glass Erlenmeyer flask (closed vessel) placed in water bath
- Type (delete if not applicable): closed
- Volume of solution: 50 mL
- 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
- Source/preparation of dilution water: Dechlorinated tap water, aerated sufficiently and temperature-controlled, was used. It was confrrmed that the concentration of residual chlorine in dilution water was <= 0.02 mg/L at the use.
- Total organic carbon: < 0.5 mg/L
- pH: 7.8 (23.5 °C)
- Particulate matter: < 1 mg/L suspended solids
- Chlorine: < 0.02 mg/L
- Alkalinity: 42 mg/L CaCO3
- Conductivity: 17 mS/m (at 25 °C)
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16-hour light/8-hour dark
- Light intensity: room light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations about immobility and symptoms of .the test organisms were conducted at 24 and 48 hours after exposure. Daphnids were considered immobile when they were not able to swim within 15 seconds after gentle agitation of the test vessel. Dissolved oxygen, pH, and temperature were measured at the start and end of exposure. Appearance of test solution was monitored at the start and end of exposure.

VEHICLE CONTROL PERFORMED: No

RANGE-FINDING STUDY
- Conducted: Yes
- Test concentrations: 5, 10, 50, and 100% content of a stock solution (100 mg/L nominal, 1.6 mg/L arithmetic mean measured)
- Results used to determine the conditions for the definitive study: 0, 10, 100, 100% mortality of daphnids after 48-h exposure, respectively

Reference substance (positive control):

yes

Remarks:

A 48-hour acute immobilization study with the reference substance potassium dichromate with the test organisms was periodically conducted. The latest data of testing was April 5, 2021.

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.177 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities:Observed abnormal responses during exposure were immobilization (including lethargic) and reduced activity. In the control, no abnormal response (discolor of body, trapping at the surface of the water and so on) was observed during exposure.

Results with reference substance (positive control):

- ECx: The 48-h EC50 with potassium dichromate was 0.18 mg/L

Reported statistics and error estimates:

The ECso values were calculated by Binomial test. The ECso was estimated using a computer program (running on Microsoft software "Excel") developed by the test facility. The results of this study were estimated based on the time-weighted mean of the measured concentrations of the test solution during exposure as the test concentrations.

Any other information on results incl. tables

Table 1. Immobilisation of Daphnia magna after an 24 h and 48 h exposure to DEHF

Measured Concentration [mg/l]	Immobile daphnids
	absolute		%
	24 h	48 h	24 h	48 h
Control	0	0	0	0
	0	0
	0	0
	0	0
0.068	0	0	0	0
	0	0
	0	0
	0	0
0.128	1	1	5	5
	0	0
	0	0
	0	0
0.270	1	5	5	100
	0	5
	0	5
	0	5
0.503	5	5	95	100
	5	5
	4	5
	5	5
0.802	5	5	100	100
	5	5
	5	5
	5	5

 

Table 2.Cumulative biological observations

Measured Concentration [mg/l]	Observations (n=20)
	24 h	48 h
Control	-	-
0.0648	-	-
0.128	IM (1); RA (1)	IM (1); LETH (1); RA (1)
0.270	IM (1); RA (9)	IM (20); LETH (17)
0.503	IM (19); LETH (16); RA (1)	IM (20); LETH (20)
0.802	IM (20); LETH (20)	IM (20); LETH (20)

-: Normal response

IM: Immobilization

RA: Reduced activity

LETH: Lethargic, the severe symptom including mortality in immobilization

 

Table 3: Validity criteria for OECD 202.

Criterion from the guideline	Outcome	Validity criterion fulfilled
In the control, including the control containing the solubilising agent, not more than 10% of the daphnids should have been immobilized.	0% of daphnids were immobilized in the control	Yes
The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.	Dissolved oxygen in all vessels was 7.7 – 7.9 mg/l	Yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See Table 3 in 'any other information on results incl. tables'