N,N-bis(2-ethylhexyl)formamide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 - 20 May 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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

March 23, 2006

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: "Algal Growth Inhibition Test" stipulated in the "Testing Methods for New Chemical Substances" of Japanese Chemical Substances Control Law

Version / remarks:

March 31, 2011

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 23 (second edition), "Guidance Document on Aqueous-phase Aquatic 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, 2.56, 6.40, 16.0, 40.0, 100% of stock solution content
- Sampling method: Approximately 10 mL of every test concentration were sampled from another solution from the preparation container at the start. Approximately 100 mL from all test concentrations were sampled from each test vessel at 24, and 48 hours after start of the exposure. Approximately 12 mL were sampled at the end of exposure from all concentrations.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test sample (0.100 g) and OECD medium (1000 mL) were mixed to prepare 100 mg/L nominal concentration, the solution was gently stirred for 48 hours. A second batch was prepared in exactly the same manner. After stirring, the solution was settled for 1 hour, the middle layer was collected as the stock solution. The stock solution was accordingly diluted to prepare the test solution.
- Controls: The test medium without test item, which was treated in the same manner as the stock solution (taking out of middle layer was not conducted)
- Test concentration separation factor: a geometric series with a factor of 2.5
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All test solutions were colorless and clear at the beginning of the study

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection (supplied on June 30, 1995)
- Method of cultivation: Passage cultured under sterile conditions in the test facility

ACCLIMATION
- Acclimation period: The used algae cells originated from a culture maintained at the test facility

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.3 - 22.6 °C

pH:

7.8 - 9.5

Nominal and measured concentrations:

- Nominal: Control, 2.56, 6.40, 16.0, 40.0, 100% of stock solution content
- Measured (start): < LOQ, 0.0248, 0.103, 0.238, 0.455, 1.23 mg/L
- Measured (24 hours): < LOQ, 0.00867, 0.030, 0.111, 0.326, 0.994 mg/L
- Measured (48 hours): < LOQ, 0.00758, 0.0222, 0.102, 0.308, 0.929 mg/L
- Measured (end): < LOQ, 0.0176, 0.0598, 0.142, 0.359, 1.06 mg/L
- Measured (time-weighted mean): < LOQ, 0.0117, 0.0410, 0.131, 0.345, 1.02 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask (closed) placed in incubator with temperature and light control under continous shaking
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 100 mL
- Aeration: No
- Initial cells density: 0.5 x 10^4 cells/mL
- Control end cells density: 37 x 10^4 cells/mL (mean of six replicates)
- No. of vessels per concentration (replicates): 3 (one additional replicate for analytical determination of the test item was set for 24, 48 hours)
- No. of vessels per control (replicates): 6 (one additional replicate for measuring the background was set for control)


GROWTH MEDIUM
- Standard medium used: yes, OECD medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continous illumination
- Light intensity and quality: 89 µmol/m2 x s, 400 - 700 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: biomass (cell concentration) was determined by an electronic particle counter every 24 hours after start of the exposure. The pH at start and end of the exposure. In the beginning the pH was measured in another solution sampled separately from the preparation container. Temperature and light intensity were measured at the start of the exposure and every 24-hours thereafter in the incubator.
- Other: Blank correction was conducted by measuring the value of blank solution of test vessel prepared for background

TEST CONCENTRATIONS
- Spacing factor for test concentrations: a geometric series with a fcator of 2.5
- Range finding study: yes
- Test concentrations: 0.3, 1.0, 10, 100% of a stock solution with a nominal conecntration of 100 mg/L (1.34 mg/L measured arithmetic mean)
- Results used to determine the conditions for the definitive study: Growth inhibition rate based on growth rate (0-3 d): 0.79, -0.24, -6.0, 58%, respectively

CULTURING APPARATUS
-Details on culturing apparatus used: Incubator with temperature and light control

Reference substance (positive control):

yes

Remarks:

An algal growth inhibition study with the reference substance potassium dichromate is periodically conducted. The latest date of testing was March 1, 2021.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.02 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.131 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Unusual cell shape: In the 1.02 mg/L test concentration many thin and small cells were observed (cell miniaturization)

Results with reference substance (positive control):

- EC50: 1.1 mg/L based on growth rate

Reported statistics and error estimates:

In order to determine the NOEC for growth rate, first a Bartlett's test on variance homogeneity was run followed by a Step-down Jonckheere-Terpstra trend test.

Any other information on results incl. tables

Table 1. Cell counts

Measured concentration (mg/L)*	Cell concentration (x 10^4 cells/mL)
	0 hours	24 hours	48 hours	72 hours
Control	0.50	2.7	12	36
	0.50	2.7	13	37
	0.50	2.4	12	37
	0.50	2.5	12	36
	0.50	2.7	12	37
	0.50	2.8	12	36
Mean	0.50	2.7	12	37
0.0117	0.50	2.9	13	38
	0.50	2.7	13	36
	0.50	2.7	13	35
Mean	0.50	2.8	13	36
0.0410	0.50	2.8	13	36
	0.50	2.9	14	37
	0.50	2.6	13	37
Mean	0.50	2.8	13	37
0.131	0.50	2.5	12	31
	0.50	2.7	13	38
	0.50	2.4	11	32
Mean	0.50	2.5	12	34
0.345	0.50	2.1	8.3	27
	0.50	2.1	7.8	23
	0.50	1.9	7	23
Mean	0.50	2.0	7.7	24
1.02	0.50	0.96	2.3	6.3
	0.50	1.1	3	6.3
	0.50	0.99	2	5.9
Mean	0.50	1.0	2.5	6.1

* = time-weighted average

 

Table 2. Growth rate and Growth inhibition rate

Measured concentration (mg/L)*	Growth rate (0-3d)	Growth inhibition rate (%)	Statistical results
Control	1.43	-	-
	1.43	-	-
	1.43	-	-
	1.43	-	-
	1.43	-	-
	1.43	-	-
Mean	1.43	-	-
0.0117	1.44	-0.93	n.s.
	1.42	0.55
	1.41	1.2
Mean	1.43	0.27
0.0410	1.43	0.12	n.s.
	1.43	0.017
	1.43	-0.050
Mean	1.43	0.028
0.131	1.38	3.6	n.s.
	1.44	-0.67
	1.39	3.2
Mean	1.40	2.0
0.345	1.33	7.1	**
	1.28	11
	1.27	11
Mean	1.29	9.6
1.02	0.843	41	**
	0.843	41
	0.821	43
Mean	0.836	42

* = time-weighted average; n.s. = not significant difference; ** = significant difference (p<0.01) (Step-down Jonckheere-Terpstra trend test

Table 3. Validity criteria OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	Increase of a factor of 73	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	The mean coefficient of variation for section-by-section specific growth rates = 20.86%	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	The coefficient of variation of average specific growth rates = 0.34%	Yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

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