N,N-bis(2-ethylhexyl)formamide

Bioaccumulation: aquatic / sediment

Administrative data

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Jun - 15 Sep 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: "Bioconcentration test of chemical substances in fish and shellfish" (Japanese notification, Yakushokuhatsu 0331 No.7, Heiei 23.03.29 Seikyoku No 110331009

Version / remarks:

2019

Deviations:

not specified

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test

Version / remarks:

2012

Deviations:

no

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

Radiolabelling:

no

Sampling and analysis

Details on sampling:

- Sampling intervals/frequency for test organisms: 4 fish were sampled from the exposure group on days 11, 25, 39, 49, 60of the exposure period
- Sampling intervals/frequency for test medium samples: The test water in the exposure group was sampled on days 0 (before introduction of fish), 11, 25, 39, 49, 60 of the exposure period
- Sample storage conditions before analysis: samples were not stored and analyzed immediatly

Test solutions

Vehicle:

yes

Remarks:

Dimethyl sulfoxide

Details on preparation of test solutions, spiked fish food or sediment:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was consecutively diluted in Dimethyl sulfoxide to a final stock solution concentration of 10 mg/L. The stock solution and dilution water were delivered by individuale pumps to a mixing tube, were the final exposure concentration of 0.0005 mg/L test substance and 50 ppm (v/v) Dimetyhl sulfoxide
- Controls: Dimethyl sulfoxide and and dilution water were delivered by individuale pumps to a mixing tube, were the final exposure concentration of 50 ppm (v/v) Dimethyl sulfoxide
- Chemical name of vehicle: Dimethyl sulfoxide
- Concentration of vehicle in test medium (final test solution and in control): 50 ppm (v/v)
- Evidence of undissolved material: none reported

Test organisms

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp
- Source: Kitamura-yourijou, Kumamoto, Japan
- Lot: 19-K-0910
- Age at study initiation: Yearling , all fish have the same source and age
- Length at study initiation: 8 ± 4 cm
- Weight at study initiation: 6.65 g (mean of fish for lipid content determination)
- Weight at termination: 9.51 g (mean of all fish taken from exposure and control group)
- Lipid content at test initiation: 5.1 % (n = 3, 3.8 - 6.4%)
- Description of housing/holding area: Tanks made of glass with a capacity of 50 L were employed.
- Feeding during test
- Food type: Babygold, Kyorin Co. Ltd., Japan
- Amount: 1.5% of fish weight
- Frequency: Daily

ACCLIMATION
- Acclimation period: 13 May - 19 June 2020
- Acclimation conditions: same as testing
- Type and amount of food: Babygold, Kyorin Co. Ltd., Japan; approx. 1.5% of body weight
- Feeding frequency: Daily
- Health during acclimation: < 5% mortality during acclimation period

Study design

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

60 d

Test conditions

Hardness:

48 - 50 mg CaCO3/L

Test temperature:

23.8 - 24.6°C

pH:

7.2 - 7.4

Dissolved oxygen:

7.6 - 8.3 mg/L

TOC:

17.4 - 19.7 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Tanks made of glass with a capacity of 50 L were employed
- Aeration: Continuous
- Type of flow-through: Conitnous flow-through dilution system
- Renewal rate of test solution: 800 L/day (16 volumes/day turnover)
- No. of organisms per vessel: Exposure group: 28 fish; Control group: 12 fish
- No. of vessels per concentration (replicates): 1
- No. of vessels per vehicle control: 1
- Biomass loading rate: ≥ 1 L/day/g at start of the exposure period

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water (Yokohama city) treated with activated charcoal filter and sodium thiosulfate solution
- The quality of the test water was measured periodically (about every 6 months) and fulfilled the criteria specified in the standard operating procedure of the testing facility
- Particulate matter: < 1.0 mg/L
- Metals: < 0.04 mg/L
- Pesticides: < 0.002 mg/L
- Chlorine: < 1.0 mg/L
- Alkalinity: 33 mg CaCO3/L (pH 4.8)
- Conductance: 13 mS/m

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: approx. 16 h/day, Hf fluorescent lamp (wavelength 400 - 700 nm)


RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations:
A preliminary test with two test concentrations (0.005 and 0.0005 mg/L) showed no dependency of the BCF on the test substance concentrations (BCF 208 and 183, respectively, after 13 days)
A short-term toxicity test on fish showed a LC50 of 0.67 mg/L after 96 h
- Results used to determine the conditions for the definitive study: A perliminary water solubility test (Dilution water, 24°C; Flask method) showed a water solubility of 0.25 mg/L

Nominal and measured concentrations:

Nominal 0.0005 mg/L
Mean concentrations in test water: 0.000423 - 0.000506 mg/L

Reference substance (positive control):

no

Details on estimation of bioconcentration:

BASIS FOR CALCULATION OF BCF
- Calculation: Obtain the uptake curve of the test substance by plotting its concentration in fish (or specified tissues) in the uptake phase against time on arithmetic scales. If the curve has reached a plateau, that is, become approximately asymptotic to the time axis (changing extent did not exceed ±20%), calculate the steady state BCFss from: BCFss = Cf,s(steady-state)/Cw,s( steady - state)
Cf,s: mean concentration of test substance in test fish at steady-state (mg/kg);
Cw,s: mean concentration of test substance in test water at steady-state (mg/L).

Steady-state: Steady-state is attained when variations of the test substance concentration in the test fish in four successive measurements conducted at intervals of 48 h or longer are within 20%. Cf,s is calculated from the last four consecutive values. Cw,s is the average of all values during the steady-state period.

Results and discussion

Lipid content

Lipid content 1

Lipid content:

5.1 %

Time point:

start of exposure

Remarks on result:

other:

Remarks:

n = 3, 3.8 - 6.4%

Lipid content 2

Lipid content:

4.3 %

Time point:

end of exposure

Remarks on result:

other:

Remarks:

n = 3, 3.5 - 4.9%

Bioaccumulation factor

Conc. / dose:

0.001 mg/L

Temp.:

23.8 - 24.6 °C

pH:

7.3

Type:

BCF

Value:

210 - <= 657 L/kg

Basis:

not specified

Calculation basis:

steady state

Remarks on result:

other:

Remarks:

BCF values were in a range of < 210 - 657 L/kg. On day 60, the BCF value was < 210 L/kg

Details on results:

- Mortality of test organisms: 3.6% mortality in the exposure grou
- Behavioural abnormalities: none reported
- Observations on feeding behavior: none reported
- Mortality and/or behavioural abnormalities of control: 0% in the control group
- Loss of test substance during test period: At day 0, before induction of fish, the measured test substance concentration in water was 0.000506 mg/L (0.0005 mg/L nominal). During the exposure, test substance concentration ranged between 0.000423 - 0.000435 mg/L, with a mean concentration of 0.000423 mg/L (84.6% of nominal)

Reported statistics:

The mean concentrations in the test water and BCF were calculated according to the JIS Handbook "Guide to the handling of numbers". JIS Z9041-1:1999, Statistical interpretation of data - Part 1: Statistical presentation of data. JIS Z8401:1999, Guide to the rounding of numbers

Any other information on results incl. tables

Table 1: Water quality parameters

Measurement value	Contents
	Dissolved Oxygen [mg/L]	Water temperature [°C]	Hardness [mg CaCO3/L]	Total Organic Carbon [mg/L]	pH
	7.6 – 8.3	23.8 – 24.6	48 - 50	17.4 – 19.7*	7.2 – 7.4

* = The TOC concentration attributable to the solvent Dimentyl sulfoxide is 17.1 mg/L

 

Table 2: Mean concentration of test substance in water and fish and BCF

	Replicate	Exposure period
		Day 11	Day 25	Day 39	Day 49	Day 60
Mean concentration of test substance in water [mg/L]		0.000435	0.000427	0.000432	0.000424	0.000423
Concentration of test substance in fish [mg/kg]	1	< 0.087	< 0.087	0.116	< 0.087	< 0.087
	2	< 0.087	< 0.087	0.284	0.122	< 0.087
BCF [L/kg]	1	< 210	< 210	268	< 210	< 210
	2	< 210	< 210	657	287	< 210

 

Table 3: Validity criteria of OECD 305 (2012)

Criterion from the guideline	Outcome	Validity criterion fulfilled
The temperature variation is less than ± 2 °C	23.8 – 24.6°C	yes
The concentration of dissolved oxygen does not fall below 60% saturation	7.6 – 8.3 mg/L (≥60% saturation)	yes
The concentration of the test substance in the chambers is maintained within ± 20% of the mean of the measured values during the uptake phase	≥84.6 % of nominal	yes
The concentration of the test substance is below its limit of solubility in water, taking into account the effect that the test water may have on effective solubility	Water solubility is determined to be 0.25 mg/L in a preliminary test	yes
The mortality or other adverse effects/disease in both control and treated fish is less than 10% at the end of the test; where the test is extended over several weeks or months, death or other adverse effects in both sets of fish should be less than 5% per month and not exceed 30% in all.	0% mortality in the control group 3.6 % mortality in the exposure group No further adverse effects reported	yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Conclusions:

As all BCF values were less than 1000 L/kg, it was concluded that the bioconcentration potential of the test substance DEHF is low.