N,N'-bis{3-[di-(branched and cyclic) alkylamino]alkyl}alkanediamide

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 27, 2016 to Nov 9, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: New Chemical Substances “Bioconcentration test of chemical substances in fish and shellfish

Version / remarks:

Japanese notification, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kanpokihatsu No.110331009, March 31, 2011; latest revision, December 21, 2015

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, dark, air-tight
- Stability: After the end of the experiment, the infrared absorption spectrum of the test substance was measured. The spectrum obtained was consistent with that measured before starting the experiment, indicating that the test substance was stable under these storage conditions.
- Stability under test conditions: stable

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms: samples were collected on day 7, 14, 28, 42 and 60
- Sampling intervals/frequency for test medium samples: samples were collected on day 7, 14, 28, 42 and 60
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
Method for Analysis of Test Water
High concentration level
Test water 0.75 mL -> mix with tetrahydrofuran 0.75ml -> LC/MS analysis
Low concentration level
Test water 100ml pass through an Empore disk SDB-XC 47mm (pre-conditioned with approx. 5 mL tetrahydrofuran and approx. 10 mL ultra pure water) -> eluate with Tetrahydrofuran, fill up to 10 ml with Tetrahydrofuran, take 0.75 ml and mix with 0.75 ml Ultra pure water-> LC/MS analysis

Method for Analysis of Test Fish
Weigh two fish, cut into pieces, Homogenize 4 times for approx. 1 min at approx. 8QQ0 rpm-> take 2g portion of the fish slurry -> Homogenize for approx. 3 min at approx. 8000 rpm in 20 mL of Tetrahydrofuran / Acetonitrile = 8/2 (v/v) -> filter with suction through a filter paper in a 40-mm Kirivama funnel->Tetrahydrofuran / Acetonitrile layer was diluted in 50ml of Tetrahydrofuran -> take 5 ml portion -> dilute with Tetrahydrofuran by factor of 4 -> 1ml portion and further dilute with Tetrahydrofuran by factor of 10 -> pass through an InertSep C8 -> take 0.75 ml portion and dilute with 0.75ml Ultra pure water -> LC/MS analysis

Vehicle:

yes

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

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Continuous flow-through dilution system, test substance dissolved in Tetrahydrofuran and dilute in water.
- Controls: Tetrahydrofuran
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Tetrahydrofuran
- Concentration of vehicle in test medium (stock solution and final test solution(s) at different concentrations and in control(s)):
Control group: vehicle concentration: Tetrahydrofuran 25 ppm (v/v)
Low dose group: vehicle concentration: Tetrahydrofuran 25 ppm (v/v)
High dose group: vehicle concentration: Tetrahydrofuran 24 ppm (v/v)

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp
- Source: Kitamura-yourij ou, Japan
- Age at study initiation (mean and range, SD): Less than 1 year
- Length at study initiation (length definition, mean, range and SD): 8±4 cm
- Weight at study initiation (mean and range, SD): Approximately 9 g
- Lipid content at test initiation (mean and range, SD):
4.2% (n=3, 3.8 to 4.5%) at the start of the exposure period
5.1% (n=3, 3.8 to 5.9%) at the end of the exposure period
- Health status: No occurrence of mortality or abnormality was observed.
- Description of housing/holding area:
- Feeding during test
- Food type: Babygold, Kyorin
- Amount: The food was given daily equivalent to 1.5% of the body weight.

ACCLIMATION
- Acclimation period: April 27, 2016 to June 9,2016
- Acclimation conditions (same as test or not): same
- Type and amount of food: The food was given daily equivalent to 1.5% of the body weight.
- Health during acclimation (any mortality observed): Mortality 1 week before testing: <5%

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: The concentrations of the test substance in the test water and fish were measured periodically, and their ratios were used to calculate the bioconcentration factor (BCF) to evaluate the bioconcentration potential of the test substance in fish.

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

61 d

Total depuration duration:

0 d

Hardness:

48-52 mg CaCO3/L

Test temperature:

24.0 - 24.4°C

pH:

7.0 - 7.5

Dissolved oxygen:

> 60% of the saturation (6.2 - 8.3 mg/L)

TOC:

13.4 - 15.8 mg/L

Salinity:

NA

Conductivity:

13 mS/m

Details on test conditions:

TEST SYSTEM
- Test vessel: Open vessel (lid: plastic plate)
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 47 fish in 50-L test water (>1 L/day/g)
- Aeration: Continuous
- Type of flow-through (e.g. peristaltic or proportional diluter): Continuous flow-through dilution system
- Renewal rate of test solution (frequency/flow rate): 16 volumes/day
- No. of organisms per vessel: 47 fish
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated water processed by treating Yokohama city tap water with an activated charcoal filter and sodium thiosulfate was used as the dilution water. Water quality parameters of the dechlorinated water have been measured periodically (about every 6 months) by MC Evolve Technologies Corporation. As each measurement value met the criteria specified in the standard operating procedure of the testing facility, the quality of this water was considered to be acceptable for use as the dilution water.
- Chlorine: < 0.1 mg/L
- Alkalinity (pH4.8) 34 mg CaCO3
- Ca/mg ratio: 15mg/L: 3.7mg/L
- Holding medium different from test medium: none
- Intervals of water quality measurement: every 6 months
- Intervals of test medium replacement: 16 volumes/day

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: Room light (16hr light / 8hr dark)
- Light intensity: Hf fluorescent lamp

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations:
An acute toxicity test was performed to confirm that the nominal concentrations used in the bioconcentration test were less than 1% and 0.1% of the 96-hr LC50.
- Results used to determine the conditions for the definitive study: The 96-hr LC50 of the test substance to Medaka in the acute toxicity test was calculated to be >100 mg/L.

Nominal and measured concentrations:

High concentration level: 1 mg/L
Low concentration level: 0.1 mg/L
Control: 0 mg/L

Reference substance (positive control):

no

Details on estimation of bioconcentration:

Not relevant

Lipid content:

4.2 %

Time point:

start of exposure

Lipid content:

5.1 %

Time point:

end of exposure

Key result

Conc. / dose:

1 mg/L

Temp.:

24 °C

pH:

6

Type:

BCF

Value:

<= 489 L/kg

Basis:

whole body w.w.

Calculation basis:

kinetic

Remarks on result:

other:

Remarks:

Result for Component A of the test material

Key result

Conc. / dose:

0.1 mg/L

Temp.:

24 °C

pH:

6

Type:

BCF

Value:

< 1 400 L/kg

Basis:

whole body w.w.

Calculation basis:

kinetic

Remarks on result:

other:

Remarks:

Result for Component A of the test material

Key result

Conc. / dose:

1 mg/L

Temp.:

24 °C

pH:

6

Type:

BCF

Value:

140 L/kg

Basis:

whole body w.w.

Calculation basis:

kinetic

Remarks on result:

other:

Remarks:

Result for Component B of the test material

Key result

Conc. / dose:

0.1 mg/L

Temp.:

24 °C

pH:

6

Type:

BCF

Value:

< 1 400 L/kg

Basis:

whole body w.w.

Calculation basis:

kinetic

Remarks on result:

other:

Remarks:

Result for Component B of the test material

Metabolites:

Not identified.

Results with reference substance (positive control):

Not examined.

Details on results:

- Mortality of test organisms: no effect.
- Behavioural abnormalities: no effect
- Observations on feeding behavior: no effect
- Observations on body length and weight: no effect
- Reproduction during test period: not relevant
- Other biological observations: no effect
- Mortality and/or behavioural abnormalities of control: no effect
- Loss of test substance during test period: no effect

Reported statistics:

The correlation coefficient was calculated by the least squares method according to JIS Handbook “General rules for chemical analysis”.
The mean concentrations in the test water and BCF were calculated according to the JIS Handbook “Guide to the handling of numbers”.

Validity criteria fulfilled:

yes

Conclusions:

The bioconcentration potential of the test substance in fish is not high.

Executive summary:

Methods:

This study was conducted in accordance with the Test Method Relating to New Chemical Substances “Bioconcentration test of chemical substances in fish and shellfish” (Japanese notification, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kanpokihatsu No.110331009, March 31, 2011; latest revision, December 21, 2015).

Test substance in test water (nominal concentrations)

High concentration level: 1 mg/L

Low concentration level: 0.1 mg/L

Control: 0 mg/L

Vehicle in test water

High concentration level: Tetrahydrofuran 24 ppm (v/v)

Low concentration level: Tetrahydrofuran 25 ppm (v/v)

Control: Tetrahydrofuran 25 ppm (v/v)

Test water supply: Continuous flow-through dilution system (800 L/day)

Exposure period: 61 days

Analytical method:

Pre-treated samples were analyzed for the test substance with a liquid chromatography mass spectrometer (LC/MS)

As the test substance contained a number of components with the molecular weight below 1000, it was difficult to target all of these components for the LC/MS analysis. Therefore, 2 components referred to as A (m/z\ 480.5), and B (m/z\ 536.6) were determined and evaluated for their bioconcentration potential. Six unquantifiable components referred to as C (m/z: 494.5), D (m/z: 508.5), E (m/z: 520.6), F (m/z: 522.7), G (m/z: 607.7) and H (m/z: 790.8) were monitored for their respective ions. When the monitored ions were detected as significant peaks in the analysis of fish samples, their bioconccntration potential was evaluated as much as possible.

The concentrations of each component are expressed in terms of the weight concentrations of the test substance without regard to the relative proportion of the component in the composition of the test substance.

Lipid content of carp:

4.2% (n=3, 3.8 to 4.5%) at the start of the exposure period

5.1% (n=3, 3.8 to 5.9%) at the end of the exposure period

96-hr LC50 to Medaka: >100 mg/L

 

Results:

The study results are shown below.

Analyzed components

Component A

	High concentration level	Low concentration level
Mean cone, in test water (mg/L)	0.933	0.0923
BCFss . BCF	BCF ≤ 489	BCF < 1400*

 

Component B

	High concentration level	Low concentration level
Mean cone, in test water (mg/L)	0.916	0.0907
BCFss . BCF	BCF < 140*	BCF < 1400*

*: below detection limit

 

Monitored components

The monitored component C, D, E, F, G and H were confirmed for the fish samples taken during the exposure period (days 28 to 60). No significant peak attributable to any of the monitored component was detected at either concentration level.

 

Discussions:

Due to a lack of analytical sensitivity, BCF could not be confirmed to be less than 1000 L/kg in any component at the low concentration level. Meanwhile, BCFs of all components at the high concentration level were below 489 L/kg, BCFs of all components at the low concentration level were less than 1400 L/kg, and no components detected. Therefore, it can be concluded that the bioconcentration potential of the test substance in fish is not high.

 

Description of key information

The potential for the test material to bioaccumulate was evaluated in a study conducted in accordance with the Test Method Relating to New Chemical Substances “Bioconcentration test of chemical substances in fish and shellfish” (Japanese notification, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kanpokihatsu No.110331009, March 31, 2011; latest revision, December 21, 2015). Carp were exposed to concentrations up to 1 mg/L for 61 days in a flow-through study. Due to a lack of analytical sensitivity, BCF could not be confirmed to be less than 1000 L/kg in any component at the low concentration level. Meanwhile, BCFs of all components at the high concentration level were below 489 L/kg, BCFs of all components at the low concentration level were less than 1400 L/kg, and no components detected. Therefore, it can be concluded that the bioconcentration potential of the test substance in fish is not high.