(2Z)-2-phenylhex-2-enenitrile

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:

10-10-2017 to 12-01-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JAPAN: Method for Testing the Degree of Accumulation of Chemical Substances in Fish Body

Deviations:

no

GLP compliance:

yes

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms: Days 5, 13, 21, 25 and 28
- Sampling intervals/frequency for test medium samples: Days 5, 13, 21, 25 and 28
- Sample storage conditions before analysis: On autosampler
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
Test water analysis: The test water of Levels 1 and 2 were analysed once before the uptake phase and at the same time as analysis of test organisms. The test water for the Control was analysed only one time, at the end of uptake phase. The test water analysis before the uptake phase was not conducted since no interfering peak was observed in the blank test. Single replicates were analysed at each sampling time. An aliquot of the test water was taken from each test tank and pre-treated for GC-MS analysis.
Test organism analysis: The test fish of Levels 1 and 2 were analysed five times during the uptake phase. Four organisms per treatment level in two sub-groups. Sampling was carried out at intervals of more than 48 hours, and the final analysis was performed after 28 days. Control was analysed only one time, at the end of uptake phase. After measurement of weight and body length the organism tissue was processed and then extract supernatant was then subjected to GC-MS analysis.

Vehicle:

yes

Remarks:

Dimethylformamide [CAS 68-12-2]

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

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Level 1: 50 µg/L ; 2.5 g test item was dissolved in dimethylformamide to prepare a 2500 mg/L : 1 L stock solution. Level 2: 5 µg/L ; a 2500 mg/L stock solution (as prepared for level 1), 100 mL was diluted with dimethylformamide to 250 mg/L : 1 L stock solution.
- Controls: Dimethylformamide [CAS 68-12-2] (blank) control was utilised.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide was utilised to prepare stock solutions.
- Concentration of vehicle in test medium (stock solution and final test solution(s) at different concentrations and in control(s)): The flow rate of the stock solutions was as follows: Level 1: 50 µg/L : 0.04 mL/minute of 2500 mg/L stock solution and 2000 mL/minute dilution water (2880 L/day) – 1 L bottle renewed 1-2 times every two weeks ; Level 2: 5 µg/L : same as level 1 with corresponding 250 mg/L stock solution. 1 L bottle renewed 1-2 times every two weeks
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported.

PREPARATION OF SPIKED FISH FOOD
- Details on fish food (source, fat content as supplied, etc): Not applicable.
- Details of spiking (e.g. i) liquid test material (neat); ii) with a vehicle (corn or fish oil); or iii) using an organic solvent: Not applicable.
- Quantity of corn or fish oil vehicle, if used, per unit mass of fish food: Not applicable.
- Chemical name of vehicle (organic solvent), if used: Not applicable.
- Method of mixing: Not applicable.
- Equilibration time: Not applicable.
- Method for removal of solvent, if used: Not applicable.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Common carp (Cyprinus carpio)
- Strain: Not reported.
- Source: In-house breeding
- Age at study initiation (mean and range, SD): Yearlings
- Length at study initiation (length definition, mean, range and SD): 7.2-8.7 cm (at the beginning of uptake phase 7.3-7.8 cm)
- Weight at study initiation (mean and range, SD): Not reported (see ‘length’ above ; which was within the expected range for species/strain).
- Weight at termination (mean and range, SD): Individual fish weight data was incorporated into the calculation of the growth rate constant (kg)
- Method of breeding: Not reported.
- Lipid content at test initiation (mean and range, SD): 6.03% (at the beginning of uptake phase 5.32%) (applicant assessment indicates: presume to be mean lipid contents (n=6) control fish).
- Health status: Healthy.
- Description of housing/holding area: Indicated in the full study report.
- Feeding during test
- Food type: Recognised supplier feed
- Amount: amount corresponding to 2% of total body weight was fed twice a day in halve
- Frequency: amount corresponding to 2% of total body weight was fed twice a day in halve

ACCLIMATION
- Acclimation period: 47 days
- Acclimation conditions (same as test or not): Yes.
- Type and amount of food: Recognised supplier feed ; amount corresponding to 2% of total body weight was fed twice a day in halve
- Feeding frequency: amount corresponding to 2% of total body weight was fed twice a day in halve
- Health during acclimation (any mortality observed): < 5% during acclimatisation.

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: International regulatory requirements

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Hardness:

Level 1: 16.0 mgCaCO3/L and Control 16.0 mgCaCO3/L

Test temperature:

Before the uptake phase :
Level 1: 24.3-24.5°C and Level 2: 24.5°C ; Control: 24.9-25.0°C
During the uptake phase :
Level 1: 24.2-24.6°C and Level 2: 24.5-25.0°C ; Control: 24.8-25.0°C

pH:

Before the uptake phase :
Level 1: 6.9 and Level 2: 6.9 ; Control 7.0
During the uptake phase (at start and end of the study)
Level 1: start: 7.0 end: 7.2 and Level 2: start: 7.0 end: 7.2 ; Control: start: 7.2 end: 7.1

Dissolved oxygen:

Before the uptake phase :
Level 1: 6.9 mg/L and Level 2: 6.9 mg/L ; Control: 7.5 mg/L
During the uptake phase
Level 1: 6.4-6.6 mg/L and Level 2 6.3-6.5 mg/L ; Control: 7.1-7.3 mg/L

TOC:

Before the uptake phase (before 48 h, 24 h):
Level 1: 9.7, 9.8 mgC/L and Level 2: 9.9, 10.0 mgC/L ; Control: 9.8, 9.9 mgC/L
During the uptake phase
Level 1: 9.5-9.8 mgC/L and Level 2: 10.0-10.4 mgC/L ; Control: 9.9-10.2 mgC/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 70 L glass tank – flow-through water system
- Type (delete if not applicable): for suspected ‘volatile test item’ (presume: ‘closed’ and/or minimum headspace)
- Material, size, headspace, fill volume: glass, 70 L; 70 L fill volume
- Aeration: No.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not reported.
- Renewal rate of test solution (frequency/flow rate): The flow rate of the stock solutions was as follows: Level 1: 50 µg/L : 0.04 mL/minute of 2500 mg/L stock solution and 2000 mL/minute dilution water (2880 L/day) – 1 L bottle renewed 1-2 times every two weeks ; Level 2: 5 µg/L : same as level 1 with corresponding 250 mg/L stock solution. 1 L bottle renewed 1-2 times every two weeks
- No. of organisms per vessel: 26 per concentration (Level 1 and Level 2) ; 16 control
- No. of vessels per concentration (replicates): ca. 4 fish per sampling point, approximately 26 per concentration (including spares)
- No. of vessels per control / vehicle control (replicates): see above.
- Biomass loading rate: Not reported.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Groundwater from the premises of test laboratory (regularly tested twice per annum) ; most recent analysis appended to the full study report.
- Particulate matter: < 1 mg/L
- Metals: All metals are indicated at < 0.1 mg/L down to < 0.00001 mg/L
- Pesticides: all pesticides indicated at < 0.001 mg/L down to < 0.0001 mg/L
- Chlorine: < 0.02 mg/L
- Alkalinity: 110 mg/L
- Ca/mg ratio: 16 mg/L
- Conductance: 290 uS/cm
- Holding medium different from test medium: No.
- Intervals of water quality measurement: See above. Dissolved oxygen: Once before the uptake phase, and once or twice a week during the uptake phase ; pH: Once before the uptake phase, and twice during the uptake phase ; Total organic carbon: Twice before the uptake phase (24h and 48h before uptake phase), and
once a week during the uptake phase and Total Hardness: Once during the uptake phase for Control and Level 1
- Intervals of test medium replacement: Flow through method.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 14 hours light / 10 hours dark
- Light intensity: Not reported ; artificial white fluorescent lamp

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: Not applicable.
- Results used to determine the conditions for the definitive study: Not applicable.
- Other justification for choice of test concentrations: Nominal concentrations of test item would be based on any preceding short term fish toxicity information (see full study report for details, as applicable).

Nominal and measured concentrations:

Level 1: Nominal: 50 µg/L / Measured: 48.3 µg/L (average of 28 days)
Level 2: Nominal: 5 µg/L / Measured: 5.16 µg/L (average of 28 days)
Control: 0 µg/L

Reference substance (positive control):

no

Lipid content:

6.03 %

Time point:

start of exposure

Lipid content:

5.32 %

Time point:

end of exposure

Key result

Conc. / dose:

5 µg/L

Temp.:

>= 24.2 - <= 24.6 °C

pH:

7

Type:

BCF

Value:

300 L/kg

Basis:

whole body w.w.

Time of plateau:

21 d

Calculation basis:

steady state

Remarks:

the average concentration of the test item in test fish at 13, 21, 25 and 28 days of the uptake phase were within ± 20% of each other. Indicating a steady state had been reached.

Remarks on result:

other: BCF (as steady state) = 300 L/kg (Level 2)

Key result

Conc. / dose:

50 µg/L

Temp.:

>= 24.5 - <= 25 °C

pH:

7

Type:

BCF

Value:

270 L/kg

Basis:

whole body w.w.

Time of plateau:

20 d

Calculation basis:

steady state

Remarks:

the average concentration of the test item in test fish at 13, 21, 25 and 28 days of the uptake phase were within ± 20% of each other. Indicating a steady state had been reached.

Remarks on result:

other: BCF (as steady state) = 270 L/kg (Level 1)

Key result

Rate constant:

growth rate constant (d-1)

Remarks:

Level 2: Nominal: 5 µg/L / Measured: 5.16 µg/L (average of 28 days)

Value:

0.008

Key result

Rate constant:

growth rate constant (d-1)

Remarks:

Level 1: Nominal: 50 µg/L / Measured: 48.3 µg/L (average of 28 days)

Value:

0.007

Key result

Rate constant:

growth rate constant (d-1)

Remarks:

Control

Value:

0.007

Metabolites:

None reported.

Details on results:

- Mortality of test organisms: None reported.
- Behavioural abnormalities: None reported.
- Observations on feeding behaviour: None reported.
- Observations on body length and weight: None reported.
- Reproduction during test period: None reported.
- Other biological observations: None reported.
- Organ specific bioaccumulation: None reported.
- Bound residues forming a plateau: Not reported.
- Mortality and/or behavioural abnormalities of control: None.
- Loss of test substance during test period: Level 1: Nominal: 50 µg/L / Measured: 48.3 µg/L (average of 28 days) ; Level 2: Nominal: 5 µg/L / Measured:5.16 µg/L (average of 28 days). Concentrations of the test item in test water before uptake phase were 49.5 μg!L (Level 1) and 4.88 μg!L (Level 2). Differences of nominal to measured were not indicated.
- Non-eliminated residues (NER) at the end of elimination phase: Not reported.
- Results with vehicle control: No interfering peak was observed at the peak positions of the test item in the GC-MS chromatogram for the Control fish at the end of the uptake phase.

Validity criteria fulfilled:

yes

Conclusions:

The test item steady state bioaccumulation concentration factor (BCF) at two concentration levels was determined to be: Nominal: 5 µg/L: BCF = 270 L/Kg and Nominal: 50 µg/L: BCF = 300 L/Kg whole body wet weight. Under the conditions of this study, the BCF was 270 to 300 L/Kg.

Executive summary:

The bioconcentration factor of the test item to common carp (Cyprinus carpio) was determined in a 28-day test according to OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) - I: Aqueous Exposure Bioconcentration Fish Test and JAPAN: Method for Testing the Degree of Accumulation of Chemical Substances in Fish Body under GLP. Groups of test organisms were exposed to the test item in a flow-through test system for 28-days. Test item was prepared from stock solutions in dimethylformamide. Exposures were at nominal concentrations Level 1: 50 µg/L and Level 2: 5 µg/L. The corresponding measured concentrations were 48.3 µg/L and 5.16 µg/L averaged over 28-days. A control group was also utilised exposed to dimethylformamide only. The test item in test water and test organisms was analysed by GC-MS by external calibration and linear regression. The analytical method that was suitably validated. The total lipid content of fish before the uptake phase was 6.03% and at the end of the uptake phase 5.32%. The growth rate constants for Level 1: 50 µg/L and Level 2: 5 µg/L were: 0.00753 and 0.00722 /day and in the control 0.0733 /day. The average concentration of the test item in test fish at 13, 21, 25 and 28 days of the uptake phase were within ± 20% of each other. Indicating a steady state had been reached. All validity criteria were considered to have been met. The test item steady state bioaccumulation concentration factor (BCF) at two concentration levels was determined to be: Nominal: 5 µg/L: BCF = 270 L/Kg and Nominal: 50 µg/L: BCF = 300 L/Kg whole body wet weight. Under the conditions of this study, the BCF was 270 to 300 L/Kg.

Description of key information

BCF (fish) = 270 – 300 L/Kg whole body w.w., steady state within 28-days, OECD TG 305, 2018

Key value for chemical safety assessment

BCF (aquatic species):

300 L/kg ww

Additional information

Key Study : OECD 305, 2018 : The bioconcentration factor of the test item to common carp (Cyprinus carpio) was determined in a 28-day test according to OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) - I: Aqueous Exposure Bioconcentration Fish Test and JAPAN: Method for Testing the Degree of Accumulation of Chemical Substances in Fish Body under GLP. Groups of test organisms were exposed to the test item in a flow-through test system for 28-days. Test item was prepared from stock solutions in dimethylformamide. Exposures were at nominal concentrations Level 1: 50 µg/L and Level 2: 5 µg/L. The corresponding measured concentrations were 48.3 µg/L and 5.16 µg/L averaged over 28-days. A control group was also utilised exposed to dimethylformamide only. The test item in test water and test organisms was analysed by GC-MS by external calibration and linear regression. The analytical method that was suitably validated. The total lipid content of fish before the uptake phase was 6.03% and at the end of the uptake phase 5.32%. The growth rate constants for Level 1: 50 µg/L and Level 2: 5 µg/L were: 0.00753 and 0.00722 /day and in the control 0.0733 /day. The average concentration of the test item in test fish at 13, 21, 25 and 28 days of the uptake phase were within ± 20% of each other. Indicating a steady state had been reached. All validity criteria were considered to have been met. The test item steady state bioaccumulation concentration factor (BCF) at two concentration levels was determined to be: Nominal: 5 µg/L: BCF = 270 L/Kg and Nominal: 50 µg/L: BCF = 300 L/Kg whole body wet weight. Under the conditions of this study, the BCF was 270 to 300 L/Kg.