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Environmental fate & pathways

Bioaccumulation: aquatic / sediment

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Reference
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07-08-2017 to 16-10-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
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 3, 7, 14, 21 and 28 of the exposure period
- Sampling intervals/frequency for test medium samples: Days 0 (before fish were introduced), 3, 7, 14, 21 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 from each concentration level will be sampled on days 0 (before introduction of the fish), 3, 7, 14, 21 and 28 of the exposure period, and analysed for the test substance. This is done for the test water 100 mL for high concentration level, 1000 mL for low concentration level, passage through an Empore disk C18 47 mm with aspiration. Passed with approx. 5 mL of acetone and approx. 10 mL of ultrapure water beforehand for conditioning). The elute with acetone ca. 4.8 mL x 2, dilute to 10 mL with acetone is then subject to GC-MS analysis.
Test organism analysis: Four fish will be sampled from each concentration level on days 3, 7, 14, 21 and 28 of the exposure period, and analysed for the test substance two by two using the method described (in the full study report), terminal procedures. The fish remaining from analysis at the end of the exposure period and back-up samples will be stored in a freezer for three months, and may be discarded after that period, unless additional analysis is necessary.
Lipid content: At the start and end of the exposure period, lipid contents of the fish is measured. Five fish are sampled from the acclimation aquarium at the start of the exposure period, and three fish each are sampled from the test aquariums at the end of the exposure period. In these samples, lipid contents of three fish are sampled from the acclimation aquarium and the control aquarium are measured individually according to the procedure described (in the full study report). The two fish remaining from analysis that are sampled upon initiation of exposure and three fish each sampled from each concentration level upon completion of exposure may be used as back-up samples.
Vehicle:
yes
Remarks:
Dimethyl sulfoxide 50 ppm (v/v)
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Feed solution and the dilution water are delivered to a mixing glass tube through individual metering pumps with a certain flow rate each, and then supplied continuously to respective test aquariums.
- Controls: Dimethyl sulfoxide 50 ppm (v/v) without test item
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethyl sulfoxide 50 ppm (v/v).
- Concentration of vehicle in test medium (stock solution and final test solution(s) at different concentrations and in control(s)): see above.
- 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): feed documented in the full study report.
- 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: Recognised supplier (documented in the full study report)
- Age at study initiation (mean and range, SD): Yearlings
- Length at study initiation (length definition, mean, range and SD): at the beginning of uptake phase 8 +/- 4 cm
- Weight at study initiation (mean and range, SD): at the beginning of uptake phase ca. 8 g
- Weight at termination (mean and range, SD): Not reported.
- Method of breeding: Not reported.
- Lipid content at test initiation (mean and range, SD): 3.7% (n=3, 2.4 to 4.4%) at the start of the exposure period
- 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 1.5% of total body weight was fed once daily
- Frequency: amount corresponding to 1.5% of total body weight was fed once daily

ACCLIMATION
- Acclimation period: > 14 days
- Acclimation conditions (same as test or not): Yes.
- Type and amount of food: Recognised supplier feed ; amount corresponding to 1.5% of total body weight was fed once daily
- Feeding frequency: amount corresponding to 1.5% of total body weight was fed once daily
- 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:
53 - 56 mgCaCO3/L
Test temperature:
24.0-24.3°C
pH:
pH 7.0
Dissolved oxygen:
7.1-8.8 mgO2/L
TOC:
15.1-24.5 mgC/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 56 L glass tank
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 56 L; 50 L fill volume
- Aeration: Yes.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not reported.
- Renewal rate of test solution (frequency/flow rate): Continuous flow-through dilution system (800 L/day) ; turnover 16 times per day.
- No. of organisms per vessel: 28 per concentration ; 12 in control
- No. of vessels per concentration (replicates): One.
- No. of vessels per control / vehicle control (replicates): see above.
- Biomass loading rate: > 1 L/day/g

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: water from the premises of test laboratory (regularly tested per annum) ; most recent analysis appended to the full study report.
- Holding medium different from test medium: No.
- Intervals of water quality measurement: See above.
- Intervals of test medium replacement: Flow through method.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16 hours light / 8 hours dark
- Light intensity: Not reported ; artificial white fluorescent lamp (400 – 700 nm))

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: Based on the physico-chemical properties of the substance and/or preceding ecotoxicology testing.
Nominal and measured concentrations:
Level 1: Nominal: 0.01 mg/L / Measured: 0.00748 mg/L (average of 28 days)
Level 2: Nominal: 0.001 mg/L / Measured: 0.000701 mg/L (average of 28 days)
Control: 0 mg/L
Reference substance (positive control):
no
Lipid content:
3.7 %
Time point:
start of exposure
Remarks on result:
other: n=3 ; 2.4 to 4.4%
Lipid content:
3.4 %
Time point:
end of exposure
Remarks on result:
other: n= 3 ; 3.0 to 3.7%
Lipid content:
3.55 %
Time point:
other: mean
Remarks on result:
other: start and end of exposure
Conc. / dose:
0.01 mg/L
Temp.:
>= 24 - <= 24.3 °C
pH:
7
Type:
BCF
Value:
<= 14 L/kg
Basis:
whole body w.w.
Time of plateau:
7 d
Calculation basis:
steady state
Remarks:
the variation was within ± 20% of the mean value in the steady-state confirmation period (Days 7-28). Indicating a steady state had been reached.
Conc. / dose:
0.001 mg/g food
Temp.:
>= 24 - <= 24.3 °C
pH:
7
Type:
BCF
Value:
<= 73 L/kg
Basis:
whole body w.w.
Time of plateau:
7 d
Calculation basis:
steady state
Remarks:
the variation was within ± 20% of the mean value in the steady-state confirmation period (Days 7-28). Indicating a steady state had been reached.
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: 0.01 mg/L / Measured: 0.00748 mg/L (average of 28 days). Level 2: Nominal: 0.001 mg/L / Measured: 0.000701 mg/L (average of 28 days). 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.
Reported statistics:
The calibration curve correlation coefficient was calculated using least squares methodology according to:
JIS Handbook "General rules for chemical analysis". · JIS K0050, General rules for chemical analysis
Calculations of Mean Concentration in Test Water and BCF was conducted according to:
JIS Handbook "Guide to the handling of numbers"
JIS Z9041-1: 1999, Statistical interpretation of data - Part I :Statistical presentation of data
JIS Z8401-1:1999, Guide to the rounding of numbers

Applicant recalculation of the reported BCF (steady state) accounting for normalisation to Lipid content of 5% in accordance with OECD TG 305: paragraph 75 and Annex 5 and ECHA R7.b (2017) :

Mean lipid content (start and end of exposure period): 3.55%

Worst case BCF (steady state) measured = 73 L/Kg whole body wt. weight

BCF (lipid-normalised 5%) = [0.05 / 0.0355] x 73 L/Kg whole body wt. weight

BCF (lipid-normalised 5%) = 103 L/Kg whole body wet weight.

 

References:

1. OECD TG 305 (2012) : Annex 5

Validity criteria fulfilled:
yes
Conclusions:
The test item steady state bioaccumulation concentration factor (BCF) at two concentration levels was determined to be: Nominal: 0.01 mg/L: BCF < or = 14 L/Kg and Nominal: 0.001 mg/L: BCF = or < 73 L/Kg whole body wet weight. Under the conditions of this study, the BCF was 14 to 73 L/Kg. Based on a mean lipid content at start and end of exposure (3.55%), the BCF (lipid-normalised 5%) is < or = 103 L/Kg whole body wet weight.
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 Dimethyl sulfoxide 50 ppm (v/v). Exposures were at nominal concentrations Nominal: 0.01 mg/L and Nominal: 0.001 mg/. The corresponding measured concentrations were 0.00748 mg/L and 0.000701 mg/L averaged over 28-days. A control group was also utilised exposed to Dimethyl sulfoxide 50 ppm (v/v) only. The test item in test water and test organisms was analysed by GC-MS. The total lipid content of fish before the uptake phase was mean 3.4% to 3.7%. At both concentration levels, the steady-state was considered to have been reached within the 28-day exposure period since all values were less than 100 L/kg (wet wt.) (observed < or = 14 L/kg (wet wt.) and< 73 L/kg (wet wt.) at the high- and low-exposure concentrations, respectively). In view of these results, it was not necessary to conduct a depuration phase. 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: 0.01 mg/L: BCF < or = 14 L/Kg and Nominal: 0.001 mg/L: BCF = or < 73 L/Kg whole body wet weight. Under the conditions of this study, the BCF was 14 to 73 L/Kg. Based on the mean lipid content (start and end of exposure period): 3.55%, the applicant recalculates the reported value for 5% lipid content as BCF (lipid-normalised 5%) is < or = 103 L/Kg whole body wet weight.

Description of key information

1. BCF (fish: steady state) : 14 – 73 L/Kg whole body w.w., steady state within 28-days, OECD TG 305, 2018

The corresponding BCF (fish: steady state : lipid normalised 5%) = 103 L/Kg whole body w.w. based on 73 L/Kg whole body w.w. starting value.

2. BCF (fish: in vitro – in vivo extrapolation (IVIVE)) : 650 L/kg ww Fucalc and 271 L/kg ww Fu=1.0, predicted BCF including in vitro biotransformation using Rainbow Trout (Oncorhynchus mykiss) Liver S9 Fraction, eq. or similar to OECD TG 319B, 2019

Key value for chemical safety assessment

BCF (aquatic species):
103 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 Dimethyl sulfoxide 50 ppm (v/v). Exposures were at nominal concentrations Nominal: 0.01 mg/L and Nominal: 0.001 mg/. The corresponding measured concentrations were 0.00748 mg/L and 0.000701 mg/L averaged over 28-days. A control group was also utilised exposed to Dimethyl sulfoxide 50 ppm (v/v) only. The test item in test water and test organisms was analysed by GC-MS. The total lipid content of fish before the uptake phase was mean 3.4% to 3.7%. At both concentration levels, the steady-state was considered to have been reached within the 28-day exposure period since all values were less than 100 L/kg (wet wt.) (observed < or = 14 L/kg (wet wt.) and< 73 L/kg (wet wt.) at the high- and low-exposure concentrations, respectively). In view of these results, it was not necessary to conduct a depuration phase. 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: 0.01 mg/L: BCF < or = 14 L/Kg and Nominal: 0.001 mg/L: BCF = or < 73 L/Kg whole body wet weight. Under the conditions of this study, the BCF was 14 to 73 L/Kg. Based on the mean lipid content (start and end of exposure period): 3.55%, the applicant recalculates the reported value for 5% lipid content as BCF (lipid-normalised 5%) is < or = 103 L/Kg whole body wet weight.

 

Key study : equivalent or similar to OECD TG 319B, 2019 : A study was performed to assess the in vitro stability of test item in fish liver S9 fractions using a method equivalent or similar to OECD TG 319B. The method followed was a standardised assay, previously prevalidated by a consortium under the coordination of HESI/ILSI and under corresponding peer reviewed methodology. The method was recently internationally validated and OECD accepted. Full details of the in vitro assay methodology are provided in the full study report and are presented by the applicant. Very rapid enzymatic turnover of the major constituent of test item by trout liver S9 fractions was observed. Biotransformation was independent on the addition of cofactors indicating the involvement of a carboxylesterase. The equivalent alcohol and cyclopropanecarboxylic acid were identified as metabolites, which are potential degradation products of an esterase catalysed cleavage. Substrate depletion assays using RT-S9 are considered to be a reliable and adequate method to assess biotransformation of chemicals in fish. As per the published literature. Predicted BCFs based on in vitro biotransformation rates in RT-S9 showed a better agreement to in vivo measured BCFs for chemicals compared to the in silico predictions based on solely log Kow (H. Laue et al, 2014 : Predicting the bioconcentration of fragrance ingredients by rainbow trout using measured rates of in vitro intrinsic clearance. Environ. Sci. Technol., 2014. 48(16): p. 9486-9495). The biotransformation rate determined for test item in fish liver S9 fractions in the main experiment (8.42/h) was used as input into an in vitro - in vivo extrapolation model to predict the BCF using the measured log Kow (5.6 , cited measured value). The partitioning based BCF assuming no metabolism for test item based on the measured log Kow value was calculated as part of the in vitro-in vivo extrapolation model (BCF 13344 L/Kg). Including the in vitro biotransformation rate of test item in trout S9 fractions and other parameters (cited in the full study report), the predicted BCFs were 650 L/kg wet weight assuming different binding to serum in vivo vs. in vitro (Fu calc) and 271 L/kg wet weight setting Fu = 1.0. These predicted BCFs based on in vitro biotransformation rates are significantly lower compared to the BCF value calculated with an assumed turnover rate of 0. Especially for chemicals with higher log Kow values, a better correlation of predicted BCFs calculated with the in vitro-in vivo extrapolation model using an assumed Fu of 1.0 and measured in vivo BCFs was shown in several studies (cited) than when Fu calc is used. One possible explanation may be that the assumption of the extrapolation model that only freely dissolved chemicals are available for metabolic turnover is incorrect. Chemicals bound to proteins may desorb rapidly and thus contribute to the metabolic turnover of the chemicals. In an available in vivo Fish BCF study (2018) with common carp (Cyprinus carpio), according to or equivalent to OECD TG 305 on the test item, the lipid normalised (5%) steady state BCF of test item was determined to be ≤ 103 L/kg (wet wt.) for the low test concentration and ≤ 20 L/kg (wet wt.) for the high test concentration. The predicted BCFs based on the RT-S9 biotransformation rates using a theoretically calculated Fu were 6.3-fold higher and 2.6-fold higher using an assumed Fu of 1.0 than the highest in vivo BCF value (≤ 103 L/kg). A similar trend of overprediction of BCFs based on in vitro biotransformation and IVIVE especially for Fu calculated was also observed previously in several peer reviewed studies (cited by the report author). Biotransformation rates determined following OECD TG 319B may be utilised as an important tool in an ITS (Intelligent Testing Strategy) tiered testing regime to determine the bioaccumulation potential of chemicals.