5-bromo-1,3-dichloro-2-fluorobenzene

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Remarks:

The study was conducted to meet the national regulatory requirements in a non-EEA country.

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according 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)

Radiolabelling:

no

Details on sampling:

Sampling of water: two replicates were taken at each sampling occasion from the control and treated groups; sampling occasions were on the day before start of treatment and on day 0, 1, 2, 5, 7, 9 13, 16, 21 and 23 during the exposure period; water samples were taken at the start of the depuration phase
Sampling of fish (chemical analysis): 5 individuals were sampled for chemical analysis at the start of the test; 4 individuals were samled from the control and treatment groups at each further sampling occasion at day 1, 2, 5, 7, 9, 13, 16, 21 and 23 of the exposure phase and on day 0.25, 1, 3, 6, 10, 12 and 14 of the depuration phase
Sampling of fish (lipid content): 5 individuals were sampled at the sart of the uptake phase and the end of the uptake phase and depuration phase

Vehicle:

no

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

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: a stock solution was prepared by mixing 50 mg substance into 50 mL DMF; this stock solution was further diluted to a concentration of 180 mg/OL using DMF; this solution was diluted using dilution water to obtain a final application solution with a concentration of 1.8 mg/L
- Controls: solvent control
- Chemical name of vehicle: dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) at different concentrations and in control(s)): application solution had a DMF concentration of 1%
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: Bluegill fish
- Strain: Lepomis macrochirus
- Source: Osage Catfisheries Inc, 1170 Nichols Road, Osage Beach, USA
- Age at study initiation (mean and range, SD): juvenile
- Length at study initiation (length definition, mean, range and SD): 3.9 to 5.1 cm
- Weight at study initiation (mean and range, SD): 936 to 1910 mg
- Lipid content at test initiation (mean and range, SD): mean lipid content at the start and end of the uptake phase was 11.0 and 9.6%, and at the end of the depuration phase was 10.9%, resulting in a mean of 10.3% (SD 2.3%) over the study period
- Health status: healthy
- Description of housing/holding area: Glass aquarium containing ca. 30 L of test medium, placed in a controlled environment room in a ventilated area
- Feeding during test
- Food type: 1:1 mixture of Inicio Plus G and Tetra min
- Amount: about 1% of body weight
- Frequency: daily

ACCLIMATION
- Acclimation period: 83 days
- Acclimation condition: same as main study
- Type and amount of food: same as main study
- Feeding frequency: same as main study
- Health during acclimation: 3 fish died during acclimatin (mortality <1%)

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: most suitable due to volatility of test substance and possibility to conduct at low concentrations (based on low water solubility of test substance and available sensitive analytical method)

Test type:

flow-through

Water / sediment media type:

other: reconstituted water

Total exposure / uptake duration:

23 d

Total depuration duration:

14 d

Hardness:

250 mg/L as CaCO3

Test temperature:

21 ± 2 °C

pH:

7.5 to 7.6

Dissolved oxygen:

At least 60% of the saturation value, at Day 1 of the uptake the saturation value was once below 60% (59.4%).

TOC:

14.7 to 40.3 mg/L

Salinity:

not specified

Conductivity:

not specified

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquarium
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass containing ca. 30 L test medium, headspace not specified
- Aeration: due to volatility of the test substance, aeration of the tank was reduced
- Type of flow-through: an application solution at a concentration of 1.8 mg/L and dilution water was pumped into a mixing unit with a constant flow rate by a diaphragm metering pump; application solution and dilution water were continuously mixed with a magnetic stirrer; the mixing unit was connected with the aquarium by a tube
- Renewal rate of test solution (frequency/flow rate): fivefold per day
- No. of organisms per vessel: 85
- No. of vessels per concentration (replicates): one
- No. of vessels per control / vehicle control (replicates): one
- Biomass loading rate: 0.78 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water prepared from deionised water (conductivity <10 µS/cm) by addition of analytical grade salts
- Particulate matter: total solids 6 mg/L
- Metals: not specified
- Pesticides: not specified
- Chlorine: not specified
- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4:1 (based on molarity)
- Conductance: not specified
- Holding medium different from test medium: no
- Intervals of water quality measurement: not specified
- Intervals of test medium replacement: continuous

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h darkness
- Light intensity: 400 - 800 lux (light intensity was measured weekly at three positions)

Nominal and measured concentrations:

Nominal: 0.007 mg/L
Measured: 0.0063 mg/L (time-weighted average; all concentrations were within ±17% of the mean measured concentration during the uptake phase)

Reference substance (positive control):

no

Lipid content:

11 %

Time point:

start of exposure

Lipid content:

9.6 %

Time point:

end of exposure

Key result

Conc. / dose:

0.006 mg/L

Temp.:

>= 20.2 - <= 22.3 °C

pH:

7.5

Type:

BCF

Value:

1 838 L/kg

Basis:

whole body w.w.

Time of plateau:

9 d

Calculation basis:

steady state

Key result

Conc. / dose:

0.006 mg/L

Temp.:

>= 20.2 - <= 22.3 °C

pH:

7.5

Type:

BCF

Value:

892 L/kg

Basis:

whole body w.w.

Time of plateau:

9 d

Calculation basis:

other: steady state corrected for 5% lipid content

Key result

Conc. / dose:

0.006 mg/L

Temp.:

>= 20.2 - <= 22.3 °C

pH:

7.5

Type:

BCF

Value:

1 709 L/kg

Basis:

whole body w.w.

Time of plateau:

9 d

Calculation basis:

kinetic

Key result

Conc. / dose:

0.006 mg/L

Temp.:

>= 20.2 - <= 22.3 °C

pH:

7.5

Type:

BCF

Value:

1 679 L/kg

Basis:

whole body w.w.

Time of plateau:

9 d

Calculation basis:

kinetic, corrected for growth

Key result

Conc. / dose:

0.006 mg/L

Temp.:

>= 20.2 - <= 22.3 °C

pH:

7.5

Type:

BCF

Value:

815 L/kg

Basis:

whole body w.w.

Time of plateau:

9 d

Calculation basis:

other: growth corrected kinetic BCF corrected for 5% lipid content

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

1.22 d

Key result

Rate constant:

overall uptake rate constant (L kg-1 d-1)

Value:

974.8

Key result

Rate constant:

overall depuration rate constant (d-1)

Value:

0.57

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

0.581

Details on results:

- Mortality of test organisms: no mortality occurred
- Behavioural abnormalities: no abnormalities observed
- Observations on body length and weight: no abnormalities
- Other biological observations: no adverse effects
- Organ specific bioaccumulation: not reported
- Mortality and/or behavioural abnormalities of control: no mortality occurred
- Loss of test substance during test period: minimised by applying flow-through regime
- Non-eliminated residues (NER) at the end of elimination phase: full elimination observed in depuration phase
- Results with vehicle control: no adverse effects observed

Validity criteria fulfilled:

yes

Conclusions:

The substance showed a limited bioconcentration potential in fish over a 23 day exposure period, with BCF values determined in the range from 815 to 1838 L/kg. At steady state, a lipid normalised BCF of 892 L/kg was determined. The growth corrected and lipid normalised kinetic BCF was 815 L/kg. The substance was eliminated quickly from the fish with approximately 97% reduction in body burden after 6 days, 98% reduction after 10 days and 100% reduction after 12 days of depuration.

Executive summary:

The bioconcentration of the substance in fish was studied in accordance with OECD TG 305 (2012) under GLP in a flow-through study using juvenile bluegill fish (Lepomis macrochirus). The test consisted of an exposure phase of 23 days and a depuration phase of 14 days. During the uptake phase, one group of fish was exposed to the substance in treated water under flow-through conditions at a nominal concentration of 0.007 mg/L, which was chosen on the basis of the 96-hour LC50 value observed in an acute fish toxicity study with rare minnow. A control group treated with water containing dilution water treated with the same amount of organic solvent used in the treatment group, was also included in the test. After the exposure period of 23 days, the treated fish were transferred to a medium free of test item for the depuration phase. All tests were conducted in aquaria containing about 30 L test medium with 5-fold water exchange per day, which were placed in a well ventilated room with controlled environmental conditions. Test water had a temperature of 21 °C ± 2 °C, a cycle of 16 hours light per day and 8 hours darkness per day was applied, light intensity was between 400 and 800 lux, fish were fed commercially available fish diet daily at a rate of approximately 1% of their body weight. Test water and fish were sampled throughout the exposure and depuration phases and the test substance concentrations were determined by GC-MS/MS analysis.

The overall mean concentration of the substance in the test water was 0.0063 mg/L over the course of the study, with a standard deviation of 7%. Overall, all measured concentrations were within ±17% of the time weighted average measured concentration. At day 1 of the study, the mean concentration of test substance in fish was 5.6 mg/kg wet weight. During the exposure phase, the analysed concentration in whole fish samples increased until day 9 to 14.1 mg/kg wet weight, and an apparent plateau was reached. As such, steady state calculations were based on the mean tissue concentrations measured on days 9, 13, 16, 21 and 23 of the uptake phase. The mean measured concentration of test substance in whole fish samples was 11.9 mg/kg wet weight. The bioconcentration factor determined for steady state (BCFss) was 1838 L/kg, and the BCFss normalised for a lipid content of 5% was 892 L/kg. Furthermore, the kinetic bioconcentration factor (BCFk) was also calculated and was 1709 L/kg (95% confidence interval of 1551 to 1868 L/kg), and the BCFk normalised to a lipid content of 5% was 829.8 L/kg. The BCFk corrected for growth (BCFkg) was determined to be 1679 L/kg. Finally, the BCFkg normalised to a lipd content of 5% was 815.2 L/kg.

The depuration phase after cease of exposure resulted in a reduction in whole body concentrations in fish of 44% on day 1, 97% reduction after 6 days of depuration and 100% reduction after 12 days of depuration. The calculated DT50 value was thus 1.22 days.

The validity criteria were fulfilled: water temperature was 21 °C, with less than 2 °C variation, dissolved oxygen was between 62 and 93%, except on a single occasion at day 1 when dissolved oxygen was 59.4%, all measured concentrations were within ±17% of the mean average measured concentration of 0.0063 mg/L, the test concentration was clearly below the measured water solubility of 15 mg/L and no mortality or adverse effects were observed in the control and the treated fish. A few small deviations from the study plan occurred, e.g. slightly higher water temperature of 21 instead of 20 °C or lower feeding rate of control group on one day, which had no negative impact on the study results.

Description of key information

No significant bioaccumulation (BCF <2000 L/kg) was observed in an in-vivo study with fish conducted in a flow-through regime and performed under GLP and according to OECD TG 305.

Key value for chemical safety assessment

BCF (aquatic species):

1 679 L/kg ww

Additional information

Kinetic bioconcentration factor corrected for growth but not normalised to lipid content, based on in vivo study in fish in a flow-through regime applying aqueous exposure