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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:
From 21 August 2008 to 22 October 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study was performed according to OECD Guideline 305 without GLP statement. All validity criteria were fulfilled but restrictions should be taken into account. The BCF result was not expressed as normalised to a fish with 5% lipid content, therefore the BCF was recalculated. In addition, this results seems to be overestimated because the BCF was based upon total radiolabelled residues without taking into account the potential of degradation of the test substance and the total radioactive residues in fish were not measured between 28 days (maximum accumulation in fish) and 35 days (total depuration).
Qualifier:
according to
Guideline:
OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)
Version / remarks:
, 14 June 1996
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): ST 11 C 08
- Water solubility: 1.88 mg/L
Radiolabelling:
yes
Details on sampling:
- Sampling intervals/frequency for test organisms and sample storage conditions: See table 5.3.1/1 in "Any other information on materials and methods incl. tables". On each sampling occasion, four fish were collected randomly from each exposure tank, rinsed with water, sacrificed in 1.5% (v/v) 2-phenoxy-ethanol in purified water and blotted dry. Four fish were analysed immediately following sacrifice. At one time interval (after 28 days of accumulation) eight fish were sampled and stored at about -20 °C for possible additional analyses. Additionally, ten fish were sampled for lipid determination.
- Sampling intervals/frequency for test medium samples: See table 5.3.1/1 in "Any other information on materials and methods incl. tables". Daily during the uptake phase and at selected time points during the depuration phase, duplicate samples of appropriate volumes (10 mL) were removed from the treated tank and as far as appropriate from the corresponding mixture chambers. Radioactivity was directly determined. Additionally at the last time interval, duplicate samples of 500 mL were taken and stored at -20 °C for possible additional analyses.
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods): Duplicate samples of 10 mL water were directly analysed for total radioactivity by LSC. Four fish were individually analysed for total radioactivity at each sampling point. The fish were weighed and solubilized at 40 °C for 48 - 96 hours with the tissue solubilizer Solvable (Perkin Elmer, about 1 mL solubilizer per 100 mg fish weight). Thereafter, duplicate solubilized subsamples (corresponding to 100 or 200 mg) were measured by LSC. The 2 x 5 fish sampled on day 28 were used to determine the fish lipid/wet weight ratio at the end of the accumulation period by means of chloroform/methanol. The additional eight fish sampled on day 28 were weighed and stored at -20 °C. However, as the average BCF value was below 1000, these fish were not further analysed and remained stored at -20 °C.
Vehicle:
yes
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Based on the target concentration of 10 μg/L and based on a total volume of 7750 L (assuming a flow-through volume of 250 L per day during 3 days pre-treatment, and 28 days treatment), an amount of 78 mg 14C-ST 11 C 08 at a specific radioactivity of 0.527 MBq/mg was needed. Including an appropriate excess, 110 mg at 0.527 MBq/mg were prepared (approx. 60 MBq).
- Stock solution 1: The total amount of the available material (14.42 mg at 5.27 MBq/mg) was dissolved in 10 mL ethanol. The radioactivity was determined and an amount of 15.60 mg was measured.
- Stock solution 2: Based on the required amount of about 11 mg 14C-ST 11 C 08 at 5.27 MBq/mg, 7.20 mL of stock solution 1 were added to and 99.0573 mg unlabelled test item in a volumetric flask (weighed amount: 102.65 unlabelled material with a purity of 96.5 %). The volume was filled up to 100 mL with ethanol. Using liquid scintillation counting (LSC), the amount was determined to be 109.9675 mg at a specific activity 0.5229 MBq/mg. The final concentration in stock solution 2 was 1.0997 mg/mL.
- Preparation of application solution: Aliquot of the stock solution 2 were diluted in ethanol. Before initiating the test, level in the treated tank was adjusted by adding 0.68 mL of stock solution 2 to the tank. Throughout the entire test, the system was equilibrated with tap water at a flow-through volume of 250 L/day, to which 14C-ST 11 C 08 was added from the application solutions at a rate of 25 mL/24 h to achieve final target concentrations of 10 μg/L.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Strain: no data
- Source: Forellenzucht Hohler, Zeiningen, Switzerland.
- Age at study initiation (mean and range, SD): no data
- Length at study initiation (lenght definition, mean, range and SD): no data
- Weight at study initiation (mean and range, SD): 2.5-3.5g
- Weight at termination (mean and range, SD): no data
- Method of breeding: no data
- Health status: good
- Description of housing/holding area: no data
- Feeding during test: yes
The fish were fed daily (H.U. Hofmann AG, HOKOVIT, “Forellenfutter”, diet of known lipid and total protein content), based on about 3% of the average fish body weight during acclimation and during the study, taking into account increasing body weights and the decreasing number of fish per sampling interval.

ACCLIMATION
- Acclimation period: at least two weeks
- Acclimation conditions (same as test or not): same as test
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
28 d
Total depuration duration:
14 d
Hardness:
Water hardness measured once during the test (on day 6 of exposure) was 12 °d (21.36 °f) or 2.136 mmol/L for the treated tank.
Test temperature:
Temperature was monitored from day 0 to day 28 and measurements ranged from 13.0 – 15.4°C.
pH:
pH was monitored from day 0 to day 28 and measurements ranged from 7.9 - 8.3.
Dissolved oxygen:
Oxygen concentration was monitored from day 0 to day 28 and measurements ranged from 6.7 – 9.2 mg/L.
TOC:
TOC values were not measured within this study. However, historical TOC values for tank water at the Test Facility are < 2 mg C/L.
Salinity:
Not applicable
Details on test conditions:
TEST SYSTEM
- Test vessel: One aerated and temperature controlled tank containing 75 L. The tank was dosed with the 14C-labelled test item from an application solution with a flow-through volume of 250 L/24 hours (i.e. about 3 times the tank volume), delivered via a Hamilton dispenser unit into a mixing flask where the application solution was pre-diluted with tap water (Figure 6). Before placing the fish in the tank, the flow-through system was run for 2-3 times 24 hours with labelled test item (i.e. about 7-10 tank volumes) to ensure a stabilized concentration of the test item at the onset of the uptake phase.
- No. of organisms per vessel: 60
- No. of vessels per concentration (replicates): not applicable
- No. of vessels per control / vehicle control (replicates): not applicable
- Biomass loading rate: 0.55 g/L/day, based on a daily flow-through volume of 250 L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Local tap water (non chlorinated well water of drinking water quality), reduced for total hardness by ion exchange

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light
- Light intensity: approximately 300-400 lux

RANGE-FINDING / PRELIMINARY STUDY
None
Nominal and measured concentrations:
- Nominal concentration: 10 µg/L
- Average exposure concentration: 9.46 µg/L. See table 5.3.1/2 in "Any other information on results incl. tables"
Reference substance (positive control):
no
Details on estimation of bioconcentration:
Not applicable
Lipid content:
76.1 other: mg/g fish
Time point:
end of exposure
Remarks on result:
other: none
Lipid content:
78.4 other: mg/g fish
Time point:
end of exposure
Remarks on result:
other: none
Type:
BCF
Value:
864 dimensionless
Basis:
other: average of total radioactivity in parent equivalents in the fish during exposure related to the concentration of total radioactivity in the water.
Time of plateau:
3 d
Calculation basis:
steady state
Remarks on result:
other: Conc.in environment / dose:9.46 µg eq/L
Type:
BCF
Value:
11 189 dimensionless
Basis:
total lipid content
Time of plateau:
3 d
Calculation basis:
steady state
Remarks on result:
other: Conc.in environment / dose:9.46 µg eq/L
Key result
Type:
BCF
Value:
559.4 dimensionless
Basis:
normalised lipid fraction
Time of plateau:
3 d
Calculation basis:
steady state
Remarks on result:
other: recalculated
Remarks:
Conc.in environment / dose:9.46 µg eq/L
Key result
Elimination:
yes
Parameter:
DT50
Depuration time (DT):
2 d
Details on kinetic parameters:
No data
Metabolites:
Not measured
Results with reference substance (positive control):
Not applicable
Details on results:
- Mortality of test organisms: none
- Behavioural abnormalities: none
- Observations on body length and weight: no data
- Other biological observations: none
- Organ specific bioaccumulation: no data
- Bound residues forming a plateau: no data
- Mortality and/or behavioural abnormalities of control: no data
- Loss of test substance during test period: See table 5.3.1/3 in "Any other information on results incl. tables". See Figure in "Illustration".
- Results with vehicle control: not applicable
Reported statistics:
None

Table 5.3.1/2: Actual concentration of total radioactivity of 14C-ST 11 C 08 in the exposure water during accumulation period.

Time interval (days)

Exposure tank (mean of duplicates)

comments

Dose (µg eq/L)

-3

09:30 a.m.

10.08

-3

11:30 a.m.

9.18

-3

01:30 p.m.

9.20

-2

07:15 a.m.

6.59

-2

09:30 a.m.

10.0

-2

01:30 p.m.

9.17

-2

15:00 p.m.

8.56

-1

08:30 a.m.

7.51

-1

11:00 a.m.

10.01

-1

13:00 p.m.

11.29

-1

15:00 p.m.

12.03

0

Before adding fish

11.18

0

After adding fish

9.49

0

 

7.92

1

 

8.13

1

 

8.65

2

 

9.16

3

 

10.08

4

 

10.49

5

 

10.41

6

 

9.90

7

 

9.68

8

 

10.00

9

 

10.04

10

 

9.58

11

 

9.95

12

 

9.86

13

 

10.55

14

 

9.51

15

 

9.43

16

 

9.13

17

 

9.34

18

 

10.34

19

 

9.64

20

 

9.73

21

 

8.91

22

 

8.91

23

 

8.91

24

 

8.97

25

 

8.84

26

 

9.01

27

 

9.15

28

 

9.03

Mean

(Day 1 to 28)

9.46

± Standard Deviation

 

0.65

Table 5.3.1/3: Concentration of radioactivity in the whole fish during the test at an average dose level of 9.46 µg/L

Phase

Time interval

Concentration of radioactivity in fish

Days after the onset of accumulation

Days after the onset of depuration

Dose

µg eq/g

%*

Accumulation period

3

5

7

14

21

28

-

-

-

-

-

-

7.498

8.082

7.353

8.089

8.094

9.932

-

-

-

-

-

-

Mean

± Standard Deviation

8.175

0.392

-

-

Depuration period

35

42

7

14

0.740

0.326

9.1

4.0

* Residual radioactivity in fish in percent of the average concentration in fish during exposure.

Validity criteria fulfilled:
yes
Conclusions:
The BCF value of 560 (recalculated, expressed as normalised to a fish with 5% lipid content) and the depuration half-life of 2.0 days indicate that the test substance did not bioaccumulate in the rainbow trout. In addition, this results seems to be overestimated because the BCF was based upon total radiolabelled residues without taking into account the potential of degradation of the test substance and the total radioactive residues in fish were not measured between 28 days (maximum accumulation in fish) and 35 days (total depuration).
Executive summary:

This study was performed according to OECD Guideline 305 without GLP statement, to assess the bioconcentration and depuration characteristics of the test substance in the rainbow trout under dynamic flow through system.

The fish were continuously exposed to 14C-ST 11 C 08 at an average dose level of 9.46 μg eq/L for 28 days. After the exposure, the fish were transferred to flowing untreated water and the depuration of radioactivity was followed for further 14 days. Temperature, pH and oxygen concentration were monitored from day 0 to day 42 and were within acceptable limits; measurements ranged from 13.0-15.4 °C, 7.9-8.3 and 6.7-9.2 mg/L, respectively.

The plateau levels could be determined as average concentration level in the fish. The radioactive residues in fish during exposure amounted on average to 8.175 ± 0.392 μg eq/g. The radioactive residues during depuration decreased to 0.740 μg eq/g on day 35 and to 0.326 μg eq/g on day 42.

Depuration half-life calculations was performed based on the concentration of radioactivity in fish on day 28 (exposure) and the values measured during 14 days of depuration. For the dose level selected, a depuration half-life of 2.0 days was calculated (correlation R² = 0.999).

For the dose level selected, based on the radioactivity levels in fish after exposure to 14C-ST 11 C 08 at an average dose level of 9.46 μg eq/L, the average steady state bioconcentration factor (BCF) amounted to 864 ± 41.

BCFlipid based on the lipid content measured in representative fish amounted to 11189.

All validity criteria were fulfilled but restrictions should be taken into account. The BCF result was not expressed as normalised to a fish with 5% lipid content, therefore the BCF was recalculated, at 560. In addition, this results seems to be overestimated because the BCF was based upon total radiolabelled residues without taking into account the potential of degradation of the test substance and the total radioactive residues in fish were not measured between 28 days (maximum accumulation in fish) and 35 days (total depuration).

In conclusion, the BCF value of 560 indicated that the test substance did not bioaccumulate in the rainbow trout.

Description of key information

in vivo non GLP-OECD Guideline 305 test and in vitro biotransformation assay using liver S9 fractions and hepatocytes from rainbow trout, performed on the registered substance:

- in vivo test: BCF = 560 L/kg (recalculated, expressed as a normalised value to a fish with 5% lipid content) . This results seems to be overestimated because the BCF was based upon total radiolabelled residues without taking into account the potential of degradation of the substance and the total radioactive residues in fish were not measured between 28 days (maximum accumulation in fish) and 35 days (total depuration).

-in vitro test: BCF = 363 L/kg and 196 L/kg for the RT-S9 and RT-HEP assays, respectively.

AVERAGE BCF VALUE = 373 L/kg

Key value for chemical safety assessment

BCF (aquatic species):
373 L/kg ww

Additional information

One experimental study was performed according to OECD Guideline 305 without GLP statement to assess the bioconcentration and depuration characteristics of the registered substance in the rainbow trout under dynamic flow through system.

The fish were continuously exposed to 14C-test substance at an average dose level of 9.46 μg eq/L for 28 days. After the exposure, the fish were transferred to flowing untreated water and the depuration of radioactivity was followed for further 14 days. Temperature, pH and oxygen concentration were monitored from day 0 to day 42 and were within acceptable limits; measurements ranged from 13.0-15.4 °C, 7.9-8.3 and 6.7-9.2 mg/L, respectively.

The plateau levels could be determined as average concentration level in the fish. The radioactive residues in fish during exposure amounted on average to 8.175 ± 0.392 μg eq/g. The radioactive residues during depuration decreased to 0.740 μg eq/g on day 35 and to 0.326 μg eq/g on day 42.

Depuration half-life calculations was performed based on the concentration of radioactivity in fish on day 28 (exposure) and the values measured during 14 days of depuration. For the dose level selected, a depuration half-life of 2.0 days was calculated (correlation R² = 0.999).

For the dose level selected, based on the radioactivity levels in fish after exposure to 14C-test substance at an average dose level of 9.46 μg eq/L, the average steady state bioconcentration factor (BCF) amounted to 864 ± 41.

BCFlipid based on the lipid content measured in representative fish amounted to 11189.

All validity criteria were fulfilled but restrictions should be taken into account. The BCF result was not expressed as normalised to a fish with 5% lipid content, therefore the BCF was recalculated, at 560. In addition, this results seems to be overestimated because the BCF was based upon total radiolabelled residues without taking into account the potential of degradation of the test substance and the total radioactive residues in fish were not measured between 28 days (maximum accumulation in fish) and 35 days (total depuration).

In conclusion, the BCF value of 560 indicated that the test substance did not bioaccumulate in the rainbow trout.

In addition, an in vitro biotransformation assay using liver S9 fractions and hepatocytes from rainbow trout (Oncorhynchus mykiss) was performed on the registered substance. Based on the results of this study, the predicted in vivo BCF values using the model of Nichols et al., 2013, were determined at 363 L/kg and 196 L/kg for the RT-S9 and RT-HEP assays, respectively. The study was finalised, submitted to Journal of Applied Toxicology, but not yet published. The dossier will be updated as soon as the report is made available.