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Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 319B: Determination of in vitro intrinsic clearance using rainbow trout liver S9 sub-cellular fraction (RT-S9)
Version / remarks:
27 June 2018
Deviations:
yes
Remarks:
adapted and optimized for difficult test conditions and substances
GLP compliance:
not specified
Remarks:
publication
Specific details on test material used for the study:
stored at +4°C
Purity : 95%
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Source: obtained from a commercial fish farm were kept in the fish facilities of the Centre for Fish and Wildlife Health, University of Bern.
- Weight at study initiation (mean and range, SD): Female trout weighting 400 to 700 g wet weight in the nonreproductive stage were used for the liver S9 and hepatocyte preparations.
- Description of housing/holding area: The fish were maintained in 1500‐L tanks in a recirculation system for cold‐water fish (10–15 °C) equipped with a ultraviolet unit and a biofilter and kept under natural photoperiod conditions.

- Food type: The fish were fed a commercially available extruded diet (Trout Gourmet 4.5 mm; Hokovit).

Preparation of the hepatic S9 fractions (RT-S9) :
Fish were euthanized using tricaine methanesulfonate (MS‐222, concentration 250 mg/L); subsequently, the liver was perfused for blood clearance and dissected.
Hepatic S9 supernatants were prepared
Aliquots of the S9 fractions were stored at −80 °C. The protein content of the S9 fractions was determined using the Bradford assay. Enzymatically, the liver S9 fractions were characterized by measuring the 7‐ethoxyresorufin‐O‐deethylase activity and the glutathione‐S‐transferase activity, using 1‐chloro‐ 2,4‐dinitrobenzene as the substrate

Preparation of rainbow trout hepatocytes (RT‐HEP):
the fish were anesthetized by 250mg/L MS‐222. Heparin was injected into the caudal vein to prevent blood clotting.
The body cavity was opened to access the liver. A sterile canula connected to a peristaltic pump was inserted into the portal vein. The blood was removed from the liver using a clearing solution. After 8 to 10 min of perfusion, the solution was replaced by a collagenase solution for 15 to 20min.To clear the liver from the enzymatic solution, the liver was again rinsed for 8min using the clearing solution. Afterward, the liver tissue was excised, placed in culture medium, cut into small pieces, and passed gently through a series of nylon meshes. The resulting cell suspension was centrifuged and washed twice by centrifugation at 52 × g, 4 °C, for 5min. The cell pellets were resuspended, counted in a hemocytometer, and assessed for viability using the trypan blue exclusion assay. Only isolates with >90% viability were used.
Route of exposure:
aqueous
Justification for method:
other:
Hardness:
not specified
Test temperature:
18 °C
pH:
not specified
Dissolved oxygen:
not specified
TOC:
not specified
Salinity:
not specified
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
Key result
Type:
BCF
Value:
195.8 dimensionless
Remarks on result:
other: Predicted BCF based on RT‐HEP assay results
Key result
Type:
BCF
Value:
363.4 dimensionless
Remarks on result:
other: Predicted BCF based on RT‐S9 assay results
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 results:
The measured decline of the test chemical concentration over time was log10‐transformed and plotted against time. From the linear regression slope a first‐order elimination rate constant (1/h; ke) was calculated. The in vitro intrinsic clearance values (CLin vitro, int) were obtained by dividing ke by the nominal S9 protein concentration (milliliters per hour per milligram) or by the hepatocyte cell number (milliliters per hour per 106 cells; Organisation for Economic Co‐operation and Development 2018a, 2018b). To obtain in vivo intrinsic clearance estimates for comparison purposes, the CLin vitro, int values were multiplied by the hepatocellularity (500 × 106 hepatocytes/g liver) or by the milligram S9 liver content (163 mg/g liver; Nichols et al. 2013).


The BCF values were calculated using the predictive model provided by Nichols et al. (2013), which allows for incorporation of in vitro clearance data derived from liver S9 fractions or isolated hepatocyte assays. This model predicts the BCF for a “standardized” rainbow trout, defined as a 10‐g fish containing 5% whole‐body lipid. For each test chemical, model inputs included the log octanol–water partition coefficient (KOW) and the initial concentration of the test chemical, the average firstorder elimination rate constant (ke), and the hepatocyte or hepatic S9 concentration. The binding effect (fu; unitless) was set at 1.0, assuming that the chemical availability is the same in vitro and in vivo.

Validity criteria fulfilled:
not specified
Conclusions:
The in-vitro predicted BCF values with the RT-S9 assays is 363.4
The in-vitro predicted BCF values with the RT-HEP assays is 195.8
Executive summary:

 A publication about in vitro biotransformation assay using liver S9 fractions (RT-S9) and hepatocytes (RT-HEP)  from rainbow trout (Oncorhynchus mykiss) was performed on the registered substance. The present study explored the suitability of the RT-HEP and RT-S9 assays for difficult test chemicals and the in vitro-based predictions were compared to in silico-based predictions and in vivo-measured bioconcentration factors (BCFs). 


The RT-S9 assay was performed as described by Johanning et al (2012) and OECD test guideline 319 B with slight modifications. The RT-HPE assay was performed as described by Fay et al (2014) and in OECd test guideline 319 A with slight modifiction for volatile chemicals.



The BCF values were calculated using the predictive model provided by Nichols et al. (2013), which allows for incorporation of in vitro clearance data derived from liver S9 fractions or isolated hepatocyte assays. This model predicts the BCF for a “standardized” rainbow trout, defined as a 10‐g fish containing 5% whole‐body lipid. For each test chemical, model inputs included the log octanol–water partition coefficient (KOW) and the initial concentration of the test chemical, the average firstorder elimination rate constant (ke), and the hepatocyte or hepatic S9 concentration. The binding effect (fu; unitless) was set at 1.0, assuming that the chemical availability is the same in vitro and in vivo.


Based on the results of this study, the predicted in vivo BCF values were determined at 363 L/kg and 196 L/kg for the RT-S9 and RT-HEP assays, respectively.

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
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
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

Read across on Weight of Evidence approach, geometric mean of results:


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 source substance:


- in vivo test: BCF = 560  (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  and 196  for the RT-S9 and RT-HEP assays, respectively.


AVERAGE BCF VALUE = 373 

Key value for chemical safety assessment

BCF (aquatic species):
373 dimensionless

Additional information

Two experimental studies were performed to assess bioaccumulation of the source substance. The source substances is the racemic form of the registered/target substance.


One experimental study was performed according to OECD Guideline 305 without GLP statement to assess the bioconcentration and depuration characteristics of the source 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.


BCF lipid 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 source 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  and 196 for the RT-S9 and RT-HEP assays, respectively. 


 





















in vivo BCF560
in vitro BCF for the RT-S9 assays363 
in vitro BCF for RT-HEP assays196
Geometric mean373

 


The results of the available reliable data for the source and target substances are identical for biodegradation in water and short-term toxicity to aquatic invertebrates. No acute effects were observed for both compounds up to the attainable limit of solubility in test medium (see ECHA disseminated dossier of the source substance EC#223 -118 -6). The similarity of the structural, physico-chemical properties and ecotoxicity profiles between both substances is pronounced. On this basis, it's considered suitable and scientifically justified to read-across the data between the two substances to fill the bioaccumulation endpoint in the present dossier (see IUCLID section 13 for justification).