1-Butanone, 3-(dodecylthio)-1-[(1R,2S)-2,6,6-trimethyl-3-cyclohexen-1-yl]-, rel-

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:

2014-10-31 to 2014-11-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD Guideline 305-I with GLP statement. All validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Specific details on test material used for the study:

- Vapor pressure: 7.5×10^-6 (25 °C) (OECD 104, SafePharn 2006)
- Solubility in water: 0.0494 - 0.114 μg/L (25 °C; column eluting, measured in Kurume Laboratory)
- 1-Octanol-water partition coefficient logPow: 9.5 ± 0.3 (Begnaud F et al. (2015))
- Melting point: 19 °C
- Boiling point: 294 °C (at 1013 kPa as measurement pressure)
- Density 0.92 g/cm3 (20 °C)

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms: For both concentration levels 1 and 2, the analysis of test fish was performed 5 times during exposure period. Four fish were sampled for one analysis, but analytical sensitivity was not sufficient, therefore they were separated into two groups (two fish in a group), and each group was determined in analysis.
For last consecutive three tests, test fish were sampled over the interval of more than 48 hours, and the final analysis was performed on 28 days.
For the control level, the analysis of test fish was performed before the start of experiment and after the completion of experiment. Four fish were sampled for one analysis, and they were separated into two groups (two fish in a group), and each group was determined in analysis.
- Sampling intervals/frequency for test medium samples: For both concentration levels 1 and 2, the analysis of test water was performed once before the first test fish analysis and then at the same time of test fish analysis during the exposure period. Single sample was used for each analysis.
- Details on analysis of test organisms and test media samples: The analysis of the test substance in test water and test fish was performed by liquid chromatography-tandem-mass spectroscopy (LC-MS/MS).

Vehicle:

yes

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

PREPARATION AND APPLICATION OF TEST SOLUTION
Acute toxicity test:
- Preparation of stock solution: Due to the very low solubility of the test item, the solvent N,N-dimethylformamide, was used to prepare the stock solutions. 500 mL of 1000mg/L test substance and 50.0 g/L HCO-40 stock solution were prepared by dissolving 500 mg of the test sample provided by the sponsor and 25.0 g of HCO-40 in 500 mL of N,N-dimethylformamide. The stock solution was visually assessed to be colorless and transparent.
Bioconcentration test:
Preparation of stock solution
a) Concentration level 1: The amount of 100 mg of the test sample provided by the sponsor and 5.0 g of HCO-40 corresponding to 50-times amount of the test sample were dissolved in N,N-dimethylformamide to prepare 100 mL of a test substance solution at the test substance concentration of 1000 mg/L. Then this solution was further diluted with N,N-dimethylformamide to prepare 1 L of a stock solution at the test substance concentration of 10.0 mg/L . Stock solutions as well as test solutions were assessed visually to be colorless and transparent.
b) Concentration level 2: The test substance solution of 1000 mg/L prepared above (Concentration level 1) was diluted with N,N-dimethylformamide to prepare 1 L of a stock solution at the test substance concentration of 1.00 mg/L.
- Controls: The amount of 500 mg of HCO-40 was dissolved in N,N-dimethylformamide to prepare 1 L of a stock solution at the concentration HCO-40 of 500 mg/L.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp
- Source: Kurume Laboratory, Chemicals Evaluation and Research Institute, Japan .
- Lot number: TFC-141002 (acute toxicity test); TFC-140902 (bioconcentration test)
- Age at study initiation: One-year-old fish
- Length at study initiation: 3.5 to 3.9 cm (acute toxicity test); 6.9 to 8.9 cm (6.9 to 7.6 cm at the start of experiment) (bioconcentration test)
- Lipid content at test initiation: 3.43%
- Health status: Normal
- Feeding during test
- Food type: Formula feed for juvenile carp breeding; Protein 43.0% or more; Lipid 3.0% or more
- Method : The test fish were fed twice a day (fed once on holidays). The total daily amount of feed corresponded to about 3% of body weight. However, the fish were not fed for 24 hours prior to sampling.

ACCLIMATION
- Acclimation period: 22 days
- Acclimation conditions (same as test or not): Fish were treated by a medicated bath using OTC (oxytetracycline hydrochloride, Kyoritsu Seiyaku Corporation) for aquaculture vaccination and sodium chloride (The Salt Industry Center of Japan), and then were acclimatized.
- Water temperature: 25 ± < 2 °C
- Health during acclimation (any mortality observed): Total mortality was less than 5% during acclimatization period.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Hardness:

No data

Test temperature:

Acute toxicity test:
24.2 °C at the start of exposure
24.6 °C before renewal of water (at first renewal)

Bioconcentration test:
Concentration level 1: 24.3-24.8 °C
Concentration level 2: 24.5-24.8 °C
Control: 24.5-24.8 °C

pH:

Acute toxicity test:
8.1 at the start of exposure
8.1 before renewal of water (at first renewal)

Bioconcentration test:
Concentration level 1: 7.6 and 7.8
Concentration level 2: 7.6 and 7.8
Control: 7.6 and 7.8

Dissolved oxygen:

Acute toxicity test:
8.1 mg/L at the start of exposure
7.0 - 7.1 mg/L before renewal of water (at first renewal)

Bioconcentration test:
Concentration level 1: 6.9 – 7.6 mg/L
Concentration level 2: 6.8 – 7.5 mg/L
Control: 6.8 – 7.4 mg/L

TOC:

No data

Salinity:

No data

Conductivity:

No data

Details on test conditions:

TEST SYSTEM
- Method of supplying test solution: Flow-through system made by Kurume Laboratory
- Test vessel: 70 L glass aquarium
- Flow volume of test solution: The flow of 2880 L/day of test solution was supplied to the test vessel at a rate of 0.04 mL/min of the stock solution and a rate of 2000 mL/min of water for the test.
- Stock solution tank: 1 L brown glass bottle
- Aeration: None
- Renewal rate of test solution (frequency/flow rate): Exchange frequency once/2 weeks
- No. of test fish: 24 fish at the start of experiment in Concentration level 1 and level 2
- No. of control fish: 14 fish at the start of experiment in control.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Ground water pumped in the premises of Kurume Laboratory

OTHER TEST CONDITIONS
- Photoperiod: 14 hours light/10 hours dark (artificial lighting using white fluorescent lamps)

OBSERVATION
Observation of test fish: Test fish were observed visually twice a day and once on holidays during experiment.
Test temperature: Measured daily during experiment.
Flow volume of test solution: Measured once a day during experiment.
Dissolved oxygen concentration: Measured one or twice a week during experiment.
pH: Measured two times during experiment.

RANGE-FINDING / PRELIMINARY STUDY
- Based on preliminary test results for the 96 h LC50 value and analytical detection limits, test concentrations of the test item were decided as follows. The control was set as a blank test.
Concentration level 1: 0.2 μg/L
Concentration level 2: 0.02 μg/L

Nominal and measured concentrations:

Concentration level 1: 0.2 μg/L
Concentration level 2: 0.02 μg/L

Reference substance (positive control):

no

Details on estimation of bioconcentration:

Calculation of bioconcentration factor:
BCF = Cf/Cw
BCF: Bioconcentration factor
Cf: Concentration of test substance in test fish (after subtraction of FB) (ng/g)
Cw: Average concentration of test substance in test water for calculation of a bioconcentration factor (μg/L)
FB: Average of the concentrations or apparent (blank) concentrations of test substance in test fish before and after the experiment for control (ng/g)

The average bioconcentration factor at the m-th analysis
BCFm = (BCFa + BCFb) / n

BCFm : Average of bioconcentration factor at the m-th analysis (number of groups is two (a, b).)
BCFa,b : Bioconcentration factor for each group at the m-th analysis
n : Number of groups analyzed at the m-th analysis
When the test substance is not detected on a measurement day, an average of bioconcentration factor on such the measurement day is not determined.

Lipid content:

3.43 %

Time point:

start of exposure

Lipid content:

3.21 %

Time point:

end of exposure

Key result

Conc. / dose:

0.2 µg/L

Temp.:

25 °C

pH:

7.8

Type:

BCF

Value:

< 3.9 other: folds

Basis:

total lipid content

Time of plateau:

28 d

Calculation basis:

steady state

Remarks on result:

other: Concentration level 1

Key result

Conc. / dose:

0.02 µg/L

Temp.:

25 °C

pH:

7.8

Type:

BCF

Value:

< 38 other: folds

Basis:

total lipid content

Time of plateau:

28 d

Calculation basis:

steady state

Remarks on result:

other: Concentration level 2

Results with reference substance (positive control):

No reference substance was reported for bioconcentration potential.
The 48-hour LC50 of the reference substance, sodiumpentachlorophenolate was determined using the test fish of the same lot. The result was 0.327 mg/L and it was within the acceptable range of the historical data.

Details on results:

Concentration of test substance in test water: The concentrations of test substance in test water were kept at 71% and above of the nominal concentration, and their variation rates were maintained within ±20% of the average of measured values.

Bioconcentration factors during exposure period are less than 3.9 to 22 folds in Concentration level 1, and less than 38 folds in Concentration level 2.
Bioconcentration factor at steady state:
From the results Table 5.3.1/2, bioconcentration factors under a steady state could not be calculated because no-detected values were included in each three consecutive results on 14 days, 19 days, and 28 days. However, since all bioconcentration factors were below 100 folds, it was regarded that a steady state had been achieved after 28 days.

Lipid content in test fish:
The average lipid contents in test fish are as follows. Variation rate of the lipid content (average value) after the experiment was - 6% with respect to the lipid content before the experiment, and fell within ±25%.
Lipid content before the experiment: 3.43%
Lipid content after the experiment: 3.21%

External observation, etc., of test fish
No abnormality was observed.

Reported statistics:

No data

Table 5.3.1/1: Test substance concentration in test water

 

Concentration Level	After 4 days	After 5 days	After 10 days	After 14 days	After 19 days	After 28 days	Average (Standard deviation)
1	0.181	0.149	0.167	0.151	0.156	0.156	0.160 (0.0122)
2	0.0169	0.0143	0.0179	0.0166	0.0169	0.0162	0.0165 (0.00118)

 

Table 5.3.1/2: Bioconcentration factor

 

Concentration Level	After 5 days	After 10 days	After 14 days	After 19 days	After 28 days
1	<3.9 <3.9	22 15 (19)	<3.9 17	21 13 (17)	<3.9 <3.9
2	<38 <38	<38 <38	<38 <38	<38 <38	<38 <38

 

Validity of the test:

This test meets the validity stipulated in the test method as follows, and it is considered that the bioconcentration of the test substance can be validly assessed.

1) The water temperature variation rate was within ±2 °C with respect to the setting temperature of 25 °C.

2) The concentration of dissolved oxygen was 60% or more with respect to the saturated concentration of 8.1 mg/L at 25 °C.

3) The variation rate of the test substance concentration was maintained within ±20% with respect to the average of the measured values during the exposure period.

4) No mortality or no other adverse effects/disease were noted in test fish in the control group or the test groups .

 

Recovery rate for test water

In the recovery test for test water, the average of recovery rate was 66.3%. Although type of solvent and method for extraction was examined in the preliminary examination, a recovery rate higher than the recovery rate achieved in the pretreatment procedure adopted in this test was not obtained. However, since the difference between the two measured recovery rates was within ±10% while these rates were low, it was judged that precise analysis was possible and consequently this pretreatment procedure was adopted.

 

Recovery rate for test fish:

In the recovery test for test fish, the average of recovery rate was 61.5%. As a result of examination of the procedure of a pretreatment method in the preliminary examination, it was clear that the low recovery rate was caused by extraction loss during solid phase extraction. Therefore type of solid-phase and solvent for extraction was examined. However, a recovery rate higher than the recovery rate achieved in the pretreatment procedure adopted in this test was not obtained. However, since the difference between the two measured recovery rates was within ±10% while these rates were low, it was judged that precise analysis was possible and consequently this pretreatment procedure was adopted.

 

Nominal concentration:

Solubility of the test substance in water was 0.0494 – 0.114 μg/L, and the nominal concentration in the main test were 0.2μg/L for concentration level 1, 0.02μg/L for concentration level 2. For the concentration level 1, the concentration exceeded water solubility in the main test. Pretreatment method of the test water was examined in the preliminary examination but it was difficult to establish the lower nominal concentration. However, the bioconcentration factors were less than 3.9 to 22 folds in the concentration level 1, less than 38 folds in the concentration level 2 and these were low level, and the concentration-dependency was not noted. Therefore it is considered that the result of this study is valid to determine the bioaccumulation level of the test substance.

 

Concentration of the test water:

The test substance concentrations in test water in the main test were 74 - 91% of the nominal value in the concentration level 1, 71 – 920% in the concentration level 2. Adsorption to the test system by dodecyl is considered to be the reason for the low retention level of the concentration. However the test substance concentration in test water in the main test was kept above 71% of the nominal value in the concentration level 1 and 2, and the variation was within ±20%. Based on these it is considered that there was no problem in the homogeneity in the test water.

Validity criteria fulfilled:

yes

Conclusions:

Bioconcentration factors during exposure period are less than 3.9 to 22 folds in Concentration level 1, and less than 38 folds in Concentration level 2. Bioconcentration factors under a steady state could not be calculated because no-detected values were included in each three consecutive results on 14 days, 19 days, and 28 days. However, since all bioconcentration factors were below 100 folds, it was regarded that a steady state had been achieved after 28 days.

Executive summary:

The Bioconcentration test was performed according to the OECD Guideline No. 305-I and in compliance with GLP. The study was carried out to determine the extent of accumulation of test item by the carp, Cyprinus carpio during the exposure period of 28 days.   

In preliminary acute toxicity test (semi-static method), Carp (Cyprinus carpio) was exposed to test substance at concentrations of 500 and 1000 μg/L for 96 hours. The 96-hour LC50 for the test substance was > 1000 μg/L. Based on preliminary test results for the 96 h LC50 value and analytical detection limits, test concentrations of the test item were decided as follows: Concentration level 1: 0.2 μg/L; Concentration level 2: 0.02 μg/L. Bioconcentration test was performed according to Flow-through method. Carp (Cyprinus carpio) was exposed to test substance at concentrations (Concentration level 1: 0.2 μg/L; Concentration level 2: 0.02 μg/L) for 28 days. Analyses of test water was conducted on Days 4, 5, 10, 14, 19 and 28 after exposure using Liquid chromatography-tandem-mass spectrometry. Bioconcentration factor (BCF) was calculated.

 

The concentrations of test substance in test water were kept at 71% and above of the nominal concentration, and their variation rates were maintained within ±20% of the average of measured values.

 

The average lipid contents in test fish are as follows. Variation rate of the lipid content (average value) after the experiment was - 6% with respect to the lipid content before the experiment, and fell within ±25%. Lipid content before the experiment: 3.43%; Lipid content after the experiment: 3.21%.

 

Bioconcentration factors during exposure period are less than 3.9 to 22 folds in Concentration level 1, and less than 38 folds in Concentration level 2. Bioconcentration factors under a steady state could not be calculated because no-detected values were included in each three consecutive results on 14 days, 19 days, and 28 days. However, since all bioconcentration factors were below 100 folds, it was regarded that a steady state had been achieved after 28 days.

Description of key information

GLP-OECD Guideline 305 study (performed for a Japan notification):

Bioconcentration factors during exposure period are less than 3.9 to 22 fold in Concentration level 1, and less than 38 fold in Concentration level 2.

According to this result, the registered substance is not considered as bioaccumulable.

Key value for chemical safety assessment

BCF (aquatic species):

38 dimensionless

Additional information

To assess the bioaccumulation potential of the registered substance, one experimental study was performed according to OECD Guideline 305 with GLP compliance. This study was carried out for a Japanese notification on the registered substance, to determine the extent of accumulation of the substance by the carp, Cyprinus carpio, during the exposure period of 28 days. Bioconcentration test was performed according to Flow-through method. Carp was exposed to test substance at two concentrations: 0.2 μg/L (Concentration level 1) and 0.02 µg/L (Concentration level 2). Bioconcentration factors during exposure period are less than 3.9 to 22 folds in Concentration level 1, and less than 38 folds in Concentration level 2. Bioconcentration factors under a steady state could not be calculated because no-detected values were included in each three consecutive results on days 14, 19, and 28. However, since all bioconcentration factors were below 100 fold, it was regarded that a steady state had been achieved after 28 days. According to this result, the registered substance is not considered as bioaccumulable.