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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD and GLP guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)
Deviations:
yes
Remarks:
but the deviations did not affect the integrity of the study
Qualifier:
according to guideline
Guideline:
EU Method C.13 (Bioconcentration: Flow-through fish test)
Deviations:
yes
Remarks:
but the deviations did not affect the integrity of the study
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Identification: 14C-TBzTD
- Molecular formula: C30H28N2S4
- Molecular weight: 545.54 (at this specific activity)
- Batch: 2999MJG019-1
- Radiochemical purity: 96.5% (HPLC)
- Chemical purity: 98.9% (HPLC)
- Test substance storage: freezer - Expiry date: 2 October 2009, allocated by Notox, 1 year after receipt of the substance
- Specific activity: 817 MBq/mmol (1.50 MBq/mg)
- Total activity: 195.6 MBq
- Supplier: Selcia Limited, Fyfield Business and Research Park, Fyfield road, Ongar, Essex, CM5 0GS, UK
- Date received: 02 October 2008
Radiolabelling:
yes
Details on sampling:
- TBzTD conc was determined in samples of the test media taken after 1, 3, 7, 14, 21 and 28 days of exposure.
Vehicle:
yes
Details on preparation of test solutions, spiked fish food or sediment:
TBzTD: 0.5 and 5 μg/L, these test concentrations were chosen taking the water solubility limit of TBzTD and the limit of detection of the analytical method into account.
Control:: thest medium without test substance including acetone in the same amount as used in the treated solutions.

Stock solutions were prepared in acetone. The concentrations in these stocks were a factor 10 000 higher than the final concentrations in water: 5 and 50 mg/L.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
ORGANISMS
- Sex: both male and female organisms
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, the Netherlands
- Initial fish length: 3.9 +/- 0.2 cm, based on the measurement of a total of 10 fish from the batch used in this test
- Initial fish weight: 1.90 +/- 0.26g based on weighing of a total of 10 fish from the batch used for this test
- Characteristics: F1 from a single parent-pair bred in UV-treated water

HOLDING
- Quarantine/acclimatisation: at least 12 days after delivery
- Medium: adjusted ISO medium, formulated using RO-water (tap water purified by reverse osmosis, GEON Waterbehandeling, Berkel-Enschot, The netherlands) with the following composition: CaCl2.2H2O: 211.5 mg/L, MgSO4.7H2O: 88.8 mg/L, NaHCO3: 46.7 mg/L, KCl: 4.2 mg/L
- Feeding: daily with pelleted fish food (Cyprico Crumble Excellent (300-500 μm), Coppens International bv, Helmond, The Netherlands
- Validity of batch: in the batch of fish used for the test, mortality during seven days prior to the start of the test was less than 5%
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
28 d
Total depuration duration:
28 d
Hardness:
Ranged from 179-196 mg calcium carobonate per litre
Test temperature:
Ranged within the ranges described by the protocol (20-25°C, constant within +/- 2°C)
pH:
ranged from 7.1 to 7.9
Dissolved oxygen:
generally above 5 mg/L (which is 60% saturation)
on the 6th and 20th day of the uptake phase oxygen levels of both target conc had dropped below 5 mg/L for unknown causes, on the sixth day aeration was introduced and maintained for one day, on the 20th day vessels were refreshed
TOC:
Ranged from 21-37 mg C/L in samples from the acetone control during the uptake phase, except on day 6 when a value of 68 mg/L was obtained. During the depuration phase T(D)OC was low (below 1 mg/L). Based on the acetone conc of 100 mg/L, the T(D)OC was expected to be at a max of 62 mg C/L during the uptake phase. The extra 6 mg C/L on day 6 can therefore not be related to the presence of acetone.
Salinity:
na
Details on test conditions:
DOSING
During the uptake phase, the stock solutions were dosed via a computer-controlled system (DaVinci) consisting of micro-dispensers (Gilson). Via this system the dosed volume from the stock entered a mixing flask separately from the medium supply. The medium was supplied via a flow meter and the flow rate was 13 L/h, allowing approximately five volume replacements through each aquarium each day. In the mixing flask the dosed stock volume and the medium were mixed under continuous stirring at a ratio of 1 : 10 000. The whole system was checked daily.
During the depuration phase the medium was supplied directly via the same flow meters at a flow rate of 13 L/h. The system was checked daily.

SPECIFIC TEST CONDITIONS
- Test vessels: 64 litres (40x40x40 cm) consisting of stainless steel and covered by a removable Perspex plate.
- Test medium: adjusted ISO medium
- Total number of fish: 150
- Number of fish per test group: 42 for the control, 54 for each test concentration
- Max fish loading: 0.33g fish/litre/day
- Illumination: 16h light
- Aeration: after 6d of exposure, maintained for 1 day
- Feeding: pelleted fish food (2% of body weight per day)
- Euthanasia: with 1.2% ethylene glycol in monophenylether in water

During the bioconcentration test, samples were taken from the test medium and from the fish for several purposes and at several time points:
-Quantitative and or qualitative analysis of water: 24 samples
- Quantitative analysis of fish: 100 fish
- Fish lipid extraction: 40 fish
Nominal and measured concentrations:
TBzTD: 0.5 and 5 μg/L, these test concentrations were chosen taking the water solubility limit of TBzTD and the limit of detection of the analytical method into account.
Control:: thest medium without test substance including acetone in the same amount as used in the treated solutions.
Reference substance (positive control):
no
Lipid content:
9 %
Time point:
start of exposure
Remarks on result:
other: unexposed fish
Lipid content:
8 %
Time point:
end of exposure
Remarks on result:
other: for controls and lowest concentration
Lipid content:
7 %
Time point:
end of exposure
Remarks on result:
other: highest concentration
Type:
BCF
Value:
ca. 118
Time of plateau:
7 d
Calculation basis:
steady state
Remarks on result:
other: individual BCFs varied within +/- 20% window of the mean BCF
Remarks:
Conc.in environment / dose:0.5 μg/L
Type:
BCF
Value:
ca. 27
Time of plateau:
7 d
Calculation basis:
steady state
Remarks on result:
other: individual BCFs varied within +/- 20% window of the mean BCF
Remarks:
Conc.in environment / dose:5 μg/L
Type:
BCF
Value:
131
Calculation basis:
kinetic
Remarks on result:
other: Conc.in environment / dose:0.5 μg/L
Type:
BCF
Value:
29
Calculation basis:
kinetic
Remarks on result:
other: Conc.in environment / dose:5 μg/L
Elimination:
yes
Parameter:
other: DT50 (lowest conc)
Depuration time (DT):
3.3 d
Elimination:
yes
Parameter:
other: DT50 (highest conc)
Depuration time (DT):
3 d
Details on kinetic parameters:
The kinetic parameters were estimated with Modelmaker

- Uptake rate constant (k1, Ku): for the 0.5 and 5 microg/L have uptake rate constants of 27 and 6.7 d-1
- Depuration (loss) rate constant (k2: Kd): for the 0.5 and 5 microg/L have depuration rates of 0.21 and 0.23 d-1
- Indication of bi- or multiphasic kinetics: no
- Computation / data analysis: Modelmaker
Metabolites:
Medium (based on the information from the analytical report),
- organic phase: two relevant degradation products were detected (> 10% of applied) with retention times 19.1 and 25.4 minutes.
- aqueous residues: these were not analysed on HPLC because the conc in these samples were below the detection limit of TBzTD. However, based on the results of the validation of the concentration method, it can be concluded that the aqueous residues do not containt TBzTD, but metabolites thereof. Because these metabolites were not partitioned to the organic phase, they can be characterized as polar metabolites.

Results on quantitative and qualitative analysis of the test medium

QUANTITATIVE

- although the test was performed with the radioactive version of the substance, all conc were expressed as TBzTD

- mean conc were 0.41 +/- 0.025 microg/L and 4.7 +/- 0.38 microg/L at respectively the low and high test concentration

- the measured conc varied within the 20% window of the mean conc

QUALITATIVE

- measurements in stock solutions indicated 99 -100% recovery

- no TBzTD was detected in the test medium samples taken from the lowest target test concentration. Based on HPLC analysis the mean conc of parental TBzTD were below the limit of detection (0.2 microg/L) at the lowest target conc of 0.5 microg/L and 2.9 +/- 0.86 microg/L at the 5 microg/L respectively. Two relevant degradation products (> 10% of the applied total radioactivity) were detected.

Table: measured conc of TBzTD in fish

 Time (days) Time (days)   Target conc 0.5 µg/L    Target conc 5 µg/L  
 Uptake phase  Conc (µg/g fish)  BCF Conc (µg/g fish)   BCF
   1  0.017  42  0.044  9
   3  0.035  96  0.090  22
   7  0.056  133  0.13  30
   14  0.048  111  0.12  24
   21  0.048  114  0.13  28
   28  0.049  114  0.12  24
 Depuration phase  30  0.026    0.064  
   35  0.013    0.028  
   42  0.0064    0.018  
   56  0.0031    0.0070  

Table: Effect parameters

 Target concentration (μg/L) BCFss   Kd (d-1)  Ku (d-1)  BCFk  DT50 (d)
 0.5  118  0.21  27  131  3.3
 5  27  0.23  6.7  29  3.0
Validity criteria fulfilled:
yes
Conclusions:
The bioconcentration study investigated the possible bioconcentration of TBzTD in carp exposed to two different concentrations for a period of 28 days in a flow through system followed by a depuration phase of 28 days. The BCFss values of TBzTD at target concentrations of 0.5 and 5 μg/I were 118 ± 10 and 27 ± 3, respectively. The BCFk values of TBzTD at target concentrations of 0.5 and 5 μg/I were 131 and 29, respectively.
For both target concentrations the BCFk was similar to the BCFss indicating a relatively rapid depuration. The DT50 values for depuration of TBzTD at target concentrations of 0.5 and 5 μg/I were 3.3 and 3.0 days, respectively. Based on these results TBzTD accumulation in fish is low and appears to be concentration related.
Executive summary:

This report describes a flow-through bioconcentration test in carp with [14C]-TBzTD. For this bioconcentration test [14C]-TBzTD was used. Analysis of water and fish samples was based on the radiochemical substance. Stock solutions were prepared in acetone and dosed via a computer-controlled system consisting of micro-dispensers into a mixing flask separately from the medium supply. Medium was supplied via a flow meter at a flow rate of 13 L/h. In the mixing flask the dosed stock volume and the dilution water were mixed under continuous stirring at a ratio of 1 : 10,000. The final target concentrations were 0.5 and 5 μg/1. At the start of the bioconcentration test, 54 fish per concentration were exposed to the target concentrations of 0.5 and 5 μg TBzTD per litre and 42 fish were exposed to a control. The uptake phase lasted for 28 days, during which samples were taken from the test medium for both quantitative and qualitative analysis and from the fish for quantitative analysis. During the subsequent depuration phase, which lasted for 30 days only fish were sampled (for quantitative analysis). The mean concentrations of TBzTD (i.e. total radioactivity) in the test medium were 0.41 ± 0.025 μg/L and 4.7 ± 0.38 μg/L at target concentrations of 0.5 and 5 μg/L, respectively. The measured concentrations varied within the ± 20% window of the mean concentration at both target concentrations. No TBzTD was detected in the test medium samples taken from the 0.5 μg/L target concentration. Based on HPLC analysis the mean concentrations of parental TBzTD were below the limit of detection (i.e. 0.2 μg/L) at the target concentration of 0.5 μg/L and 2.9 ± 0.86 μg/L at the target concentration of 5 μg/L respectively. Two relevant degradation products (>10% of applied total radioactivity) were detected. The steady state concentration in fish was reached after 7 days of exposure for both target concentrations. During steady state the mean concentrations of TBzTD in fish at target concentrations of 0.5 and 5 IJg/1 were 0.050 ± 0.0041 μg/g and 0.13 ± 0.0049 μg/g, respectively. The measured concentrations varied within the ± 20% window of the mean concentration in fish at both target concentrations. After 28 days of depuration TBzTD concentrations in the fish were reduced to 6% of the concentrations at the start of the depuration phase. The BCFss values of TBzTD at target concentrations of 0.5 and 5 μg/L were 118 ± 10 and 27 ± 3, respectively. The BCFk values of TBzTD at target concentrations of 0.5 and 5 μg/L were 131 and 29, respectively. For both target concentrations the BCFk was similar to the BCFss indicating a relatively rapid depuration. The DTso values for depuration of TBzTD at target concentrations of 0.5 and 5 μg/L were 3.3 and 3.0 days, respectively. Based on these results TBzTD accumulation in fish is low and appears to be concentration related.

Description of key information

The bioconcentration study investigated the possible bioconcentration of TBzTD in carp exposed to two different concentrations for a period of 28 days in a flow through system followed by a depuration phase of 28 days. The BCFss values of TBzTD at target concentrations of 0.5 and 5 μg/L were 118 ± 10 and 27 ± 3, respectively. The BCF values of TBzTD at target concentrations of 0.5 and 5 μg/L were 131 and 29, respectively.
For both target concentrations the BCFk was similar to the BCFss indicating a relatively rapid depuration. The DT50 values for depuration of TBzTD at target concentrations of 0.5 and 5 μg/L were 3.3 and 3.0 days, respectively. Based on these results TBzTD accumulation in fish is low and appears to be concentration related.

Key value for chemical safety assessment

BCF (aquatic species):
131

Additional information

This report describes a flow-through bioconcentration test in carp with [14C]-TBzTD. For this bioconcentration test [14C]-TBzTD was used. Analysis of water and fish samples was based on the radiochemical substance. Stock solutions were prepared in acetone and dosed via a computer-controlled system consisting of micro-dispensers into a mixing flask separately from the medium supply. Medium was supplied via a flow meter at a flow rate of 13 L/h. In the mixing flask the dosed stock volume and the dilution water were mixed under continuous stirring at a ratio of 1 : 10,000. The final target concentrations were 0.5 and 5 μg/1. At the start of the bioconcentration test, 54 fish per concentration were exposed to the target concentrations of 0.5 and 5 μg TBzTD per litre and 42 fish were exposed to a control. The uptake phase lasted for 28 days, during which samples were taken from the test medium for both quantitative and qualitative analysis and from the fish for quantitative analysis. During the subsequent depuration phase, which lasted for 30 days only fish were sampled (for quantitative analysis). The mean concentrations of TBzTD (i.e. total radioactivity) in the test medium were 0.41 ± 0.025 μg/L and 4.7 ± 0.38 μg/L at target concentrations of 0.5 and 5 μg/L, respectively. The measured concentrations varied within the ± 20% window of the mean concentration at both target concentrations. No TBzTD was detected in the test medium samples taken from the 0.5 μg/L target concentration. Based on HPLC analysis the mean concentrations of parental TBzTD were below the limit of detection (i.e. 0.2 μg/L) at the target concentration of 0.5 μg/L and 2.9 ± 0.86 μg/L at the target concentration of 5 μg/L respectively. Two relevant degradation products (>10% of applied total radioactivity) were detected. The steady state concentration in fish was reached after 7 days of exposure for both target concentrations. During steady state the mean concentrations of TBzTD in fish at target concentrations of 0.5 and 5 IJg/1 were 0.050 ± 0.0041 μg/g and 0.13 ± 0.0049 μg/g, respectively. The measured concentrations varied within the ± 20% window of the mean concentration in fish at both target concentrations. After 28 days of depuration TBzTD concentrations in the fish were reduced to 6% of the concentrations at the start of the depuration phase. The BCFss values of TBzTD at target concentrations of 0.5 and 5 μg/L were 118 ± 10 and 27 ± 3, respectively. The BCFkvalues of TBzTD at target concentrations of 0.5 and 5 μg/L were 131 and 29, respectively. For both target concentrations the BCFkwas similar to the BCFss indicating a relatively rapid depuration. The DTso values for depuration of TBzTD at target concentrations of 0.5 and 5 μg/L were 3.3 and 3.0 days, respectively. Based on these results TBzTD accumulation in fish is low and appears to be concentration related.

The highest value has been used for the chemical safety assessment.

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