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Bioaccumulation: aquatic / sediment

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Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 April 2009 to 15 May 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
other: read-across target
Qualifier:
according to guideline
Guideline:
OECD Guideline 305 B (Bioaccumulation: Semi-static Fish Test)
Deviations:
yes
Remarks:
(please refer to "Principles of method if other than guideline" for further information)
Principles of method if other than guideline:
1. The measured concentrations in water at the target concentration of 0.25 µg/I varied within the ± 35% of the mean DOT concentration. The results obtained at this target concentration should be considered as indicative.
2. The volume of the test vessels was 64 litres.
3. The water flow rate was 10 I/h and allowed for approximately four volume replacements through each vessel each day.
4. Volume of water samples was 25 ml. These were taken directly from the test vessels.
It was considered that these deviations do not affect the reliability of the study result.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
The registered substance is predicted to hydrolyse into MOT within minutes to hours of solution in water.

PHYSICO-CHEMICAL PROPERTIES
- Water solubility: Unsolvable (<0.1mg/l)


OTHER PROPERTIES (if relevant for this endpoint)
none reported.

Radiolabelling:
no
Details on sampling:
During the bioconcentration test, water and fish samples were taken for several purposes and at several timepoints:
Analysis of water (DOT and MOT): 14 samples (see Table 1)
Analysis of fish (DOT and MOT): 60 fish (see Table 1)
Analysis of fish (total tin): 10 fish (see Table 1)
Fish lipid extraction: 40 fish (see Table 2)

See 'Any other information on materials and methods including tables' section for Tables.
Vehicle:
yes
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
The standard test procedures required generation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogenous mixtures or dispersions. The testing of concentrations that ditrub the test system should be prevented (e.g. film of the test substance on the water surface).
Stock solutions were prepared in acetone (>99.8%, VWR, Leuven, Belgium). The concnetrations in these stocks were a factor 10,000 higher than the final concentrations in water: 2.5 and 25 mg/l.
During the uptake phase, the stock colutions 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 10 l/h, allowing approximately 4 volume replacements (64 litres) through each aquarium each day. 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 whole system was checked daily.

- Controls:
negative cntrol.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
acetone

- Concentration of vehicle in test medium (stock solution and final test solution(s) at different concentrations and in control(s)):
not reported.

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
none reported.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name:
Rainbow trout

- Strain:
not applicable.

- Source:
Fraunhofer Institit für Molekularbiologie und Angewandte Oekologie (IME), Schmallenberg, Germany.

- Age at study initiation (mean and range, SD):
not reported.

- Length at study initiation (lenght definition, mean, range and SD):
4.5±0.3 cm, based on measurement of a total of 20 fish from the batch used for this test.

- Weight at study initiation (mean and range, SD):
1.74±0.33g, based on weighing of a total of 20 fish from the batch used for this test.

- Weight at termination (mean and range, SD):
not reported.

- Method of breeding:
not applicable

- Health status:
Healthy at study initiation.

- Description of housing/holding area:
Test vessels: 64 litres (40x40x40cm) consisting of stainless steel and covered by a removable Perspex plate.
Test medium: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7±0.3.

- Feeding during test
Pelleted fish food (lipid and protein content appended to the report). Ration was 2% of bodyweight per day. Recalculation of the feeding rate was performed after each sampling point.


ACCLIMATION
- Acclimation period:
at least 12 days after delivery.

- Acclimation conditions (same as test or not):
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

- Type and amount of food:
Daily with pelleted fish food (Cyprico Crumble Excellent (300-500 um), Coppens International bv, Helmond, The Netherlands).

- Feeding frequency:
- Health during acclimation (any mortality observed):
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
30 d
Hardness:
180 mg CaCO3
Test temperature:
13-17ºC, constant within 2 ºC
pH:
7.7 ± 0.3
Dissolved oxygen:
8.5-9.8 mg/L
TOC:
24-36mg C/L
Salinity:
not applicable.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type:
Closed

- Material, size, headspace, fill volume:
64 litres (40x40x40 cm) consisting of stainless steel and covered by a removable Perspex plate.

- Aeration:
The test material was aerated continuously.

- Type of flow-through (e.g. peristaltic or proportional diluter):
Proportional diluter

- Renewal rate of test solution (frequency/flow rate):
10 l/h

- No. of organisms per vessel:
58

- No. of vessels per concentration (replicates):
2

- No. of vessels per control / vehicle control (replicates):
control: 1

- Biomass loading rate:
Maximum loading: 0.42g fish/litre/day

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The netherlands).

- Particulate matter:
not reported.

- Metals:
as for acclimation.

- Pesticides:
not reported.

- Chlorine:
not reported.

- Alkalinity:
not reported.

- Ca/mg ratio:
CaCO3: 180mg/l

- Conductance:
not reported.

- Holding medium different from test medium:
no



OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:
- Light intensity:


RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations:
- Results used to determine the conditions for the definitive study:
Nominal and measured concentrations:
DOT: 0.25 & 2.5 µg/L
Reference substance (positive control):
no
Details on estimation of bioconcentration:
Evaluation of the results started with obtaining the uptake curve by plotting the measured concentration in/on fish against time on arithmetic scales. Since all measured concentrations, based on both DOT and MOT, were below the limit of quantification a 'plateau' could not be seen. A mean BCF, based on DOT was calculated as follows:

BCFmean = Cf,mean / Cw,mean

where:
Cf,mean=mean concentration in fish
Cw,mean=mean concentration in water
Lipid content:
4 %
Time point:
start of exposure
Remarks on result:
other: This percentage was in agreement with other lipid extractions during bioconcentration studies.
Lipid content:
6 %
Time point:
end of exposure
Remarks on result:
other: Control group
Lipid content:
6 %
Time point:
end of exposure
Remarks on result:
other: 0.25 µg/l concentration
Lipid content:
6 %
Time point:
end of exposure
Remarks on result:
other: 2.5 µg/l copncentration
Conc. / dose:
0.25 µg/L
Type:
BCF
Value:
1 294
Basis:
whole body w.w.
Time of plateau:
1 d
Calculation basis:
steady state
Remarks on result:
other: Results for DOT based upon LOQ of analytical method.
Conc. / dose:
2.5 µg/L
Type:
BCF
Value:
99
Basis:
whole body w.w.
Time of plateau:
1 d
Calculation basis:
steady state
Remarks on result:
other: Results for DOT based upon LOQ of analytical method.
Conc. / dose:
0.25 µg/L
Type:
BCF
Value:
1 294
Basis:
whole body w.w.
Time of plateau:
1 d
Calculation basis:
steady state
Remarks on result:
other: Results for MOT based upon LOQ of analytical method.
Conc. / dose:
2.5 µg/L
Type:
BCF
Value:
99
Basis:
whole body w.w.
Time of plateau:
1 d
Calculation basis:
steady state
Remarks on result:
other: Results for MOT based upon LOQ of analytical method.
Details on kinetic parameters:
not applicable.
Metabolites:
not reported.
Results with reference substance (positive control):
not applicable.
Details on results:
WATER CONCENTRATIONS
The results of the analysis of water samples taken during the bioconcentration test are described in Table 3 (DOT) and Table 4 (MOT) in the remarks on results section.

Analyses were based on both DOT and MOT. Results were based on DOT, due to the fact that MOT-concentrations were below the Limit Of Quantification (LOQ) for the target concentration of 0.25 µg/I. Also for the target concentration of 2.5 µg/l MOT-concentrations were below LOQ for three out of seven sampling points. Note that procedural recoveries for DOT at the start of the test (day 0) were very low (0.25 µg/l: 2.1 and 1.8%; 2.5 µg/I: 6.3 and 5.9%). Therefore, the actual test concentrations were corrected with the means of these low recoveries. Furthermore, the outlying high value obtained for the lowest treatment group on day 7 was not expected to represent the actual situation and was therefore excluded from the calculation of mean exposure concentrations.

The mean concentrations of DOT were 0.19 ± 0.048 µg/I and 2.6 ± 0.35 µg/I at target concentrations of 0.25 and 2.5 µg/I, respectively. The measured concentrations varied within the ± 20% window of the mean concentration at the target concentration of 2.5 µg/I but not at the target concentration of 0.25 µg/I. See also sections 6.15.2 and 6.16.1.

FISH CONCENTRATIONS AND BIOCONCENTRATION FACTORS
Analyses of the concentrations of both DOT and MOT in fish tissues during the uptake phase showed measured concentrations that were always below the Limit of Quantification, i.e. <0.25 mg/kg.

Following a worst-case-scenario, the LOQ of 0.25 mg/kg was used as the concentration in fish in order to calculate the BCF at each sampling point. Subsequently, the mean BCF's of DOT at target concentrations of 0.25 and 2.5 µg/I were calculated to be 1294 and 99, respectively. Table 4 summarizes the measured concentration of DOT in the fish and the bioconcentration factor (BCF) on each sampling point.

Based on the fact that the LOQ for measurement of DOT in fish was 0.25 mg/kg a bioaccumulation factor of 100 (for the 2.5 µg/I test group) was the lowest that could be measured. Measured concentrations in fish in both test groups were always below 0.25 mg/kg, resulting in BCF's close to 1300 and 100 for respectively the 0.25 and 2.5 µg/I test group.

However, concentrations in fish exposed to the lower target concentration will most probably have been lower than the concentrations in fish exposed to the higher target concentration. The high BCF value of 1294 was in fact due to the analytical methodology limit, which could not be further improved. With a more sensitive method, i.e. a method that would be able to measure concentrations <<< 0.25 mg/kg fish and with exact figures, BCF values of less than 100 would have been found.

Since the concentrations of both DOT and MOT in fish were below the LOQ during the uptake phase, a depuration phase was not considered to be significant. Therefore, the bioconcentration test was terminated after 30 days of exposure, at which moment fish samples were taken for analysis on total tin and lipid.

TOTAL TIN IN FISH
In order to confirm that BCF values were below the cut off criterion of 2000, further analytical determinations were done based on analysis of total tin (therefore encompassing all tin species) in fish.

The results of the analyses of fish samples for total tin at the end of the uptake phase are described in Table 10 of Appendix IV.

After 30 days of exposure the concentrations of total tin in fish at target concentrations of 0.25 and 2.5 µg/l were 0.027 and 0.054 mg/kg, respectively.

Total tin concentrations in water samples of day 30 were not analysed. However, based on the known percentages of tin in both DOT and MOT (29.5 and 37.3%, respectively) total tin concentrations in water could be calculated. Mean measured concentrations of both DOT and MOT in water were corrected for the respective percentages of tin and then added. Note that for MOT concentrations in water that were below LOQ, the LOQ was used. Mean concentrations of total tin in water were thus calculated to be 0.15 and 0.92 µg/I at target concentrations of 0.25 and 2.5 µg/I, respectively.

The BCF's of total tin at target concentrations of 0.25 and 2.5 µg/I were 178 and 58, respectively.
Reported statistics:
Not reported.

Table 3. Measured concentration of dioctyltin species in water samples.

Time of sampling (day) Date of sampling (dd-mm-yy) Date of analysis (dd-mm-yy) Concentration
Target (µg/l) Analysed (µg/l) Relative to target (%)
-1 14-04-09 14-04-09 , n.d. n.a.
0.25 0.175 70
2.5 2.32 93
0 15-04-09 15-04-09 0 n.d. n.a.
0.25 0.00273 1.1
2.5 0.183 7.3
1 16-04-09 16-04-09 0 n.d. n.a.
0.25 0.18 72
2.5 2.91 116
3 18-04-09 18-04-09 0 n.d. n.a.
0.25 0.157 63
2.5 2.1 84
7 22-04-09 22-04-09 0 n.d. n.a.
0.25 0.623 249
2.5 2.96 118
14 29-04-09 29-04-09 0 n.d. n.a.
0.25 0.169 68
2.5 2.49 100
21 06-05-09 06-05-09 0 n.d. n.a.
0.25 0.239 96
2.5 2.68 107
28 13-05-09 13-05-09 0 n.d. n.a.
0.25 0.259 104
2.5 2.29 92

Table 4. Concentration of monoctyltin species in water samples.

Time of sampling (day) Date of sampling (dd-mm-yy) Date of analysis (dd-mm-yy) Concentration
Target (µg/l) Analysed (µg/l)
-1 14-04-09 14-04-09 0 n.a.
0.25 <LOQ
2.5 <LOQ
0 15-04-09 15-04-09 0 n.a.
0.25 n.a.
2.5 n.a.
1 16-04-09 16-04-09 0 <LOQ
0.25 <LOQ
2.5 0.669
3 18-04-09 18-04-09 0 <LOQ
0.25 <LOQ
2.5 <LOQ
7 22-04-09 22-04-09 0 n.a.
0.25 <LOQ
2.5 0.32
14 29-04-09 29-04-09 0 <LOQ
0.25 <LOQ
2.5 0.577
21 06-05-09 06-05-09 0 n.a.
0.25 <LOQ
2.5 0.396
28 13-05-09 13-05-09 0 n.a.
0.25 <LOQ
2.5 <LOQ

n.d. = not detected

n.a. = not applicable

LOQ = The limit of quantification was 0.25µg/l

Validity criteria fulfilled:
yes
Conclusions:
The bioconcentration study investigated the possible bioconcentration of Dioctyltin bis(2-ethylhexyl thioglycolate) in rainbow trout exposed to two different concentrations for a total period of 30 days in a flow through system.

The mean BCF-values based on dioctyltin at target concentrations of 0.25 and 2.5 µg/L were 1294 and 99, respectively. The mean BCF-values based on total tin at target concentrations of 0.25 and 2.5 µg/L were 178 and 58, respectively.

The high value found at the target concentration of 0.25 µg/l was in fact due to the analytical methodology limit, which could not be further improved.

Based on the result found for the highest target concentration of 2.5 µg/I, the BCF is at least less than 100 and therefore the substance does not bioconcentrate.
Executive summary:

The study procedure described in this report was based on the OECD guideline No. 305, 1996. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.13, 2008.

 

The batch of Dioctyltin bis(2-ethylhexyl thioglycolate) used was a colourless liquid with a purity of 97.5 area %. Water solubility was indicated to be below 0.1 mg/L. Stock solutions for the bioconcentration test 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 10 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 0.25 and 2.5 µg/L (based on water solubility and detection limits of the analytical methods used).

 

At the start of the Bioconcentration test 58 fish per concentration were exposed to the target concentrations of 0.25 and 2.5 µg Dioctyltin bis(2-ethylhexyl thioglycolate) per litre and 42 fish were exposed to a control. The uptake phase lasted for 30 days, during which samples were taken from the test medium and from the fish.

 

Analyses were based on both dioctyltin (DOT) and monooctyltin (MOT). Results were based on DOT, due to the fact that MOT-concentrations were below the Limit Of Quantification (LOQ) for the target concentration of 0.25 µg/L. The mean concentrations of DOT were 0.19 ± 0.048 µg/L and 2.6 ± 0.35 µg/L at target concentrations of 0.25 and 2.5 µg/L, respectively. The measured concentrations varied within the ± 20% window of the mean concentration at the target concentration of 2.5 µg/L but not at the target concentration of 0.25 µg/L.

 

Analyses of the concentrations of both DOT and MOT in fish tissues during the 30-day uptakephase showed measured concentrations that were always below the Limit of Quantification, i.e.< 0.25 mg/kg.

Since the concentrations of both DOT and MOT in fish were constantly below the LOQ duringthe uptake phase, a depuration phase was not considered to be significant. Therefore, the bioconcentration test was terminated after 30 days of exposure. In addition to the analytical work already performed on tin species, fish samples taken after 30 days of exposure were analysed on total tin.

 

The mean BCF-values based on dioctyltin at target concentrations of 0.25 and 2.5 µg/L were 1294 and 99, respectively. The mean BCF-values based on total tin at target concentrations of 0.25 and 2.5 µg/L were 178 and 58, respectively.

 

The high value found at the target concentration of 0.25 µg/L was in fact due to the analytical methodology limit, which could not be further improved.

 

Based on the result found for the highest target concentration of 2.5 µg/L, the BCF is at leastless than 100 and therefore the substance does not bioconcentrate.

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
Read Across to Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) (EC Number 248-227-6 and CAS No 27107-89-7) based on structural similarity and hydrolytical behaviour, see attached justification.
Reason / purpose for cross-reference:
read-across source
Lipid content:
4 %
Time point:
start of exposure
Remarks on result:
other: This percentage was in agreement with other lipid extractions during bioconcentration studies.
Lipid content:
6 %
Time point:
end of exposure
Remarks on result:
other: control group
Lipid content:
6 %
Time point:
end of exposure
Remarks on result:
other: 0.25 µg/L concentration
Lipid content:
6 %
Time point:
end of exposure
Remarks on result:
other: 2.5 µg/L concentration
Conc. / dose:
0.25 µg/L
Type:
BCF
Value:
1 294
Basis:
whole body w.w.
Time of plateau:
1 d
Calculation basis:
steady state
Remarks on result:
other: Results for DOT based upon LOQ of analytical method.
Conc. / dose:
2.5 µg/L
Type:
BCF
Value:
99
Basis:
whole body w.w.
Time of plateau:
1 d
Calculation basis:
steady state
Remarks on result:
other: Results for DOT based upon LOQ of analytical method.
Conc. / dose:
0.25 µg/L
Type:
BCF
Value:
1 294
Basis:
whole body w.w.
Time of plateau:
1 d
Calculation basis:
steady state
Remarks on result:
other: Results for MOT based upon LOQ of analytical method.
Conc. / dose:
2.5 µg/L
Type:
BCF
Value:
99
Basis:
whole body w.w.
Time of plateau:
1 d
Calculation basis:
steady state
Remarks on result:
other: Results for MOT based upon LOQ of analytical method.

Description of key information

Read across to DOTE: Dioctyltin bis(2-ethylhexyl thioglycolate) (CAS 15571-58-1)

Based on the result found for the highest target concentration of 2.5 µg/L, the BCF is at least less than 100 and therefore the substance does not bioconcentrate.

Key value for chemical safety assessment

BCF (aquatic species):
99 dimensionless

Additional information

Read across to DOTE: Dioctyltin bis(2-ethylhexyl thioglycolate)

The study procedure described in this report was based on the OECD guideline No. 305, 1996. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.13, 2008.

 

The batch of Dioctyltin bis(2-ethylhexyl thioglycolate) used was a colourless liquid with a purity of 97.5 area %. Water solubility was indicated to be below 0.1 mg/L. Stock solutions for the bioconcentration test 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 10 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 0.25 and 2.5 µg/L (based on water solubility and detection limits of the analytical methods used).

 

At the start of the Bioconcentration test 58 fish per concentration were exposed to the target concentrations of 0.25 and 2.5 µg Dioctyltin bis(2-ethylhexyl thioglycolate) per litre and 42 fish were exposed to a control. The uptake phase lasted for 30 days, during which samples were taken from the test medium and from the fish.

 

Analyses were based on both dioctyltin (DOT) and monooctyltin (MOT). Results were based on DOT, due to the fact that MOT-concentrations were below the Limit Of Quantification (LOQ) for the target concentration of 0.25 µg/L. The mean concentrations of DOT were 0.19 ± 0.048 µg/L and 2.6 ± 0.35 µg/L at target concentrations of 0.25 and 2.5 µg/L, respectively. The measured concentrations varied within the ± 20% window of the mean concentration at the target concentration of 2.5 µg/L but not at the target concentration of 0.25 µg/L.

 

Analyses of the concentrations of both DOT and MOT in fish tissues during the 30-day uptake phase showed measured concentrations that were always below the Limit of Quantification, i.e.< 0.25 mg/kg.

Since the concentrations of both DOT and MOT in fish were constantly below the LOQ duringthe uptake phase, a depuration phase was not considered to be significant. Therefore, the bioconcentration test was terminated after 30 days of exposure. In addition to the analytical work already performed on tin species, fish samples taken after 30 days of exposure were analysed on total tin.

 

The mean BCF-values based on dioctyltin at target concentrations of 0.25 and 2.5 µg/L were 1294 and 99, respectively. The mean BCF-values based on total tin at target concentrations of 0.25 and 2.5 µg/L were 178 and 58, respectively.

 

The high value found at the target concentration of 0.25 µg/L was in fact due to the analytical methodology limit, which could not be further improved.

 

Based on the result found for the highest target concentration of 2.5 µg/L, the BCF is at leastless than 100 and therefore the substance does not bioconcentrate.