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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:
2012-01-30 to 2012-05-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch No.: 20111009#1
Purity: 99.29%
Radiolabelling:
no
Details on sampling:
- Sampling intervals/frequency for test organisms: Days 5, 17, 31, 38, 47, 61, 69 of exposure phase; days 6, 19, 22, 25 of depuration phase.
- Additionally 5 fish were sampled for lipd content on day 0 of exposure phase and day 25 of depuration phase.
- Sampling intervals/frequency for test medium samples: The concentration and stability of the test item in the test solutions were verified by chemical analysis on Days -1, 0, 5, 17, 31, 38, 45, 47, 61 and 69 of the exposure phase. Water samples were also taken on Days 6, 19, 22 and 25 of the depuration phase.
- Sample storage conditions before analysis: Duplicate water samples were taken on all sampling occasions and stored at approximately -20 °C for further analysis if necessary.
- Details on sampling and analysis of test organisms and test media samples:
- Sample Preparation - Water Analysis
A volume (500 mL) of test sample was extracted with dichloromethane (2 x 50 mL). The extracts were filtered through anhydrous sodium sulphate and washed through with a further volume (10 ml) of dichloromethane. The combined extracts were evaporated to dryness and the residue re-dissolved in methanol (3 mL).
- Sample Preparation - Fish Analysis
A whole fish was homogenised to a paste and a portion (approximately 5 g) was transferred to a beaker with the addition of n-hexane (150 mL). This mixture was macerated for 1 minute at high speed. After allowing the solids to settle the n-hexane extract was decanted into a separating funnel. The fish solids and the macerator were washed with a further volume (50 mL) of n-hexane and added to the initial extract. The combined n-hexane extracts were then washed with 1 M sodium hydroxide (2 x 100 mL) and water* (2 x 250 mL), filtered through anhydrous sodium sulphate and washed through with a further volume (10 mL) of n-hexane. The n-hexane extract was then evaporated to dryness and the residue redissolved in methanol (20 mL).
- Sample Preparation - Solvent Stock Solution Analysis
A volume of solvent stock solution (3 mL - 1.5 mg/L, 2 mL - 15 mg/L) was pipetted into a volumetric flask (50 Ml - 1.5 mg/L, 250 mL - 15 mg/L) and evaporated to dryness with nitrogen. The residue was redissolved in methanol and diluted to volume.
- Lipid Determination
A whole fish was homogenised for approximately two minutes, 100 mL of chloroform and 100 mL of methanol was added and the resultant fish mixture was further homogenised for approximately two minutes. The fish mixture was filtered, the filter paper and residue were washed with chloroform: methanol (80:40 v/v). These extracts were transferred to a separating funnel containing 400 mL of sodium chloride 10% (w/v) in water and shaken to induce two immiscible layers. The layers were allowed to separate and the lower chloroform layer filtered through anhydrous sodium sulphate into a tared flask, evaporated to dryness and dried in an oven for a minimum of two hours at approximately 105 °C. The flask was allowed to cool in a desiccator prior to weighing the Iipid residue.
- Standards
Standard solutions of test item were prepared in methanol at nominal concentrations of 0.0025, 0.0050, 0.010, 0.025, 0.050, 0.075, 0.10 and 0.15 mg/L.
Vehicle:
no
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Source: Brow Well Fisheries Ltd, Hebden, Skipton, North Yorkshire, United Kingdom
- Length at study initiation: Mean 8.1 cm (sd = 0.4 cm)
- Weight at study initiation: Mean 8.26 g(sd = 0.62 g)
- Description of housing/holding area: Glass fibre tank with a single pass water renewal system.
- Feeding during test
- Food type: Commercial trout pellets (lipid content 15 %; protein content 54 %)
- Amount: 2 % body weight/day

ACCLIMATION
- Acclimation period: 2012-01-27 to 2012-02-10
- Acclimation conditions: Same as test
- Type and amount of food: 2 % body weight/day of commercial trout pellets (lipid content 15 %; protein content 54 %)
- Health during acclimation: < 1 % mortality in 7 d prior to start of test.
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
69 d
Total depuration duration:
25 d
Hardness:
Approx. 140 mg/L as CaCO3
Test temperature:
Approx. 14 °C.
pH:
Mean 7.497, (range 7.020 - 7.870; within prescribed range 6.5 - 9.5)
Dissolved oxygen:
≥ 10.6 mg/L
TOC:
Mean 0.92 mg/L (range <0.130 to 2.020; no abnormal changes).
Salinity:
Chloride mean 21.052 mg/L (range 9.660 to 50.800). Fluoride mean 0.331 mg/L (range 0.270 to 0.414).
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 L glass exposure vessels, covered to reduce evaporation.
- Type of flow-through (e.g. peristaltic or proportional diluter): Peristaltic pump
- Renewal rate of test solution (frequency/flow rate): Diluent was supplied at a rate of 800 ml/minute
- No. of organisms per vessel: 52
- No. of vessels per concentration (replicates): 2


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water, dechlorinated by passage through an activated carbon filter and softened


OTHER TEST CONDITIONS
- Photoperiod: 16 hrs light / 8 hrs dark


VEHICLE
- Acetone
Nominal and measured concentrations:
Nominal 0.000050 mg/L: on Days 5, 17, 31 , 38, 45, 47, 61 and 69 of the exposure phase, measured < LOQ (0.0000034 mg/L) to 108% of nominal
Nominal 0.00050 mg/L: Days 5, 17, 31, 38, 45, 47, 61 and 69 of the exposure phase measured 38 to 81% of nominal
Analysis of the test solutions on Day -1 showed measured concentrations of 196% and 140% of nominal for the 0.000050 and 0.00050 mg/L test concentrations respectively. However, analysis of the test solutions on Day 0 showed measured concentrations of 108% and 113%.
Reference substance (positive control):
no
Lipid content:
6.7 %
Time point:
start of exposure
Remarks on result:
other:
Remarks:
The mean percentage lipid content
Lipid content:
8.5 %
Time point:
end of exposure
Remarks on result:
other:
Remarks:
The mean percentage lipid content
Key result
Conc. / dose:
0 mg/L
Type:
BCF
Value:
0 dimensionless
Basis:
other: whole fish
Time of plateau:
69 d
Calculation basis:
steady state
Key result
Conc. / dose:
0.001 mg/L
Type:
BCF
Value:
9 dimensionless
Basis:
other: whole fish
Time of plateau:
69 d
Calculation basis:
steady state
Key result
Conc. / dose:
0 mg/L
Type:
BCF
Basis:
other: whole fish
Calculation basis:
kinetic
Remarks on result:
other: not calculated due to BCF was 0
Key result
Conc. / dose:
0.001 mg/L
Type:
BCF
Value:
11 dimensionless
Basis:
other: whole fish
Calculation basis:
kinetic
Key result
Conc. / dose:
0 mg/L
Type:
BCF
Value:
0 dimensionless
Basis:
other: A function of lipid content
Time of plateau:
69 d
Calculation basis:
steady state
Key result
Conc. / dose:
0.001 mg/L
Type:
BCF
Value:
132 dimensionless
Basis:
other: A function of lipid content
Time of plateau:
69 d
Calculation basis:
steady state
Key result
Conc. / dose:
0 mg/L
Type:
BCF
Basis:
other: A function of lipid content
Calculation basis:
kinetic
Remarks on result:
other: not calculated due to BCF was 0
Key result
Conc. / dose:
0.001 mg/L
Type:
BCF
Value:
161 dimensionless
Basis:
other: A function of lipid content
Calculation basis:
kinetic
Details on kinetic parameters:
- Uptake rate constant (k1):
10.6814 for the 0.00050 mg/l test concentration (in the Whole Fish)
161.1 for the 0.00050 mg/l test concentration (in the Whole Fish as a Function of Lipid Content)
K1 and K2 for 0.000050 mg/I not calculated due to BCF was 0
- Depuration (loss) rate constant (k2):
1.0000 for the 0.00050 mg/l test concentration (in the Whole Fish)
1.0000 for the 0.00050 mg/l test concentration (in the Whole Fish as a Function of Lipid Content)
K1 and K2 for 0.000050 mg/I not calculated due to BCF was 0
- Computation/data analysis:
The rate constants k1 and k2 and hence the bioconcentration factor were estimated by the use of non-linear parametric estimation given the sequential time concentration data and the model using the SAS computer software package and the equation as follows: Cf = Cw × (k1 ÷ k2) × (1 - e^-k2t)
Details on results:
Analysis of Test Water Samples
Analysis of the test solutions on Day -1 showed measured concentrations of 196% and 140% of nominal for the 0.000050 and 0.00050 mg/I test concentrations respectively. However, analysis of the test solutions on Day 0 showed measured concentrations of 108% and 113% of nominal for the 0.000050 and 0.00050 mg/L test concentrations respectively, therefore it was considered that the system was dosing correctly. Analysis of the 0.000050 mg/I test solutions on Days 5, 17, 31 , 38, 45, 47, 61 and 69 of the exposure phase showed measured test concentrations to range from less than the limit of quantitation (LOQ) of the analytical method (assessed as 0.0000034 mg/I) to 108% of nominal. Analysis of the 0.00050 mg/I test solutions on Days 5, 17, 31 , 38, 45, 47, 61 and 69 of the exposure phase showed measured test concentrations to range from 38% to 81 % of nominal. A gradual decline in measured concentration was observed up to Day 38 after which the test concentrations remained at approximately 40% of nominal with the exception of the 0.000050 mg/L test concentrations which showed measured concentrations of less than the LOQ on Days 61 and 69. A review of the data indicated that the correct stock solutions had been prepared and that the test system was dosing correctly. It was therefore considered possible that the test item may have been taken up by the fish but rapidly metabolised and excreted hence resulting in a lower concentration of the parent test item. These low values were considered not to affect the integrity of the study given that the test item was not shown to significantly accumulate in the fish tissues.
Analysis of the solvent control water samples throughout the test showed no test item to be present with the exception of Day 17 when a measured concentration of 0.0000259 mg/L was shown. Analysis of the duplicate sample, stored at approximately -20 °C prior to analysis, showed a measured concentration of less than the LOQ. It was therefore considered that the initial result may have been due to post sampling contamination. No test item was detected in the water samples taken during the depuration phase of the test.
Analysis of Test Fish Samples
Analysis of the test fish on Days 5, 17, 31, 38, 47, 61 and 69 of the exposure phase showed the test item not to significantly accumulate, with mean measured values for whole fish of 0.0040 mg/kg on Day 5 to 0.0020 mg/kg on Day 69 for the 0.00050 mg/L test concentration. In general, concentrations less than the limit of quantitation (LOQ) of the analytical method (assessed as 0.0023 mg/kg) were determined for the 0.000050 mg/L test concentration. On Days 5, 17, 31, 38 and 47 of the exposure phase for the 0.000050 mg/L test concentration measured concentrations were found in a single fish at each sample timing. These concentrations ranged from 0.0103 to 1.33 mg/kg. Given that the remaining fish at each sample timing showed measured concentrations of less than the LOQ, and the concentrations were in excess of any observed throughout the duration of the test, it was considered that these measured concentrations were due to contamination from an unknown source rather than a true reflection of the concentration of the test item in the fish tissues. These results were therefore not included in the BCF calculations. This was considered not to affect the integrity of the test.
At the end of the depuration period 100% of the test item was eliminated from the fish tissues for the 0.00050 mg/L test concentration.
The mean percentage lipid content at the start of the test was 6.7% and 8.5% at the end of the test.

Physico-Chemical Measurements

The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The pH and the dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The temperature was also monitored approximately every hour in the solvent control using a temperature logger.

Total Organic Carbon (TOC) analysis was performed on the solvent control and each test concentration 2 days and 1 day prior to the initiation of the test, on day 0 of the test and once per week thereafter during the exposure and depuration phases.

The water hardness in the solvent control and the highest test concentration was measured at the start of the test and was determined using the methods described in Field and On-Site Test Methods for the Analysis of Waters (British Standards Institution 1993).

 

Evaluation of data

BCF at steady state is calculated as follows, not by the time-weighted average concentration of the substance in water.

The concentration of test item in the fish tissues was plotted graphically to show the rate of uptake, the plateau concentration and the rate of depuration.

When the uptake curve had reached a plateau (steady-state) the BCFss was calculated as follows:

BCFss = Cf at steady - state (mean)/Cw at steady - state (mean)

where:

BCFss =Bioconcentration factor at steady-state

Cf = Mean concentration in fish at steady-state

Cw = Mean concentration in water at steady-state

 

The BCF will be expressed both as a function of the total wet weight of fish, and for high lipophilic materials (log Pow >3), as a function of the lipid content.

The BCF values were calculated directly from the kinetic rate constants k1 and k2 and termed the kinetic bioconcentration factor as follows:

BCFk=K1/K2

where:

BCFk = Kinetic bioconcentration factor

K1 = Uptake rate constant

K2 = Depuration rate constant

 

The rate constants k1 and k2 and hence the bioconcentration factor were estimated by the use of non-linear parametric estimation given the sequential time concentration data and the model using the SAS computer software package and the equation as follows:

Cf = Cw × (k1 ÷ k2) × (1 - e^-k2t)

Validation criteria

For a test to be valid the following conditions apply:

The temperature variation is less than ± 2°C.

The concentration of dissolved oxygen does not fall below 60% Air Saturation Value.

The concentration of the test item in the chambers is maintained within ±20% of the mean of the measured values during the uptake phase. However, if bioconcentration of the test item into the fish tissues is very high, resulting in a significant deviation from 20% of the mean measured concentration then this factor will be taken into consideration.

The mortality or other adverse effects/disease in both control and treated fish is less than 10% at the end of the test; where the test is extended over several weeks or months, deaths or other adverse effects in both sets of fish should be less than 5% per month and not exceed 30% in all.

The contribution to the organic carbon content in the test water from the test fish excreta and from the food residues should be as low as possible. The concentration of organic carbon in the test vessels should not exceed the concentration of organic carbon originating from the test item, and, if used, the solubilising agent by more than 10 mg/L (±20%).

Validity criteria fulfilled:
yes
Remarks:
See under "remarks on results including tables and figures" above
Conclusions:
The test substance was shown not to significantly accumulate in fish tissue and as observed to be eliminated from the fish tissues over a 25 day depuration period. At the end of the depuration period 100% of test substance was eliminated from the fish tissues for the 0.00050 mg/L test concentration. Based on this information the biological half life of the test item is considered to be between 6 to 25 days.
Executive summary:

A study was performed to assess the bioconcentration potential of the test item in rainbow trout (Oncorhynchus mykiss) according to OECD Guideline 305.

Rainbow trout were exposed, in groups of 52, to an aqueous solution of the test item at nominal concentrations of 0.000050 and 0.00050 mg/L for a period of 69 days under dynamic test conditions. Samples of test fish were taken from the solvent control, 0.000050 and 0.00050 mg/L test groups on days 5, 17, 31, 38, 47, 61 and 69 of the uptake phase and the concentration of test item in the fish tissues determined. After 69 days exposure the remaining fish were subjected to a depuration period of 25 days. Samples of test fish were taken from the solvent control, 0.000050 and 0.00050 mg/L test groups on days 6, 19, 22 and 25 of the depuration phase and the concentration of test item determined.

 

The Bioconcentration Factors (BCF) for the test item in the whole fish after 69 days were calculated to be 0 at a concentration of 0.000050 mg/L and 9 at a concentration of 0.00050 mg/L.

The Kinetic Bioconcentration Factor (BCFk) after 69 days for the 0.00050 mg/L test concentration was calculated to be 11. A BCFk was not calculated for the 0.000050 mg/L test concentration given that the BCF was 0.

The uptake and depuration rate constants, k1 and k2, were determined to be 10.6814 and 1.0000 for the 0.00050 mg/L test concentration.

As the test item had a log Kow of greater than 6.5, following the recommendations of the test guidelines, the BCF values were also calculated as a function of the lipid content. These BCF values were higher than those determined in the whole fish thereby suggesting that the accumulation of the test item was into the lipid content of the fish. However, as the values were below 2000, the test item would not be classed as bioaccumulative according to the PBT assessment criteria.

The Bioconcentration Factors (BCF) for the test item as a function of lipid content after 69 days were calculated to be 0 and 132 at concentrations of 0.000050 and 0.00050 mg/L.

The Kinetic Bioconcentration Factor (BCFk) was also calculated for the 0.00050 mg/L test concentration and was shown to be 161 after 69 days. A BCFk was not calculated for the 0.000050 mg/L test concentration given that the BCF was 0.

The uptake and depuration rate constants, k1 and k2, were determined to be 161.1 and 1.0000 for the 0.00050 mg/L test concentration.

Analysis of the test solutions on Day -1 showed measured concentrations of 196% and 140% of nominal for the 0.000050 and 0.00050 mg/L test concentrations respectively. However, analysis of the test solutions on Day 0 showed measured concentrations of 108% and 113% of nominal for the 0.000050 and 0.00050 mg/L test concentrations respectively, therefore it was considered that the system was dosing correctly. Analysis of the 0.000050 mg/L test solutions on Days 5, 17, 31 , 38, 45, 47, 61 and 69 of the exposure phase showed measured test concentrations to range from less than the limit of quantitation (LOQ) of the analytical method (assessed as 0.0000034 mg/L) to 108% of nominal. Analysis of the 0.00050 mg/L test solutions on Days 5, 17, 31 , 38, 45, 47, 61 and 69 of the exposure phase showed measured test concentrations to range from 38% to 81% of nominal. A gradual decline in measured concentration was observed up to Day 38 after which the test concentrations remained at approximately 40% of nominal with the exception of the 0.000050 mg/L test concentrations which showed measured concentrations of less than the LOQ on Days 61 and 69. A review of the data indicated that the correct stock solutions had been prepared and that the test system was dosing correctly. It was therefore considered possible that the test item may have been taken up by the fish but rapidly metabolised and excreted hence resulting in a lower concentration of the parent test item. These low values were considered not to affect the integrity of the study given that the test item was not shown to significantly accumulate in the fish tissues.

Analysis of the solvent control water samples throughout the test showed no test item to be present. No test item was detected in the water samples taken during the depuration phase of the test.

Analysis of the test fish on Days 5, 17, 31, 38, 47, 61 and 69 of the exposure phase showed the test item not to significantly accumulate, with mean measured values for whole fish of 0.0040 mg/kg on Day 5 to 0.0020 mg/kg on Day 69 for the 0.00050 mg/L test concentration. Concentrations less than the limit of quantitation (LOQ) of the analytical method (assessed as 0.0023 mg/kg) were determined for the 0.000050 mg/L test concentration.

 

The test substance was shown not to significantly accumulate in fish tissue and as observed to be eliminated from the fish tissues over a 25 day depuration period. At the end of the depuration period 100% of test substance was eliminated from the fish tissues for the 0.00050 mg/L test concentration. Based on this information the biological half-life of the test item is considered to be between 6 to 25 days.

Description of key information

100% of test substance was eliminated from fish tissues for the 0.00050 mg/L test concentration. Half life considered to be between 6 to 25 days (OECD 305).

Key value for chemical safety assessment

Additional information

A study was performed to assess the bioconcentration potential of the test item in rainbow trout (Oncorhynchus mykiss) according to OECD Guideline 305.

Rainbow trout were exposed, in groups of 52, to an aqueous solution of the test item at nominal concentrations of 0.000050 and 0.00050 mg/L for a period of 69 days under dynamic test conditions. Samples of test fish were taken from the solvent control, 0.000050 and 0.00050 mg/L test groups on days 5, 17, 31, 38, 47, 61 and 69 of the uptake phase and the concentration of test item in the fish tissues determined. After 69 days exposure the remaining fish were subjected to a depuration period of 25 days. Samples of test fish were taken from the solvent control, 0.000050 and 0.00050 mg/L test groups on days 6, 19, 22 and 25 of the depuration phase and the concentration of test item determined.

 

The Bioconcentration Factors (BCF) for the test item in the whole fish after 69 days were calculated to be 0 at a concentration of 0.000050 mg/L and 9 at a concentration of 0.00050 mg/L.

The Kinetic Bioconcentration Factor (BCFk) after 69 days for the 0.00050 mg/L test concentration was calculated to be 11. A BCFk was not calculated for the 0.000050 mg/L test concentration given that the BCF was 0.

The uptake and depuration rate constants, k1 and k2, were determined to be 10.6814 and 1.0000 for the 0.00050 mg/L test concentration.

As the test item had a log Kow of greater than 6.5, following the recommendations of the test guidelines, the BCF values were also calculated as a function of the lipid content. These BCF values were higher than those determined in the whole fish thereby suggesting that the accumulation of the test item was into the lipid content of the fish. However, as the values were below 2000, the test item would not be classed as bioaccumulative according to the PBT assessment criteria.

The Bioconcentration Factors (BCF) for the test item as a function of lipid content after 69 days were calculated to be 0 and 132 at concentrations of 0.000050 and 0.00050 mg/L.

The Kinetic Bioconcentration Factor (BCFk) was also calculated for the 0.00050 mg/L test concentration and was shown to be 161 after 69 days. A BCFk was not calculated for the 0.000050 mg/L test concentration given that the BCF was O.

The uptake and depuration rate constants, k1 and k2, were determined to be 161.1 and 1.0000 for the 0.00050 mg/L test concentration.

Analysis of the test solutions on Day -1 showed measured concentrations of 196% and 140% of nominal for the 0.000050 and 0.00050 mg/L test concentrations respectively. However, analysis of the test solutions on Day 0 showed measured concentrations of 108% and 113% of nominal for the 0.000050 and 0.00050 mg/L test concentrations respectively, therefore it was considered that the system was dosing correctly. Analysis of the 0.000050 mg/L test solutions on Days 5, 17, 31 , 38, 45, 47, 61 and 69 of the exposure phase showed measured test concentrations to range from less than the limit of quantitation (LOQ) of the analytical method (assessed as 0.0000034 mg/L) to 108% of nominal. Analysis of the 0.00050 mg/L test solutions on Days 5, 17, 31 , 38, 45, 47, 61 and 69 of the exposure phase showed measured test concentrations to range from 38% to 81% of nominal. A gradual decline in measured concentration was observed up to Day 38 after which the test concentrations remained at approximately 40% of nominal with the exception of the 0.000050 mg/L test concentrations which showed measured concentrations of less than the LOQ on Days 61 and 69. A review of the data indicated that the correct stock solutions had been prepared and that the test system was dosing correctly. It was therefore considered possible that the test item may have been taken up by the fish but rapidly metabolised and excreted hence resulting in a lower concentration of the parent test item. These low values were considered not to affect the integrity of the study given that the test item was not shown to significantly accumulate in the fish tissues.

Analysis of the solvent control water samples throughout the test showed no test item to be present. No test item was detected in the water samples taken during the depuration phase of the test.

Analysis of the test fish on Days 5, 17, 31, 38, 47, 61 and 69 of the exposure phase showed the test item not to significantly accumulate, with mean measured values for whole fish of 0.0040 mg/kg on Day 5 to 0.0020 mg/kg on Day 69 for the 0.00050 mg/L test concentration. Concentrations less than the limit of quantitation (LOQ) of the analytical method (assessed as 0.0023 mg/kg) were determined for the 0.000050 mg/L test concentration.

 

The test substance was shown not to significantly accumulate in fish tissue and as observed to be eliminated from the fish tissues over a 25 day depuration period. At the end of the depuration period 100% of test substance was eliminated from the fish tissues for the 0.00050 mg/L test concentration. Based on this information the biological half-life of the test item is considered to be between 6 to 25 days.