Registration Dossier

Environmental fate & pathways

Bioaccumulation: aquatic / sediment

Currently viewing:

Administrative data

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:
21/07/1998 - 27/09/1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 305 in compliance with GLP, without deviations that influence the quality of the results.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
EPA OPPTS 850.1730 (Fish Bioconcentration Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)
Deviations:
no
GLP compliance:
yes
Radiolabelling:
yes
Details on sampling:
- Sampling intervals/frequency for test organisms: organisms were sampled after 4 hours and after 1, 3, 7, 14 and 21 days of the exposure period and on days 1, 3, 7, 10 and 14 of the depuration period.
- Sampling intervals/frequency for test medium samples: water was sampled sampled after 0 and 4 hours and after 1, 3, 7, 14 and 21 days of the exposure period and on days 1, 3, 7, 10 and 14 of the depuration period.
- Sample storage conditions before analysis: All water samples were stored frozen. All fish samples were subsequently stored frozen at -20 °C.
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):

Water was sampled throughout the study. On sampling days during the exposure and depuration periods, water samples of approximately 3,000-mL and 500-mL were collected from each aquarium and analyzed for total 14C-residues, respectively. Water samples of approximately 500-mL were also collected from each mixing chamber and analyzed for total 14C-residues on the sampling days during the exposure periods. The water samples collected from the treated aquaria on 0, 14 and 21 days of the exposure period and the water samples collected from the mixing chamber on 14 and 21 days of the exposure period were used for metabolite/degradate characterization. All water samples were collected prior to adding the daily feed to the test aquaria. Water samples from the control aquarium were stored in 32-oz. polyethylene bottles, while samples from the treated aquaria were stored in 32-oz. exterior plastic-coated amber glass bottles.

On sampling Day 21 of the exposure period, forty-eight fish were removed from each aquarium, analyzed for total 14C-residues, and used for metabolite/degradate characterization. On the remaining sampling days, four fish were collected from each aquarium and analyzed for TRR only.
All fish samples were collected prior to adding the daily feed to the test aquaria. Fish were netted from the aquaria, immediately rinsed with fresh well water, blotted dry, humanely sacrificed, representative group measured (total length and wet weight), dissected into fillet (edible: body, muscle, skin, and skeleton) and viscera (non-edible: fins, head, and internal organs), and weighed (to determine percentages of whole fish). To prevent contamination, the control fish were dissected first followed by the low and high treatment groups. Separate scalpels and dissecting plates were used for the control and each treatment groups. The tissues were immediately placed in preweighed labeled polyethylene containers, that contained liquid nitrogen. The liquid nitrogen was allowed to evaporate before weight determinations.
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:

Preparation and Analysis of Nonradiolabeled Test Substance
The standard solution of supplied nonradiolabeled test compound was prepared in dimethylformamide (DMF). Nonradiolabeled OTNE (1006.2 mg, ABC Reference No. TS-9601) was transferred into a 10-mL volumetric flask and brought to volume with dimethylformamide. The solution was assigned ABC Reference No. PC-00914. The concentration of nonradiolabeled OTNE in PC-00914 was 100.62 mg/mL.

Preparation of Stock Solution and Radiochemical Purity Determination of 14C-OTNE
The 14C-OTNE (ABC Reference No. RS-10759) was supplied as liquid in four ampules by Wizard Laboratories, Inc. (West Sacramento, CA) and received by ABC Laboratories on August 13, 1998. The amount of radioactivity and specific activity of 14C-OTNE were declared to be 7.92 mCi (4 x 1.98 mCi per ampule) and 29.9 mCi/mmol, respectively. The contents in the ampules were quantitatively transferred to a 10-mL volumetric flask and brought to volume with DMF. The solution was assigned ABC Reference No. PR-01149. To quantitate the total radioactivity and mass of [14C]OTNE in Stock Solution (PR-01149), two identical dilutions were prepared (25 µL diluted to 5 mL) with acetonitrile (R-02238-A & B) and analyzed by LSC (5 x 25 µL). To determine the radiochemical purity of the test substance, aliquots of the diluted Stock Solution (R-02238-A) were analyzed using HPLC Method #44799-A. Eluate fractions were collected at half minute intervals, fortified with 5 mL of scintillation fluid and analyzed by LSC. The radioactivity in the eluate also was followed by radioactivity flow-cell monitoring. The radiochemical purity of the test substance was also estimated by duplicate analyses of aliquots from R-02238-A using 1D-TLC. The adhering test compound in the caps of the ampules was also quantitatively transferred to a 10-mL volumetric flask and brought to volume with DMF. The solution was assigned ABC Reference No. PR-01151. To quantitate the total radioactivity and mass of [14C]OTNE in Stock Solution (PR-01151), a dilution was prepared (25 µL diluted to 10 mL) with acetonitrile (R-02243) and analyzed by LSC (5 x 25 µL). The test substance in the stock solution (PR-01 149) was used to prepare dose solutions (R-02241 & R-02242) for an earlier definitive study. The In-Life phase of the earlier definitive study was aborted on Day 16 of the uptake phase since the low and high treatment stock dose solutions were inadvertently delivered to the incorrect graduated cylinders that hold thestock dose solutions on Day 14 of the uptake phase. The remaining contents in the flasks containing the stock solution PR-01149 and PR-01151 were thoroughly rinsed with DMF and the rinsates were quantitatively transferred to another 10-mL volumetric flask and brought up to the desired volume with DMF (PR-01163). A dilution was prepared (10 µL to 5 mL) with DMF (R-02283) and analyzed by LSC (5 x 25 µL). The total radioactivity in stock solution rinsate (PR-01163) was determined to be 0.226 mCi.

Low Dose Solution Preparation
The low dose solution (R-02281) was prepared by combining: 500 mL of the remaining aliquots of low dose solution (subaliquots R-02241-24 thru -33) measured with a 500-mL volumetric flask), the entire contents in stock solution rinsate (PR-01163) and 0.16 mL of non-radiolabeled OTNE solution PC-00914 (measured with a 250-µL positive displacement pipet) in a 2-liter Erlenmeyer flask. The total volume of the mixed solution was brought up to approximately 1800 mL by adding 1290 mL of DMF (part of the 1290 mL DMF was used to rinse the 10-mL volumetric flask holding the PR-01163 solution and the 500-mL volumetric flask used for measuring the R-02241 solution). The prepared low dose solution was assigned ABC Reference No. R-02281. The solution was subaliquoted into thirty-four 50-mL amber bottles (ABC Reference No. R-02281-1 thru -34) and stored in the freezer at -20°C until use. To quantitate the total radioactivity in the low dose solution (R-02281), five 100 µL aliquots were analyzed by LSC. To determine the radiochemical purity of the test substance, one 25-µL aliquot of low dose Solution (R-02281) was analyzed using HPLC. The HPLC eluate was followed by monitoring the radioactivity and the ultraviolet light absorption (UV = 213 nm) and half minute fractions were collected and the radioactivity was quantitated by LSC. The radiochemical purity of the test substance was also determined by analyzing two 20-µL aliquots of R-02281 using 1D-TLC. The specific activity of low dose solution was determined gravimetrically.

High Dose Solution Preparation
The high dose solution (R-02282) was prepared by combining: 550 mL of previous high dose solution (subaliquots R-02242-24 thru -34 measured with 500-mL and 50-mL volumetric flasks) and 1.70 mL of non-radiolabeled OTNE solution PC-00914 (measured with a 250-µL positive displacement pipet) in a 2-liter Erlenmeyer flask. The total volume of the mixed solution was brought up to approximately 1801.7 mL by adding 1250 mL of DMF (part of the 1250 mL DMF was used to rinse the 500-mL and 50-mLvolumetric flasks used for measuring the R-02242 solution). The prepared high dose solution was assigned ABC Reference No. R-02282. The solution was subaliquoted into thirty-four 50-mL amber bottles (ABC-Reference No. R-02282-1 thru -34) and stored in the freezer at -20 °C until use. To quantitate the total radioactivity in the high dose solution (R-02282), five 25 µL aliquots were analyzed by LSC. To determine the radiochemical purity of the test substance, one 10-µL aliquot of high dose Solution (R-02282) was analyzed using HPLC. The HPLC eluate was followed by monitoring the radioactivity and the ultraviolet light absorption(UV = 213 nm) and half minute fractions were collected. The radioactivity in the eluate fractions was quantitated by LSC. The radiochemical purity of the test substance was also determined by analyzing two 10-µL aliquots of R-02282 using ID-TLC. The specific activity of low dose solution was determined gravimetrically.

- Controls: Yes, blanks
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): DMF
- Concentration of vehicle in test medium (stock solution and final test solution(s) at different concentrations and in control(s)): No data
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No
Test organisms (species):
Lepomis macrochirus
Details on test organisms:
TEST ORGANISM
- Common name: Bluegill
- Strain: No data
- Source: Osage Catfisheries Inc. (Osage Beach, Missouri)
- Age at study initiation (mean and range, SD): +/- 1 year
- Length at study initiation (lenght definition, mean, range and SD): 63 +/- 2.5 mm
- Weight at study initiation (mean and range, SD): 4.239 +/- 0.543 grams
- Weight at termination (mean and range, SD): 6.390 +/- 2.186 grams
- Method of breeding: As described by Brauhn and Schoettger
- Health status: No mortality during test
- Description of housing/holding area: As described by Brauhn and Schoettger
- Feeding during test
- Food type: salmon starter (Rangen Inc., (Buhl, ID)
- Amount: 2% total fish weight per aquarium.
- Frequency: daily


ACCLIMATION
- Acclimation period: 63 days
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: flake food (Worldwide Aquatics, Inc., San Francisco, CA, ABC Lot #FF398), salmon starter (Rangen, Inc., Buhl, ID, ABC Lot #SS198 and SS298), and brine shrimp (Mackay Marine Brine Shrimp Co., Providence, UT, ABC Lot #44519) ad libitum
- Feeding frequency: No data
- Health during acclimation (any mortality observed): Healthy
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
21 d
Total depuration duration:
14 d
Hardness:
138 - 144 mg/l CaCO3
Test temperature:
20 - 22 degrees Celsius
pH:
7.9 - 8.3
Dissolved oxygen:
Each aquarium was aerated throughout the test period to maintain dissolved oxygen levels at or above 74 % saturation, except on Day 12 of the exposure period when the dissolved oxygen levels in the TL and TH aquaria were <60% saturation. The low dissolved oxygen levels were for a short duration (few hours) and resulted when aeration was not provided following daily siphoning. During water quality measurements, the dissolved oxygen concentrations for all test aquaria ranged between 6.3 and 8.4 mg/L and stayed between 74 and 99% saturation at 21 °C, respectively, which was considered adequate for testing.
TOC:
< 50 mg/l in aquaria during the exposure period and < 1.3 mg/l during the depuration period.
< 1.0 mg/l in dilution water, 44 mg/l for the dilution water fortified with DMF.
Salinity:
Not relevant
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 90l, filled with 70l medium
- Aeration: yes
- Renewal rate of test solution (frequency/flow rate): 70L was replaced 7.8 times per 24 hours
- No. of organisms per vessel: 130
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: No data


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: All water used for the in-life phase of the study was from an uncontaminated, deep well source. Blended water was used for testing fish during the conduct of the study. Well water and blended water used for culturing fish prior to the study were obtained from the same source as that used during the study.
- Particulate matter: < 1.0 mg/l
- Holding medium different from test medium: Yes, see above
- Intervals of water quality measurement: Measured ater 0 and 2-6 hours and after 1, 3, 7, 14 and 21 days of exposure as well as after 1, 3, 7, 10 and 14 days of depuration
- Intervals of test medium replacement: Continuous, see above


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16/8 hours light/dark with 30 minute transition periods
- Light intensity: No data


PRELIMINARY STUDY
- Test concentrations: 1.3 and 13 ug/L
- Results used to determine the conditions for the definitive study: yes, test substance was soluble, same concentration levels used
Nominal and measured concentrations:
Nominal: 1.3 and 13 ppb
Measured: 1.3 and 13 ppb
Reference substance (positive control):
no
Details on estimation of bioconcentration:
BASIS FOR CALCULATION OF BCF
- Estimation software: DOW Biofac
Lipid content:
7.37 %
Time point:
start of exposure
Remarks on result:
other: Control treatment - day 0
Lipid content:
8.02 %
Time point:
end of exposure
Remarks on result:
other: Control treatment - day 21
Lipid content:
8.1 %
Time point:
end of exposure
Remarks on result:
other: Low treatment - day 21
Lipid content:
7.89 %
Time point:
end of exposure
Remarks on result:
other: High treatment - day 21
Key result
Type:
BCF
Value:
603
Basis:
whole body w.w.
Remarks:
OTNE concentration
Time of plateau:
3.6 d
Calculation basis:
steady state
Remarks on result:
other: average over day 14 and 21
Remarks:
Conc.in environment / dose:1.3 ug/l
Type:
BCF
Value:
747
Basis:
whole body w.w.
Remarks:
total radioactivity
Time of plateau:
3.6 d
Calculation basis:
steady state
Remarks on result:
other: average over day 14 and 21, based on concentration in water in mixing chamber
Remarks:
Conc.in environment / dose:1.3 ug/l
Type:
BCF
Value:
593
Basis:
whole body w.w.
Remarks:
OTNE concentration
Time of plateau:
4.2 d
Calculation basis:
steady state
Remarks on result:
other: average over day 14 and 21
Remarks:
Conc.in environment / dose:13 ug/l
Type:
BCF
Value:
736
Basis:
whole body w.w.
Remarks:
Total radioactivity
Time of plateau:
4.2 d
Calculation basis:
steady state
Remarks on result:
other: average over day 14 and 21, based on concentration in water in mixing chamber
Remarks:
Conc.in environment / dose:13 ug/l
Type:
BCF
Value:
750
Basis:
whole body w.w.
Time of plateau:
3.6 d
Calculation basis:
kinetic
Remarks on result:
other: +/- 130
Remarks:
Conc.in environment / dose:1.3 ug/l
Type:
BCF
Value:
860
Basis:
whole body w.w.
Time of plateau:
4.2 d
Calculation basis:
kinetic
Remarks on result:
other: +/- 120
Remarks:
Conc.in environment / dose:13 ug/l
Type:
BCF
Value:
720
Basis:
whole body w.w.
Remarks:
Total radioactivity
Time of plateau:
3.6 d
Calculation basis:
steady state
Remarks on result:
other: Conc.in environment / dose:1.3 ug/l
Type:
BCF
Value:
850
Basis:
whole body w.w.
Remarks:
Total radioactivity
Time of plateau:
4.2 d
Calculation basis:
steady state
Remarks on result:
other: Conc.in environment / dose:13 ug/l
Elimination:
yes
Parameter:
DT50
Depuration time (DT):
1.2 d
Details on kinetic parameters:
- Uptake rate constant (k1): 480 +/- 60 kg^-1d^-1 at 1.3 ug/l; 470 +/- 47 kg^-1d^-1 at 13 ug/l
- Depuration (loss) rate constant (k2): 0.64 +/- 0.082 d^-1 at 1.3 ug/l; 0.55 +/- 0.056 at 13 ug/l
- Indication of bi- or multiphasic kinetics: No data
- Computation / data analysis: The concentration factor (BCFk) will be calculated as the ratio ki/k2, the two first-order kinetic constants. The depuration rate constant (k2) is determined from the depuration curve. The uptake rate constant (ki) is then calculated given k2 and a value of Cr which is derived from the uptake curve. The BCFk and the rate constants, ki and k2, will be calculated using non-linear parameter estimation methods. If the depuration curve is obviously not first-order, then more complex models may be employed. The BCF will be determined and expressed as a function of the total wet weight of the fish. To reduce variability in test results as a result of differences in lipid content of the test fish, the BCF will also be expressed in relation to lipid content in addition to whole body weight.

Steady state is that condition under which no significant difference in test substance concentration in the test organism, over three consecutive sampling points (at intervals of at least two days), is observed. Steadystate conditions will be determined by appropriate graphical and/or statistical techniques for the data set. If sufficient uptake and depuration data are available, the uptake rate constant (K), depuration rate constant (K2), and kinetic BCF will be determined on a whole organism basis by the graphical methods presented by ASTM (4) and Blanchard, et al. (5) or by an appropriate computer program such as BIOFAC. If the BCF value for the whole fish is > 1000, the bioconcentration potential of the test substance will be estimated based on lipid content.

Mean and standard deviation for data analysis will be calculated and reported whenever appropriate. Outlying replicates may be tested using
a Q-test or t-test. No other statistical analyses are anticipated.
Metabolites:
Metabolism by the fish was extensive. In fish fillet, OTNE was the major radioactive residue. In total 6 components, including OTNE, were detected. In fish viscera, OTNE (approximately 50% of TRR) as well as two major polar metabolites (comprising 30% to 35% TRR) were observed. In viscera approximately 7-10 components were detected. Most of the metabolites present in the viscera samples were also present in the water in the aquaria.
Results with reference substance (positive control):
Not relevant
Details on results:
- Mortality of test organisms: No mortality during test
- Observations on body length and weight: The fish remained healthy throughout the biological phase of the study. Fish measurements at the end of the exposure period demonstrated normal weight and length growth. Exposure of the fish in the low and high treatment groups to 14C-OTNE did not produce mortality and/or adverse health effects.
- Mortality and/or behavioural abnormalities of control: No mortality in controls
- Loss of test substance during test period: test substances concentrations maintained at nominal levels through flow-through setup
- Results with vehicle control: No adverse effects of vehicle reported
Reported statistics:
Mean and standard deviation for data analysis will be calculated and reported whenever appropriate. Outlying replicates may be tested using a Q-test or t-test. No other statistical analyses are anticipated.

Most of the metabolites present in the viscera samples were also present in the water in the aquaria, indicating that these were actively excreted from the fish into water. Analysis of the water suggested that the metabolism of OTNE in fish tissues was relatively rapid. OTNE levels in water at steady state were less than 40%, in spite of the high daily turnover rate of 7.3 – 8 times for the test solution. This implies that around 60% of the test material can be attributed to metabolites.

Mass balances were calculated based on the loss of radioactivity k2 multiplied with the plateau concentration TRR at day 21 for the whole fish. Multiplication of this ‘loss’ by the average fish-to-water loading rate (0.93 g/l day-1) gives the nominal concentration of ‘lost’ radioactivity in water leaving the system: 0.65 μg/l in the low treatment and 5.60 μg/l in the high treatment. Comparing these concentrations to the average measured concentrations of metabolites in water (day 14 and 21): 0.7 μg/l for the low treatment and 7.02 μg/l for the high treatment, shows that 93% and 80% of the totally applied radioactivity can be accounted for.

Validity criteria fulfilled:
yes
Remarks:
Concentration within 20% of nominal, oxygen saturation > 60%, temperature variation less than +/- 2 degrees Celsius, mortality < 10%
Conclusions:
The BCF of OTNE is approximately 600. Conversion to a 5% standard lipid content the result becomes: 391.
Executive summary:

Bioconcentration and metabolism of OTNE in Lepomis macrochirus was investigated according to international guidelines (OPPTS 850.1730 / OECD Guideline 305) under GLP. Validity criteria for the test were met. OTNE was extensively metabolised by the fish, therefore the BCF was determined both by the kinetic approach (k1/k2) and by the steady state approach (Cf/Cw). There is no significant difference between these BCFs, nor between the BCFs for the high and low dose treatment. In the steady state approach, the BCF was also calculated with the measured OTNE concentration in the premix chamber.

The results show that the BCF is 603 and 593 in the low and high treatment. Based on total radioactivity in fish and the premix chamber containing no metabolites, the corresponding BCF value is 747 and 736. This indicates that part of the extracted radioactivity in the fish is to be attributed to metabolites. The DT50 was found to be 1.2 day. Conversion to a 5% standard lipid content the result becomes: 391, which value will be used in the risk assessment.

OTNE was extensively metabolised by the fish. A total of 6 components including OTNE were detected in fish fillet. In fish viscera, OTNE (approximately 50% of TRR) as well as two major polar metabolites (comprising 30% to 35% TRR) were observed. In viscera approximately 7-10 components were detected. Most of the metabolites present in the viscera samples were also present in the water in the aquaria, indicating that these were actively excreted from the fish into water.

Description of key information

Key value for chemical safety assessment

BCF (aquatic species):
391 dimensionless

Additional information

Bioconcentration and metabolism of OTNE in Lepomis macrochirus was investigated according to international guidelines (OPPTS 850.1730 / OECD Guideline 305) under GLP. Validity criteria for the test were met. OTNE was extensively metabolised by the fish, therefore the BCF was determined both by the kinetic approach (k1/k2) and by the steady state approach (Cf/Cw). There is no significant difference between these BCFs, nor between the BCFs for the high and low dose treatment. In the steady state approach, the BCF was also calculated with the measured OTNE concentration in the premix chamber.

The results show that the BCF is 603 and 593 in the low and high treatment. Based on total radioactivity in fish and the premix chamber containing no metabolites, the corresponding BCF value is 747 and 736. This indicates that part of the extracted radioactivity in the fish is to be attributed to metabolites. The DT50 was found to be 1.2 day.

OTNE was extensively metabolised by the fish. A total of 6 components including OTNE were detected in fish fillet. In fish viscera, OTNE (approximately 50% of TRR) as well as two major polar metabolites (comprising 30% to 35% TRR) were observed. In viscera approximately 7-10 components were detected. Most of the metabolites present in the viscera samples were also present in the water in the aquaria, indicating that these were actively excreted from the fish into water.

The average lipid content of the fish was 7.7%. For subsequent calculations the empirical BCF of 603 was converted to a standard fish with a lipid content of 5%. Thus a BCF of 603/7.7 * 5 = 391 is used for the further evaluations. 

Mass balance calculations based on the study report indicate that 80 -93% of the residual radio-activity in the water can be explained by the presence of excreted metabolites.

Mass balance (further calculations based on study report):

Most of the metabolites present in the viscera samples were also present in the water in the aquaria, indicating that these were actively excreted from the fish into water (Madsen et al 1999). Analysis of the water suggested that the metabolism of OTNE in fish tissues was relatively rapid. OTNE levels in water at steady state were less than 40%, in spite of the high daily turnover rate of 7.3 – 8 times for the test solution. This implies that around 60% of the test material can be attributed to metabolites.

Mass balances were calculated based on the loss of radioactivity k2multiplied with the plateau concentration TRR at day 21 for the whole fish. Multiplication of this ‘loss’ by the average fish-to-water loading rate (0.93 g/l day-1) gives the nominal concentration of ‘lost’ radioactivity in water leaving the system: 0.65 μg/l in the low treatment and 5.60 μg/l in the high treatment. Comparing these concentrations to the average measured concentrations of metabolites in water (day 14 and 21): 0.7 μg/l for the low treatment and 7.02 μg/l for the high treatment, shows that 93% and 80% of the totally applied radioactivity can be accounted for.