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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:
2008-04-10 to 2010-03-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study following recent test guidelines and GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)
Deviations:
yes
Remarks:
only one concentration was tested, at saturation determined by a pre-test and feasibility study
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
yes
Details on sampling:
- Sampling intervals/frequency for test organisms: uptake Day 0, 3,7, 13, 21, 28, 33, depuration days 3, 7, 10, 17, 31, 45, 60 for radiaactivity
Parent: uptake days 21, 28, 33, depuration days 60
- Sampling intervals/frequency for test medium samples: Days: -1 (pre-test), uptake Day 0, 3,7, 13, 21, 28, 33, depuration days 3, 7, 10
Parent: Day 33 of uptake
Lipid analysis: day 0 and 35 of uptake, day 60 of depuration
- Sample storage conditions before analysis:
Water samples were analysed immediately without storage, or storage at 4 deg. C. Sample on day 33 for parent analysis: frozen
- tissue samples: frozen after processing.
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
Collection and Analysis of Stock Solutions and Water Samples
Samples of the stock solution was collected on Day -1 and analyzed for parent AN2 and total radioactivity. The stock solution sample was sampled using a gas tight syringe and placed in a 20 mL glass scintillation vial containing scintillation cocktail. Water samples were collected and analyzed for AN2 (based on total radioactivity), on Pretest Day –1, uptake Days 0, 3, 7, 13, 21, 28, and 33 and on depuration Days 3, 7 and 10. At each water sampling interval, one water sample was collected from the solvent control and two samples were collected from the AN2 treatment group. All samples were analyzed for total radioactivity. All water samples were collected from mid-depth of each test chamber using a volumetric pipette. Water samples were analyzed as soon as possible after collection without storage. On uptake Days 28 and 33, additional water samples were collected from the solvent control and the AN2 treatment group. At each of these intervals, one sample was collected from the solvent control and two from the AN2 treatment group. These samples were collected from mid-depth from each test chamber using a volumetric pipette. Water samples were stored refrigerated (~4°C) after collection for possible analysis, but only the Day 33 treatment group samples were analyzed for parent AN2. The Day 28 samples or the solvent control sample from Day 33 were not analyzed.
Collection and Analysis of Tissue Samples
Tissue samples were collected on uptake Days 3, 7, 13, 21, 28 and 33, and on depuration Days 3, 7, 10, 17, 31, 45, and 60. At each tissue sampling interval, a sufficient number of fish were collected to provide at least two replicate samples of solvent control fish and four replicate samples of each AN2 treatment group. Fish were impartially removed from the test chambers and euthanized by making an incision from just posterior to the base of the pectoral fin dorsally through the spinal cord. The fish were blotted dry and measured for total length and wet weight within approximately 15 minutes of collection, when possible. Each fish was then dissected into edible and non-edible tissue fractions. The head, fins and viscera were removed from the body and were considered to be non-edible tissue. The remaining tissue was considered the edible tissue. Tissue samples were transferred to pre-weighed glass vials and weighed. All tissue samples were processed immediately or stored frozen.
The concentrations of the test substance in freshwater and fish tissue were verified by liquid scintillation counting (LSC). Aqueous samples were extracted with dichloromethane, evaporated to dryness and reconstituted with acetonitrile. The extract of the samples was transferred into scintillation vials to which approximately 15 milliliters of Ultima Gold XR scintillation cocktail were added and samples were counted by LSC. Tissue samples were weighed and combusted in a Packard sample oxidizer prior to LSC analysis. A Packard 2500TR Liquid Scintillation Analyzer was used to determine disintegrations per minute (dpm).

Working Stock Analyzed by LSC
Concentrations of the working AN2 stock solution were determined using an Agilent Model 1100/1200 High Performance Liquid Chromatograph (HPLC) equipped with an Agilent Series 1100 Variable Wavelength Detector. Chromatographic separations were achieved using a YMC Pack ODS-AM column (150 mm x 4.6 mm, 3-µm particle size).

Background Radioactivity
In addition to the test samples, 15 mL samples of Ultima Gold XR scintillation cocktail were analyzed for aqueous samples at each interval to determine background radioactivity. For tissue samples, a blank cone was oxidized at each interval to determine background activity.

Water Samples Analyzed for Parent AN2 by HPLC/UV
The concentrations of the parent AN2 in the Day 33 uptake test solutions were verified by High Performance Liquid Chromatography with Ultraviolet Detection (HPLC/UV). Samples were extracted with dichloromethane, rotary evaporated and reconstituted with acetonitrile. The reconstituted samples were transferred to autosampler vials with HPLC-grade bottled water and submitted for analysis.

Concentrations of AN2 in the samples were determined using an Agilent Series 1100/1200 High Performance Liquid Chromatograph (HPLC) equipped with an Agilent Series 1100 Variable Wavelength Detector. Chromatographic separations were achieved using a Zirchrom-Carb column (150 mm x 4.6 mm, 5-m particle size).
Water and Tissue Samples Analyzed by LSC
The analytical methods used to analyze the test samples were based upon methodology developed by Wildlife International, Ltd. Freshwater and edible and non-edible fish tissue samples were collected from a test designed to determine the bioconcentration potential of AN2 in rainbow trout. The analyses of these samples were performed at Wildlife International, Ltd. using liquid scintillation counting (LSC).


Tissue Samples Analyzed for Parent AN2 by Fractionation HPLC/LSC
To differentiate between AN2 and metabolite concentrations, a fractionation procedure was employed. A combined fish tissue sample was prepared for each uptake Day 21, 28 and 33 and depuration Day 60 by combining all remaining edible or non-edible tissue from each of the fish sampled for those days. Edible tissue sample composites were extracted in duplicate where a single non-edible sample composite was processed at each specified interval. Tissues were extracted with methanol. An aliquot was analyzed by LSC for total extractable radioactivity. Fractionation of each extract yielded ten regions consisting of polar metabolites, parent AN2 and non-polar metabolites.

From the combined tissue samples, an approximate one-gram sample was weighed into a 40-mL Oakridge Teflon centrifuge tube, and 10-mL of methanol was volumetrically added to each sample. Each sample was sonic disrupted for approximately 5 minutes, and then the samples were centrifuged at 10000 rpm for approximately 10 minutes. The organic extract was decanted to a pear-shaped evaporation flask. The extraction was repeated with an additional 10 mL of methanol, and all the extracts were combined in the evaporation flask. The extract was rotary evaporated to 1-2 mL, and quantitatively transferred to a graduated centrifuge tube. The extract was evaporated to less than one milliliter under a stream of nitrogen, and then adjusted to final volume with methanol. The extract was centrifuged at 14000 rpm for approximately 5 minutes.

The extract was transferred to an amber HPLC autosampler vial for fractionation of component regions of AN2. Each extract was fractionated using reverse-phase, gradient elution high performance liquid chromatography. A non-radiolabelled standard of AN2 in methanol was injected to verify the elution profile of AN2. Each extract was injected and the predetermined regions were collected into 20-mL scintillation vials. Approximately 10-15 mL of Ultima Gold XR was added to each sample and the fractions were analyzed by LSC. The eluant from the UV detector was collected directly into scintillation vials. Ten fractions reflecting 0-2.0, 2.0-3.5, 3.5-5.0, 5.0-6.5, 6.5-8.0, 8.0-9.5, 9.5-11.5 (fraction containing AN2), 11.5-13.0, 13.0-14.5 and 14.5-16.0 minutes were collected and analyzed by LSC. Radioactivity detected in the first three fractions represented very polar metabolites. Radioactivity in the next three fractions reflected moderately polar metabolites. Radioactivity measured in the 9.5-11.5 minute fractions reflected AN2 and the last three fractions represented non-polar metabolites.

Lipid Analyses
Vials containing tissue samples for lipid analysis were removed from the freezer and allowed to thaw. Tare weights of the vials had been pre-determined. HPLC-grade bottled water (10 mL) was added to each sample and samples were homogenized for approximately one minute using a hand-held tissue homogenizer. The homogenizer was rinsed with the appropriate solvent(s) in between samples. Each homogenate was transferred to a 250 mL separatory funnel containing 25 mL of chloroform and 50 mL of methanol. Each vial was rinsed with an additional 10 mL of bottled water and the rinse was poured into its respective separatory funnel. The funnels were then shaken with venting for approximately one minute, and 50 mL of chloroform followed by 50 mL of saturated sodium chloride was added to each separatory funnel. Funnels were swirled briefly with venting and the phases were allowed to separate. For each sample, the chloroform layer was drained through a powder funnel packed with Teflon wool and anhydrous sodium sulfate into a 250-mL roundbottom flask. An additional 50-mL aliquot of chloroform was added to each separatory funnel and the extraction and draining procedures were repeated. The extracts were rotary-evaporated to near dryness in a waterbath maintained at approximately 40C. Each sample was then transferred to a pre-weighed, labeled scintillation vial. Each 250-mL roundbottom flask was rinsed with a small volume of chloroform and the rinse was transferred to its respective scintillation vial. The remaining solvent in each vial was evaporated under a gentle stream of nitrogen or clean dry air. Each vial was reweighed and the weight recorded. The lipid content was calculated from the difference in the vial tare weight and the vial weight containing the lipid. Percent lipids were calculated from the ratio of lipid content to total tissue sample weight.


Vehicle:
no
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION OF SPIKED WATER
- Details of spiking:
The primary stock solution of 14C-AN2 was prepared by weighing a 1.07 mg aliquot of the test material and bringing to a 50 mL volume with DMF. Three 0.100 mL aliquots were transferred to Ultima Gold scintillation cocktail and counted by LSC analysis. These analyses resulted in a mean recovery of 103% of the expected radioactivity, corresponding to a mean concentration of 22.0 mg/L. An appropriate volume (mL) of a primary stock was used in the preparation of the test solution stock, resulting in a nominal radioactivity of 1.08 x 105 dpm/mL, which corresponds to a nominal stock concentration of 0.25 µg/mL.

The stock solution was delivered to the diluter mixing chamber (at a rate of 35.0 L/minute) where they were mixed with dilution water (at a rate of 350 mL/minute) to achieve the desired test concentration 0.025 μg/L, which corresponds to a nominal radioactivity of 10.8 dpm/mL.
- Controls:
The solvent control was prepared by injecting DMF into the mixing chamber for the solvent control. The concentration of DMF in the solvent control and the AN2 treatment group was 0.10 mL/L. The test solutions appeared clear and colorless in the diluter mixing chambers. The test solution in the test chambers at test initiation and termination appeared clear and colorless.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethyl formamide
- Concentration of vehicle in final test solution: 0.1 ml/L
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Source: Thomas Fish Company Anderson, Ca
- Length at study initiation : mean total: 65.5 mm, range: 64-67 mm
- Weight at study initiation: 3.02 g range 2.79 to 3.3 g
- Weight at termination (mean and range, SD):
- Health status: checked, good
- Description of housing/holding area: well water, 14 to 14.7 deg. C, pH 8.0 to 8.4, diss. Oxygen: 8.9 to 10 mg?l > 89% saturatin
- Feeding during test once daily
- Food type:salmon starter, Zeigler Brothjers

ACCLIMATION
- Acclimation period: 7 weeks
- Acclimation conditions (same as test or not):yes
- Type and amount of food: see above
- Feeding frequency: once daily
- Health during acclimation (any mortality observed): no good health
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
35 d
Total depuration duration:
60 d
Hardness:
138 mg/L: (CO3)
Test temperature:
15 deg C
pH:
8.1 control, 8.2 test
Dissolved oxygen:
8.1 mg/L control, 8.0 mg/l test
TOC:
18.45 mg C/L, dilutiomn water: 0.83 mgC/l
Salinity:
n.a.
Details on test conditions:
TEST SYSTEM
- Test vessel: 106 l stainlees steel aquaria
- Type (delete if not applicable): in plexiglas ventilation hood
- fill volume: 80 l
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter): continous flow diluter, syringe pump
- Renewal rate of test solution (frequency/flow rate): 6.3 volume additions every 24 h
- No. of organisms per vessel: 85
- No. of vessels per concentration (replicates): 2
- No. of vessels per control / vehicle control (replicates): 2


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Well water filtered through a sand filter and through a 0.45 mico-m filter to remove microbes and particles
- Alkalinity: 180 mg/L as CaCO3
- Conductance: 364 mico-S/cm
- Holding medium different from test medium: no
- Intervals of water quality measurement: periodic analysis followign SOP


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:
0.025 mico-g/l nominal, 0.018 mico-g/L measured
Reference substance (positive control):
no
Lipid content:
ca. 6.5 %
Time point:
other: mean measured concentration
Lipid content:
ca. 6.7 %
Time point:
other: end of depuration phase
Remarks on result:
other: day 60 of depuration
Lipid content:
ca. 7.2 %
Time point:
end of exposure
Remarks on result:
other: day 35
Lipid content:
ca. 5.6 %
Time point:
start of exposure
Type:
BCF
Value:
ca. 600 L/kg
Basis:
whole body w.w.
Time of plateau:
21 d
Calculation basis:
steady state
Remarks on result:
other: Based on test substance
Remarks:
Conc.in environment / dose:0.018 micro-g/L
Type:
BCF
Value:
ca. 552 L/kg
Basis:
edible fraction
Time of plateau:
21 d
Calculation basis:
steady state
Remarks on result:
other: based on test substance
Remarks:
Conc.in environment / dose:0.018 micro-g/l
Type:
BCF
Value:
ca. 666 L/kg
Basis:
non-edible fraction
Time of plateau:
21 d
Calculation basis:
steady state
Remarks on result:
other: based on test substance
Remarks:
Conc.in environment / dose:0.018 micro-g/l
Elimination:
yes
Parameter:
DT50
Depuration time (DT):
18 d
Details on kinetic parameters:
These calculations are based on the total radioactivity, not parent compound
- Uptake rate constant (k1): 105 Dayexp-1
- Depuration (loss) rate constant (k2): 0.083 dayexp-1
- Indication of bi- or multiphasic kinetics: yes
- Computation / data analysis: Simultaneous method (fits best whole data and steady state as measured)

using the OPPTS 850.1730
- Uptake rate constant (k1): 44.5 dayexp-1
- Depuration (loss) rate constant (k2): 0.019 day-1
- Indication of bi- or multiphasic kinetics: yes
- Computation / data analysis: OPPTS 850.1730

Do not fit the total curve very well
Metabolites:
In the edible fractions radioactive metabolites with lower retention time than the parent were detected on days 21, 28, and 33 of uptake. At the end of the depuration period the total radioactivity had decreased considerably, but the residual radioactivity was mainly from parent compound.
Details on results:
- Mortality of test organisms: There were no mortalities in control and treated animals during the uptake phase, motalities or losses during the depuration phase were not treatment related and occurred more frequently in controls. (overall 15% in controls, 2.4% in test group).
- Behavioural abnormalities: no treatment related clincial signs were observed
- Observations on body length and weight: no treatment related changes
- Other biological observations: no treatment related observations
.
Reported statistics:
Whole fish concentrations were calculated based on the sum of the edible and non-edible tissue concentrations for each fish. The steady-state bioconcentration factor (BCF) values were determined from the mean tissue concentrations at apparent steady-state divided by the average water concentration. Tissue concentrations were considered to be at apparent steady-state if three or more consecutive sets of tissue concentrations were not significantly different (p  0.05). Tissue concentrations were evaluated for normality and homogeneity of variance using the Shapiro-Wilk’s test and Bartlett’s test, respectively. Since the data passed the assumptions of normality and homogeneity, analysis of variance (ANOVA) was used to determine whether or not statistically significant differences existed between days at the end of the uptake phase of the test (p = 0.05). Treatment means that were significantly different between days were identified using Tukey’s test (p ≤ 0.05). All statistical tests were performed using a personal computer with TOXSTAT (6) or SAS (7) software.

Calculation of the Kinetic BCF values and Equations for Reaching 90% Steady-State and 50% ClearanceThe main requirement of the both the OECD and OPPTS guidelines is to report the steady state BCF. In addition, the OPPTS and OECD guideline give two methods for calculating rate constants (uptake rate (k1) and depuration rate (k2)) to determine kinetic BCF (BCFK) values. One method is the graphical method and the other method is non-linear regression. Both guidelines also provide equations for calculating the half-life for clearance in tissue (t½), time to reach 90% of steady-state (t90), and BCFK values using the equations and graphical methods .

 

The graphical method is typically based on two data points, the last day of uptake and the last day of depuration using the equations outlined in the draft OPPTS 850.1730 Guidance Document (Appendix 11). Since the parent analysis of AN2 was only conducted on fish tissue at steady-state Days 21, 28 and 33 and Day 60 depuration, kinetic BCF values could not be calculated using nonlinear regression in the computer program described by Newman (8). Instead, the kinetic BCF values for edible, non-edible and whole fish tissue based on total radioactivity were calculated using the equations outlined in the draft OPPTS 850.1730 Guidance Document (Appendix 11) using Day 33 of uptake and Day 60 of depuration.

 

The non-linear regression method uses all the uptake and depuration data in a computer program to build a regression equation to fit the data. This method was used for total radioactivity data since this was the method used to analyze samples throughout the study. The kinetic uptake rate (k1) and depuration rate (k2) were calculated for edible, non-edible and whole fish using SAS computer code described by Newman (8). These rate constants were used to calculate a kinetic bioconcentration factor (BCFK =k1/k2) and also to calculate half-life for clearance in tissue (t½), time to reach 90% of steady-state (t90), and BCFK values using the equations outlined in the draft OPPTS 850.1730 Guidance Document and OECD 305E .

The two procedures used within the non-linear regression method were: the simultaneous and sequential procedures described by Newman (8). In the simultaneous procedure, nonlinear regression was used to simultaneously solve for k1and k2using fish tissue data from the uptake phase. In the sequential method, data from the depuration (elimination) phase were used to first estimate k2, and then using both the k2estimate and fish tissue from the uptake phase to estimate k1. Both methods generally give similar results. However, in this study the results were different, and the simultaneous method was used because the residual error from regression was lower than in the sequential method. Additionally, the simultaneous method appeared to give results more consistent with both the graphical method and stead-state BCF values. Therefore kinetic parameters estimated by the simultaneous method based on non-linear regression were assumed to be better estimates of rate constants than the sequential method and were used to calculate half-lives and time to 90% of steady-state for total radioactivity data. 

 

Because declining concentrations of the test material in tissues is not consistent with the assumption of first order kinetics (required for valid kinetic estimates of BCF), and it is clear that a steady-state had been achieved in this study, the steady state estimates of the BCF was considered to be the most accurate of the available BCF estimates from this study. 

Results based onm total radioactivity:

BCF steady state

edible tissue: 815

non-edible tissue: 1644

whole fish: 1146

 

Validity criteria fulfilled:
yes
Conclusions:
Because declining concentrations of the test material in tissues is not consistent with the assumption of first order kinetics (required for valid kinetic estimates of BCF), and it is clear that a steady-state had been achieved in this study, the steady-state estimates of the BCF were considered to be the most accurate of the available BCF estimates from this study.

Steady-state concentrations of 2,2’,6,6’-Tetra-tert-butyl-4,4-methylenediphenol (AN2) were achieved in the tissues of rainbow trout (Oncorhynchus mykiss) after 21 days. The mean measured water concentration based on total radioactivity was 0.016 μg/L, and on parent was 0.018 μg/L. Steady-state BCF values based on total radioactivity concentrations were 815, 1644 and 1146 in edible, non-edible and whole fish tissue, respectively. The substance depurated slowly in fish tissue and was 25 – 38% of steady-state values by Day 60 depuration. Steady-state BCF values based on parent concentrations were 552, 666 and 600 in edible, non-edible and whole fish tissue, respectively.
Executive summary:

A standard BCF study following OECD guidlein 305 and GLP in rainbow trout under flow through conditions was performed at saturated measured test concentrations of 0.018 micro-g/l. Because declining concentrations of the test material in tissues is not consistent with the assumption of first order kinetics (required for valid kinetic estimates of BCF), and it is clear that a steady-state had been achieved in this study, the steady-state estimates of the BCF were considered to be the most accurate of the available BCF estimates from this study.

Steady-state concentrations of 2,2’,6,6’-Tetra-tert-butyl-4,4-methylenediphenol (AN2) were achieved in the tissues of rainbow trout (Oncorhynchus mykiss) after 21 days. The mean measured water concentration based on total radioactivity was 0.016 μg/L, and on parent was 0.018 μg/L. Steady-state BCF values based on total radioactivity concentrations were 815, 1644 and 1146 in edible, non-edible and whole fish tissue, respectively. The substance depurated slowly in fish tissue and was 25 – 38% of steady-state values by Day 60 depuration. Steady-state BCF values based on parent concentrations were 552, 666 and 600 in edible, non-edible and whole fish tissue, respectively.

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Test according to recently approved guidelines in development, only partly available to the registrant, not all details available. In particular ciritcal detailed results are missing and it is for example unclear if concnetrations of the test substance remaining in the intestines were reported and deduced from the concentrations in fish. It is critical that the intestines are removed before fish analysis for accurate measurements of the absorbed concentrations.
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 305 dietary study
Deviations:
yes
Remarks:
stability not analysed, diet analyses only once, feeding rate higher than recommended, incorrect formula for assimilation efficiency used, lipid content was normalized, k2g not calcultated, BMF calculated with wrong assimilation efficiencies.
GLP compliance:
not specified
Radiolabelling:
no
Vehicle:
yes
Details on preparation of test solutions, spiked fish food or sediment:
Preparation of the diet:
The test substance and reference standard were dissolved in solvent and added to Alaskan pollack liver oil*1. After mixing, formula feed for carp fry development*2 was added and shaken. The mixture was air dried for 24 hours to prepare the feed. The concentration of each test compound in the feed was set at 100 μg/g. The reference standard concentration in the feed was also set at 100 μg/g.

Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: Carp
- Source: not given
- Age at study initiation: not given
- Length at study initiation: not given
- Weight at study initiation: 2.75 - 3.6 g test, 2.18-3.61 HCB
- Weight at termination : 8.24 to 10.42 g test S, 9.71 - 11.46 g
- Description of housing/holding area: 100 l glass tanks
- Feeding during test
- Food type: Formula feed for carp fry development Nippon Formula Feed Manufacturing Company
- Amount: 3% of fish body weight
- Frequency: 2 times per day 30 min apart
ACCLIMATION not stated
.
Route of exposure:
feed
Test type:
other: feeding study
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
ca. 10 d
Total depuration duration:
>= 37 d
Hardness:
not given
Test temperature:
24.2 to 25 deg. C
pH:
7.5 to 8.1
Dissolved oxygen:
7.1 to 7.6
TOC:
no data
Salinity:
n.a.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type
- Material, size, headspace, fill volume: 100 l glass
- Renewal rate of test solution (frequency/flow rate): 1600 ml/min
- No. of organisms : 110
.

TEST MEDIUM / WATER PARAMETERS: no data

OTHER TEST CONDITIONS
- Photoperiod: 14 h light, 10 h dark
. artificial fluorescent lamp

.
Nominal and measured concentrations:
Test substance 100 micro-g/g, 95.2 micro-g/g feed
HCB: 100 micro-g/g. 96.8 micro-g/g feed
Reference substance (positive control):
yes
Remarks:
Hexachlorobenzene
Lipid content:
ca. 2.37 - ca. 3.9 %
Time point:
start of exposure
Lipid content:
ca. 3.65 - ca. 3.9 %
Time point:
end of exposure
Lipid content:
ca. 4.08 - 4.41 %
Time point:
other: 7 days of depuration
Lipid content:
ca. 4.04 - 4.74 %
Time point:
other: 10 days of depuration
Lipid content:
ca. 4.62 - ca. 4.79 %
Time point:
other: 14 days of depuration
Lipid content:
ca. 4.58 - ca. 5.6 %
Time point:
other: 28 days of depurtaion
Lipid content:
ca. 5.09 - ca. 6.23 %
Time point:
other: 37 days of depuration
Type:
BMF
Value:
ca. 10.7 dimensionless
Basis:
other: I x alpha x kdepuration exp-1
Calculation basis:
other: dietary biomagnification
Remarks on result:
other: the data are insufficiently reported to judge the validity
Remarks:
Conc.in environment / dose:95.2 micro-g/g
Type:
BMF
Value:
ca. 2.38 dimensionless
Basis:
other: lipid normalized dietary BMF
Calculation basis:
other: dietary lipid corrected BMF
Remarks on result:
other: the data are insufficiently reported to judge the validity
Remarks:
Conc.in environment / dose:95.2 micro-g/g feed
Details on kinetic parameters:

- Depuration (loss) rate constant (koverall) Test substance: - 0.0495, HBC: - 0.095
. C0 depuration: test substance 2.04, HCB: 2.79
Metabolites:
not determined
Results with reference substance (positive control):
BMF HCB: 11.6, lipid corrected: 2.56

Growth, Fish weights

Sample

Exposure Start Date

Exposure after 10 days

Excretion 1 day later

Excretion 7 day later

Excretion 10 day later

Excretion 14 day later

Excretion 28 day later

Excretion 37 day later

Average

3.27

4.37

4.05

5.33

5.47

5.92

8.01

9.2

STD

0.349

0.812

0.162

0.285

0.449

0.192

0.587

0.853

Average

3.16

4.36

4.25

5.38

5.36

5.67

8.01

10.19

STD.

0.583

0.343

0.438

0.346

0.448

0.411

0.727

0.774

STD standard deviation.

Concentrations is fish [micro-g/g]

Plot

 

Exposure after 10 days

Excretion 1 day later

Excretion 7 day later

Excretion 10 day later

Excretion 14 day later

Excretion 28 day later

Excretion 37 day later

 

TBMD

6.81

7.52

4.82

5.33

4.18

1.81

1.21

 

 

6.01

6.41

5.34

5.29

4.00

1.95

1.22

   HCB  13.3  15.3  7.29  6.28  5.11  1.37  0.611
     11.4  15.7  8.05  6.89  3.73  0.778  0.435
Conclusions:
Due to some uncertainties and missing details in the data and questions on the evaluation in this test system no firm conclusions can be drawn from this study. In particular no single analytical data are avaialble and it remains unclear how and if intestinal contents of the substance were considered.
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2005-11-28 to 2006-04-16
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, applicant has only obtained part of the results
Qualifier:
according to guideline
Guideline:
OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)
Version / remarks:
1996
GLP compliance:
yes
Radiolabelling:
no
Details on sampling:
- Sampling intervals/frequency for test organisms: 1. exposure concentration 6 samples (duplicates), second concentration 5 samples (duplicates), controls at begin and end of the study two additional fish were added for lipid analysis, 4 times in the elimination test (no further data available)
- Sampling intervals/frequency for test medium samples: not given
- Sample storage conditions before analysis: samples were not stored
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
Following an extraction flow chart. Water: extraction with Hexane after addition of ammonoium sulfate, Filtration, removal of solvent in rotary evaporator at 40 deg. C. Dissolution in Methanol with ultrasonication.

Fish: 1 g tissues (outer skin, head, internal organs, edible parts): treatment with sodium sulfate, HCl. Extraction with Acetonitril in reflux at 50 deg. C 60 min, supernatant filtrated, rotary evaporation, dissolution in hexane (ultrasonificatin), evaportaion of hexane rotarry evaporator 40 deg. C, dissolution in Methanol ultrasonication.
Vehicle:
yes
Details on preparation of test solutions, spiked fish food or sediment:

REPARATION OF SPIKED WATER
- Details of spiking: A stock solution of 100 mg/L was prepared in 2-methoxy ethanol. It was diluted with methoxyethanol to 1 mg/l and 0.1 mg/l respectively. 0.2 ml/min of stockl solution was mixed with 2000 ml/min of test water. 2880 l/day supplied to the test tank during exposure and elimination (without test substance in the latter case).
- Controls: vehicle treated in same way
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):2-Methoxyethanol
- Concentration of vehicle in final test solution: 0.1 ml/L
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: carp
- Source: Fukuoka Fisheries and Marine Technology Center, Inland Water Fisheries institute, Yamada
- Age at study initiation (mean and range, SD): 1 year
- Length at study initiation (lenght definition, mean, range and SD): 7 to 11 cm
- Weight at study initiation (mean and range, SD):
- Weight at termination (mean and range, SD):
- Method of breeding:
- Health status:
- Description of housing/holding area: 70 l glass tank
- Feeding during test
- Food type:Composite feed for Carp growth >=43% protein, >=3% lipid, Nippon Formula Feed Man. Co.
- Amount: an amount corresponding to ca. 2% of the body wieight.
- Frequency: twice per day

ACCLIMATION
- Acclimation period: 27 days in cultivatin tank in running water aty 25 +- 2 deg C, thereafter 37 d in test tank in running water at 25 +- 2 deg C.
fish with abormalities were removed
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
ca. 67 d
Total depuration duration:
ca. 54 d
Test temperature:
24.0 to 24.6 deg. C
pH:
7.4 - 8.0
Dissolved oxygen:
Test solutions: 6.8 to 7.6 mg/L, ciontrol: 7.1 to 7.7 mg/l
TOC:
not given
Salinity:
n.a.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: glass, 70 l volume
- Renewal rate of test solution (frequency/flow rate): 2880 L/day
- No. of organisms per vessel: 47, 12 controls
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Kurkume municipal water
.
Nominal and measured concentrations:
Level 1 nominal : 0.1 micro-g/l 0.06 to 0.109 micro-g/l
Level 2 nominal 0.01 micro-g/l 0.0059 to 0.0097 micro-g/l
Lipid content:
ca. 5.03 %
Time point:
other: 28 d
Remarks on result:
other: Level 2
Lipid content:
ca. 5.43 %
Time point:
other: 42 days
Remarks on result:
other: Level 2
Lipid content:
ca. 6.64 %
Time point:
other: 60 days
Remarks on result:
other: Level 2
Lipid content:
ca. 5.7 %
Time point:
other: average whole period
Remarks on result:
other: Level 2
Type:
BCF
Value:
4 941 L/kg
Basis:
whole body w.w.
Time of plateau:
42 d
Calculation basis:
steady state
Remarks on result:
other: Conc.in environment / dose:0.1 micro-g/l
Type:
BCF
Value:
9 200 L/kg
Basis:
whole body w.w.
Time of plateau:
28 d
Calculation basis:
steady state
Remarks on result:
other: Conc.in environment / dose:0.01 micro-g/l
Type:
BCF
Value:
9 000 L/kg
Basis:
whole body w.w.
Time of plateau:
28 d
Calculation basis:
steady state
Remarks on result:
other: lipid corrected
Remarks:
Conc.in environment / dose:0.01 micro-g/L
Elimination:
yes
Parameter:
other: residual % after 54 d depuration time: at 0.1 micro-g/g: 17-25%
Depuration time (DT):
54 d
Elimination:
yes
Parameter:
other: residual % after 54 d depuration time at 0.01 micro-g/L: 40-42%
Depuration time (DT):
54 d
Elimination:
yes
Depuration time (DT):
19 d
Elimination:
yes
Depuration time (DT):
28 d
Details on kinetic parameters:
not calculated
Metabolites:
not identified
Details on results:
No information available

Test substance concentration in test water

Level

After 5 days

After 11 days

After 17 days

After 28 days

After 42 days

After 60 days

After 67 days

Mean (standard deviation)

Table

Figure

1

0.0805

0.0611

0.0781

0.0876

0.0923

0.0981

0.109

0.0867

(0.01546)

11

6

2

0.00695

0.00589

0.00703

0.00750

0.00885

0.00968

 

0.00765

(0.001381)

12

Bioconcentration factor at different time points The numbers in parentheses are mean values

Level

After 11 days

After 17 days

After 28 days

After 42 days

After 60 days

After 67 days

Table

Figure

1

2000

1900

(1900)

2200

2500

(2300)

3600

2900

(3300)

3900

4500

(4200)

5800

6000

(5900)

4100

5200

(4700)

14

9

2

4000

4100

(4100)

4700

5000

(4800)

8000

7500

(7700)

11000

8600

(9700)

13000

13000

(13000)

 

15

10

Variations in bioconcentration factor at 0.1 mirog/l nominal (values expressing the results up to 5 digits)

Level

 

After 28 days

After 42 days

After 60 days

After 67 days

Mean of 3 measurements

1

Mean bioconcentration factor

 

4181.8

5907.5

4654.9

4914.7

Rate of divergence from mean of 3 (%)

 

14.913

20.199

5.2861

 

2

Mean bioconcentration factor

7722.8

9689.7

12859

 

10090

Rate of divergence from mean of 3 (%)

23.465

3.9730

27.438

 

 

 

Based on the above results, variations in the bioconcentration factor (mean) in Level 1 after 42, 60, and 67 days were reproted to be within 20% of the mean bioconcentration factor of the three analyses, and it was therefore judged that a steady state had been reached. The bioconcentration factor in a steady state was calculated using these results.

At 0.01 micro-g/l, variations in the bioconcentration factor (mean) exceeded 20% in the last three consecutive analyses. The bioconcentration factor corrected for the lipid content was therefore determined, and whether or not a steady state had been reached was verified as follows. The bioconcentration factor for an average lipid content of 5.70% (see Table 9) was calculated. The following table shows the bioconcentration factor corrected for the average lipid content at 0.01 microg/l.. Furthermore, the lipid content of the test fish was measured after 28, 42, and 60 days, and the average lipid content was calculated from these results.

Bioconcentration factor corrected for average lipid content The numbers in parentheses are mean values

Level

After 28 days

After 42 days

After 60 days

0.01 microg/l

9600

8000

(8800)

11000

9700

(10000)

10000

12000

(11000)

 

The following table shows variations in the bioconcentration factor corrected for the average lipid content in order to confirm whether or not a steady state had been reached.

Variations in bioconcentration factor (corrected for average lipid content) (values expressing the results up to 5 digits)

Level

 

After 28 days

After 42 days

After 60 days

Mean of 3 measurements

0.01 micro-g/l

Mean bioconcentration factor

8806.1

10136

11108

10017

Rate of divergence from mean of 3 (%)

12.089

1.1901

10.899

 

 

 It was found from these results that the variation in the bioconcentration factor (mean) corrected for the average lipid content was within 20% of the mean of the bioconcentration factor in three analyses after 28, 42, and 60 days, and it was therefore judged that a steady state had been reached. The bioconcentration factor in a steady state was accordingly calculated using the uncorrected (not corrected for lipid content) values after 28, 42, and 60 days.

 Test substance concentration in test water in a steady state (Unit: µg/L)

Level

After 28 days

After 42 days

After 60 days

After 67 days

Mean

1

 

0.0923

0.0981

0.109

0.0998

2

0.00750

0.00885

0.00968

 

0.00868

Validity criteria fulfilled:
not specified
Conclusions:
Due to the limitaiton of the data available to the registrant, no firm conclusion on the results of this study can be drawn.
Endpoint:
bioaccumulation in aquatic species, other
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
1992
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Only summary data available
Qualifier:
no guideline available
GLP compliance:
not specified
Route of exposure:
aqueous
Test type:
static
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
ca. 6 wk
Nominal and measured concentrations:
Nominal concentrations 1 mg/L and 0.1 mg/l with solubiliser
Type:
BCF
Value:
ca. 3.4 - ca. 13.4 L/kg
Basis:
whole body w.w.
Calculation basis:
steady state
Remarks on result:
other: no further data available
Remarks:
Conc.in environment / dose:1 mg/l
Type:
BCF
Value:
ca. 14 - 64.5 L/kg
Basis:
whole body w.w.
Calculation basis:
steady state
Remarks on result:
other: no further data available
Remarks:
Conc.in environment / dose:0.1 mg/l
Conclusions:
No conclusions can be drawn from this study, as no details are available, a solubilizer was used and the concentrations reproted were far above the water solubility.
Endpoint:
bioaccumulation: aquatic / sediment
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The validity of the (Q)SAR model is characterised according to the OECD principles.
Justification for type of information:
QSAR prediction: migrated from IUCLID 5.6
Principles of method if other than guideline:
Original formulation of the base-line concept – it is published in Dimitrov S., Dimitrova N., Parkerton T., Comber M., Bonnell M., Mekenyan O. (2005) Base-line model for identifying the bioaccumulation potential of chemicals, SAR and QSAR in Environmental Research, 16, 6, 531-554.
GLP compliance:
no
Details on estimation of bioconcentration:
The base-line modeling concept presented in this work is based on the assumption of a maximum bioconcentration factor (BCFmax) with mitigating factors (Fi) that reduce the BCF. The maximum bioconcentration potential should have chemicals which are small sized (to passively penetrate skin
membrane), non-ionized and not metabolized. Hence, BCFmax was described by the multi-compartment partitioning model for passive diffusion. The significance of different mitigating factors associated either with interactions with an organism (e.g., metabolism) or bioavailability (ionization,
size) were investigated. The most important mitigating factor was found to be metabolism. Accordingly, a simulator for fish liver was used in the model, which has been trained to reproduce fish metabolism based on related mammalian metabolic pathways. Other significant mitigating factors, depending on the chemical structure, e.g. molecular size and ionization were also taken into account in the model.
Type:
other: log BCF
Value:
1.61 dimensionless
Type:
BCF
Value:
41 dimensionless

The validity of the (Q)SAR model is characterised according to the OECD principles.

Validity criteria fulfilled:
yes
Conclusions:
The log BCF value for 2,2',6,6'-tetra-tert-butyl-4,4'-methylenediphenol (CAS 118-82-1) was predicted by making use of base-line model.
The predicted value is log BCF 1.61 (BCF 41).
Executive summary:

Original formulation of the base-line concept – it is published in Dimitrov S., Dimitrova N., Parkerton T., Comber M., Bonnell M., Mekenyan O. (2005) Base-line model for identifying the bioaccumulation potential of chemicals, SAR and QSAR in Environmental Research, 16, 6, 531-554.

The log BCF value for 2,2',6,6'-tetra-tert-butyl-4,4'-methylenediphenol (CAS 118-82-1) was predicted by making use of base-line model. The predicted value is log BCF 1.61 (BCF 41). In parallel with the model prediction the modeling system provides warning message for extremely low water solubility of the target chemical 0.00014 - 0.0005 mg/l .

Description of key information

For aquatic bioaccumulation, several studies are available for the substance. The key study, a flow through study in rainbow trout conducted at the saturation concentration yielded a steady state BCF of 600 for the test substance and 1146 based on total radioactivity. However, other studies report BCFs of 4000 to 9000. The value of BCF from the key study has been taken as the key value for the chemical safety assessment.

Key value for chemical safety assessment

BCF (aquatic species):
600 L/kg ww

Additional information

For aquatic bioaccumulation, several studies are available for the substance. The key study, a flow through study in rainbow trout conducted at the saturation concentration yielded a steady state BCF of 600 for the test substance and 1146 based on total radioactivity. However, other studies report BCFs of 4000 to 9000 - but these are noted as being wholely unreliable studies with a Klimisch score of 4 in each case. As only one reliable study is available, the results of it should be taken as the key value for the chemical safety assessment for the the assessment of the aquatic bioaccumulation. Therefore, it can be concluded that, based on the reliable evidence presented, the BCF of the test substance is 600, and it does not meet the requirements for being considered as bioaccumulative in the aquatic compartment.

 

Additionally to this, a study conducted by Wildlife International (Project No.: 471A-126) assessed juvenile rainbow trout induced with 30 μg AN2/kg fish wet weight through intra-peritoneal injections at four time points over the course of a week. After the induction period, the livers were harvested, pooled and homogenized to allow for the hepatic microsomal fraction to be extracted for the assay. An assay was carried out to measure metabolism. A positive control of fluroxypyr-methylheptyl ester and three negative controls of heat-inactivated, no-NADPH, and buffer control groups were included. Samples were analysed using HPLC β-RAM. The results showed that there was no metabolism of the test substance, making bioaccumulation of the test substance possible as it is not being broken down within the liver. However, scientific literature indicates variable validity of hepatic microsomal fractions for indicating metabolic potential for some xenobiotics.