6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol

Bioaccumulation: aquatic / sediment

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:

29 September 2017 and 20 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was conducted in accordance with the requirements of OECD Chemicals Testing Guideline No.305 Bioaccumulation in Fish: Aqueous and Dietary Exposure (October 2012), section III: Dietary Exposure Bioaccumulation Fish Test. Attached justification is below.

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -III: Dietary Exposure Bioaccumulation Fish Test

Deviations:

yes

Remarks:

. On one occasion, the dark period was recorded as 4.5 rather 8 hours. No impact was observed after this deviation.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further information.

Radiolabelling:

yes

Remarks:

on phenyl ring.

Details on sampling:

Total [14C]-Residue Analysis
Total Radioactive Residues (TRR) in Diet
Samples of the prepared diet were taken for determination of [14C]-residues on the day of preparation, Day 0 (day of fish addition) and Day 14 (end of uptake/start of depuration). Solvent control samples were taken on Day 0 but no sample was available for testing at Day 14.

Total Radioactive Residues (TRR) in Fish

To determine the concentration of [14C]-residues in fish, five fish from each vessel were taken on Days 0, 4, 7, 11, 19, 28 and 38 of the depuration phase.
On each sampling occasion, the fish (selected at random) were removed from each tank, rinsed quickly with water using a wash bottle and gently blotted dry. They were then concussed (by striking the cranium) and killed by severing the spinal cord above the opercular region followed by destruction of the brain. Each fish was then wet weighed and the total length measured. Sampling was conducted at a similar time at each sampling occasion (± 1 hour) immediately prior to the next feed.
Samples were homogenised by maceration. For solvent control samples, homogenised tissue was then combusted in oxygen using a Harvey Biological Sample Oxidiser, model OX 501. Homogenised tissue from fish fed diet at the test concentration was extracted to recover radioactivity before also being combusted as above.

Lipid Analysis on Fish (Gravimetric Method)
Lipid analysis was performed on the fish (five fish per timepoint) removed from the stock fish on Day 0 and from the solvent control vessel on Day 0 of the depuration phase and at the end of the test. A weighed sub-sample (ca 1.0 g) was removed from the homogenised tissue of each fish and placed in a glass culture tube. Extraction solvent (chloroform:methanol, 1:1 v/v, approximately 10 mL) was added to the homogenised tissue sample, shaken and left to stand at room temperature for approximately 3 hours. The sample was centrifuged (2500 rpm for 15 minutes), the supernatant removed and decanted into a 50 mL centrifuge tube. A second extraction solvent (chloroform:ethanol, 2:1 v/v, approximately 10 mL) was added to the homogenised tissue and, following shaking, the sample was left to stand at room temperature for approximately 24 hours. Following centrifugation (2500 rpm for 15 minutes), the supernatant was removed and added to the first extract. The extraction was repeated (chloroform:ethanol, 2:1 v/v, approximately 10 mL, shaken well then left at room temperature for approximately 24 hours). The sample was then centrifuged once more (2500 rpm for 15 minutes), the supernatant removed and added to the previous extracts.

Approximately 8.0 mL of HPLC grade water was added to the pooled supernatants and the contents shaken well. Following centrifugation (2500 rpm for 15 minutes), the upper aqueous layer was removed and discarded. A solvent (methanol:chloroform:water, 48:5:47 v/v/v, 20 mL) was added to the lower organic layer and shaken well. The mixture was centrifuged (2500 rpm for 15 minutes) and the lower layer transferred to a weighed scintillation vial. The upper layer was discarded. The sample was concentrated to dryness under a gentle stream of nitrogen. The lipid residue in the vial was determined after placing in a vacuum desiccator for approximately 20 minutes and drying to a constant weight.

Vehicle:

no

Details on preparation of test solutions, spiked fish food or sediment:

Preparation of Test Diet
The radio-labelled spiking solution was prepared by adding 22.6 mL of [14C] 6,6’ di tert-butyl-4,4’-butylidenedi-m-cresol Stock Solution 2 to acetone (100 mL) to give a solution containing 8.085 MBq, equivalent to 1.5 mg of test substance. The diet was dosed by adding ca 100 g of fish feed to a round bottom flask to which the spiking solution was added. The diet/spiking solution mix was then rotary evaporated to dryness to give the 15 mg/kg test concentration.
Storage of the spiked diet for the definitive test was under refrigerated conditions (2 to 8 ºC).
The solvent control was prepared in a similar manner using acetone (100 mL) only, applied to ca 100 g of fish feed.
Preliminary testing of Spiked Diet
A nominal concentration of test substance selected was 15 mg/kg of diet to allow chromatographic analysis of diet and fish tissue if required. A stock solution of [14C] 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in acetone (Stock Solution 2, 65 µg/mL) was prepared by dissolving a portion (5.48 mg) of the supplied solid in acetone (85 mL) and used for the preparation of spiked diet. A treatment solution was prepared by adding 5 mL of [14C]-6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol Stock Solution 2 to 20 mL of acetone. The diet was dosed by adding 20.027 g of fish feed to a round bottom flask to which the treatment solution was added. The diet/solvent stock solution mix was then rotary evaporated to dryness to give a 15 mg/kg test concentration.
Three samples from the diet were taken to assess recovery of radioactivity by solubilisation but insufficient radioactivity (63%) was recovered to allow this to be the quantification method.
Weighed ca 3 g subsamples of the spiked feed were taken after 0, 7 and 14 days refrigerator storage. The subsamples were extracted using multiple 5 mL aliquots of acetonitrile and acetone. Concentrated acetonitrile and acetone extracts were produced by rotary evaporation and analysed by HPLC.
Solid residues after extraction were aliquoted in triplicate (ca 0.3 g), combusted using a Harvey Oxidiser. The combusted products were absorbed in Permafluor® E+ and Carbo-Sorb® E (2:1 v:v) and the radioactivity absorbed was determined by LSC.
Palatability Test
A nominal test substance concentration of 15 mg/kg was selected for the study. A stock solution of [14C]-6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in acetone (Stock Solution 2) was created and used for the preparation of spiked diet. The diet (10 g) was mixed with an aliquot of the stock solution (2.3 mL) and further acetone (12.5 mL) and the solvent removed by rotary evaporation.
A pre-test was conducted whereby seven fish were fed spiked diet at a concentration of 15 mg/kg over a period of 14 days. A solvent control group, consisting of fish fed diet free from the test substance, were also included for comparison. The aim of the test was to ensure that the spiked diet was palatable and did not cause any adverse effects on the fish.
Definitive Test
The definitive test was therefore conducted at a nominal test substance concentration of 15 mg/kg.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

The Oncorhynchus mykiss (rainbow trout) used in the definitive test were obtained from Northern Trout (formerly Brow Well Fisheries), Hebden, Skipton, North Yorkshire, United Kingdom details of whom are maintained in Smithers Viscient (ESG) Ltd. records. On arrival at the test facility, a unique batch number was assigned and the health status of the fish was assessed prior to use.

The test vessels were glass aquaria with capacities of 81 and 82.7 L for the solvent control and test vessel respectively. Treated mains water was continuously supplied, from the laboratory treated mains water supply, at a nominal flow rate of 350 ± 10% mL/minute. The flow rates of the treated mains water into each test vessel were checked daily during the test.

The fish were held in a temperature controlled room under artificial light, with a 16 hour light: 8-hour dark photo-period, in holding tanks at a density appropriate to their size, under continuous water renewal (flow-through) conditions. On one occasion, the dark period was recorded as 4.5 rather 8 hours. No impact was observed after this deviation.

The fish were acclimated to the laboratory conditions for at least 14 days before being used in tests.

The fish were fed with a proprietary fish food, which was added to the holding tank in quantities dictated by the size of the fish. Uneaten food and debris was siphoned or cleaned from the tanks as required.

Fish mortalities in the holding tank were recorded as they occurred. Use of the fish for testing was considered acceptable if the cumulative mortality in the batch was <5% during the 7 day period preceding the proposed test start date. If the mortality exceeded 5% mortality in this same period, or disease was apparent, the batch of fish was not to be used for testing and acclimation would be increased for at least another 7 days before mortality rate re-assessment for testing.

At the start of the test, sixty Oncorhynchus mykiss, randomly selected from a holding stock, were added to the solvent control and test vessel. At the start of the test, the fish had a mean total length of 7.7 cm and a mean wet weight of 4.94 g. The fish were considered acceptable for use on the test, as the fish weight to volume ratio was 0.59 g/L/24 hours.
Waste was siphoned daily from the tanks, generally within 1 hour after feeding.
Throughout the test all fish were observed on a daily basis and records were made on their condition and general behaviour.

Water Source and Treatment
The water used for the holding of fish stocks and for the toxicity tests was laboratory mains supply. The water was pumped to the laboratory through an activated carbon filter.

Quality of Laboratory Mains Supply
A typical Certificate of Analysis of the laboratory mains supply water is available for inspection.

Route of exposure:

feed

Justification for method:

dietary exposure method used because stable, measurable water concentrations cannot be maintained

Remarks:

Given the low water solubility (< 4 μg/l at 20 deg C), it was not possible to demonstrate a stable and fully dissolved concentration of the test substance in water. Hence the dietary route was considered to be the most appropriate route of dosing.

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

14 d

Total depuration duration:

38 d

Hardness:

The water hardnesses at Day 0 in the solvent control and 15 mg/kg test vessels were 128 and 123 mg/L as CaCO3, respectively.

Test temperature:

15 ± 2ºC

pH:

7.6

Dissolved oxygen:

Diissolved oxygen remained at ≥80% of air saturation value (ASV).

TOC:

2 mg/l

Salinity:

Not applicable.

Conductivity:

205 uS/cm

Details on test conditions:

Test Organism
Healthy Oncorhynchus mykiss (rainbow trout) were supplied by Northern Trout (formerly Brow Well Fisheries), United Kingdom, details of which are maintained in Smithers Viscient (ESG) Ltd. records.

The fish were acclimatised to the test conditions for at least 14 days prior to testing. The holding tanks were maintained under flow-through conditions and the fish were fed a proprietary food at 1.5% of body weight per day, which was considered to contain no contaminants capable of influencing the outcome of the test. Details of stock fish holding conditions are presented in Appendix 3. The mortality rate of the stock batch of fish was 0% in the seven days prior to the definitive test and the fish were free from disease or any visual abnormalities.

Test water
The water used in the study was treated mains water (treated via particulate and activated carbon filters to remove the chlorine).

Food
The food used for the test was the same proprietary food as used during the acclimatisation period. The crude protein and lipid content of the food was 56% and 18%, respectively.

Preliminary testing of Spiked Diet
A nominal concentration of test substance selected was 15 mg/kg of diet to allow chromatographic analysis of diet and fish tissue if required. A stock solution of [14C] 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in acetone (Stock Solution 2, 65 µg/mL) was prepared by dissolving a portion (5.48 mg) of the supplied solid in acetone (85 mL) and used for the preparation of spiked diet. A treatment solution was prepared by adding 5 mL of [14C]-6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol Stock Solution 2 to 20 mL of acetone. The diet was dosed by adding 20.027 g of fish feed to a round bottom flask to which the treatment solution was added. The diet/solvent stock solution mix was then rotary evaporated to dryness to give a 15 mg/kg test concentration.
Three samples from the diet were taken to assess recovery of radioactivity by solubilisation but insufficient radioactivity (63%) was recovered to allow this to be the quantification method.
Weighed ca 3 g subsamples of the spiked feed were taken after 0, 7 and 14 days refrigerator storage. The subsamples were extracted using multiple 5 mL aliquots of acetonitrile and acetone. Concentrated acetonitrile and acetone extracts were produced by rotary evaporation and analysed by HPLC.
Solid residues after extraction were aliquoted in triplicate (ca 0.3 g), combusted using a Harvey Oxidiser. The combusted products were absorbed in Permafluor® E+ and Carbo-Sorb® E (2:1 v:v) and the radioactivity absorbed was determined by LSC.

Palatability Test
A nominal test substance concentration of 15 mg/kg was selected for the study. A stock solution of [14C]-6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in acetone (Stock Solution 2) was created and used for the preparation of spiked diet. The diet (10 g) was mixed with an aliquot of the stock solution (2.3 mL) and further acetone (12.5 mL) and the solvent removed by rotary evaporation.
A pre-test was conducted whereby seven fish were fed spiked diet at a concentration of 15 mg/kg over a period of 14 days. A solvent control group, consisting of fish fed diet free from the test substance, were also included for comparison. The aim of the test was to ensure that the spiked diet was palatable and did not cause any adverse effects on the fish.

Definitive Test
The definitive test was therefore conducted at a nominal test substance concentration of 15 mg/kg.

Preparation of Test Diet
The radio-labelled spiking solution was prepared by adding 22.6 mL of [14C] 6,6’ di tert-butyl-4,4’-butylidenedi-m-cresol Stock Solution 2 to acetone (100 mL) to give a solution containing 8.085 MBq, equivalent to 1.5 mg of test substance. The diet was dosed by adding ca 100 g of fish feed to a round bottom flask to which the spiking solution was added. The diet/spiking solution mix was then rotary evaporated to dryness to give the 15 mg/kg test concentration.
Storage of the spiked diet for the definitive test was under refrigerated conditions (2 to 8 ºC).
The solvent control was prepared in a similar manner using acetone (100 mL) only, applied to ca 100 g of fish feed.

Test Vessels
The test vessels were glass aquaria with capacities of 81 and 82.7 L for the solvent control and test vessel respectively. Treated mains water was continuously supplied, from the laboratory treated mains water supply, at a nominal flow rate of 350 ± 10% mL/minute. The flow rates of the treated mains water into each test vessel were checked daily during the test.
The test vessels were maintained in a temperature and light controlled laboratory with a 16:8 hour light:dark cycle and an approximate 30 minute dawn/dusk transition period.

Fish Addition and Observations
At the start of the test, sixty Oncorhynchus mykiss, randomly selected from a holding stock, were added to the solvent control and test vessel. At the start of the test, the fish had a mean total length of 7.7 cm and a mean wet weight of 4.94 g. The fish were considered acceptable for use on the test, as the fish weight to volume ratio was 0.59 g/L/24 hours.
Waste was siphoned daily from the tanks, generally within 1 hour after feeding.
Throughout the test all fish were observed on a daily basis and records were made on their condition and general behaviour.
Uptake phase
During the uptake phase, the fish in the test concentration were fed the diet spiked with the test substance. A solvent control group was also included which were fed diet prepared with the solvent carrier only. The test fish were fed 1.5% body weight per day. The amount of food required was recalculated after each sample timing based on the wet weights of the fish sampled.
The start of the uptake phase began at the time the fish were first fed the spiked diet (Day 0). The fish were fed at 12:20 ± 1 hour for the duration of the test. The water flow was suspended during feeding. No uneaten food was visible in the tanks within 30 minutes of feeding therefore it was considered that all the food had been consumed.
The duration of the uptake phase was 14 days after which the depuration phase was initiated.

Depuration phase
During the depuration phase, the fish were fed diet free from the test substance. The depuration phase commenced when the fish were first fed the unspiked diet (Day 14 uptake/Day 0 depuration). The feeding rate and time were the same as those used for the uptake phase except on Day 29 when the fish were fed at 11:15. This deviation was not considered not to affect the integrity of the test given that the fish were in the depuration phase and no longer being fed the spiked diet.
The duration of the depuration phase was 38 days.

Water Quality Determinations
The temperature was monitored continuously in the test area throughout the duration of the test. The temperature in each vessel was measured daily. The dissolved oxygen content and pH were measured in each vessel on Day 0 (pre-fish addition), and then weekly throughout the duration of the test.
Total hardness in the each test vessel was determined on Day 0. Total organic carbon (TOC) was measured on Day 0.

Total [14C]-Residue Analysis

Total Radioactive Residues (TRR) in Diet
Samples of the prepared diet were taken for determination of [14C]-residues on the day of preparation, Day 0 (day of fish addition) and Day 14 (end of uptake/start of depuration). Solvent control samples were taken on Day 0 but no sample was available for testing at Day 14.
Total Radioactive Residues (TRR) in Fish
To determine the concentration of [14C]-residues in fish, five fish from each vessel were taken on Days 0, 4, 7, 11, 19, 28 and 38 of the depuration phase.
On each sampling occasion, the fish (selected at random) were removed from each tank, rinsed quickly with water using a wash bottle and gently blotted dry. They were then concussed (by striking the cranium) and killed by severing the spinal cord above the opercular region followed by destruction of the brain. Each fish was then wet weighed and the total length measured. Sampling was conducted at a similar time at each sampling occasion (± 1 hour) immediately prior to the next feed.
Samples were homogenised by maceration. For solvent control samples, homogenised tissue was then combusted in oxygen using a Harvey Biological Sample Oxidiser, model OX 501. Homogenised tissue from fish fed diet at the test concentration was extracted to recover radioactivity before also being combusted as above.

Lipid Analysis on Fish (Gravimetric Method)

Lipid analysis was performed on the fish (five fish per timepoint) removed from the stock fish on Day 0 and from the solvent control vessel on Day 0 of the depuration phase and at the end of the test. A weighed sub-sample (ca 1.0 g) was removed from the homogenised tissue of each fish and placed in a glass culture tube. Extraction solvent (chloroform:methanol, 1:1 v/v, approximately 10 mL) was added to the homogenised tissue sample, shaken and left to stand at room temperature for approximately 3 hours. The sample was centrifuged (2500 rpm for 15 minutes), the supernatant removed and decanted into a 50 mL centrifuge tube. A second extraction solvent (chloroform:ethanol, 2:1 v/v, approximately 10 mL) was added to the homogenised tissue and, following shaking, the sample was left to stand at room temperature for approximately 24 hours. Following centrifugation (2500 rpm for 15 minutes), the supernatant was removed and added to the first extract. The extraction was repeated (chloroform:ethanol, 2:1 v/v, approximately 10 mL, shaken well then left at room temperature for approximately 24 hours). The sample was then centrifuged once more (2500 rpm for 15 minutes), the supernatant removed and added to the previous extracts.

Approximately 8.0 mL of HPLC grade water was added to the pooled supernatants and the contents shaken well. Following centrifugation (2500 rpm for 15 minutes), the upper aqueous layer was removed and discarded. A solvent (methanol:chloroform:water, 48:5:47 v/v/v, 20 mL) was added to the lower organic layer and shaken well. The mixture was centrifuged (2500 rpm for 15 minutes) and the lower layer transferred to a weighed scintillation vial. The upper layer was discarded. The sample was concentrated to dryness under a gentle stream of nitrogen. The lipid residue in the vial was determined after placing in a vacuum desiccator for approximately 20 minutes and drying to a constant weight.

Conditions for HPLC Analysis with UV Detection

Instrumentation: Agilent 1100 or 1200 with LabLogic β-ram Radiodetector
Column: Agilent Eclipse XBF-C8 150 mm x 4.6 mm
Mobile phase A: 0.01% Trifluoroacetic acid in water
Mobile phase B: 0.01% Trifluoroacetic acid in acetonitrile
Gradient: Time (min) %A %B
0 90 10
3 90 10
8 0 100
28 0 100
28.1 90 10
32 90 10
Flow rate: 1.0 mL/min (eluent), 2 mL/min (scintillant)
Operating temperature: 40°C
UV wavelength: 225 nm
CALCULATION OF RESULTS
Determination of Percent of Applied Radioactivity in Samples

Total radioactivity (kBq) applied to sample
(1 kBq = 60 x 103 dpm) = T
Initial sample weight (g) = I
Sub-sample weight (g) = SS
Total weight of sample for LSC assay (g) = A
Weight (g) of aliquots assayed by LSC = W
Background corrected radioactivity (dpm) in aliquots assayed by LSC = D
Concentration (dpm/g) of radioactivity in sample (C) = D/W

The individual estimates of concentration derived from the duplicate aliquots were averaged to provide a mean concentration of radioactivity in the sample.
Radioactivity (kBq) in assayed sample (B) = C x A / 60,000
Percent of applied represented by sample (X) = B/T x I/SS x 100%

Nominal and measured concentrations:

A nominal test substance concentration of 15 mg/kg was selected for the study

Reference substance (positive control):

not required

Details on estimation of bioconcentration:

BCF is estimated from the BMF and associated data using the OECD Section 3 Software: Environmental Fate and Behaviour (Softwares for TG 305 and TG 318). This is available at:

http://www.oecd.org/chemicalsafety/testing/section-3-environmental-fate-behaviour-software-tg-305.htm

All 3 method outputs are considered by the tool and the output results are listed below.

Lipid content:

6.83 %

Time point:

start of exposure

Lipid content:

7.53 %

Time point:

other: Day 0 of the depuration phase

Lipid content:

8.7 %

Time point:

other: End of the study (depuration phase)

Lipid content:

7.69 %

Time point:

other: mean value used for lipid correction of the BMF.

Key result

Conc. / dose:

15 other: mg/kg food

Temp.:

15 °C

pH:

7.2

Type:

BMF

Value:

0.016 dimensionless

Basis:

whole body w.w.

Time of plateau:

38 d

Calculation basis:

steady state

Key result

Conc. / dose:

15 other: mg/kg food

Temp.:

15 °C

pH:

7.2

Type:

BCF

Value:

>= 14.2 - <= 158.3 L/kg

Basis:

other: Calculation from BMF using OECD Software tool for OECD 305

Remarks:

Method 1

Time of plateau:

38 d

Calculation basis:

steady state

Key result

Conc. / dose:

15 other: mg/kg food

Temp.:

15 °C

pH:

7.2

Type:

BCF

Value:

28.1 L/kg

Basis:

other: Calculation from BMF using OECD Software tool for OECD 305

Remarks:

Method 2

Time of plateau:

38 d

Calculation basis:

steady state

Key result

Conc. / dose:

15 other: mg/kg food

Temp.:

15 °C

pH:

7.2

Type:

BCF

Value:

684.7 L/kg

Basis:

other: Calculation from BMF using OECD Software tool for OECD 305

Remarks:

Method 3

Time of plateau:

38 d

Calculation basis:

steady state

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

10.8 d

Remarks on result:

other: growth corrected half-life of 19.1 days

Key result

Rate constant:

other: k2, depuration rate constant

Value:

0.064

Key result

Rate constant:

other: kg, growth rate constant

Value:

0.028

Key result

Rate constant:

other: k2g , growth-corrected depuration rate constant

Value:

0.036

Details on kinetic parameters:

See "any other information" below.

Metabolites:

Not measured

Results with reference substance (positive control):

Not measured.

Details on results:

Homogeneity
Triplicate analysis of the spiked diet showed concentrations with a maximum deviation from the mean of 1.4%, based on total radioactive residues (TRR) of 13.8 mg/kg (equivalent to 92% of nominal value) indicating that the dose preparation method resulted in a homogenous sample. Results are presented in Table 1.

Stability of Spiked Diet
The results from the stability assessment are presented in Table 1. Analysis of the diet showed recoveries as parent of 95.4, 96.9 and 96.0% for the 0, 7 and 14 day samples respectively, indicating that the test substance had not degraded and could be considered stable in the diet for a period of 14 days under refrigerated storage conditions.

Palatability Test
During the palatability test, no adverse effects or mortality were observed in the fish fed the spiked diet. In addition, no abnormal feeding behaviour was observed compared to the control fish fed diet free from the test substance indicating that the test diet was palatable. It was therefore considered that a test concentration of 15 mg/kg was appropriate for the definitive test.

Definitive Test
Fish Observations
There were no treatment related mortalities in the solvent control or test concentration during the study. One mortality was observed in the solvent control vessel presenting only damage to gill covers. In addition, uneaten food was not observed in the test vessels after each feeding.

Water Quality
Environmental measurements during the uptake and depuration phases are presented in Table 2. The environmental conditions in the test vessels remained within acceptable limits throughout the duration of the study. The temperature was maintained at 15 ± 2ºC and dissolved oxygen remained at ≥80% of air saturation value (ASV). The water hardnesses at Day 0 in the solvent control and 15 mg/kg test vessels were 128 and 123 mg/L as CaCO3, respectively.

Concentrations in the Spiked Diet
The concentrations in the dosed diet are presented in Table 3. There was no detectable radioactivity in the solvent control diet.
The total radioactive residue (TRR) concentrations at the time of dosing of the diet, on Day 0 (fish addition) and Day 14 (end of uptake phase) were 13.8 and 14.0 mg/kg, respectively. In addition, corresponding HPLC analysis (see Appendix 2) showed chemical purity to be 94.1% and 94.6%, respectively. These results indicated that the diet had been dosed appropriately and no degradation of the test substance occurred in the diet over the duration of the uptake phase.

Concentrations in the Fish
The wet weight and total lengths of the fish taken at each sample timing are presented in Table 4. The [14C] residues in whole tissues as parent equivalents are presented in Table 5. The results are also presented graphically in Figure 1. There was no detectable radioactivity in the solvent control fish (sampled at the same time as the dosed diet).

At the start of the depuration phase the mean concentration in the fish tissues was 0.283 mg/kg. The concentrations in the tissues decreased over the depuration period to a mean concentration in the tissues of 0.017 mg/kg at Day 28 and were 0.021 mg/kg at Day 38 indicating that a plateau had been reached.

HPLC analysis of extracts of fish tissues from days 0, 4 and 7 of depuration confirmed [14C] 6,6’ di tert-butyl-4,4’-butylidenedi-m-cresol to be the major component present.

Lipid Analysis
Expressed as a percent of body weight, the lipid content of the fish at the start of the test (Day 0), on Day 0 of the depuration phase and end of the test were 6.83, 7.53 and 8.70%, respectively (see Table 6). A mean value of 7.69% was used for lipid correction of the BMF.

Biomagnification of [14C]-Residues
The food ingestion rate constant (I) was 0.015 mg/g/day based on a daily feeding rate of 1.5% of body weight. The overall depuration rate constant (k2) was estimated to be 0.0643.

The mean concentration in the fish on Day 0 of the depuration phase (C0) was 0.131 mg/kg. The mean concentration in the food (Cfood) was 13.9 mg/kg and the duration of the feeding period (t) was 14 days. Therefore, the assimilation efficiency (α) was calculated to be 0.0678.
Based on a mean lipid content in the fish of 7.69% and a diet lipid content of 18%, the lipid correction factor (Lc) was calculated to be 0.427.

Validity Criteria
All validity criteria were satisfied therefore the test is considered valid:
• Water temperature variation - (≤ ± 2ºC)
• Dissolved oxygen concentration – (> 60% of the air saturation value)
• Concentration of the test substance in the fish food before and at the end of the uptake phase (within ± 20%)
• Homogeneity of the test substance in the spiked diet - (not more than ± 15% from the mean)
• Test substance not detected in the un-spiked diet or solvent control fish
• The mortality or other adverse effects in both solvent control and treated fish at the end of the test – (< 10%)

The biomagnification factor (BMF) of [14C]-6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in the fish tissue was determined to be 0.0158 based on an uptake phase of 14 days and a depuration phase of 38 days. The BMF values corrected for growth and lipid were calculated to be 0.0281 and 0.0371, respectively. The results indicated that biomagnification of the test substance in the fish was low when dosed via the diet. Subsequently, the [14C]-6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was rapidly depurated from the fish with a half-life of 10.8 days (growth corrected half-life of 19.1 days).
Using the OECD Software tool for OECD 305, the following results were obtained for the 3 methods available:

Method 1: Estimated BCF Range of 14.2 to 158.3
Method 2: Estimated BCF of 28.1
Method 3: Estimated BCF of 684.7.

All 3 values are below the the 2000 L/kg threshold, and the substance is considered to be not bioaccumulative.

The rate constants were as follows;

k2	kg	k2g
0.0643	0.0281	0.0362

 k₂= depuration rate constant

k_g= growth rate constant
k_2g= growth-corrected depuration rate constant

The biomagnification factors for whole fish tissue were as follows:

 

BMF	BMFG	BMFL	t1/2	t1/2g
0.0158	0.0281	0.0371	10.8	19.1

BMF = biomagnification factor

BMF_G= growth corrected biomagnification factor

BMF_L= lipid corrected biomagnification factor corrected

t_1/2= half-life (days)

t_1/2g= growth corrected half-life (days)

Table 1
Diet homogeneity and stability analysis

Homogeneity analysis

Nominal concentration (mg/kg)	Nominal concentration (dpm/g)	Sample	Dpm per g	Mean dpm per g	% deviation from mean	Calculated concentration (mg/kg)	% nominal value
15	4860000	1	4542260	4480994	1.4	13.83	92.2
		2	4451505		0.7
		3	4449217		0.7

  

Stability analysis

 

Day		Extracts (summed)		Recovery (%)		Recovery as parent (%)
0		Acetonitrile		94.4*		95.4
		Acetone		2.1*		NA
		Residue		0.9		NA
		Total		97.4		95.4
7		Acetonitrile		71.4		70.8
		Acetone		26.2		26.1
		Residue		1.9		NA
		Total		99.5		96.9
14		Acetonitrile		79.5		78.7
		Acetone		17.3		17.3
		Residue		5.4		NA
		Total		101.9		96.0
Overall mean		Total		99.6		96.1

NA – Not applicable
* Samples combined during concentration

 

Table 2
Water quality determinations during the definitive test

 

Parameter	Nominal concentration (mg/kg)	Day
		Uptake phase			Depuration phase
		0	7	14	7	14	21	28	35	38
pH	Solvent control	7.75	7.28	7.29	7.55	7.17	7.30	7.45	7.32	7.23
	15	7.70	7.26	7.22	7.42	7.14	7.24	7.40	7.22	7.18
Dissolved oxygen % ASV	Solvent control	81	87	87	84	90	84	87	80	84
	15	80	82	88	86	89	84	86	81	84

ASV       Air saturation value (mg/L value presented in brackets)

 

Temperatures (ºC) during the uptake phase

 

Nominal concentration (mg/kg)	Day
	0		1		2		3		4		5		6
Solvent control	14.7		15.0		15.1		15.4		14.7		14.7		14.8
15	14.8		14.9		15.0		15.4		14.7		14.7		14.8
Nominal concentration (mg/kg)	Day
	7	8		9		10		11		12		13		14
Solvent control	15.1	15.2		15.2		15.2		15.1		15.2		15.0		15.2
15	15.2	15.2		15.2		15.2		15.2		15.2		15.1		15.2

 Table 2 continued
Water quality determinations during the definitive test

 

Temperatures (ºC) during the depuration phase

 

Nominal concentration (mg/kg)	Day
	0	1	2	3	4	5	6
Solvent control	15.2	15.3	15.2	15.2	15.2	15.1	15.2
15	15.2	15.3	15.3	15.3	15.3	15.2	15.2
Nominal concentration (mg/kg)	Day
	7	8	9	10	11	12	13
Solvent control	15.4	15.2	15.2	15.3	15.2	15.2	15.2
15	15.4	15.2	15.2	15.6	15.2	15.2	15.2
Nominal concentration (mg/kg)	Day
	14	15	16	17	18	19	20
Solvent control	15.4	15.1	15.1	15.0	15.1	15.1	15.1
15	15.4	15.2	15.1	15.1	15.2	15.2	15.2
Nominal concentration (mg/kg)	Day
	21	22	23	24	25	26	27
Solvent control	15.1	15.1	15.1	15.1	15.1	15.1	15.1
15	15.2	15.2	15.2	15.2	15.1	15.2	15.3
Nominal concentration (mg/kg)	Day
	28	29	30	31	32	33	34
Solvent control	15.1	15.1	14.7	14.6	14.7	15.0	14.4
15	15.2	15.2	14.8	14.7	14.8	15.1	14.5
Nominal concentration (mg/kg)	Day
	35	36	37	38
Solvent control	14.3	13.6	13.6	14.2
15	14.3	13.8	13.8	14.2

Maximum water temperature – 15.6°C

Minimum water temperature – 13.6°C

 

The test area maximum temperature was - 15.9°C

The test area minimum temperature was - 13.6°C

Table 3
Definitive test diet analysis

 

Day	Nominal concentration (mg/kg)	Aliquot weight (g)	dpm in aliquot	Background dpm	dpm per g	Calculated concentration (mg/kg)	% nominal value
0	Solvent control	0.1002	54	53	10	ND	NA
		0.1054	57		38
		0.1129	62		80
	15	0.1071	486535	59	4542260	13.8	92
		0.1110	494176		4451505
		0.1085	482799		4449217
14	15	0.1034	465631	42	4502795	14.0	93
		0.1015	465058		4581438
		0.1060	474647		4477406

ND – Not Detected

NA – Not applicable

  

	Recovery of radioactivity (%)
Day	Initial extract	Extracted residue	Total		As parent by HPLC
0	95.0	5.1	100.1		94.1
14	96.5	3.9	100.4		94.6

 

Table 4
Length and weights of fish sampled during the test

Timepoint (Day)	Fish number	Solvent control		15 mg/kg
		Total length (cm)	Wet weight (g)	Total length (cm)	Wet weight (g)
0 uptake	1	6.7	2.8	6.7	2.8
	2	8.4	6.5	8.4	6.5
	3	7.5	4.5	7.5	4.5
	4	7.4	4.5	7.4	4.5
	5	8.3	6.4	8.3	6.4
	Mean	7.7	4.94	7.7	4.94
	SD	0.7	1.54	0.7	1.54
14 uptake/0 depuration	1	9.7	9.0	7.3	3.6
	2	8.8	7.0	8.3	5.7
	3	8.3	6.0	9.7	9.5
	4	9.1	7.3	8.6	7.1
	5	8.4	6.1	7.5	5.3
	Mean	8.9	7.08	8.3	6.24
	(SD)	0.6	1.21	1.0	2.21
4 depuration	1	6.4	3.3	8.9	7.7
	2	8.5	7.1	9.5	9.1
	3	8.8	8.1	8.4	6.6
	4	8.0	6.5	8.5	8.9
	5	8.7	7.3	6.7	4.2
	Mean	8.1	6.46	8.4	7.30
	SD	1.0	1.86	1.0	2.00
7 depuration	1	9.2	8.2	8.6	6.9
	2	9.3	8.4	9.1	7.9
	3	8.4	6.3	8.4	6.8
	4	9.7	9.4	7.8	5.2
	5	9.1	7.8	8.8	6.6
	Mean	9.1	8.02	8.5	6.68
	SD	0.5	1.13	0.5	0.97
11 depuration	1	9.5	9.3	9.0	7.5
	2	7.7	5.3	9.1	7.9
	3	9.5	9.8	8.1	5.4
	4	9.7	10.2	10.9	12.8
	5	9.2	8.9	10.0	10.0
	Mean	9.1	8.70	9.4	8.72
	SD	0.8	1.96	1.1	2.80
19 depuration	1	11.8	18.2	8.7	6.8
	2	9.0	7.6	10.1	10.8
	3	10.7	11.9	10.2	13.2
	4	10.3	12.3	8.9	6.9
	5	9.3	9.7	10.7	14.0
	Mean	10.2	11.94	9.7	10.34
	SD	1.1	3.97	0.9	3.40
28 depuration	1	9.5	10.5	18.8	16.7
	2	10.2	10.8	9.8	9.8
	3	11.8	18.0	12.3	19.7
	4	1.1	16.0	10.4	12.6
	5	9.5	10.2	12.1	18.6
	Mean	8.4	13.10	12.7	15.48
	SD	4.2	3.64	3.6	4.17
38 depuration	1	10.8	12.7	11.1	14.8
	2	12.5	23.9	12.2	20.0
	3	11.3	15.6	10.4	12.7
	4	10.8	13.9	11.4	14.1
	5	11.6	16.3	12.3	20.4
	Mean	11.4	16.48	11.5	16.40
	SD	0.7	4.38	0.8	3.55

     SD – Standard deviation

Table 5
Concentrations found in the fish samples during the definitive test

Timepoint (days)	Fish number	Calculated concentration in fish (mg/kg)	Mean calculated concentration (mg/kg)
0 depuration	1	0.3890	0.2833
	2	0.2567
	3	0.2447
	4	0.2122
	5	0.3140
4 depuration	1	0.0668	0.1000
	2	0.0596
	3	0.0669
	4	0.1910
	5	0.1157
7 depuration	1	0.0874	0.0690
	2	0.1148
	3	0.0553
	4	0.0334
	5	0.0538
11 depuration	1	0.0731	0.0552
	2	0.0744
	3	0.0277
	4	0.0702
	5	0.0305
19 depuration	1	0.0140	0.0327
	2	0.0264
	3	0.0473
	4	0.0169
	5	0.0587
28 depuration	1	0.0110	0.0166
	2	0.0235
	3	0.0085
	4	0.0238
	5	0.0162
38 depuration	1	0.0111	0.0211
	2	0.0297
	3	0.0304
	4	0.0210
	5	0.0135

 

The table represents concentration found in fish fed spiked diet. No radioactivity was recovered from fish from the solvent control vessel.

Table 6
Lipid content of fish sampled during the definitive test

Timepoint (days)	Fish number	Lipid (%)	Mean Lipid (%)
0 uptake	1	8.1393	6.8288
	2	7.3103
	3	5.1139
	4	6.9971
	5	6.5835
0 depuration	1	7.2718	7.5260
	2	7.8252
	3	6.8173
	4	7.0260
	5	8.6898
38 depuration	1	6.5537	8.7003
	2	10.5000
	3	9.0575
	4	10.9289
	5	6.4615
Overall mean	-	-	7.69

 

Validity criteria fulfilled:

yes

Conclusions:

The biomagnification factor (BMF) of [14C]-6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in the fish tissue was determined to be 0.0158 based on an uptake phase of 14 days and a depuration phase of 38 days. The BMF values corrected for growth and lipid were calculated to be 0.0281 and 0.0371, respectively. The results indicated that biomagnification of the test substance in the fish was low when dosed via the diet. Subsequently, the [14C]-6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was rapidly depurated from the fish with a half-life of 10.8 days (growth corrected half-life of 19.1 days).
Using the OECD Software tool for OECD 305, the following results were obtained for the 3 methods available:

Method 1: Estimated BCF Range of 14.2 to 158.3
Method 2: Estimated BCF of 28.1
Method 3: Estimated BCF of 684.7.

All 3 values are below the the 2000 L/kg threshold, and the substance is considered to be not bioaccumulative.

Executive summary:

The biomagnification factor (BMF) of [¹⁴C]-6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in the fish tissue was determined to be 0.0158 based on an uptake phase of 14 days and a depuration phase of 38 days. The BMF values corrected for growth and lipid were calculated to be 0.0281 and 0.0371, respectively. The results indicated that biomagnification of the test substance in the fish was low when dosed via the diet. Subsequently, the [¹⁴C]-6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was rapidly depurated from the fish with a half-life of 10.8 days (growth corrected half-life of 19.1 days).

Using the OECD Software tool for OECD 305, the following results were obtained for the 3 methods available:

 

Method 1: Estimated BCF Range of 14.2 to 158.3

Method 2: Estimated BCF of 28.1

Method 3: Estimated BCF of 684.7.

 

All 3 values are below the the 2000 L/kg threshold, and the substance is considered to be not bioaccumulative.

Description of key information

Significant accumulation is not to be expected.

Key value for chemical safety assessment

BCF (aquatic species):

684.7 L/kg ww

BMF in fish (dimensionless):

0.016

Additional information

The biomagnification factor (BMF) of [14C]-6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in the fish tissue was determined to be 0.0158 based on an uptake phase of 14 days and a depuration phase of 38 days. The BMF values corrected for growth and lipid were calculated to be 0.0281 and 0.0371, respectively. The results indicated that biomagnification of the test substance in the fish was low when dosed via the diet. Subsequently, the [14C]-6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was rapidly depurated from the fish with a half-life of 10.8 days (growth corrected half-life of 19.1 days).

Using the OECD Software tool for OECD 305, the following results were obtained for the 3 methods available:

Method 1: Estimated BCF Range of 14.2 to 158.3

Method 2: Estimated BCF of 28.1

Method 3: Estimated BCF of 684.7.

All 3 values are below the the 2000 L/kg threshold, and the substance is considered to be not bioaccumulative.

Two different QSAR calculations with different models have been performed, and are considered suitable supporting evidence to the full study as follows:

(1) CAESAR: BCF = 41 L/kg

(2) EPISuite BCFBAF: BCF = 758.9 L/kg

Significant accumulation is not to be expected.