4-sec-butyl-2,6-di-tert-butylphenol

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:

February 01, 2011 to August 22, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)

Version / remarks:

OECD Guideline for Testing of Chemicals No. 305, "Bioconcentration: Flow-through Fish Test", Proposal adopted June 14, 1996.

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Radiolabelling:

yes

Remarks:

[14C ring] phenol

Details on sampling:

For the sampling schedule, six sampling points during the uptake phase and four depuration sampling points were spaced throughout the duration of the experiment, assuming a total period of 120 days.

Water
Water samples were taken from the central area of the respective test tank before feeding and immediately before fish sampling.

Treated Tanks:
Daily during the uptake phase and at selected time points during the depuration phase, duplicate samples of 10 mL were removed from the treated tanks and as far as appropriate from the corresponding mixture chambers. Radioactivity was directly determined.
Additionally at selected time intervals, duplicate samples of approximately 1000 mL (both dose levels) were taken stored at approximately -20°C until analysis. One sample per time interval was partitioned into dichloromethane and stored at approximately -20 °C until analyses by HPLC. The second sample was stored as reserve sample.

Control Tank:
Duplicate background samples were taken at the beginning, in the middle and at the end of the uptake phase and at the end of depuration phase from the control tank and directly analyzed by liquid scintillation counting (LSC).

Fish
Treated Tanks:
On each sampling occasion, six fish were collected randomly from each exposure tank, rinsed with water, sacrificed in 1.5% (v/v) 2-phenoxy-ethanol in purified water and blotted dry. Four fish were analyzed immediately following sacrifice. At two time intervals (after 40 and 60 days of accumulation) eight fish were sampled and stored at approximately -20 °C to allow analyses of metabolites.

Control Tank:
At the beginning, at the end of the accumulation period and at the end of the depuration period, six control fish were taken and four were analyzed for total radioactivity.
At two selected time points during the uptake phase ten fish were additionally sampled for lipid determination.

Vehicle:

yes

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

Preparation of Stock Solution
For the low dose (target concentration 1 μg/L), based on a total volume of 15750 L (assuming a flow-through volume of 250 L per day during 3 days pre-treatment and 60 days of treatment) and an appropriate reserve, an amount of 23 mg 2,6-Di-tert-butyl-4-sec-butyl [14C ring]phenol at a specific radioactivity of 2.85 MBq/mg (total 65.55 MBq) was needed.
Analogously, for the high concentration (target concentration 10 μg/L), an amount of 230 mg 2,6-Di-tert-butyl-4-sec-butyl [14C ring]phenol at a specific radioactivity of 0.57 MBq/mg (total 131.1 MBq) was needed.
Stock solution 1: An amount of approx. 35 mg of the labelled test item was needed. Because it was difficult to take this amount out of the test item ampoule, the total of initial material was diluted in 20 mL ethanol. The radioactivity was determined by liquid scintillation counting (LSC). Based on this measurement, the total amount of radiolabelled test item was determined to be 64.3 mg.
Stock Solution Low Dose: Based on the required amount, a volume of 3.6 ml of stock solution 1 containing 11.5 mg 2,6-Di-tert-butyl-4-sec-butyl [14C ring]phenol were added to 11.7 mg unlabelled test item in a volumetric flask. The volume was filled up to 250 mL with ethanol. By LSC, the amount was determined to be to 23.4205 mg at a new specific activity of 2.8525 MBq/mg). The final concentration in this stock solution was 0.0937 mg/mL.
Stock Solution High Dose: Based on the required amount, a volume of 7.2 ml of stock solution 1 containing 23 mg 2,6-Di-tert-butyl-4-sec-butyl [14C ring]phenol were added to 207.2 mg unlabelled test item in a volumetric flask. The volume was filled up to 250 mL with ethanol. By LSC, the amount was determined to be 230.2768 mg at a new specific activity of 0.5712 MBq/mg. The final concentration in this stock solution was 0.9211 mg/mL.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

The study was performed with common rainbow trouts (Oncorhynchus mykiss) supplied by Forellenzucht Hohler, Zeiningen, Switzerland. After receipt, the health status of the fish was evaluated in an expert statement (Appendix 1).
For the study, a total of 217 fish were used. Before starting the experiment, the fish were allowed to acclimatize to the laboratory environment in tap water for at least two weeks. The fish length at the start of the accumulation as determined in 10 representative fish was 4.0-5.0 cm (1.01-1.68 g).

Feeding:
The fish were fed daily (H.U. Hofmann AG, HOKOVIT, “Forellenfutter”, diet of known lipid and total protein content), based on about 2% of the average fish body weight during acclimatisation and during the study, taking into account increasing body weights and the decreasing number of fish per sampling interval.

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: Recognized by the OECD Guideline as an appropriate system to measure the bioconcentration of substances in fish and thus to contribute to the assessment of the possible impact in the event that the test item would enter natural waters.

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

60 d

Total depuration duration:

60 d

Hardness:

Water hardness measured once during the test (on day 17 of depuration) ranged from 10.4-10.6 °d (18.6-18.9 °f) or 1.9 mmol/L for all three tanks.

Test temperature:

Temperature was monitored from day 0 to day 120 and measurements ranged from 12.8-16.2 °C

pH:

pH was monitored from day 0 to day 120 and measurements ranged from 7.3-8.2

Dissolved oxygen:

Oxygen concentration was monitored from day 0 to day 120 and measurements ranged from 6.2-10.2 mg/L

TOC:

The TOC value of untreated water before the study was on average 0.08 mg/L. During depuration values ranged from 0.09 to 3.22 mg/L. These are normal values for the test system used. TOC values during the uptake phase ranged from 15.91-26.72 mg/L for the two test item tanks and the control tank. The values represented TOC resulting from the added ethanol, test item in the test tanks and fish excreta.

Salinity:

Not applicable

Conductivity:

Not specified

Details on test conditions:

Study Design
For the treated tanks in the main test the minimal duration of the uptake phase was calculated according to the OECD Guideline 305. Based on the log Pow, the expected depuration rate constant (k2) and the optimal duration of the uptake phase (u = 95% of steady state) are defined as:
log k2 = -0.414 log (Pow) + 1.47
and
u = 3.0/k2
Based on a log POW value of 6.43, a theoretical k2 of 0.064 was calculated. Based on k2 approximately 47 days would be needed to reach 95% of “steady state”. Therefore, an accumulation period of 60 days was selected which seemed to be sufficient to reach steady state (= plateau level).
To reach 95 % depuration, theoretically a depuration period of 47 days would be needed (ln 0.05/-k2). Therefore, a depuration period of 60 days seemed to be appropriate.
To initiate depuration, the fish were transferred to a flow-through system with untreated tap water and the elimination of the radioactivity from the fish was determined.
Conditions:
Illumination: Photoperiod of 16 hours light, intensity at light period approximately 300-400 Lux.
Average target temperature: 13-17 °C
Tap water: Local tap water (non chlorinated well water of drinking water quality), reduced for total hardness by ion exchange.

Application of the Test Item
Before initiating the main test, levels in the treated tanks were adjusted by adding 0.80 mL of the low dose stock solution and 0.81 mL of the high dose stock solution to the respective tank.
Throughout the entire test, the system was equilibrated with tap water at a flow-through volume of 250 L/day, to which 2,6-Di-tert-butyl-4-sec-butyl [14C ring]phenol was added from the application solutions at a rate of 25 mL/24 h to achieve final target concentrations of 1.0 μg/L at 2.85 MBq/mg and 10 μg/L at 0.57 MBq/mg for the low and high dose levels, respectively. The final solvent concentration in the test tanks and the control tank was 0.01%.

Water
Water samples were taken from the central area of the respective test tank before feeding and immediately before fish sampling.

Treated Tanks:
Daily during the uptake phase and at selected time points during the depuration phase, duplicate samples of 10 mL were removed from the treated tanks and as far as appropriate from the corresponding mixture chambers. Radioactivity was directly determined.
Additionally at selected time intervals, duplicate samples of approximately 1000 mL (both dose levels) were taken stored at approximately -20°C until analysis. One sample per time interval was partitioned into dichloromethane and stored at approximately -20 °C until analyses by HPLC. The second sample was stored as reserve sample.

Control Tank:
Duplicate background samples were taken at the beginning, in the middle and at the end of the uptake phase and at the end of depuration phase from the control tank and directly analyzed by liquid scintillation counting (LSC).

Fish
Treated Tanks:
On each sampling occasion, six fish were collected randomly from each exposure tank, rinsed with water, sacrificed in 1.5% (v/v) 2-phenoxy-ethanol in purified water and blotted dry. Four fish were analyzed as described in section 3.5 immediately following sacrifice. At two time intervals (after 40 and 60 days of accumulation) eight fish were sampled and stored at approximately -20 °C to allow analyses of metabolites.

Control Tank:
At the beginning, at the end of the accumulation period and at the end of the depuration period, six control fish were taken and four were analyzed for total radioactivity.
At two selected time points during the uptake phase ten fish were additionally sampled for lipid determination.

Sample Preparation
Water:
Duplicate samples of 10 mL water were directly analyzed for total radioactivity by LSC.
One of the duplicate water samples of 1000 mL was partitioned with dichloromethane under neutral and acidic conditions. The pooled dichloromethane fraction was concentrated after adding a small volume of DMSO. Appropriate aliquots of the DMSO1 samples were analyzed by HPLC.
Fish:
Of the six fish collected from each exposure tank four fish were individually analyzed for total radioactivity at each sampling point. The remaining two fish were stored at approximately -20 °C as reserve. The fish were weighed and solubilized at ca. 40 °C for 24 - 96 hours with the tissue solubilizer Solvable (Perkin Elmer, about 100 mg/mL solubilizer). Thereafter, duplicate solubilized subsamples (corresponding to 100 or 200 mg) were measured by LSC.
The 10 fish sampled from the control tank (after 5 and 60 days of accumulation) were used to determine the fish lipid/wet weight ratio at the beginning and at the end of the assumed plateau phase by means of dichloromethane/methanol.
For lipid determination the fish pools were thawed and homogenized in a Waring Blendor in the presence of dry-ice. Thereafter, the homogenates were extracted once with methanol/dichloromethane (1:1; v/v). The ratio of fish against MeOH/CH2Cl2 was 1:8 (w/v). The first extraction step was performed by gentle shaking (220 rpm) overnight at room temperature. A subsequent extraction step was performed with dichloromethane (fish: CH2Cl2; 1:2, w/v) by shaking for about 30 min (220 rpm) at room temperature. The two extracts were combined in a separation funnel and after addition of 10% purified water, the dichloromethane phases could be separated. After drying the dichloromethane phases with sodium sulphate they were concentrated to dryness under reduced pressure. The fish lipid residue was determined by means of an analytical balance after drying at approximately 105 °C and subsequent cooling in an exicator to constant weight.
Extraction of radioactivity from fish: 8 fish collected at the end of the uptake phase (low and high concentration) were pooled and homogenized, and extracted three times with CH2Cl2/MeOH (1+1, v/v) followed by once extraction with methanol. These extracts were pooled and partitioned.
Partitioning was performed as follows: The extracts were pooled and the solvent was evaporated. The residue was dissolved in hexane. Then 5-6 partitionings with acetonitrile were performed. The radioactivity in the acetonitrile phases and the remaining hexane phase was determined. The ACN phases were pooled, concentrated and analyzed by HPLC.
Fish Samples:
Fish were solubilized at about 40 °C for 24-48 hours with the tissue solubilizing agent Solvable (1 mL/100 mg fresh weight). Solubilized fish samples of 1.0 mL (containing 100 mg tissue) were measured in 10 mL scintillation fluid A.
Organic Samples:
Appropriate aliquots of the diluted stock solutions were measured in 10 mL of scintillation fluid A. Organic extracts were routinely measured in 10 mL scintillation fluid A.

Nominal and measured concentrations:

Based on the available solubility information, the target high dose concentration was selected to be 10 μg/L. The low dose concentration of 1 μg/L was approximately 220 times the LOD.

Reference substance (positive control):

no

Details on estimation of bioconcentration:

Bioconcentration factors (BCF) were calculated from the concentration of total radioactivity in parent equivalents (μg/kg) in fish at plateau levels (Cfss, Cfss, fit) related to the average concentration of total radioactivity (Cw):

BCFss = Cfss / Cw

BCFk = Cfss, fit / Cw = k1 / k2

BCF was also expressed as lipid normalized BCFL. The normalization will be done based on the actual measured lipid content in the fish and an assumed normal fish lipid content of 5%.

Key result

Conc. / dose:

0.55 µg/L

Temp.:

>= 12.8 - <= 16.2 °C

pH:

7.75

Type:

BCF

Value:

4 718 dimensionless

Basis:

other: Radioactivity levels

Time of plateau:

40 d

Calculation basis:

steady state

Key result

Conc. / dose:

4.44 µg/L

Temp.:

>= 12.8 - <= 16.2 °C

pH:

7.75

Type:

BCF

Value:

5 765 dimensionless

Basis:

other: Radioacitivity levels

Time of plateau:

40 d

Calculation basis:

steady state

Key result

Conc. / dose:

0.55 µg/L

Temp.:

>= 12.8 - <= 16.2 °C

pH:

7.75

Type:

BCF

Value:

4 333 dimensionless

Basis:

other: Radioactivity levels

Time of plateau:

40 d

Calculation basis:

kinetic

Key result

Conc. / dose:

4.44 µg/L

Temp.:

>= 12.8 - <= 16.2 °C

pH:

7.75

Type:

BCF

Value:

5 270 dimensionless

Basis:

other: Rdioactivity levels

Time of plateau:

40 d

Calculation basis:

kinetic

Key result

Elimination:

not specified

Parameter:

DT50

Depuration time (DT):

10.7 d

Remarks on result:

other: Low dose

Key result

Elimination:

not specified

Parameter:

DT50

Depuration time (DT):

11.9 d

Remarks on result:

other: High dose

Details on kinetic parameters:

Half-life calculations were performed based on the Cf value on day 60 (of the exposure) and the values measured during 60 days of depuration. For the low dose, a depuration half-life of 10.7 days was calculated (correlation r2: 0.987). For the high dose, a half-life of 11.9 days was obtained (correlation r2: 0.999).

Details on results:

Observations
No adverse effects in fish were noticed in the exposure tanks and in the control tank. Only two dead fish each were observed in the control tank and in the high dose tank. Thus, the mortality rate was <10%, and thus acceptable according to the test guideline.

Temperature, pH, Oxygen, Water Hardness and TOC Concentration in the Water
Temperature, pH and oxygen concentration were monitored from day 0 to day 120 and measurements ranged from 12.8-16.2 °C, 7.3-8.2 and 6.2-10.2 mg/L, respectively.
Water hardness measured once during the test (on day 17 of depuration) ranged from 10.4-10.6 °d (18.6-18.9 °f) or 1.9 mmol/L for all three tanks.
The TOC value of untreated water before the study was on average 0.08 mg/L. During depuration values ranged from 0.09 to 3.22 mg/L. These are normal values for the test system used. TOC values during the uptake phase ranged from 15.91-26.72 mg/L for the two test item tanks and the control tank. The values represented TOC resulting from the added ethanol, test item in the test tanks and fish excreta.

Levels of Radioactivity in Water during Equilibration, Exposure and Depuration
During the equilibration slightly too low and variable levels were observed, even though in the mixing chambers, values close to the target values were measured.
During the accumulation period (day 0-60) in both exposure tanks, total radioactivity levels remained sufficiently constant to show equilibrium. Total radioactivity levels amounted on average to 0.55 ± 0.07 μg/L and 4.44 ± 0.78 μg/L in the low dose tank and high dose tank, respectively. These values represented approximately 50 % of the target values, meaning that part of the radioactivity evaporated form the fish tanks under test conditions.
The radioactivity values measured in the mixing chambers were 1.05 ± 0.15 μg/L and 9.71 ± 1.08 μg/L. These results were close to the target values and demonstrated that the test item was correctly dosed to the fish tanks.
It can be concluded that the radioactivity is stripped out of the fish tanks based on the following facts:
- The measurements of the mixing chambers demonstrated that the correct amount of radioactivity (100%) was dosed to the tanks.
- After bubbling air through a 0.5 L aqueous solution of the test item under stirring simulating test conditions, recovery after 2 and 4 hours were only 2.8% and 2.1 %, respectively, of the total radioactivity in the sample.
- Significant amounts of radioactivity could be trapped in charcoal filters placed approximately 10 cm above the fish tanks.
During depuration, the radioactivity level in the low concentration tanks was 0.119 μg/L at depuration day 5 and decreased to a values
Level of Radioactivity in Fish during Exposure (Bioconcentration) and Depuration
The residues were expressed in μg parent equivalents/g according to the total radioactivity found in whole fish and their fresh weights.

Level of Radioactivity in Tank 2 (Low Dose):
The radioactive residues during exposure in whole fish increased from 1.383 on day 5 to 2.750 μg eq/g on day 60. The radioactive residues during depuration decreased to 2.161 μg eq/g on day 65 (day 5 of depuration) and 0.205 μg eq/g on day 120 (day 60 of depuration).

Level of Radioactivity in Tank 3 (High Dose):
The radioactive residues during exposure in whole fish increased from 13.749 on day 5 to 28.515 μg eq/g on day 60. The radioactive residues during depuration decreased to 20.980 μg eq/g on day 65 and 1.618 μg eq/g on day 120.

Level of Radioactivity in Tank 1
Taking into account the specific radioactivity of 2.8525 MBq/mg for the low dose level and 0.5712 MBq/mg for the high dose level, all values for whole fish were
Plateau Levels:
The plateau levels could be determined as average concentration level in the fish of the last three time intervals of the uptake phase (days 40-60). The plateau levels were 2.595 μg eq/g for the low dose and 25.956 μg eq/g for the high dose.

Bioconcentration Factor BCF
Approximately at day 40 of exposure a constant level of test item in fish was obtained. Therefore, the BCF value was calculated based on the average values of the last three time intervals of the uptake phase.
Low dose: Based on the radioactivity levels in fish after exposure to 2,6-Di-tert-butyl-4-sec-butyl [14C ring]phenol at an average dose level of 0.55 μg/L, the bioconcentration factor at plateau level (BCF) amounted to 4718.
High dose: Based on the radioactivity levels in fish after exposure to 2,6-Di-tert-butyl-4-sec-butyl [14C ring]phenol at an average dose level of 4.44 μg/L, the bioconcentration factor at plateau level (BCF) amounted to 5765.
The BCF obtained by fitting of the uptake data resulted in very similar BCF's of 4333 and 5270 for the low and the high concentration, respectively.
BCF based on the lipid content measured in representative fish and normalized to a 5% lipid content in fish amounted to 10578 in the low dose and 12926 in the high dose.

Partitioning and Chromatographic Analyses of Exposure Water
The radioactivity from exposure water of the both dose levels was characterized by HPLC.

Partitioning of Exposure Water
At the low dose level, radioactivity partitioned into dichloromethane (and ethyl acetate; day 5) ranged from 77.8 to 93.3% during the 60 days exposure period. Accordingly, low amounts of radioactivity (6.7 to 22.2%) remained in the aqueous phase.
At the high dose level, radioactivity partitioned into dichloromethane ranged from 87.4 to 95.7% during the 60 days exposure period. Accordingly, low amounts of radioactivity (4.3 to 12.6%) remained in the aqueous phase.

Pattern of Radioactivity in Exposure Water
At the low dose level, radioactive fraction W3 (parent) was found in amounts of 10.6-57.9% of the radioactivity recovered. Besides parent, up to 4 metabolite fractions (W4, W5, W7 and W9) were detected. Fraction W4 occurred in amounts ranging from 24.3 to 55.4% of the total radioactivity recovered. The other fraction occurred in amounts of 7.9 to 22.7%.
At the high dose level, radioactive fraction W3 (parent) was found in amounts of 22.2-78.5% of the radioactivity recovered. Up to 8 metabolite fractions (W1, W2, W4-W9) were detected. Fraction W4 occurred in amounts ranging from 6.6 to 32.5% of the total radioactivity recovered. The other fraction occurred in amounts of 4.6 to 19.5%.

Extraction and Chromatographic Analyses of Fish Samples
The radioactivity from fish samples collected on day 60 of exposure of the both dose levels was characterized by HPLC analyses.

Extraction of Fish Samples
Low Concentration
99.3% of total radioactive residue (TRR) or 2.732 μg/g could be extracted with dichloromethane/methanol and methanol. Then 95.2% or 2.618 μg/g could be partitioned into acetonitrile. Only a small amount of 4.1% or 0.114 μg/g remained in the hexane phase.
High Concentration
99.4% or 28.342 μg/g could be extracted with dichloromethane/methanol and methanol. Then 95.6% or 27.270 μg/g could be partitioned into acetonitrile. Only a small amount of 3.8% or 1.072 μg/g remained in the hexane phase.

Pattern of Radioactivity in Fish Extracts
Low dose level: Radioactive fraction F2 (parent) was found in an amount of 97.8% TRR or 2.579 μg/g. Besides F2, up to 2 metabolite fractions (F1 and F3) were detected. hese two fractions were measured in very small amounts of below 1% (0.6-0.9% TRR).
High dose level: Radioactive fraction F2 (parent) was found in an amount of 98.1% TRR or 26.915 μg/g. Besides F2, up to 2 metabolite fractions (F1 and F3) were detected. These two fractions were measured in very small amounts of below 1% (0.6-0.7% TRR).

Characterized Radioactive Fractions Identical to Reference Items
2,6-Di-tert-butyl-4-sec-butylphenol
2,6-Di-tert-butyl-4-sec-butylphenol was detected as main fraction in tank water (W3) and in fish extracts (F2).

HPLC Behavior:
Radioactive fraction W3 with a representative retention time 20.6 min in HPLC showed a similar HPLC behavior as the unlabelled test item. After spiking with unlabelled test item, radioactive fraction W3 co-chromatographed with the unlabelled test item.
Radioactive fraction F2 with a representative retention time 20.0 min showed a similar HPLC behavior as the unlabelled test item. After spiking with unlabelled test item, radioactive fraction F1 co-chromatographed with the unlabelled test item.
In conclusion, based on HPLC analyses, radioactive fractions W3 and F2 proved to be identical to the test item.

Temperature values (A), pH values (B) and total hardness (C) in control and exposure tanks during accumulation and depuration period.

 

A)

Time interval		Tank
Phase	(days)	Control (°C)	Low dose (°C)	High dose (°C)
A A	0-30 31-60	14.3-15.9 13.9-16.0	13.8-16.2 13.3-15.8	14.0-15.6 13.6-15.8
D D	61-90 91-120	13.4-15.2 12.8-14.1	13.8-15.2 13.3-14.4	14.1-15.4 14.3-14.9

 

B)

Time interval		Tank
Phase	(days)	Control (pH)	Low dose (pH)	High dose (pH)
A A	0-30 31-60	7.4-7.7 7.4-8.2	7.6-8.0 7.5-8.2	7.8-8.1 7.6-8.2
D D	61-90 91-120	7.3-7.8 7.4-7.6	7.6-7.8 7.7	7.7-7.9 7.7-7.8

 

C)

Time interval		Tank
Phase	(day)	Control			Low and high dose
		(mmol/L)	(°d)	(°f)	(mmol/L)	(°d)	(°f)
D	17	1.9	10.6	18.9	1.9	10.4	18.6

 

A: Accumulation period

D: Depuration period

°d: German degrees

°f: French degrees

 

Oxygen values (A) and TOC values (B) in control and exposure tanks during accumulation and depuration period.

 

A)

Time interval		Tank
Phase	(days)	Control (mg/L)	Low dose (mg/L)	High dose (mg/L)
A A	0-30 31-60	6.2-9.3 8.6-10.2	6.7-9.3 7.3-9.6	6.8-9.3 8.2-9.7
D D	61-90 91-120	9.7-10.2 8.8-9.3	8.1-8.9 6.7-7.1	8.2-9.1 7.4

 

B)

Time interval		Tank
Phase	(days)	Control (ppm)	Low dose (ppm)	High dose (ppm)
--	Untreated water (mean of 5 values):		0.08
E E	-2 -1	22.6 18.33	25.80 23.21	31.20 19.14
A A A A	10 20 40 60	25.88 19.78 21.26 20.43	22.44 24.69 19.21 18.44	26.72 20.48 21.23 15.91
D D	90 120	0.09 1.42	2.65 3.22	1.52 1.33

 

E: Equilibration period

A: Accumulation period

D: Depuration period

 

Actual concentration of total radioactivity in the exposure water during accumulation at target dose levels of 1 µg/L and 10 µg/L of 2,6-Di-tert-butyl-4-sec-butyl [¹⁴C ring] phenol

Time interval	Exposure tanks (mean of duplicates)
(days)	Comments	Low dose (µg/L)	High dose (µg/L)
-3 -3 -3 -2 -2   -1 -1   -1 0	After 1.5 hours After 3 hours After 5 hours 8 a.m. 1.15 p.m., after spiking the tanks with additional test item 7.30 a.m. 9.30 a.m., after spiking the tanks with additional test item 2.40 p.m. Before adding the fish	0.58 0.46 0.38 0.31 0.71   0.34 0.69   0.49 0.39	5.58 4.73 3.99 3.14 3.43   2.60 4.92   3.44 3.11
0 0 1 2 3   4 5 6 7 8 9 10 11 12 13 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60	After adding the fish 1 p.m. 3.15 p.m.     Additionally spiked with test item after the measurement	0.35 0.37 0.50 0.54 0.55   0.65 0.45 0.49 0.52 0.53 0.52 0.69 0.59 0.50 0.56 0.52 0.63 0.58 0.52 0.56 0.57 0.52 0.50 0.58 0.54 0.63 0.55 0.53 0.60 0.58 0.54 0.93* 0.56 0.60 0.54 0.70 0.63 0.56 0.64	2.94 3.71 4.04 4.72 3.41   5.96 3.83 4.43 4.66 3.85 3.83 3.98 5.10 4.65 5.05 6.60 5.00 5.55 4.83 3.95 4.59 4.91 3.30 4.40 4.41 4.80 4.16 4.99 4.68 3.39 3.23 4.69 4.27 5.88 4.55 3.73 4.62 3.76 4.54
Mean S.D.		0.55 0.07	4.44 0.78

S.D.: Standard deviation

*: Outlier value, not used for the calculation of the mean.

 

Actual concentration of total radioactivity in the mixing chambers during accumulation

Time interval	Exposure tanks (mean of duplicates)
(days)	Comments	Low dose (µg/L)	High dose (µg/L)
-3 -3 -3 -2 -2   -1 0	After 1.5 hours After 3 hours After 5 hours 8 a.m. 1.15 p.m., after spiking the tanks with additional test item 7.30 a.m. Before adding the fish	0.88 0.90 0.99 1.06 1.02   1.01 1.11	9.25 11.02 10.71 9.92 9.13   9.56 10.14
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60	After adding the fish 1 p.m.	1.24 1.01 0.97 1.00 1.09 1.08 0.99 1.20 1.04 0.99 0.99 0.98 0.98 1.09 1.10 1.08 0.88 0.94 1.03 1.04 0.94 0.94 0.91 0.92 1.14 0.86 0.99 0.96 1.07 1.07 0.94 1.33 1.58 1.50 0.91 1.01 1.13 1.07	9.33 8.62 11.11 9.30 10.09 8.97 9.23 10.52 10.36 9.07 10.17 9.52 10.32 8.94 8.52 9.08 10.74 9.14 9.64 8.66 8.84 8.70 8.76 10.94 10.38 9.52 10.29 9.39 11.21 8.44 10.87 7.58 13.61 8.78 9.60 10.99 9.59 9.74
Mean S.D.		1.05 0.15	9.71 1.08

S.D.: Standard deviation

 

Concentration of total radioactivity in the exposure tanks during depuration (A) and in the control tanks during the whole test (B).

 

A)

Time interval		Exposure tanks (mean of duplicates)
I	II	Low dose		High dose
		(dpm/10 mL)	(µg/L)	(dpm/10 mL)	(µg/L)
65 70 90 120	5 10 30 60	204 96 49 13	0.119 0.056 0.029 0.08*	379 165 103 26	1.106 0.480 0.299 0.076

 

B)

Time interval		Control tank (mean of duplicates)
I	II	(dpm/10 mL)	µg/L (low dose)	µg/L (high dose)
-3 0 40 60 120	- - - 0 60	16 10 6 2 3	0.009* 0.006* 0.003* 0.001* 0.002*	0.045* 0.029* 0.016* 0.004* 0.009*

 

I: Days after the onset of accumulation

II: Days after the onset of depuration

*: <LOQ

 

Concentration of radioactivity in the whole fish during the test at average dose levels of 0.55 µg/L and 4.44 µg/L.

Phase	Time interval		Concentration of radioactivity in fish (Cf)		Concentration of radioactivity in fish (Cf)
	I	II	Low dose		High dose
			(µg/g)	(%*)	(µg/g)	(%*)
A A A A A A	5 10 20 40 50 60	-- -- --   -- --	1.383 1.690 1.785 2.588 2.448 2.750	-- -- --   -- --	13.749 17.208 20.523 26.960 21.313 28.515	-- -- --   -- --
Plateau ± S.D.			2.595 0.151	-- --	25.956 3.790	-- --
D D D D	65 70 90 120	5 10 30 60	2.161 1.326 0.399 0.205	83 51 15 8	20.980 15.673 4.964 1.618	82 61 19 6

Values given in parent equivalents on fresh weight basis, i.e. µg/g

Plateau: average of days 40-60

*: Radioactivity residues in fish in percent of the average concentration in fish during exposure.

I: Days after the onset of accumulation

II: Days after the onset of depuration

A: Accumulation period

D: Depuration period

S.D.: Standard deviation

Bioconcentration factors (BCF) in fish during the test at average dose levels of 0.55 µg/L and of 4.44 µg/L.

Phase	Time interval	BCF*
	I	Low dose	High dose
A A A A A A	5 10 20 40 50 60	3515 3073 3245 4705 4451 5000	3097 3876 4622 6072 4800 6422
BAFssat plateau level (days 40 – 60)		4718	5765

*: BCF = Cf / Cw

A: Accumulation period

I: Days after the onset of accumulation

 

Bioconcentration factors (BCF) calculated for the average lost dose level of 0.55 µg/L (A) and the average high dose level of 0.44 µg/L (B) based on lipid content in control fish as determined on days 5 and 60.

 

A) Low dose

	Lipid (mg/g fish)	Cf (µg/g fish)	BCF	BCFL*
Day 5 Day 60	19.5 22.3	1.383 2.750	2515 5000	6449 11211
Plateau, BCFss	22.3	2.595	4718	10578
Plateau, fit, BCFk	22.3	2.383	433	9715

 

B) High dose

	Lipid (mg/g fish)	Cf (µg/g fish)	BCF	BCFL*
Day 5 Day 60	19.5 22.3	13.749 28.515	3097 6422	7941 14399
Plateau, BCFss	22.3	25.956	5765	12926
Plateau, fit, BCFk	22.3	23.398	5270	11816

 

*: Lipid normalized to a lipid content of 5%

Validity criteria fulfilled:

yes

Conclusions:

The study conducted using rainbow trout was performed to determine the bioconcentration potential of 2,6-Di-tert-butyl-4-sec-butylphenol at exposure concentrations of 0.55 μg/L (low dose) and 4.44 μg/L (high dose).
The plateau levels could be determined as the average concentration levels in the fish of the last three time intervals of the uptake phase, i.e. 2.595 μg eq/g for the low dose and 25.956 μg eq/g for the high dose.
Depuration half-life calculations were performed based on the Cf values from day 60 (end of exposure) to day 120 (end of depuration). For the low dose, a depuration half-life of 10.7 days was calculated. For the high dose, a similar half-life of 11.9 days was obtained.
BCFss steady state values of 4718 and 5765 were calculated for the low dose and high dose level, respectively. The corresponding BCFk were 4333 and 5270.
For both dose levels, the parent item was detected as major fraction in all tank water samples analyzed. The amounts of parent item (W3) were 10.6-57.9% for the low dose and 22.2-78.5% for the high dose. Besides the test item, up to 8 metabolite fractions were detected in water (W1, W2, W4-W9). Metabolite fraction W4 ranged from 6.6 to 55.4%. All other metabolite fractions occurred in amounts of 4.6-22.7% of the radioactivity in the tank water.
In fish extracts, almost exclusively the parent item was detected in the samples analyzed. The amount of parent item (F2) was 97.8% TRR for the low dose and 98.1% TRR for the high dose. Besides the test item, up to 2 metabolite fractions were detected (F1, F3). These two metabolite fractions were below 1% of TRR in fish.

Executive summary:

The bioconcentration and depuration characteristics of 2,6-Di-tert-butyl-4-sec-butylphenol in the rainbow trout were investigated by means of a dynamic flow-through system. The bioconcentration in whole fish was calculated.

 

The fish were continuously exposed to 2,6-Di-tert-butyl-4-sec-butyl [¹⁴C ring]phenol at an average low dose concentration of 0.55 μg/L and an average high dose concentration of 4.44 μg/L for 60 days. After the exposure, the fish were transferred to flowing untreated water and the depuration of radioactivity was followed for further 60 days. Temperature, pH and oxygen concentrations were monitored from day 0 to day 120 and were within acceptable limits; measurements ranged from 12.8 - 16.2 °C, 7.3 - 8.2 and 6.2 - 10.2 mg/L, respectively.

 

The plateau levels could be determined as average concentration level in the fish of the last three time intervals of the uptake phase, i.e. 2.595 μg eq/g for the low dose and 25.956 μg eq/g for the high dose.

 

For the low dose, the radioactive residues in fish on day 60 of exposure amounted to 2.750 μg eq/g. The radioactive residues during depuration decreased to 1.326 μg eq/g on day 70 and 0.205 μg eq/g on day 120. For the high dose, the radioactive residues in fish on day 60 of exposure amounted to 28.515 μg eq/g. The radioactive residues during depuration decreased to 15.673 μg eq/g on day 70 and 1.618 μg eq/g on day 120.

 

Depuration half-life calculations were performed based on the Cf values on day 60 (exposure) and the values measured during 60 days of depuration. For the low dose, a depuration half-life of 10.7 days was calculated. For the high dose, a similar half-life of 11.9 days was obtained.

 

For the low dose, based on the radioactivity levels in fish and an average concentration level of 0.55 μg/L in the tank water, the steady state bioconcentration factor (BCF_ss) was 4718. For the high dose, based on the radioactivity levels in fish and an average concentration level of 4.44 μg eq/L in the tank water, the BCF_ss amounted to 5765. The corresponding BCF_k were 4333 and 5270. BCF_L based on the normalized lipid content of 5% amounted to 10578 for the low dose and 12926 for the high dose.

 

For both dose levels, the parent item was detected as major fraction in most of the tank water samples analyzed. The amounts of parent item (W3) were 10.6-57.9% for the low dose and 22.2-78.5% for the high dose. Besides the test item, up to 8 metabolite fractions were detected in water (W1, W2, W4-W9). Metabolite fraction W4 ranged from 6.6 to 55.4%. All other metabolite fractions occurred in amounts of 4.6-22.7% of the radioactivity in the tank water.

 

In fish extracts, almost exclusively the parent item was detected in the samples analyzed. The amount of parent item (F2) was 97.8% TRR for the low dose and 98.1% TRR for the high dose. Besides the test item, up to 2 metabolite fractions were detected (F1, F3). These two metabolite fractions were below 1% of TRR in fish.

Description of key information

For the low dose, based on the radioactivity levels in fish and an average concentration level of 0.55 μg/L in the tank water, the steady state bioconcentration factor (BCF_ss) was 4718. For the high dose, based on the radioactivity levels in fish and an average concentration level of 4.44 μg eq/L in the tank water, the BCF_ssamounted to 5765. The corresponding BCF_kwere 4333 and 5270. BCF_Lbased on the normalized lipid content of 5% amounted to 10578 for the low dose and 12926 for the high dose.

Key value for chemical safety assessment

BCF (aquatic species):

5 765 dimensionless

Additional information

The bioconcentration and depuration characteristics of 2,6-Di-tert-butyl-4-sec-butylphenol in the rainbow trout were investigated by means of a dynamic flow-through system. The bioconcentration in whole fish was calculated.

 

The fish were continuously exposed to 2,6-Di-tert-butyl-4-sec-butyl [¹⁴C ring]phenol at an average low dose concentration of 0.55 μg/L and an average high dose concentration of 4.44 μg/L for 60 days. After the exposure, the fish were transferred to flowing untreated water and the depuration of radioactivity was followed for further 60 days. Temperature, pH and oxygen concentrations were monitored from day 0 to day 120 and were within acceptable limits; measurements ranged from 12.8 - 16.2 °C, 7.3 - 8.2 and 6.2 - 10.2 mg/L, respectively.

 

The plateau levels could be determined as average concentration level in the fish of the last three time intervals of the uptake phase, i.e. 2.595 μg eq/g for the low dose and 25.956 μg eq/g for the high dose.

 

For the low dose, the radioactive residues in fish on day 60 of exposure amounted to 2.750 μg eq/g. The radioactive residues during depuration decreased to 1.326 μg eq/g on day 70 and 0.205 μg eq/g on day 120. For the high dose, the radioactive residues in fish on day 60 of exposure amounted to 28.515 μg eq/g. The radioactive residues during depuration decreased to 15.673 μg eq/g on day 70 and 1.618 μg eq/g on day 120.

 

Depuration half-life calculations were performed based on the Cf values on day 60 (exposure) and the values measured during 60 days of depuration. For the low dose, a depuration half-life of 10.7 days was calculated. For the high dose, a similar half-life of 11.9 days was obtained.

 

For the low dose, based on the radioactivity levels in fish and an average concentration level of 0.55 μg/L in the tank water, the steady state bioconcentration factor (BCF_ss) was 4718. For the high dose, based on the radioactivity levels in fish and an average concentration level of 4.44 μg eq/L in the tank water, the BCF_ssamounted to 5765. The corresponding BCF_kwere 4333 and 5270. BCF_Lbased on the normalized lipid content of 5% amounted to 10578 for the low dose and 12926 for the high dose.

 

For both dose levels, the parent item was detected as major fraction in most of the tank water samples analyzed. The amounts of parent item (W3) were 10.6-57.9% for the low dose and 22.2-78.5% for the high dose. Besides the test item, up to 8 metabolite fractions were detected in water (W1, W2, W4-W9). Metabolite fraction W4 ranged from 6.6 to 55.4%. All other metabolite fractions occurred in amounts of 4.6-22.7% of the radioactivity in the tank water.

 

In fish extracts, almost exclusively the parent item was detected in the samples analyzed. The amount of parent item (F2) was 97.8% TRR for the low dose and 98.1% TRR for the high dose. Besides the test item, up to 2 metabolite fractions were detected (F1, F3). These two metabolite fractions were below 1% of TRR in fish.