Reaction mass of 4-tert-butyl-2,5-xylenol and 6-tert-butyl-2,4-xylenol

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Apr 2017 - 13 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Details on sampling:

- Sampling intervals/frequency for test organisms: uptake phase: at days 0.6, 1, 3, 7, 11, 14 and 15; depuration phase: at days 15.6, 16, 18, 20, 24 and 28.
- Sampling intervals/frequency for test medium samples: uptake phase: at days -3, -2, -1, 0, 0.6, 1, 3, 7, 11 and 14; depuration phase: at day 20.
- Sample storage conditions before analysis: At the end of the uptake and depuration phase eight fish were additionally sampled from each test concentration and stored at < -18ºC for lipid determination.
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
Fish: The fish were collected randomly from each exposure and control tank, rinsed quickly with water, sacrificed, blotted dry and wet weight and body length were measured. The fish samples were coded in order to link body weight and length to the analysed test item concentration in each individual fish.
Water: Analytical sampling was performed at each concentration level and control. Test water was sampled before adding the fish and during uptake and depuration phase at the same time as the fish. It was taken approximately from the middle of the aquaria before feeding on the sampling day.
Test item solutions: 10 mL per water sample were taken at each sampling date for direct measurement of total radioactivity by means of LSC (Liquid Scintillation Counting).
Control solution: No specific analyses on parent test item were conducted in the control.

For detail information on sampling schedule see table 1 on "any other information on materials and methods incl. tables"

Vehicle:

no

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

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test media were prepared by dissolving the test item in test medium and added to a water flow using rotary piston pumps. The test item (stock) solutions were spiked with radiolabelled test item for analytical purposes.
Further information on preparation of test solutions is included on table 2 of "any other information on materials and methods incl. tables"

PREPARATION OF SPIKED FISH FOOD
- Details on fish food (source, fat content as supplied, etc): commercial trout food with a content of 63 % protein and 11 % fat

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Walbaum
- Source: Forellenzucht Störk, Wagenhausen, 88348 Bad Saulgau, Germany
- Length at study initiation (length definition, mean, range and SD): 8 ± 4 cm
- Weight at study initiation (mean and range, SD): 0.62 ± 0.08 g
- Weight at termination (mean and range, SD): 0.96 ± 0.16 g
- Lipid content at test initiation (mean and range, SD): 0.0409 g per g fish (4.1±0.18 %)
- Health status: Only fish in good health and free from any apparent malformations were used. The fish were not treated with any medication before use in the test.
- Feeding during test
- Food type: commercial trout food
- Amount: 1.0 % of fish body weight
- Frequency: daily

ACCLIMATION
- Acclimation period: 44 days
- Acclimation conditions (same as test or not): same as test

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

15 d

Total depuration duration:

13 d

Hardness:

Control (day 0): 178 mg/L (as CaCO3)
Group 5 μg/L (day 0): 196 mg/L (as CaCO3)

Test temperature:

Control (mean test period): 14.4ºC
Group 0.5 μg/L (mean test period): 14.5ºC
Group 5 μg/L (mean test period): 14.6ºC

pH:

Control (mean test period): 7.99
Group 0.5 μg/L (mean test period): 8.01
Group 5 μg/L (mean test period): 8.05

Dissolved oxygen:

Control (mean test period): 94 (as % air saturation)
Group 0.5 μg/L (mean test period): 95 (as % air saturation)
Group 5 μg/L (mean test period): 96 (as % air saturation)

TOC:

<2 mg/L (over the test period in each group and control)

Details on test conditions:

TEST SYSTEM
- Test vessel: 200 L test aquarium
- Type of flow-through (e.g. peristaltic or proportional diluter): peristaltic
- Renewal rate of test solution (frequency/flow rate): 25.2 mL/h (stock solution) and 16.7 L/h (test medium)
- No. of organisms per vessel: 80
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: <1.0 g fish per liter and day

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The medium was composed of dechlorinated drinking water and deionised water. Further information on preparation of dilution water is included on table 2 of "any other information on materials and methods incl. tables"
- Particulate matter: < 2 mg/L
- Intervals of water quality measurement:
Oxygen saturation: 6 times during the test period.
pH: 6 times during the test.
Temperature: daily in all test vessels. In addition temperature was monitored continuously in the control test vessel.
TOC: 24 and 48 h prior to initiation of uptake phase, once a week during uptake and depuration phase.
Particulate matter (dry mass not passing a 0.45 μm filter): once during the test in water inflow.
Water hardness: once at test start in the control and in the highest test item concentration.
Monitoring of flow rate and as far as appropriate adjustment to pre-set values on each day.
Monitoring of fish health status including mortality daily during the study period.
- Intervals of test medium replacement: Five test chamber volumes per 24 h.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h daily

Nominal and measured concentrations:

Nominal: 0 (control), 0.5 and 5 μg/L
Measured (during uptake phase): mean concentration corresponding to 110 ± 6 % of the nominal value was measured in the test solution at the concentration level of 5.00 μg/L and 107 ± 5 % of nominal was measured at the concentration level of 0.5 μg/L.

Reference substance (positive control):

no

Details on estimation of bioconcentration:

BASIS INFORMATION
- Monitoring data: The BCF-values at time t were calculated on basis of the actually measured test item concentrations at time t.

BASIS FOR CALCULATION OF BCF
- Estimation software:
Model parameters (k1 and k2) were optimised simultaneously using the RStudio Server v1.0.143 software. Different starting values for k1 and k2 were tested in the optimization process to find the best fit for a non-linear regression using the Marquardt method.

Lipid content:

4.1 %

Time point:

start of exposure

Lipid content:

2.1 %

Time point:

end of exposure

Key result

Conc. / dose:

0.5 µg/L

Temp.:

14.5 °C

pH:

8.01

Type:

BCF

Value:

505 L/kg

Basis:

whole body w.w.

Time of plateau:

3 d

Calculation basis:

steady state

Key result

Conc. / dose:

0.5 µg/L

Temp.:

14.5 °C

pH:

8.01

Type:

BCF

Value:

814 L/kg

Basis:

normalised lipid fraction

Time of plateau:

3 d

Calculation basis:

steady state

Key result

Conc. / dose:

0.5 µg/L

Temp.:

14.5 °C

pH:

8.01

Type:

BCF

Value:

514 L/kg

Basis:

whole body w.w.

Time of plateau:

3 d

Calculation basis:

kinetic

Key result

Conc. / dose:

0.5 µg/L

Temp.:

14.5 °C

pH:

8.01

Type:

BCF

Value:

829 L/kg

Basis:

normalised lipid fraction

Time of plateau:

3 d

Calculation basis:

kinetic

Key result

Conc. / dose:

0.5 µg/L

Temp.:

14.5 °C

pH:

8.01

Type:

BCF

Value:

520 L/kg

Basis:

whole body w.w.

Time of plateau:

3 d

Calculation basis:

kinetic, corrected for growth

Key result

Conc. / dose:

0.5 µg/L

Temp.:

14.5 °C

pH:

8.01

Type:

BCF

Value:

838 L/kg

Basis:

normalised lipid fraction

Time of plateau:

3 d

Calculation basis:

kinetic, corrected for growth

Key result

Conc. / dose:

5 µg/L

Temp.:

14.6 °C

pH:

8.05

Type:

BCF

Value:

512 L/kg

Basis:

whole body w.w.

Time of plateau:

3 d

Calculation basis:

steady state

Key result

Conc. / dose:

5 µg/L

Temp.:

14.6 °C

pH:

8.05

Type:

BCF

Value:

826 L/kg

Basis:

normalised lipid fraction

Time of plateau:

3 d

Calculation basis:

steady state

Key result

Conc. / dose:

5 µg/L

Temp.:

14.6 °C

pH:

8.05

Type:

BCF

Value:

510 L/kg

Basis:

whole body w.w.

Time of plateau:

3 d

Calculation basis:

kinetic

Key result

Conc. / dose:

5 µg/L

Temp.:

14.6 °C

pH:

8.05

Type:

BCF

Value:

823 L/kg

Basis:

normalised lipid fraction

Time of plateau:

3 d

Calculation basis:

kinetic

Key result

Conc. / dose:

5 µg/L

Temp.:

14.6 °C

pH:

8.05

Type:

BCF

Value:

516 L/kg

Basis:

whole body w.w.

Time of plateau:

3 d

Calculation basis:

kinetic, corrected for growth

Key result

Conc. / dose:

5 µg/L

Temp.:

14.6 °C

pH:

8.05

Type:

BCF

Value:

832 L/kg

Basis:

normalised lipid fraction

Time of plateau:

3 d

Calculation basis:

kinetic, corrected for growth

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

0.603 d

Remarks on result:

other: low dose

Elimination:

yes

Parameter:

DT90

Depuration time (DT):

2 d

Remarks on result:

other: low dose

Elimination:

yes

Parameter:

DT90

Depuration time (DT):

1.92 d

Remarks on result:

other: high dose

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

0.578 d

Remarks on result:

other: high dose

Key result

Rate constant:

growth rate constant (d-1)

Value:

0.013

Remarks on result:

other: low dose

Key result

Rate constant:

overall uptake rate constant (L kg-1 d-1)

Value:

592

Remarks on result:

other: low dose

Key result

Rate constant:

overall depuration rate constant (d-1)

Value:

1.15

Remarks on result:

other: low dose

Key result

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

1.14

Remarks on result:

other: low dose

Key result

Rate constant:

growth-corrected half-life (d)

Value:

0.608

Remarks on result:

other: low dose

Key result

Rate constant:

growth rate constant (d-1)

Value:

0.013

Remarks on result:

other: high dose

Key result

Rate constant:

overall uptake rate constant (L kg-1 d-1)

Value:

614

Remarks on result:

other: high dose

Key result

Rate constant:

overall depuration rate constant (d-1)

Value:

1.2

Remarks on result:

other: high dose

Key result

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

1.19

Remarks on result:

other: high dose

Key result

Rate constant:

growth-corrected half-life (d)

Value:

0.582

Remarks on result:

other: high dose

Metabolites:

not determined

Results with reference substance (positive control):

Not applicable

Details on results:

- Mortality of test organisms: No lethal or sublethal effects were observed at the fish in the control and at 5.00 μg/L until the end of the study after 28 days. At 0.50 μg/L one dead fish on day 14 was observed.
- Behavioural abnormalities: none recorded.
- Observations on body length and weight: Growth was assessed to be similar in control and test item groups.

Table 3: Steady-state Bioconcentration factors at the concentration of 5.00 μg/L

Day	BCFSS(L/kg)^1)	Mean BCFSS(L/kg)^1)of days 3 - 14	BCFL(L/kg) (corrected by lipid normalization factor)^2)	Mean BCFL(L/kg) of days 3 - 14
0.6	273	-	440	-
1	369		596
3	632	512	1020	826
7	456		735
11	508		819
14	453		731

1) The BCF-values were calculated on basis of the mean measured test item concentration

Table 4: Steady-state Bioconcentration factors at the concentration of 0.50 μg/L

Day	BCFSS(L/kg)^1)	Mean BCFSS(L/kg)^1)of days 3 - 14	BCFL(L/kg) (corrected by lipid normalization factor)^2)	Mean BCFL(L/kg) of days 3 - 14
0.6	269	-	434	-
1	359		578
3	494	505	797	814
7	535		862
11	486		784
14	505		814

1) The BCF-values were calculated on basis of the mean measured test item concentration

Table 5: Summary of all calculated biological results for the low dose (0.50 µg/L)

Parameter	Description	Value
kg	overall growth rate constant [day^-1]	0.0132
k1	overall uptake rate constant [L kg^-1day^-1]	592
k2	overall depuration rate constant [day^-1]	1.15
k2g	growth-corrected depuration rate constant [day^-1]	1.14
Cf,SS	chemical concentration in fish at steady-state [µg kg^-1]	270
Cw	Mean measured chemical concentration in the water [µg L^-1]	0.535
Ln	lipid normalisation factor	1.613
BCFSS	steady-state BCF [L kg^-1]	505
BCFL	lipid-normalised steady-state BCF [L kg^-1]	814
BCFk	kinetic BCF [L kg^-1]	514
BCFkL	lipid-normalised kinetic BCF [L kg^-1]	829
BCFkg	growth-corrected kinetic BCF [L kg^-1]	520
BCFkLg	lipid-normalised growth-corrected kinetic BCF [L kg^-1]	838
DT50	half-life (time of 50% depuration) [day]	0.603
DT50g	growth-corrected half-life [day]	0.608
DT90	time of 90% depuration [day]	2.00
DT90g	growth-corrected time of 90% depuration [day]	2.02

Table 6: Summary of all calculated biological results for the high dose (5.00 µg/L)

Parameter	Description	Value
kg	overall growth rate constant [day^-1]	0.0132
k1	overall uptake rate constant [L kg^-1day^-1]	614
k2	overall depuration rate constant [day^-1]	1.20
k2g	growth-corrected depuration rate constant [day^-1]	1.19
Cf,SS	chemical concentration in fish at steady-state [µg kg^-1]	2818
Cw	Mean measured chemical concentration in the water [µg L^-1]	5.50
Ln	lipid normalisation factor	1.613
BCFSS	steady-state BCF [L kg^-1]	512
BCFL	lipid-normalised steady-state BCF [L kg^-1]	826
BCFk	kinetic BCF [L kg^-1]	510
BCFkL	lipid-normalised kinetic BCF [L kg^-1]	822
BCFkg	growth-corrected kinetic BCF [L kg^-1]	516
BCFkLg	lipid-normalised growth-corrected kinetic BCF [L kg^-1]	832
DT50	half-life (time of 50% depuration) [day]	0.578
DT50g	growth-corrected half-life [day]	0.582
DT90	time of 90% depuration [day]	1.92
DT90g	growth-corrected time of 90% depuration [day]	1.93

Table 7: Measured concentrations of 4-(tert-butyl)-2,5-dimethylphenol in water during the uptake phase

Test item	Sampling	4-(tert-butyl)-2,5-dimethylphenol
nominal (µg/L)	(d)	(µg/L)	% of nominal	Mean %± Std. dev.
5.00	0	5.9	118	110 ± 6
	0.6	5.48	110
	1	5.34	107
	3	5.2	104
	7	5.88	118
	11	5.36	107
	14	5.32	106
0.50	0	0.558	112	107 ± 5
	0.6	0.548	110
	1	0.542	108
	3	0.51	102
	7	0.566	113
	11	0.496	99
	14	0.536	107

Table 8: Measured concentrations of 4-(tert-butyl)-2,5-dimethylphenol in fish during the uptake phase (0.50 µg/L)

Test item nominal (µg/L)	Sampling (d)	Fish replicate	4-(tert-butyl)-2,5-dimethylphenol in fish	4-(tert-butyl)-2,5-dimethylphenol in fish
			(µg/kg ww*) measured	Mean (µg/kg ww*) measured
0.50	0.6	1	125	144
		2	137
		3	170
		4	144
	1	1	216	192
		2	218
		3	157
		4	178
	3	1	277	264
		2	236
		3	284
		4	261
	7	1	423	286
		2	233
		3	271
		4	217
	11	1	227	260
		2	323
		3	250
		4	239
	14	1	209	270
		2	297
		3	362
		4	212

* ww = wet weight

Table 9: Measured concentrations of 4-(tert-butyl)-2,5-dimethylphenol in fish during the uptake phase (5.00 µg/L)

Test item nominal (µg/L)	Sampling (d)	Fish replicate	4-(tert-butyl)-2,5-dimethylphenol in fish	4-(tert-butyl)-2,5-dimethylphenol in fish
			(µg/kg ww*) measured	Mean (µg/kg ww*) measured
5.00	0.6	1	1364	1499
		2	1478
		3	1897
		4	1257
	1	1	1937	2031
		2	2016
		3	2142
		4	2030
	3	1	3810	3478
		2	3900
		3	4672
		4	1531
	7	1	2105	2506
		2	2212
		3	4010
		4	1697
	11	1	2058	2794
		2	2459
		3	3491
		4	3167
	14	1	2148	2492
		2	1874
		3	2323
		4	3623

* ww = wet weight

Table 10: Measured concentrations of 4-(tert-butyl)-2,5-dimethylphenol in fish during the depuration phase (0.50 µg/L)

Test item nominal (µg/L)	Sampling (d)	Fish replicate	4-(tert-butyl)-2,5-dimethylphenol in fish	4-(tert-butyl)-2,5-dimethylphenol in fish
			(µg/kg ww*) measured	Mean (µg/kg ww*) measured
0.50	15	1	209	270
		2	297
		3	362
		4	212
	15.6	1	128	174
		2	196
		3	97.1
		4	275
	16	1	84.7	55.1
		2	60.7
		3	46.4
		4	28.5
	18	1	6.3	29.1
		2	8.7
		3	49.2
		4	52
	20	1	5.7	4.8
		2	4.9
		3	4.2
		4	4.4
	24	1	3.5	3.9
		2	4
		3	4.6
		4	3.5
	28	1	2.9	3.1
		2	3.1
		3	2.9
		4	3.5

* ww = wet weight

Table 11: Measured concentrations of 4-(tert-butyl)-2,5-dimethylphenol in fish during the depuration phase (5.00 µg/L)

Test item nominal (µg/L)	Sampling (d)	Fish replicate	4-(tert-butyl)-2,5-dimethylphenol in fish	4-(tert-butyl)-2,5-dimethylphenol in fish
			(µg/kg ww*) measured	Mean (µg/kg ww*) measured
5.00	15	1	2148	2492
		2	1874
		3	2323
		4	3623
	15.6	1	1262	1600
		2	2341
		3	800
		4	1995
	16	1	486	929
		2	728
		3	1413
		4	1088
	18	1	45	89
		2	54
		3	204
		4	54
	20	1	63	49
		2	49
		3	33
		4	50
	24	1	21	92
		2	36
		3	117
		4	193
	28	1	31	25
		2	28
		3	23
		4	17

* ww = wet weight

Validity criteria fulfilled:

yes

Conclusions:

Based on data obtained in a bioconcentration fish test, no long term accumulation of 4-(tert-butyl)-2,5-dimethylphenol could be observed. The lipid normalised and growth corrected kinetic BCFkLg ranged from 832 L/Kg (at 5.00 μg/L) to 838 L/Kg (at 0.50 μg/L). A DT50 of 0.603 days (at 0.50 μg/L) and 0.578 days (at 5.00 μg/L) were calculated.

Executive summary:

The bioconcentration of 4-(tert-butyl)-2,5-dimethylphenol in fish was assessed under flow-through conditions according to OECD guideline 305-I: Aqueous Exposure Bioconcentration Fish Test, and GLP conditions. The duration of the test was 28 days, with an uptake phase of 15 days and a depuration phase of 13 days. The test was performed in Rainbow trout Oncorhynchus mykiss. The nominal test concentrations were 0.50 and 5.00 μg/L and a control. Test media were prepared by dissolving the test item in test medium (dechlorinated drinking water and deionised water) and added to a water flow using rotary piston pumps. The test item (stock) solutions were spiked with radiolabelled test item for analytical purposes. Each test concentration and the control contained 80 fish. Oxygen saturation, pH, temperature, TOC, particulate matter and water hardness were measured in all test item treatments and in the control during the test. Fish health status including mortality was monitored during the study period. The concentrations of total radioactivity in the test solutions and in whole fish were regularly analysed during the uptake and depuration period by a validated analylical method. A mean measured concentrations of 0.535 μg/L (107 ± 5 % of low dose nominal value) and 5.50 μg/L (110 ± 6 % of high dose nominal value) were obtained during the uptake phase. BCF calculations were based on measured concentrations in the test vessels. 

The accumulation of the test item in fish was fast during the uptake phase. The approximate plateau concentrations were already reached after 3 days of exposure in the fish for the high and low concentration levels.

A mean BCFss during uptake of 512 L/Kg was observed at 5.00 μg/L and a mean BCFss of 505 L/Kg at 0.50 μg/L. The corresponding BCFL (steady state BCFss normalised to a fish lipid content of 5%) ranged from 826 L/Kg (at 5.00 μg/L) to 814 L/Kg (at 0.50 μg/L). A kinetic BCFk of 510 L/Kg was observed at the high concentration level  and a BCFk of 514 L/Kg at the lower concentration level. The lipid normalised and growth corrected kinetic BCFkLg (growth corrected kinetic BCFkg normalised to a fish lipid content of 5%) ranged from 832 L/Kg (at 5.00 μg/L) to 838 L/Kg (at 0.50 μg/L). 

4-(tert-butyl)-2,5-dimethylphenol showed a fast removal from fish. A DT50 of 0.578 days was calculated for the high concentration level. The DT50 for the low concentration level was determined as 0.603 days. Based on these data no long term accumulation of 4-(tert-butyl)-2,5-dimethylphenol could be observed.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Aug 1995 - 20 Nov 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 C (Bioaccumulation: Test for the Degree of Bioconcentration in Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: "Bioaccumulation test of a chemical substance in fish or shellfish" provided in "the Notice on the Test Method Concerning New Chemical Substances"

Deviations:

no

GLP compliance:

yes

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms: Analyses of the test substance in test fish were conducted after the start of exposure on Week 2, 4, 6 and 8 (4 times in total) for both Group 1 and 2, and 2 fish were sampled for each analysis. Analyses for control were conducted after the start of exposure and at the end of exposure. 2 fish were sampled for each analysis.
- Sampling intervals/frequency for test medium samples: twice a week during exposure period (16 times in total) for Group 1 and 2, and one analytical sample was used for each analysis.
- Sample storage conditions before analysis: Based on the preliminary examination before the start of exposure, the test substance was confirmed to be stable under the test condition.

Vehicle:

yes

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

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test substance is mixed with 10-fold its amount of surfactant HCO-40 and dissolved in deionised water to prepare a test solution at 1000 mg/L. Afterwards the following stock solutions were prepared from the 1000 mg/L test solution in a 25 L glass tank:
Group 1: 8 mg/L stock solution
Group 2: 0.8 mg/L stock solution
Control group: 80 mg/L of surfactant HCO-40.
Above solutions were supplied to test vessels.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): surfactant HCO-40
- Concentration of vehicle in test medium (stock solution and final test solution(s) at different concentrations and in control(s)):
Group 1: 80 mg/L stock solution
Group 2: 8 mg/L stock solution
Control group: 80 mg/L

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Common carp (Cyprinus carpio)
- Source: Sugishima Yogyojo, 123-2 Gunchiku-Ichiban-cho, Yatsushiro-shi, Kumamoto 866, Japan. Receipt of test fish: May 9, 1995
- Length at study initiation (length definition, mean, range and SD): 9.6 cm (mean)
- Weight at study initiation (mean and range, SD): 23.2 g (mean)
- Lipid content at test initiation (mean and range, SD): 3.9% (mean)
- Health status: After abnormal fish were removed by visual observation at the time of receipt, the fish were placed in receiving vessel and treated by medicated bath. Then, the fish were cultivated for 2 days in flow-through water
- Feeding during test
- Food type: Pelleted formulated feed for carp. Nippon Shoku Feed Co., Ltd.
- Amount: 2% of body weight/daily
- Frequency: The test fish were fed twice a day with the total daily amount of feed. However, the fish were not fed for 24 hours prior to sampling.

ACCLIMATION
- Acclimation period: 66 days + 34 days
- Acclimation conditions (same as test or not): After cultivation, the fish were moved to acclimatization vessel after treated by medicated bath to exterminate parasites, treated by medicated bath again and acclimatization was continued. During this period, abnormal fish were removed. Fish were acclimatized for 66 days in flow-through water at 25+/-2ºC. The fish were again transferred to test vessel, treated by medicated bath and acclimatized for 34 days at the same temperature in flow-through water.
Completion of acclimatization: July 20, 1995

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

8 wk

Hardness:

Not specified

Test temperature:

25 ± 2ºC

pH:

Not specified

Dissolved oxygen:

Group 1: 7.0 – 7.9 mg/L
Group 2: 6.9 – 7.9 mg/L
Control: 7.4 – 7.9 mg/L

TOC:

Not specified

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 L glass aquarium
- Type of flow-through (e.g. peristaltic or proportional diluter): The test water was supplied using the flow-through apparatus assembled at Kurume Laboratory.
- Renewal rate of test solution (frequency/flow rate): The stock solution (2 mL/min) and test medium/water (800 mL/min) were supplied to the test vessel at a rate of 1155 L/day.
- No. of organisms per vessel:
Group 1 and 2: 11 fish (at the start of exposure)
Control: 5 fish (at the start of exposure)
- 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: Ground water pumped up in the premises of the Kurume laboratory.
- Intervals of water quality measurement: Analysis of water quality parameters was performed periodically every six months at Kurume laboratory according to the water quality standard of the Japanese Ministry of Health and Welfare.

RANGE-FINDING / PRELIMINARY STUDY
- Results used to determine the conditions for the definitive study: A preliminary toxicity test in fish was conducted in order to set the concentrations of test substance for the bioaccumulation test. The LC50-48 h was determined to be 6.58 mg/L.

Nominal and measured concentrations:

Nominal: 0 (control), 0.02 (Group 1) and 0.002 (Group 2) mg/L
Measured: 0.0188-0.0193 mg/L (Group 1) and 0.00196-0.00201 mg/L (Group 2)

Reference substance (positive control):

no

Details on estimation of bioconcentration:

BASIS FOR CALCULATION OF BCF
The bioconcentration factor was calculated (equations not included in the test report) and the calculation results were rounded to 2 significant digits when the result was below 100, and to 3 significant digits when the result was 100 or above.
According to the lower limit of quantification for the test substance concentration in test fish determined, calculation of bioconcentration factor becomes possible when bioconcentration exceeds the factors shown below.
Group 1: 2.2
Group 2: 21

Lipid content:

3.9 %

Time point:

start of exposure

Key result

Conc. / dose:

0.02 mg/L

Temp.:

25 °C

Type:

BCF

Value:

>= 107 - <= 213 dimensionless

Basis:

whole body w.w.

Calculation basis:

other: From start to the end of experiment

Key result

Conc. / dose:

0.002 mg/L

Temp.:

25 °C

Type:

BCF

Value:

>= 53 - <= 263 dimensionless

Basis:

whole body w.w.

Calculation basis:

other: From start to the end of experiment

Metabolites:

Not determined.

Results with reference substance (positive control):

Not applicable

Details on results:

- Mortality of test organisms: none recorded.
- Behavioural abnormalities: No abnormality was observed.

Table 1: Bioconcentration factor

	Week 2	Week 4	Week 6	Week 8
Group 1	172	107	184	160
	213	129	162	135
Group 2	188	93	159	58
	166	147	263	97

NOTE: Tables & figures referenced along the study report were not included.

Validity criteria fulfilled:

yes

Conclusions:

The bioconcentration factor of 6-(tert-butyl)-2,4-dimethylphenol was determined to be 107 – 213 at 0.02 mg/L and 58 – 263 at 0.002 mg/L in an experimental test performed in fish during 8 weeks.

Executive summary:

The bioconcentration of 6-(tert-butyl)-2,4-dimethylphenol in fish was assessed under flow-through conditions according to OECD guideline 305 C and GLP conditions. The duration of the test was 8 weeks. The test was performed in common carp Cyprinus carpio. A preliminary toxicity test in fish was conducted resulting in LC50-48 h = 6.58 mg/L. The nominal test concentrations were 0 (control), 0.02 (group 1) and 0.002 mg/L (group 2). Analyses of test water in aquaria were conducted twice a week during the exposure period. Analyses of the test substance in test fish were conducted after the start of exposure on Week 2, 4, 6 and 8, and 2 fishes were sampled for each analysis. These analyses were performed by a validated HPLC. Measured values obtained in test water samples ranged 0.0188-0.0193 mg/L (Group 1) and 0.00196-0.00201 mg/L (Group 2) during the test, which represent 90% or more of nominal values. All validity criteria were fulfilled.The bioconcentration factor values obtained during the 8-week test ranged from 107 to 213 in group 1 and from 58 to 263 in group 2. On the basis of these results, the test substance is not considered to be bioaccumulative.

Description of key information

Key study: Experimental result. Test according to OECD guideline 305-I. GLP study. No long term accumulation of 4-(tert-butyl)-2,5-dimethylphenol was observed. The lipid normalised and growth corrected kinetic BCFkLg ranged from 832 L/Kg (at 5.00 μg/L) to 838 L/Kg (at 0.50 μg/L). A DT50 of 0.603 days (at 0.50 μg/L) and 0.578 days (at 5.00 μg/L) were calculated.

Key study: Experimental result. Test according to OECD guideline 305 C. GLP study. 6-(tert-butyl)-2,4-dimethylphenol is not considered to be bioaccumulative. The bioconcentration factor of the test item was determined to be 107 – 213 at 0.02 mg/L and 58 – 263 at 0.002 mg/L in an experimental test performed in fish during 8 weeks.

Key value for chemical safety assessment

BCF (aquatic species):

838 L/kg ww

Additional information

Key study: The bioconcentration of 4-(tert-butyl)-2,5-dimethylphenol in fish was assessed under flow-through conditions according to OECD guideline 305-I: Aqueous Exposure Bioconcentration Fish Test, and GLP conditions. The duration of the test was 28 days, with an uptake phase of 15 days and a depuration phase of 13 days. The test was performed in Rainbow trout Oncorhynchus mykiss. The nominal test concentrations were 0.50 and 5.00 μg/L and a control. Test media were prepared by dissolving the test item in test medium (dechlorinated drinking water and deionised water) and added to a water flow using rotary piston pumps. The test item (stock) solutions were spiked with radiolabelled test item for analytical purposes. Each test concentration and the control contained 80 fish. Oxygen saturation, pH, temperature, TOC, particulate matter and water hardness were measured in all test item treatments and in the control during the test. Fish health status including mortality was monitored during the study period. The concentrations of total radioactivity in the test solutions and in whole fish were regularly analysed during the uptake and depuration period by a validated analylical method. A mean measured concentrations of 0.535 μg/L (107 ± 5 % of low dose nominal value) and 5.50 μg/L (110 ± 6 % of high dose nominal value) were obtained during the uptake phase. BCF calculations were based on measured concentrations in the test vessels. 

The accumulation of the test item in fish was fast during the uptake phase. The approximate plateau concentrations were already reached after 3 days of exposure in the fish for the high and low concentration levels.

A mean BCFss during uptake of 512 L/Kg was observed at 5.00 μg/L and a mean BCFss of 505 L/Kg at 0.50 μg/L. The corresponding BCFL (steady state BCFss normalised to a fish lipid content of 5%) ranged from 826 L/Kg (at 5.00 μg/L) to 814 L/Kg (at 0.50 μg/L). A kinetic BCFk of 510 L/Kg was observed at the high concentration level  and a BCFk of 514 L/Kg at the lower concentration level. The lipid normalised and growth corrected kinetic BCFkLg (growth corrected kinetic BCFkg normalised to a fish lipid content of 5%) ranged from 832 L/Kg (at 5.00 μg/L) to 838 L/Kg (at 0.50 μg/L). 

4-(tert-butyl)-2,5-dimethylphenol showed a fast removal from fish. A DT50 of 0.578 days was calculated for the high concentration level. The DT50 for the low concentration level was determined as 0.603 days. Based on these data no long term accumulation of 4-(tert-butyl)-2,5-dimethylphenol could be observed.

Key study: The bioconcentration of 6-(tert-butyl)-2,4-dimethylphenol in fish was assessed under flow-through conditions according to OECD guideline 305 C and GLP conditions. The duration of the test was 8 weeks. The test was performed in common carp Cyprinus carpio. A preliminary toxicity test in fish was conducted resulting in LC50-48 h = 6.58 mg/L. The nominal test concentrations were 0 (control), 0.02 (group 1) and 0.002 mg/L (group 2). Analyses of test water in aquaria were conducted twice a week during the exposure period. Analyses of the test substance in test fish were conducted after the start of exposure on Week 2, 4, 6 and 8, and 2 fishes were sampled for each analysis. These analyses were performed by a validated HPLC. Measured values obtained in test water samples ranged 0.0188-0.0193 mg/L (Group 1) and 0.00196-0.00201 mg/L (Group 2) during the test, which represent 90% or more of nominal values. All validity criteria were fulfilled.The bioconcentration factor values obtained during the 8-week test ranged from 107 to 213 in group 1 and from 58 to 263 in group 2. On the basis of these results, the test substance is not considered to be bioaccumulative.