Tris(2,4-ditert-butylphenyl) phosphite

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:

weight of evidence

Study period:

06-Feb-2008 to 24-Jul-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

GLP, according to recent background document (test guideline not available)

Guideline:

other: -Anonymous (2004). Background document to the fish dietary study protocol, Working Group on identification of PBT and vPvB substances. Ispra (IT): European Chemicals Bureau.

Deviations:

no

Principles of method if other than guideline:

The study was conducted in order to determine the dietary accumulation of the test item to the freshwater fish Danio rerio (Zebrafish). One concentration of the test item in fish food was prepared, and the test animals were exposed to this concentration as well as to a negative control without the test item and a positive control treated with 14C-hexachlorobenzene (14C-HCB) for a test period of 15 days (uptake phase). During the test period the test medium was renewed in a semi-static test system. In this study, an elimination (depuration) phase was not foreseen.

To measure the actual concentrations of the test item and the reference item in fish, food and water as well as in faeces and uneaten food, samples were taken from the test and used for analysis of test item concentrations.

The requirement of the study is based on:

- Council Regulation 793/93 of 23 March 1993 on the Evaluation and Control of the Risks of Existing Substances, OJ L 84, 5.4.1993, p. 1.

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Details on sampling:

Samples from stock solution, test medium (water), fish, food and faeces were analysed to determine total radioactive residues (TRR) in comparison to the nominally applied concentrations. A sampling schedule (Table 1) gives further details.

Samples of the stock solutions S14C (test item and reference item solved in suitable solvent) were taken before the start of the test. The stock solution and the solutions used for spiking the fish food were sampled directly before spiking.
Samples from controls, and the test medium (water), fish and food were taken as described in the sampling schedule (Table 1).

Samples of trapping solutions were taken at the end of the uptake period for quantification of evaporated radioactivity.

Samples of control matrix (fish and food) were provided in order to determine the recovery of the analytical method with the specific test medium (fortifications) and to determine the lipid content, and the fish dry weight.

Each sample was analysed independently from each other.



Sampling Procedures of Samples for TRR:

Stock solution: An appropriate volume (1 – 500 µL) of stock solution was mixed with Liquid Scintillation (LSC)-cocktail in a polyethylene LSC vial, and total radioactivity was measured directly by LSC.

Water: 5 mL samples of water were mixed with LSC-cocktail in a polyethylene LSC vial, and total radioactivity was measured directly by LSC.

Fish: At each sampling date the sampled fish were sacrificed with a solution of MS222. The viscera were separated from the remaining tissue (carcass and fillet), and the portions transferred into pre-weighed LSC-vials, weighed (to the nearest 1 mg), and stored at  - 18°C until further processing.

Food: An appropriate sub-sample (0.13 – 11.1 mg) of fish food was weighed (to the nearest 1 mg), wrapped in a piece of cellulose tissue of suitable size and transferred to LSC-vials, and stored at - 18°C or lower until further processing.

Faeces: The filter paper with the collected faeces and food was stored in the refrigerator during the uptake period. Thereafter the filter paper was stored at - 18°C or lower. For further processing, faeces samples were dried overnight at 60 ± 5°C, and weighed to the nearest 1 mg. The dried samples were stored in a desiccator at room temperature until further processing.

Vessels: After sampling, the vessels were washed with cyclohexane (26 - 62 mL) to dissolve any potentially adsorbed test item. The resulting volume of the solvent was volumetrically measured, a sample of each washing solution was taken, mixed with LSC-cocktail, and analysed for total 14C-activity by LSC. Additonally, the vessels were cleaned with a piece of cellulose tissue, which was submerged in LSC-cocktail, and analysed for total 14C-activity by LSC.

Trapping solutions: At the end of the uptake phase, the total volume of solution was determined for each washing bottle. 5-mL samples of each of the trap contents was sampled, mixed with LSC-cocktail, and analysed for total 14C-activity by LSC.

Fortification samples: Control food and fish samples were fortified with the test item in the range between the quantification limit and the test concentration. Therefore, two replicates with a low and a high amount of 14C-labelled test item were analysed using the same methods of analytical procedures.

Vehicle:

yes

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

Preparation of Food:
The food used for the negative control was prepared in the same manner as the treated food, except for the presence of the test item or 14C-HCB.
The test item, and the 14C-labelled HCB solved in cyclohexane were added to a carrier (fish oil). The spiked fish oil was mixed with the dry food using a steel spatula to achive a homogeneous mixture (0.1 mL oil per gram fish food). Food was prepared on days 0 and 11 of the study. The spiked food was stored in a refrigerator.

Details on fish food and carrier (fish oil) are given below:
Fish food
- supplier Tetra GmbH, Melle, Germany
- type of fish food TetraMin, ground fish food flakes

Carrier (fish oil)
- supplier Caesar & Loretz GmbH, Hilden, Germany
- type of fish food Caelo cod liver oil, cod liver oil
- composition/specification Cod liver oil 100%

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The test system used in this study was Danio rerio Hamilton 1822 (Zebrafish). Juvenile fish (weight range: 200 - 400 mg wet weight per fish) was used in this test.

Holding Conditions

 Fish Species: Danio rerio (Zebrafish)
 Supplier: Aquaristik Rothe, 65199 Wiesbaden, Germany
 Date of arrival: January 31, 2008
 Material of stock vessel: glass
 Amount of water per stock vessel: approximately 150 L
 Depth of water in the stock vessel: approximately 35 cm
 Estimated number of fish kept as a stock per holding vessel (batch size): approx. 70 - 80
 Water: reconstituted water (see section 14.4) supplemented with 0.1% of artificial seawater
 Renewal of water: once per week
 Temperature of water: 26 ± 2°C; min/max 25.3 - 26.1 (14 days before start of exposure)
 Food: Artemia sp. and TetraMin® (fish food)
 Amount of food: ad libitum
 Feeding frequency: daily ration fed in 3 portions on workdays
 Photoperiod: 12 h light / 12 h dark
 Light intensity: 100 - 1000 lx
 Mortalities during the period of 14 d before start of the test: 0%

Route of exposure:

feed

Test type:

semi-static

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

15 d

Total depuration duration:

0

Hardness:

mean 240.2 +- 1.7 mg/L as CaCO3, min 237.5 mg/L, max 242.9 mg/L

Test temperature:

mean 26.5 +- 0.3 °C, min 25.8 °C, max 27.0 °C

pH:

7.4 - 7.8

Dissolved oxygen:

mean 7.4 +-0.6 mg/L, min 5.6 mg/L, max 8.4 mg/L

Details on test conditions:

Test Units

Glass aquaria with 15 L volume covered with glass plates were used as test vessels.

All glass aquaria designated for contact with the test item were washed with tap water, rinsed with deionised water, dried, rinsed with ethanol, washed twice with deionised water and dried afterwards. Before adding the test organisms, the test solutions were adapted to test temperature.

Exposure Conditions

- Amount of test solution per test vessel: 15 L
- Depth of test solution in the test vessels: approx. 20 cm
- Number of fish per test vessel at start of exposure: 10
- Average weight of fish: 0.26 ± 0.04 g (n = 90, day -9 of exposure)
- Fish loading: 0.22 g per L (maximum value calculated with the sum of weights measured during exposure period)
- Renewal of test solution and control water during the test period: three times per week, at least 50% of test solution
- Feeding: daily, 3% of fish body mass, fed in 2 - 3 portions, separated by 2 - 5 hours; On weekends, the daily ration was fed in 1 portion
- Photoperiod: light/dark - 12 h/12 h
- Light intensity: 100 - 1000 lx; measured: 668 ± 68 (SD) lx
- Temperature, online measurements (min / max): 26.0 / 28.2 °C
- Temperature, manual measurements (min / max): 25.8 / 27.0 °C
- Aeration: permanent

Results of measurements of physico-chemical parameter throughout the test see Table 3.

Nominal and measured concentrations:

The treated food had a mean measured concentration of 102244463 dpm/g food dry weight; the corresponding mean concentration of the test item was approx. 0.45 mg/g food dry weight based on the specific radioactivity of the tracer used. Based on the mean measured lipid content in food at the end of exposure the test item concentration on a lipid basis was calculated to 571198117 dpm/g lipid; the correponding concentration of the test item was approx. 2.51 mg/g lipid.

Reference substance (positive control):

yes

Remarks:

14C-hexachlorobenzene

Details on estimation of bioconcentration:

Data Assessment and Statistical Evaluation of Results

1. Background Correction

The radioactivity measured by LSC in individual samples (expressed in dpm) was corrected by the mean radioactivity measured in samples of the respective control matrix (background activity), i.e. fish, food, water and faeces-containing filter paper.

2. Calculation of Dietary Biomagnification Factor (BMF)
The concentration in fish and food samples was obtained by dividing the amount of test compound in the sample (dpm, corrected for background activity) by the sample weight in g, and was expressed in dpm/g based on wet weight or dry weight, respectively. For each fish, the amounts of 14C-activity in viscera and remaining tissue (carcass and fillet) was added and divided by the total wet weight of the respective fish. The concentration in total fish samples was then divided by the concentration in the food of the corresponding sampling vessel to result in the dietary biomagnification factor (BMF) for each fish at a specific sampling date of the uptake phase. The mean BMF per sampling date was calculated as arithmetic mean of the sample BMFs. The overall mean BMF was calculated as arithhmetic mean of the mean BMFs per sampling date.

The wet-weight based dietary BMF was corrected by the lipid content of fish and food to calculate a lipid-normalised dietary BMF.

3. Balance and Assimilation Efficiency

A mass balance was calculated for each replicate taking into account the activity added to the test system via food, and the determined activity in fish (viscera and remaining tissue), faeces, uneaten food, water, trapping solutions and adsorption onto the glass walls of the test vessels. In some cases measured activities below the LOQ were also used to calculate the mass balance.

The assimilation efficiency was calculated by dividing the activity measured in each fish (viscera and remaining tissue) by the radioactivity added to the test system via food divided by the number of fish per replicate. This calculation was only done with fish from the positive control sampled after an exposure period of one day.

Lipid content:

8.5 other: % of wet weight (ww)

Time point:

start of exposure

Remarks on result:

other: in fish

Lipid content:

9.8 other: % of wet weight (ww)

Time point:

end of exposure

Remarks on result:

other: in fish

Lipid content:

9.2 other: % of wet weight (ww)

Time point:

other: over all mean

Remarks on result:

other: in fish

Lipid content:

17.5 other: % of wet weight (ww)

Time point:

other: before start

Remarks on result:

other: in food

Lipid content:

17.9 other: % of wet weight (ww)

Time point:

end of exposure

Remarks on result:

other: in food

Lipid content:

17.7 other: % of wet weight (ww)

Time point:

other: over all

Remarks on result:

other: in food

Key result

Type:

BMF

Value:

0.003 dimensionless

Basis:

normalised lipid fraction

Remarks on result:

other: no plateau reached

Key result

Type:

BMF

Value:

0.002 dimensionless

Basis:

whole body w.w.

Remarks on result:

other: no plateau was reached

Elimination:

no

Results with reference substance (positive control):

The highest BMF for the positive control was 0.1930 as determined on day 15 of exposure, and 0.3777 for the lipid-normalised BMF. The BMFs for the positive control on days 1 and 15 were statistically different (ANOVA; p ≤ 0.05).

Details on results:

Biomagnification Factor (test item)

The codes C0, C0+ and C1 are used for the concentration levels and a,b,c,d for replicated test vessels. The data concerning total radioactive residues and the Biomagnification Factor (BMF) for the test item (tris(2,4-di-tert-butyl[U-14C]-phenyl)phosphite) are shown in Table 6 and Table 7 (see section below). In Table 3 the BMF related to whole fish is calculated from the measured concentration in whole fish on a wet weight basis and the concentration in the food (102244463 dpm/g).
In addition the BMF was calculated on a lipid basis. Using the measured lipid concentration in food and fish the measurements were normalised to the lipid content using the mean lipid contents for fish as measured at start and end of exposure, and for food as measured at the end of exposure, dividing the measurements in Table 7 by a factor of 0.511.
The highest mean BMF per sampling date for the test item was 0.0028 based on wet weight, and 0.0055 for the lipid-normalised BMF. These values were determined on day 3 of exposure. No statistically significant difference (ANOVA; p ≤ 0.05) between the test item BMFs for each sampling date was determined.

Results related to the growth of Fish: The weight of the fish was recorded on each sampling date; the growth was calculated in comparison to the initial fish weight measured on day -1. A weight increase of 4% between of the initial fish wet weight (day -1) and the end of exposure (day 15) was determined. Therefore a growth dilution was not assessed.

Results related to the mortality of fish: In the controls and the treated fish no mortality was observed.

Results related to the Behaviour of Fish: In the fish treated with test item, behavioural differences from the control fish were not observed.

Table 4: Positive control (¹⁴C-hexachlorobenzene): Total radioactive residues in fish (dpm/g wet weight), and Biomagnification Factor (BMF) as based on wet weight; mean concentration in food: 1750258 dpm/g.

			total radioactive residues in dpm/g wet weight			Biomagnification Factor (BMF)
	sample code^a	day	total fish			BMF	mean	sd	n
wet weight-based	C0+a	1	20014			0.0114
	C0+b	1	37797			0.0216
	C0+c	1	40817			0.0233
	C0+d	1	26925			0.0154	0.0179	0.0055	4
	C0+a	15	267269			0.1527
	C0+b	15	337796			0.1930
	C0+c	15	227072			0.1297
	C0+d	15	199733			0.1141	0.1474	0.0343	4

^aa - d = samples of individual fish taken out of one replicate

Table 5: Positive control (¹⁴C-hexachlorobenzene): Total radioactive residues in fish (dpm/g lipid weight), and Biomagnification Factor (BMF) as based on lipid weight; mean concentration in food: 9755462 dpm/g lipid.

 

			total radioactive residues in dpm/g lipid weight			Biomagnification Factor (BMF)
	sample code^a	day	total fish			BMF	mean	sd	n
lipid weight-based	C0+a	1	218314			0.0224
	C0+b	1	412291			0.0423
	C0+c	1	445229			0.0456
	C0+d	1	293698			0.0301	0.0351	0.0108	4
	C0+a	15	2915341			0.2988
	C0+b	15	3684644			0.3777
	C0+c	15	2476882			0.2539
	C0+d	15	2178674			0.2233	0.2884	0.0671	4

^a a - d = samples of individual fish samples taken out of one replicate

 

Table 6: Test item: Total radioactive residues in fish (dpm/g wet weight), and Biomagnification Factors (BMF) as based on total fish wet weight; mean radioactive concentration of test item in food: 102244463 dpm/g (seesection14.7).

 

			total radioactive residues in dpm/g wet weight			Biomagnification Factor (BMF)
	replicate code	day	viscera	remaining tissue	total fish	BMF	mean	sd	n
wet weight-based	C1a	3	1670038	25277	481463	0.0047
	C1b	3	7083299	36574	1799201^a	--
	C1c	3	1516716	20328	157790	0.0015
	C1d	3	2980478	54316	225571	0.0022	0.0028	0.0017	3
	C1a	8	1821644	23035	318454	0.0031
	C1b	8	195562	10077	60838	0.0006
	C1c	8	130613	7007	23976	0.0002
	C1d	8	108745	7934	35624	0.0003	0.0011	0.0014	4
	C1a	15	476164	10104	98238	0.0010
	C1a	15	576312	8450	55580	0.0005
	C1b	15	839626	12373	168651	0.0016
	C1b	15	1084129	11195	97054	0.0009
	C1c	15	549436	7206	48138	0.0005
	C1c	15	174641	15311	41613	0.0004
	C1d	15	1709156	8375	179873	0.0018
	C1d	15	1389470	13622	142654	0.0014	0.0010	0.0005	8
	overall mean BMF						0.0016	0.0010	3

^a: outlier as confirmed by Outlier-Test after Dixon; not used for further calculations

 

Table 7: Test item: Total radioactive residues in fish (dpm/g lipid), and Biomagnification Factors (BMF) as based on lipid weight; mean radioactive concentration of test item in food: 569882774 dpm/g lipid.

 

			total radioactive residues in dpm/g lipid weight			Biomagnification Factor (BMF)
	replicate code	day	total fish			BMF	mean	sd	n
lipid weight-based	C1a	3	5251754			0.0092
	C1b	3	19625516^a			--
	C1c	3	1721161			0.0030
	C1d	3	2460502			0.0043	0.0055	0.0033	3
	C1a	8	3473661			0.0061
	C1b	8	663620			0.0012
	C1c	8	261526			0.0005
	C1d	8	388585			0.0007	0.0021	0.0027	4
	C1a	15	1071576			0.0019
	C1a	15	606261			0.0011
	C1b	15	1839630			0.0032
	C1b	15	1058660			0.0019
	C1c	15	525090			0.0009
	C1c	15	453914			0.0008
	C1d	15	1962043			0.0034
	C1d	15	1556060			0.0027	0.0020	0.0010	8
	overall mean BMF						0.0032	0.0020	3

^a: outlier as confirmed by Outlier-Test after Dixon; not used for further calculations

 

Summary Biomagnification Factors 

Table 8: Biomagnification Factors (BMF) calculated for the testitem (tris(2,4-di-tert-butyl[U-¹⁴C]-phenyl)phosphite) and the positive control (¹⁴C-hexachlorobenzene) based on wet weight (ww), and lipid weight-corrected; mean values, standard deviation and confidence limits (n = 4 per day, for day 3: n = 3).

 

treatment / day	mean BMF (ww)				mean BMF (lipid corrected; ww)
	mean	SD	95%lo	95%up	mean	SD	95%lo	95%up
test item / day 3	0.0028	0.0017	-0.0015	0.0071	0.0055	0.0033	-0.0029	0.014
test item / day 8	0.0011	0.0014	-0.0011	0.0033	0.0021	0.0027	-0.0022	0.0064
test item / day 15	0.0010	0.0005	0.0006	0.0015	0.0020	0.0010	0.0011	0.0029
positive control /day 1	0.0179	0.0055	0.0091	0.0268	0.0351	0.0108	0.0177	0.0525
positive control /day 15	0.1474	0.0343	0.0922	0.2026	0.2884	0.0671	0.1804	0.3965

mean: mean value

SD: standard diviation

95%lo: confidence interval (lower limit)

95%up: confidence interval (upper limit)

 

No statistically significant difference (ANOVA; p≤0.05) between the test item BMFs for each sampling date was determined. The BMFs for the positive control on days 1 and 15 were statistically different (ANOVA; p≤0.05).

 

 

Balance of TRR in the test system

Table 9: Mass balance (mean values) as based on total amount of radioactivity added to the test system during exposure period (15 d); all values expressed in per cent of total applied radioactivity.

 

replicate	C0+	C1a	C1b	C1c	C1d
water renewal	10.1	29.1	28.3	30.8	31.2
adsorption to the test vessels	0.0	7.7	3.1	4.4	4.4
filter (faeces and food residues)	3.3	36.1	63.7	56.9	43.4
fish	56.2	0.4	0.4	0.4	0.4
losses	30.4	26.7	4.5	7.5	20.6
balance	69.6	73.3	95.5	92.5	79.4

water renewal: radioactivity removed from the system during water renewal, adsorption: residues adsorbed to vessel walls, filter: collected faeces and uneaten food, fish: calculated activity based on the weights of fish at each sampling date; losses: difference between total applied radioactivity and measured residues; balance; sum of measured radioactivity.

 

Remarks: The measured activities in the trapping solutions were negligible. Therefore they were not included in the mass balance calculation. The mass balance for the treatment ranged between 73.3% - 95.5%, the mass balance for the positive control was 69.6%; seeAppendix A (section23.2) for an example of balance calculation.

 

 

 

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were fulfilled: The dissolved oxygen level at any point in the study in the control did not fall below 60% of air saturation value. The mortality in the controls did not exceed 10% .

Conclusions:

Under the experimental conditions, the test item tris(2,4-di-tert-butyl[U-14C]-phenyl)phosphite was not accumulated by the test organisms within the test period of 15 days. The biomagnification factor (BMF) of the test item as measured in fish and food was always lower than 0.01.

Executive summary:

The overall mean biomagnifaction factor (BMF) calculated for the test item based on total radioactivity (TRR) for the whole exposure phase was 0.0016 based on wet weight and 0.0032 based on lipid weight. On day three of exposure, the biomagnification factor (BMF) for the test item based on mean total radioactivity (TRR) was 0.0028 based on wet weight and 0.0055 based on lipid weight. On day 15 of exposure the BMF showed mean values of 0.0010 based on wet weight, and 0.0020 based on lipid weight. No statistically significant difference between the BMFs for each sampling date was determined (p≤0.05). Therefore bioaccumulation was not observed for the test item. The assimilation efficency measured on day one in the positive control (¹⁴C-HCB) was 65.2% of the administered test item. 

 

Conclusion:

Under the experimental conditions, the test itemtris(2,4-di-tert-butyl[U-¹⁴C]-phenyl)phosphitewas not accumulated by the test organisms within the test period of 15 days. The biomagnification factor (BMF) of the test item as measured in fish and food was always lower than 0.01.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Remarks:

The substance does not completely fall into the applicability domain of the submodels Biotransformation rate in fish (kM) and Arnot & Gobas BCF/BAF, since the log Pow is slightly higher than the substances of the training set. However, this suggest that the calculated uptake limitation is even stronger than indicated by the QSAR calculation. Thus, the QSAR calculation is considered to be a valid prove of limited uptake into aquatic organisms.

Justification for type of information:

QMRF and QPRF are attached

Principles of method if other than guideline:

Calculation based on BCFBAF v3.01, Estimation Programs Interface Suite™ for Microsoft® Windows v 4.10. US EPA, United States Environmental Protection Agency, Washington, DC, USA.

GLP compliance:

no

Test organisms (species):

other: fish

Route of exposure:

aqueous

Test type:

other: calculation

Water / sediment media type:

natural water: freshwater

Details on estimation of bioconcentration:

BASIS FOR CALCULATION OF BCF
- Estimation software: BCFBAF v3.01
- Result based on calculated log Pow of: 10.52 (KOWWIN v1.68)

Type:

BCF

Value:

18.62 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: Upper trophic, incl. biotransformation estimates; The substance is not fully within the applicability domain of the BCFBAF submodel: Arnot & Gobas BAF and steady-state BCF Arnot & Gobas, 2003).

Key result

Type:

BCF

Value:

37.73 L/kg

Basis:

whole body w.w.

Remarks on result:

other: The substance is within the applicability domain of the BCFBAF submodel: Bioconcentration factor (BCF; Meylan et al., 1997/1999).

Reference 3

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Bioconcentration of a chemical substance in fish body prescribed in the "Guidelines for testing of a new chemical" (Kanhogyo No.5, Yakuhatsu No. 615, 49 Kikyoku No. 392)

Version / remarks:

July 13, 1974

GLP compliance:

yes

Specific details on test material used for the study:

- Test substance: K-794
- LOT No: AP01
- light orange-yellow crystalline mass
- solubility in water: 35 mg/L
- log Pow: 5.19
- storage: at a cool, dark place
- stability under test conditions: confirmed in a preliminary test

Details on sampling:

The test water was analyzed twice a week (50 mL in 0.02 mg/L treatment and 500 mL in 0.002 mg/L treatment), 16 times in total, during the exposure period for both groups (one each sample).The test fish were analyzed at 2, 4, 6 and 8 weeks after initiation of exposure, 4 times in total, for both groups (2 fish each at a time).

Vehicle:

yes

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

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance and HCO-40 in 10-time portion were dissolved in acetone. Acetone was evaporated subsequently and the residue was dissolved in ion-exchanged water to obtain a stock solution containing 1000 mg/L.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): HCO-40

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: carp
- Source: Sugishima Fish Farm, Kumamoto, Japan
- Length at study initiation (length definition, mean, range and SD): 9.9 cm (mean)
- Weight at study initiation (mean and range, SD): 24.9 g (mean)
- Method of breeding: Fish with macroscopically detectable abnormalities at the time of receipt were removed and subjected to a medicated bath in the receiving vessel and held in flow-through water for 1 day. A medicated bath with 50 mg/L solution of terramyc in powder for aquatic use (Taito Pfizer) and 7 g/L sodium chloride solution was given for 24h in static water
- Lipid content at test initiation (mean and range, SD): 4.7% (mean)
- Feeding during test
- Food type: formulated pellet food (Nippon Formula Feed Manufacturing Co., Ltd, Japan)
- Amount/Frequency: An amount equivalent to about 2%of the body weight of test fish was given in 2 divided portions per day. Feeding was stopped on the previous day of sampling the test fish

ACCLIMATION
- The fish were acclimated in the acclimation vessel where any fish with abnormalities were removed and they were finally kept in flow-through water at 25 +- 2 °C for 24 days.The fish were then transferred to the test water vessel and kept in flow-through water at the same temperature for 7 days.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Remarks:

groundwater pumped up on the premises of Kyushu Research Laboratories

Total exposure / uptake duration:

8 wk

Test temperature:

25 +- 2 °C

pH:

7.9 - 8.3

Dissolved oxygen:

6.4 - 7.5 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 L glass vessel
- Type of flow-through (e.g. peristaltic or proportional diluter): 1158 L/d of water was supplied to the test water vessels in the ratio of 4 mL/min of the stock solution and 800 mL/min of the test water
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 15

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: groundwater
- Holding medium different from test medium: no
- Intervals of water quality measurement: Water temperature, pH and DO were monitored continuously at this lab.

OTHER TEST CONDITIONS
- Adjustment of pH: no

Nominal and measured concentrations:

Nominal: 0.02 and 0.002 mg/L

Lipid content:

4.7 %

Time point:

start of exposure

Conc. / dose:

0.002 mg/L

Temp.:

25 °C

pH:

8

Type:

BCF

Value:

>= 135 - <= 360 dimensionless

Basis:

whole body w.w.

Time of plateau:

8 wk

Calculation basis:

steady state

Conc. / dose:

0.02 mg/L

Temp.:

25 °C

pH:

8

Type:

BCF

Value:

>= 128 - <= 436 dimensionless

Basis:

whole body w.w.

Time of plateau:

8 wk

Calculation basis:

steady state

Validity criteria fulfilled:

not specified

Conclusions:

The test substance does not significantly accumulate in fish.

Reference 4

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Jul. 1, 1977 - Jan. 31, 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Fish bioaccumulation test over 60 days

GLP compliance:

no

Radiolabelling:

no

Vehicle:

yes

Remarks:

Tween 20

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

500 mg of the test substance was dissolved in acetone in order to create a stock solution of 5 g/L. The stock solution was added to 4 L of test water containing 3 mg/L Tween 20 and was dispersed with ultrasonic waves.

Test organisms (species):

Oryzias latipes

Details on test organisms:

TEST ORGANISM
- Common name: carp
- Length at study initiation: 10.1 cm
- Weight at study initiation (mean and range, SD): 24.3 g
- Lipid content at test initiation (mean and range, SD): 3.9 ± 0.2 %

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

60 d

Test temperature:

24 +- 1 °C

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 L tank
- Aeration: constantly

Nominal and measured concentrations:

Nominal: 0.15 and 1 mg/L
Measured: 0.16 and 0.98 mg/L

Details on estimation of bioconcentration:

Amount accumulated in fish body = Test substance concentration × Volume of constant volume solution
Concentration in fish body = Accumulation in fish × 100 / ( fish weight x recovery rate)
Concentration factor = concentration in fish/concentration in medium

Lipid content:

>= 3.7 - <= 4.1 %

Conc. / dose:

0.98 mg/L

Temp.:

24 °C

Type:

BCF

Value:

4.66 dimensionless

Basis:

whole body w.w.

Time of plateau:

60 d

Calculation basis:

steady state

Key result

Conc. / dose:

0.16 mg/L

Temp.:

24 °C

Type:

BCF

Value:

>= 2.88 - <= 6.25 dimensionless

Basis:

whole body w.w.

Time of plateau:

60 d

Calculation basis:

steady state

Reference 5

Endpoint:

bioaccumulation in aquatic species, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Remarks:

The substance does not completely fall into the applicability domain of the QSAR, since the log Pow and the molecular weight are a bit higher than the substances of the training set. However, this suggest that the calculated uptake limitation is even stronger than indicated by the QSAR calculation. Thus, the QSAR calculation is considered to be a valid prove of limited uptake into aquatic organisms.

Justification for type of information:

QMRF and QPRF are attached

Principles of method if other than guideline:

Calculation based on BCFBAF v3.01, Estimation Programs Interface Suite™ for Microsoft® Windows v 4.10. US EPA, United States Environmental Protection Agency, Washington, DC, USA.

GLP compliance:

no

Test organisms (species):

other: fish

Route of exposure:

aqueous

Test type:

other: calculation

Water / sediment media type:

natural water: freshwater

Details on estimation of bioconcentration:

BASIS FOR CALCULATION OF BCF
- Estimation software: BCFBAF v3.01
- Result based on calculated log Pow of: 16.16 (KOWWIN v1.68)

Key result

Type:

BCF

Value:

3.16 L/kg

Basis:

whole body w.w.

Remarks on result:

other: The substance is not fully within the applicability domain of the BCFBAF submodel: Bioconcentration factor (BCF; Meylan et al., 1997/1999).

Key result

Type:

BCF

Value:

0.89 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: Upper trophic, incl. biotransformation estimates; The substance is not fully within the applicability domain of the BCFBAF submodel: Arnot & Gobas BAF and steady-state BCF Arnot & Gobas, 2003).

Type:

BCF

Value:

0.89 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: Upper trophic, incl. biotransformation rate of zero; The substance is not fully within the applicability domain of the BCFBAF submodel: Arnot & Gobas BAF and steady-state BCF Arnot & Gobas, 2003).

Type:

BAF

Value:

1.19 L/kg

Basis:

whole body w.w.

Remarks on result:

other: Upper trophic, incl. biotransformation estimates; The substance is not fully within the applicability domain of the BCFBAF submodel: Arnot & Gobas BAF and steady-state BCF Arnot & Gobas, 2003).

Details on kinetic parameters:

Biotransformation half-life (days): 7.27e+004
Biotransformation rate (kM, normalised to 10 g fish at 15 °C): 9.541e-006
The substance is within the applicability domain of the BCFBAF submodel: Biotransformation rate in fish (kM; Arnot et al., 2008a/b).

Conclusions:

The substance does not accumulate in aquatic organisms.

Description of key information

Tris(2,4-di-tert-butylphenyl) phosphite does not significantly accumulate in organisms.

Key value for chemical safety assessment

BCF (aquatic species):

6.25 L/kg ww

BMF in fish (dimensionless):

0.003

Additional information

Experimental and QSAR data evaluating the bioaccumulation potential of Tris(2,4-ditert-butylphenyl) phosphite and its transformation products are available. Therefore, all available and relevant data is combined in a Weight of Evidence (WoE), which is in accordance to the REACh Regulation (EC) No 1907/2006, Annex XI General rules for adaptation of the standard testing regime set out in Annexes VII to X, 1.2, to cover the data requirements of Regulation (EC) No. 1907/2007 Annex VIII.

A dietary accumulation study has been carried out with the test substance using Zebrafish (Danio rerio) (Ciba, 2008). The semi-static study design was similar to OECD 305 with some deviations but in accordance with GLP. Under the experimental conditions, the test item tris(2,4-di-tert-butyl[U-14C]phenyl)phosphite was not accumulated by the test organisms within the test period of 15 days. Fish were sampled on day 3, 8 and 15 and the biomagnification factor (BMF) was calculated based on wet weight as well as based on lipid weight. The overall mean BMF for the whole exposure phase was 0.0016 (wet weight) and 0.0032 (lipid weight). On day three of exposure, the biomagnification factor (BMF) for the test item based on mean total radioactivity (TRR) was 0.0028 based on wet weight and 0.0055 based on lipid weight. On day 15 of exposure the BMF showed mean values of 0.0010 based on wet weight, and 0.0020 based on lipid weight. The BMF of the test item was always lower than 0.01 and it can be concluded that the test item was not accumulated by the test organism.

 

In addition, one study is available investigating the bioaccumulation potential of the possible transformation product 2,4-ditert-butylphenol in carp (Cyprinus carpio, Kyushu Research Laboratories 1987). The test was conducted according to "Guidelines for testing of a new chemical" (Kanhogyo No.5, Yakuhatsu No. 615, 49 Kikyoku No. 392). Fish were exposed to 0.002 mg/L and 0.02 mg/L of the test item for 8 weeks. The test water was analyzed twice a week, fish were analyzed at 2, 4, 6 and 8 weeks after start of the exposure and the bioconcentration factor (BCF) was calculated. A steady state BCF of 135-360 L/kg whole body w.w. (0.002 mg/L) and 128-436 L/kg whole body w.w. (0.02 mg/L) was determined. This study indicates that this transformation product also does not significantly accumulate in fish.

 

In a second study the bioconcentration potential of tris(2,4-ditert-butylphenyl)phosphite was investigated using the Japanese rice fish (Oryzias latipes) as test organism (Gakushuin University 1978). Oryzias latipes was exposed to 0.98 mg/L and 0.16 mg/L of the test item for 60 days. A BCF of 4.66 L/kg respectively 2.88-6.25 L/kg was determined based on whole body wet weight confirming that the test item does not significantly bioaccumulate via dermal (gill) uptake.

 

The bioaccumulation potential of further transformation products was calculated using BCFBAF v3.01. The calculations are considered to be valid and reliable based on the validity criteria checked and listed in the attached QMRF/QPRF. QSAR calculations using BCFBAF v3.01 were performed with the metabolites Bis(2,4-ditert-butylphenyl)phosphate (CAS 69284-93-1) and Tris(2,4-ditert-butylphenyl)phosphate (CAS 95906-11-9). None of the substances showed a high potential for bioaccumulation. For CAS 69284-93-1 the BCF was 37.73 L/kg whole body w.w. (regression based estimate) and 18.62 L/kg whole body w.w. (Arnot & Gobas upper trophic) while the BCF for CAS 95906-11-9 was even lower at 3.16 L/kg whole body w.w (regression based estimate) and 0.89 L/kg whole body w.w. (Arnot & Gobas upper trophic). Tris(2,4-ditert-butylphenyl) phosphate does not completely fall into the applicability domain of the QSAR, since the log Pow and the molecular weight are a bit higher than the substances of the training set. However, this suggests that the calculated uptake limitation is even stronger, and the calculation can be interpreted as worst case assumption. Thus, the QSAR calculation is considered to be a valid prove of limited uptake into aquatic organisms.

Based on the reliable experimental data and supported by BCF values, calculated using BCFBAF v3.01 it can be concluded that tris(2,4-ditert-butylpheyl) phospite and its relevant transformation products do not significantly accumulate in aquatic organisms.