Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol

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:

2016-04-14 to 2016-07-04 with the definitive exposure phase from 2016-04-14 to 2016-05-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD TG 305 published in 2012

Deviations:

yes

Remarks:

See 'Principles of method if other than guideline'

Principles of method if other than guideline:

The study was conducted as a combined study intended to encompass the two endpoints of bioaccumulation and endocrine (oestrogenic) activity (ECHA decision 2014). Exposure was for 14 days via food spiked in parallel with the test material and positive controls HCB (for bioaccumulation) and 17beta-estradiol (for oestrogenic activity).

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): Novares LA 300, Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol
- Appearance: light yellow to colourless liquid
- Source and lot/batch No.of test material: RÜTGERS Novares GmbH, batch No. 38900
- Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LA 300_phenol, methylstyrenated
- Expiry date: 2017-02-13

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms:
Fish were collected for chemical analysis, 10 individuals were taken from each treatment (T = 0, 7, 14 days in the uptake phase and T = 1, 2, 5, 15, 21, 28 days in the depuration phase).
For tissue specific analysis of the test item, 5 additional fish samples were collected at the end of the uptake phase (T = 14 day). For the tissue specific analysis, guts were dissected, and the remaining carcasses served as base for the tissue specific analysis.
For lipid analysis, 3 additional fish were sampled (T= 0, 14 uptake phase and T = 28 depuration phase).
The collected fish were euthanised according to laboratory SOPs and in line with the animal welfare act (TierSchG, 2017). The fish were rinsed, dried and either extracted immediately or stored for analysis at -18 °C.
- Sampling intervals/frequency for test medium samples:
Fish food (T = 0, 7, 14 days uptake phase and T = 4 day of the depuration phase) and water samples (T = 14 day uptake phase) were collected for analysis of the test item and the reference substances.
- Sample storage conditions before analysis:
Water samples, fish extracts, food samples and prepared fish samples were stored in a refrigerator at 6 ± 2 °C, and fish samples were stored in a freezer at -20 ± 2 °C, if necessary. Prepared water samples were stored in the auto-sampler at room temperature prior to analysis.
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
- Sample preparation of whole fish tissue
A euthanised and weighed fish was homogenised with a spatula in 50 mL centrifugation tubes. Afterwards the samples were shaken for 15 min on the rotary mixer with 15 mL cyclohexane and centrifuged for 5 min at 3000 rpm. The supernatant was poured into a 50 mL graduated flask. The procedure of extraction was repeated twice, and the combined supernatants were filled up to the mark with cyclohexane and dried with sodium sulphate.
- Sample preparation of specific tissue
Weighed tissues in 50 mL centrifugation tubes were shaken for 15 min on the rotary mixer with 7 mL cyclohexane and centrifuged for 5 min at 3000 rpm. The supernatant was poured into a 25 mL graduated flask. The procedure of extraction was repeated twice, the combined supernatants were filled up to the mark with cyclohexane and dried with sodium sulphate.
Further details on analytical methods are listed in the ‘Details on analytical methods’ section.

Vehicle:

yes

Remarks:

Sunflower oil (Eden, organic grade, HEIRLER CENOVIS GMBH)

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

- Details on preparation of spiked fish food:
Spiked fish food was prepared by adding the desired amount of the mixture containing the test item and sunflower oil (150 mg test item /2g sunflower oil mixed by ultrasonication for 5 minutes) into 200 g of fish food VITAL fish, Coppens international GmbH to reach a nominal concentration of 500 µg/g wet weight (ww).
The reference items (HCB and 17β estradiol) were added into the food as described above, to reach a concentration of 100 µg/g ww. Briefly, 50 mg of each reference item were mixed into 20 g sunflower oil and ultrasonicated for 15 minutes, followed by addition of 3.2 mL of acetone to dilute the reference items. The mixture was further sonicated for 10 minutes before it was added to the fish food. The prepared food was left overnight and then transferred to a rotary evaporator for the acetone to evaporate.
Control feed was subject to the same treatment as the spiked feed, without the addition of the reference items.
The final pellet size of the fish food remained between 0.8-1.22 mm and the total fat content was 14 %.

Test organisms (species):

Pimephales promelas

Details on test organisms:

- Details on fish used in the experiment:
Fish, aged 12 to 13 months were used in the exposures, and a total of 170 animals were used in each treatment, of which at least 20 % were male (weighing 2-5 g fresh weight) and > 10 % female (weighing 1-3 g fresh weight).
- Source of fish:
The fish were obtained from a single breeding stock reared at the testing laboratory (originally from Umweltbundesamt, Schichauweg 58, 12307 Berlin, Germany).
- Holding and acclimation conditions:
Holding was performed at the test facility at 21 ± 2 °C, diffuse light (0.1-10 µmol·m-2·s-1, diurnal light with 12-16 h light / 8-12 h dark will be provided) and under flow-through conditions. The water exchange was at least 5 times the aquarium volume per day. The dissolved oxygen concentration was more than 80 % of the air saturation value.
The fish were kept in dechlorinated tap water (pH range 6 – 8.5, hardness 10-250 mg CaCO3/L, temperature 21 ± 2 °C) and fed VITAL fish food as specified above.
Only fathead minnow with at least 12 days of acclimation and mortality < 5 % within the last 7 days before the study started were used in the test. No disease treatments were administered throughout holding and testing. The stock population was fed the same type of food to be used during the test. Feeding was carried out ad libitum.

Route of exposure:

feed

Justification for method:

dietary exposure method used for following reason: The test item is a UVCB, not water soluble, therefore, route of exposure via water fraction was deemed not feasible.

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Remarks:

dechlorinated tap water

Total exposure / uptake duration:

ca. 14 d

Total depuration duration:

ca. 28 d

Hardness:

In the control exposures:
Start of the experiment (day 0) = 62 mg CaCO3/L
End of the experiment (day 42) = 58 mg CaCO3/L

Test temperature:

Control exposures: 21.5 ± 0.24 °C (n = 14, mean ± SD)
Reference material exposures: 21.4 ± 0.24 (n = 14, mean ± SD)
Test material exposures: 21.5 ± 0.26 °C (n = 14, mean ± SD)

pH:

Control exposures: 7.30 ± 0.19 (n = 14, mean ± SD)
Reference material exposures: 7.40 ± 0.13 (n = 14, mean ± SD)
Test material exposures: 7.33 ± 0.15 (n = 14, mean ± SD)

Dissolved oxygen:

Control exposures: 93 ± 5.22 % (n = 14, mean ± SD)
Reference material exposures: 95 ± 4.16 % (n = 14, mean ± SD)
Test material exposures: 94 ± 3.74 % (n = 14, mean ± SD)

TOC:

Measured weekly in the water supply tank throughout the uptake and depuration phase of the experiment, mean value of total TOC 1.28 ± 0.39 mg/L (n= 7, mean ± standard deviation)

Salinity:

Freshwater

Conductivity:

No data. Residual chlorine in the water supply tank throughout the exposures was < 0.01 mg/L.

Details on test conditions:

The exposures were carried out in 175 L glass aquaria filled with 145 L water and covered with glass lids. Flow-through test system with aeration were used, where the flow rate was approximately at 35 ± 1 L/h. Diurnal light, 16:8 (light:dark) h cycle with a measured light intensity of 1.56, 1.48, 1.81 µmol photons·m2/s in the control, reference material and test material group, respectively.

Nominal and measured concentrations:

Nominal concentration of the test material was 500 µg/g Food(ww). This nominal value corresponded to nominal concentrations of 195 µg Dimers/g; 115 µg Trimers/g; 46.1 µg p-CU-Phenol/g; 118 µg Di-Cu-Phenol/g; 6.01 µg Tri-Cu-Phenol/g food;
Since the test material is an UVCB substance, the measured concentrations were made on the components. The measured concentrations of the test concentrations are provided in the section “Any other information on materials and methods incl. tables”.

Reference substance (positive control):

yes

Remarks:

The reference substance was hexachlorobenzene (HCB) at 100 µg/g food(ww). The study was a combined study, including an endpoint to assess endocrine disruption, another reference material was spiked into the fish food, 17β-estradiol at 100 µg/g food(ww).

Details on estimation of bioconcentration:

The biomagnification factor was estimated, based on the calculations detailed in the OECD TG 305. Biomagnification was also normalised to the lipid content, based on the OECD TG 305.
Since the dietary study does not allow the derivation of a bioconcentration factor, a weight of evidence approach was utilised to derive a range of bioconcentration factors, based on published methodologies. The detailed methods are described in the document on “Bioaccumulation assessment” attached in the section “Attached background information”.

Lipid content:

ca. 6.2 %

Time point:

start of exposure

Remarks on result:

other: Control group

Lipid content:

ca. 9.3 %

Time point:

end of exposure

Remarks on result:

other: Control group

Lipid content:

ca. 3.7 %

Time point:

start of exposure

Remarks on result:

other: Test material group

Lipid content:

ca. 9.7 %

Time point:

end of exposure

Remarks on result:

other: Test material group

Lipid content:

ca. 5.5 %

Time point:

start of exposure

Remarks on result:

other: Reference material group

Lipid content:

ca. 7.6 %

Time point:

end of exposure

Remarks on result:

other: Reference material group

Key result

Conc. / dose:

ca. 195 µg/g food

Temp.:

ca. 21.5 °C

pH:

7.3

Type:

BMF

Value:

ca. 0.074 dimensionless

Basis:

normalised lipid fraction

Remarks:

Lipid factor: 0.564

Calculation basis:

kinetic, corrected for growth

Remarks on result:

other: Nominal concentration reflects the concentration of dimers in food

Key result

Conc. / dose:

ca. 115 µg/g food

Temp.:

ca. 21.5 °C

pH:

7.3

Type:

BMF

Value:

ca. 0.137 dimensionless

Basis:

normalised lipid fraction

Remarks:

Lipid factor: 0.564

Calculation basis:

kinetic, corrected for growth

Remarks:

all evaluations are based on a food ingestion rate of 0.02 g food / g fish / day

Remarks on result:

other: Nominal concentration reflects the concentration of trimers in food

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

5.3 d

Remarks on result:

other: Dimers

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

25.8 d

Remarks on result:

other: Trimers

Key result

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

0.13

Remarks on result:

other: Dimers

Key result

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

0.027

Remarks on result:

other: Trimers

Key result

Rate constant:

overall depuration rate constant (d-1)

Value:

0.136

Remarks on result:

other: Dimers

Key result

Rate constant:

overall depuration rate constant (d-1)

Value:

0.033

Remarks on result:

other: Trimers

Details on kinetic parameters:

- Assimilation efficiency
Dimers: calculated assimilation efficiency (alpha, α) was 0.270.
Trimers: calculated assimilation efficiency (alpha, α) was 0.104.
==================================================

- Lipid correction factor
Lipid in fish at uptake day 14: 9.7 % of weight
Lipid in fish at depur. on day 28: 6.1 % of weight
=====================================
Lipid in fish, mean value: 7.9 % of weight
Lipid in diet: 14 % of weight
=====================================
Lipid factor calculation: Lipid in fish(mean) / Lipid in diet =7.9% / 14% = 0.564
Lipid factor: 0.564
===================================================================


- Uptake rate constant
Since the dietary study does not allow the calculation of an uptake rate constant, a weight of evidence approach was utilised where several published methods were used to derive uptake rate constants. Please see attached document on the “Bioaccumulation assessment” in the ‘Attached background material’.

Results with reference substance (positive control):

Experimental work was done under GLP conditions, however, data evaluation and calculation of endpoints for HCB was done as non-GLP. The results were as follows (see Report, Tab. 37):
The overall depuration rate koverall was 0.0567/d.
The growth-corrected depuration rate (kdepuration) was 0.0595/d.
The half-life depuration time (DT50) was 11.6 days.
The calculated assimilation efficiency (alpha) was 0.913.
The BMF = 0.307
The lipid-corrected BMF = 0.544
Conclusion from the weight of evidence approach using published methods in deriving a lipid-normalised BCF value for the reference substance (HCB) found the BCF to be 5040 ± 1811 (mean ± standard deviation).
The accumulation of 17β-estradiol was not assessed, since it was included to assess another endpoint.

Details on results:

- Mortality of test organisms/ - Mortality and/or behavioural abnormalities of control:
No significant mortality and no non-lethal effects (morphological and behavioural) were observed in the test material group and in the control group (a cumulative mortality of 0.6% after 42 days of the study) throughout the study. In the reference material group, a cumulative mortality of 18.2% occurred between study day 10 and 42. Furthermore, some fish showed morphological effects (internal bleeding) during day 11 to day 13 (uptake phase). These morphological effects did not occur during the depuration phase. Since a reference material group is not mandatory in the guideline OECD 305 and the combination of hexachlorobenzene and 17β-estradiol has not been tested before, this mortality is considered to have no impact on the validity and integrity of the study and the results of the control and exposure group.
- Observations on body length and weight:
The growth and weight of fish did not change significantly throughout the experiment (both uptake and depuration phase), and it did not differ between treatments (Table 3, Section ‘Any other information on results incl. tables’).
- Other biological observations:
No difference in feeding was noted in fish in any of the treatments.
- Results on other constituents of the test material:
All measurements of the phenols were below the limit of quantification (LOQ values: 0.092 µg p-cumylphenol/g; 0.236 µg di-cumylphenol/g; 0.030 µg tri-cumylphenol/g) in fish. Therefore, biomagnification of the phenolic components of the test material can be ruled out.
- Organ specific bioaccumulation:
Fish tissues without guts were analysed on day 14 and the results are reported in Table 5 (Section ‘Any other information on results incl. tables’).

Reported statistics:

The differences between two treatments were assessed using Students t-test. Where no differences were found, the data was pooled, and an overall growth rate was calculated as the overall slope of the linear correlation. For any correlation analyses, the data were ln transformed (natural logarithm) and linear least squared correlations were used. The individual fish concentration data for two main constituent groups (dimers and trimers) for the depuration period were converted to their natural logarithms and plotted versus time (day). No outlier test was carried out. In order to refine the regression line, estimated values LOD were used for calculation, but were given as The growth-corrected depuration rate was calculated by subtraction of the growth rate from the overall elimination rate (kdepuration = koverall - kgrowth). The chemical assimilation efficiency (alpha) is calculated using the equation in OECD TG 305.

Calculated BCFlipid-normalised 5% values:

Uptake and depuration data could only be quantified for the Dimers and Trimers. Based on the calculations detailed in the attached “Bioaccumulation” document (‘Attached background material’), the following lipid-normalised BCF values could be derived by calculation:

Dimers: 2564 ± 1803 (mean ± standard deviation)

Trimers: 22338 ± 23353 or range (mean ± standard deviation)

 

Growth rate data

The growth rate of fish in the control and test group were compared (Table 3).

Table 3. Growth rate in the control and test material groups.

		Control		Test Group
Growth rate  (=Slope) [1/d]		0.0073		0.0045
Stat. significance		No		No
Intercept		0.799		0.867
C.I.		0.00114 – 0.0135		-0.00166 – 0.0107

C.I. = Confidence interval, p = 95 %        

Key data are summarised in Table 4: 

Table 4. Summary of Results for Depuration of the Two Main Constituent Groups Dimers and Trimers of the Test Item (Report Table 37)

	Dimers	Trimers	Phenols	Reference substance HCB
C0, depuration^# [µg/g fish, fresh-weight]	6.5 µg/g	2.3 µg/g	<LOQ	18.2 µg/g
Koverall [d^-1]	0.136	0.0327	n.d	0.0567
α	0.270	0.104	n.d	0.913
Growth-corrected depuration kdepuration[d^-1]	0.130	0.0269	n.d	0.0595
Growth-corrected half-life [d]	5.3	25.8	n.d	11.6
BMF	0.0415	0.0775	<0.001*	0.307
BMFLipid	0.0737	0.137	<0.001*	0.544

α            = Chemical assimilation factor

BMF        = Biomagnification factor

BMF_Lipid   = Lipid-corrected biomagnification factor, based on a lipid factor of 0.564

#            = Calculated, for details see section 9

n.d.        = not determined

*            = derived empirically, no calculation was carried out

Italics     = evaluation as non-GLP

The mean concentration of the test material constituents and HCB in fish are shown in Table 5. Since the phenolic constituents of the test material were <LOQ, only the dimer and trimer results are shown in Table 5.

Concentration of test material active ingredients and the reference substance HCB.

Table 5. Mean Concentrations of the Active Ingredients of the test material and HCB in Fish during the Test.
Separate fish were analysed for test material and HCB (each five replicates). (Report, Table 25).

	Uptake phase
Active ingredient	Day 0 mean value		Day 7 mean value^*)		Day 14 mean value
Groups for evaluation	CfBody [µg/g]		CfBody [µg/g]		CfBody [µg/g]
Dimers	< LOQ		1.79		3.15
Trimers	< LOQ		0.916		2.76
HCB	< LOQ		6.19		12.8
	Depuration phase
Active ingredient	Day 1 mean value	Day 2 mean value		Day 5 mean value		Day 8 mean value
Groups for evaluation	CfBody [µg/g]	CfBody [µg/g]		CfBody [µg/g]		CfBody [µg/g]
Dimers	1.45	1.16		0.334 **		0.891
Trimers	1.56	2.30		0.986		2.59
HCB	9.55	6.77		5.62		11.7
Active ingredient	Day 15 mean value	Day 21 mean value		Day 28 mean value
Groups for evaluation	CfBody [µg/g]	CfBody [µg/g]		CfBody [µg/g]
Dimers	< LOQ	< LOQ		< LOQ
Trimers	1.13	0.915		0.773
HCB	4.64	3.27		2.57

C_fBody               =Measured concentration of the test material related to fish body wet weight,
enrichment and dilution factors taken into account

C_{F,L Body}            = Concentration test material related to fish body wet weight, lipid-normalised

L                = lipid fraction (based on wet weight), see chapter8.2.5

LOQ                  = Limit of quantification of the analytical method

test material: LOQ for the active ingredients for fish:
0.390 µg Dimers/g; 0.575 µg Trimers/g; 0.092 µg pCU-Phenol/g;
0.236 µg Di-Cu-Phenol/g; 0.030 µg Tri-Cu-Phenol/g)

            HCB: 0.505 µg HCB/g

*              = mean value of 7 replicates

**            = calculated concentration below LOQ, but > 70 % of the above values for the LOQ, therefore only for information

 

Table 6. Concentrations of the Active Ingredients of Test material and HCB in Fish on Day 14 Uptake Phase.

Fish tissues were analysed for test material and HCB, values refer to carcass after dissection of the gut. (Report, Table 26)

Groups for evaluation	Fish 6	Fish 7	Fish 8	Fish 9	Fish 10	Mean value
Active ingredient	CTissue [µg/g]	CTissue [µg/g]	CTissue [µg/g]	CTissue [µg/g]	CTissue [µg/g]]	CTissue [µg/g]
	Uptake phase – day 14
Dimers	0.803	0.812	2.45	0.834	2.47	1.47
Trimers	3.94	2.84	3.93	5.42	6.41	4.51
pCU-Phenol	0.0332	0.149	0.375	0.405	0.702	0.333
Di-Cu-Phenol	0.255	0.104	0.206	0.471	0.336	0.274
Tri-Cu-Phenol	0.457	0.174	0.317	0.680	0.740	0.474
HCB	20.7	44.1	29.4	29.5	40.4	32.8

 

Three spiked food samples and one control were analysed on day 0, day 7 and day 14 of the uptake phase and on day 4 of the depuration phase to determine the concentration of the test material (active ingredients), HCB and 17ß-estradiol (Report, Table 27).The concentration of the test material in food was 86 – 110 % of the nominal concentration throughout the test.

 

After the uptake phase (14 d), the concentrations of the active ingredients of the test material in the test media (water) were 0.126, 0.186, 0.150, 0.131 and 0.0187 µg/L for dimers, trimers, pCU-Phenol, Di-Cu-Phenol, Tri-Cu-Phenol, respectively (Report, Table 28). These values correspond to measured concentrations after enrichment of >lowest calibration level (LCL), but of <LOQ. The concentration of the reference materials were <LOQ in the water in the uptake phase day 14.

Validity criteria fulfilled:

yes

Conclusions:

The study revealed that only the Dimers and Trimers of test material (Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol (OAPP); phenol, methylstyrenated; Novares LA 300) show a potential to be biomagnified. The lipid-corrected BMF values were 0.073 and 0.1374 for the dimers and trimers, respectively, both active ingredients of the test material. The corresponding BCF values, calculated on the basis of several approaches published in the literature are in the order of ≤ and ≥ 2000 for the dimers, and ≥ 5000 for the group of the trimers (further details in the ‘Bioaccumulation assessment’ document in the ‘Attached background material’ section). Uncertainty in these values are based on uncertainties in the conversion factors employed.

Executive summary:

A dietary bioaccumulation study was conducted with adult fathead minnow (Pimephales promelas) to determine the biomagnification potential of the test material, Oligomerisation and alkylation reaction products of 2‑phenylpropene and phenol [new EC name with EC no. 700-960 -7 (previously: phenol, methylstyrenated, CAS no. 68512-30-1)]. The test material was a UVCB substance containing alkylation products of phenol with α-methylstyrene (mono-, di- and triarylalkylated phenols, i.e., ‘phenols’) and oligomerisation products of α-methylstyrene (i.e. ‘dimers’ and ‘trimers’). A flow-through test with 3 treatments (control, test material, reference material) was carried out according to the OECD TG 305 with a 14-day uptake and 28-day depuration phase. The calculated biomagnification factors (BMFs) normalised to the lipid fractions for the test material constituents, dimers, trimers and phenols were 0.073, 0.1374 and < limit of quantification, LOQ, respectively, suggesting high and immediate elimination/excretion. The study did reveal that only the dimers and trimers of test material show a potential to biomagnify. The corresponding BCF values, calculated on the basis of several approaches are in the order of ≤ and ≥2000 for the dimers, and ≥5000 for the group of the trimers. Uncertainty in these values are based on uncertainties in the conversion factors employed.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, basic data given (Jp. study report available with English results tables); test was performed by an laboratory with an high reputation for delivery of robust data.

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The test was performed by a laboratory with a high reputation for delivery of robust data.
This test was conducted in accordance with the test method "Bioaccumulation test of chemical substance in fish and shellfish" stipulated in the Order Prescribing the Items of the Test Relating to the New Chemical Substance (1974, Order of the Prime Minister, the Minister of Health and Welfare, the Minister of International Trade and Industry No.1). It corresponds to OECD TG 305 C of May 12, 1981)

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report or NITE database): 4-methyl-2,4-diphenylpent-1-ene or 2,4-diphenyl-4-methyl-1-pentene
- Dimer of methylstyrene and key constituent in OAPP
- Purity 97 % (according to data provided from client)
- No further information on test substance

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms: 2 fish every two weeks (0, 7, 14, 28, 42 and 60 days)
- Sampling intervals/frequency for test medium samples: twice a week
- Sample storage conditions before analysis: in Japanese
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods): in Japanese

Vehicle:

yes

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

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances):
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): 2-methoxyethanol and HCO-40
- Concentration of vehicle in test medium: high exposure level: 2-methoxyethanol ca. 25 ppm (v/v), HCO-40 0.2 mg/L; low exposure level: 2-methoxyethanol ca. 25 ppm (v/v), HCO-40 0.02 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no data

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM (see also CITI 1992)
- Common name: carp
- Source: Sugishima Fish Farm, Kumamoto, Japan
- Length at study initiation: 8 +/-4 cm
- Weight at study initiation: about 5 g
- Lipid content: 6.1 g (start); 7.3 g (end)
- Weight at termination: no data
- Method of breeding: flow through system at 24 ± 2°C
- Health status: only visibly healthy fish were used; after reception, fish were externaly desinfected (static conditions,
50 mg/L Terramycin (Taito Pfizer) and 7g/L sodium chloride for 24 h).
- Feeding during test:
- Food type: pelleted feed for carp (Japan Haigo Shiryo K.K.)
- Amount: 2% of total body weight
- Frequency: twice a day, half of total amount each

ACCLIMATION
- Acclimation period: at least one month
- Acclimation conditions (same as test or not): yes
- Type and amount of food: pelleted feed for carp (Japan Haigo Shiryo K.K.), 2% of total body weight
- Feeding frequency: twice a day, half of total amount each
- Health during acclimation (any mortality observed): no data, abnormal fish were removed.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

60 d

Total depuration duration:

16 d

Hardness:

--

Test temperature:

24 ± 2°C

Dissolved oxygen:

6 - 8 mg/L

Details on test conditions:

TEST SYSTEM (see also CITI 1992)
- Test vessel:
- Type (delete if not applicable): no data
- Material, size, headspace, fill volume: glas tank , volume 100 L
- Aeration: aeration of dilution water
- Type of flow-through (e.g. peristaltic or proportional diluter): no data
- Renewal rate of test solution (frequency/flow rate): 2.9 to 11.5 per day / flow rate 200 to 800 mL/min
- No. of organisms per vessel: 15 - 20
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): no data
- Biomass loading rate: 4.5 - 6 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: underground water pumped at the site of Kurume Research Laboratories
- dilution water was confirmed to meet the quality criteria fisheries
- Intervals of water quality measurement: once every six month; temperature, pH and dissolved oxygen continuously.
- Intervals of test medium replacement: flow through system, see above

RANGE-FINDING / PRELIMINARY STUDY
- Results used to determine the conditions for the definitive study: test concentrations were decided on basis of an acute toxicity test (Oryzias latipes; 48h-LC50 5.4 mg/L)

Nominal and measured concentrations:

High exposure level (level 1): 10 µg/L
low exposure level (level 2): 1 µg/L

Reference substance (positive control):

no

Details on estimation of bioconcentration:

Calculation of bioconcentration factor = concentration of test substance in fish / concentration of test substance in water

Lipid content:

>= 3 - <= 7.7 %

Time point:

other: 7 d

Remarks on result:

other: Range of individual values: see Attached Document: BCF

Lipid content:

>= 1.2 - <= 4.5 %

Time point:

other: 60 d

Remarks on result:

other: Range of individual values: see Attached Document: BCF

Key result

Conc. / dose:

1 µg/L

Type:

BCF

Value:

2 767 L/kg

Basis:

whole body w.w.

Remarks:

lipid normalised (5 %) based on individual fish

Time of plateau:

14 d

Calculation basis:

steady state

Remarks on result:

other: average of measurements during steady state (three measurements from day 28 to day 60 with two fish per measurement)

Key result

Conc. / dose:

10 µg/L

Type:

BCF

Value:

2 320 L/kg

Basis:

whole body w.w.

Remarks:

lipid normalised (5 %) based on individual fish

Time of plateau:

14 d

Calculation basis:

steady state

Remarks on result:

other: average of measurements during steady state (three measurements from day 28 to day 60 with two fish per measurement)

Conc. / dose:

1 µg/L

Type:

BCF

Value:

2 912 L/kg

Basis:

whole body w.w.

Time of plateau:

28 d

Calculation basis:

steady state

Remarks on result:

other: average of measurements during steady state (three measurements from day 28 to day 60 with two fish per measurement)

Conc. / dose:

10 µg/L

Type:

BCF

Value:

1 790 L/kg

Basis:

whole body w.w.

Time of plateau:

14 d

Calculation basis:

steady state

Remarks on result:

other: average of measurements during steady state (three measurements from day 28 to day 60 with two fish per measurement)

Conc. / dose:

1 µg/L

Type:

BCF

Value:

>= 724 - <= 4 410 L/kg

Basis:

whole body w.w.

Time of plateau:

28 d

Calculation basis:

steady state

Remarks on result:

other: minimum and maximum value of three measurements with two fish per measurement during steady state from day 28 to day 60

Conc. / dose:

10 µg/L

Type:

BCF

Value:

>= 896 - <= 3 330 L/kg

Basis:

whole body w.w.

Time of plateau:

14 d

Calculation basis:

steady state

Remarks on result:

other: minimum and maximum value of three measurements with two fish per measurement during steady state from day 28 to day 60

Elimination:

yes

Parameter:

other: DT50 (high exposure)

Depuration time (DT):

4.5 d

Elimination:

yes

Parameter:

other: DT50 (low exposure)

Remarks:

probably mistaken (see re-estimation)

Depuration time (DT):

15.7 d

Remarks on result:

other: probably mistaken (see re-estimation)

Details on kinetic parameters:

The cumulated final tissue levels in whole fish on day 60 correlate well with either exposure level: 12.2 µg/g at 10 µg/L and 1.37 at 1 µg/L, i.e. a 10-fold higher exposure concentration resulted in a 10-fold higher tissue level. This indicates that the elimination kinetics at both exposure regimens are identical, which should end in similar eliminations constants or DT50 values. The presentation of the kinetic plots (Report, Fig. 13 and 14: see Attached Document BCF) suggest poor correlations between the residual fish levels over time, demonstrated by low regression coefficients R^2: R^2 (10 µg/L) = 0.58, R^2(1 µg/L) = 0.18 (rounded). In either case, the kinetics are obviously determined each by an outlier.
At least, the comparatively long DT50 of 15.7 d seems to be erroneous. A simple comparison of the initial tissue concentration (100%) with the residual one after 9 days suggest that the elimination half-lives are as follows (assuming first-order elimination kinetics):
High exposure (10 µg/L): decrease ~95%/9d = ~4 T/2 --> DT50 = ~2.3d
Low exposure (1 µg/L): decrease ~83%/9d = ~2.7 T/2 --> DT50 = ~3.3d.

Metabolites:

no data

Results with reference substance (positive control):

not included

Details on results:

Note: In the original study, BCF was normalised to a lipid content of 4% rather than 5% (not shown: see Attached Document BCF).

Reported statistics:

--

Original data (overview) (unadjusted to lipid content):

Day		7	14	28	42	60	BCF range from 28–60 days #
High	Conc. water(µg/l)	9.95	10.07	10.33	10.54	10.48
exposure group							896 -3330
	BCF replica1	1610	957	1370	896	1080
(10 µg/L)	BCF replica2	427	1950	2810	3330	1250
Low	Conc. water(µg/l)	1.01	0.98	0.94	0.94	0.93
exposure group							724 -4410
	BCF replica1	469	1610	3730	4410	724
(1µg/L)	BCF replica2	423	1740	2270	4120	2220

# Note: Original individual BCF values as measured, NOT normalised to a lipid content of 4 or 5%: Final reported BCF values have been related to 4% lipid (see Jp. Report, Table 10) . The values published in the NITE databank are misleading, because firstly neither has explicitly been stated that they cover the range of time-point specific BCFs, and because secondly nor is clear that they are original without lipid normalisation.

Single and mean BCFs after normalisation to 5% lipid content

Exposure group		42 d	60 d
10 µg/L	BCF replica 1	1659	1200
	BCF replica 2	2378	5208
	Average	2019	3204
1 µg/L	BCF replica 1	4690	905
	BCF replica 2	3075	2582
	Average	3883	1743

Validity criteria fulfilled:

not specified

Conclusions:

Despite lipid normalisation to 5 % , single BCF replicates were still relative variable in particular on day 60, with ratios between about 2.8 and 4.3. The highest mean BCFs are approx. 3200 (10 µg/L, 60 d) and 3900 (1 µg/L, 42 d). Averaged over a longer time period (still steady state), BCF are below 3000 L/kg. Based on these values, bioaccumulation is assessed to be moderate. Note: The authors concluded "low bioaccumulation" (NITE 2002: databank). Given the BCF < 2000 at 1 µg/L after 60 d, the bioconcentration potential is considered to be low, since -moreover- the lower experimental exposure concentration is closer to potential environmental water levels and therefore is of higher environmental relevance.

Reference 3

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target substance oligomerisation and alkylation reaction products of 2-phenylpropene and phenol (OAPP) (EC no. 700-960-79) is obtained by a Lewis acid catalysed reaction between phenol and 1-methylstyrene. Products of the reaction are either methylstyrenated phenols or dimers/trimers of 1-methylstyrene (2-phenylpropene). Accordingly, the target substance OAPP consists of four basic groups of constituents. Two groups contain purely aryl-aliphatic (non-phenolic) substances differing only in the degree of oligomerisation (dimers and trimers of 2-phenylpropene). The third and fourth group contain mono-methylstyrenated and di-methylstyrenated phenols, respectively. Dimers and trimers amount together to about 45 to 80 % of OAPP, while the phenolic components contribute about 20 to 50 %.
The source substance p-cumylphenol (4-(1-methyl-1-phenylethyl)phenol) (CAS No. 599-64-4) is a main constituent of the target substance OAPP. It is a mono-methylstyrenated phenol present in OAPP and it is estimated that its bioaccumulation properties will also be representative for other phenolic components of OAPP.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance OAPP is a UVCB with a complex and variable composition of constituents. The source substance p-cumylphenol is a mono-constituent substance. It is a constituent of the target substance (mono-methylstyrenated phenol) thus contributing at least in part to the properties of the target substance.

3. ANALOGUE APPROACH JUSTIFICATION
Based on the chemical structure of the different components of OAPP, the bioaccumulation potential of the phenolic components is assessed to be lower than the bioaccumulation potential of non-phenolic components. Log Kow is somewhat lower compared to the same size non-phenolic components and the ability for biotransformation is considered to be better. Therefore, the source substance p-cumylphenol represents the part of the target substance OAPP that will bioaccumulate to a lesser extent. Bioconcentration factors determined for the source substance, represent the part of the target substance OAPP with a lower bioaccumulation potential.

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Read-across to the preceding entry:
Source substance: p-Cumylphenol (NITE, Japan, CSCL)_;
Reference: NITE National Institute of Technology and Evaluation, Japan 2002

Lipid content:

3.5 %

Time point:

start of exposure

Lipid content:

4.1 %

Time point:

end of exposure

Key result

Conc. / dose:

10 µg/L

Temp.:

25 °C

Type:

BCF

Value:

168 L/kg

Basis:

whole body w.w.

Time of plateau:

14 d

Calculation basis:

steady state

Remarks on result:

other: average of measurements during steady state (three measurements from day 28 to day 60 with two fish per measurement)

Remarks:

high exposure level

Conc. / dose:

10 µg/L

Temp.:

25 °C

Type:

BCF

Value:

>= 139 - <= 187 L/kg

Basis:

whole body w.w.

Time of plateau:

14 h

Calculation basis:

steady state

Remarks on result:

other: minimum and maximum value of three measurements with two fish per measurement during steady state from day 28 to day 60

Remarks:

high exposure level

Description of key information

The bioaccumulation potential of the substance Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol (OAPP, previously phenol, methylstyrenated, Novares LA 300) has been assessed in bioaccumulation/ bioconcentration studies in fish. The BCF of two individual constituents of OAPP, one methylstyrenated (cumyl) substituted phenol and a dimer of 2-phenylpropene were experimentally shown to be low to moderate. For dimers and trimers of 2-phenylpropene, BMF were determined. Conversion into BCF indicates that the BCF for dimers is between 2000 and 5000, while the BCF for trimers is above 5000. However, the conversion of BMF into BCF values show such a high variability/standard deviation due to uncertainties in conversion methods that the high BCF values resulting from conversion have to be considered as not very reliable. They are not suited for assessment and should be taken with caution.

Key value for chemical safety assessment

BCF (aquatic species):

3 000 L/kg ww

BMF in fish (dimensionless):

0.137

Additional information

Aquatic bioaccumulation of OAPP and its constituents has been investigated in three bioconcentration/bioaccumulation studies. The constituents diphenylmethylpentene (4-methyl-2,4-diphenylpent-1-ene) (NITE 2002) and cumylphenol (1-methyl-1-phenylethyl)phenol (NITE 1990) were subject of valid bioconcentration flow-through fish tests (OECD 305). BCFs were determined to be 2767/2320 L/kg fish ww (low/high exposure concentration, lipid normalised) and 168 L/kg fish ww (high exposure concentration) at steady state, respectively. In a bioaccumulation study (dietary exposure, OECD 305-III) (Klix 2018), BMF factors could only be determined for the non-phenolic constituents of OAPP. For the phenolic components, the concentrations in fish during the depuration phase was too low to be determined. A bioaccumulation potential was thus found only for the non-phenolic constituents of OAPP. Lipid-based and growth-corrected BMF values were 0.0737 and 0.1374 for dimers and trimers, respectively. Conversion into BCF values by calculation of k₁rate constants using various methods resulted in average values of 2564 ± 1803 and 22338 ± 23353 (mean ± SD) for dimers and trimers, respectively. Their variation is very high and the results differ very much, so that especially the higher value is considered not to be reliable. This estimation of k₁ bears a high uncertainty indicated by the high standard deviation.

Overall, BCF are below 3000 for about 75 % (w/w) of the test substance (phenolic constituents and dimers) while for approx. 25 % (trimers) a reliable BCF value suited for assessment is not yet available. An estimate derived from the experimental BMF suggests that a BCF for trimers may be above 5000. Based on currently available evidence a preliminary BCF for OAPP is set at 3000 L/kg ww.