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Bioaccumulation: aquatic / sediment

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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
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 17 beta-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 ca. d
Total depuration duration:
ca. 28 ca. 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 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;
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 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 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:
all evaluations were based on a food ingestion rate of 0.02 g food / g fish / day
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) was 0.270.
Trimers: calculated assimilation efficiency (alpha) was 0.104.

- 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:
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

 

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

0.1374

<0.001*

0.544

α            = Chemical assimilation factor

BMF        = Biomagnification factor

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

#            = Calculated, for details see section 9 of the Report

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

 

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

CF,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 0.0737 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 0.0737, 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.

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 source test material ‘Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol (OAPP) [EC no. 700-960-7] consists of a complex mixture of numerous aryl-aromatic constituents some of them containing also a phenolic structural element. The constituents can basically be divided into groups corresponding to the presence of phenolic OH-groups as well as their level of oligomerisation (dimers, trimers).
The target substance ‘Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction, alkylated and oligomerised’ (NAF-AO) [EC no. 701-299-207] consists also of a complex mixture of numerous aryl-aromatic constituents but without phenolic substituents present. Its components can basically divided into groups corresponding to their level of oligomerisation (dimers, trimers, tetramers).
The potential for aquatic bioaccumulation of the target substance NAF-AO will be characterised by the range of constituents present in the substance. Therefore, results obtained from an aquatic bioaccumulation study with the source substance OAPP that contains to a large extent the same constituents can be used in order to characterise the bioaccumulation potential of NAF-AO.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source substance OAPP is a UVCP substance obtained in an acid catalysed oligomerisation / alkylation reaction of the starting materials 2-phenylpropene and phenol. In this process, aromatic and phenolic components react with each other resulting on the one hand in oligomerisation products of 2-phenylpropene (reaction of 2-phenylpropene molecules among themselves, aryl-aliphatic substances) and on the other hand in 2-phenylprop-2-yl substituted (methylstyrenated) phenols (reaction of 2-phenylpropene with phenol, aralkyl-substituted phenols, phenolic substances). Besides dimeric products also trimeric and oligomeric products can be formed. Aromatic rings within the products are connected by methyl-substituted alkenyl carbon-chains in case of the 2-phenylpropene oligomers and by a single bridging carbon atom substituted by two methyl groups in case of methylstyrenated phenols. Besides phenyl substituents, non-phenolic oligomers of OAPP can also include indan substituents.
Based on the degree of oligomerisation four to five basic groups of constituents are formed. Two or three groups contain purely aryl-aliphatic (non-phenolic) substances differing only in the degree of oligomerisation (dimers, trimers, and higher (n ≥ 4) oligomers). Two groups comprise the alkylation products of phenol (mono or di 2-phenylprop-2-yl (methylstyryl) substituted phenols; phenolic products). Non-phenolic constituents (aryl-aliphatic oligomers) amount together to about 45 to 80% of OAPP, while the phenolic components contribute about 20 to 50%.
OAPP is a viscous oily material with a low water solubility (between 1 and 3.5 mg/L depending on its composition).
Basically, there are two centres for metabolic transformation present in both component types. On the one hand, the aliphatic structure linking aromatic/phenolic rings and especially their methyl substituents can be metabolised by oxidative transformation finally leading to carboxylic acids and eventually to decarboxylation. The second major pathway for metabolism concerns the aromatic rings. Metabolism will result in hydroxylated species like phenols or quinones.
The target substance NAF-AO is a UVCB substance as well, also obtained in an acid catalysed alkylation and oligomerisation reaction of the starting material ‘Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction’. This material comprises predominantly styrene and indene derivatives. In the production process, oligomerisation products of C8-10 aromatic hydrocarbons are formed consisting of dimeric, trimeric, and oligomeric (n ≥ 4) products. The individual constituents are the same as are produced in the production process of OAPP. Due to the composition of the starting material, no phenolic but only aryl-aliphatic products are formed. These products consist of two, three, or more aromatic rings connected by methyl-substituted alkenyl carbon-chains (dimers, trimers, oligomers). The aromatic substituents include besides benzene also indan. Based on the degree of oligomerisation, constituents are combined into three different groups (dimers, trimers and higher oligomers). Depending on the type of technical product (Novares L 100, L 700, TL 10), composition with respect to different component groups will vary (dimers from ca. 20 to 70%, trimers from ca. 10 to 30%, higher oligomers from ca. 15 to 50%).
NAF-AO is a viscous oily material with a low water solubility (between 0.6 and 1.3 mg/L depending on its composition). Thus, water solubility is somewhat lower than the water solubility of OAPP. Values for water solubility obtained by (Q)SAR (US EPA EPI suite) for individual model constituents are lower (between 0.082 and 0.038 mg/L for dimers and between 0.011 and 0.027 mg/L for trimers) indicating that measured values may overestimate the real water solubility. Overall, the water solubility of NAF-AO is low to very low depending on the technical product and its composition (higher percentages of trimers and higher oligomers will reduce the water solubility).
Basically, metabolism of NAF-AO is likely to proceed at the same two centres as in OAPP resulting in similar metabolic transformation products. Aliphatic structures with their methyl substituents linking aromatic rings can be metabolised by oxidative transformation finally leading to carboxylic acids and eventually to decarboxylation. The second sites for metabolic transformation are the aromatic rings present in the substance. Metabolism will result in hydroxylated species like phenols or quinones. Thus metabolism at aromatic rings can lead to similar products that are already present in the phenolic fraction of OAPP.

3. ANALOGUE APPROACH JUSTIFICATION
Environmental fate of complex mixtures like UVCB substances will be determined by their intrinsic properties. Under environmental conditions or during processing of the substances, environmentally available or volatile components can be released into the environment. Distribution properties and uptake/depuration will characterise the potential for aquatic bioaccumulation.
The target substance NAF-AO and the source substance OAPP are very similar. They contain to a substantial extent the same constituents. They only differ in the presence of phenolic constituents, which are lacking in NAF-AO.
In the present bioaccumulation study, test material (source substance OAPP) was applied in the diet. Time dependent changes in fish concentration were determined individually for the groups of phenolic constituents and for the groups of dimeric and trimeric aryl-aliphatic constituents. Hence, specific results are obtained for the group of dimers and the group of trimers. Data for dimers and trimers measured in the experiment with the source substance OAPP as test material can directly be used for the same components group of the target substance NAF-AO. Therefore, it is justified to use data determined for the source substance OAPP to characterise the bioaccumulation potential of the target substance NAF-AO.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Read-across to preceding entry:
Source test material: Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol;
Reference: Klix 2018
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: tetst 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:
all evaluations were based on a food ingestion rate of 0.02 g food / g fish / day
Remarks on result:
other: concentration/dose and BMF value relates to the dimeric constituents of the test material (OAPP) in food
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance NAF-AO
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: concentration/dose and BMF value relates to the trimeric constituents of the test material (OAPP) in food
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance NAF-AO
Key result
Elimination:
yes
Parameter:
DT50
Depuration time (DT):
5.3 d
Remarks on result:
other: the test result relates to the dimeric constituents of the test material OAPP
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance NAF-AO
Key result
Elimination:
yes
Parameter:
DT50
Depuration time (DT):
25.8 d
Remarks on result:
other: the test result relates to the trimeric constituents of the test material OAPP
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance NAF-AO
Key result
Rate constant:
growth-corrected depuration rate constant (d-1)
Value:
0.13
Remarks on result:
other: the test result relates to the dimeric constituents of the test material OAPP
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance NAF-AO
Key result
Rate constant:
growth-corrected depuration rate constant (d-1)
Value:
0.027
Remarks on result:
other: the test result relates to the dimeric constituents of the test material OAPP
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance NAF-AO
Key result
Rate constant:
overall depuration rate constant (d-1)
Value:
0.136
Remarks on result:
other: the test result relates to the trimeric constituents of the test material OAPP
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance NAF-AO
Key result
Rate constant:
overall depuration rate constant (d-1)
Value:
0.033
Remarks on result:
other: the test result relates to the trimeric constituents of the test material OAPP
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance NAF-AO
Details on kinetic parameters:
- Assimilation efficiency
Dimers: calculated assimilation efficiency (alpha) was 0.270
Trimers: calculated assimilation efficiency (alpha) was 0.104
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-12-14 - 2002-04-15
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, basic data given (Jp. study report available with English results tables); acceptable for assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)
Version / remarks:
May 12, 1981
Deviations:
yes
Remarks:
only two fish per sampling time
Principles of method if other than guideline:
The test was performed by a laboratory with an 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 ot 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 from May 12, 1981)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material (as cited in study report or NITE database): 2,4-diphenyl-4-methyl-1-pentene or 4-methyl-2,4-diphenylpent-1-ene
- Dimeric constituent (EC name 1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene) [CAS no. 6362-80-7] of the substance 'Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction, alkylated and oligomerised' (NAF-AO) [EC no. 701-299-7]; previous name: Hydrocarbons, C9-unsaturated, polymerized [CAS no. 71302-83-5]
- Analytical 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 at days 7, 14, 28, 42 , and 60 of the exposure period and at days 2, 4, 9, and 16 of the depuration period
- 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 (A) and HCO-40 (B) (Hydrogenated Castor Oil)
- Concentration of vehicle in test medium: high and low concentration: ca. 25 ppm (v/v) of A and 0.2 mg/L of B
- 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
Justification for method:
aqueous exposure method used for following reason: standard method according to test guideline
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
60 d
Total depuration duration:
16 d
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
- Water quality: dilution water was confirmed to meet the quality criteria of 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 (Rice fish (Oryzias latipes), LC50(96h) > 1.0 mg/L)
Nominal and measured concentrations:
Nominal concentrations: high exposure level (level 1): 10 µg/L
low exposure level (level 2): 1 µg/L
Measured concentrationc: high exposure level (level 1): 10.27 µg/L (n= 5)
low exposure level (level 2): 0.92 µg/L (n= 5)
for more details see below under 'Any other information on results incl. tables'
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 tables
Lipid content:
>= 1.2 - <= 4.5 %
Time point:
other: 60 d (end of exposure)
Remarks on result:
other: Range of individual values: see Attached Document: BCF tables
Key result
Conc. / dose:
1 µg/L
Temp.:
24 °C
Type:
BCF
Value:
ca. 3 500 L/kg
Basis:
whole body w.w.
Remarks:
lipid normalised to 5%) (conc. 1 µg/L
Calculation basis:
steady state
Remarks:
no stable plateau reached over time
Remarks on result:
other: 75th percentile of all low concentration BCF values
Key result
Conc. / dose:
1 µg/L
Temp.:
24 °C
Type:
BCF
Value:
ca. 3 880 L/kg
Basis:
whole body w.w.
Remarks:
lipid normalisation to 5%) (conc. 1 µg/L
Calculation basis:
steady state
Remarks:
no stable plateau reached over time
Remarks on result:
other: mean BCF value from two fish (day 42), highest value of all time points
Key result
Conc. / dose:
1 µg/L
Temp.:
24 °C
Type:
BCF
Value:
ca. 1 740 L/kg
Basis:
whole body w.w.
Remarks:
lipid normalisation to 5%) (conc. 10 µg/L
Calculation basis:
steady state
Remarks:
no stable plateau reached over time
Remarks on result:
other: mean BCF value from two fish (day 60)
Key result
Conc. / dose:
10 µg/L
Temp.:
24 °C
Type:
BCF
Value:
ca. 2 020 L/kg
Basis:
whole body w.w.
Remarks:
lipid normalisation to 5%) (conc. 10 µg/L
Calculation basis:
steady state
Remarks:
no stable plateau reached over time
Remarks on result:
other: mean BCF value from two fish (day 42)
Key result
Conc. / dose:
10 µg/L
Temp.:
24 °C
Type:
BCF
Value:
ca. 3 200 L/kg
Basis:
whole body w.w.
Remarks:
lipid normalisation to 5%) (conc. 1 µg/L
Calculation basis:
steady state
Remarks:
no stable plateau reached over time
Remarks on result:
other: mean BCF value from two fish (day 60), highest value of all time points
Key result
Elimination:
yes
Parameter:
DT50
Remarks:
(high exposure)
Depuration time (DT):
4.5 d
Key result
Elimination:
yes
Parameter:
DT50
Remarks:
(low exposure)
Depuration time (DT):
15.7 d
Remarks on result:
other: probably mistaken (see 'Details on kinetic parameteres')
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
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).

Bioaccumulation test results (high exposure concentration - 0.01 mg/L) extracted from study report

 

Day

Test substance concentration [µg/L]

Fish

BCF
(measured)
[L/kg]

Lipid content
[%]

BCF
(normalised)*
[L/kg]

BCF(lip 5)
(mean)
[L/kg]

7

9.95

1

1610

7.7

1045

707

 

 

2

427

5.8

368

 

14

10.07

1

957

6.6

725

1021

 

 

2

1950

7.4

1318

 

28

10.33

1

1370

6.0

1142

1722

 

 

2

2810

6.1

2303

 

42

10.54

1

896

2.7

1659

2019

 

 

2

3330

7.0

2379

 

60

10.48

1

1080

4.5

1200

3204

 

 

2

1250

1.2

5208

 

 

Bioaccumulation test results (low exposure concentration - 0.001 mg/L) extracted from study report

 

Day

Test substance concentration [µg/L]

Fish

BCF
(measured)
[L/kg]

Lipid content
[%]

BCF
(normalised)*
[L/kg]

BCF(lip 5)
(mean)
[L/kg]

7

1.013

1

469

5.8

404

555

 

 

2

423

3.0

705

 

14

0.982

1

1610

6.2

1298

1737

 

 

2

1740

4.0

2175

 

28

0.943

1

3730

5.1

3657

2675

 

 

2

2270

6.7

1694

 

42

0.944

1

4410

4.7

4691

3883

 

 

2

4120

6.7

3075

 

60

0.929

1

724

4.0

905

1743

 

 

2

2220

4.3

2581

 

 

* measured BCF values normalised to a lipid content of 5 %

 

Statistical evaluation of lipid normalised BCF values [L/kg]

 

Exposure level

Range
(day 28 to 60)

Average
(day 28 to 60)

75th percentile
(day 28 to 60)

90th percentile
(day 28 to 60)

10 µg/L

1142 - 5208

2315

2360

3794

1 µg/L

905 - 4691

2767

3512

4174

 

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, day 60) and 3900 (1 µg/L, day 42). Data indicate that a stable plateau covering different time points was not reached. At the high concentration level, mean BCF values increased from day 14 to day 60. At the low concentration level, mean BCF values reached a peak at day 42. Then, values decreased considerably from day 42 to day 60. Taking into account the intrinsic poperties of the substance (log Pow of ca 6.2 to 6.5), a steady state should have been reacht at day 28 or shortly thereafter. Based on the highest mean values, bioaccumulation is assessed to be moderate. Note: The authors concluded "low bioaccumulation" (NITE 2002: database). Given the BCF < 2000 at 1 µg/L after 60 days, 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. Still, average and 75th/90th percentile values are higher at the low than at the high exposure level.
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 source test material 2,4-diphenyl-4-methyl-1-pentene (EC name 1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene) [CAS no 6362-80-7, EC no 228-846-8] is a constituent of the target substance representing the dimeric fraction of the oligomerisation products.
The target substance ‘Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction, alkylated and oligomerised’ (NAF-AO) [EC no. 701-299-207] consists of a complex mixture of numerous aryl-aliphatic constituents. Its components can basically divided into groups corresponding to their level of oligomerisation (dimers, trimers, tetramers).
Being a constituent of the target substance NAF-AO, test results of the source substance represent at least in part the properties of NAF-AO with regard to aquatic bioaccumulation.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source substance 2,4-diphenyl-4-methyl-1-pentene is a constituent of the target substance NAF-AO. It is a dimerisation product of aryl-olefinic components (2-phenylpropene) in the starting material. Its concentration in NAF-AO is ca. 15%. It is representative of the total group of dimeric constituents in NAF-AO, which amount to about 20 to 70%. The specific analytical purity of the test material is reported in the Japanese database to be 97%. Thus, the results reported relate definitely to the test substances (2,4-diphenyl-4-methyl-1-pentene).
The target substance NAF-AO is a UVCB substance obtained in an acid catalysed alkylation and oligomerisation reaction of the starting material ‘Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction’. This material comprises predominantly styrene and indene derivatives. In the production process, oligomerisation products of C8-10 aromatic hydrocarbons are formed consisting of dimeric, trimeric, and oligomeric (n ≥ 4) products. These products consist of two, three, or more aromatic rings connected by methyl-substituted alkenyl carbon-chains (dimers, trimers, oligomers). The aromatic substituents include besides benzene also indan. Based on the degree of oligomerisation, constituents are combined into the three different groups, the dimers, trimers, and higher oligomers. Depending on the type of technical product (Novares L 100, L 700, TL 10), composition with respect to different component groups will vary (dimers from ca. 20 to 70%, trimers from ca. 10 to 30%, higher oligomers from ca. 15 to 50%).
NAF-AO is a viscous oily material with a low water solubility (between 0.6 and 1.3 mg/L depending on its composition). Values for water solubility obtained by (Q)SAR (US EPA EPI suite) for individual model constituents are lower (between 0.082 and 0.038 mg/L for dimers and between 0.011 and 0.027 mg/L for trimers). Overall, the water solubility of NAF-AO is low to very low depending on the technical product and its composition (higher percentages of trimers and higher oligomers will reduce the water solubility).

3. ANALOGUE APPROACH JUSTIFICATION
Properties of the target substance NAF-AO relevant for its environmental fate will be determined by the properties of its constituents. Under environmental conditions or during processing of the target substance, environmentally available or volatile components can be released. Out of the dimers, trimers, and oligomers, this will primarily be the dimers.
All constituents in combination will specify the distribution properties and environmental behaviour of the substance NAF-AO as such. With regard to aquatic bioaccumulation, dimers are predominant, because they are more mobile in the environment (higher water solubility) resulting in higher concentration in water. Therefore, it is justified to use data determined for a constituent of NAF-AO that represents the dimers (2,4-diphenyl-4-methyl-1-pentene), as weight of evidence to characterise the environmental fate (aquatic bioaccumulation) of NAF-AO.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Read-across to preceding entry:
Source test material: 2,4-diphenyl-4-methyl-1-pentene (NITE, Japan, CSCL);
Reference: MOE 2002, NITE National Institute of Technology and Evaluation, Japan 2002 etc.
Key result
Conc. / dose:
1 µg/L
Temp.:
24 °C
Type:
BCF
Value:
ca. 3 400
Basis:
whole body w.w.
Remarks:
lipid normalised to 5%) (conc. 1 µg/L
Calculation basis:
steady state
Remarks:
no stable plateau reached over time
Remarks on result:
other: 75th percentile of all low concentration BCF values
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance NAF-AO
Key result
Conc. / dose:
1 µg/L
Temp.:
24 °C
Type:
BCF
Value:
ca. 1 740 - ca. 3 880 L/kg
Basis:
whole body w.w.
Remarks:
normalised to a lipid content of 5%) (conc. 1 µg/L
Calculation basis:
steady state
Remarks:
no stable plateau reached over time
Remarks on result:
other: range of BCF values (mean of two fish) covering day 28 to day 60 (considered to represent steady state)
Remarks:
the test results of the source substance are adopted as weight of evidence for the target substance NAF-AO
Key result
Conc. / dose:
10 µg/L
Temp.:
24 °C
Type:
BCF
Value:
>= 1 720 - <= 3 200 L/kg
Basis:
whole body w.w.
Remarks:
normalised to a lipid content of 5%) (conc. 10 µg/L
Calculation basis:
steady state
Remarks:
no stable plateau reached over time
Remarks on result:
other: range of BCF values (mean of two fish) from day 28 to day 60 (considered to represent steady state)
Remarks:
the test results of the source substance are adopted as weight of evidence for the target substance NAF-AO

Description of key information

The bioaccumulation potential of the substance ‘Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction, alkylated and oligomerised’ (NAF-AO) [EC no. 701-299-7] has been assessed based on data obtained with the supporting (source) substance ‘Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol’ (OAPP) [EC no. 700-960-7] and with the substance 2,4-diphenyl-4-methyl-1-pentene (EC name 1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene) [CAS no. 6362-80-7, EC no. 228-846-8], which is a dimeric constituent of NAF-AO.

In the study with OAPP (feeding study), BMF values were obtained for each of the groups of dimeric and trimeric constituents (0.0737 and 0.1374, respectively). In the bioaccumulation study with 2,4-diphenyl-4-methyl-1-pentene, BCF were determined related to this test substance. Individual fish BCF, normalised to 5% lipid, ranged from 1142 to 5208 L/kg (average 2302L/kg; high exposure concentration - 10 µg/L) and from 905 to 4691 L/kg (average 2767 L/kg; low exposure concentration - 1 µg/L). The 75th percentile derived from the BCF values of the low exposure group is selected as key value (see below).

Key value for chemical safety assessment

BCF (aquatic species):
3 500 dimensionless
BMF in fish (dimensionless):
0.137

Additional information

For the substance NAF-AO, no data on aquatic bioaccumulation could be located. Instead, data obtained with the substances OAPP and 2,4-diphenyl-4-methyl-1-pentene are used. OAPP is produced in a similar process as NAF-AO producing overlapping constituents in both substances. 2,4-diphenyl-4-methyl-1-pentene is one of the dimeric aryl-aliphatic constituents of NAF-AO representing this type of constituents. Study result of both substances are considered to adequately represent the bioaccumulation potential of NAF-AO. Hence, data obtained with the source substances are used to assess the potential for aquatic bioaccumulation of the target substance NAF-AO.

The bioaccumulation potential in aquatic species was investigated in a GLP compliant study according to OECD TG 305 (dietary exposure) with OAPP as test material (Klix 2017). Fish were kept in a flow-through system for an uptake period of 14 days and for a depuration period of 28 days. The concentration of the dimeric and trimeric constituents in the feed was 195 and 115 µg/g food, respectively. Concentrations of dimers and trimers in fish as well as growth and lipid content of fish were determined at day 0, 7, and 14 of the uptake phase and at day 1, 2, 5, 15, 21, and 28 of the depuration phase. Evaluation of the data according to TG 305 resulted in a growth and lipid corrected BMF of ca. 0.0737 for the dimers fraction and of 1.374 for the trimers fraction.

Due to the type of test method (oral uptake), reliable BCF values were not obtained in this study. Therefore, the BMF values determined are used as key information. The higher of the two BMF values obtained for trimers (0.1374) is used as key value in the chemical safety assessment.

Elimination half-lives (DT50) were determined to be 5.3 days for the dimers and 25.8 days for the trimers.

The bioaccumulation potential of the substance 2,4-diphenyl-4-methyl-1-pentene representing the dimer fraction of NAF-AO was investigated in a study under GLP conditions according to a Japanese test guideline (“The Notice on the Test Method Concerning New Chemical Substances”) similar to OECD TG 305 (MOE/NITE 2002). The uptake phase lasted 60 days followed by a depuration phase of 16 days. Two test material concentrations were tested (high exposure level: 10 µg/L, low exposure level: 1 µg/L). Concentrations in water and fish and the lipid content of fish were determine at day 7, 14, 28, 42, and 60 of the uptake phase and at day 0, 2, 4, 9, and 16 of the depuration phase. Lipid normalised BCF values during the uptake phase at day 28, 42, and 60 fluctuate between 1722 and 3204 L/kg (low concentration level, each value mean of two fish) and between 1743 and 2675 L/kg (high concentration level, each value mean of two fish). Average values were 2767 and 2315 L/kg for the low and high exposure level, respectively. Overall, BCF values were higher for the low exposure level compared to the high exposure level. The low exposure concentration correlates better with environmental concentrations. It is concluded that these data better represent aquatic environmental bioaccumulation than data resulting from the high exposure level. In order to account for the variability of data, the 75th percentile of the BCF values at the low exposure level (3500 L/kg ww) is selected as key value.

Half-lives (DT50 values) for high and low exposure were determined as 4.5 and 15.7 days, respectively. Compared with the half-life for dimers (5.3 days) determine in the feeding study above, the lower value for high exposure seems to have a higher reliability. The value for low exposure seems to be erroneous.

Uptake was very similar in the high and low exposure groups (final tissue concentration of 12.2 and 1.37 mg/L at 10 and 1 µg/L exposure concentration). This indicates that the elimination kinetics at both exposure regimens should also be similar resulting in similar elimination constants or DT50 values. The much higher regression coefficient r² in the data analysis of the high exposure group compared to low exposure (depuration data, pp. 36 and 37 of the Report) indicates that the high exposure half-life is more valid.

The data available indicate, that bioconcentration factors in fish of dimeric constituents are located within the range from < 2000 to > 3000, but < 5000. Valid BCF values are not available for the trimeric constituents of NAF-AO. But the higher BMF value for trimers from the feeding study with OAPP indicates that BCF values may exceed 5000. On the other hand, a BMF of 0.1374 for the trimers is moderate and does not indicate a strong accumulation potential in the aquatic environment.