bis(2-ethylhexyl) 4,4’-{6-[4-tert-butylcarbamoyl)anilino]-1,3,5-triazine-2,4-diyldiimino}dibenzoate

Biodegradation in water and sediment: simulation tests

Administrative data

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26/10/2020 - 18/03/2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)

Deviations:

yes

Remarks:

See any other information on materials and methods incl. tables section for details on deviations.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

421-450-8

EC Name:

-

Cas Number:

154702-15-5

Molecular formula:

C44H59N7O5

IUPAC Name:

2-ethylhexyl 4-[(4-{[4-(tert-butylcarbamoyl)phenyl]amino}-6-[(4-{[(2-ethylhexyl)oxy]carbonyl}phenyl)amino]-1,3,5-triazin-2-yl)amino]benzoate

Radiolabelling:

yes

Remarks:

The isotopic labelling is on the triazine ring carbons. See any other information on materials and methods incl. tables section for more details.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

other: Natural sediment and overlying water

Details on source and properties of surface water:

Fluvial
- Details on collection (e.g. location, sampling depth, contamination history, procedure): Via Molino Nuovo, 20080 Moncucco di vernate (MI), Italy; 10 cm depth
- Temperature (°C) at time of collection: 14 degrees C
- pH at time of collection: 8.0
- Oxygen concentration (mg/l) initial/final: 8.9 mg/L


Marine
- Details on collection (e.g. location, sampling depth, contamination history, procedure):17015 Celle Ligure (SV), Italy; 10 cm depth
- Temperature (°C) at time of collection: 22 degrees C
- pH at time of collection: 8.1
- Oxygen concentration (mg/l) initial/final: 8.5 mg/L

Details on source and properties of sediment:

Fluvial
- Details on collection (e.g. location, sampling depth, contamination history, procedure): Via Molino Nuovo, 20080 Moncucco di vernate (MI), Italy – 45°18’26’’ N, 9°02’04’’ E, 10 cm depth
- Textural classification (i.e. %sand/silt/clay): Fluvial - Sand: 93.8%, Silt: 4.%, Clay: 2.19%;
- pH at time of collection: 6.4
- Organic carbon (%): 3.01%
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): 3 x 10^5 CFU
-- Sediments were sampled and stored at 5 ± 3°C until use (12 days).
- Dry weight: 81.6%
- Sediments were sampled and stored at 5 ± 3°C until use (12 days).

Marine
- Details on collection (e.g. location, sampling depth, contamination history, procedure): - 17015 Celle Ligure (SV), Italy – 44°20’30’’ N, 8°32’54’’ E, 10 cm depth
- Textural classification (i.e. %sand/silt/clay): Marine - Sand: 95.9%, Silt: 4.0%, Clay: 0.16%
- pH at time of collection: 6.6
- Organic carbon (%): <0.1%
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): 3 x 10^4 CFU
- Dry weight: 82.8%

Duration of test (contact time):

100 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

3 mg/L

Based on:

test mat.

Remarks:

For sediment

Initial test substance concentration 2

Initial conc.:

1 mg/L

Based on:

test mat.

Remarks:

For water

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

TEST CONDITIONS
- Mass of sediment: 50 g (dry weight basis)
- Volume of overlying water: 150 mL
- Water:sediment ratio: 3:1
Application solvent (type and concentration if used): methanol 0.1% (relative to volume of water)
- Test temperature: 20 +/- 2 degrees C
- Acclimation period: 18 days
- pH during study : Fluvial – water: 6.6-7.9, sediment: 6.1-6.3; Marine – water: 7.5-8.0, sediment: 6.3-7.06
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: glass flask
- Number of culture flasks/concentration: 2 replicates for each aquatic sediment system at each sampling point (total of 20 samples for each aquatic sediment system); 1 control sample for each aquatic sediment system at each sampling point (total of 10 samples for each aquatic sediment system); 2 analytical method recovery check at each extraction date (total of 10 samples for each aquatic sediment system) that after separation in water and sediment were treated with amount of analyte at LOQ and 10xLOQ in order to check the performance of the analytical method; 10 untreated samples for each aquatic sediment system to follow up the acclimation; 10 treated samples for physical-chemical properties determinations during the experiment. Spare samples were also made.
- Method used to create aerobic conditions: exposure to air
- Measuring equipment: electrochemical cell



SAMPLING
- Sampling frequency:
Fluvial: Days 0, 1, 3, 7, 16, 30, 44, 60, 77, 100*, 102*
* Data obtained from T9 samplings gave anomalous results (not reported) that were verified at T9 RE (2 days after), obtaining results in accordance with the behaviour expected from the results of other samplings
Marine: Days 0, 1, 3, 7, 16, 30, 44, 60, 77, 100
- Sampling method used per analysis type: The first operation was the separation of the water from the sediment. This step was done avoiding the perturbation of the system, purging out the water from the vessel with the use of a syringe. Water and sediment samples were extracted immediately and analysed after the extraction or stored.
- Sample storage before analysis: If needed, stored until analysis in the dark in refrigerated conditions for a time shorter than the maximum storage time of 7 days, verified during the validation of the analytical method (for sampling at T0 and T1).


DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Inoculum blank: untreated


STATISTICAL METHODS: The error associated to the determination was estimated during the validation of the analytical method and expressed as %RSD. The values of %RSD found both at application rate and at the method LOQ was <5%, with recovery near to 100%, demonstrating that the method performances are enough to determine the amount of residual analyte according to the performances requested by the guidelines.

Reference substance

Reference substance:

other: Diethylhexyl Butamido Triazone - isotopic labelled according to OECD guidelines page 7.

Results and discussion

Mean total recovery

Mean total recovery 1

Compartment:

natural water / sediment: marine

% Recovery:

106

Mean total recovery 2

Compartment:

other: Fluvial aquatic sediment system

% Recovery:

101

% Degradation

% Degradation 1

Parent/product:

parent

Compartment:

total system

Key result

% Degr.:

0

Parameter:

test mat. analysis

Sampling date:

2021

Sampling time:

100 d

Remarks on result:

not determinable

Remarks:

It was not possible to determine a percent degradation. It was not possible to determine actual degradation from measurement noise due to the low if any degradation.

% Degradation 2

Parent/product:

parent

Reference

Reference substance name:

Unnamed

IUPAC name:

2-ethylhexyl 4-[(4-{[4-(tert-butylcarbamoyl)phenyl]amino}-6-[(4-{[(2-ethylhexyl)oxy]carbonyl}phenyl)amino]-1,3,5-triazin-2-yl)amino]benzoate

Inventory number:

InventoryMultipleMappingImpl [inventoryEntryValue=EC 421-450-8]

CAS number:

154702-15-5

Identity:

Diethylhexyl butamido triazone

Identity:

Dioctylbutamidotriazone

Identity:

Iscotrinizol

Identity:

RA 3643

Identity:

Bis(2-ethylhexyl) 4,4’-{6-[4-tert-butylcarbamoyl)anilino]-1,3,5-triazine-2,4-diyldiimino}dibenzoate

Molecular formula:

C44H59N7O5

Molecular weight:

765.983

SMILES notation:

CCCCC(CC)COC(=O)c1ccc(cc1)Nc2nc(Nc3ccc(cc3)C(=O)OCC(CC)CCCC)nc(Nc4ccc(cc4)C(=O)NC(C)(C)C)n2

Compartment:

sediment

% Degr.:

0

Parameter:

TOC removal

Sampling date:

2021

Sampling time:

102 d

Remarks on result:

other: aquatic sediment system - fluvial origin

Other kinetic parameters:

other: Kinetic analysis was not performed as no degradation was noticed.

Transformation products:

no

Details on transformation products:

Transformation products were not investigated since no degradation of the test substance higher than the 10% of the initial amount was observed.

Details on results:

TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): In the sample 2 T4 C (untreated control sample of the marine aquatic sediment system) the residue of analyte was < LOQ instead of < LOD both in the aqueous fraction and in the sediment fraction. During the whole storage period (120 days) there were few cases in which the temperature exceeded the range stated in the Study Plan (20 ± 2°C), due to door opening events.

Results with reference substance:

Analysis of radiolabelled reference substance metabolites was not performed due to the lack of degradation.

Any other information on results incl. tables

Storage data

Storage temperature (°C)	Mean temperature (°C)	Min temperature (°C)	Max temperature (°C)	Storage start (dd/mm/yyyy)	Storage end (dd/mm/yyyy)
20 ± 2	20.5	19.3	22.9 *	29/10/2020	26/02/2021

Acclimation monitoring

Parameter	Acclimation monitoring
	29/10/2020 (Start of acclimation)	02/11/2020	04/11/2020	06/11/2020	09/11/2020	11/11/2020	13/11/2020
Fluvial aquatic sediment system
Sample code	1 AC 0	1 AC 1	1 AC 2	1 AC 3	1 AC 4	1 AC 5	1 AC 6
pH of water	8.1	7.8	7.3	7.3	7.4	7.4	7.4
Oxygen concentration (mg/L)	7.86	7.56	7.34	7.73	8.21	8.24	8.13
Redox potential of water (mV)	226.6	184	395.2	361.2	242.9	250.7	211.2
TOC of water (mg/L)	5.55	/	/	/	/	/	/
pH of sediment	6.4	/	/	/	/	/	/
Redox potential of sediment (mV)	252.9	161.4	368.2	305.8	242.2	255.2	237.2
TOC of sediment (g/kg)	30.3	/	/	/	/	/	/
Marine aquatic sediment system
Sample code	2 AC 0	2 AC 1	2 AC 2	2 AC 3	2 AC 4	2 AC 5	2 AC 6
pH of water	8.1	8.0	7.8	7.8	7.8	7.8	7.8
Oxygen concentration (mg/L)	8.89	8.72	8.57	8.77	8.86	8.28	8.17
Redox potential of water (mV)	249.2	174	426.0	398.5	217.4	248.6	203.4
TOC of water (mg/L)	2.83	/	/	/	/	/	/
pH of sediment	6.3	/	/	/	/	/	/
Redox potential of sediment (mV)	261.2	163.2	356.0	202.5	239.8	247.2	227.6
TOC of sediment (g/kg)	< 1	/	/	/	/	/	/

UVASORB HEB repartition and degradation – fluvial aquatic sediment system

Fluvial aquatic sediment system
Sampling ID	Sampling Time (days)	% repartition in water	% repartition in sediment	% of spiked amount (overall system)
T0	0	95.0	5.0	106
T1	1	88.3	11.7	101
T2	3	68.3	31.7	104
T3	7	29.4	70.6	110
T4	15	5.5	94.5	105
T5	30	< 2	100	98.1
T6	44	< 2	100	97.4
T7	60	< 2	100	102
T8	77	< 2	100	106
T9 RE	102	< 2	100	101

: UVASORB HEB repartition and degradation – marine aquatic sediment system

Marine aquatic sediment system
Sampling ID	Sampling Time (days)	% repartition in water	% repartition in sediment	% of spiked amount (overall system)
T0	0	89.2	10.8	93.8
T1	1	75.4	24.6	96.1
T2	3	54.5	45.5	94.9
T3	7	23.0	77.0	106
T4	15	3.9	96.1	102
T5	30	< 2	100	94.9
T6	44	< 2	100	101
T7	60	< 2	100	102
T8	77	< 2	100	102
T9	100	< 2	100	96.0

Applicant's summary and conclusion

Validity criteria

Validity criteria:

Mass balance

Observed value:

Residual amount of UVASORB HEB found at each checkpoint were within the range of 90-110%, with values after 110 days of 101% in marine sediment and 96% in fluvial sediment

Validity criteria fulfilled:

yes

Conclusions:

No significant degradation occurred during 100 days under aerobic conditions.

Executive summary:

The objective of this study was the determination of the rate of the aerobic transformation in aquatic sediment of the test item UVASORB HEB.

 

The study was conducted according to the OECD guideline 308 “Aerobic and Anaerobic Transformation in Aquatic Sediment Systems”, operating on a fluvial aquatic sediment system and on a marine sediment system under aerobic conditions at 20°C as required by ECHA Board of Appeals (“BoA”) in Case A-004-2017 3V Sigma S.p.A.. The same decision provided for that “The 10 % threshold for identification of transformation and/or degradation products defined in paragraph 41 of OECD TG 308 should be followed” (paragraph 135).

 

As per the result of the study, no degradation in an amount at or greater than 10% was identified.

 

The obtained results showed that no degradation in the selected fluvial and marine sediment systems occur during 100 days of incubation.

 

 As no degradation occurred, no kinetic analyses of degradation were performed. In compliance with the OECD 308 guideline, in fact, the metabolites were not investigated since no degradation of the test substance higher than 10% of the initial amount was observed.

 

The analytical method (HPLC-HRMS) was validated according to SANCO/825/00 and SANCO/3029/99 (also complying with the new guideline SANTE/2020/12830 rev.1)]

 

It has to be highlighted that the result of the conducted test confirmed what was clear since the origin and at the time of the registration of the product HEB with ECHA. At that time, indeed, the product (that is completely comparable to other structurally related analogue Ethylhexyltriazone  such as “Uvinul T150”, CAS-Nr. 88122-99-0, with same reactions to degradation) was subject to the test OECD 301.B “Ready biodegradability” CO2 evolution tests. Such test showed degradation of about 6% in the first 12 days but no additional changes and/or degradations occurred in the following days until the 28^th day. As a consequence, it could be concluded already at that time, that the molecule is not biodegradable and the 6% of initial degradation was just an “assessment” of the biosystem.

 

On the contrary, as per the request of BAUA, the ECHA Board of Appeals - in Case A-004-2017 3V Sigma S.p.A -  required for the additional OECD 308 study. Such request was based on the data available from the OECD 301.B “Ready biodegradability” - CO2 evolution tests and the following QSAR estimations (EAWAG Pathway Prediction System (EAWAG PPS)) according to which the biodegradation pathway of the substance in the environment are available.

 

Nevertheless, the EAWAG Pathway Prediction System (EAWAG PPS) may suffer from limitations related to molecular databases on which the Pathway Prediction algorithm has been trained.  Results can be considered accurate only for compounds (or analogs) for which biodegradation pathways are reported in the scientific literature, if the compounds under study are located in environments with no competing chemicals or toxins, and whether the compounds under study are the sole source of energy, carbon and nitrogen (plus other essential elements) for the microbes in such environments. While EAWAG PPS returns potential metabolites, these results need to be considered with extreme caution as the query molecule falls out of the domain of applicability of the models. [1]

 

This is further proved by the OECD 301.F “Ready biodegradability” - Manometric respirometry. Under this test UVASORB HEB showed 0% degradation as showed by the structurally related analogue Ethylhexyltriazone [“Uvinul T150”, CAS-Nr. 88122-99-0] (that was subject only to this test and not even to the 301.B “Ready biodegradability” CO2 evolution tests in order to assess the biodegradability and consequent degradation[2]). All the results of the OECD “Ready biodegradability” studies show that the potential for degradation and metabolite formation was not properly evaluated by the QSAR estimations (EAWAG PPS) and that the UVASORB HEB is not subject to degradation and to metabolite formation (as per Ethylhexyltriazone [“Uvinul T150”, CAS-Nr. 88122-99-0]).

 

The abovementioned results are further confirmed under the OECD 308: in particular the observation of no aerobic degradation in fluvial or marine environments, confirms ulteriorly that UVASORB HEB is not subject to degradation and formation of metabolites.

 

[1] Please see “University of Pavia QSAR”

[2] Taking into account that the Uvasorb HEB and the Uvinul T150 are structurally analogue and showed the same answer under the OECD 301.F , we can assume that the Univil T150 should have showed the same results if subject to OECD 301.B.