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Environmental fate & pathways

Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study planned
Justification for type of information:
TESTING PROPOSAL ON BIODEGRADATION SIMULATION TESTING IN WATER ACCORDING TO OECD TG 309
NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Reactive Yellow 176 (EC 474-870-9)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies
No GLP study on the substance for the endpoint ‘biodegradation simulation testing in water’ is available. For the substance, an abiotic degradation study is available:
• Hydrolysis as a function of pH (OECD 111)
Furthermore, there is the following study from a structural analogue (Tanimoto Index 77%) available:
• Biodegradation in water: screening test (OECD 301E)
Available non-GLP studies
No non-GLP study on the substance for the endpoint ‘biodegradation simulation testing in water’ is available.
- (Q)SAR
• BIOWIN model
Reactive Yellow 176 Sulfato was tested for stability in water at different pH levels. Hydrolysis is considered a function of the pH. The test was conducted at pH 4, 7 and 9. Typically for reactive dyes, the substances was rather stable under acidic, slowly hydrolysable under neutral and instable under alkaline conditions. The mean DT50 values at 25°C were > 1 y (pH 4), 18.9 d (pH 7) and < 2.4 h (pH 9). The 25°C values at pH 4 and 7 were extrapolated from measurements at 50 – 74°C done in the main test. The 25°C value at pH 9 was derived from the instability found in the preliminary study. Identification of degradation products was not performed.
A structurally close substance was tested for its biodegradation potential in an OECD 301F guideline study in 2003. The test was conducted under GLP with an inoculum containing activated sludge from a local wastewater treatment plant cleaning predominantly domestic sewage. The total test duration was 28 d with an initial test substance concentration of 100 mg/L based on ThOD. A reference item (benzoic acid, sodium salt) was tested in parallel. The biodegradation of the structural analogue after 28 days remained < 10% based on the O2-consumption. The reference item reached a maximum degradation of ca. 90% after 14 days. Identification of degradation products was not performed. Consequently, the substance was not considered to be readily biodegradable according to OECD criteria. By the nature of their design and use, dyes are not intended to be readily biodegradable as this would assist in the rapid destruction of the dyestuff, rendering it unfit for purpose. As such, it is accepted that such substances are not readily biodegradable under relevant environmental conditions. A published study (Pagga & Brown, 1986) describes the results of the testing of 87 dyestuffs in short-term aerobic biodegradation tests. The authors of this publication concluded that dyestuffs are very unlikely to show any significant biodegradation in such tests and that 'there seems little point in carrying out such test procedures’ on dyestuffs. There are ISO, European, American (AATCC) and national standards for the colour fastness of dyes. Dyes are required to have specific fastness properties. If the dyes were readily biodegradable it would not be possible for them to pass those fastness properties.
- Grouping and read-across
The only substance with data on biodegradation simulation testing exists is Reactive Black 5 (EC 241-164-5).

The study was conducted with a water/sediment system. The structural similarity between Reactive Yellow 176 and Reactive Black 5 is relatively low with a Tanimoto Index of 50%. Reactive Black 5 is a slightly smaller molecule without the triazine and the urea group, but with two “parabase ester” groups. It is not considered a feasible read-across candidate.
Reactive Black 5 is considered inherently but not readily biodegradable based on water screening tests. The substance was found to be not hydrolytically stable at pH 7 and 9 but hydrolytically stable at pH 4. The aerobic aquatic metabolism of Reactive Black 5 — uniformly labelled 14C in the phenyl rings — was investigated to determine the rate of degradation of the dye and the identity of the major degradation products. The study was conducted according to EPA Pesticide Assessment Guidelines, Subdivision N: Environmental Fate, § 162-4. Reactive Black 5 was applied to a system of surface water and associated sediment (Nidda origin) in the laboratory with an initial concentration of 1 mg/kg. After application, the samples were incubated in the dark at 20 ± 2 °C for 112 days under oxygen admission. The incubation flasks had suitable absorbents to absorb volatile radioactive substances. After 0, 3, 7, 14, 21, 28, 42, 57, 84 and 112 days the water was separated from the sediment. Residues in the sediment were extracted with acetonitrile/water. The liquid phases were investigated by radio-DC. Non-extracted residues were quantified after burning the dried sediment.
During incubation, radioactivity in the water phase decreased from 94.4% to 8.8% (all values in this section are mean values from two series). The radioactivity extractable from the sediment decreased to 2.0% by the end of incubation. Non-extractable residues increased from 1.2 to 82.8%. The main metabolite detected was 1-hydroxy-2,7-bis-[4'-(ß-hydroxyethylsulfonyl)-phenyl-azo]-8-aminonaphthalene-3,6-disulfonic acid (maximum 41.4 %), which had decreased to 1.4 % by the end of the study. Another six metabolites occurred only sporadically or only in small concentrations. At the end of the study all metabolites had dropped to values close to or below the limit of quantification (0.1 %). The mean recovery rate related to the radioactivity recovered on day 0 was 100.6 % (96.8 -108.1%).
A "half-life" (DT50) of two days was found for Reactive Black 5.
These results show a rapid degradation of Reactive Black 5 and its metabolites with the simultaneous formation of significant amounts of residues firmly bound to the components of the sediment.
In summary, Reactive Black 5, applied to a sediment/water test system at a final concentration of 1 mg/kg in the test system was very rapidly degraded. The DT50 of the test item was 2 days. After 3 to 6 days, only 10% of the applied test substance could be found. All metabolites decreased at the end of the test period to levels around the lower level of quantification. Consequently, an accumulation of metabolites could be excluded. The majority of the metabolites of the test substance was deposited in form of non-extractable residues in sediment during test phase. After about 1 month, mineralization of the metabolites started.
- (Q)SAR: BIOWIN v.4.10 from EPISUITE v. 4.1

BIOWIN (v4.10) Program Results:
==============================
SMILES : NC(=O)Nc4cc(Nc5nc(CL)nc(Nc3ccc(cc3)S(=O)(=O)CCOS(O)(=O)=O)n5)ccc4N=Nc1cc2c(cc(cc2cc1S(O)(=O)=O)S(O)(=O)=O)S(O)(=O)=O
CHEM :
MOL FOR: C28 H24 CL1 N9 O16 S5
MOL WT : 938.31
--------------------------- BIOWIN v4.10 Results ----------------------------

Biowin1 (Linear Model Prediction) : Does Not Biodegrade Fast
Biowin2 (Non-Linear Model Prediction): Does Not Biodegrade Fast
Biowin3 (Ultimate Biodegradation Timeframe): Recalcitrant
Biowin4 (Primary Biodegradation Timeframe): Months
Biowin5 (MITI Linear Model Prediction) : Not Readily Degradable
Biowin6 (MITI Non-Linear Model Prediction): Not Readily Degradable
Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast
Ready Biodegradability Prediction: NO

------+-----+--------------------------------------------+---------+---------
TYPE | NUM | Biowin1 FRAGMENT DESCRIPTION | COEFF | VALUE
------+-----+--------------------------------------------+---------+---------
Frag | 1 | Triazine ring (symmetric) | 0.0095 | 0.0095
Frag | 1 | Aromatic chloride [-CL] | -0.1824 | -0.1824
Frag | 2 | Aromatic amine [-NH2 or -NH-] | -0.2338 | -0.4675
Frag | 3 | Sulfonic acid / salt -> aromatic attach | -0.2238 | -0.6713
Frag | 1 | Azo group [-N=N-] | -0.2418 | -0.2418
MolWt| * | Molecular Weight Parameter | | -0.4467
Const| * | Equation Constant | | 0.7475
============+============================================+=========+=========
RESULT | Biowin1 (Linear Biodeg Probability) | | -1.2527
============+============================================+=========+=========

------+-----+--------------------------------------------+---------+---------
TYPE | NUM | Biowin2 FRAGMENT DESCRIPTION | COEFF | VALUE
------+-----+--------------------------------------------+---------+---------
Frag | 1 | Triazine ring (symmetric) | -5.7252 | -5.7252
Frag | 1 | Aromatic chloride [-CL] | -2.0155 | -2.0155
Frag | 2 | Aromatic amine [-NH2 or -NH-] | -1.9070 | -3.8140
Frag | 3 | Sulfonic acid / salt -> aromatic attach | -1.0283 | -3.0849
Frag | 1 | Azo group [-N=N-] | -8.2194 | -8.2194
MolWt| * | Molecular Weight Parameter | |-13.3240
============+============================================+=========+=========
RESULT | Biowin2 (Non-Linear Biodeg Probability) | | 0.0000
============+============================================+=========+=========

A Probability Greater Than or Equal to 0.5 indicates --> Biodegrades Fast
A Probability Less Than 0.5 indicates --> Does NOT Biodegrade Fast

------+-----+--------------------------------------------+---------+---------
TYPE | NUM | Biowin3 FRAGMENT DESCRIPTION | COEFF | VALUE
------+-----+--------------------------------------------+---------+---------
Frag | 1 | Triazine ring (symmetric) | -0.2459 | -0.2459
Frag | 1 | Aromatic chloride [-CL] | -0.2066 | -0.2066
Frag | 2 | Aromatic amine [-NH2 or -NH-] | -0.1349 | -0.2699
Frag | 3 | Sulfonic acid / salt -> aromatic attach | 0.1422 | 0.4266
Frag | 1 | Azo group [-N=N-] | -0.3004 | -0.3004
MolWt| * | Molecular Weight Parameter | | -2.0735
Const| * | Equation Constant | | 3.1992
============+============================================+=========+=========
RESULT | Biowin3 (Survey Model - Ultimate Biodeg) | | 0.5295
============+============================================+=========+=========

------+-----+--------------------------------------------+---------+---------
TYPE | NUM | Biowin4 FRAGMENT DESCRIPTION | COEFF | VALUE
------+-----+--------------------------------------------+---------+---------
Frag | 1 | Triazine ring (symmetric) | -0.0575 | -0.0575
Frag | 1 | Aromatic chloride [-CL] | -0.1653 | -0.1653
Frag | 2 | Aromatic amine [-NH2 or -NH-] | -0.1084 | -0.2168
Frag | 3 | Sulfonic acid / salt -> aromatic attach | 0.0216 | 0.0649
Frag | 1 | Azo group [-N=N-] | -0.0528 | -0.0528
MolWt| * | Molecular Weight Parameter | | -1.3537
Const| * | Equation Constant | | 3.8477
============+============================================+=========+=========
RESULT | Biowin4 (Survey Model - Primary Biodeg) | | 2.0664
============+============================================+=========+=========

Result Classification: 5.00 -> hours 4.00 -> days 3.00 -> weeks
(Primary & Ultimate) 2.00 -> months 1.00 -> longer

------+-----+--------------------------------------------+---------+---------
TYPE | NUM | Biowin5 FRAGMENT DESCRIPTION | COEFF | VALUE
------+-----+--------------------------------------------+---------+---------
Frag | 1 | Triazine ring (symmetric) | 0.1168 | 0.1168
Frag | 1 | Aromatic chloride [-CL] | 0.0062 | 0.0062
Frag | 2 | Aromatic amine [-NH2 or -NH-] | -0.1577 | -0.3154
Frag | 3 | Sulfonic acid / salt -> aromatic attach | 0.0221 | 0.0664
Frag | 1 | Azo group [-N=N-] | -0.0459 | -0.0459
Frag | 11 | Aromatic-H | 0.0082 | 0.0904
Frag | 2 | -CH2- [linear] | 0.0494 | 0.0988
MolWt| * | Molecular Weight Parameter | | -2.7915
Const| * | Equation Constant | | 0.7121
============+============================================+=========+=========
RESULT | Biowin5 (MITI Linear Biodeg Probability) | | -2.0620
============+============================================+=========+=========

------+-----+--------------------------------------------+---------+---------
TYPE | NUM | Biowin6 FRAGMENT DESCRIPTION | COEFF | VALUE
------+-----+--------------------------------------------+---------+---------
Frag | 1 | Triazine ring (symmetric) | -9.3006 | -9.3006
Frag | 1 | Aromatic chloride [-CL] | -0.2191 | -0.2191
Frag | 2 | Aromatic amine [-NH2 or -NH-] | -1.2264 | -2.4527
Frag | 3 | Sulfonic acid / salt -> aromatic attach | 0.6780 | 2.0341
Frag | 1 | Azo group [-N=N-] |-10.6129 |-10.6129
Frag | 11 | Aromatic-H | 0.1201 | 1.3216
Frag | 2 | -CH2- [linear] | 0.4295 | 0.8590
MolWt| * | Molecular Weight Parameter | |-27.0877
============+============================================+=========+=========
RESULT |Biowin6 (MITI Non-Linear Biodeg Probability)| | 0.0000
============+============================================+=========+=========

A Probability Greater Than or Equal to 0.5 indicates --> Readily Degradable
A Probability Less Than 0.5 indicates --> NOT Readily Degradable


------+-----+--------------------------------------------+---------+---------
TYPE | NUM | Biowin7 FRAGMENT DESCRIPTION | COEFF | VALUE
------+-----+--------------------------------------------+---------+---------
Frag | 1 | Triazine ring (symmetric) | -0.0783 | -0.0783
Frag | 1 | Aromatic chloride [-CL] | -0.4023 | -0.4023
Frag | 2 | Aromatic amine [-NH2 or -NH-] | -0.2778 | -0.5556
Frag | 3 | Sulfonic acid / salt -> aromatic attach | -0.3768 | -1.1305
Frag | 1 | Azo group [-N=N-] | 0.0000 | 0.0000
Frag | 11 | Aromatic-H | -0.0954 | -1.0497
Frag | 2 | -CH2- [linear] | 0.0260 | 0.0520
Const| * | Equation Constant | | 0.8361
============+============================================+=========+=========
RESULT | Biowin7 (Anaerobic Linear Biodeg Prob) | | -2.3283
============+============================================+=========+=========

A Probability Greater Than or Equal to 0.5 indicates --> Biodegrades Fast
A Probability Less Than 0.5 indicates --> Does NOT Biodegrade Fast

Ready Biodegradability Prediction: (YES or NO)
----------------------------------------------
Criteria for the YES or NO prediction: If the Biowin3 (ultimate survey model) result is "weeks" or faster (i.e. "days", "days to weeks", or "weeks" AND the Biowin5 (MITI linear model) probability is >= 0.5, then the prediction is YES (readily biodegradable). If this condition is not satisfied, the prediction is NO (not readily biodegradable). This method is based on application of Bayesian analysis to ready biodegradation data (see Help). Biowin5 and 6 also predict ready biodegradability, but for degradation in the OECD301C test only; using data from the Chemicals Evaluation and Research Institute Japan (CERIJ) database.
None of the models predicted Reactive Yellow 176 to be biodegradable. However, the outcome of the QSAR model for ionic substances like salts is questionable. Furthermore, the model cannot calculate with Na as counterion. The result of the QSAR calculation is not considered conclusive for further assessment of the environmental fate and behaviour of Reactive Yellow 176.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
According to Column 2 in Annex IX, the biotic degradation study in water does not need to be conducted if:
- the substance is highly insoluble in water; or
- the substance is readily biodegradable.
According to Column 2 in Annex IX, the identification of degradation products does not need to be conducted if:
- the substance is readily biodegradable

As none of the above-mentioned exemptions of the standard information requirements are true for Reactive Yellow 176, a simulation testing on ultimate degradation in surface water shall be conducted. DyStar Colours Distribution GmbH therefore proposes an OECD 309 guideline study under GLP to assess both ultimate degradation in surface water and identity of degradation products simultaneously.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: OECD 309
Due to the high water solubility of the substance and no potential for exposure to other compartments than water, no simulation testing on degradation in sediment and soil is considered scientifically feasible.
DyStar Colours Distribution GmbH proposes the following study design:
- Simulation testing on ultimate degradation in surface water (Annex IX, Section 9.2.1.2.; test method: Aerobic mineralisation in surface water - simulation biodegradation test, EU C.25.l; OECD TG 309)
- at a temperature of 12°C
- including the identification of the degradation products (Annex IX, Section 9.2.3.) using the registered substance Reactive Yellow 176. The biodegradation of each relevant constituent present in concentration at or above 0.1% (w/w) or, if not technically feasible, in concentrations as low as technically detectable shall be assessed.
- This can be done simultaneously during the same study.
DyStar Colours Distribution GmbH welcomes any suggestions from ECHA on study plan details for a successful performance of the OECD 309 study.

Data source

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Reactive Yellow 176 Ester

Results and discussion

Applicant's summary and conclusion