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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 Red 239 (EC 402-420-3)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies
There are no adequate GLP studies on water/sediment simulation testing on the test substance.
The following environmental studies on the substance are available:
· Biodegradation in water: screening test (OECD 301E)
· Hydrolysis as a function of pH (OECD 111)
- Available non-GLP studies: There are no adequate non-GLP studies on water/sediment simulation testing on the test substance.
- (Q)SAR
• BIOWIN model

The substance was tested for its biodegradation potential in an OECD 301E guideline study in 1987. At that time, the relevant guideline was EEC Directive 79/831, Part C: Methods for the Determination of Ecotoxicity, Nr. 5.2: Degradation, ENV 278/80, which is, however, equivalent to OECD 301E (Modified OECD Screening Test). This equivalence is mentioned in the study report. 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 32 d with an initial test substance concentration of 70 mg/L based on DOC. A reference item (sodium acetate) was tested in parallel. The biodegradation of Reactive Red 239 after 28 days remained < 20% based on the DOC removal. The reference item reached a maximum degradation of ca. 97.7% after 6 days. Identification of degradation products was not performed. Consequently, Reactive Red 239 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.
Reactive Red 239 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 96 d (pH 4), 9.6 d (pH 7) and < 24 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.

- Grouping and read-across
Similar studies are available for the following reactive dyes at DyStar:
· Reactive Red H112339 (EC 413-550-5)
Based on structural similarity, Reactive Red H112339 would be a possible candidate for read-across. The dye split in halves between the triazine groups (red marker) closely represents Reactive Red 239. Reactive Red H112339 was tested for ready biodegradability and stability in water (hydrolysis as a function of pH). In the GLP OECD 301C guideline study, no biodegradability
could be determined after 28 days. The mean oxygen depletion was 0% based on an initial test item concentration of 112 mg/L. The substance was less hydrolysable than Reactive Red 239. The lowest DT50 at 25°C was found at pH 4 (ca. 54 days). However, given the differing hydrolysis rates and the fact, that Reactive Red H112339 more hydrolyses at lower pH than under neutral to alkaline conditions, it has turned out as inappropriate for read-across purposes concerning environmental fate. There is no higher tier study on biodegradation such as a simulation test available for Reactive Red H112339.

· Reactive Black 5 (EC 241-164-5)
The only substance with data on biodegradation simulation testing exists is Reactive Black 5. The study was conducted with a water/sediment system. The structural similarity between Reactive Red 239 and Reactive Black 5 is low. Reactive Black 5 is a considerably smaller molecule without a triazine system. 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-hydroxyethylsulfony1)-phenyl-l'-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 dose 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 : c1cc2c(S(=O)(=O)O)cccc2c(S(=O)(=O)O)c1N=Nc3c(S(=O)(=O)O)cc4cc(S(=O)(=
O)O)cc(c4c3O)Nc5nc(CL)nc(n5)Nc6ccc(cc6)S(=O)(=O)CCOS(=O)(=O)O
CHEM : 1,5-Naphthalenedisulfonic acid, 2-[[8-[[4-chloro-6-[[4-[[2-(sulfooxy)
ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]am
MOL FOR: C31 H24 CL1 N7 O19 S6
MOL WT : 1026.38
--------------------------- BIOWIN v4.10 Results ----------------------------

************************************************************************
* WARNING - The entered structure contains Na, K or Li *
* ---- The metal WAS REMOVED for these calculations ---- *
* Option of Removal/Non-Removal available from entry screen menu-bar. *
* For additional information, see the BIOWIN Help file. *
************************************************************************

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) : Does Not Biodegrade Fast
Biowin6 (MITI Non-Linear Model Prediction): Does Not Biodegrade Fast
Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast
Ready Biodegradability Prediction: NO

------+-----+--------------------------------------------+---------+---------
TYPE | NUM | Biowin1 FRAGMENT DESCRIPTION | COEFF | VALUE
------+-----+--------------------------------------------+---------+---------
Frag | 1 | Aromatic alcohol [-OH] | 0.1158 | 0.1158
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 | 4 | Sulfonic acid / salt -> aromatic attach | -0.2238 | -0.8951
Frag | 1 | Azo group [-N=N-] | -0.2418 | -0.2418
MolWt| * | Molecular Weight Parameter | | -0.4886
Const| * | Equation Constant | | 0.7475
============+============================================+=========+=========
RESULT | Biowin1 (Linear Biodeg Probability) | | -1.4026
============+============================================+=========+=========

------+-----+--------------------------------------------+---------+---------
TYPE | NUM | Biowin2 FRAGMENT DESCRIPTION | COEFF | VALUE
------+-----+--------------------------------------------+---------+---------
Frag | 1 | Aromatic alcohol [-OH] | 0.9086 | 0.9086
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 | 4 | Sulfonic acid / salt -> aromatic attach | -1.0283 | -4.1132
Frag | 1 | Azo group [-N=N-] | -8.2194 | -8.2194
MolWt| * | Molecular Weight Parameter | |-14.5747
============+============================================+=========+=========
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 | Aromatic alcohol [-OH] | 0.0564 | 0.0564
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 | 4 | Sulfonic acid / salt -> aromatic attach | 0.1422 | 0.5688
Frag | 1 | Azo group [-N=N-] | -0.3004 | -0.3004
MolWt| * | Molecular Weight Parameter | | -2.2682
Const| * | Equation Constant | | 3.1992
============+============================================+=========+=========
RESULT | Biowin3 (Survey Model - Ultimate Biodeg) | | 0.5335
============+============================================+=========+=========

------+-----+--------------------------------------------+---------+---------
TYPE | NUM | Biowin4 FRAGMENT DESCRIPTION | COEFF | VALUE
------+-----+--------------------------------------------+---------+---------
Frag | 1 | Aromatic alcohol [-OH] | 0.0397 | 0.0397
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 | 4 | Sulfonic acid / salt -> aromatic attach | 0.0216 | 0.0865
Frag | 1 | Azo group [-N=N-] | -0.0528 | -0.0528
MolWt| * | Molecular Weight Parameter | | -1.4808
Const| * | Equation Constant | | 3.8477
============+============================================+=========+=========
RESULT | Biowin4 (Survey Model - Primary Biodeg) | | 2.0007
============+============================================+=========+=========

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 | Aromatic alcohol [-OH] | 0.0382 | 0.0382
Frag | 1 | Triazine ring (symmetric) | 0.2345 | 0.2345
Frag | 1 | Aromatic chloride [-CL] | -0.0392 | -0.0392
Frag | 2 | Aromatic amine [-NH2 or -NH-] | -0.1273 | -0.2546
Frag | 4 | Sulfonic acid / salt -> aromatic attach | 0.1384 | 0.5536
Frag | 1 | Azo group [-N=N-] | 0.1165 | 0.1165
Frag | 12 | Aromatic-H | 0.0004 | 0.0048
Frag | 2 | -CH2- [linear] | 0.0255 | 0.0511
MolWt| * | Molecular Weight Parameter | | -1.6186
Const| * | Equation Constant | | 0.5544
============+============================================+=========+=========
RESULT | Biowin5 (MITI Linear Biodeg Probability) | | -0.3593
============+============================================+=========+=========

------+-----+--------------------------------------------+---------+---------
TYPE | NUM | Biowin6 FRAGMENT DESCRIPTION | COEFF | VALUE
------+-----+--------------------------------------------+---------+---------
Frag | 1 | Aromatic alcohol [-OH] | 0.2723 | 0.2723
Frag | 1 | Triazine ring (symmetric) |-14.5419 |-14.5419
Frag | 1 | Aromatic chloride [-CL] | -0.7609 | -0.7609
Frag | 2 | Aromatic amine [-NH2 or -NH-] | -1.3170 | -2.6340
Frag | 4 | Sulfonic acid / salt -> aromatic attach | 0.0760 | 0.3041
Frag | 1 | Azo group [-N=N-] |-12.8694 |-12.8694
Frag | 12 | Aromatic-H | 0.0342 | 0.4103
Frag | 2 | -CH2- [linear] | 0.2345 | 0.4690
MolWt| * | Molecular Weight Parameter | |-17.7565
============+============================================+=========+=========
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 | Aromatic alcohol [-OH] | 0.0807 | 0.0807
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 | 4 | Sulfonic acid / salt -> aromatic attach | -0.3768 | -1.5073
Frag | 1 | Azo group [-N=N-] | 0.0000 | 0.0000
Frag | 12 | Aromatic-H | -0.0954 | -1.1452
Frag | 2 | -CH2- [linear] | 0.0260 | 0.0520
Const| * | Equation Constant | | 0.8361
============+============================================+=========+=========
RESULT | Biowin7 (Anaerobic Linear Biodeg Prob) | | -2.7199
============+============================================+=========+=========

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 Red 239 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 Red 239.

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 Red 239, 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 Red 239. 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

Results and discussion

Applicant's summary and conclusion