Reactive Orange 16

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

other: read across from similar substance

Adequacy of study:

key study

Study period:

13. June 1989 to 17. Sep. 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.4300 (Aerobic Aquatic Metabolism)

GLP compliance:

yes

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water / sediment

Details on source and properties of surface water:

- Details on collection (e.g. location, sampling depth, contamination history, procedure): Nidda (Altarm), 6230 Frankfurt a.M. 80
- Storage length: 6 days
- pH at time of collection: 6.6
- Redox potential (mv): 186
- Oxygen concentration (mg/l) initial/final: 8.5/4.7
- Biomass (aerobic bacteria/ml): 49
- Water filtered: yes

Details on source and properties of sediment:

- Details on collection (e.g. location, sampling depth, contamination history, procedure): Nidda (Altarm), 6230 Frankfurt a.M. 80
- Storage length: 6 days
- Textural classification (%sand/silt/clay): 42.1/44.2/13.7
- pH at time of collection: 6.6
- Organic carbon (%): 1.98
- Redox potential (mv) initial/final: 186/163.4
- CEC (meq/100 g): 12.75
- Bulk density (g/cm³): 1.11
- Biomass (e.g. in mg microbial C/100 mg, CFU or other):
- Sediment samples sieved: yes (2 mm sieve)

Details on inoculum:

the sediment/water sample was suspended and sieved through a 2 mm sieve. Thereafter, the sediment was separated from its water by filtration. The remaining moistness was determined by drying of aliquots. 20 g of the sediment was mixed with 180 g of its respective surface water in a 500 ml ground-neck Erlenmeyer flask.
The flasks were kept at 18 to 22°C in the dark

Duration of test (contact time):

112 d

Initial conc.:

1 other: mg/kg

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: 0.201 mg/200 g water/sediment
- Composition of medium: 20 g sediment + 180 g surface water
- Solubilising agent: TS was dissolved in methanol; aliquots containing 201 µg Reactive Black 5 in 510 µL were added drop by drop to the sediment/water test system
- Test temperature: 18°C-22°C
- pH: 6.6
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: erlenmeyer flask with a ground neck
- Number of culture flasks/concentration: 20 + 4 reserve
- Method used to create aerobic conditions:
- Method used to create anaerobic conditions:
- Measuring equipment:


- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used: 1. glass wool with paraffin oil
2. glass wool
3. soda-lime for adsorption of CO2
4. glass wool
5. soda-lime for adsorption of airborne CO2


SAMPLING
- Sampling frequency: Day 0, 3, 7, 14, 21, 28, 42, 57, 84, 112

SAMPLE PREPARATION
14CO2 was transmitted to a mixture of 2-amino ethanol/methanol and a scintillation cocktail was added, afterwards, the 14CO2 was measured by means of a liquid scintillation counter (LCS). Other volatile radioactivity was measured directly with a LCS after addition of a scintillation cocktail to the glass wool.
The sediment was separated from the surface water by centrifugation. Afterwards, the sediment was extracted with acetonitrile/water (4:1). The extracts were pooled and adjusted to a defined volume. Aliquots of the extract and of the water phase were mixed with a scintillation cocktail and the radioactivity was counted. The not extractable radioactivity in sediment was determined by combustion of aliquots of the dried sediment.



PARAMETERS MEASURED:
- Radioactivity in
- Traps for CO2 and volatile metabolites
- Surface water
- Sediment extract
- extracted sediment
- Identity of radioactive residues in water and sediment extraxt
- Assessment of the half life (DT50) of the test substance

Compartment:

other: water, material (mass) balance

% Recovery:

9

Compartment:

other: sediment, material (mass) balance

% Recovery:

85

% Degr.:

95 - 96

Parameter:

radiochem. meas.

Sampling time:

112 d

Compartment:

entire system

DT50:

2 d

Remarks on result:

other: DT50; DT90=3 to 6 days

Other kinetic parameters:

first order rate constant

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

No.:

#4

No.:

#5

No.:

#6

No.:

#7

Details on transformation products:

- Pathways for transformation: see attachment

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

yes

Details on results:

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
- M1: 10.2% to 19.5% Day 3 ----> 0.9% to 1% Day 112
- M2: 15.9% to 20.4% Day 3 ----> only traces Day 14
- M3: 12.7% to 16.1% Day 3 ----> < 0.1% Days 7 to 21 ----> 0.5% to 2% Day 28 to 57 ----> < 0.1% from Day 84 onwards
- M4: 5.1% to 5.2% Day 0
- M5: 40.3% to 42.5% Days 7 to 14 ----> 1.2% to 1.5% Day 112
- M6: 2.0% to 2.4% from Day 84 onwards
- M7: 1.4% to 1.9% from Day 28 onwards ----> 7.6% to 8.8% Day 42 ----> 2.1% to 2.5% Day 112
- Range of maximum concentrations in % of the applied amount at end of study period: 7% to 17% on 3rd day of incubation
4% to 5% on the 112th day of incubation


MINOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
- Range of maximum concentrations in % of the applied amount at end of study period:


TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:


EXTRACTABLE RESIDUES
- % of applied amount at day 0:
- % of applied amount at end of study period:


NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0:
- % of applied amount at end of study period:


MINERALISATION
- % of applied radioactivity present as CO2 at end of study:


VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study:


STERILE TREATMENTS (if used)
- Transformation of the parent compound:
- Formation of transformation products:
- Formation of extractable and non-extractable residues:
- Volatilization:


RESULTS OF SUPPLEMENTARY EXPERIMENT (if any):

Results with reference substance:

NA

Validity criteria fulfilled:

not applicable

Conclusions:

The test substance was rapidly degraded. Metabolites were bound to sediment in non-extractable form. After about one month, mineralization started.

Executive summary:

The substance was applied to a sediment/water test system at a final concentration of 1 mg/kg in the test system. It was very rapidly degraded. The DT50 was of 2 days. After 3 to 6 days, only 10% of the applied TS 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 TS was deposited in form of non-extractable residues in sediment during test phase. After about 1 month, mineralization of the metabolites started.

Description of key information

OECD 309, similar substance 2, DT50 (sediment)= 2 day, DT90 (sediment)= 3 to 6 days

Key value for chemical safety assessment

Half-life in freshwater:

1.9 d

at the temperature of:

25 °C

Half-life in freshwater sediment:

2 d

at the temperature of:

18 °C

Additional information

The DT50 (whole system) of similar substance 2 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. After about 1 month, mineralization of the metabolites started.