N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]phenyl]acetamide

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:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is taken from experimental study report performed as per the standard test guideline.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 13th April 2004

Deviations:

yes

Remarks:

Deviation done in temperature (as per request from European authority)

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Remarks:

Radiolabelling was done at N-acetylphenyl ring-U-14C position.

Oxygen conditions:

aerobic

Inoculum or test system:

natural water

Details on source and properties of surface water:

- Details on collection (e.g. location, sampling depth, contamination history, procedure):
Location: The surface water was collected from Kaveri River, Sangama, Ramnagar District, Karnataka State, India in a thoroughly cleansed container.
The sampling site for collection of the surface water was selected ensuring that no known history of its contamination with the test item or its structural analogues within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The pH and temperature of the water was measured at the site of collection and the depth of sampling and the appearance of the water sample. (e.g. color and turbidity) was also noted. Oxygen concentration of the surface layer was measured in order to demonstrate aerobic conditions.
Depth of sampling was 1 feet and surface water was clear with no turbidity.
- Storage conditions: The test water was stored at 4°C with continuous aeration prior use for a period not more than 4 weeks.
- Storage length: not more than 4 weeks
- Temperature (°C) at time of collection: 21.1°C
- pH at time of collection: 6.73
- Oxygen concentration (mg/l) initial/final: 5.1 mg/l
- Dissolved organic carbon (%) leachable: 2.4 mg/kg dm
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): 4000 CFU/ml
- Water filtered: yes, prior to use, the coarse particles were removed by filtration through a 100 μm mesh sieve.
- Type and size of filter used, if any:
- Other:
Total organic carbon (TOC): 2.6 mg/l
Nitrate (NO3- ): 3 mg/l
Nitrite (NO2- ): <0.005 mg/l
P: 0.3 mg/l
Orthophosphates (PO43-): 0.22 mg/l
Total ammonia tot (NH4+ ): <0.3 mg/l
BOD: <2.0 mg/l

Details on source and properties of sediment:

Not applicable

Details on inoculum:

Not applicable

Duration of test (contact time):

60 d

Initial conc.:

10 µg/L

Based on:

test mat.

Remarks:

(low concentration)

Initial conc.:

100 µg/L

Based on:

test mat.

Remarks:

(high concentration)

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment:
- Solubilising agent (type and concentration if used): 1% acetonitrile was used as a solubilising agent.
- Test temperature: 12±2°C
- pH: 6.73
- Suspended solids concentration: 15 mg/l
- Continuous darkness: yes, study was performed under continuous darkness.
- Any indication of the test material adsorbing to the walls of the test apparatus: The average recovery of 14C-test chemical after 24 hours of equilibration period was 99.2% from the test solutions of 100 μg/L (0.1 μg/mL) prepared in test water which indicated no significant adsorption (<5%).
- Other: An investigation of adsorption of 14C-test chemical to test apparatus (conical flasks) at 100 μg/L (0.1 μg/mL) concentration was carried out.
Triplicate 25 μL aliquot of 14C-test chemical stock solution was analyzed by LSC, and the concentration was 101.676 μg/mL. A 100 μL aliquot of this solution was transferred to a 20 mL scintillation vial and 0.9 mL of acetonitrile was added and mixed well. Triplicate 25 μL aliquot of this solution was analyzed by LSC, and the concentration was 10.220 μg/mL.
A 1.0 mL aliquot of the above 10.220 μg/mL treatment solution was dispensed into separate triplicate 100 mL volumetric flask and the volume was made up to the mark with test water. The solutions were mixed thoroughly to get solutions of nominal concentration of 100 μg/L (0.1 μg/mL) and transferred to 250 mL capacity conical flasks. Triplicate conical flasks containing 100 mL of test water without test item were also included in the experiment. Triplicate 1 mL aliquots from each vessel were analyzed by LSC.
The solutions thus prepared in conical flasks were agitated for 24 hours on a shaking incubator at 20ºC. Following 24 hours of shaking, triplicate 1000 μL aliquots from each container were submitted for LSC.

Another experiment was conducted using test water at a nominal highest concentration of 500 μg/L (0.5 μg/mL) with 1% acetonitrile as co-solvent for metabolite identification.

TEST SYSTEM
- Culturing apparatus: Conical flasks of 250 ml
- Number of culture flasks/concentration: Duplicates
- Method used to create aerobic conditions: Aerobic condition was maintained in the test system by continuous shaking.
- Method used to create anaerobic conditions: not applicable
- Method used to control oxygen conditions: Agitation was provided to facilitate oxygen transfer from the headspace to the liquid so that aerobic conditions were adequately maintained.
- Test performed in closed vessels: yes, test vessel was covered with cotton plugs.
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: Test systems consisted of treated surface water in sterile conical flasks under continuous stirring and connected to ethylene glycol and 1N KOH traps to collect the volatile 14CO2.
- Other: The test vessels were sterilized by maintaining 121ºC and 15 PSI for 20 minutes in an autoclave before use to avoid microbial contamination. Test vessel was kept in an incubator shaker at 12 ± 2°C in dark.

SAMPLING
- Sampling frequency: Duplicate test vessels from each test concentration were removed at each sampling occasion and analyzed at zero-time (immediately following test chemical application), day 1, day 3, day7, day 14, day 28, day 45 and day 60, respectively.
- Sampling method used per analysis type: Samples were removed at regular intervals, measured pH and oxygen concentration. After that the samples were diluted at 1:1, v/v ratio with methanol to prevent further degradation prior to LC-MS/MS analysis. Shaking was continued at 12 ± 2°C in dark with 14CO2 trapping for using in other sampling intervals.

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
Abiotic sterile control:
A 100 mL aliquot sterile water treated with test chemical at 10 µg/L (0.010 µg/mL) concentration was transferred into 20 Conical flask of 250 mL capacity.
A 100 mL aliquot sterile water treated with test chemical at 100 µg/L (0.100 µg/mL) concentration was transferred into 20 Conical flask of 250 mL capacity.
A 100 mL aliquot sterile water treated with test chemical at 100 µg/L (0.100 µg/mL) concentration with 1% acetonitrile as a co-solvent was transferred into 20 Conical flask of 250 mL capacity.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 1 blank test vessel containing only the test water for all sampling intervals was included.
- Abiotic sterile control: yes, 1 blank test vessel containing only the sterile test water was also treated at 100 µg/L (0.1 µg/mL) conc..
- Other: Duplicates test vessels with reference (aniline) and 1 blank test vessel containing only the test water with co-solvent was also kept in the study.

STATISTICAL METHODS: The data were assessed using simple first order (SFO) model using the CAKE version 3.5 (Release) software.

Reference substance:

aniline

Remarks:

(conc. 10 μg/l i.e. 0.01 mg/l)

Compartment:

natural water

% Recovery:

0

Remarks on result:

other: Recovery of test chemical conc. of 10 μg/l at Day 60

Compartment:

natural water

% Recovery:

46.7

Remarks on result:

other: Recovery of test chemical conc. of 100 μg/l at Day 60

Key result

% Degr.:

90

Parameter:

test mat. analysis

Sampling time:

23.9 d

Remarks on result:

other: at test chemical conc. of 10 μg/l

Key result

% Degr.:

90

Parameter:

test mat. analysis

Sampling time:

150 d

Remarks on result:

other: at test chemical conc. of 100 μg/l

Key result

Compartment:

natural water

DT50:

7.2 d

Temp.:

12 °C

Remarks on result:

other: at test chemical conc. of 10 μg/l

Key result

Compartment:

natural water

DT50:

45.3 d

Temp.:

12 °C

Remarks on result:

other: at test chemical conc. of 100 μg/l

Transformation products:

yes

No.:

#1

Details on transformation products:

- Formation and decline of each transformation product during test:
Following application of 14C-test chemical to test water at 10 μg/L (0.01 μg/mL), the average amount of test chemical declined from 98.8% AR on day 0 to 0.0% AR on day 28. One metabolite was observed during the incubation period of 60 days. The maximum unidentified component (identified as ‘Others’) of 1.0% AR was observed on day 1 sampling interval.
Following application of 14C-test chemical to test water at 100 μg/L (0.1 μg/mL), the average amount of test chemical declined from 101.7% AR on day 0 to 46.7% AR on day 60. One metabolite was observed during the incubation period of 60 days. The maximum unidentified component (identified as ‘Others’) of 4.3% AR was observed on day 45 sampling interval.
Following application of 14C-test chemical to sterile test water at 100 μg/L (0.1 μg/mL), the average amount of Solvent Blue 122 declined from 100.9% AR on day 0 to 60.6% AR on day 60. One metabolite was observed during the incubation period of 60 days. No unidentified component (as ‘Others’) was observed in any sampling interval.
There is one major unknown transformation product accounting for greater than or equal to 10% of initial applied radioactivity, or >5% AR in two sequential measurements or >5% AR at the end of incubation.

- Pathways for transformation:
Test chemical is unstable in natural water and test chemical is completely converted into degradation product (1-hydroxy-4-(phenylamino)anthracene-9,10-dione) by end of incubation period of 60 days.
The pathway of degradation was given in the attached document of this template.

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

not specified

Details on results:

Analysis of the Day 0 samples at 10 μg/L and 100 μg/L test concentrations demonstrated quantitative recovery of test chemical.
The average amount of test chemical present was 98.8% and 0% at Day 0 and Day 60, respectively following application of test chemical to test water at 10 μg/L (low dose).
The average amount of test chemical present was 101.7% and 46.7% at Day 0 and Day 60, respectively following application of test chemical to test water at 100 μg/L (high dose).
The average amount of test chemical present was 100.9% and 60.6% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 100 μg/L.

TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Yes, test conditions were maintained during the study.

TRANSFORMATION PRODUCTS
Test chemical is unstable in natural water and test chemical is completely converted into degradation product (1-hydroxy-4-(phenylamino)anthracene-9,10-dione) by end of incubation period of 60 days.
Mass balances for test water treated at 10 μg/L (0.01 μg/mL) before stripping 14CO2 ranged from 95.5% to 98.8% AR with a mean of 96.8% AR.
Mass balances for test water treated at 100 μg/L (0.1 μg/mL) before stripping 14CO2 ranged from 97.6% to 101.7% AR with a mean of 99.1% AR.
Mass balances for sterile test water treated at 100 μg/L (0.1 μg/mL) before stripping 14CO2 ranged from 98.0% to 100.9% AR with a mean of 99.0% AR.
Analysis of the day 0 samples from 10 μg/L (0.01 μg/mL) and 100 μg/L (0.1 μg/mL) test concentrations demonstrated quantitative recovery of test chemical before stripping 14CO2.
Following application of 14C-test chemical to test water at 10 μg/L (0.01 μg/mL), the average amount of test chemical declined from 98.8% AR on day 0 to 0.0% AR on day 28. One metabolite was observed during the incubation period of 60 days. The maximum unidentified component (identified as ‘Others’) of 1.0% AR was observed on day 1 sampling interval.
Following application of 14C-test chemical to test water at 100 μg/L (0.1 μg/mL), the average amount of test chemical declined from 101.7% AR on day 0 to 46.7% AR on day 60. One metabolite was observed during the incubation period of 60 days. The maximum unidentified component (identified as ‘Others’) of 4.3% AR was observed on day 45 sampling interval.
Following application of 14C-test chemical to sterile test water at 100 μg/L (0.1 μg/mL), the average amount of test chemical declined from 100.9% AR on day 0 to 60.6% AR on day 60. One metabolite was observed during the incubation period of 60 days. No unidentified component (as ‘Others’) was observed in any sampling interval.
There is one major unknown transformation product accounting for greater than or equal to 10% of initial applied radioactivity, or >5% AR in two sequential measurements or >5% AR at the end of incubation.


TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:
Following application of 14C-test chemical to test water at 10 μg/L (0.01 μg/mL), the maximum unidentified component (identified as ‘Others’) of 1.0% AR was observed on day 1 sampling interval.
Following application of 14C-test chemical to test water at 100 μg/L (0.1 μg/mL), the maximum unidentified component (identified as ‘Others’) of 4.3% AR was observed on day 45 sampling interval.
Following application of 14C-test chemical to sterile test water at 100 μg/L (0.1 μg/mL), no unidentified component (as ‘Others’) was observed in any sampling interval.

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: Result was provided in the atttached document of this template.

VOLATILIZATION: not applicable

STERILE TREATMENTS (if used)
The average amount of test chemical present was 100.9% and 60.6% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 100 μg/L.
Mass balances for sterile test water treated at 100 μg/L (0.1 μg/mL) before stripping 14CO2 ranged from 98.0% to 100.9% AR with a mean of 99.0% AR.
Following application of 14C-test chemical to sterile test water at 100 μg/L (0.1 μg/mL), the average amount of test chemical declined from 100.9% AR on day 0 to 60.6% AR on day 60. One metabolite was observed during the incubation period of 60 days. No unidentified component (as ‘Others’) was observed in any sampling interval.

Results with reference substance:

The percent recovery of reference substance aniline was observed to be 0.0% on day 13, thereby indicating that its degradation in the test surface water within the expected time interval of two weeks. Therefore, the validity of the test is acceptable.

Mass Balance

Mass balances for test watertreated at 10mg/L (0.01mg/mL) before stripping 14CO2 ranged from 95.5%to 98.8% AR with a mean of96.8% AR.

Mass balances for test watertreated at 100mg/L (0.1mg/mL) before stripping14CO2 ranged from 97.6%to 101.7% AR with a mean of99.1%AR.

Mass balances for sterile test watertreated at 100mg/L (0.1mg/mL) before stripping 14CO2 ranged from 98.0%to 100.9% AR with a mean of99.0% AR.

Mineralization of Test Item to 14CO2

When the samples from different test systems were subjected to CO2 stripping, there was no significant loss of CO2.

Distribution and Composition of Radioactivity

Analysis of the day 0 samples from 10mg/L (0.01mg/mL) and 100mg/L(0.1mg/mL) test concentrations demonstrated quantitative recovery of test chemical before stripping 14CO2.

Following application of 14C-test chemical to test water at 10mg/L
(0.01mg/mL), the average amount of Solvent Blue 122 declined from 98.8% AR (on day 0) to <LOD (by day 28). One degradation product was observed during the incubation period of 60 days. The average amount of degradation product increased from <LOD (on day 0) to 95.2% AR (end of incubation on day 60). The maximum unidentified component (identified as ‘Others’) of 1.0% AR was observed on day 1 sampling interval.

Following application of 14C-test chemical to test water at 100mg/L
(0.1mg/mL), the average amount of test chemical declined from 101.7 % AR (on day 0) to 46.7% AR (by end of incubation on day 60). One degradation product was observed during the incubation period of 60 days. The average amount of degradation product increased from <LOD (on day 0) to 51.4 % AR (end of incubation on day 60). The maximum unidentified component (identified as ‘Others’) of 4.3% AR was observed on day 45 sampling interval.

Following application of 14C-test chemical to sterile test water at 100mg/L
(0.1mg/mL), the average amount of Solvent Blue 122 declined from 100.9% AR (on day 0) to 60.6% AR (by end of incubation on day 60). One degradation product was observed during the incubation period of 60 days. The average amount of degradation product increased from <LOD (on day 0) to 37.2 % AR (end of incubation on day 60). No unidentified component (as ‘Others’) was observed in any sampling interval

There is one major unknown transformation product accounting for greater than or equal to 10% of initial applied radioactivity, or>5% AR in two sequential measurements or>5% AR at the end of incubation.

Based on the results, the test item solvent blue 122 is unstable in natural surface water and solvent blue 122 is converted into degradation product (1-hydroxy-4-(phenylamino)anthracene-9,10-dione) by end of incubation period of 60 days. 

 Identification of Test Item

Unchanged 14C-test chemical in the test samples was identified using HPLC by comparing the retention time of the radioactive peak with that of a reference standard. The identification of unchanged 14C-test chemical was also confirmed by LC-MS/MS analysis of a day 60 sampling interval. Comparison of the mass spectra of test chemical standard and 14C-test chemical from representative samples confirms the identity of unchanged 14C-test chemical. The spectrum of the test chemical standard contained an (M-H)^-ion at 371.0 which was comparable to the spectra of the treated sample which contained an (M-H)^-ion at 373.1 (two mass units extra confirms the labelled test item). The matching HPLC retention time with the standard and the mass spectrometry results, confirm the identity of unchanged 14C-test chemical.

KINETIC ANALYSIS OF DATA

The data generated for test chemical from day 0, day 1, day 3 day 7, day 14, day 28, day 45 and day 60 after test chemical application to test water was used for degradation kinetics by CAKE version 3.5 (Release) software.

The details of optimized parameters, Chi square and r²values from each model are included. The fit model and its calculated DT50 and DT90 values were shown in Table. 

The calculated DT50 (Days) and DT90 (Days) for each concentrationare summarized in the table below:

Sample	Test conc.	DT50(days)	DT90(days)	c^2error	Model
Test water	10 µg/L	7.2	23.9	6.92	SFO
Test water	100 µg/L	45.3	150	5.62	SFO
Sterile test water	100 µg/L	74.8	249	3.53	SFO

Plots of the observed and fitted data and parameter estimates from the best-fit model for test water treated with test chemical.

TABLE:          Adsorption to Test Vessels

Test conc. and replication	Amount of test item at ‘0’ h (µg), (A)	Amount of test item at ‘24’ h (µg), (B)	Test item adsorbed (µg), (C)	Adsorption at 24 h (%)	Mean adsorption at 24 h (%)
Test water treated at 100mg/L, Rep 1	10.413	9.133	1.280	12.3	12.2
Test water treated at 100mg/L, Rep 2	10.498	9.261	1.237	11.8
Test water treated at 100mg/L, Rep 3	10.515	9.203	1.312	12.5

Test item adsorbed (µg), (C) = A – B

Adsorption (%) =	C	×100
	A

 

Test conc. and replication	Amount of test item in washings (µg), (D)	Test item adsorbed after washing (µg), (E)	Adsorption after washings (%)	Overall Mean adsorption (%)
Test water treated at 100mg/L, Rep 1	1.197	0.083	0.8	0.8
Test water treated at 100mg/L, Rep 2	1.189	0.048	0.5
Test water treated at 100mg/L, Rep 3	1.195	0.117	1.1

Adsorption after washings(µg) E=A-(B+D)

TEST ITEM RECOVERY

 

Test concentration and replication	% Recovery at 24 h	Average recovery (%)	Overall % recovery after washings	Average recovery (%)
Test water at 100mg/L, Rep 1	87.7	87.8	99.2	99.2
Test water at 100mg/L, Rep 2	88.2		99.5
Test water at 100mg/L, Rep 3	87.5		98.9

% Recovery =		Amount of test item at completion of experiment (B)		× 100
		Amount of test item at beginning of experiment (A)

 

Overall % Recovery after washings =	Amount of test item at 24 h (b) + Amount of test item from washings (d)	x 100
	Amount of test item at beginning of experiment (a)

TABLE: Summary of Kinetic Data for 14C-test chemical in Test Water

Concentration and components modeled		Fit model		Optimised parameters±standard error		c^2error		r^2		DT50 (days)		DT90 (days)
10 µg/L, parent only		SFO		M0 (%AR) = 103.3 ± 2.953 k (d^-1) = 0.09633 ± 0.007922		6.92		0.9828		7.2		23.9
100 µg/L, parent only		SFO		M0 (%AR) = 98.52 ± 2.199 k (d^-1) = 0.01532 ± 0.001273		5.62		0.9358		45.3		150
100 µg/L (sterile), parent only		SFO		M0 (%AR) = 98.96 ± 1.465 k (d^-1) = 0.009263 ± 6.90E-004		3.53		0.9393		74.8		249

TABLE: Recovery of Aniline from Test Water Treated at 100 µg/L

Sampling interval	Rep.	% Recovery	Average recovery (%)
Day 0	1	103.0	103.4
	2	103.7
Day 1	1	105.5	102.3
	2	99.1
Day 3	1	82.3	83.7
	2	85.1
Day 5	1	67.4	64.9
	2	62.4
Day 7	1	51.8	49.2
	2	46.5
Day 10	1	17.9	18.2
	2	18.4
Day 13	1	Not detected	-

TABLE: Measurement of Oxygen Concentration and pH of Sterile Test Water Treated at 100 µg/L

Sampling interval	Test conc. (µg/L)	Rep.	Oxygen (mg/L)	Mean	pH	Mean
Day 0	100	1	4.7	4.5	6.68	6.85
		2	4.3		7.01
Day 1	100	1	3.9	3.8	6.44	6.48
		2	3.6		6.51
Day 3	100	1	2.6	2.7	6.86	6.94
		2	2.8		7.01
Day 7	100	1	2.1	2.3	7.42	7.35
		2	2.5		7.28
Day 14	100	1	1.9	2.0	7.48	7.44
		2	2		7.39
Day 28	100	1	1.9	1.8	7.36	7.28
		2	1.6		7.19
Day 45	100	1	1.7	1.6	7.73	7.96
		2	1.4		8.18
Day 60	100	1	1.2	1.3	8.27	8.29
		2	1.4		8.31

 

TABLE: Mass Balance of Radioactivity (% AR) from Test Water Treated with    ¹⁴C-Solvent Blue 122 Before¹⁴CO₂Stripping

Test concentration	Sampling interval (day)	Rep.	Radioactivity recovered (dpm)	Applied radioactivity (dpm)	% Recovery	Mean
10 µg/L	0	1	385000	386800	99.5	98.8
		2	379600	386800	98.1
	1	1	381480	386800	98.6	98.7
		2	381920	386800	98.7
	3	1	376750	386800	97.4	97.3
		2	375980	386800	97.2
	7	1	374000	386800	96.7	96.6
		2	373010	386800	96.4
	14	1	370370	386800	95.8	95.5
		2	368390	386800	95.2
	28	1	370590	386800	95.8	96.2
		2	373450	386800	96.5
	45	1	367070	386800	94.9	95.4
		2	370810	386800	95.9
	60	1	368830	386800	95.4	95.2
		2	367070	386800	94.9
	Mean				96.7	96.7
100 µg/L	0	1	3875500	3826600	101.3	101.7
		2	3906300	3826600	102.1
	1	1	3787850	3826600	99.0	99.1
		2	3791370	3826600	99.1
	3	1	3820080	3826600	99.8	100.0
		2	3828660	3826600	100.1
	7	1	3820630	3826600	99.8	100.0
		2	3835150	3826600	100.2
	14	1	3720420	3826600	97.2	97.6
		2	3748910	3826600	98.0
	28	1	3688850	3826600	96.4	97.7
		2	3783560	3826600	98.9
	45	1	3753200	3826600	98.1	98.2
		2	3762990	3826600	98.3
	60	1	3738460	3826600	97.7	98.1
		2	3765300	3826600	98.4
	Mean				99.0	99.1

 

TABLE: Mass Balance of Radioactivity (% AR) from Sterile Test Water Treated with¹⁴C-Solvent Blue 122 Before¹⁴CO₂Stripping

Test concentration	Sampling interval (day)	Rep.	Radioactivity recovered (dpm)	Applied radioactivity (dpm)	% Recovery	Mean
100 µg/L	0	1	3840500	3826600	100.4	100.9
		2	3876900	3826600	101.3
	1	1	3828000	3826600	100.0	99.6
		2	3794450	3826600	99.2
	3	1	3820630	3826600	99.8	99.9
		2	3824150	3826600	99.9
	7	1	3799180	3826600	99.3	99.2
		2	3788400	3826600	99.0
	14	1	3766950	3826600	98.4	98.0
		2	3730100	3826600	97.5
	28	1	3795000	3826600	99.2	98.5
		2	3739120	3826600	97.7
	45	1	3731750	3826600	97.5	97.8
		2	3751330	3826600	98.0
	60	1	3726030	3826600	97.4	97.8
		2	3759470	3826600	98.2
	Mean				98.9	99.0

 

TABLE :  Mass Balance of Radioactivity (% AR) from Test Water Treated with¹⁴C-Solvent Blue 122 After¹⁴CO₂Stripping

Test concentration	Sampling interval (day)	Rep.	Radioactivity recovered (dpm)	Applied radioactivity (dpm)	% Recovery	Mean
10 µg/L	0	1	377100	386800	97.5	97.6
		2	377500	386800	97.6
	1	1	379610	386800	98.1	96.6
		2	367730	386800	95.1
	3	1	375540	386800	97.1	97.0
		2	374770	386800	96.9
	7	1	368060	386800	95.2	95.7
		2	371800	386800	96.1
	14	1	371800	386800	96.1	95.8
		2	369270	386800	95.5
	28	1	365970	386800	94.6	95.2
		2	370260	386800	95.7
	45	1	372130	386800	96.2	96.4
		2	373450	386800	96.5
	60	1	368390	386800	95.2	95.2
		2	368170	386800	95.2
	Mean				96.2	96.2
100 µg/L	0	1	3760500	3826600	98.3	98.6
		2	3785400	3826600	98.9
	1	1	3735820	3826600	97.6	97.1
		2	3697760	3826600	96.6
	3	1	3824920	3826600	100.0	99.4
		2	3775640	3826600	98.7
	7	1	3752870	3826600	98.1	97.8
		2	3728780	3826600	97.4
	14	1	3740880	3826600	97.8	97.4
		2	3710410	3826600	97.0
	28	1	3699300	3826600	96.7	96.9
		2	3711070	3826600	97.0
	45	1	3739670	3826600	97.7	97.3
		2	3708100	3826600	96.9
	60	1	3725260	3826600	97.4	97.3
		2	3715580	3826600	97.1
	Mean				97.7	97.7

TABLE: Transformation of 14C-test chemical in Test Water (% AR) Treatedat 10 µg/LBefore14CO2 Stripping

Parameter	R*	Sampling interval (days)
		0	1	3	7	14	28	45	60
Solvent Blue 122	1	99.5	95.7	79.1	60.8	19.3	0	0	0
	2	98.1	94.7	79.1	59.7	23.9	0	0	0
Mean	-	98.8	95.2	79.1	60.3	21.6	0.0	0.0	0.0
Un Known	1	<LOD	2.9	18.3	35.9	76.5	95.8	94.9	95.4
	2	<LOD	2.1	18.1	36.7	71.3	96.5	95.9	94.9
Mean	-	<LOD	2.5	18.2	36.3	73.9	96.2	95.4	95.2
Others	1	0	0	0	0	0	0	0	0
	2	0	1.9	0	0	0	0	0	0
Mean	-	0.0	1.0	0.0	0.0	0.0	0.0	0.0	0.0
Total recovery	1	99.5	98.6	97.4	96.7	95.8	95.8	94.9	95.4
	2	98.1	98.7	97.2	96.4	95.2	96.5	95.9	94.9
Mean	-	98.8	98.7	97.3	96.6	95.5	96.2	95.4	95.2

*Replication

Note: The total value may differ from the mass balance table due to rounding off.

<LOD = below the limit of detection as no peak was observed during HPLC analysis.

TABLE: Transformation of 14C-test chemical in Test Water (% AR) Treatedat 100 µg/LBefore 14CO2 Stripping

Parameter	R*	Sampling interval (days)
		0	1	3	7	14	28	45	60
Solvent Blue 122	1	101.3	99	95.3	90.6	70	54.3	52.3	46.5
	2	102.1	99.1	95.5	91.5	69.6	58.9	51	46.8
Mean	-	101.7	99.1	95.4	91.1	69.8	56.6	51.7	46.7
Un Known	1	<LOD	<LOD	4.5	9.2	27.2	42.1	41.5	51.2
	2	<LOD	<LOD	4.6	8.7	28.4	40	43	51.6
Mean	-	<LOD	<LOD	4.6	9.0	27.8	41.1	42.3	51.4
Others	1	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD	4.3	<LOD
	2	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD	4.3	<LOD
Mean	-	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD	4.3	<LOD
Total recovery	1	101.3	99	99.8	99.8	97.2	96.4	98.1	97.7
	2	102.1	99.1	100.1	100.2	98	98.9	98.3	98.4
Mean	-	101.7	99.1	100.0	100.0	97.6	97.7	98.2	98.1

*Replication

Note: The total value may differ from the mass balance table due to rounding off.

<LOD = below the limit of detection as no peak was observed during HPLC analysis.

 

TABLE:  Transformation of 14C-SOLVENT BLUE 122 in Sterile Test Water (% AR) Treatedat 100 µg/LBefore 14CO2 Stripping

Parameter	R*	Sampling interval (days)
		0	1	3	7	14	28	45	60
Solvent Blue 122	1	100.4	100	99.8	92.8	79	71.8	65.8	59.4
	2	101.3	99.2	99.9	92.3	80.1	71.9	67.7	61.8
Mean	-	100.9	99.6	99.9	92.6	79.6	71.9	66.8	60.6
Un Known	1	<LOD	<LOD	<LOD	6.5	19.4	27.4	31.7	38
	2	<LOD	<LOD	<LOD	6.7	17.4	25.8	30.3	36.4
Mean	-	<LOD	<LOD	<LOD	6.6	18.4	26.6	31.0	37.2
Others	1	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD
	2	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD
Mean	-	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD
Total recovery	1	100.4	100	99.8	99.3	98.4	99.2	97.5	97.4
	2	101.3	99.2	99.9	99	97.5	97.7	98	98.2
Mean	-	100.9	99.6	99.9	99.2	98.0	98.5	97.8	97.8

*Replication

Note: The total value may differ from the mass balance table due to rounding off.

<LOD = below the limit of detection as no peak was observed during HPLC analysis.

Note: Additional result tables and graphs are provided in the attached document of this template.

Validity criteria fulfilled:

yes

Conclusions:

Analysis of the Day 0 samples at 10 μg/L and 100 μg/L test concentrations demonstrated quantitative recovery of test chemical. The average amount of test chemical present was 98.8% and 0% & 101.7% and 46.7% at Day 0 and Day 60, respectively following application of test chemical to test water at 10 μg/L (low dose) and 100μg/L (high dose). The average amount of test chemical present was 100.9% and 60.6% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 100μg/L (high dose). The DT50 value was determined to be 7.2 d and 45.3 d at test chemical conc. of 10 μg/l and 100 μg/l at 12°C, respectively. 90% degradation of test chemical in natural surface water was determined after 23.9 d and 150 d at test chemical conc. of 10 μg/l and 100 μg/l, respectively. Test chemical was unstable in natural water and test chemical was completely converted into degradation product (1-hydroxy-4-(phenylamino)anthracene-9,10-dione) by end of incubation period of 60 days. Based on the these results, test chemical was considered to be not persistent in water.

Executive summary:

Aerobic mineralisation of test chemical in water was studies as per the principles of the OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) (Adopted 13th April 2004) under aerobic conditions. The surface water was collected from Kaveri River, Sangama, Ramnagar District, Karnataka State, India in a thoroughly cleansed container. The sampling site for collection of the surface water was selected ensuring that no known history of its contamination with the test item or its structural analogues within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The pH and temperature of the water was measured at the site of collection and the depth of sampling and the appearance of the water sample. (e.g. color and turbidity) was also noted. Oxygen concentration of the surface layer was measured in order to demonstrate aerobic conditions. Depth of sampling was 1 feet and surface water was clear with no turbidity. The test water was stored at 4°C with continuous aeration prior use for a period not more than 4 weeks. Temperature (°C) at time of collection was 21.1°C, pH of temperature was 6.73, Oxygen concentration (mg/l) of 5.1 mg/l,  Dissolved organic carbon (%) of 2.4 mg/kg dm, colony count consists of 4000 CFU/ml, Total organic carbon (TOC) of 2.6 mg/l, Nitrate (NO3- ) of 3 mg/l, Nitrite (NO2- ) of <0.005 mg/l, P of 0.3 mg/l, Orthophosphates (PO43-) of 0.22 mg/l, Total ammonia tot (NH4+ ) of <0.3 mg/l and BOD of <2.0 mg/l, respectively. Prior to use of surface water, the coarse particles were removed by filtration through a 100 μm mesh sieve. Test chemical conc. used in the study was 10 μg/L as low dose and 100 μg/L as high dose, respectively. The surface water was also treated at 500 µg/L (0.5 µg/mL) which was used for identification of degradation products. Study was performed in duplicates in a 250 ml conical flasks which was covered with cotton plugs under continuous darkness. Test conditions involve a temperature of 12±2°C, pH of  6.73. Test vessel was kept in an incubator shaker at 12 ± 2°C in dark. Aerobic condition was maintained in the test system by continuous shaking. Agitation was provided to facilitate oxygen transfer from the headspace to the liquid so that aerobic conditions were adequately maintained. Additional to test vessels, 1 blank test vessel containing only the test water for all sampling intervals was included, 1 blank test vessel containing only the sterile test water was also treated at 10 µg/L (0.01 µg/mL) and 100 µg/L (0.1 µg/mL) conc., 1 blank test vessel containing only test chemical with co-solvent and duplicate test vessels with reference (aniline) (conc. 10 μg/l i.e. 0.01 mg/l) was also kept in the study. All experiments were performed in duplicates. The concentration of test chemical residues in samples collected at different pre-determined interval zero-time (immediately after treatment day 0), day 1, day 3 day 7, day 14, day 28, day 45 and day 60 were diluted suitably with acetonitrile and at each sampling occasion, triplicate aliquots from each test concentration were subjected to total radioactivity analysis by LSC and the components were quantified by reverse phase radio-HPLC with on-line radiochemical detection. Additionally, an aliquot of each sample was subjected for 14CO2 determination by indirect method followed by LSC analysis and trapped 14CO2 in KOH and ethylene glycol by LSC analysis. Each sample was analyzed by HPLC-UV detection with on-line radiochemical detection. High performance liquid chromatograph (Exion HPLC) equipped with a mass spectrometer (TQ 5500) was used with a column of Column: Shimpack C18(2), 250 mm × 4.6 mm i.d., 5 µm, column oven temperature of 30°C, mobile phase consists of Solvent A : 5 mM ammonium formate in Milli-Q® water and Solvent B : Acetonitrile in a ratio of 30 : 70, v/v, flow rate of 0.5 mL/min with splitter, respectively. Detection method involve the use of MS. Using the method of Currie L. A. (1968), the LOD and LOQ of the LSC analyses were 28 and 111 dpm, respectively. During method validation, acceptable recoveries were generated for the samples fortified at LOQ and 10 LOQ level. The % RSD (precision) was ≤20% at each fortification level. Recovery data from these samples demonstrated that test chemical was unstable during analysis. The identification and quantification of the degradation product was carried out using mass spectrometry. Analysis of the Day 0 samples at 10 μg/L and 100 μg/L test concentrations demonstrated quantitative recovery of test chemical. The average amount of test chemical present was 98.8% and 0% & 101.7% and 46.7% at Day 0 and Day 60, respectively following application of test chemical to test water at 10 μg/L (low dose) and 100μg/L (high dose). The average amount of test chemical present was 100.9% and 60.6% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 100μg/L (high dose). The DT50 value was determined to be 7.2 d and 45.3 d at test chemical conc. of 10 μg/l and 100 μg/l at 12°C, respectively. 90% degradation of test chemical in natural surface water was determined after 23.9 d and 150 d at test chemical conc. of 10 μg/l and 100 μg/l, respectively. Test chemical was unstable in natural water and test chemical was completely converted into degradation product (1-hydroxy-4-(phenylamino)anthracene-9,10-dione) by end of incubation period of 60 days. Based on these results, test chemical was considered to be not persistent in water.

Description of key information

Aerobic mineralisation of test chemical in water was studies as per the principles of the OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) (Adopted 13th April 2004) under aerobic conditions. The surface water was collected from Kaveri River, Sangama, Ramnagar District, Karnataka State, India in a thoroughly cleansed container. The sampling site for collection of the surface water was selected ensuring that no known history of its contamination with the test item or its structural analogues within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The pH and temperature of the water was measured at the site of collection and the depth of sampling and the appearance of the water sample. (e.g. color and turbidity) was also noted. Oxygen concentration of the surface layer was measured in order to demonstrate aerobic conditions. Depth of sampling was 1 feet and surface water was clear with no turbidity. The test water was stored at 4°C with continuous aeration prior use for a period not more than 4 weeks. Temperature (°C) at time of collection was 21.1°C, pH of temperature was 6.73, Oxygen concentration (mg/l) of 5.1 mg/l,  Dissolved organic carbon (%) of 2.4 mg/kg dm, colony count consists of 4000 CFU/ml, Total organic carbon (TOC) of 2.6 mg/l, Nitrate (NO3- ) of 3 mg/l, Nitrite (NO2- ) of <0.005 mg/l, P of 0.3 mg/l, Orthophosphates (PO43-) of 0.22 mg/l, Total ammonia tot (NH4+ ) of <0.3 mg/l and BOD of <2.0 mg/l, respectively. Prior to use of surface water, the coarse particles were removed by filtration through a 100 μm mesh sieve. Test chemical conc. used in the study was 10 μg/L as low dose and 100 μg/L as high dose, respectively. The surface water was also treated at 500 µg/L (0.5 µg/mL) which was used for identification of degradation products. Study was performed in duplicates in a 250 ml conical flasks which was covered with cotton plugs under continuous darkness. Test conditions involve a temperature of 12±2°C, pH of  6.73. Test vessel was kept in an incubator shaker at 12 ± 2°C in dark. Aerobic condition was maintained in the test system by continuous shaking. Agitation was provided to facilitate oxygen transfer from the headspace to the liquid so that aerobic conditions were adequately maintained. Additional to test vessels, 1 blank test vessel containing only the test water for all sampling intervals was included, 1 blank test vessel containing only the sterile test water was also treated at 10 µg/L (0.01 µg/mL) and 100 µg/L (0.1 µg/mL) conc., 1 blank test vessel containing only test chemical with co-solvent and duplicate test vessels with reference (aniline) (conc. 10 μg/l i.e. 0.01 mg/l) was also kept in the study. All experiments were performed in duplicates. The concentration of test chemical residues in samples collected at different pre-determined interval zero-time (immediately after treatment day 0), day 1, day 3 day 7, day 14, day 28, day 45 and day 60 were diluted suitably with acetonitrile and at each sampling occasion, triplicate aliquots from each test concentration were subjected to total radioactivity analysis by LSC and the components were quantified by reverse phase radio-HPLC with on-line radiochemical detection. Additionally, an aliquot of each sample was subjected for 14CO2 determination by indirect method followed by LSC analysis and trapped 14CO2 in KOH and ethylene glycol by LSC analysis. Each sample was analyzed by HPLC-UV detection with on-line radiochemical detection. High performance liquid chromatograph (Exion HPLC) equipped with a mass spectrometer (TQ 5500) was used with a column of Column: Shimpack C18(2), 250 mm × 4.6 mm i.d., 5 µm, column oven temperature of 30°C, mobile phase consists of Solvent A : 5 mM ammonium formate in Milli-Q® water and Solvent B : Acetonitrile in a ratio of 30 : 70, v/v, flow rate of 0.5 mL/min with splitter, respectively. Detection method involve the use of MS. Using the method of Currie L. A. (1968), the LOD and LOQ of the LSC analyses were 28 and 111 dpm, respectively. During method validation, acceptable recoveries were generated for the samples fortified at LOQ and 10 LOQ level. The % RSD (precision) was ≤20% at each fortification level. Recovery data from these samples demonstrated that test chemical was unstable during analysis. The identification and quantification of the degradation product was carried out using mass spectrometry. Analysis of the Day 0 samples at 10 μg/L and 100 μg/L test concentrations demonstrated quantitative recovery of test chemical. The average amount of test chemical present was 98.8% and 0% & 101.7% and 46.7% at Day 0 and Day 60, respectively following application of test chemical to test water at 10 μg/L (low dose) and 100μg/L (high dose). The average amount of test chemical present was 100.9% and 60.6% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 100μg/L (high dose). The DT50 value was determined to be 7.2 d and 45.3 d at test chemical conc. of 10 μg/l and 100 μg/l at 12°C, respectively. 90% degradation of test chemical in natural surface water was determined after 23.9 d and 150 d at test chemical conc. of 10 μg/l and 100 μg/l, respectively. Test chemical was unstable in natural water and test chemical was completely converted into degradation product (1-hydroxy-4-(phenylamino)anthracene-9,10-dione) by end of incubation period of 60 days. Based on the these results, test chemical was considered to be not persistent in water.

Key value for chemical safety assessment

Half-life in freshwater:

7.2 d

at the temperature of:

12 °C

Additional information

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

Aerobic mineralisation of test chemical in water was studies as per the principles of the OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) (Adopted 13th April 2004) under aerobic conditions. The surface water was collected from Kaveri River, Sangama, Ramnagar District, Karnataka State, India in a thoroughly cleansed container. The sampling site for collection of the surface water was selected ensuring that no known history of its contamination with the test item or its structural analogues within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The pH and temperature of the water was measured at the site of collection and the depth of sampling and the appearance of the water sample. (e.g. color and turbidity) was also noted. Oxygen concentration of the surface layer was measured in order to demonstrate aerobic conditions. Depth of sampling was 1 feet and surface water was clear with no turbidity. The test water was stored at 4°C with continuous aeration prior use for a period not more than 4 weeks. Temperature (°C) at time of collection was 21.1°C, pH of temperature was 6.73, Oxygen concentration (mg/l) of 5.1 mg/l,  Dissolved organic carbon (%) of 2.4 mg/kg dm, colony count consists of 4000 CFU/ml, Total organic carbon (TOC) of 2.6 mg/l, Nitrate (NO3- ) of 3 mg/l, Nitrite (NO2- ) of <0.005 mg/l, P of 0.3 mg/l, Orthophosphates (PO43-) of 0.22 mg/l, Total ammonia tot (NH4+ ) of <0.3 mg/l and BOD of <2.0 mg/l, respectively. Prior to use of surface water, the coarse particles were removed by filtration through a 100 μm mesh sieve. Test chemical conc. used in the study was 10 μg/L as low dose and 100 μg/L as high dose, respectively. The surface water was also treated at 500 µg/L (0.5 µg/mL) which was used for identification of degradation products. Study was performed in duplicates in a 250 ml conical flasks which was covered with cotton plugs under continuous darkness. Test conditions involve a temperature of 12±2°C, pH of  6.73. Test vessel was kept in an incubator shaker at 12 ± 2°C in dark. Aerobic condition was maintained in the test system by continuous shaking. Agitation was provided to facilitate oxygen transfer from the headspace to the liquid so that aerobic conditions were adequately maintained. Additional to test vessels, 1 blank test vessel containing only the test water for all sampling intervals was included, 1 blank test vessel containing only the sterile test water was also treated at 10 µg/L (0.01 µg/mL) and 100 µg/L (0.1 µg/mL) conc., 1 blank test vessel containing only test chemical with co-solvent and duplicate test vessels with reference (aniline) (conc. 10 μg/l i.e. 0.01 mg/l) was also kept in the study. All experiments were performed in duplicates. The concentration of test chemical residues in samples collected at different pre-determined interval zero-time (immediately after treatment day 0), day 1, day 3 day 7, day 14, day 28, day 45 and day 60 were diluted suitably with acetonitrile and at each sampling occasion, triplicate aliquots from each test concentration were subjected to total radioactivity analysis by LSC and the components were quantified by reverse phase radio-HPLC with on-line radiochemical detection. Additionally, an aliquot of each sample was subjected for 14CO2 determination by indirect method followed by LSC analysis and trapped 14CO2 in KOH and ethylene glycol by LSC analysis. Each sample was analyzed by HPLC-UV detection with on-line radiochemical detection. High performance liquid chromatograph (Exion HPLC) equipped with a mass spectrometer (TQ 5500) was used with a column of Column: Shimpack C18(2), 250 mm × 4.6 mm i.d., 5 µm, column oven temperature of 30°C, mobile phase consists of Solvent A : 5 mM ammonium formate in Milli-Q® water and Solvent B : Acetonitrile in a ratio of 30 : 70, v/v, flow rate of 0.5 mL/min with splitter, respectively. Detection method involve the use of MS. Using the method of Currie L. A. (1968), the LOD and LOQ of the LSC analyses were 28 and 111 dpm, respectively. During method validation, acceptable recoveries were generated for the samples fortified at LOQ and 10 LOQ level. The % RSD (precision) was ≤20% at each fortification level. Recovery data from these samples demonstrated that test chemical was unstable during analysis. The identification and quantification of the degradation product was carried out using mass spectrometry. Analysis of the Day 0 samples at 10 μg/L and 100 μg/L test concentrations demonstrated quantitative recovery of test chemical. The average amount of test chemical present was 98.8% and 0% & 101.7% and 46.7% at Day 0 and Day 60, respectively following application of test chemical to test water at 10 μg/L (low dose) and 100μg/L (high dose). The average amount of test chemical present was 100.9% and 60.6% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 100μg/L (high dose). The DT50 value was determined to be 7.2 d and 45.3 d at test chemical conc. of 10 μg/l and 100 μg/l at 12°C, respectively. 90% degradation of test chemical in natural surface water was determined after 23.9 d and 150 d at test chemical conc. of 10 μg/l and 100 μg/l, respectively. Test chemical was unstable in natural water and test chemical was completely converted into degradation product (1-hydroxy-4-(phenylamino)anthracene-9,10-dione) by end of incubation period of 60 days. Based on the these results, test chemical was considered to be not persistent in water.

Biodegradation in water: sediment simulation testing

In accordance with Annex IX column 2 of REACH regulation, test for this endpoint is scientifically not necessary and does not need to be conducted, since the substance is readily biodegradable i.e. not persistent based on the experimental result of surface water simulation biodegradation study.