Tetrabutylammonium bromide

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

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

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

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 1-2 feet and surface water was clear with no turbidity.
- Storage conditions: The test water was stored at 4 to 6°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: 21.8°C
- pH at time of collection: 6.83
- Oxygen concentration (mg/l) initial/final: 4.8 mg/l
- Dissolved organic carbon (%) leachable: 3.9 mg/l
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): 4500 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): 3.8 mg/l
Nitrate (NO3- ): 4.5 mg/l
Nitrite (NO2- ): 0.62 mg/l
P: <0.1 mg/l
Orthophosphates (PO43-): 0.19 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
- Test temperature: 12±2°C
- pH: 6.83
- Suspended solids concentration: 15 mg/l
- Continuous darkness: yes, study was performed under continuous darkness. yes, study was performed under continuous darkness.
- Any indication of the test material adsorbing to the walls of the test apparatus: no

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.
- Measuring equipment:
- 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: no
- Other: 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 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.

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 10 µg/L (0.01 µg/mL) and 100 µg/L (0.1 µg/mL) conc..
- Other: Duplicate test vessels with reference (aniline) 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:

2.8

Remarks on result:

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

Compartment:

natural water

% Recovery:

3

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:

33.7 d

Remarks on result:

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

Key result

% Degr.:

90

Parameter:

test mat. analysis

Sampling time:

34.5 d

Remarks on result:

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

Key result

Compartment:

natural water

DT50:

10.2 d

Temp.:

12 °C

Remarks on result:

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

Key result

Compartment:

natural water

DT50:

10.4 d

Temp.:

12 °C

Remarks on result:

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

Transformation products:

not measured

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 107.5% and 2.8% 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 96.9% and 3.0% 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 105.1% and 61.6% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 10 μg/L (low dose).
The average amount of test chemical present was 108.0% and 59.1% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 100 μg/L (high dose).

Based on the above results, test chemical was degraded in surface water and sterile surface water. However, the degradation was obsered to be slow in sterile surface water than compared to non-sterile surface water.

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

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.

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 in Appendix 3. 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:

Concentration	DT50(Days)	DT90(Days)	Rate constant (d^-1)
10 µg/L	10.2	33.7	0.06829 ± 0.002918
100 µg/L	10.4	34.5	0.0667 ± 0.004021
10 µg/L (sterile)	87.7	291	0.007902 ± 8.66E-004
100 µg/L (sterile)	78.2	260	0.008866 ± 6.81E-004

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

TABLE: SUMMARY OF KINETIC DATA FOR 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/Lfor parent only		SFO		M0 (%AR) = 103.6 ± 1.509 k (d^-1) =0.06829 ± 0.002918		3.79		0.9943		10.2		33.7
100 µg/Lfor parent only		SFO		M0 (%AR) = 96.17 ± 1.975 k (d^-1) =0.0667 ± 0.004021		3.57		0.9885		10.4		34.5
10 µg/L (sterile)for parent only		SFO		M0 (%AR) = 95.87 ± 1.864 k (d^-1) = 0.007902± 8.66E-004		4.51		0.8743		87.7		291
100 µg/L (sterile)for parent only		SFO		M0 (%AR) = 100.2 ± 1.483 k (d^-1) = 0.008866± 6.81E-004		3.19		0.9365		78.2		260

TABLE:   METHOD VALIDATION - LINEARITY OF DETECTOR RESPONSE AND RANGE

X (Conc.) µg/mL	Y (Peak area) Quantification mass ion transition m/z (244.1->143.2)	Y (Peak area) Confirmation mass ion transition m/z (244.1->101.1)
0.00026	53136	11056
0.00026	56968	11682
0.00026	55802	12094
0.00052	104499	22203
0.00052	105075	21056
0.00052	106000	21397
0.00103	213486	43374
0.00103	210079	43433
0.00103	213934	43221
0.00516	1053127	217833
0.00516	1044555	219484
0.00516	1040951	216123
0.01032	1040615	216647
0.01032	2036364	435455
0.01032	2016776	421064
0.02064	3715682	795802
0.02064	3772490	800808
0.02064	3730412	799004
Slope	194000000	40500000
Intercept	5160	992
r	0.9986	0.9989

TABLE:  METHOD VALIDATION – ACCURACY AND PRECISION

Quantification mass ion transition m/z(244.1->101.1)

 

Concentration (µg/mL)	Test item recovered (µg)	Test item added (µg)	Accuracy as % Recovery	Mean ± s.d	Precision as % RSD
0.001 (LOQ)	0.00109	0.00103	105.8	100.0 ± 8.6	8.6
	0.00108	0.00103	104.9
	0.00109	0.00103	105.8
	0.00036	0.00103	87.4
	0.00105	0.00103	101.9
0.01 (10 LOQ)	0.01060	0.01032	102.7	101.8 ± 1.0	1.1
	0.01059	0.01032	102.6
	0.01051	0.01032	101.8
	0.01031	0.01032	99.9
	0.01052	0.01032	101.9

Confirmation mass ion transition m/z(244.1->143.2)

 

Concentration (µg/mL)	Test item recovered (µg)	Test item added (µg)	Accuracy as % Recovery	Mean ± s.d	Precision as % RSD
0.001 (LOQ)	0.00109	0.00103	105.8	101.5 ± 6.2	6.1
	0.00108	0.00103	104.9
	0.00109	0.00103	104.9
	0.00036	0.00103	92.2
	0.00105	0.00103	103.9
0.01 (10 LOQ)	0.01060	0.01032	102.5	101.9 ± 1.2	1.2
	0.01059	0.01032	103.2
	0.01051	0.01032	102.1
	0.01031	0.01032	99.9
	0.01052	0.01032	101.8

TABLE:  DETERMINATION OF TEST CHEMICAL IN TEST WATER SAMPLES TREATED AT 10 µg/L

Test concentration	Sampling intervals	Rep	Recovery of Tetra butyl ammonium bromide (%)	Mean recovery of Tetra butyl ammonium bromide (%)
10 µg/L	Day-0	1	107.4	107.5
		2	107.6
	Day-1	1	93.4	92
		2	90.6
	Day-3	1	81.7	86.1
		2	90.4
	Day 7	1	63.3	62.6
		2	61.8
	Day 14	1	39.2	39.5
		2	39.8
	Day 28	1	17.3	17.2
		2	17.1
	Day 45	1	5.0	5.0
		2	5.0
	Day 60	1	2.6	2.8
		2	2.9

TABLE: DETERMINATION OF TEST CHEMICAL IN TEST WATER SAMPLES TREATED AT 100 µg/L

Test concentration	Sampling intervals	Rep	Recovery of Tetra butyl ammonium bromide (%)	Mean recovery of Tetra butyl ammonium bromide (%)
10 µg/L	Day-0	1	91	96.9
		2	102.8
	Day-1	1	92	92.3
		2	92.6
	Day-3	1	73.1	77.2
		2	81.3
	Day 7	1	61.4	55.8
		2	50.2
	Day 14	1	40.2	40.0
		2	39.8
	Day 28	1	16.1	16.4
		2	16.7
	Day 45	1	5.2	5.3
		2	5.4
	Day 60	1	3.0	3.0
		2	3.0

TABLE:  DETERMINATION OF TEST CHEMICAL IN TEST WATER SAMPLES (STERILE) AT 10 µg/L

Test concentration	Sampling intervals	Rep	Recovery of Tetra butyl ammonium bromide (%)	Mean recovery of Tetra butyl ammonium bromide (%)
10 µg/L	Day-0	1	105.8	105.1
		2	104.4
	Day-1	1	98	97.1
		2	96.2
	Day-3	1	92.4	90.4
		2	88.3
	Day 7	1	82.9	83.7
		2	82.7
	Day 14	1	82.9	82.8
		2	82.7
	Day 28	1	78.3	78.2
		2	78.1
	Day 45	1	68.3	67.1
		2	65.9
	Day 60	1	59.8	61.6
		2	63.3

TABLE: DETERMINATION OF TEST CHEMICAL IN TEST WATER SAMPLES (STERILE) AT 100 µg/L

Test concentration	Sampling intervals	Rep	Recovery of Tetra butyl ammonium bromide (%)	Mean recovery of Tetra butyl ammonium bromide (%)
100 µg/L	Day-0	1	109	108.0
		2	107
	Day-1	1	96.4	96.3
		2	96.2
	Day-3	1	94.4	94.4
		2	94.4
	Day 7	1	89.2	91.1
		2	93
	Day 14	1	89.8	88.3
		2	86.7
	Day 28	1	76.3	80.1
		2	83.9
	Day 45	1	66.1	66.7
		2	67.3
	Day 60	1	57.8	59.1
		2	60.4

TABLE: MEASUREMENTS OF pH AND OXYGEN CONCENTRATIONS OF SURFACE TEST WATER TREATED WITH TEST CHEMICAL.

Sampling interval	Sample	Oxygen conc. (mg/L)	pH
Day 0	TW, LD, R1	4.2	6.7
	TW, LD, R2	3.9	6.9
	TW, HD, R1	4.0	6.7
	TW, HD, R2	4.1	6.8
Day 1	TW, LD, R1	3.7	6.8
	TW, LD, R2	4.1	7.0
	TW, HD, R1	4.5	6.4
	TW, HD, R2	3.8	6.8
Day 3	TW, LD, R1	3.8	7.3
	TW, LD, R2	3.6	7.0
	TW, HD, R1	3.4	6.8
	TW, HD, R2	3.9	7.1
Day 7	TW, LD, R1	3.4	7.2
	TW, LD, R2	3.2	7.3
	TW, HD, R1	3.6	7.3
	TW, HD, R2	3.8	7.4
Day 14	TW, LD, R1	2.7	7.3
	TW, LD, R2	3.1	7.7
	TW, HD, R1	3.0	7.2
	TW, HD, R2	2.7	7.5
Day 28	TW, LD, R1	2.1	7.5
	TW, LD, R2	2.6	7.5
	TW, HD, R1	2.4	8.1
	TW, HD, R2	2.3	7.9
Day 45	TW, LD, R1	1.9	8.0
	TW, LD, R2	1.6	7.8
	TW, HD, R1	1.4	7.9
	TW, HD, R2	1.7	7.9
Day 60	TW, LD, R1	1.7	8.1
	TW, LD, R2	1.6	8.2
	TW, HD, R1	1.4	8.6
	TW, HD, R2	1.9	8.3

TABLE:  MEASUREMENTS OF pH AND OXYGEN CONCENTRATIONS OF STERILE TEST WATER TREATED WITH TEST CHEMICAL

Sampling interval	Sample	Oxygen conc. (mg/L)	pH
Day 0	SW, LD, R1	3.8	6.91
	SW, LD, R2	4.1	6.75
	SW, HD, R1	4	6.94
	SW, HD, R2	3.7	6.73
Day 1	SW, LD, R1	3.9	6.96
	SW, LD, R2	3.7	7.04
	SW, HD, R1	4	6.92
	SW, HD, R2	4.1	7.16
Day 3	SW, LD, R1	3.6	7.06
	SW, LD, R2	3.7	7.24
	SW, HD, R1	3.8	6.92
	SW, HD, R2	3.5	7.06
Day 7	SW, LD, R1	3.1	7.41
	SW, LD, R2	3.3	7.64
	SW, HD, R1	3.1	7.08
	SW, HD, R2	2.8	7.24
Day 14	SW, LD, R1	2.6	7.34
	SW, LD, R2	2.8	7.59
	SW, HD, R1	2.4	7.31
	SW, HD, R2	2.1	7.48
Day 28	SW, LD, R1	2.2	7.65
	SW, LD, R2	2.5	8.04
	SW, HD, R1	2.1	7.41
	SW, HD, R2	2	7.36
Day 45	SW, LD, R1	1.8	7.9
	SW, LD, R2	1.6	7.65
	SW, HD, R1	1.9	8.03
	SW, HD, R2	2.2	8.17
Day 60	SW, LD, R1	1.2	8.37
	SW, LD, R2	1.6	7.95
	SW, HD, R1	1.8	8.01
	SW, HD, R2	1.5	8.32

TABLE:   DATA USED FOR KINETIC CALCUALTIONS.

Sampling Intervals	Tetra butyl ammonium bromide (%)
	Low Dose (10 µg/L) in Test Water	High Dose (100 µg/L) in Test Water	High Dose (10 µg/L) in Sterile Water	High Dose (100 µg/L) in Sterile Water
Day 0	107.4	91.0	105.8	109.0
	107.6	102.8	104.4	107.0
Day 1	93.4	92.0	98.0	96.4
	90.6	92.6	96.2	96.2
Day 3	81.7	73.1	92.4	94.4
	90.4	81.3	88.3	94.4
Day 7	63.3	61.4	82.9	89.2
	61.8	50.2	82.7	93.0
Day 14	39.2	40.2	82.9	89.8
	39.8	39.8	82.7	86.7
Day 28	17.3	16.1	78.3	76.3
	17.1	16.7	78.1	83.9
Day 45	5.0	5.2	68.3	66.1
	5.0	5.4	65.9	67.3
Day 60	2.6	3.0	59.8	57.8
	2.9	3.0	63.3	60.4

 

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 107.5% and 2.8% & 96.9% and 3.0% 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 105.1% and 61.6% & 108.0% and 59.1% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 10 μg/L (low dose) and 100μg/L (high dose). The DT50 value was determined to be 10.2 d and 10.4 d at test chemical conc. of 10 μg/l and 100 μg/l at 12°C, respectively. 90% of test chemical in natural surface water was determined after 33.7 d and 34.5 d at test chemical conc. of 10 μg/l and 100 μg/l, respectively. Based on the these results, test chemical was degraded in surface water and sterile surface water. Hence, 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 1-2 feet and surface water was clear with no turbidity. The test water was stored at 4 to 6°C with continuous aeration prior use for a period not more than 4 weeks. Temperature (°C) at time of collection was 21.8°C, pH of temperature was 6.83, Oxygen concentration (mg/l) of 4.8 mg/l,  Dissolved organic carbon (%) of 3.9 mg/l, colony count consists of 4500 CFU/ml, Total organic carbon (TOC) of 3.8 mg/l, Nitrate (NO3- ) of 4.5 mg/l, Nitrite (NO2- ) of 0.62 mg/l, P of <0.1 mg/l, Orthophosphates (PO43-) of 0.19 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. 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  6.83. 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. and duplicate test vessels with reference (aniline) (conc. 10 μg/l i.e. 0.01 mg/l) was also kept in the study. 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, duplicate aliquots from each test concentration were subjected to analysis by a validated LC-MS/MS method. Simutaneously, 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 for using in other sampling intervals. The surface water samples were analyzed for the residues of test item by liquid chromatography with positive-ion electrospray ionization (ESI) tandem mass spectrometry using the mass ion transition m/z 244.1 -> 143.2 for primary quantification and the mass ion transition m/z 244.1 -> 101.1 for qualitative confirmation. High performance liquid chromatograph (Exion HPLC) equipped with a mass spectrometer (TQ 5500) was used with a column of  Phenomenex Luna, C18 (2), 4.6mm×150mm i.d., 3.0µm, column oven temperature of 40°C, mobile phase consists of Solvent A : 5 mM ammonium formate in Milli-Q® water and Solvent B : Acetonitrile in a ratio of 15 : 85, v/v, flow rate of 0.6 mL/min with splitter, respectively. Detection method involve the use of MS. Linearity range was evaluated to be in the range of 0.00026-0.02064 µg/ml, 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 stable during analysis. The recoveries of all the samples analyzed were in the range of 70-110% with %RSD ≤ 20%. 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 107.5% and 2.8% & 96.9% and 3.0% 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 105.1% and 61.6% & 108.0% and 59.1% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 10 μg/L (low dose) and 100μg/L (high dose). The DT50 value was determined to be 10.2 d and 10.4 d at test chemical conc. of 10 μg/l and 100 μg/l at 12°C, respectively. 90% of test chemical in natural surface water was determined after 33.7 d and 34.5 d at test chemical conc. of 10 μg/l and 100 μg/l, respectively. Based on the these results, test chemical was degraded in surface water and sterile surface water. Hence, test chemical was considered to be not persistent in water.

Reference 2

Endpoint:

biodegradation in water: sediment simulation testing

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is readily biodegradable

Description of key 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 1-2 feet and surface water was clear with no turbidity. The test water was stored at 4 to 6°C with continuous aeration prior use for a period not more than 4 weeks. Temperature (°C) at time of collection was 21.8°C, pH of temperature was 6.83, Oxygen concentration (mg/l) of 4.8 mg/l,  Dissolved organic carbon (%) of 3.9 mg/l, colony count consists of 4500 CFU/ml, Total organic carbon (TOC) of 3.8 mg/l, Nitrate (NO3- ) of 4.5 mg/l, Nitrite (NO2- ) of 0.62 mg/l, P of <0.1 mg/l, Orthophosphates (PO43-) of 0.19 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. 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  6.83. 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. and duplicate test vessels with reference (aniline) (conc. 10 μg/l i.e. 0.01 mg/l) was also kept in the study. 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, duplicate aliquots from each test concentration were subjected to analysis by a validated LC-MS/MS method. Simutaneously, 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 for using in other sampling intervals. The surface water samples were analyzed for the residues of test item by liquid chromatography with positive-ion electrospray ionization (ESI) tandem mass spectrometry using the mass ion transition m/z 244.1 -> 143.2 for primary quantification and the mass ion transition m/z 244.1 -> 101.1 for qualitative confirmation. High performance liquid chromatograph (Exion HPLC) equipped with a mass spectrometer (TQ 5500) was used with a column of  Phenomenex Luna, C18 (2), 4.6mm×150mm i.d., 3.0µm, column oven temperature of 40°C, mobile phase consists of Solvent A : 5 mM ammonium formate in Milli-Q® water and Solvent B : Acetonitrile in a ratio of 15 : 85, v/v, flow rate of 0.6 mL/min with splitter, respectively. Detection method involve the use of MS. Linearity range was evaluated to be in the range of 0.00026-0.02064 µg/ml, 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 stable during analysis. The recoveries of all the samples analyzed were in the range of 70-110% with %RSD ≤ 20%. 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 107.5% and 2.8% & 96.9% and 3.0% 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 105.1% and 61.6% & 108.0% and 59.1% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 10 μg/L (low dose) and 100μg/L (high dose). The DT50 value was determined to be 10.2 d and 10.4 d at test chemical conc. of 10 μg/l and 100 μg/l at 12°C, respectively. 90% of test chemical in natural surface water was determined after 33.7 d and 34.5 d at test chemical conc. of 10 μg/l and 100 μg/l, respectively. Based on the these results, test chemical was degraded in surface water and sterile surface water. Hence, 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.

Key value for chemical safety assessment

Half-life in freshwater:

10.2 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 1-2 feet and surface water was clear with no turbidity. The test water was stored at 4 to 6°C with continuous aeration prior use for a period not more than 4 weeks. Temperature (°C) at time of collection was 21.8°C, pH of temperature was 6.83, Oxygen concentration (mg/l) of 4.8 mg/l,  Dissolved organic carbon (%) of 3.9 mg/l, colony count consists of 4500 CFU/ml, Total organic carbon (TOC) of 3.8 mg/l, Nitrate (NO3- ) of 4.5 mg/l, Nitrite (NO2- ) of 0.62 mg/l, P of <0.1 mg/l, Orthophosphates (PO43-) of 0.19 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. 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  6.83. 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. and duplicate test vessels with reference (aniline) (conc. 10 μg/l i.e. 0.01 mg/l) was also kept in the study. 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, duplicate aliquots from each test concentration were subjected to analysis by a validated LC-MS/MS method. Simutaneously, 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 for using in other sampling intervals. The surface water samples were analyzed for the residues of test item by liquid chromatography with positive-ion electrospray ionization (ESI) tandem mass spectrometry using the mass ion transition m/z 244.1 -> 143.2 for primary quantification and the mass ion transition m/z 244.1 -> 101.1 for qualitative confirmation. High performance liquid chromatograph (Exion HPLC) equipped with a mass spectrometer (TQ 5500) was used with a column of  Phenomenex Luna, C18 (2), 4.6mm×150mm i.d., 3.0µm, column oven temperature of 40°C, mobile phase consists of Solvent A : 5 mM ammonium formate in Milli-Q® water and Solvent B : Acetonitrile in a ratio of 15 : 85, v/v, flow rate of 0.6 mL/min with splitter, respectively. Detection method involve the use of MS. Linearity range was evaluated to be in the range of 0.00026-0.02064 µg/ml, 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 stable during analysis. The recoveries of all the samples analyzed were in the range of 70-110% with %RSD ≤ 20%. 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 107.5% and 2.8% & 96.9% and 3.0% 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 105.1% and 61.6% & 108.0% and 59.1% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 10 μg/L (low dose) and 100μg/L (high dose). The DT50 value was determined to be 10.2 d and 10.4 d at test chemical conc. of 10 μg/l and 100 μg/l at 12°C, respectively. 90% of test chemical in natural surface water was determined after 33.7 d and 34.5 d at test chemical conc. of 10 μg/l and 100 μg/l, respectively. Based on the these results, test chemical was degraded in surface water and sterile surface water. Hence, 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.