Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: sediment simulation testing

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Transformation products:

no

Reference 2

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 June 2019 to 23 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

April 2004

Deviations:

no

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: The test water and sediment was collected from Brandywine Creek, Chadds Ford, PA (GPS: 39.865 N, 75.592 W) on 7 October 2019. Water was collected from a depth of ca. 20 cm above sediment.
- Temperature (°C) at time of collection: 17.5 °C (water); ca. 23 °C (air)
- pH at time of collection: 7.4
- Dissolved oxygen at time of collection: 8.4 mg/L
- Storage conditions: The water was transported in a sealed container with enough headspace to provide access to air and was stored refrigerated.
- Preparation of surface water: The water was filtered through a 0.2 mm sieve and the sediment was filtered through a 2 mm sieve to remove coarse particulate matter and debris.

Water Characterisation:
- Total suspended solids: 6 ppm
- Dissolved Organic Carbon: 10.5 ppm
- Ammoniacal Nitrogen: 1.8 ppm
- Total Nitrogen: 2.1 ppm
- Nitrate-Nitrogen: 0.3 ppm
- Nitrite-Nitrogen: < LOD (0.1 ppm)
- Total Phosphorus: 0.2 ppm

Total Organic Carbon:
- Start of study: 13.94 ppm
- End of study: 12.98 ppm

Details on source and properties of sediment:

- Details on collection: The sediment was collected from Brandywine Creek, Chadds Ford, PA (GPS: 39.865 N, 75.592 W) on 7 October 2019. A depth of approximately 20 cm sediment was collected.
- Redox potential: 253 mV
- Sediment samples sieved: no

Sediment characterisation:
- Sand: 24%
- Silt: 50%
- Clay: 26%
- USDA Textural Class (hydrometer method): Loam

- Bulk density (disturned): 0.67 g/cc
- Cation Exchange Capacity: 8.6 meq/100 g

- Moisture at 1/3 Bar: 59.3%
- Organic matter (Walkley Black): 8.7%
- Organic carbon: 5.1%
- pH in 1:1 soil:water ratio: 5.5

Base Saturation Data
Cations: calcium (30.6%), magnesium (11.4%), sodium (2.8%), potassium (1.3%), hydrogen (53.9%)

Duration of test (contact time):

60 d

Initial conc.:

9.7 µg/L

Based on:

test mat.

Initial conc.:

98.8 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

TEST CONDITIONS
- All samples were placed on orbital shakers for continuous gentle shaking (50 rpm) in a constant temperature room maintained at 12 ± 2°C during the incubation period. The temperature of the constant temperature room was continuously monitored with a REES temperature monitoring system.

TEST SYSTEM
Approximately six aliquots of Brandywine creek sediment (1 g each) were transferred into six aliquots of filtered Brandywine creek water (one litre each), creating water amended with sediment. The six bottles of water amended with sediment were continuously mixed using a stir plate and stir bar. Samples were prepared by measuring 100 mL of water amended with sediment via a graduated cylinder and dispensing into 250 mL amber bottles. The remaining water amended with sediment was placed in the autoclave for 30 minutes at 250°F (15 psi) to be sterilised for the sterile sample set. The sterilised water amended with sediment was dispensed using sterilised graduated cylinders into 250 mL sterilised amber glass bottles (which had also been autoclaved at 250°F and 15 psi for 30 minutes).

NUMBER OF REPLICATES
- 16 treated samples to be dosed at the high dose rate (98.8 µg/L), sufficient for 6 sampling intervals in duplicate and four extra samples
- 16 treated samples to be dosed at the low dose rate (9.7 µg/L), sufficient for 6 sampling intervals in duplicate and four extra samples
- 6 treated samples to be dosed with [14C]benzoic acid as a reference control substance (100.9 µg/L), sufficient for 2 sampling intervals in duplicate and two extra samples
- 8 sterile treated samples dosed at the high dose rate (98.8 µg/L), sufficient for 3 sampling intervals in duplicate and two extra samples
- 4 untreated incubated samples for TOC measurement

DETAILS ON EXPERIMENTAL SET UP
The high dose and low dose viable samples were connected to trapping vessels for continuous trapping throughout the study. A peristaltic pump was used to draw ambient air first through a vial containing deionised water to moisturise the air, followed by the sample container and then to a series of traps containing a foam plug trap to collect organic volatiles and two caustic traps (10% aqueous sodium hydroxide) traps to collect 14C-carbon dioxide. The samples were connected via Teflon tubing threaded through the septum caps and connected to manifolds. Trap solutions were housed in glass vials (40 mL capacity) fitted with open top caps with Teflon-lined silicon septa through which the Teflon tubing was threaded in the same fashion as the samples.
The setup of the reference control samples (dosed with [14C]benzoic acid) was similar to that of the high dose/low dose viable test material samples, except foam plugs were not used in the trapping system.
For sterile sample set, all glassware was autoclaved at 250°F and 15 psi for 30 minutes prior to use. The sterile samples were prepared with a sterilised foam plug inserted into the opening of the test vessel, and no traps were required or connected for volatile/CO2 collection since no mineralisation was expected in these samples.
The untreated incubated samples were capped with Teflon lined silicon caps and connected to a humidifier (D.I. water) as well as a peristaltic pump for air flow.

SAMPLING
- Sampling frequency: High and low dose samples were sacrificed after 0, 7, 14, 20, 40, and 60 days of incubation. Sterile samples were sacrificed after 7, 40, and 60 days of incubation. Benzoic acid reference samples were sacrificed after 7 and 14 days of incubation. Duplicate samples for each set were removed as applicable from the constant temperature room at each time point.
- Sample preparation and processing: At each interval, samples which were taken for processing were allowed to sit at ambient temperature for approximately one hour. Sample DO and pH of each water sample was measured. The water samples, contained in amber glass bottles, were decanted into 250 mL fluorinated centrifuge bottles and centrifuged for 10 minutes at 3500 rpm. High dose, low dose, and high dose sterile water supernatants were then decanted into 250 mL graduated cylinders which contained 10 mL of THF. Reference water supernatants were decanted into 100 mL graduated cylinders. The volumes of the centrifuged water were measured and recorded, transferred to new 250 mL fluorinated centrifuge bottles, and triplicate aliquots (1 mL) were taken for LSC radioassay. The original amber bottle and the remaining pellet (sediment film) in the 250 mL centrifuge bottles were then rinsed with 20 mL of THF by hand shaking the vessels and sonicating. The rinses were kept separate, volumes were measured, and triplicate aliquots (1 mL) were taken for radioassay by LSC. When direct analysis of the water and rinses was not feasible, an aliquot of the sample was concentrated by Savant speed-vac or under nitrogen to a desired volume (0.5 – 1 mL) and the concentration vessel was rinsed with an aliquot of THF.
Foam plugs were soaked in 20 mL of ACN for at least one hour. Volumes were measured for all traps where applicable, and triplicate aliquots (0.5 – 1.0 mL) were taken, quenched with methanol (for sodium hydroxide traps), and radioassayed by LSC.
High dose, low dose, and high dose sterile water samples, and THF amber bottle and sediment film rinses which exceeded 5% AR were analysed by HPLC utilising co-chromatography with reference standard solution. Reference samples were not analysed by HPLC.
- Sampling for Evolved 14CO2: Due to the large amount of 14CO2 evolved in the reference control samples, the sodium hydroxide #1 traps were exchanged with fresh solutions after 10 days of application.

CALCULATIONS
- Material Balance
Total DPM Recovered in Solution = [ (Raw dpm - background dpm) / Aliquot volume ] x Total volume
Percent Recovery = (Total DPM Recovered / Applied DPM) x 100

- Quantitation of Components by HPLC
Percent of Test Substance and Degradates Based on Applied Radiocarbon:
% As = IAs x (E.P.)s/100
where:
As = compound A in sample S
IAs = % of compound A from HPLC integration of sample S
(E.P.)s = percent of applied radiocarbon recovered in sample S

- Determination of Degradation Kinetics
Single First-Order Model (SFO)
M = M0 e-kt
where:
M = chemical concentration (compound expressed as percent of applied radiocarbon)
t = time
M0 = initial concentration
k = degradation rate constant

STATISTICAL METHODS
Statistical analyses included calculations of means, standard deviations and relative standard deviations for the interpretation and summarization of results. Many tables were constructed with Microsoft® Excel software and calculations may include some variation in rounding.

Reference substance:

other: [7-14C]benzoic acid

Compartment:

natural water / sediment

DT50:

362 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: HIgh Dose

Compartment:

natural water / sediment

DT50:

72.5 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: Low Dose

Transformation products:

no

Details on results:

- Radiochemical Purity of the Test Material
The radiochemical purity of the test material in the high and low dose solutions was determined to be 98.2 and 98.8%, respectively, following the application processes, confirming the stability of the test material during application.

- Application Rate
The target dose rate was 100 μg/L (high dose and high dose sterile) and 10 μg/L (low dose). This corresponds to 10 and 1.0 μg/100 mL sample, respectively. The achieved dose rates were 0.0988 ppm or 9.88 μg/sample (98.8 μg/L) for high dose and high dose sterile samples, and 0.0097 ppm or 0.97 μg/sample (9.7 μg/L) for low dose samples. The homogeneity of the dose solutions during the dosing procedure was confirmed by radioassay of aliquots of the dose solution taken at least before and after application. The relative standard deviations for these aliquots were ≤ 0.82%.

- Properties of Test System
Dissolved oxygen (DO) content measurements ranged from 6.24 – 6.80 ppm, demonstrating that the test systems were maintained under aerobic conditions throughout the study. Sample pH measurements ranged from 7.09 to 7.40. All samples were incubated at 12 ± 2°C in a temperature-controlled room throughout the study.

- HPLC Column Recovery
HPLC column recoveries were determined by directly counting an aliquot of the injected sample and comparing to the radioactivity eluted from the column. HPLC recoveries averaged 101.1 ± 6.4% throughout the study.

- Mass Balance
High Dose
Acceptable mass balance (90 – 110% AR) was achieved in all high dose samples. Individual sample mass ranged 90.7 to 95.0% AR. Average recoveries in high dose water samples decreased from 60.6% AR at time 0 to 6.5% AR at day 60. Average THF bottle rinse recoveries increased from 4.8% AR at time 0 to a maximum of 27.9% AR at day 40, and then declined to 7.5% AR at day 60. Average THF sediment film rinse recoveries increased from 28.3% AR at time 0 to a maximum of 71.1% AR at day 60. A second THF sediment film rinse was required for the 20 and 60 day samples to achieve acceptable mass balance and averaged 1.9 and 8.0% AR, respectively. Recoveries in the foam plugs was below limits of detection (< 0.1% AR). Recoveries in the sodium hydroxide traps were insignificant and averaged 0.1% AR for the 40 and 60 day samples.

Low Dose
Acceptable mass balance was achieved in all low dose samples except for the 20 and 60 day samples. Individual sample mass balance ranged from 83.1 to 96.1% AR. Average recoveries in low dose water samples decreased from 60.6% at time 0 to 7.3% AR at day 60. Average THF bottle rinse recoveries increased from 2.6% AR at time 0 to a maximum of 24.1% AR at day 40, and then declined to 10.0% AR at day 60. Average THF sediment film rinse recoveries increased from 31.6% AR at time 0 to a maximum of 58.7% AR at day 60. A second THF sediment film rinse was required for all samples starting at day 7 to achieve acceptable mass balance. Recoveries in the THF sediment film rinse #2 averaged 1.7% to 7.6% AR. Recoveries in the foam plugs and sodium hydroxide traps were below limits of detection (< 0.1% AR). No significant difference was observed in the recovery in NaOH traps between high dose and low dose viable samples, demonstrating that the mineralisation of the test material was not concentration dependent.

High Dose Sterile
Acceptable mass balance was achieved in all high dose sterile samples. Individual sample mass balance ranged 90.4 to 97.6% AR. Average recoveries in high dose sterile water samples decreased from 47.6% AR at day 7 to 30.6% AR at day 60. Average THF bottle rinse recoveries increased from 3.0% AR at day 7 to a maximum of 17.1% AR at day 60. Average THF sediment film rinse recoveries decreased from 46.7% AR at time 0 to 43.5% AR at day 60. Recoveries in the foam plug (neck) traps were insignificant and averaged 0.2% AR at day 60. No volatile traps were used for the high dose sterile samples.

- Product Balance
The product balance is a composite of the water and THF bottle and/or sediment film rinse product distribution as determined by HPLC analysis of components that exceeded 5% AR.
High Dose
HPLC analysis of high dose samples throughout the study indicated that the majority of radiocarbon analysed was test material. At time 0, the test material represented an average of 83.0% AR in the total system and decreased to an average of 72.2% AR at day 60. In the high dose water alone, the test material represented an average of 56.9% AR at time 0 and decreased to 4.8% AR at day 60. Several uncharacterised regions were detected, but no individual region exceeded 8.3% AR in any single sample.

Low Dose
HPLC analysis of low dose samples throughout the study indicated that the majority of radiocarbon analysed was test material. At time 0, the test material represented an average of 81.9% AR in the total system and decreased to 44.6% AR at day 60. In the low dose water alone, the test material represented an average of 55.9% AR at time 0 and decreased to 1.2% AR at day 60. Several uncharacterised regions were detected, but no individual region exceeded 6.2% AR in any single sample.

Sterile
HPLC analysis of high dose sterile samples throughout the study indicated that the majority of radiocarbon analysed was test material. The test material represented an average of 87.1% AR in the total system after 7 days of incubation and represented an average of 72.1% AR by day 60. In the high dose sterile water alone, the test material represented an average of 45.6% AR at day 7 and decreased to 24.6% AR at day 60. Several uncharacterised regions were detected, but no individual region exceeded 4.6% AR in any single sample.

- Confirmation of test material
The identification of the test material was based on HPLC retention times and co-elution with the reference standard. The HPLC/β-ram assignments of the test material in the surface water amended with sediment were confirmed by TLC analysis of representative samples with the reference standard.

- Proposed Degradation Pathway of the test material
The degradation pathway of the test material in natural water amended with sediment under aerobic conditions cannot be elucidated based on the results of this study.

- Kinetic Analysis
Degradation of the test material was slow in surface water. DT50 and DT90 of the degradation of the test material in surface water were calculated using CAKE software version 3.3 according to FOCUS kinetics. The rate of degradation of the test material over 60 days of incubation at 12 ± 2°C was determined according to the SFO (single first order) model, as follows:
> High dose (Total System): DT50 = 362 days, DT90 = 1,200 days (Chi² = 4.66 Err%, R² = 0.2653)
> Low dose (Total System): DT50 = 72.5 days, DT90 = 241 days (Chi² = 4.61 Err%, R² = 0.8723)

Results with reference substance:

- Radiochemical Purity of the Reference Substance
The purity of [14C]benzoic acid in the reference control substance dose solution was determined to be 96.4% following application.

Application Rate
The target dose rate was 100 μg/L (reference). This corresponds to 10 μg/100 mL sample, respectively. The achieved dose rate was 0.1009 ppm or 10.09 μg/sample (100.9 μg/L) for reference samples.

- Microbial Activity
The reference samples were treated with [14C]benzoic acid at a concentration of 10.09 μg/sample. Duplicate samples were sacrificed after 7 and 14 days of incubation. An average of 71.0% of the applied radiocarbon was converted to 14CO2 (recovered in NaOH traps) after 7 days of incubation, and an average of 87.2% AR of the applied radiocarbon was converted to 14CO2 after 14 days of incubation. This demonstrated the viability of the test system and showed that the microbial activity of the water used in the study was sufficient to conduct the test.

- Mass Balance
Acceptable mass balance was achieved in all reference control substance (benzoic acid) samples. Mass balance averaged 92.0 and 93.1% AR for the 7 and 14 day samples, respectively. Average recoveries in reference control substance water samples decreased from 20.4% at day 7 to 5.3% AR at day 14. Average THF rinse recoveries (bottle and sediment film) averaged ≤ 0.4% AR for all samples. Average NaOH trap recoveries increased from 71.0% AR at day 7 to 87.2% at day 14. The recovery in the NaOH traps indicate the viability of the test system.

Table 1: Mass Balance (Average)

	Time point (day)	Water	THF Rinse (Bottle)	THF Rinse (Sediment Film)	THF Rinse (Sediment Film #2)	Foam Plug (Trap)	NaOH Traps	Total Recovery (%AR)
High dose (dpm applied: 2,646,826)	0	60.6	4.8	28.3	NA	NA	NA	93.7
	7	25.5	11.4	56.5	NA	0.0	0.0	93.4
	14	15.8	13.0	64.8	NA	0.0	0.0	93.5
	20	9.2	13.5	67.9	1.9	0.0	0.0	92.5
	40	11.6	27.9	52.9	NA	0.0	0.1	92.4
	60	6.5	7.5	71.1	8.0	0.0	0.1	93.1
Low dose (dpm applied: 258,768)	0	60.6	2.6	31.6	NA	NA	NA	94.7
	7	26.1	9.5	52.7	3.0	0.0	0.0	91.2
	14	18.1	16.2	56.7	1.7	0.0	0.0	92.6
	20	12.7	15.0	55.9	2.3	0.0	0.0	85.8
	40	12.9	24.1	48.7	4.3	0.0	0.0	90.0
	60	7.3	10.0	58.7	7.6	0.0	0.0	83.5
		Water	THF Rinse (Bottle)	THF Rinse (Sediment Film)		Foam Plug (Neck)		Total Recovery (%AR)
High Dose Sterile (dpm applied: 1,645,809)	7	47.6	3	46.7		0.1		97.4
	40	34.9	16.6	40.4		0.1		91.9
	60	30.6	17.1	43.5		0.2		91.3
		Water	THF Rinse (Bottle)	THF Rinse (Sediment Film)			NaOH Traps	Total Recovery (%AR)
Reference Control (dpm applied: 10,651,070)	7	20.4	0.3	0.4			71	92
	14	5.3	0.4	0.3			87.2	93.1

Table 2: Product Balance (Average % AR)

		Incubation time (days)
		0	7	14	20	40	60
High Dose (Total System)	Test material	83.0	74.1	81.7	68.3	72.8	72.2
	Others	8.9	19.3	11.8	22.4	19.5	20.9
	Foam plug trap	NA	0.0	0.0	0.0	0.0	0.0
	NaOH	NA	0.0	0.0	0.0	0.1	0.1
High Dose (Water)	Test material	56.9	16.8	13.3	6.3	10.1	4.8
	Others	3.8	8.7	2.5	3.0	1.5	1.8
High Dose (Sediment Rinse)	Test material	23.8	47.5	57.2	51.9	40.9	62.2
	Others	4.5	9.0	7.6	16.1	12.0	16.9
Low Dose (Total System)	Test material	81.9	71	69.1	57.4	56.5	44.6
	Others	10.3	17.3	21.9	26.1	31.9	38.9
	Foam plug trap	NA	0.0	0.0	0.0	0.0	0.0
	NaOH	NA	0.0	0.0	0.0	0.0	0.0
Low Dose (Water)	Test material	55.9	20.8	15.2	7.9	9.4	1.2
	Others	4.7	5.4	3.0	4.8	3.5	6.1
Low Dose (Sediment Rinse)	Test material	26.1	42.3	41.3	36.7	28.8	38.1
	Others	5.6	10.4	15.4	19.2	22.6	28.2
High Dose Sterile (Total System)	Test material		87.1			68.4	72.1
	Others		7.2			23.4	19.1
	Foam plug trap		0.1			0.1	0.2
High Dose Sterile (Water)	Test material		45.6			30.7	24.6
	Others		2.0			4.2	6.0
High Dose Sterile (Sediment Rinse)	Test material		41.5			24.7	33.2
	Others		5.2			15.7	10.4

Others: This column comprises several regions present in the HPLC analysis which do not co-elute with reference standards

Validity criteria fulfilled:

yes

Conclusions:

Degradation of the test material was slow in surface water. The rate of degradation (DT50) of the test material over 60 days of incubation at 12 ± 2°C was determined to be 72.5 (low dose) to 362 days (high dose) (SFO model).
Minimal 14CO2 production (at most 0.1% AR) was observed in the high and low dose samples, demonstrating that the mineralisation of the test material was not concentration dependent.

Executive summary:

An aerobic mineralisation study was conducted with the test material in surface water amended with sediment (at 1 g sediment per litre of water) from Brandywine Creek, Pennsylvania, USA for up to 60 days at two concentrations, 98.8 and 9.7 μg/L. The study was conducted in accordance with the standardised guideline 309, under GLP conditions. The samples were incubated in the dark on orbital shakers under aerobic conditions at 12 ± 2 °C for 60 days. In addition, reference and sterile control samples were incubated under the same conditions to confirm the microbial activity of the test water with amended sediment and to examine possible abiotic degradation, respectively. The reference control samples were treated with [14C]benzoic acid at a concentration of 100.9 μg/L, and the sterile control samples were treated with test material at 98.8 μg/L. The test was performed in flow-through systems which allowed humidified air to pass over the sample headspace and through traps to collect volatile organic components (foam plug) and 14C-carbon dioxide (aqueous sodium hydroxide). Reference samples were connected to a series of aqueous sodium hydroxide traps to collect 14C-carbon dioxide. Sterile samples were not connected to a flow-through system, but instead passively aerated with a foam plug in the neck of the sample bottle.

Samples were sacrificed at designated intervals: at time 0 and 7, 14, 20, 40, 60 days after application. Sample pH and dissolved oxygen (DO) measurements were made from the sacrificed samples. Following measurement, the water and the suspended sediment were separated by centrifugation. The sample bottle and remaining pellet (sediment film) were rinsed with tetrahydrofuran (THF). The amount of radioactivity in the water, traps for volatiles (if applicable), and rinses were determined by liquid scintillation counting (LSC), and those high dose, low dose, and sterile subsamples which represented > 5% of the applied radiocarbon (AR) were analysed by high-performance liquid chromatography (HPLC) coupled with flow-through β-ram radiodetector.

The samples containing the reference control substance [14C]benzoic acid were sacrificed after 7 and 14 days of incubation. The mineralisation of benzoic acid to 14CO2 observed after 14 days of incubation (average of 87.2% AR recovered in NaOH traps) confirmed the microbial activity of the test system. The sterile control samples were sacrificed after 7, 40, and 60 days of incubation to examine possible abiotic degradation.

Acceptable mass balance (90 – 110% AR) was achieved in all experimental sets except for the low dose samples, which averaged 85.8% and 83.5% AR at day 20 and 60, respectively. As recovery in the water declined, the recovery in the THF bottle and sediment film rinse increased. Most notably, the majority of the radiocarbon in the day 60 high dose samples was recovered in the THF sediment film rinse (average of 71.1% AR).

The product distribution for all samples analysed throughout the study indicated that the majority of radiocarbon analysed was test material. In the time 0 high dose samples, the test material represented an average of 83.0 % AR in the total system and decreased to 72.2% AR at day 60. In the high dose water alone, the test material represented an average of 56.9% AR at time 0 and decreased to 4.8% AR at day 60. In the time 0 low dose samples, the test material represented an average of 81.9% AR in the total system and decreased to 44.6% AR at day 60. In the low dose water alone, the test material represented an average of 55.9% AR at time 0 and decreased to 1.2% AR at day 60. Several uncharacteridsed regions were detected in the high and low dose samples, but no individual region exceeded 8.3% and 6.2% AR, respectively, in any single sample.

In the sterile samples, the test material represented an average of 87.1% AR in the total system after 7 days of incubation and represented an average of 72.1% AR by day 60. In the sterile water alone, the test material represented an average of 45.6% AR at day 7 and decreased to 24.6% AR at day 60.

Degradation of the test material was slow in surface water. The rate of degradation (DT50) of the test material over 60 days of incubation at 12 ± 2°C was determined to be 72.5 (low dose) to 362 days (high dose) (SFO model).

Minimal 14CO2 production (at most 0.1% AR) was observed in the high and low dose samples, demonstrating that the mineralisation of the test material was not concentration dependent.

Reference 3

Endpoint:

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

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Reason / purpose for cross-reference:

read-across source

Compartment:

natural water / sediment

DT50:

362 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: HIgh Dose

Compartment:

natural water / sediment

DT50:

72.5 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: Low Dose

Transformation products:

no

Description of key information

Simulation testing on ultimate degradation in surface water

Degradation of the test material was slow in surface water. The rate of degradation (DT50) of the test material (read-across from a representative component) over 60 days of incubation at 12 ± 2°C was determined to be 72.5 (low dose) to 362 days (high dose) (SFO model).

Minimal 14CO2 production (at most 0.1% AR) was observed in the high and low dose samples, demonstrating that the mineralisation of the test material was not concentration dependent.

Identification of degradation products

In accordance with REACH Annex XI, Identification of degradation products (Annex IX, 9.2.3.)

using an appropriate test method with the registered substance does not need to be conducted as testing is not technically possible.

The complex UVCB nature of the registered substance meant that the Simulation testing on ultimate degradation

in surface water study (OECD Guideline 309) could not be conducted on the substance itself and further advice from experienced testing laboratories was that it would be technically impossible to monitor any degradation products because of the complexity

of tracking potentially such a high number of substances with multiple similar structures. 

The OECD 309 study was instead conducted on a representative component of the registered substance and this was

found to have a very slow rate of degradation (rate of degradation (DT50) of over 60 days of incubation in surface water). Given the stability of the representative component tested it is not relevant to monitor and identify any degradation products and such an exercise would not be technically feasible at the very low levels involved.

 

Additional information

Simulation testing on ultimate degradation in surface water

An aerobic mineralisation study was conducted with the test material in surface water amended with sediment (at 1 g sediment per litre of water) from Brandywine Creek, Pennsylvania, USA for up to 60 days at two concentrations, 98.8 and 9.7 μg/L. The study was conducted in accordance with the standardised guideline 309, under GLP conditions. The samples were incubated in the dark on orbital shakers under aerobic conditions at 12 ± 2 °C for 60 days. In addition, reference and sterile control samples were incubated under the same conditions to confirm the microbial activity of the test water with amended sediment and to examine possible abiotic degradation, respectively. The reference control samples were treated with [14C]benzoic acid at a concentration of 100.9 μg/L, and the sterile control samples were treated with test material at 98.8 μg/L. The test was performed in flow-through systems which allowed humidified air to pass over the sample headspace and through traps to collect volatile organic components (foam plug) and 14C-carbon dioxide (aqueous sodium hydroxide). Reference samples were connected to a series of aqueous sodium hydroxide traps to collect 14C-carbon dioxide. Sterile samples were not connected to a flow-through system, but instead passively aerated with a foam plug in the neck of the sample bottle.

Samples were sacrificed at designated intervals: at time 0 and 7, 14, 20, 40, 60 days after application. Sample pH and dissolved oxygen (DO) measurements were made from the sacrificed samples. Following measurement, the water and the suspended sediment were separated by centrifugation. The sample bottle and remaining pellet (sediment film) were rinsed with tetrahydrofuran (THF). The amount of radioactivity in the water, traps for volatiles (if applicable), and rinses were determined by liquid scintillation counting (LSC), and those high dose, low dose, and sterile subsamples which represented > 5% of the applied radiocarbon (AR) were analysed by high-performance liquid chromatography (HPLC) coupled with flow-through β-ram radiodetector.

The samples containing the reference control substance [14C]benzoic acid were sacrificed after 7 and 14 days of incubation. The mineralisation of benzoic acid to 14CO2 observed after 14 days of incubation (average of 87.2% AR recovered in NaOH traps) confirmed the microbial activity of the test system. The sterile control samples were sacrificed after 7, 40, and 60 days of incubation to examine possible abiotic degradation.

Acceptable mass balance (90 – 110% AR) was achieved in all experimental sets except for the low dose samples, which averaged 85.8% and 83.5% AR at day 20 and 60, respectively. As recovery in the water declined, the recovery in the THF bottle and sediment film rinse increased. Most notably, the majority of the radiocarbon in the day 60 high dose samples was recovered in the THF sediment film rinse (average of 71.1% AR).

The product distribution for all samples analysed throughout the study indicated that the majority of radiocarbon analysed was test material. In the time 0 high dose samples, the test material represented an average of 83.0 % AR in the total system and decreased to 72.2% AR at day 60. In the high dose water alone, the test material represented an average of 56.9% AR at time 0 and decreased to 4.8% AR at day 60. In the time 0 low dose samples, the test material represented an average of 81.9% AR in the total system and decreased to 44.6% AR at day 60. In the low dose water alone, the test material represented an average of 55.9% AR at time 0 and decreased to 1.2% AR at day 60. Several uncharacteridsed regions were detected in the high and low dose samples, but no individual region exceeded 8.3% and 6.2% AR, respectively, in any single sample.

In the sterile samples, the test material represented an average of 87.1% AR in the total system after 7 days of incubation and represented an average of 72.1% AR by day 60. In the sterile water alone, the test material represented an average of 45.6% AR at day 7 and decreased to 24.6% AR at day 60.

Degradation of the test material was slow in surface water. The rate of degradation (DT50) of the test material over 60 days of incubation at 12 ± 2°C was determined to be 72.5 (low dose) to 362 days (high dose) (SFO model).

Minimal 14CO2 production (at most 0.1% AR) was observed in the high and low dose samples, demonstrating that the mineralisation of the test material was not concentration dependent.