Benzotriazole

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

Study period:

10 March 2020- 11 Mai 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Version: 13 April 2004

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: American Radiolabeled Chemicals ARC. INC; Lot Nr.: 191212

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 99.9 %
- Lot Number: 191212
- Specific activity: 95 mCi/mmol (29.02 MBq/mg)
- Locations of the label: into Benzolring

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: < -18 °C
- Chemical stability (Water/Light): Stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Solid radiolabeled 14C-Benzotriazole was dissolved in acetonitrile to prepare a stocksolution, followed by dilution to the final test concentrations (10 µg/L, 50 µg/L). The samples required < 1 % of solvent as required by the OECD-guideline 309. The actual concentration of 14C-labelled Benzotriazole in this test solutions were determined by LSC measurement and based on these results the exact application volumes were calculated for each concentration level. 0.5 L Erlenmeyer flasks, were filled with 250 mL surface water and the specified amount of 14C-Bezotriazole were added.

Radiolabelling:

yes

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): The surface water was sampled from Biggesee (Bigge reservoir), Germany (51°4’41.96” N, 7°50’3.1” E) at depth of 10 – 20 cm.
- Storage conditions: stored at < 8 °C for a maximum of 8 days.
- Water Temperature (°C) at time of collection: 8.6 °C
- pH at time of collection: 6.9
- Redox potential (mv) initial/final: 208.3 mV / 210.0 mV
- Oxygen concentration (mg/l) initial/final: 9.68 (15.7 °C) / 8.85 (13.7 °C)
- Hardness (CaCO3): not specified
- Dissolved organic carbon (%): 2.139 mg/L
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): not specified
- Water filtered: yes filtered through a 100 µm mesh filter prior storing
- Nitrogen (total, mg/L) 3.4
- Nitrate (mg/L) 13.0
- Nitrite (mg/L) 0.009
- Ammonium-N (mg/L) < 0.01
- total P (µg/L) 14.9

Duration of test (contact time):

62 d

Initial conc.:

10 µg/L

Based on:

test mat.

Remarks:

exact quantity determined analytically: 10.3 µg/L

Initial conc.:

50 µg/L

Based on:

test mat.

Remarks:

exact quantity determined analytically: 51.3 µg/L

Details on study design:

TEST CONDITIONS
- Culturing apparatus: flow-through system on orbital shakers at controlled test temperature, 0.5L Erlenmeyer flasks combined with two traps of sodium hydroxide solution and saturated with air.
- amount of test solution/treatment per 250 mL surface water: C14 Benzotriazole (10 µg/l): 74.7 kBq C14 Benzotriazole (50 µg/l): 372 kBq
- Additional substrate: No
- Solubilising agent (type and concentration if used): Acetonitrile (in Sample < 1% as required by the OECD-guideline 309)
- Test temperature: 12 ± 2 °C (Test samples and controls)
- pH: stable between 6.83 - 7.65
- oxygen: stable between 8.41 mg/L – 9.67 mg/L
- pH adjusted: no
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: No

TEST SYSTEM
- Test flask (type, material, size): Erlenmeyer glass flasks, 0.5 L
- Amount of test solution per flask: 250 mL
- Study treatments: two test item concentrations (10 µg/l, 50 µg/l Benzotriazole), abiotic sterile control for examining possible abiotic degradation or other non-biological removal of Benzotriazole, reference item control for microbial activity, solvent control of acetonitrile, Blank control
- No. of replicates per treatment: two (duplicate flask)
- Method used to create aerobic conditions: flow through system on orbital shakers
- Method used to control oxygen conditions: measured regularly on weekly basis
- Test performed in closed vessels due to significant volatility of test substance: No
- Details of trap for CO2 and volatile organics:
- Traps for samples treated with 14C-Benzotriazole: one glass trap per replicate were filled with 2M sodium hydroxide and installed on vessels. Exhaust gas were sampled at the same time as the water samples.
- Traps for samples treated with the reference item sodium benzoate: two glass traps per reference were filled with 2M sodium hydroxide and installed in series.

SAMPLING
- Surface water samples treated with 14C-Benzotriazole, were taken in duplicates at day 0, 7, 14, 21, 28, 42 and 62. Whole flasks were sampled.
- Surface water sample of reference control, were taken at day 35 after application. The volume of surface water was determined by weighting of the vessels and two aliquots of 1 mL were taken for LSC measurement. To strip-off dissolved 14CO2 in the water phases, 2 mL of concentrated formic acid were added, and the samples were incubated again in the test system.
- Surface water samples from Abiotic sterile control, were taken in duplicates after 62d.
- Trap sample (treated with 14C-Benzotriazole) at day 0, 7, 14, 28, 42 and 62 after application. The absorption solution with exhaust gas was sampled, and Traps not removed. The volume and pH of each trap was measured. The total radioactivity in each solution was measured by LSC.
- Trap sample (reference control) at day 1, 3, 6, 8, 14, 21, 35 after application. At sampling, the CO2 traps were removed from the flow-through system and replaced by new traps. The volume and pH of NaOH was measured. The total radioactivity in each solution was measured by LSC. The formation of 14CO2 was monitored until 38 days.
- Sample storage before analysis: not specified

STATISTICAL METHODS:
- software “CAKE” version 3.3 on R version 3.0.0 (2013-04-03)
- considered kinetics: single first order, first order multi compartment, hockey stick and double first order in parallel

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
Abiotic sterile control: For preparation, the pre filtered Biggesee surface water, test vessels, and all required equipment was autoclaved two times at 121 °C for 20 minutes. An aliquote of both test solutions (10 µg/l, 50 µg/l Benzotriazole) were sterile filtered and add to the pre-treated surface water.
Biological Activity control (reference control): 250 mL duplicate flasks were prepared. Sodium benzoate was provided as a solution in ethanol. Therefore, the solvent was evaporated to dryness under a gentle stream of nitrogen before re-dissolving in ultrapure water. The actual concentration of 14C-labelled sodium benzoate was determined analytically and based on these results the exact application volumes were calculated. An 0.5 L Erlenmeyer flasks, were filled with 250 mL surface water and 14C-sodium benzoate was added to a total of 10 µg/L.
Solvent Control for acetonitrile: For solvent control, a duplicate of the biological activity control samples was prepared containing additionally acetonitrile.
Blank control: Blank control samples (2 replicates) remained untreated in order to monitor the sample conditions throughout the incubation period.

Reference substance:

benzoic acid, sodium salt

Remarks:

Reference for microbial activity; Purity: 99 % Lot/Batch Nr.:171201, Exp Date: Dec 2022 Specific activity: 33.4 MBq/mg State of matter and appearance: Solution in ethanol Storage conditions: <-15 °C

Compartment:

natural water

% CO2:

>= 90 - <= 110

Remarks on result:

other: the recovery range mentioned above refers to the total recovery of applied radioactivity for all individual samples

Parent/product:

parent

Compartment:

total system

% Degr.:

< 1

Remarks on result:

other: The degradation of <1 % AR refers to all trap samples taking within 62 days

Key result

Compartment:

natural water

DT50:

831 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: SFO model

Remarks:

geometric mean

Compartment:

natural water

DT50:

658 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: SFO model

Remarks:

test concentration: 50 µg/L

Compartment:

natural water

DT50:

1 050 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: SFO model

Remarks:

test concentration: 10 µg/L

Mineralization rate (in CO2):

1 other: %

Other kinetic parameters:

first order rate constant

Transformation products:

no

Remarks:

Amounts below 1.0 % AR were detected in the sodium hydroxide traps. The results show nearly no mineralisation of the test item during the aerobic incubation for up to 62 days.

Details on results:

TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): No

MAJOR TRANSFORMATION PRODUCTS
- No transformation products were found in the water samples. Only parent Benzotriazole were detected by radio-HPLC in the SPE-eluates in all samples. Consequently, the amount of parent compound in the SPE-eluates was between 94.4 – 98.6 % AR immediately after start of incubation and 92.9 – 96.1 % AR (concentration level of 50 µg/L) at final sampling time point (62 d).

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: The results show nearly no mineralisation of Benzotriazole during the aerobic incubation for up to 62 days. Amounts below 1.0 % AR were detected in the sodium hydroxide traps.

RECOVERY
- The total recoveries ranged between 90 and 110 % of applied radioactivity for all individual samples.The amount of radioactivity in the SPE-eluates of the surface water did not change significantly during the incubation time. Amounts of radioactivity remaining in the water phase after the SPE treatment were < 1.0 % AR for all individual samples over the total incubation time of 62 d.

STERILE TREATMENTS (if used)
- Sterile samples show no differences between biotic and abiotic degradation of Benzotriazole. Radioactive amounts in the organic eluates and aqueous phases were found in ranges which are comparable to the respective microbial active samples.
- Recovery of the parent Compound: 14C-Benzotriazol was found > 97 % at the end (62 d)
-Transformation of the parent compound: No
- Formation of transformation products: No
- Formation of extractable and non-extractable residues: No
- Volatilization: No

Results with reference substance:

Mineralisation (approx. 60 % AR, 12 °C no addition of organic solvent and >70 % 12 °C with addition of organic solvent) of the reference substance (14C- sodium benzoate) within the incubation time of 63 days under test conditions confirmed appropriate biological activity in the surface water.

Validity criteria:

The validity of the test according to OECD 309 criteria was confirmed by rapid mineralisation of the reference substance 14C- sodium benzoate with > 50 % AR evolved as 14C-carbon dioxide within 7 days of incubation.

Validity criteria fulfilled:

yes

Conclusions:

This study fulfilled the validity criterion .The DT50 values of Benzotriazole based on SFO kinetics (best fit) are in the range of 658 to 1050 days. The geometric mean DT50 of the recommended optimisation was found to be 831 days for the parent compound.

Executive summary:

In this GLP study, according to OECD guideline 309, radiolabelled ¹⁴C-Benzotriazol was incubated in test water for 62 day at two different low concentrations (10, 50 µg/L) to determine biodegradation of the test item in aerobic natural water based on Mineralisation, and transformation products.

Based on experimental observations Benzotriazole was mostly stable in surface water during the incubation time of 62 days and did not mineralize significantly (< 1 % in 62 Days). No transformation products were detected. The DT₅₀ values of Benzotriazole based on SFO kinetics (best fit) are in the range of 658 to 1050 days. The geometric mean DT₅₀ of the recommended optimization was found to be 831 days for the parent compound.

Description of key information

Under the test conditions of the OECD 309 study (12 °C, two concentrations) no significant degradation was observed. After 62 days of incubation approximately 1 % of 1H-Benzotriazole was detected in the CO2 traps. No transformation products could be detected. Assuming a (pseudo) first order degradation a geometric mean half-life of the substance in fresh water was estimated. Further testing on degradation behaviour in sediment was not performed, as the findings in the simulation study with surface water fulfils the criteria for persistency in Annex XIII of the REACH Regulation.

Key value for chemical safety assessment

Half-life in freshwater:

831 d

at the temperature of:

12 °C