A mixture of: isomers of 2-(2H-benzotriazol-2-yl)-4-methyl-(n)-dodecylphenol; isomers of 2-(2H-benzotriazol-2-yl)-4-methyl-(n)-tetracosylphenol; isomers of 2-(2H-benzotriazol-2-yl)-4-methyl-5,6-didodecyl-phenol. n=5 or 6

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019 - 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Organisation for Economic Cooperation and Development - OECD
Guidelines for Testing of Chemicals
Aerobic and Anaerobic Transformation in Aquatic Sediment Systems No. 308:
24 Apr 2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Remarks:

14C-2-(2H-benzotriazole-2-yl)-4-methyl-6-(1-methylundecyl)phenol was used as test material. A representative Isomer was synthesized and labeled.

Oxygen conditions:

aerobic

Inoculum or test system:

natural water / sediment: freshwater

Details on source and properties of surface water:

Origin water/sediment system 1 (test system 1 or TS1):
Stream named “Ranschgraben” west from the city of Schifferstadt 67105, near the federal road L 532 surrounded by a forest. The sediment smelled earthy and appeared brownish grey. The water was clear and colourless and without any specific odour (GPS coordinate 49.4040036/8.4021280).

Origin water/sediment system 1 (test system 2 or TS2:
Side arm of the river Rhein named “Berghäuser Altrhein” south from the city of Speyer 67346, near the industrial park south surrounded by a forest. The sediment smelled earthy-mouldy and appeared grey. The water was clear and colourless and without any particular odour (GPS coordinate 49.2861252/8.4710159).

Collection: 13 August 2019, sunny, outside temperature was between 17.1 - 19.9°C, atmospheric pressure of 1018 hPa. No particular remarks were made during the sampling.
Depth of sediment collection: 5- 10 cm for each test system
Storage of the test systems: Test systems were stored in the laboratory for one day because the sediment from the water/sediment system “Berghäuser Altrhein” was required to be settled down after sieving.
Acclimatization phase: 26 days of acclimatization (15 August 2019 – 10 September 2019)

Reason for selection of test systems:
Test guideline OECD 308 [1] recommendation: Two sediments should differ at least 2% with respect to organic carbon content and have different textures. One sediment should have a high organic carbon content (2.5-7.5%) and a fine texture, the other sediment should have a low organic carbon content (0.5-2.5%) and a coarse texture. "Fine texture" sediment has clay + silt content (<50 µm particle size fraction) of >50% and "coarse texture" is defined as a clay + silt content of <50%. The difference in [clay + silt] content for the two sediments should normally be at least 20%. Selected test systems: Test system 1 has a coarse texture with a clay and silt content of 0.81% and test system 2 has fine texture with 51.2% of clay and silt content with a difference of 50.4 % between each other. TOC of TS1 is 0.2%* and of TS2 is 3.4%. Difference in texture of the sediments: 50.4%. Both sediments differ in TOC by 3.2%.



Water from test system 1 “Ranschgraben”
Temp. pH value TOC O2 Redox potential
[°C] [mg/L] [mg/L] [mV]*
Sampling (13 Aug 2019) 16.8 7.7 --- 8.7 ---
Start of Acclimation in CA1 (15 Aug 2019) 21.5 7.9 1.9 8.5 312
During acclimation in CA1 (21 Aug. 2019) --- 8.1 3.1 8.1 382
During acclimation in CA1 (26 Aug. 2019) --- 8.4 6.7 8.3 402
During acclimation in CA1 (06 Sep. 2019) --- 8.1 6.4 7.9 434
Start of exposure in CA1 (10 Sep. 2019) --- 8.6 4.9 8.2 235
During exposure in CA1 (12 Sep. 2019) --- 8.6 --- 8.3 216
During exposure in CA1 (18 Sep. 2019) --- 8.4 --- 7.9 162
During exposure in CA1 (23 Oct. 2019) --- 8.5 --- 8.1 253
During exposure in CA1 (13 Nov. 2019) --- 8.2 --- 8.3 475
End of Exposure in the CA1 (19 Dec. 2019) --- 7.6 3.6 8.4 204
End of exposure in the CA2 (19 Dec. 2019) --- 7.1 2.3 8.3 208

odour: neutral; color: clear; *Measured without a GLP status



Water from water/sediment system 2 “Berghäuser Altrhein”
Temp pH value TOC O2 Redox potential
[°C] [mg/L] [mg/L] [mV]*
Sampling (13 Aug 2019) 22.9 7.8 --- 7.6 ---
Start of Acclimation in CA3 (15 Aug 2019) 21.3 7.8 1.5 8.3 284
During acclimation in CA3 (21 Aug. 2019) --- 8.7 1.6 8.3 411
During acclimation in CA3 (26 Aug. 2019) --- 8.9 6.0 7.9 402
During acclimation in CA3 (06 Sep. 2019) 8.4 4.8 8.2 483
Start of exposure in CA3 (10 Sep. 2019) --- 8.6 3.7 7.9 360
During exposure in CA3 (12 Sep. 2019) --- 8.5 --- 8.1 284
During exposure in CA3 (18 Sep. 2019) --- 8.4 --- 7.8 475
During exposure in CA3 (23 Oct. 2019) --- 8.5 --- 8.1 353
During exposure in CA3 (13 Nov. 2019) --- 8.2 --- 8.1 373
End of Exposure in the CA3 (19 Dec. 2019) --- 8.5 6.8 8.2 193
End of exposure in the CA4 (19 Dec. 2019) --- 8.6 7.5 8.3 196

Odour: neutral; color: clear; *Measured without a GLP status

Details on source and properties of sediment:

Origin water/sediment system 1 (test system 1 or TS1):
Stream named “Ranschgraben” west from the city of Schifferstadt 67105, near the federal road L 532 surrounded by a forest. The sediment smelled earthy and appeared brownish grey. The water was clear and colourless and without any specific odour (GPS coordinate 49.4040036/8.4021280).

Origin water/sediment system 1 (test system 2 or TS2):
Side arm of the river Rhein named “Berghäuser Altrhein” south from the city of Speyer 67346, near the industrial park south surrounded by a forest. The sediment smelled earthy-mouldy and appeared grey. The water was clear and colourless and without any particular odour (GPS coordinate 49.2861252/8.4710159).

Collection: 13 August 2019, sunny, outside temperature was between 17.1 - 19.9°C, atmospheric pressure of 1018 hPa. No particular remarks were made during the sampling.
Depth of sediment collection: 5- 10 cm for each test system
Storage of the test systems: Test systems were stored in the laboratory for one day because the sediment from the water/sediment system “Berghäuser Altrhein” was required to be settled down after sieving.
Acclimatization phase: 26 days of acclimatization (15 August 2019 – 10 September 2019)

Reason for selection of test systems:
Test guideline OECD 308 [1] recommendation: Two sediments should differ at least 2% with respect to organic carbon content and have different textures. One sediment should have a high organic carbon content (2.5-7.5%) and a fine texture, the other sediment should have a low organic carbon content (0.5-2.5%) and a coarse texture. "Fine texture" sediment has clay + silt content (<50 µm particle size fraction) of >50% and "coarse texture" is defined as a clay + silt content of <50%. The difference in [clay + silt] content for the two sediments should normally be at least 20%. Selected test systems: Test system 1 has a coarse texture with a clay and silt content of 0.81% and test system 2 has fine texture with 51.2% of clay and silt content with a difference of 50.4 % between each other. TOC of TS1 is 0.2%* and of TS2 is 3.4%. Difference in texture of the sediments: 50.4%. Both sediments differ in TOC by 3.2%.



Sediment from water/sediment system 1 “Ranschgraben”

Parameter Depth of layer pH Particle size distrib. TOC Microbial biomass Redox potential
[cm] [g/100g] [cfu/g] [mV]*
Sampling b (13 August 2019) 5-10 --- --- --- --- ---
Receipt in lab – post handling (13 Aug 2019) --- 7.1 a ≥2 mm =3.2% 0.2 a 7.8*10^5 c ---
<2 mm = 96.9%
<50 µm = 0.8%
Start of Acclimation in CA1 (15 Aug 2019) --- 7.1 --- 0.0 --- 94
During acclimation in CA1 (21 Aug. 2019) --- 7.2 --- --- --- -105
During acclimation in CA1 (26 Aug. 2019) --- 7.9 --- --- --- -102
During acclimation in CA1 (06 Sep. 2019) --- 6.8 --- --- --- -121
Start of exposure in CA1 (10 Sep. 2019) --- 7.9 --- 0.0 8.5*106 -118
During exposure in CA1 (12 Sep. 2019) --- 7.8 --- --- --- -132
During exposure in CA1 (18 Sep. 2019) --- 6.7 --- --- --- -168
During exposure in CA1 (23 Oct. 2019) --- 7.9 --- --- --- -123
During exposure in CA1 (13 Nov. 2019) --- 7.1 --- --- --- -140
End of Exposure in the CA1 (19 Dec. 2019) --- 6.4 --- 0.0 1.7*106 -109
End of exposure in the CA2 (19 Dec. 2019) --- 6.6 --- 0.0 5.7*105 -115

a This parameter was measured in the 2 mm sieved fraction.; b Sediment smelled as fresh earth and appeared greyish brown. Water and sediment phases were taken separately on 13.08.2019 and transported separately after sampling. c measured in the settled sediment from 14.08.2019; CA = control assay
*Measured without a GLP status.



Sediment from water/sediment system 2 “Berghäuser Altrhein”

Parameter Depth of layer pH Particle size distrib. TOC Microbial biomass Redox potential
[cm] [g/100g] [cfu/g] [mV]*
Sampling (13 Aug 2019) 5-10 --- --- --- --- ---
Receipt in lab – post handling (13 Aug 2019) --- 6.8 a ≥2 mm =34.8% 3.4 a 9.4*106 c --- ---
<2 mm = 65.2%
<50 µm = 51.2%
Start of Acclimation in CA3 (15 Aug 2019) --- 7.2 --- 3.4 --- 118
During acclimation in CA3 (21 Aug. 2019) --- 7.0 --- --- --- -108
During acclimation in CA3 (26 Aug. 2019) --- 7.2 --- --- --- -110
During acclimation in CA3 (06 Sep. 2019) --- 6.9 --- --- --- -109
Start of exposure in CA3 (10 Sep. 2019) --- 7.2 --- 0.3 2.3*107 -121
During exposure in CA3 (12 Sep. 2019) --- 7.1 --- --- --- -118
During exposure in CA3 (18 Sep. 2019) --- 7.0 --- --- --- -218
During exposure in CA3 (23 Oct. 2019) --- 7.3 --- --- --- -137
During exposure in CA3 (13 Nov. 2019) --- 7.2 --- --- --- -198
End of Exposure in the CA3 (19 Dec. 2019) --- 7.2 --- 2.3 6.9*105 -162
End of exposure in the CA4 (19 Dec. 2019) --- 7.4 --- 2.1 2.5*106 -138

a This parameter was measured in the 2 mm sieved fraction; b Sediment smelled musty-earthy and appeared grey. b Sediment smelled as fresh earth and appeared greyish. Water and sediment phases were taken separately on 13.08.2019 and transported separately after sampling. c measured in the settled sediment from 14.08.2019; CA = control assay *Measured without a GLP status.

Details on inoculum:

please refer to water and sediment descriptions

Duration of test (contact time):

100 d

Initial conc.:

281 µg/L

Based on:

test mat.

Remarks:

Test System: Ranschgraben; corresponds to 466 µg test substance per kg sediment (wet weight)

Initial conc.:

281 µg/L

Based on:

test mat.

Remarks:

Test System: Berghäuser Altrhein; corresponds to 438 µg test substance per kg sediment (wet weight)

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

TEST CONDITIONS (ACTUAL SPECIFICATIONS)
Selection of the water/sediment system:
Two different water/sediments combinations according to OECD 308 [1] and Commission Regulation (EC) No 440/2008-C.24.
The test systems were checked for the contamination of test substance. No presence was detected, and the results are present in the analytical report (see appendix 2, section xxx). Therefore, it is assumed that the natural water/sediment systems used in this test were not contaminated with the test substance within the last 4 years.

Number of water/sediment systems: 2
TOC of the sediments at arrival in the laboratory:
First water/sediment system “Ranschgraben”:
Sediment: TOC = 0.2 g/100 g corresponding to 0.2% TOC

Second water/sediment system “Berghäuser "Altrhein":
Sediment: TOC = 3.4/100 g corresponding to 3.4% TOC

The difference in TOC content of the two water/sediment blends was 3.2% (3.4 % to 0.2 %) which is >2% mentioned in the guideline.

Texture:
Water/sediment system 1: Coarse texture with a clay and silt content of 0.81% (< 50 µm fraction).
Water/sediment system 2: Fine texture with clay and silt content of 51.2% (< 50 µm fraction)
Difference in texture of the sediments: 50.4 % (51.2 % to 0.81 %). The difference in clay and silt content of the two water/sediment systems was > 20 %.

Dry matter content of the sediments at arrival in the laboratory:
Sediment 1 of TS1 has 78.6 g DM per 100g wet weight (mean value of 78.4%/78.8%) and sediment 2 from TS2 contained 65.8 g DM per 100g wet weight (mean value of 64.9%/66.6%).

Test vessels:
Standard cylindrical reagent bottles of 1L volume from Schott, narrow necked with standard ground socket (NS 29/32), material glass soda glass, outer diameter ~ 9.6 cm and 12 cm height.

Test duration: 100 days
Number of replicates (test item assays) per water/sediment system and sampling day: 2
Number of replicates (control assays) per water/sediment system: 2
Sampling times: 8

Number of test vessels:
24 vessels per water/sediment system: Among this, 16 test vessels (TA) for regular sampling and 2 test vessels for water sampling for dissipation measurement (AS) with test substance assays. Furthermore, 2 control assays and 2 inhibition assays and 2 solvent control assays

Water volume of water/sediment system 1 in test vessels: Approx. 499 mL
Mass of sediment water/sediment system 1 in test vessels: Approx. 236 g Dw, equivalent to approx. 300 g wet sediment (78.6% DM)
Thickness of water: water/sediment system 1 in test vessels: About 5.8 cm
Thickness of sediment: water/sediment system 1 in test vessels: Approx. 2.3 cm
Volume ratio in the test vessels of water to sediment in the test vessels of the water/sediment system 1: Approx. 3:1
Water volume of water/sediment system 2 in test vessels: Approx. 499 mL
Mass of sediment water/sediment system 2 in test vessels: Approx. 210 g Dw, equivalent to approx. 320 g wet sediment (65.8% DM)
Thickness of water in water/sediment system 2 in test vessels: Approx. 5.8 cm
Thickness of sediment water/sediment system 2 in test vessels: Approx. 2.5 cm
Volume ratio in the test vessels of water to sediment in the test vessels of the water/sediment system 2: Approx. 3:1

Gas flow-through:
Gas flow was checked daily from the start of the experimental phase (including the acclimatization and exposure phase).

Way of ventilation:
Bubbling in the water layer with 2-3 bubbles/second

Air supply: Moistened pressurized air
Stirring of the water phases: No stirring
Test temperature: Temperature in incubation room was ranged between 21.3 to 22.0°C (mean value 21.6 °C) during the experimental exposure
Measurement of the test temperature: 11 times during the exposure
Illumination: None, incubation in the dark.
Application of the test substance to the test vessels: With a pipette from the acetone stock solution
Absorption liquids: 2x100 mL 1 M sodium hydroxide, 50 mL ethylene glycol and 50 mL of 0.1 M sulfuric acid
Extraction method of sediment phase: 3 times x 50 mL methanol, followed by 3x 50 mL chloroform, ASE extraction with hexane, alkaline extraction at the end.

Compartment:

natural water / sediment: freshwater

% Recovery:

100

Remarks on result:

other:

Remarks:

The mean recovery rate of the applied radioactivity was between 90-110% % TAR during the exposure in both test systems TS1 and TS2. The validity criterion is fulfilled.

% Degr.:

> 0.4 - < 1.8

Parameter:

CO2 evolution

Sampling time:

100 d

Remarks on result:

other:

Remarks:

Total applied radioactivity in test system 1. Mineralization to CO2

% Degr.:

> 0.1 - < 0.3

Parameter:

CO2 evolution

Sampling time:

100 d

Remarks on result:

other:

Remarks:

Total applied radioactivity in test system 2. Mineralization to CO2

Key result

Compartment:

entire system

DT50:

108 d

Type:

other: single first order (SFO)

Temp.:

21.6 °C

Remarks on result:

other:

Remarks:

Test System 1

Key result

Compartment:

entire system

DT50:

6.2 d

Type:

other: first order multi compartment (FOMC)

Temp.:

21.6 °C

Remarks on result:

other:

Remarks:

Test System 2

Compartment:

natural water: freshwater

DT50:

2.01 d

Type:

other: single first order (SFO)

Temp.:

21.6 °C

Remarks on result:

other:

Remarks:

Test System 1

Compartment:

natural water: freshwater

DT50:

1.57 d

Type:

other: single first order

Temp.:

21.6 °C

Remarks on result:

other:

Remarks:

Test System 2

Compartment:

natural sediment: freshwater

DT50:

141 d

Type:

other: single first order

Temp.:

21.6 °C

Remarks on result:

other:

Remarks:

Test System 1

Compartment:

natural sediment: freshwater

DT50:

124 d

Type:

other: single first order

Temp.:

21.6 °C

Remarks on result:

other:

Remarks:

Test System 2

Transformation products:

yes

Remarks:

The structure of 4 metabolites could be identified (M10, M11, M14 and M15)

Details on transformation products:

No major metabolites (> 10% TAR) were identified during the experimental exposure. A total of 31 minor metabolites above 0.1% TAR were identified in the water as well as sediment phase of both TS1 and TS2. Among these, 13 metabolites were present in ≥0.1% TAR in at least two consecutive sampling dates in the degradation experiment and others were degraded to a concentration below LOQ level on Day 100. Out of 13 metabolites, M9, M19 and M21 of TS1 and M4 and M14 metabolite of TS2 exhibited an increasing tendency toward the end of exposure. Although present in ≥0.1% TAR, rest of the metabolites M4, M10, M13, M14 and M15 in TS1 and M2 in TS2 exhibited a degradation tendency towards the end of exposure.The structures of M10, M11,M14 an M15 could be identified (see smiles codes below). The other metabolites were degraded to a level below the LOQ and therefore could not be identified by structure.

SMILES CODES OF IDENTIFIED METABOLITES:
M10 CC(CCCC(O)=O)c1cc(C)cc(N2Nc3ccccc3N2)c1O
M11 CC(CC(O)=O)c1cc(C)cc(N2Nc3ccccc3N2)c1O
M14 Cc1ccc(O)c(C(C(O)CCCCCCCC(O)=O)c1CN1Nc2ccccc2N1
M15 Cc1ccc(O)c(C(C)(O)CC(O)=O)c1CN1Nc2ccccc2N1

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

yes

Details on results:

In water/sediment laboratory incubations under aerobic conditions in the dark,14C2-(2H-benzotriazole-2-yl)-4-methyl-6-(1-methylundecyl) phenol exhibited a fast removal from the water phase to the sediment compartment in both test systems. By Day 8, the radioactivity declined to ≤15% TAR in TS1 and ≤5% in TS2 and levelled off during the rest of exposure period. Mineralization of the 2H-benzo-U-C1414C radiolabels to carbon dioxide accounted for 0.4-1.8% TAR and 0.1-0.3% TAR after 83–100 days in TS1 and TS2, respectively. Formation of organic or inorganic volatile were absent during the exposure.
At the end of exposure, significant portion of applied radioactivity was found in the sediments phases of both test systems with about 86.7% in sediment of system 1 and 98.5% in TS2 from which 46.8% and 29.7% TAR were readily desorbable with methanol extraction. The formation of unextractable residues for this radiolabel accounted for about 29.8 - 39.9% TAR and 61.9-68.9 % TAR after 83-100 days. Together, solvent extraction with chloroform and hexane together recovered a significant amount (mean value) of sediment bound activity in TS2 (35.8% TAR) but a lower amount in TS1 (4.2%TAR). The rest of the nonextractable residues (NER) were extracted as humic matter with alkaline extraction and this recovered 27.2% TAR in TS1 and about 31.3% TAR in TS2. A higher alkali soluble NER fraction was found in TS1 (22.5% TAR) but the insoluble humin fraction was only 4.3%TAR. Contradictorily, alkali soluble portion was less in TS2 (14.2%) but humins were present in a very high amount (16.9% TAR). About 90-100% of the sediment bound activity was recovered using the solvent extractions and as NER fractions.
The biomass and the other physico-chemical parameters indicated that the condition inside the test systems were stable and the microbiology was active during the exposure time. No inhibition effect of test substance to microorganisms were found. Overall, the mass balances in both systems showed a good mean recovery rate within the limits of 90-110% on every sampling day over the test duration.

Validity criteria:

The limit of detection (LOD) of the method of analysis should be at least 0.01 mg/kg in water or sediment (as test substance) or if this value is lower 1% of the inserted amount of the test item. The limit of detection (LOQ) must be known.

Observed value:

LOD < 0.094 µg/kg (both test systems) in sediment, water and extracts

Validity criteria fulfilled:

yes

Validity criteria:

Repeatability and sensitivity of the method of analysis

Observed value:

About 96 to 98% of the injected radioactivity was also recovered using this method of analysis at the end of storage indicating the storage stability and sensitivity of the analysis which is in the required range of 90-100%.

Validity criteria fulfilled:

yes

Validity criteria:

The mean recovery rate of the test substance during the exposure should be in the range of 90-110 % of the activity of the labelled test substance in water/sediment systems.

Observed value:

The mean recovery rate of the applied radioactivity was between 90-110% % TAR during the exposure in both test systems TS1 and TS2.

Validity criteria fulfilled:

yes

Conclusions:

The results of the study showed that the behavior of the test material in the water/sediment systems is characterized by a fast dissipation from water phase to sediment due to adsorption. During the experimental exposure, no significant mineralization to CO2 was observed indicating that biodegradation
of the test material is a slow environmental fate process in both types of water-sediment systems. At the end of exposure, the mean degree of degradation as mineralization was only about 1.8% TAR in TS1 “Ranschgraben” and 0.3 %TAR in TS2 “Berghäuser Altrhein”. The biomass and the other parameters measured in the control at the end of exposure showed that the systems were stable and active during the exposure time. No inhibition effect to microorganism in the test vessels were found in the inhibition test vessels.

Executive summary:

In this study, the biodegradation rates of¹⁴C-2-(2H-benzotriazole-2-yl)-4-methyl-6-(1methylundecyl) phenol in water/sediment systems and the identification of its biotransformation products and their distribution into water and sediment, according to OECD 308 [1] was tested. 

 

Methods: Two different natural water/sediment systems were used in this study. One system (test system 1 hereafter mentioned as TS1) was taken from a small stream named Ranschgraben, near Schifferstadt, Germany) and the other system (test system 2 hereafter mentioned as TS2) was taken from a branch of the river Rhein named Berghäuser Altrhein near Speyer, Germany. About 140 µg which corresponds to 1 MBq of radiolabelled test item¹⁴C-2-(2H-benzotriazole-2-yl)-4-methyl-6-(1-methylundecyl) phenol was applied per test vessel (total applied radioactivity or TAR) resulting in a final nominal concentration of about 280 µg/L water. The activity of the microbial biomass and other parameters of the test system were monitored in control assays for both water/sediment systems. The test vessels were incubated in dark conditions at a temperature range of 21.3 to 22.0°C with a mean value of 21.6 °C (11 measurements) for 100 days. The test vessels were attached to a continuous air flow system and absorption traps. During sampling, two replicates of test substance from each water-sediment system were taken at the start of exposure (Day 0) and afterwards on Day 2, Day 8, Day 27, Day 43, Day 64, Day 82 and at the end of exposure (Day 100). The base value for measuring the actual total applied activity in the experimental test vessels was determined by the direct measurement of stock solution aliquots and a mean value of 59872325 dpm (1 MBq) was obtained.

The water and sediment were processed separately for the analysis. The whole water phase was decanted into a bottle and was extracted with chloroform. The sediment was extracted with methanol as a first solvent extraction step to release the readily sorbed analytes from the sediment. After the extraction, the sediment was dried at 60 °C until constant weight was achieved. A part of this dried sediment phase was later extracted with chloroform and further extracted with hexane using accelerated solvent extraction method (ASE). The water phase, the pooled sediment extracts, the washing solution after washing the test vessels and the absorption liquids were measured for radioactivity in a liquid scintillation counter (LSC). The radioactivity of the dried sediments was measured after combustion by LSC analysis. The mass balance in the test assays were calculated with these radio-activities. The nonextractable residues (NER) in the sediments were further characterized by separating into fulvic acid, humic acid, humins, adsorbed to the sediment after strong alkali and acid extractions series. 

Additionally, the water phase chloroform extracts, remaining water after chloroform extraction, sediment extracts (methanol, chloroform and hexane) were analyzed by radioHPLC, as multi-site study under GLP at NOACK Institute for substance specific analysis and for the identification and characterisation of metabolites. 

Results: In water/sediment laboratory incubations under aerobic conditions in the dark,¹⁴C2-(2H-benzotriazole-2-yl)-4-methyl-6-(1-methylundecyl) phenol exhibited a fast removal from the water phase to the sediment compartment in both test systems. By Day 8, the radioactivity declined to ≤15% TAR in TS1 and ≤5% in TS2 and levelled off during the rest of exposure period. Mineralization of the 2H-benzo-U-C14¹⁴C radiolabels to carbon dioxide accounted for 0.4-1.8% TAR and 0.1-0.3% TAR after 83–100 days in TS1 and TS2, respectively. Formation of organic or inorganic volatile were absent during the exposure. At the end of exposure, significant portion of applied radioactivity was found in the sediments phases of both test systems with about 86.7% in sediment of system 1 and 98.5% in TS2 from which 46.8% and 29.7% TAR were readily desorbable with methanol extraction. The formation of unextractable residues for this radiolabel accounted for about 29.8 - 39.9% TAR and 61.9-68.9 % TAR after 83-100 days. Together, solvent extraction with chloroform and hexane together recovered a significant amount (mean value) of sediment bound activity in TS2 (35.8% TAR) but a lower amount in TS1 (4.2%TAR). The rest of the nonextractable residues (NER) were extracted as humic matter with alkaline extraction and this
recovered 27.2% TAR in TS1 and about 31.3% TAR in TS2. A higher alkali soluble NER fraction was found in TS1 (22.5% TAR) but the insoluble humin fraction was only 4.3%TAR. Contradictorily, alkali soluble portion was less in TS2 (14.2%) but humins were present in a very high amount (16.9% TAR). About 90-100% of the sediment bound activity was recovered using the solvent extractions and as NER fractions. The biomass and the other physico-chemical parameters indicated that the condition inside the test systems were stable and the microbiology was active during the exposure time. No inhibition effect of test substance to microorganisms were found. Overall, the mass balances in both systems showed a good mean recovery rate within the limits of 90-110% on every sampling day over the test duration.

A total of 31 minor metabolites at a %TAR of >= 0.1% TAR were identified in the water as well as sediment phase of both TS1 and TS2. No major metabolites (> 10% TAR) were identified during the experimental exposure. Among these metabolites, M4, M9, M10, M13, M14, M15, M19 and M21 metabolites were present in >=0.1% TAR in at least two consecutive sampling dates in an increasing pattern in the degradation experiment. M13, M14, M15, were present above 1% TAR. Other metabolites were degraded to a concentration below LOQ level on Day 100. Metabolites such as M9, M19 and M21 of TS1 and M4 and M14 metabolite of TS2 exhibited an increasing tendency toward the end of exposure. Although present in >=0.1% TAR on day 100, rest of the metabolites M4, M10, M13, M14 and M15 in TS1 and M2 in TS2 exhibited a degradation tendency towards the end of exposure.

Kinetic analyses of parent compound were performed without a GLP status, using all available kinetic models, such as single first order (SFO), first order multi compartment (FOMC), hockey stick (HS), and double first order in parallel (DFOP). Selection of the most appropriate kinetic model was based on the criteria, such as the error value at which the X² (chi2)-test is passed by the best-fit model (SFO, FOMC, HS, or DFOP) should be below 15%,t-test should be passed at the significance level of 10% (i.e. p<0.1) and the fit must be visually acceptable.

In sediment compartment of both TS1 and TS2, TS1 whole system at P-I level, estimated DegT50 and DT90 in TS1 and TS2 were not reached within the experimental period of 100 days and these parameters are extrapolated beyond the experimental period. Similarly, in TS2 whole system too, DT90 was not reached within the experimental period. Kinetics evaluation of only M13 metabolite was conducted in M-I level and were plausible.

Description of key information

The results of the study showed that the behavior of ¹⁴C-2-(2H-benzotriazole-2-yl)-4-methyl6-(1-methylundecyl) in the water/sediment systems is characterized by a fast dissipation from water phase to sediment due to adsorption. During the experimental exposure, no significant mineralization to CO₂ was observed indicating that biodegradation of ¹⁴C-2-(2Hbenzotriazole-2-yl)-4-methyl-6-(1-methylundecyl) is a slow environmental fate process in both types of water-sediment systems. At the end of exposure, the mean degree of degradation was only about 1.8% TAR in TS1 “Ranschgraben” and 0.3 TAR in TS2 “Berghäuser Altrhein”. The biomass and the other parameters measured in the control at the end of exposure showed that the systems were stable and active during the exposure time. No inhibition effect to microorganism in the test vessels were found in the inhibition test vessels.    

 

An exponential decline of activity in the water phases in both test system was measured initially. By Day 8, the radioactivity declined to ≤15% TAR in TS1 and ≤5% in TS2 and levelled off. At the end of exposure, significant portion of applied radioactivity was found in the sediments phases of the test systems with about 86.9% in sediment of system 1 and 98.7% in TS2 from which 46.9% and 29.7% TAR were readily desorbable with methanol extraction. This left a 40% TAR in sedment of TS1 and a comparable higher amount of radiopacity with 69% TAR in TS2. In total, presence of 31 metabolites in the water as well as sediment phase of both TS1 and TS2 were identified and all these metabolites appeared to be polar and present in minor quantities. On Day 100, 13 metabolites were present ≥0.1% and others were degraded (transient metabolites). Metabolites formation profile indicated a biologically mediated reaction. No other organic or inorganic volatile were formed during the exposure. A build of bound residues or NER were visible in both systems and this was significantly higher in TS2. Humic matter or NER extraction recovered 27.3% TAR in TS1 and about 31.4% TAR in TS2. Acid soluble fulvic acid was higher in TS1 (12.8% TAR) but the part of sediment that remained insoluble in sodium hydroxide solutions and estimated as humin fraction was lower. Contradictorily, soluble part was less in TS2 but humins were present in a very high amount (16.9% TAR) compared to TS1 (3.8%TAR). Humic acid was present in comparably lower amount in both TS1 and TS2 on Day 100. About 90-100% of the sediment bound activity was recovered using various solvent extractions and as NER fractions.    

 

Kinetic analyses of parent compound were performed using all available kinetic models without a GLP status, such as single first order (SFO), first order multi compartment (FOMC), hockey stick (HS), and double first order in parallel (DFOP). Selection of the most appropriate kinetic model was based on the criteria, such as the error value at which theχ² (chi2)-test is passed by the best-fit model (SFO, FOMC or DFOP) should be below 15%, and the fit must be visually acceptable. DFOP provided the best visually acceptable fit withχ² value 3.8 (r²0.81) and 11.1 (r²0.94) for TS1 and TS2 respectively for DegT₅₀/₉₀evaluation in whole system. DegT₅₀of 149 days and DegT₉₀ of 495 days were estimated in TS1. In TS2 312 days and 601 days were calculated DegT₅₀ and DegT₉₀, respectively. Estimated DT₅₀and DT₉₀values were not reached within the experimental period and these parameters are extrapolated beyond the experimental period.   

 

The disappearance of parent substance in water columns of TS1 and TS2 was described by the SFO kinetic model as theχ² value of 3.3 (r²0.99) and1.4 (r²0.98), respectively. The observed DT₅₀value were 2.0 days in TS1 and 1.5 days in TS2. The DT₉₀value were also closer in TS1 and TS2, with 6.5 days in TS1 and 5 days in TS2. Both half-lives were shorter than the entire exposure period. In sediment SFO was again found to be comparably good fit withχ² value 4.3 and r²value of 0.90 in TS1. Although a χ² greater than the suggested 15% level recommended by FOCUS was derived in sediment TS2, SFO was chosen as the best fit. The estimated DT₅₀ and DT₉₀ were respectively, 165 days and 549 days in TS1 and 142 days and 472 days in TS2. These values were extrapolated beyond the experimental period. Kinetics evaluation results of the metabolites were plausible to report due to insufficient recovery and limited data points representing the different phases of formation with high variability.

Key value for chemical safety assessment

Half-life in freshwater:

2.01 d

at the temperature of:

21.6 °C

Half-life in freshwater sediment:

141 d

at the temperature of:

21.6 °C

Whole System

Half-life in whole system:

108 d

at the temperature of:

21.6 °C

Type of system:

fresh water and sediment