3-({[(4-methylphenyl)sulfonyl]carbamoyl}amino)phenyl 4-methylbenzenesulfonate;N-(p-toluenesulfonyl)-N'-(3-(p-toluenesulfonyloxy)phenyl)urea

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:

19 November 2020 - 11 March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A pelagic OECD 309 test was requested by ECHA (Decision number CCH-D-2114484966-27-01/F)

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on source and properties of surface water:

One non-contaminated water system was sampled from natural surface water (Schoonrewoerdsewiel, Leerdam, The Netherlands). This surface water originated as a result of a dike (levee) failure in the 16th century. The force of the water flowing through the dike created a low area behind the dike. After closure of the dike, a small freshwater pond remained. "Schoonrewoerdse Wiel" (SW) is located in the province Zuid-Holland, in Leerdam, the Netherlands (N51.9168, E005.1331).
Water was sampled on 18 Nov 2020 from the upper layer (up to 1 meter depth) at the bank of the pond. At sampling, water temperature, oxygen content and pH were measured.
The water was transported to the laboratory at ambient temperature. Upon arrival in the laboratory, the water was sieved through a 150 μm sieve. The sieved water was stored refrigerated under aerobic conditions (open lid) in the dark until use.
Surface water characteristics were determined at Smithers, UK and shown in Table 1.

Even though the OECD 309 Guideline does not indicate an amount of suspended solids (SS) in the surface water, in the ECHA Decision CCH-D-2114484966-27-01/F, an amount of SS between 10 and 20 mg SPM dw/L was recommended. The current pelagic study was conducted with surface water containing a smaller amount of suspended solids (Total SS, as determined by the spectrophotometer method, of 4 mg/L). Having less suspended solids in the surface water than recommended is not deemed to be of influence on the interpretation of the test results. On the one hand less SS in surface water normally indicates that fewer microorganisms are present (worst case for biodegradation); on the other hand,  less binding of substances with high adsorptive potential can take place to the solids (thus increasing the bioavailability for degradation to the test substance).
As the parent substance has a low adsorption potential and the same is considered applicable to the transformation product TP-1, overall the lower amount of SS in this study is deemed to provide a worst-case situation for biodegradation.

Duration of test (contact time):

60 d

Initial conc.:

90.1 µg/L

Based on:

test mat.

Initial conc.:

19.1 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

test mat. analysis

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: 300 mL/flask
- Composition of medium: surface water
- Test temperature: 12.1 - 12.7 ºC
- pH: 7.7
- pH adjusted: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 1 L amber-coloured conical flasks
- Number of culture flasks/concentration: 7 (+ 1 sterile control + one reserve vessel)
- Method used to create aerobic conditions: The flasks were ventilated gently with humidified air. The air was passed through the flasks just above the water surface. The water was kept in continuously agitation by a stirring bar and a magnetic stirrer.
- Method used to control oxygen conditions: two flasks with SW surface water were prepared to monitor oxygen and pH weekly.
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: the metabolism flasks were connected to a series of traps; a polyurethane foam (PUF) plug inserted in the neck of the metabolism flask, a liquid trap containing 2-propanol (IPA) and two liquid traps containing 2 N NaOH.
- Spike solution: the spike solution for the high concentration was prepared by dissolving radiolabeled Pergafast 201 in acetonitrile. Based on LSC of three 10 µL aliquots, the spike solution contained 0.450 MBq/mL (RSD 0.27 %), which is equivalent to 131 mg/L. The spike solution for the low concentration was prepared by diluting the spike solution for the high concentration in acetonitrile by a factor of 5 to 0.87 MBq/mL (RSD 2.0 %, which is equivalent to 25.3 mg/L). The spike solutions were prepared on the day of spiking. The radiochemical purity of the spike solutions was determined by LC on the same day (and was 95.8%).
- Spiking procedure: Spiking took place on 04 Dec 2020. A volume of 229.7 μL of the low concentration spike solution, was added to nine flasks (low test concentration). To another nine flasks, 222.4 μL high concentration spike solution was added (high concentration). To two flasks, 119 μL reference spike solution was added. The spike volume was chosen based on a water layer volume of 300 mL. Two flasks were treated with 229.7 μL of acetonitrile; one flask was used for monitoring pH and oxygen concentration, the other flask was used as blank for the identification of transformation products.
In the beginning, halfway through and at the end of the spiking procedure, the same volume of spike solution was analysed on LSC (by pipetting the spike solution into separate 5 mL volumetric flasks, which were made up to volume using acetonitrile). The results showed that 19.7 kBq (n=3, RSD 0.39 %) had been added to the low test concentration flasks (equivalent to 19.1 μg/L), 93 kBq (n=3, RSD 0.24 %) had been added to the high test concentration flasks (equivalent to 90.1 μg/L) and 431 kBq (n=3, RSD 1.4 %) had been added to the reference control flasks (equivalent to 49.1 μg/L).

SAMPLING
Two flasks (one low concentration and one high concentration) were sacrificed and sampled 0, 3, 7, 14, 26, 42, and 60 days after spiking. The two sterile controls were sacrificed and sampled 60 days after spiking. NaOH traps for the reference controls were sampled at each sampling interval up to Day 28. On Day 26, radioactivity in the surface water in the reference control flasks was determined.
The NaOH traps (first trap only) were replaced after 14 and 42 days of incubation.

PROCESSING OF SAMPLES FOR ANALYSIS
The water layer was extracted two times for 1 minute with 100 mL dichloromethane. The dichloromethane extracts were combined, weighed and total radioactivity was determined in a 200 μL weighed aliquot. After addition of 200 μL keeper solution (1% glycerol in acetone), the dichloromethane was evaporated on a rotary evaporator (40°C). The residue was dissolved in 5 mL acetonitrile, radioactivity was determined in a 200 μL weighed aliquot and 20 or 50 μL was analyzed on LC. Radioactivity in the residual aqueous phase after extraction with dichloromethane was determined by LSC in a 1 mL weighed aliquot. On day 14 and 60 re-dissolving in ACN resulted in procedural recoveries around 85%. These samples were redissolved a second time using ACN to obtain recoveries >90%. If this could not be achieved with two re-dissolving steps in ACN, acidified ACN was used.
On day 42 and 60 the aqueous residue, after the extraction of the water layer, was concentrated. Hereto a subsample of approximately 150 mL of aqueous residue was weighted and concentrated on a rotary evaporator (60°C) to a volume of approximately 2 mL. Radioactivity was determined by LSC in a 200 μL weighed aliquot and 50 μL was analyzed on LC. Radioactivity of the condensed residue was determined by LSC in a 1 mL weighed aliquot. Also, a 500 μL aliquot of the aqueous residue was analyzed directly on the LC. These direct injection results are used in the calculations. The concentrated samples were used for further identification of the transformation products.
The procedural recoveries were checked after each concentration step to show that no radioactivity was lost during process. On a few occasions (Day 0 low concentration, Day 14 high concentration, Day 42 and Day 16 low concentration) the procedural recovery of the
dichloromethane extraction or the re-dissolvement of extract, was below 90%, with a minimum of 85%. The overall procedural recovery was always between 90-110% meaning that during the total processing, no activity was lost.

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Abiotic sterile control: Two flasks were filled with sterile SW water. Sterilisation was performed by autoclaving for 20 minutes at 121°C. There was a sterile control for each treatment level.

DT50 and DT90 calculations:
The DT50 and DT90 values were calculated using the amounts of parent and transformation products as determined by LC. The choice of models is based on the FOCUS guidance document on estimating degradation kinetics. Optimisations were performed using the program CAKE version 3.4. The parent data (not averaged) were fitted to single first order kinetics (SFO) and to the Gustafson and Holden model (FOMC). If the SFO fit was acceptable and better than the FOMC fit (based on visual assessment, t-test, and χ2 test), no further work was done.
If the SFO fit was not acceptable or the FOMC fit was better, exclusion of outliers, constraining M0 or weighting was tried provided this could be justified by the data. If the SFO results were not improved by these adjustments, the data were also fitted to the hockey stick model (HS) and the double first-order in parallel model (DFOP).

Reference substance:

other: [ring-14C(U)]Benzoic acid

Test performance:

Radiochemical purity of the spike solution was 95.8 % on the day of spiking as determined by LC.
During incubation, the pH ranged between 8.4 and 8.6 (mean 8.5) indicating stable, slightly alkaline conditions. The dissolved oxygen concentrations in the water layer fluctuated between 8.1 and 10.0 mg/L (mean 8.9 mg/L). The measurements indicate aerobic conditions in the water layer throughout the incubation period.

Preliminary test: Pergafast 201 was incubated under aerobic conditions in the laboratory in surface water (Schoonrewoerdse Wiel [SW]) at 12 ± 2 °C in the dark for 7 days. The initial Pergafast 201 concentration in the test system was 92 μg/L. Two flasks were prepared. Samples were taken after 1, 2, 4 and 7 days. Volatiles were trapped by polyurethane foam (PUF), 2- propanol (IPA) and NaOH traps. At the end of the incubation period, the test systems were analysed. Radioactivity in the traps was determined by LSC (IPA and NaOH were directly counted;
PUF after extraction with acetonitrile). The surface water was analysed by LSC and LC.
Results: No significant amounts of radioactivity were trapped in the PUF and IPA traps. In the NaOH traps 1.1 % of radioactivity was trapped as CO2 after 7 days of incubation. The radioactivity
in the water layer remained steady at 100.9 % and 99.8 % after 7 days of incubation.
Mass balances were in the range 101-102 %, indicating that no radioactivity was missing after
7 days of incubation. Based on the results of this preliminary test, the incubation conditions, sampling times and aliquot sizes for the definitive study were determined.

Key result

Compartment:

natural water: freshwater

DT50:

29 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: transformation product

Key result

Compartment:

natural water: freshwater

DT50:

306 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: high concentration (parent)

Key result

Compartment:

natural water: freshwater

DT50:

212 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: Low concentration (parent)

Transformation products:

yes

No.:

#1

Details on transformation products:

Tables 5 and 6: One transformation product (TP-1) was considered a major transformation product as it exceeded 10 % of applied radioactivity at both test concentrations, i.e. 14 % and 11 % of applied radioactivity at the low and high concentration, respectively, after 42 days of incubation.
Transformation product TP-1 was tentatively identified by LC-PDA-RAD/MS. The proposed structure of this transformation product is shown in Figure 1.

Evaporation of parent compound:

no

Volatile metabolites:

no

Remarks:

In the NaOH traps of the low and high concentration, respectively 1.9 % and 0.2% of AR was recovered at the end of the incubation period (60 days). < 0.5 % AR was detected in the polyurethane foam plugs or the 2-propanol traps.

Details on results:

The distribution of the radioactivity between the various fractions of the test system is summarised in Table 2 and Table 3 (low and high test concentration, respectively). The amount of radioactivity in the surface water remained relatively constant throughout the incubation period: 98.1 % (low concentration) and 101.7 % (high concentration) of applied radioactivity after 60 days of incubation.
In the sterile controls, 99.0 % of applied radioactivity (low concentration) and 101.0 % of applied radioactivity (high concentration) was recovered in the surface water after 60 days of incubation.

Mass balances (% of AR recovered in total water layer plus from PUF, IPA and NaOH traps) were between 98 % and 101 % of AR (low test concentration) and between 101 % and 106 % of AR (high test concentration).

Tables 5 and 6: Pergafast 201 degraded to 81 % (low test concentration) and 94 % (high test concentration) of applied radioactivity in the surface water by Day 60. In the sterile control flasks (abiotic conditions), Pergafast 201 did not degrade during the course of the study, with 99.0 % and 101.2 % of applied radioactivity remaining at the low and high concentration, respectively.

Results with reference substance:

The distribution of the radioactivity for the test systems treated with the reference control is summarised in Table 4. In the NaOH traps connected to the test systems spiked with the reference control significant amounts of radioactivity were detected, increasing to 70.9 % and 69.5 % of applied radioactivity after 26 days of incubation. These results indicate sufficiently viable conditions for the test system (SW water).

 

Table 2: Low Concentration: Distribution of Radioactivity in SW Water System

Time [days]	Percentage of applied [%]						Mass Balance^3
	Volatiles			Water layer
	PUF	IPA	NaOH^1	Total water^2	Concentrated extract	Aqueous residue
0	n.a.	n.a.	n.a.	101.1	87.7	3.6	101
3	0.1	0.0	0.1	100.0	93.6	4.3	100
7	0.1	0.0	0.0	100.4	86.4	3.6	100
14	0.4	0.1	0.4	100.4	91.3	6.2	101
26	0.1	0.0	0.1	99.6	87.3	6.8	100
42	0.0	0.4	0.2	97.9	74.6	13.5	98
60	0.0	0.0	1.9	98.1	75.8	20.2	100
60 days (sterile)	0.1	0.1	0.1	99.0	87.1	3.0	99

n.a.: not applicable

PUF: polyurethane foam

¹      Total percentage NaOH measured

²      Water = mean percentage measured in duplicate water sample

³      Mass balance = PUF + IPA + total NaOH + water

 

Table 3: High Concentration: Distribution of Radioactivity in SW Water System

Time [hours / days]	Percentage of applied [%]						Mass Balance^3
	Volatiles			Water layer
	PUF	IPA	NaOH^1	Water^2	Concentrated extract	Aqueous residue
0	n.a.	n.a.	n.a.	105.0	99.4	3.0	105
3	0.0	0.0	0.0	105.1	94.8	5.2	105
7	0.0	0.0	0.0	105.3	93.4	3.9	105
14	0.0	0.0	0.2	105.8	91.5	5.0	106
26	0.0	0.1	0.0	102.6	89.5	9.9	103
42	0.2	0.0	0.1	103.2	92.2	12.5	103
60	0.0	0.0	0.2	101.7	95.3	10.5	102
60 days (sterile)	0.0	0.0	0.1	101.0	95.5	2.7	101

n.a.: not applicable

PUF: polyurethane foam

¹      Total percentage NaOH measured

²      Water = mean percentage measured in duplicate water sample

³      Mass balance = PUF + IPA + total NaOH + water

 

Table 4: Reference Control: Distribution of Radioactivity in SW Water System

Time [days]	Percentage of applied [%]			Mass Balance^3
	Volatiles		Water layer
	IPA	NaOH^1	Water^2
3	n.a.	11.4	n.a.	n.a.
	n.a.	14.2	n.a.	n.a.
7	n.a.	39.5	n.a.	n.a.
	n.a.	37.2	n.a.	n.a.
14	n.a.	60.7	n.a.	n.a.
	n.a.	63.2	n.a.	n.a.
26	0.00	70.9	27.3	98.2
	0.01	69.5	17.4	86.9
26 (IPA)	n.a.	n.a.	n.a.	n.a.
	n.a.	n.a.	n.a.	n.a.

n.a. Not applicable

¹      Total percentage NaOH measured

²      Water = mean percentage measured in duplicate water sample

³      Mass balance = total NaOH + water

 

Table 5: Low Concentration: Parent and Transformation Products in SW Water
(% of applied radioactivity)

Time [days]	code	Parent	TP-1	TP-2	TP-3	Others^2
0	Water	101.1	n.d.	n.d.	n.d.	n.d.
	Concentrated extract	85.9	n.d.	n.d.	0.8	1.0
3	Water	100.0	n.d.	n.d.	n.d.	n.d.
	Concentrated extract	90.2	n.d.	n.d.	2.5	0.9
7	Water	96.8	n.d.	n.d.	n.d.	3.6
	Concentrated extract	83.1	n.d.	n.d.	1.6	1.6
14	Water	90.0	n.d.	n.d.	n.d.	10.4
	Concentrated extract	85.8	n.d.	n.d.	5.4	n.d.
26	Water	99.6	n.d.	n.d.	n.d.	n.d.
	Concentrated extract	87.3	n.d.	n.d.	n.d.	n.d.
42	Water	86.0	8.5	n.d.	n.d.	3.4
	Concentrated extract	68.6	n.d.	n.d.	3.9	2.2
	Aqueous residue^1	n.d.	13.5	n.d.	n.d.	n.d.
60	Water	81.4	13.6	n.d.	n.d.	3.1
	Concentrated extract	71.1	n.d.	n.d.	n.d.	4.8
	Aqueous residue^1	n.d.	14.1	3.6		2.5
60 days (sterile)	Water	99.0	n.d.	n.d.	n.d.	n.d.
	Concentrated extract	79.0	n.d.	n.d.	6.5	n.d.

n.d. Not detectable

¹Aquaous residue analyzed using direct 500 µL injections

²Individual transformations product were <10%

 

Table 6: High Concentration: Parent and Transformation Products in SW Water

(% of applied radioactivity)

Time [days]	code	Parent	TP-1	TP-2	TP-3	Others
0	Water	105.0	n.d.	n.d.	n.d.	n.d.
	Concentrated extract	96.7	n.d.	n.d.	1.5	1.2
3	Water	103.4	1.2	0.5	n.d.	n.d.
	Concentrated extract	90.1	n.d.	n.d.	2.7	2.0
7	Water	101.9	0.7	1.2	n.d.	1.4
	Concentrated extract	86.1	n.d.	n.d.	5.4	1.9
14	Water	104.4	1.5	n.d.	n.d.	n.d.
	Concentrated extract	86.1	n.d.	n.d.	4.5	0.9
26	Water	96.2	6.4	n.d.	n.d.	n.d.
	Concentrated extract	88.1	n.d.	n.d.	1.0	0.4
42	Water	87.8	8.2	n.d.	n.d.	7.2
	Concentrated extract	90.4	n.d.	n.d.	1.1	0.6
	Aqueous residue^1	1.4	11.1	n.d.	n.d.	n.d.
60	Water	93.7	5.8	1.0	n.d.	1.2
	Concentrated extract	89.1	n.d.	0.6	2.0	3.5
	Aqueous residue^1	1.3	5.2	1.8	n.d.	2.1
60 days (sterile)	Water	101.2	n.d.	n.d.	0.9	1.5
	Concentrated extract	91.6	n.d.	n.d.	2.7	1.3

n.d. Not detectable

¹Aquaous residue analyzed using direct 500 µL injections

Validity criteria:

The results of the reference controls (benzoic acid) should show that the test system was sufficiently viable. The expected time interval for degradation of sodium benzoate is usually less than 2 weeks.

Observed value:

62 % CO2 formation at 14 days

Validity criteria fulfilled:

yes

Validity criteria:

The analytical method is sufficiently validated (refer to CRL 20248639)

Observed value:

The performance check of the analytical method for the analysis of 14C-labeled test item in surface water was successfully performed. LOD = 0.3- 0.6% of AR. LOQ = 0.7 - 1.4% of AR

Validity criteria fulfilled:

yes

Validity criteria:

the radiolabelled mass balance should range from 90% to 110%

Observed value:

Mass balances were between 98 % and 101 % of AR (low test concentration) and between 101 % and 106 % of AR (high test concentration).

Validity criteria fulfilled:

yes

Conclusions:

Pergafast 201 degraded to 81.4 % (low test concentration) and 93.7 % (high test concentration) of applied radioactivity in the surface water by Day 60.
One transformation product (TP-1) was considered a major transformation product as it exceeded 10 % of applied radioactivity at both test concentrations, i.e. 13.5 % and 11.1 % of applied radioactivity at the low and high concentration, respectively, after 42 days of incubation. Mineralization was not a major route of degradation since a maximum of 1.9 % of applied radioactivity was recovered as CO2. At both test concentrations negligible organic volatiles were detected (≤ 0.5 %).
The DT50 values of Pergafast 201 in surface water were between 212 and 306 days, for the low and high test concentration, respectively. The DT50 of the major transformation product TP-1 in surface water was 29 days.

Executive summary:

The mineralisation and rate of degradation of 14C- labeled Pergafast 201 in surface water under aerobic conditions at 12°C was determined in accordance with OECD 309 and GLP principles. Incubation with initial Pergafast 201 concentrations of 19 µg/L and 90 µg/L was in the dark for 60 days. Dedicated flasks were sampled immediately after spiking and 3, 7, 14 , 26, 42, and 60 days after spiking. Volatiles were trapped by polyurethane foam, 2-propanol and NaOH traps. The surface water was analysed by LSC and LC.

Pergafast 201 degraded to 81 % (low test concentration) and 94 % (high test concentration) of applied radioactivity in the surface water by Day 60. One transformation product (TP-1) was considered a major transformation product as it exceeded 10 % of applied radioactivity at both test concentrations, i.e. 14 % and 11 % of applied radioactivity at the low and high concentration, respectively, after 42 days of incubation. TP-1 was tentatively identified by LC-PDA-RAD/MS.

Mineralization was not a major route of degradation since a maximum of 2 % of applied radioactivity was recovered as CO₂. At both test concentrations negligible organic volatiles were detected (≤ 0.5 %).

DT50 values of the parent substance were 212 days and 306 days for the low and high test concentrations, respectively. DT50 value of TP1 was 29 days.

The study is considered reliable without restrictions.

Description of key information

The results of the aerobic mineralisation study of radiolabeled Pergafast 201 at 12 ºC in surface water show that the DT50 values are between 212 days (low test concentration) and 306 days (high test concentration). Geomean measured value is 255 days.

As these values indicate that the substance is Persistent in water, no further studies are required for the other compartments.

Key value for chemical safety assessment

Half-life in freshwater:

255 d

at the temperature of:

12 °C