O,O,O-tris(2(or 4)-C9-10-isoalkylphenyl) phosphorothioate

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

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:

adopted in 2002

Deviations:

yes

Remarks:

TOC content of TS1 was slightly below the recommended 0.5 %. The difference in TOC content between TS 1 and TS 2 was 1.8% at arrival at the lab, but > 2% at the beginning of acclimatization and at the beginning and end of the exposure

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: 1281-1012
- Purity, including information on contaminants, isomers, etc.: 100%

RADIOLABELLING INFORMATION
- Radiochemical purity: 100%
- Specific activity (a.i.): 2.57 MBq/mg
- Locations of the label: phenyl-ring-U-C14

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Test substance in acetonitrile: stored at <= -18 °C in the dark
Test substance stock solution in acetone: stored at 4.2°C - 12.5°C in the dark
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage:
The stability of the test substance under storage conditions was confirmed by measurement of the test substance dissolved in acetonitrile before exposure (99.9%) and after the end of experimental exposure in acetone stock solutions (100%)

Radiolabelling:

yes

Remarks:

Phenyl-ring-U-C14

Oxygen conditions:

aerobic

Inoculum or test system:

natural water / sediment

Details on source and properties of surface water:

1) Test system 1 "Ranschgraben" (hereafter "TS 1")

- Details on collection (e.g. location, sampling depth, contamination history, procedure):
Stream named “Ranschgraben” west from the city of Schifferstadt, near the federal road L 532 surrounded by a forest (GPS coordinate 94.403855, 8.402153). The water was colourless, but slightly cloudy and without any specific odour.

- Temperature (°C) at time of collection: 13.1 °C
- pH at time of collection: 7.5
- Redox potential (mv): Start of Exposure: 94 mV; End of Exposure: 271 mV in control assays
- Oxygen concentration (mg/l): Start of Exposure 7.8 mg/L; End of Exposure: 8.45 mg/L in control assays
- Water filtered: no


2) Test system "Berghäuser Altrhein" (hereafter "TS 2")

- Details on collection (e.g. location, sampling depth, contamination history, procedure):
Side arm of the river Rhein named “Berghäuser Altrhein” south from the city of Speyer, near the industrial park south surrounded by a forest (GPS coordinate 49.292256, 8.448619). The water was clear and colourless and without any particular odour.

- Temperature (°C) at time of collection: 16.6 °C
- pH at time of collection: -
- Redox potential (mv): Start of Exposure: 176 mV; End of exposure: 265.5 mV in control assays
- Oxygen concentration (mg/l): Start of Exposure: 7.3 mg/L; End of Exposure: 8.4 mg/L in control assays
- Water filtered: no

Details on source and properties of sediment:

1) Test system "Ranschgraben" (TS 1)

- Details on collection (e.g. location, sampling depth, contamination history, procedure):
Stream named “Ranschgraben” west from the city of Schifferstadt, near the federal road L 532 surrounded by a forest (GPS coordinate 94.403855, 8.402153). The sediment smelled slightly mouldy and appeared grey/brown.

- Textural classification (i.e. %sand/silt/clay): Coarse Texture with 0.33% clay and silt
- pH at time of collection: 7.6
- Organic carbon (%): 0.2% TOC (start of exposure)
- Redox potential (mv): Start of Exposure: -107 mV; End of exposure: -169 mV in control assays
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): Start of Exposure 1.2*10^6 cfu/g; End of exposure 1.2*10^5 cfu/g in control assays
- Sediment samples sieved: yes, first through a 20 mm sieve and afterwards through a 2 mm sieve

2) Test system "Berghäuser Altrhein" (TS 2)

- Details on collection (e.g. location, sampling depth, contamination history, procedure):
Side arm of the river Rhein named “Berghäuser Altrhein” south from the city of Speyer, near the industrial park south surrounded by a forest (GPS coordinate 49.292256, 8.448619). The sediment smelled neutral and appeared grey/brown.

- Textural classification (i.e. %sand/silt/clay): Fine texture with 52.1% clay and silt
- pH at time of collection: 7.6
- Organic carbon (%): 2.3% TOC (start of exposure)
- Redox potential (mv): Start of Exposure: -153 mV; End of exposure - 183 mV in control assays
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): Start of Exposure 2.1*10^7 cfu/g; End of Exposure 7.4*10^5 cfu/g
- Sediment samples sieved: yes, first through a 20 mm sieve and afterwards through a 2 mm sieve

Duration of test (contact time):

100 d

Initial conc.:

622 µg/L

Based on:

test mat.

Remarks:

equivalent to ~0.8 MBq in 500 mL water phase

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Details on study design:

ACCLIMATIZATION

After filling the test vessels with sediment and water phase, the bottles were closed with a lid containing gas inlet tube attached with the air system that supplied moistened pressurised air (3 bubbles/second in water layer) and incubated under dark conditions at 20 ±2 °C. The test systems were further allowed to equilibrate for 20 days (acclimatization from 05th May 2020 – 25th May 2020) based on the continuous measurements of parameters along with the observation of macroscopic separation of phases in the test vessels.

TEST CONDITIONS
- Volume of test solution/treatment: Approx. 500 mL water with a sediment layer of 252 g dry weight/319 g wet weight (TS1) and 109 g dry weight/236 g wet weight (TS2)
- Composition of medium: TS1: Approx. 6.7 cm of water and 2.6 cm of wet sediment; TS2: Approx. 6.6 cm of water and 2.4 cm of wet sediment; Ratio Volume Water/Sediment approx. 3:1
- Solubilising agent: Acetone
- Test temperature: 21.8 - 22.7 °C
- pH: TS1 water: 8.6 - 8.8; TS1 sediment: 7.1-7.4; TS2 water: 7.6 - 8.2; TS2 sediment: 7.3 - 7.4
- pH adjusted: no
- CEC (meq/100 g): -
- Aeration of dilution water: yes
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: no


TEST SYSTEM
- Culturing apparatus: 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.
- Number of culture flasks/concentration: In total 44 test vessels were used for the test. For each test system 16 test substance assays (TA), 2 inhibition assays (IC), 2 control assays (CA) and 2 solvent control assays (CAS) were tested.
- Method used to create aerobic conditions: The test vessels were closed with a lid containing gas inlet tube attached with the air system that supplied moistened pressurised air (3 bubbles/second in water layer) the air bubbles were as small as possible and the water layer in the test vessels was slightly moved.

- Details of trap for CO2 and volatile organics if used: After the acclimatization phase, the test bottles were connected serially to 4x 250 mL absorption flasks filled with absorption liquids. The first and second trap directly in contact with the test vessel were filled with 100 mL 1 M sodium hydroxide solution for the adsorption of carbon dioxide from biodegradation processes. Third trap consists of 50 mL ethylene glycol to absorb volatile organic substances, and the last flask was filled with 50 mL 0.1 M sulphuric acid for volatile alkali substances.

SAMPLING
- Sampling frequency: Two replicates of test vessels with the test substance of each test system were taken after 0 hr, 2d, 8d, 21d, 49d, 65d, 85d and 100d.

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 control assays without the test item
- Toxicity control: 2 inhibition assays for measuring the toxic or inhibition effects of the test substance on the microbial activity
- Solvent Control: 2 additional control assays with the solvent to measure adverse effects of solvent on the microbial activity of the test system

Test performance:

No inhibition of microorganisms was observed in the inhibition Controls and the solvent controls.

Compartment:

natural water / sediment

% Total extractable:

>= 97.4 - <= 101.1

% Non extractable:

>= 0.3 - <= 0.5

% CO2:

0

% Other volatiles:

0

% Recovery:

99.7

St. dev.:

1.8

Remarks on result:

other: TS 1 - Day 0 - TA 1 and TA 2

Compartment:

natural water / sediment

% Total extractable:

>= 63.3 - <= 78

% Non extractable:

>= 4.6 - <= 4.9

% CO2:

>= 0.2 - <= 0.3

% Other volatiles:

0

% Recovery:

77

St. dev.:

7.6

Remarks on result:

other: TS 1 - day 100 - TA 15 and TA 16

Compartment:

natural water / sediment

% Total extractable:

>= 94.4 - <= 100.3

% Non extractable:

>= 0.2 - <= 0.5

% CO2:

0

% Other volatiles:

0

% Recovery:

97.8

St. dev.:

2.7

Remarks on result:

other: TS 2 - Day 0 - TA 17 and TA 18

Compartment:

natural water / sediment

% Total extractable:

>= 63.6 - <= 85.9

% Non extractable:

>= 4.4 - <= 8

% CO2:

>= 0.1 - <= 0.2

% Other volatiles:

0

% Recovery:

82.1

St. dev.:

12.6

Remarks on result:

other: TS 2 - day 100 - TA 31 and TA 32

Compartment:

natural sediment

% Total extractable:

>= 6.8 - <= 7.9

% Non extractable:

>= 0.3 - <= 0.5

Remarks on result:

other: TS 1 - Day 0 - TA 1 and TA 2

Compartment:

natural sediment

% Total extractable:

>= 62.9 - <= 77.5

% Non extractable:

>= 4.6 - <= 4.9

Remarks on result:

other: TS 1 - day 100 - TA 15 and TA 16

Compartment:

natural sediment

% Total extractable:

>= 3.5 - <= 8.3

% Non extractable:

>= 0.2 - <= 0.5

Remarks on result:

other: TS 2 - Day 0 - TA 17 and TA 18

Compartment:

natural sediment

% Total extractable:

>= 63.5 - <= 85.5

% Non extractable:

>= 4.4 - <= 8

Remarks on result:

other: TS 2 - day 100 - TA 31 and TA 32

Compartment:

natural water / sediment: freshwater

% Total extractable:

>= 92.1 - <= 94.6

% Non extractable:

>= 0.3 - <= 1.3

Remarks on result:

other: TS 1 - Day 0 - TA 1 and TA 2

Compartment:

natural water / sediment: freshwater

% Total extractable:

0.2

% Non extractable:

0.2

Remarks on result:

other: TS 1 - day 100 - TA 15 and TA 16

Compartment:

natural water / sediment: freshwater

% Total extractable:

>= 90.7 - <= 97.8

% Non extractable:

>= 0.2 - <= 0.3

Remarks on result:

other: TS 2 - Day 0 - TA 17 and TA 18

Compartment:

natural water / sediment: freshwater

% Total extractable:

0 - <= 0.1

% Non extractable:

0.1

Remarks on result:

other: TS 2 - day 100 - TA 31 and TA 32

Parent/product:

parent

Compartment:

total system

Key result

% Degr.:

>= 0.1 - <= 0.3

Parameter:

CO2 evolution

Sampling time:

100 d

Key result

Compartment:

natural sediment

DT50:

> 1 000 d

Type:

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

Temp.:

22.3 °C

Remarks on result:

other: Test System 2 - Berghäuser Altrhein

Key result

Compartment:

natural water: freshwater

DT50:

1.05 d

Type:

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

Temp.:

22.3 °C

Remarks on result:

other: Test System 2 - Berhäuser Altrhein

Key result

Compartment:

natural water / sediment

DT50:

439 d

Type:

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

Temp.:

22.3 °C

Remarks on result:

other: Total Test System 2 - Berghäuser Altrhein

Key result

Compartment:

natural sediment

DT50:

434 d

Type:

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

Temp.:

22.3 °C

Remarks on result:

other: Test System 1 - Ranschgraben

Key result

Compartment:

natural water: freshwater

DT50:

ca. 3 d

Type:

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

Temp.:

22.3 °C

Remarks on result:

other: Test System 1 - Ranschgraben

Key result

Compartment:

natural water / sediment

DT50:

304 d

Type:

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

Temp.:

22.3 °C

Remarks on result:

other: Total Test System 1 - Ranschgraben

Other kinetic parameters:

first order rate constant

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

Details on transformation products:

Transformation Product #1
- Retention time: 24.1 min
- Chemical formula: C45H69O4P
- m/z: 705.5006 HESI+
- Formed amount: Minor < 5% TAR

Transformation Product #2
- Retention time: 15.5 min
- Chemical formula: C15H24O
- m/z: 219.175 HESI-
- Formed amount: Minor < 5% TAR

Transformation Product #3:
- Retention time: 1.1 min
- Formed amount: Minor < 3% TAR

The third potential degradation product (retention time 1.1 min) was detected but could not be identified for the following reasons:
- It was not possible to allocate a clear corresponding mass manually
- A comparable isotope pattern to 14C labelled O,O,O-tris(2(or 4))-C9-C10-isoalkylphenyl) phosphothoriate was not archived by compound discoverer
- A reasonable mass was principally found by a compound discoverer search for typical degradation products but the retention time of the proposed mass was not always conform to the retention time of the detector for radioactivity
- There was no evidence for mass which confirmed a connection to 14C labelled O,O,O-tris(2(or 4))-C9-C10-isoalkylphenyl) phosphothoriate
- The fragmentation experiment did not confirm an expected structure

Degradation products of high polarity and very short retention times (1.1 min) are often difficult to identify since the separation process is limited and co-eluting several components is possible

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

yes

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): Variations observed in the acetone LSC measurements due to the stability and homogeneity of the acetone extracts, which resulted in low recoveries. Several treatment of acetone extracts such as resolving concentrated acetone extract in methanol or dichloromethane and DMSO addition were applied to solve the issue. Additionally adsorption issues in the HPLC vials for some of the samples were observed.



MINOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
Transformation Product #1: 0.0 - 0.3% Day 65 (TS 1); 3.6 - 4.3% Day 49 (TS 2)
Transformation Product #2: 0.0 - 0.5% Day 85 (TS 1); 1.7 - 4.0% Day 49 (TS 2)
Transformation Product #3: 1.0 - 2.2% Day 85 (TS 1); 0.6 - 1.1% Day 49 (TS 2)
- Range of maximum concentrations in % of the applied amount at end of study period:
Transformation Product #1: 0.0 - 0.0% (TS1); 1.6 - 2.5% (TS 2)
Transformation Product #2: 0.0 - 0.0% (TS1); 1.2 - 1.4% (TS 2)
Transformation Product #3: 0.7 - 1.1% (TS1); 0.7 - 1.2% Day 85 (TS 2)

EXTRACTABLE RESIDUES
- % of applied amount at day 0 (sediment): 6.8 - 7.9%% (TS 1, Ranschgraben); 3.5 - 8.5% (TS 2, Berghäuser Rheinarm)
- % of applied amount at end of study period (sediment): 62.9 - 77.5% (TS 1, Ranschgraben); 63.5 - 85.8% (TS 2, Berghäuser Rheinarm)


NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: 0.3 - 0.5% (TS1, Ranschgraben); 0.2 - 0.5% (TS 2, Berghäuser Rheinarm)
- % of applied amount at end of study period: 4.6 - 4.9% (TS1, Ranschgraben); 4.4 - 8.0% (TS 2, Berghäuser Rheinarm)


MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 0.1 - 0.3%

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: 0%

Validity criteria:

Accuracy of the transformation data

Observed value:

See attached document "126019-82-7_OECD 308_Quality Criteria"

Validity criteria:

Repeatability and sensitivity of the method of analysis

Observed value:

See attached document "126019-82-7_OECD 308_Quality Criteria"

Validity criteria:

Recovery Rate

Observed value:

See attached document "126019-82-7_OECD 308_Quality Criteria"

Conclusions:

The substance is considered to be (very) persistent in the water/sediment systems according to REACh relevant criteria.

Executive summary:

One study investigating the biodegradability of O,O,O-tris(2(or 4)-C9-10-isoalkylphenyl) phosphorothioate in the environment is available. In this study, the biodegradation rates of the test substance in a water/sediment system and the identification of its biotransformation products and their distribution into water and sediment was investigated according to OECD guideline 308 under GLP conditions using radiolabeled test material (BASF 2021). Two different natural water/sediment systems were used differing in TOC content of the sediment (TS1: 0.2% and TS2: 2.3% AR). The test vessels were incubated in dark conditions at a temperature of 22.3 °C for 100 days. Whole system mineralization of the parent compound to carbon dioxide accounted for 0.2 – 0.3% TAR after 100 days incubation. Formation of organic or inorganic volatile were absent during the exposure. At the end of exposure very few radioactivity was found in water (TS 1: 0.4 - 0.5% TAR, TS 2: 0.1% TAR). The remaining radioactivity was found in sediment (TS1: 76.3%; TS2: 81.5%). NERs in sediment was dependent upon the TOC content of the sediment: TS1: 4.6% - 4.9% TAR and 4.4% - 8.0% TAR.

Half-life of the parent compound for both test systems were calculated and showed a very slow dissipation of the substance in TS 1 (DT50: 304d, total system; 434d, sediment) and TS 2 (DT50: 439d, total system; > 1000d, sediment).

Three minor metabolites were detected, two of them could be identified as tris(nonylphenyl)phosphate (RT = 24.1 min) and Nonylphenol (branched) (RT = 15.5 min) whereas it was not possible to identify the third metabolite (RT = 1.1 min). For tris(nonylphenyl)phosphate a maximum activity of 4.3% TAR (TS 2) was determined, for Nonylphenol (branched) a maximum activity of 4.0% (TS 2) was detected. The rest of the activity can be attributed to the parent compound. 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. Based on the half-life of O,O,O-tris(2(or 4)-C9-10-isoalkylphenyl) phosphorothioate in sediment and the total system, the substance can be considered very persistent in the environment.

Description of key information

DT50 > 120d in surface water and sediment (OECD 308)

Key value for chemical safety assessment

Half-life in freshwater sediment:

1 000 d

at the temperature of:

22.3 °C

Whole System

Half-life in whole system:

439 d

at the temperature of:

22.3 °C

Type of system:

other: Freshwater and sediment TS 2

Half-life in whole system:

304 d

at the temperature of:

22.3 °C

Type of system:

other: Freshwater and sediment TS 1

Additional information

One study investigating the biodegradability of O,O,O-tris(2(or 4)-C9-10-isoalkylphenyl) phosphorothioate in the environment is available. In this study, the biodegradation rates of the test substance in a water/sediment system and the identification of its biotransformation products and their distribution into water and sediment was investigated according to OECD guideline 308 under GLP conditions using radiolabeled test material (BASF 2021). Two different natural water/sediment systems were used differing in TOC content of the sediment (TS1: 0.2% and TS2: 2.3% AR). The test vessels were incubated in dark conditions at a temperature of 22.3 °C for 100 days. Whole system mineralization of the parent compound to carbon dioxide accounted for 0.2 – 0.3% TAR after 100 days incubation. Formation of organic or inorganic volatile were absent during the exposure. At the end of exposure very few radioactivity was found in water (TS 1: 0.4 - 0.5% TAR, TS 2: 0.1% TAR). The remaining radioactivity was found in sediment (TS1: 76.3%; TS2: 81.5%). NERs in sediment was dependent upon the TOC content of the sediment: TS1: 4.6% - 4.9% TAR and 4.4% - 8.0% TAR.

Half-life of the parent compound for both test systems were calculated and showed a very slow dissipation of the substance in TS 1 (DT50: 304d, total system; 434d, sediment) and TS 2 (DT50: 439d, total system; > 1000d, sediment).

Three minor metabolites were detected, two of them could be identified as tris(nonylphenyl)phosphate (RT = 24.1 min) and Nonylphenol (branched) (RT = 15.5 min) whereas it was not possible to identify the third metabolite (RT = 1.1 min). For tris(nonylphenyl)phosphate a maximum activity of 4.3% TAR (TS 2) was determined, for Nonylphenol (branched) a maximum activity of 4.0% (TS 2) was detected. The rest of the activity can be attributed to the parent compound. 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. Based on the half-life of O,O,O-tris(2(or 4)-C9-10-isoalkylphenyl) phosphorothioate in sediment and the total system, the substance can be considered very persistent in the environment.