O,O,O-triphenyl phosphorothioate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-11-04 to 2008-12-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Bacteria collected from activated sludge of the sewage treatment plant of Romanshorn, Switzerland
- Preparation of inoculum for exposure: The preparation was carried out according to the method described in the guideline.
- Concentration of sludge: a 30 mg solids/mL inoculum solution (24.5 g wet sludge with 50 mL mineral medium) was added to mineral medium directly after preparation.

Duration of test (contact time):

29 d

Initial conc.:

0.26 µg/L

Based on:

other: radiolabelled test material

Initial conc.:

0.27 µg/L

Based on:

other: non-radiolabelled test material (used in toxicity control)

Parameter followed for biodegradation estimation:

CO2 evolution

Parameter followed for biodegradation estimation:

radiochem. meas.

Remarks:

HPLC-RAM

Details on study design:

TEST CONDITIONS
2400 mL of the mineral solution and 3 mL inoculum were aerated for 24 hours in the test vessel. In the 2403 mL mineral solution, 110 mL of the radiolabelled test substance application stock solution (final concentration 0.26 µg/L) were added and homogenized. This solution was given to the test vessel, filled up to 3000 mL with mineral medium and immediately connected to the CO2 traps.
- Test temperature: 22 ± 2°C

STOCK SOLUTIONS
- Radiolabelled test item: A small amount (covering the tip of a spatula) was dissolved in 200 mL methanol. More test item had to be added as the first activity measured by liquid scintillation counting (LSC) was too low. LSC measurements determined a concentration of 19.8 µg/mL test substance in the stock solution. An application stock solution was prepared by transferring 50 mL of the stock solution into a 500 mL volumetric lfask. The methanol was evaporated and the test item resolved in 500 mL mineral medium. The final application stock solution was measured with LSC and was determined to have a concentration of 0.0072 µg/mL.
- Toxicity control: 79.59 mg of the non-radiolabelled test item were dissolved in 10 mL methanol. One mL of this first stock solution was diluted to 100 mL with methanol. To obtain the final application stock solution, 1.0 mL of the second stock solution was evaporated under a stream of nitrogen to complete dryness and resolved in 100 mL mineral medium. The toxicity control also contained the reference compound. Final concentration of the non-radiolabelled test item was 0.27 µg/L.
- Reference item: For the procedure control and the toxicity control two stock solutions were prepared on day 0 by dissolving 102.74 and 102.94 mg of sodium benzoate in 250 mL mineral medium, respectively. Final concentration was 20 mg TOC/L.
- Other:

TEST SYSTEM
- Culturing apparatus: 5 Liter flasks equipped with gas inlet and magnetic stirrer.
- Number of culture flasks/concentration: 2 for test suspension with radiolabelled test item, 2 for the inoculum blanks, 1 for procedure control and 1 for toxicity control
- Method used to create aerobic conditions: aerated with air free of carbon dioxide.
- Measuring equipment: carbon analyzer; HPLC-RAM was used for measuring the radioactivity present in the test suspension
- Details of trap for CO2 and volatile organics if used: Determination of the radioactivity from the KOH traps and determination of the absorbed CO2 in the Ba(OH)2 traps on the days 1, 2, 3, 6, 8, 10, 14, 17, 22, 28 and 29

SAMPLING
- Sampling frequency: 1, 2, 3, 6, 8, 10, 14, 17, 22, 28 and 29 days

CALCULATIONS:
- The biodegradation was calculated on the basis of the theoretical carbon content of the test substance and the cumulative quantities of carbon dioxide determined on the days of measurements. For the calculation the formula given in the guideline was used. For the blank values, the blank control with the vehicle was used.

Reference substance:

benzoic acid, sodium salt

Remarks:

20 mg TOC/L (procedure control)

Reference substance:

benzoic acid, sodium salt

Remarks:

20 mg TOC/L (toxicity control)

Key result

Parameter:

% degradation (CO2 evolution)

Value:

17.8 - 19.3

Sampling time:

29 d

Remarks on result:

other: Dose: 0.26 µg/L

Parameter:

% degradation (radiochem. meas.)

Value:

39.2 - 48.5

Sampling time:

29 d

Remarks on result:

other: Results are % of radioactivity present after 29 days referring to transfomation products of the radiolabelled test item

Details on results:

The biodegradation calculated as percentage of measured amount of carbondioxide over the theory was:
0.26 µg test substance/L = 17.8 - 19.3% in 29 days.

In addition, the transformation of the radiolabelled test item into its transformation and degradation product was measured as radioactivity via HPLC-RAM. After 29 days only 51.5% and 60.8% of the radioactivity present consisted of the radiolabelled test item. The remaining radioactivity, i.e. 48.5% and 39.2%, consisted of metabolites. From the HPLC-RAM chromatogram of the two test suspensions the detected metabolites are considered to be phenole (at a retention time of 2.6 minutes both), phenylthiophosphate (at a retention time of 10.3 and 12.8 minutes) and phenylphosphates (at a retention time of 14.5 and 14.8 minutes).

Results with reference substance:

procedure control = 90.6% in 14 days.
toxicity control = 98.1% in 29 days (87.7% in 14 days)

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-09-02 to 2008-10-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Deviations:

yes

Remarks:

, radiolabelled substance was used. Aeration was not done with purified and humidified air. Losses due to evaporation were replaced by filling up with deionized water.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): obtained from the sewage treatment plant Romanshorn, Switzerland
- Preparation of inoculum for exposure: centrifuged at 7380 rpm and 20°C for 5 minutes. Supernatant was discarded and the procedure repeated until sufficient sludge was concentrated. The sludge was washed twice with mineral medium and the supernatant discarded. The moisture content was determined by drying.
- Concentration of sludge: To obtain a final concentration of 0.2 g solid/L in the test systems, approximately 3.65 g of the activated sludge were added to the test vessels
- Water filtered: no

Duration of test (contact time):

28 d

Initial conc.:

0.26 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

Remarks:

LSC analysis of applied radioactivity

Details on study design:

TEST CONDITIONS
- Composition of medium: as described in DOC Die-away, CO2 evolution, Manometric Respirometry and Modified OECD Screening methods of Guideline 301 (adopted 1992) for determining ready biodegrability
- Test temperature: 20 - 25°C
- Aeration of dilution water: yes
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Glass beakers with volume of 2 L, equipped with magnetic stirrer and glass tube to introduce purified air.
- Number of culture flasks/concentration: 2 for control and test substance, 1 for reference substance
For the test, a super stock solution was prepared by adding a small amount (covering a small spatula) of the test item to 200 mL methanol. The super stock solution was analyzed by liquid scintillation counting (LSC) to determine the total radioactive residue (TRR). To obtain a final test concentration of 0.24 to 0.28 µg/L, corresponding to 85000 an 10000 dpm/L, more test item had to be added to the stock solution. This procedure was repeated until the appropriate concentration was obtained. The final super stock concentration was 21.5 µg/200 mL.
To prepare the application stock solution, 15 mL of the super stock solution was transferred to a 500 mL volumetric flask and evaporated to complete dryness under a stream of nitrogen. The flask was filled with mineral medium to the mark. The solution was submitted to ultrasonic treatment for approximately 10 minutes before it was mixed for 30 minutes at 60°C. The total radioactivity was determined during the mixing process. The application stock solution had a final concentration of 0.0030 µg/mL. Prior to application the stock solution was mixed over night at room temperature.

SAMPLING
- Sampling frequency: after 3,5 hours, 2, 8, 12, 16, 23, 27 and 28 days
- Sampling method: 6 mL of samples were used for determination of degradation. Samples of the blank controls were filtered by special membrane filters, discarding the first mL. The resulting 5 mL were used for DOC determination. One drop of H3PO4 was added to each sample before they were stored in the freezer until use. The samples containing the test item were not filtered as the samplings of day 0 showed that the radioactivity was bound to the filter. Liquid-Scintillation-Counting (LSC) measurements were performed with the resulting 5 mL after filtration. Additionally, LSC was measured from samples without filtration. The results showed that the radioactivity was bound to the filter. Therefore, no filtration was performed from samples of the test suspensions at the remaining sampling intervals. As the test item concentration was too low for DOC measurements, no DOC was determined.
Since the radioactivity in the application solution was too low for HPLC/RAM analysis, only LSC measurements were performed.

Reference substance:

ethylene glycol

Remarks:

165 mg TOC/L

Key result

Parameter:

% degradation (radiochem. meas.)

Value:

>= 59.5 - <= 66.8

Sampling time:

28 d

Remarks on result:

other: Dose: 0.26 µg/L

Details on results:

After 28 days, based on LSC analysis, recoveries of 37.1 and 29.9% of the initial applied concentration were found

Results with reference substance:

degradation based on measured DOC: 98.2% in 28 days

Validity criteria fulfilled:

yes

Description of key information

17-19 % CO2 evolution in 28 days (OECD 301B)

59-67% DOC removal in 28 days (OECD 302C)

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria

Type of water:

freshwater

Additional information

Five screening studies are available investigating the biodegradability of O,O,O-triphenyl phosphorothiate. Four studies investigated the ready biodegradability and one investigated the inherent biodegradation of the test substance.

The key study assessed the ready biodegradability of O,O,O- triphenyl thiophosphate over a 28-day period using OECD guideline 301B (Springborn Smithers 2009). The nominal test concentration was 0.26 µg a.s./L (two replicates), which was in the range of the water solubility of the test substance. CO₂-measurements showed biodegradation in 28 days of 17.8 to 19.3% based on CO2 evolution. Further investigations during the experimental phase using radiolabelled test material showed that after 28 days only 51.5% and 60.8% of the radioactivity present consisted of the parent substance, whereas the remaining radioactivity consisted of its degradation products. Hence, under conditions of this screening test the test substance is substantially biotransformed (primary degradation) of around 50% in 28 days and partially mineralized (ultimate degradation) of up to 19 %. Since phenol was identified from the chromatogram of the degradation products, one can assume that degradation pathway is via cleavage of phenol and further biodegradation of the phenol formed. 

In addition, the inherent biodegradability of radiolabelled test substance was assessed over a 28-day period using OECD guideline 302C (Springborn Smithers 2008). A nominal concentration of 0.26 µg a.s./L was tested, which is in the range of the water solubility of the test substance. The test material attained up to 59.5 and 66.8 % DOC removal after 28 days. This result indicate that the test substance is easily eliminable from water; however, DOC removal does not give a clear indication of inherent degradability.

 

In a supporting study, the ready biodegradability of O,O,O- triphenyl thiophosphate was assessed over a 28-day period by the modified Sturm test (OECD 301B) in concentrations of 10 and 20 mg/L. No biodegradation (0 -2%) was observed. In line with the key study on ready biodegradation described above, the results indicate that the test item cannot be classified readily biodegradable under conditions of the test. However, the very low solubility of the substance may have reduced the availability and hence the degradability of the substance in the test. For characterization of the persistency of the substance the test-set-up of the supporting study was not sufficient due to the low solubility of the test item. Two additional supporting studies on ready biodegradability with O,O,O-triphenylphosphorotioate in a lower purity are available. In a Closed Bottle Test according to OECD guideline 301D no degradation was observed after 28 days using 3 mg/L test substance (Huntington 1998). In a study according to OECD 301C (Institute of Ecotoxicology, Gakushuin University 1999) no degradation was observed after 28 days using 100 mg/L test item. Considering that the substance consists of several adsorptive constituents with poor water solubility, the limited bioavailability in the test design could be the reason for the lack of observable biodegradation. Overall, the results of supporting studies are not considered to be contradictory to the results of the key study. The methodology of these studies was not sensitive enough to measure the degradability.

 

In conclusion, O,O,O- triphenyl thiophosphate is not readily biodegradable but showed to be slowly biotransformed and to a certain degree mineralized in presence of suitable amounts of microorganisms. The determined degradation product phenol is readily biodegradable.