N4-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine-3′-O-[O-(2-cyanoethyl)-N,N′-diisopropylphosphoramidite]

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-18 to 2018-25-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

adopted July 17, 1992

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

(EEC Publication No. L 142/496, May 2008)

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: Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Rossdorf, Germany.
- Pre-treatment: The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was resuspended in test water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined.
- Concentration of sludge: 3.5 g dry material per litre were mixed with test water

Duration of test (contact time):

28 d

Initial conc.:

103.5 mg/L

Based on:

test mat.

Initial conc.:

206.8 mg/L

Based on:

other: ThOD(NH4)

Initial conc.:

242.4 mg/L

Based on:

other: ThOD(NH3)

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Reconstituted water: Analytical grade salts were added to pure water to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with pure water to 1000 mL volume; The pH-value was 7.4.
b) 11.25 g MgSO4 x 7 H2O filled up with pure water to 500 mL volume
c) 18.2 g CaCl2 x 2 H2O filled up with pure water to 500 mL volume
d) 0.125 g FeCl3 x 6 H2O filled up with pure water to 500 mL volume
To avoid precipitation of iron hydroxide in the stock solution d), one drop of concentrated HCl per litre was added before storage. 50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of 5000 mL with pure water.

- Test temperature: 22°C ± 1°C
- pH: 7.7 (measured at the start of the test in the control) and set to 7.5 with hydrochloric acid; The pH of the test flask was 7.7 and set to 7.4 with sodium hydroxide. 7.4 to 7.6 (measured at the end of the test)
- pH adjusted: yes, see above
- Aeration of dilution water: No
- Suspended solids concentration: 3.5 g dry material per litre were mixed with test water; final sludge concentration in test flasks: 28.7 mg sludge/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Manometric Test System with test flasks containing a volume of approximately 500 mL. BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany
- Number of culture flasks/concentration: Two replicates for the test item (103.5 mg/L), two replicates for inoculum control, one replicate for the procedure control, abiotic control, and the toxicity control.
- Preparation of Test flask: The amounts of test item and reference item were directly weighed into the test flasks. No emulsifiers or solvents were used, but the solutions were dispersed by stirring to achieve a homogeneous solution of the test item. The test flasks were closed gas-tight by a measuring head. Potassium hydroxide solution (45%) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.
- Incubation: The closed test flasks were incubated in a climatized chamber under continuous stirring.
- Measuring equipment: The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.

CONTROL AND BLANK SYSTEM
- Test item: 2 replicates
- Inoculum blank: 2 replicates
- Abiotic sterile control: 1 replicate (Poisoned with CuSO4 (stock solution of 1 g/L))
- Toxicity control: 1 replicate
- Other: Procedure control: 1 replicate

Test Parameters
- Measurement of Oxygen: The change of pressure in the test flasks was measured by means of a manometric method (BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany) each day.
- Temperature: The temperature was recorded by means of the automated software AMR Wincontrol©.
- pH-Value: pH-values were measured in procedure control, a separately prepared test flask with test item (to prevent loss of test item in the test flasks) and a separately prepared test flask without test item (control) at test start and in all flasks at the end of the test, except in the abiotic and toxicity control, using a pH-electrode WTW pH 340i

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

5

Sampling time:

28 d

Remarks on result:

other: based on ThOD(NH4)

Key result

Parameter:

% degradation (O2 consumption)

Value:

4

Sampling time:

28 d

Remarks on result:

other: based on ThOD(NO3)

Details on results:

The test item 5′-O-[bis(4-methoxyphenyl) phenylmethyl]-2′-deoxythymidine, 3′-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite contains nitrogen; therefore the evaluation of biodegradation has to be based on ThODNH4 and ThODNO3. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60%, reached within a 10-day window; the 10-day window starts
when the degradation of the test item reaches 10% degradation.
5′-O-[bis(4-methoxyphenyl) phenylmethyl]-2′-deoxythymidine, 3′-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite never reached 10% biodegradation. The mean biodegradation at test end after 28 days was 5% (based on ThODNH4, see Figure 1) and 4% (based on ThODNO3, see Figure 2).

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 73% after 14 days and to 84 % after 28 days of incubation (based on ThODNH4). The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Validity Criteria of the Study

-       Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 20 mg O2/L and thus not greater than 60 mg O2/L within 28 days as required by the test guideline. 

-       pH-Value: The pH-value of the test item flasks at the end of the test was 7.4 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.

-       Reference Item: The percentage degradation of the reference item should reach the level for ready biodegradability (> 60 %) within 14 days as required by the test guideline. The reference item sodium benzoate was degraded to more than 60 % after 7 days of incubation.

-       Test Item: The difference of duplicate values for the degradation of the test item at the plateau, at the end of the test was less than 20 %. The difference of duplicate values at day 28 differed by 10 % (based on ThODNH4) and 8 % (based on ThODNO3). The validity criterion was fulfilled.

-       Toxicity Control: If in a toxicity test, containing both the test substance and a reference compound less than 25 % biodegradation occurred within 14 days (ThODNH4), the test substance can be assumed to be inhibitory. The biodegradation was 32% (based on ThODNH4) and 29 % (based on ThODNO3) at day 14; the test item was not inhibitory. 

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The mean biodegradation of the test item 5′-O-[bis(4-methoxyphenyl) phenylmethyl]-2′-deoxythymidine, 3′-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite at test end after 28 days was 5 % (based on ThODNH4) and 4 % (based on ThODNO3).

Executive summary:

The biodegradation of 5′-O-[bis(4-methoxyphenyl) phenylmethyl]-2′-deoxythymidine, 3′-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite was studied in reconstituted water (pH 7.4 – 7.6) inoculated with activated sludge from a domestic sewage treatment plant (stock suspension of 3.5 g/L on dry matter base and a final sludge concentration in test flasks of 28.7 mg sludge/L). The test item was applied with 103.5 mg a.i./L.  The experiment was conducted in accordance with the OECD test guideline 301 F “Ready Biodegradability: Manometric Respirometry Test”, and in compliance with the OECD-GLP standards. The test system consisted of test flasks containing a volume of approximately 500 mL. The pressure decrease in the reaction vessels was measured over complete experimental phase of 28 days using the BSB/BODSensor- System, Aqualytic Dortmund, Germany. The test flasks were closed gas-tight by a measuring head. Potassium hydroxide solution (45 %) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.

 

The test item contains nitrogen; therefore the evaluation of biodegradation has to be based on ThODNH4 and ThODNO3. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60 %, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10 % degradation. The test item never reached 10 % biodegradation. The mean biodegradation of the test item at test end after 28 days was 5 % (ThODNH4) and 4 % (ThODNO3). Therefore, 5′-O-[bis(4-methoxyphenyl) phenylmethyl]-2′-deoxythymidine, 3′-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite is considered to be not readily biodegradable.

Description of key information

One study is available for biodegradation in water which can be used for read-across to cytidine, N-benzoyl-5′-O-[bis(4 methoxyphenyl)phenylmethyl]-2′-deoxy-, 3′-[2-cyanoethyl bis(1-methylethyl)phosphoramidite]:

The Study by Hammesfahr (2018) was conducted according to GLP principles and followed the OECD test guideline 301 F. The mean degradation rate of 5′-O-[bis(4-methoxyphenyl) phenylmethyl]-2′-deoxythymidine, 3′-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite in a test according to the OECD method 301 F after 28 days was 5 % (based on ThODNH4) and 4 % (based on ThODNO3). Therefore, the test item is not readily biodegradable.

 

This information is used in a read-across approach in the assessment of the target substance.

For details and justification of read-across please refer to the attached report in section 13 of IUCLID.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater