Propanoic acid, 2-[(ethoxythioxomethyl)thio]-, methyl ester

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:

from 5th july 2000 to 3rd november 2000

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:

yes

Remarks:

see below

Principles of method if other than guideline:

Deviations from the protocol:
- The activated sludge was taken from an oxidation ditch situated at Hazerswoude, the Netherlands.
- The correct concentration of sodium acetate in the contrai bottles is 64 mg/l.
- The pH of the medium was not measured at the time point t=28d.
- The correct purity of the test substance is 88%.
These deviations are assumed not to have affected the results of the study.

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

A sample of activated sludge was taken on 5 July 2000 from an oxidation ditch in the municipality of Hazerswoude, the Netherlands. The oxidation ditch is used to treat domestic sewage.
The medium was aerated with CO2-free air over night before use. The original sludge ( 4.0 g of solid substance/l) was rnixed to provide homogenity, and 187 ml of the sludge was used to inoculate 25 litres of medium to yield a final inoculum concentration of about 30 mg/l of solid substance.

Duration of test (contact time):

28 d

Initial conc.:

30 mg/L

Based on:

other: dry weight

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: A medium with a higher nitrogen content than that specified in the Guidelines.
The modified composition was chosen to prevent nutrient limitation of degradation during the course of the study.
Add to water 10 ml of solution a and 1 ml of solutions b to f and make up to 1000 ml.
a) Solution a contains per 1000 ml water:
KH2PO4 8.5 g
K2HPO4 21.75 g
Na2HPO4.2H2O 33.4 g
NH4Cl 1.5 g
b) Solution b contains 36.4 g of CaCl2.2H2O per 1000 ml water.
c) Solution c contains 22.5 g of MgSO4. 7H2O per 1000 ml water.
d) Solution d contains 0.025 g of FeCl3.6H2O per 100 ml water.
Prepare this solution freshly before use.
e) Trace element solution (solution e).
This solution contains per 1000 ml water:
MnSO4.4H2O 39.9 mg (30.2 mg MnSO4.H2O)
H3BO3 57.2 mg
ZnSO4.7H2O 42.8 mg
(NH4)6 Mo7 O24 34.7 mg (36.9 mg (NH4)6 Mo7 O24.4H2O)
EDTA 100 mg
f) 15 mg yeast extract dissolved in 100 ml water (solution f).
Prepare this solution freshly imrnediately before use .

- Test temperature: 20 ± 2 °C
- pH: The pH value of the medium was also determined at each sampling time, except for the last sampling time (day 28).
- pH adjusted: no
- CEC (meq/100 g): no data
- Aeration of dilution water: The medium was aerated with CO2-free air over night before use.
- Suspended solids concentration: 30 mg/L in each test vessel
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: Two-litre glass bottles (Scott Duran) closed with plastic screw caps were used for the test. A 20 ml glass vial containing the five ml C02 absorbing fluid (i.e. 0.4 or 0.6 M NaOH
solution')) was suspended from the screw cap of each bottle.
- Number of culture flasks/concentration: 3 (Blank control), 4 (inoculum activity control), 2 (inoculum toxicity control), 4 (25 and 50 mg/l)
The final (nominal) test concentrations of Rhodixan A-1 of 25 and 50 mg/l were prepared by dissolving 2.0041 g in 10 ml ethanol and applying volumes of 125 and 250 μl respectively on glass fibre filter carriers (Whatman GF/C, 0 47 mm). After being dried, the filters were added separately to one litre of inoculated medium in quadruplicate bottles. This resulted in final concentrations of 10 and 20 mg carbon per litre, respectively.
Blank control: Triplicate control bottles with glass fibre filter carriers (treated with 200 μl ethanol and dried) in 1 litre inoculated medium to determine the background CO2 production of the inoculum.
Inoculum activity control: In order to determine the inoculum activity, 64 mg (one ml of a solution of 64g/l) of the reference substance sodium acetate was added to four additional bottles containing one litre inoculated medium and blank glass fibre filter carriers. The final test concentration of sodium acetate was achieved by initially dissolving 3.4011 g of the reference substance in 50 ml of ultrapure water. From this stock solution 1 ml volumes were added to one litre of inoculated medium. The theoretical carbon dioxide evolution (ThC02) of sodium acetate is 1.07 mg CO2/mgl.
Inoculum toxicity control: In order to detect possible toxic effects of the test substance, sodium acetate ( 64 mg/l) was also added to further duplicate bottles containing 50.1 mg/l of test substance.
Before the bottles were closed, five ml of 0.6 or 0.4 M NaOH was placed in each trapping vial. The bottles were incubated in the dark on an orbital shaking machine in a temperature controlled room kept at 20 ± 2 °C for 28 days. The CO2 absorption vials were replaced by fresh ones after 7, 14, 21 and 28 days.
- Method used to create aerobic conditions: air used, orbital shaking
- Measuring equipment: The amount of C02 absorbed was determined by titration of the sodium hydroxide with 0.1 M HCl using a Metrohm 686 Titroprocessor.
- Test performed in closed vessels due to significant volatility of test substance: yes
- Details of trap for CO2 and volatile organics if used: On day 28, one ml of concentrated HCl was added to the bottles in order to remove the remaining dissolved CO2 in the medium. That CO2 was trapped
during a period of approximately one day, and determined as described above. Two of the inoculum activity bottles and three boules of each of the two test substance series were treated in the same way after 14 days of incubation.

SAMPLING
- Sampling frequency: 7, 14, 21 and 28 days
- Sampling method: No data
- Sterility check if applicable: No data
- Sample storage before analysis: No data

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: No
- Toxicity control: Yes

STATISTICAL METHODS: No data

Reference substance:

other: acétate de sodium

Test performance:

Validity criteria :
- The difference of extreme replicate values of the removal of the test compound at the plateau, at the end of the test or at the end of the 10 day window, as appropriate, is less than 20%,
- the degradation of the reference compound has reached the pass level (i.e. > 60% degradation within 14 days,
- in a toxicity test, containing both the test substance and the reference compound, the degradation is > 25% (based on total ThCO2) within 14 days,
- the total CO2 evolution in the inoculum blank should not normally exceed 40 mg/l.

These validity criteria were fulfilled as follows:
- the extreme differences at the end of the biodegradation test with test substance were more than 20% as assessed from the percentage degradation after 28 days given in Annex C. Due to the low biodegradation of the test substance the difference in percentage degradation is considered to be not relevant. The extreme differences of the controls were within the limits,
- the reference compound sodium acetate demonstrated a degradation of > 60% within 14 days,
- in the inoculum toxicity control, containing both the test substance and the reference compound, the degradation is > 25%,
- the total inoculum blank CO2 production amounted to 21.2 mg CO2/l at the end of the test.

Parameter:

% degradation (CO2 evolution)

Value:

19.4

Sampling time:

28 d

Details on results:

degradation (test substance):
6.2 % degradation after 7 d
10.9 % degradation after 14 d
14.8 % degradation after 21 d
19.4 % degradation after 28 d

Results with reference substance:

degradation (reference substance):
46.4 % degradation after 7 d
68.9 % degradation after 14 d
81.4 % degradation after 21 d
90.6 % degradation after 28 d

The test substance Rhodixan A-1 was biodegraded for 19.4% at test substance concentrations of 25 mg/l and 14.9% at 50 mg/l.

The inoculum activity and toxicity control tests with sodium acetate showed that the activity of the inoculum was sufficient, and that Rhodixan A-1 did not inhibit the degradation of the reference substance.

The results demonstrated that biodegradation of Rhodixan A-1 took place; with a maximum mean degradation of 19.3% after 28 days (degradation up to 24% was observed in single bottles).

The criteria given in the guideline (i.e. > 60% degradation reached within a 10 day period counting from the day that the level of de gradation exceeded 10%) were not met. Rhodixan A-1 is therefore assessed to be not readily biodegradable in this test.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test substance Rhodixan A-1 was biodegraded for 19.4% at test substance concentrations of 25 mg/l and 14.9% at 50 mg/l. Rhodixan A-1 therefore did not pass the criteria given in the guideline (i.e. > 60% degradation reached within a 10 day period counting from the day that the level of degradation exceeded 10% ), and is therefore assessed to be not readily biodegradable in this test.

Executive summary:

The biodegradability of Rhodixan A-1 was determined by a modified method according to the OECD Guideline 301: Ready Biodegradability, 301B CO2 Evolution Test and EEC Directive 92/69/EEG Part C, method C.4-C, using a modified trapping system. The study was carried out in conformity with the OECD principles of Good Laboratory Practice.

The CO2-evolution biodegradability test was carried out in glass bottles containing one litre inoculated medium, and a vial suspended from the screw-cap of each bottle containing a CO2 absorbing fluid. The vials containing CO2 absorbing fluid were replaced at weekly intervals and the trapped CO2 was determined by titration. Activated sludge taken from an oxidation ditch used to treat domestic sewage was used as inoculum. The inoculum concentration was 30 mg (dry weight)/l.

Rhodixan A-1 is a yellow liquid stated to be relatively soluble in water. In practice its solubility in water necessitated the use of a glass fibre filter carrier to introduce the test substance into the inoculated medium in accordance with the International Standard ISO 10634. Two concentrations of 25 and 50 mg test substance per litre medium, which corresponds to 10 and 20 mg carbon.I-1, respectively, were tested in triplicate. The test was completed with a control blank with inoculated medium only, a control on inoculum activity with sodium acetate as reference substance, and with a toxicity control with sodium acetate and 50 mg.l Rhodixan A-1.

The inoculum activity and toxicity control tests with sodium acetate showed that the activity of the inoculum was sufficient, and that Rhodixan A-1 did not inhibit the degradation of the reference substance.

The results demonstrated that biodegradation of Rhodixan A-1 took place; with a maximum mean degradation of 19.3% after 28 days (degradation up to 24% was observed in single bottles).

The criteria given in the guideline (i.e. > 60% degradation reached within a 10 day period counting from the day that the level of de gradation exceeded 10%) were not met. Rhodixan A-1 is therefore assessed to be not readily biodegradable in this test. Indications for a possible inherent biodegradability of Rhodixan A-1 were obtained.

Description of key information

The test substance Rhodixan A-1 was biodegraded for 19.4% at test substance concentrations of 25 mg/l and 14.9% at 50 mg/l after 28 days. Rhodixan A-1 therefore did not pass the criteria given in the guideline (i.e. > 60% degradation reached within a 10 day period counting from the day that the level of degradation exceeded 10% ), and is therefore assessed to be not readily biodegradable in this test (OECD guideline 301B).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

A GLP-compliant study, scored as klimisch 1 and flagged as a key study, is available on Rhodixan A-1, giving 14.9% degradation after 28 days (OECD Guideline N°301B) and revealing that Rhodixan A-1 cannot be considered to be readily biodegradable (TNO, 2000).