2-({2-[4-(dimethylamino)benzoyloxy]ethyl}(methyl)amino)ethyl 4-(dimethylamino)benzoate

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:

2006

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:

17 July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: LFC 2098
Purity: 99.69%
Solubility in water: Insoluble
Aggregate state/physicai form at room temperature: Soiid / powder
Color: White
Storage conditions: At room temperature at about 20 °C, in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz ll, FUllinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final siudge suspension was weighed. thereafter dried and the ratio of wet to dry weight was calculated.

Based on this ratio, calculated amounts of wet sludge were suspended in test water (see Section 2.4.1) to obtain a concentration equivalent to 4 g (i10%) dry material per liter. During holding, the sludge was aerated at room temperature untit use. Prior to use, the siudge was diluted with test water to a concentration of t g per liter (dry weight basis). Based on the determined dry weight of this diluted activated sludge, defined amounts were added to test water to obtain a fine! concentration of 30 mg dry material per titer.

Duration of test (contact time):

28 d

Initial conc.:

207 mg/L

Based on:

ThOD

Initial conc.:

205 mg/L

Based on:

ThOD

Initial conc.:

101 mg/L

Based on:

test mat.

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST SYSTEM
The test water was prepared according to the testing guidelines. Anatytical grade salts were dissoived in purified water to obtain the following stock solutions:
(a) KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4 x 2H20 33.40 g/L
NH4Cl 0.50 g/L

The pH of this solution was 7.4.

(b) MgSO4 x 7H20 22.50 g/L
(c) CaCl2 x 2H2O 36.40 g/L
(d) FeCl3 x 6H20 0.25 g/L, stabilized with one drop of concentrated HCl per liter

To obtain the final test water, 10 mL of stock solution (a) and 1 mL each of stock solutions (b) - (d) were combined and made up to 1000 mL with purified water. The pH was adjusted from 7.8 to 7.4 with a dituted hydrochloric acid solution.

The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz ll, FUllinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final siudge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.

Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter. During holding, the sludge was aerated at room temperature untit use. Prior to use, the siudge was diluted with test water to a concentration of 1 g per liter (dry weight basis). Based on the determined dry weight of this diluted activated sludge, defined amounts were added to test water to obtain a final concentration of 30 mg dry material per liter.

TEST CONDITIONS
Apparatus: The test flasks (500-mL Erlenmeyer flasks, labeled with the necessary information to ensure unmistakable identification) were incubated under continuous stirring in a SAPROMAT D12 (Voith GmbH, Heldenheim, Germany). Oxygen consumption was recorded manually by taking a daily reading at least on each working day.

Principle: Electro—chemlcal analysis process: The biodegradation process consumes the dissolved oxygen in the liquid and generates 002. The COZ is adsorbed by soda lime and the total pressure decreases in the airtight test flasks. The pressure drop is detected and converted into an electrical signal by means of an electrode type manometer. The consumed oxygen is replaced by electrolytically generated oxygen from a copper sulfate solution.

Test duration: 28 days

Light conditions: Darkness

Test temperature:22 °C, maintained with a built-in thermostat and checked once per week.

pH: Prior to test start, the pH was measured in each test flask before the addition of the activated sludge inoculum (Table 3). At the end of incubation. the pH was measured again in each test flask (Table 3)



The test item was weighed by means of an analytical baiance and transferred to the designated test flasks with test water. No emulsifiers or solvents were used.
The reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. A stock solution containing 2.5 g sodium benzoate per liter test water was prepared, by completely dissolving 250 mg sodium benzoate in 100 mL test water. From this stock sotutton, 10 mL aliquots were added to the corresponding test fiasks containing test water.

Finally, with the exception of the abiotic control flask, activated sludge was added to each test ftask (see Section 2.3). The final volume was 250 mL per test fiask.

CALCULATIONS

The percentage biodegradation was calculated according to the following formula:

% biodegradation = [BOD (mg O2/mg chemical) / ThOD(NH4 or NO3) (mg O2/mg chemical)] x 100%

where:

BOD = Biochemical oxygen demand ofthe test or reference item
= [ (mg O2 uptake/L by test and/or reference item) - (mg O2 uptake/L by inoculum control)] / mg test and/or reference item per liter

ThOD NH4 or NO3 = Theoretical oxygen demand of the test or reference item, without or with nitrification
(The theoretical oxygen demand is the total amount of oxygen required to oxidize a chemical completely It is calculated from the molecular formula, assuming the turnover of H into H2O, C into CO2, P into P2O5, Na into
NaO2, halogens into their corresponding hydrogen halides, and N into NH3 and/or NO3 according to EU Commission Directive 92/69 EEC C.4-D.

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Details on results:

The biochemical oxygen demand (BOD) of the test item LFC 2098 in the test media was in the normal range found for the inoculum controls throughout the study period of 28 days. Therefore. LFC 2098 was not biodegradable under the test conditions within
28 days. However, in the toxicity control an oxygen consumption exceeding the ThOD of the reference item (sodium benzoate) was observed, suggesting that the test item was co- metabolized in the presence of sodium benzoate.

In the abiotic control, oxygen consumption was measured from Exposure Day 1 until test termination after 28 days. Thus, a slight abiotic degradation process of 9% occurred under the test conditions within 28 days.

In the toxicity control, containing both LFC 2098 and the reference item sodium benzoate, no inhibitory effect on the biodegradation of the reference item was determined. Thus, LFC 2098 had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 100 mg/L.

Results with reference substance:

In the procedure controls, the reference item sodium benzoate was degraded by an average of 88% by Exposure Day 14, and reached an average biodegradation of 94% by the end of the test (Day 28), thus confirming suitability of the activated sludge.

The percent biodegradation of the test item was caiculated based on the theoretical oxygen demand of 2.05 mg O2/mg test item without nitrification (ThOD(NH4)), and 2.52 mg O2/mg test

item with nitrification (ThOD(NO3)).

The biochemical oxygen demand (BOD) of the test item LFC 2098 in the test media was in the normal range found for the inoculum controls throughout the study period of 28 days. Therefore. LFC 2098 was not biodegradable under the test conditions within 28 days. However, in the toxicity control an oxygen consumption exceeding the ThOD of the reference item (sodium benzoate) was observed, suggesting that the test item was co-metabolized in the presence of sodium benzoate.

Abiotic control: Oxygen consumption was measured from Exposure Day 1 until test termination after 28 days. Thus, a slight abiotic degradation process of 9% occurred under the test conditions within 28 days.

The percent biodegradation of the reference item sodium benzoate was calculated based on the theoretical oxygen demand (ThOD) of 1.67 mg O2/mg reference item. In the procedure controls, the reference item sodium benzoate was degraded by an average of 88% by Exposure Day 14, and reached an average biodegradation of 94% by the end of the test (Day 28), thus confirming suitability of the activated sludge.

The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the sum of the ThOD of the test item (ThOD(NH4) or

ThOD(NO3)) and the reference item.

Up to about Exposure Day 2, the course of biodegradation in the toxicity control was similar to the two procedure controls, containing only the reference item. Thus, the oxygen consumption during the first two days can primarily be attributed to the biodegradation of the reference item. Within 14 days of exposure, biodegradation amounted to 51% and 45% based on the ThOD(NH4) and the ThOD(NO3), respectively.

Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 100 mg/L because biodegradation in

the toxicity control was >25% within 14 days.

From Exposure Day 3 to the end of the test, an oxygen consumption exceeding the ThOD of the reference item (sodium benzoate) was obsewed in the toxicity control, suggesting that the test item was at least partly biodegraded in the presence of sodium benzoate. This process is described in literature as co—metabolism or co-oxidation and describes the concomitant oxidation of a non-growth substrate during growth of a microorganism on a utilizable carbon and energy source.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

LFC 2098 was not biodegradable under the test conditions within 28 days.

Executive summary:

The test item LFC 2098 was investigated for its ready biodegradability in a manometric respirometry test over 28 days according to EU Commission Directive 92/89 EEC, C.4-D

(1992) and OECD Guideline for Testing of Chemicals No. 301 F (1992).

The biochemical oxygen demand (BOD) of the test item LFC 2098 in the test media was in the normal range found for the inoculum controls throughout the study period of 28 days. Therefore. LFC 2098 was not biodegradable under the test conditions within 28 days. However, in the toxicity control an oxygen consumption exceeding the ThOD of the reference item (sodium benzoate) was observed, suggesting that the test item was co-metabolized in the presence of sodium benzoate.

In the abiotic control, oxygen consumption was measured from Exposure Day 1 until test termination after 28 days. Thus, a slight abiotic degradation process of 9% occurred under the test conditions within 28 days.

In the toxicity control, containing both LFC 2098 and the reference item sodium benzoate, no inhibitory effect on the biodegradation of the reference item was determined. Thus, LFC 2098 had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 100 mg/L.

In the procedure controls, the reference item sodium benzoate was degraded by an average of 88% by Exposure Day 14, and reached an average biodegradation of 94% by the end of

the test (Day 28), thus confirming suitability of the activated sludge.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater