Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9th October 2003 to 14th June 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted to GLP. This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

EC Number:

269-358-5

EC Name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

Cas Number:

68227-46-3

Molecular formula:

C21H35N3O4.C3H6O3

IUPAC Name:

2-hydroxypropanoic acid 2-(dimethylamino)ethyl N-[5-({[(2-ethylhexyl)oxy]carbonyl}amino)-2-methylphenyl]carbamate

Details on test material:

- Name of test material (as cited in study report): WRS-2390TX
- Physical state: Vicous liquid
- Analytical purity: 71.2% in water
- Expiration date of the lot/batch: 15th November 2004
- Storage condition of test material: The test item was stored at ambient laboratory temperature in the dark for the duration of the study.
- Sponsor's information on this batch detailed in Notebook No. 02-181-126.

Study design

Oxygen conditions:

not specified

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Samples of activated sludge were obtained from Haddington Municipal Sewage Treatment Works (a local sewage processing plant, which handles predominantly domestic sewage) on 06 October 2003 (preliminary test) and 23 October 2003 (main test).
- Preparation of inoculum for exposure:
The activated sludge was thoroughly mixed prior to sampling for solids content determination. The mean solids content of the sludge was determined as 3 g/L (preliminary test) and 3.4 g/L (main test).
After solids determination, the sludge was allowed to settle for at least 30 min prior to a sample of the supernatant being withdrawn for use as the test microbial inoculum.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

20 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
Stock Component Concentration (g/L)
Phosphate Buffer A KH2PO4 8.50
K2HPO4 21.75
Na2HPO4.2H2O 33.40
NH4Cl 0.50
Calcium Chloride B CaCl2.2H2O 36.40
Magnesium Sulphate C MgSO4.7H2O 22.50
Ferric Chloride* D FeCl3.6H2O 0.25
(* 1 drop of concentrated HCl was added to stabilise)

- Test temperature: The temperature in the laboratory was in the range 20-21°C for the duration of the test.
- pH: Day 0 = 7.4-7.5; Day 28 = 7.6-8.1; Day 29 acidified = 2.6-6.1
- pH adjusted: No
- Suspended solids concentration: The mean solids content of the sludge was determined as 3 g/L (preliminary test) and 3.4 g/L (main test).
- Continuous darkness: Yes, each bioreactor was covered with foil to block out light.


TEST SYSTEM
- Culturing apparatus: A total of 7 bioreactors (2 litre capacity) were used; 2 each for control (C1 and C2) and test item (T4 and T5) and one each for the reference item (R3), toxicity control (TC6) and abiotic control (AC7).
- Number of culture flasks/concentration: 2
- Method used to create anaerobic conditions: A totally sealed system was constructed and supplied on a positive pressure basis by CO2 free air.
- Measuring equipment: CO2 was removed by passing through self-indicating soda lime granules (Chemosorb) contained in a 2 litre capacity glass aspirator. The CO2-free air was passed through a glass manifold via an air tap, to allow fine control of airflow. The air was also passed through a secondary tube of Chemosorb prior to the bioreactor. The flow-rate in each bioreactor was adjusted to be in the range 30-100 mL/min.
- Details of trap for CO2 and volatile organics if used: Each bioreactor was connected to 3 traps, each containing 100 mL of 0.0125M Ba(OH)2. At trap collection the trap closest to the bioreactor was taken for titration and the 2 remaining traps were moved one place closer to the bioreactor. A new trap containing freshly prepared Ba(OH)2 was then placed third in line from the bioreactor.
Trap changes were conducted on Days 2, 4, 6, 9, 12, 14, 19, 24 and 29. During these trap changes the air supply was switched off for ca 15 min, each of the bioreactors being disconnected for approximately 30 s while a new trap was added. All sampled traps were sealed immediately after collection.
Each sampled trap was then titrated with a few drops of phenolphthalein indicator solution against 0.05M HCl, utilising the colour change pink to colourless.
- Other:
A 1 g/L stock solution of the reference item was prepared by dissolving sodium benzoate (0.4998 g) in 500 mL of deionised water. Aliquots of this stock (69 mL) were added to each appropriate bioreactor to give an addition rate of 20 mg DOC/L.


CONTROL AND BLANK SYSTEM
To prepare the control, test, reference and toxicity control bioreactors 1600 mL of mineral medium and 20 mL of microbial inoculum were added to each bioreactor and then purged with CO2-free air overnight. The appropriate weight of test item and/or volume of reference item stock were then added to the appropriate bioreactors. Finally, the bioreactors were made up to a final volume of 2000 mL with CO2-free mineral medium.
The abiotic control was prepared in an identical manner to the other bioreactors, without the addition of microbial inoculunm. A sterilising agent (Formaldehyde [37% formalin]) was also added to the bioreactor to ensure any degradation exhibited was not attributable to a microbial population.

Details of bioreactor preparation are given below:
Treatment Mineral Medium Test Item Reference Item Stock Microbial Inoculum Formaldehyde [37% formalin] Final Volume
(mL) (mg) (mL) (mL) (mL) (mL)
Control 1980 - - 20 - 2000
Test 1980 62.5* - 20 - 2000
Reference 1911 - 69 20 - 2000
Toxicity Control 1911 62.5* 69 20 - 2000
Abiotic Control 1960 62.5* - - 20 1980
(* Target weight)


STATISTICAL METHODS:
Calculation of Results:
The weight of CO2 evolved was calculated from the titre. The actual titre for each batch of Ba(OH)2 prepared was used for the background titre value. The mean titre value for the test, reference and control vessels were calculated and subjected to the following equation:

Weight CO2 produced (mg) = 1.1 x (background titre - ml HCl titrated)

The net CO2 production was then calculated by subtracting the control mean CO2 production value from the test and reference item CO2 production values.
The percentage biodegradation was calculated by comparing the actual CO2 evolved in test and reference vessels with the theoretical CO2 evolution.
This was calculated from the molecular weight and empirical formula of the test or reference item, as appropriate:

% Degradadtion = (mg CO2 produced / (ThCO2 x mg test substance added)) x 100

It is not possible to estimate time required for 50% biodegradation of the test item if this level of degradation is not achieved in the test. The test item is considered to be readily biodegradable if a transition from 10% to 60% (or greater) biodegradation is observed in a 10 day window during the 28 day test period.
For a valid test, the reference item should be shown to be readily biodegradable. Differences between replicate bioreactors should not exceed 20% of the mean at the end of the test, or the 10 day window.

Reference substance

Reference substance:

benzoic acid, sodium salt

Remarks:

Fisher Batch No. 0251238

Results and discussion

Preliminary study:

WRS-2390TX is not considered to be inhibitory to the microbial innoculum at an addition rate of 20 mg DOC/L as 34.8% biodegradation was observed in the toxicity control bioreactor on Day5

Test performance:

The test met all validity criteria.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

10.5

Sampling time:

28 d

Details on results:

The test item did not exhibit any notable biodegradation, the cumulative biodegradation at Day 29 was 10.5%. WRS-2390TX was therefore not readily biodegradable, under the conditions of the test.
The test item is not considered to be inhibitory to the microbial inoculum as >25% biodegradation (36.8%) was observed in the toxicity control bioreactor (test and reference items) on Day 14.
The total CO2 evolution in the control bioreactors was <40 mg/L (9.15 mg/L) at Day 29. Differences between replicate test bioreactors were within 20% of the mean (± 7.6%) at Day 29.
The test item exhibited only negligible abiotic degradation (1.6%) by the end of the test.
The pH of the bioreactors on Day 28 did not indicate any significant trends in the test.
The IC content of the test item solution as a percentage of the TC was calculated as 2.3%.

BOD5 / COD results

Results with reference substance:

The reference item was readily biodegradable as 62.0% biodegradation was achieved on Day 6.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

other: Not readily biodegradable under the conditions of the test

Conclusions:

WRS-2390TX was not readily biodegradable under the conditions of the test as 10.5% biodegradation was achieved by the end of the test. The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability & repeatability).

Executive summary:

In a study on the ready biodegradability of WRS-2390TX in the modified Sturm test (Inveresk report number: 23693) the test material can be described as not readily biodegradable under the conditions of the test.

OECD guideline 301 B (Ready Biodegradability: CO₂ Evolution Test) was followed to conduct this study.

The test item did not exhibit any notable biodegradation, the cumulative biodegradation at Day 29 was 10.5%. WRS-2390TX was therefore not readily biodegradable, under the conditions of the test.

The test item is not considered to be inhibitory to the microbial inoculum as >25% biodegradation (36.8%) was observed in the toxicity control bioreactor (test and reference items) on Day 14.

The total CO₂ evolution in the control bioreactors was <40 mg/L (9.15 mg/L) at Day 29. Differences between replicate test bioreactors were within 20% of the mean (± 7.6%) at Day 29.

The test item exhibited only negligible abiotic degradation (1.6%) by the end of the test.

The pH of the bioreactors on Day 28 did not indicate any significant trends in the test.

The IC content of the test item solution as a percentage of the TC was calculated as 2.3%.