Isobutyl 4-chloro-3,5-diaminobenzoate

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:

2016-06-23 - 2016-08-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

version dated 1992-07-17

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden

Test material

Test material information

Constituent 1

Reference substance name:

Isobutyl 4-chloro-3,5-diaminobenzoate

EC Number:

251-311-5

EC Name:

Isobutyl 4-chloro-3,5-diaminobenzoate

Cas Number:

32961-44-7

Molecular formula:

C11H15ClN2O2

IUPAC Name:

2-methylpropyl 3,5-diamino-4-chlorobenzoate

Test material form:

solid

Remarks:

dark brown

Details on test material:

- Density: 1.106 g/cm³ (25°C)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature, protected from light

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): sewage plant at Taunusstein-Bleidenstadt
- Preparation of inoculum for exposure: It was washed twice with mineral nutrient solution of the CO2-Evolution-Test to eliminate organic components and carbonates from the sludge. After resolution with mineral nutrient medium the sludge was aerated by means of compressed humidified air for about four hours. Before use as inoculum for the CO2-Evolution-Test the sludge was homogenised in a "Waring Blender" at low speed for 2 minutes and then filtered through a cotton filter previously carefully rinsed with deionised water. The filtrate was used as inoculum (1% of the final volume of the test solution) on the same day of preparation.

Duration of test (contact time):

29 d

Initial test substance concentration

Initial conc.:

19.26 mg/L

Based on:

TOC

Remarks:

According to the carbon content of the test item the amount of test item to be applied was calculated to give a final concentration of 10 to 20 mg TOC/L (Total Organic Carbon): 67.4 mg / 3.5 L / ca.10 mg TOC/L

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
For preparation of the test solutions the follo¬wing stock solutions of mineral nutrient salts were prepared:
8.50 g KH2PO4
21.75 g K2HPO4
33.40 g Na2HPO4 · 2 H2O
0.50 g NH4Cl
were diluted in 1 L Ultrapure Water [Seral, Purelab plus] (this solution may be stored for at least 3 month at +2 to +8 °C).
36.40 g CaCl2 · 2 H2O (corresp. 27.5 g CaCl2)
were diluted in 1 L Ultrapure Water [Seral, Purelab plus] (this solution may be stored for at least 3 month at +2 to +8 °C).
22.50 g MgSO4 · 7 H2O
were diluted in 1 L Ultrapure Water [Seral, Purelab plus] (this solution may be stored for at least 3 month at +2 to +8 °C).
0.25 g FeCl3 · 6 H2O
were diluted in 1 L Ultrapure Water [Seral, Purelab plus] (this solution was prepared freshly before use in the test series).

The test solutions contained in a total volume of 3500 mL: 35 mL of the inoculum, 3.5 mL of the ferrichloride solution, 3.5 mL of the magnesium sulphate solution, 3.5 mL of the calcium chloride solution, and 35 mL of the phosphate mixture solution. The test item was given directly into the test solutions. At time t0 67.4 mg of the test item were given into the first treatment and 67.7 mg of the test item was given into the second treatment at time t0. The final (calculated) concentration in the test solutions was ca 10 mg C/L.

- Test temperature: 20-21°C
- Aeration of dilution water: yes

TEST SYSTEM
- Culturing apparatus: 5-litre amber carboys served as test vessels. They were closed with stoppers with tubing for gas inlet and outlet (gas exit line). The gas outlet (exit air line) was connected to at least three CO2 absorber bottles filled with 100 mL 0.025 N Ba(OH)2 (connection in series), and bubbling of CO2 free compressed air through the solution was continued. Periodically (if necessary) the CO2 absorber nearest the test vessel was removed for analysis, and a new CO2 absorber was connected farest the carboy. Precipitation of BaCO3 in the second CO2 trap indicated that the absorber bottle nearest the test vessel had to be changed and analysed for CO2.
- Number of culture flasks/concentration: 2 (test item), 1 (Sodium Benzoate), 1 (toxicity control), 2 (blanks)
- Method used to create aerobic conditions: Before starting the test, the mineral nutrient solution with inoculum but without any test component were aerated for 24 hours with CO2-free compressed air (use of a 'CO2 scrubbing apparatus' described in the OECD Test Guideline 301 B), in order to purge the system of carbon dioxide.
- Test performed in closed vessels due to significant volatility of test substance: yes

SAMPLING
- Sampling frequency: Titrations were performed at t3d, t6d, t10d, t14d, t17d, t22d, t28d, and t29d.
- Sampling method: CO2 generated by the test item was trapped by 0.025 N Ba(OH)2 in a trap system described in the Guideline. Resting Ba(OH)2 (0.025 N) in the CO2 trap removed from the trap system was titrated with HCl (0.05 N) using phenolphthalein as an indicator.


CONTROL AND BLANK SYSTEM
- Inoculum blank: For Blank correction two test solutions were prepared in the same way as the test solutions with the test item, but without the addition of any test- or control item.
- Toxicity control: One further test solution was prepared using 123.5 mg sodium benzoate and 68.1 mg of the test item for toxicity control.
- Other: At the same time one test solution containing 123.3 mg sodium benzoate/3.5 L (ca. 20 mg C/L) of test solution was run in parallel.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

17

Sampling time:

28 d

Remarks on result:

other:

Remarks:

mean of 23% and 11% degradation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The study was conducted under GLP according to OECD 301 B without deviations on the registered substance itself. The method is to be considered scientifically reasonable and suitable for the test item, the available information allows the conclusion that the test was properly conducted, the positive control showed the appropriate results. Hence, the results can be considered as reliable to assess ready biodegradability of Isobutyl 4-chloro-3,5-diaminobenzoate. The test item attained 17% biodegradation after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Executive summary:

Isobutyl 4-chloro-3,5-diaminobenzoate was tested for biodegradability according to 'CO₂-Evolution-Test' (OECD Guideline 301B). Calculated from the organic carbon content of the test item and the measured CO₂ generation, 23 % of the theoretical CO₂ (ThCO₂) has been generated by the test item within 28d of test period in the first culture with 67.4 mg of test item. 11% of the theoretical CO₂ (ThCO₂) has been generated by the test item within 28d in the second culture with 67.7 mg of test item. The calculated mean degradation value of the test item was 17%. Thus, the test item may be regarded as “not readily biodegradable". The control item sodium benzoate was degraded 85 %, and the conditions of the "10-days-window" were met within 6 days. The total CO₂-evolution of the Blank was 115 mg CO₂. Thus the test is regarded to be valid. The results obtained with the toxicity control indicate that there was no toxicity of the test item towards microorganisms at the concentration used within the test as a degradation value of >30% was achieved in total within that treatment.