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Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27/02/2019 - 05/04/2019
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
Specific details on test material used for the study:
Date of Receipt February 15, 2019
RCC Code of Test Item 249/006
The following information was provided by the sponsor:
Identification Tyzor AC436
Colour & Appearance Yellow to Brownish Clear liquid
Chemical Name µ2– (2-oxido-2-acetato-κ1O-1,3-dicarboxylate-κ2O)-bis[(2-oxidopropane-2-carboxylato-1,3-dicarboxylate)(2- oxidopropane-1,2,3-tricarboxylate- κO)titanium](4+)
Formulation or Composition Tyzor AC 436 Contains > 44 % (w/w) Water
Lot Number 3340011167
Purity >99%
Molecular Weight 1048.3 g/mol
Molecular Formula [{Ti2(C6H7O7)( C6H6O7)}( µ2-C6H6O7 )]
Manufacture Date January, 2019
Expiry Date December, 2020
Stability The product is stable under storage at normal ambient temperature
Storage conditions Room Temperature (20 to 30°C)
Safety Precautions Aprons, masks, caps, gloves and goggles were used to ensure the health and safety of the personnel
Oxygen conditions:
Inoculum or test system:
natural water: freshwater
Details on inoculum:
The surface water was considered as source of inoculum. The surface water was sourced from Shameerpet Lake in Ranga Reddy District of Telangana State, India. The location co-ordinates correspond to 17°36’33.9”N and 78°33’38.0”E. The inoculum was aerated for 5 days followed by microbial colony forming unit count was evaluated. The domestic waste was not considered as source of inoculum because it was contaminated by chlorinating agent.
Duration of test (contact time):
28 d
Initial conc.:
95.214 mg/L
Based on:
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The experimental set up included, the Zero air cylinder which was connected to manifold from which the air devoid of oxides of carbon and nitrogen were transferred through silicon tubing at one atmospheric pressure. The air was passed to five, 5.0 litre narrow mouthed glass flasks containing mineral medium as mentioned in Section 5.1. The inlet air which comes from the manifold of Zero air cylinder was connected to the glass tube extended to the bottom of the 5.0 litre glass flasks, while the outlet at the top of 5.0 litre narrow mouth glass flask were used to vent excess air and carbon dioxide.
The inoculum, surface water 25.0 mL taken in a measuring cylinder (50 mL capacity) was added to the mineral medium. This was followed by rinsing of measuring cylinder with additional 75 mL of distilled water and the same was transferred to respective flasks. The mineral medium along with inoculum was aerated to purge out carbon dioxide at the rate of 30 mL/min for a period of 24 hours, without connecting the barium hydroxide traps.
After 24 hours of aeration with the inoculum, the test item and reference item were added in calculated amounts as mentioned in Section 5.5. The experiment was conducted with the following number of flasks labeled appropriately.
Flasks 1 & 2 - Contains both test item and inoculum in each flask (test suspension)
Flasks 3 & 4 - Contains only inoculum in each flask (inoculum blank)
Flask 5 - Contains both reference item and inoculum (reference suspension)
To flask 1 and 2 calculated volumes of around 18mg equivalent carbon per litre (Dissolved organic carbon) of the test item was added. The test item was found to be soluble in aqueous medium, therefore no extra pre work was carried out for solubilization.
To flasks 3 and 4 neither test item nor reference item was added. This serves as control for carbon dioxide evolution due to microbial interaction with mineral medium.
To the 5th flask calculated reference item, around 18mg per litre with respect to (DOC) carbon content was added. This serves as experiment validity.
All the flasks were covered with black color poly ethylene bag to avoid light transparency. To each flask, three 0.0125M Barium hydroxide (100 mL) traps were connected in series. The outlet glass tube of the test flask was connected to the inlet of the first barium hydroxide trap glass tube extended to bottom of the trap, while the outlet glass tube (extends from neck of the trap) from the first barium hydroxide trap was connected to the inlet glass tube of second trap (extends up to bottom of the trap) using silicon tubing. The same was followed for third trap.
The test was initiated by bubbling CO2 free air through the suspensions at a rate of around 30 mL/min. The study was conducted for 28 days at a temperature 22°C±2°C. The CO2 was quantified on 0th to 7th day, 11th to 12th day, 15th, 16th, 21th, 26th, 27th and 28th day. The closest barium hydroxide (trap) CO2 absorber to the test vessel was subjected to titration against 0.05M HCl using phenolphthalein indicator. The next absorber in series was connected immediate to test vessel while fresh 100 mL barium hydroxide absorber was connected to last in the series. This pattern of replacement was followed during the course of study. The volume of 0.05 M Hydrochloric acid consumed for each trap was recorded.
On the end of 28th day analysis, 1.0 mL of concentrated Hydrochloric acid was added to each test vessel and aerated them overnight to drive off the carbon dioxide present in the test suspensions.
On day 29th the last analysis of evolved carbon dioxide trapped in remaining barium hydroxide absorber and same was evaluated.
Reference substance:
benzoic acid, sodium salt
Key result
% degradation (CO2 evolution)
St. dev.:
Sampling time:
4 d
Key result
% degradation (CO2 evolution)
St. dev.:
Sampling time:
12 d
Key result
% degradation (CO2 evolution)
St. dev.:
Sampling time:
15 d
Key result
% degradation (CO2 evolution)
St. dev.:
Sampling time:
28 d
Details on results:
The microbial count evaluated for the surface water on the day of exposure to experiment bottles was found to be around 107 units of CFU.
The test item interaction with microbes in the test suspension was positive, augmented by 60% degradation within the 10 day window period from the time of initial 10% degradation. The short lag phase to achieve 10% degradation calculated in terms of evolved carbon dioxide was between 4th and 5th day. Moreover at the end of 28th day the test item bio-degraded to a level of 81.56% in terms of carbon dioxide evolved.
The details regarding test item added, theoretical carbon dioxide, and carbon dioxide produced and % biodegradation in test suspension was given in Table 1, 3 and 4.and the graphical representation was provided in Figure 1.
The bio degradation was effective for reference suspension, as the pass level of 60% degradation was attained within the 14 day window period. This substantiates the validity of the study. The details on titer value and calculations were provided in Table 5-7. The graphical representation was given in Figure 2. The inoculum blank titer value and carbon dioxide produced was presented in Table 2.
Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
The liquid test item readily undergoes biodegradation.
Executive summary:

The study was conducted to evaluate the ready bio degradability of liquid test item, AC 436 under stringent laboratory conditions. The temperature during the study period was maintained at 22±2°C for 28 days.

The surface water from Shamirpet Lake located in Telangana state of India was used as inoculum. Initially the surface water was aerated for 5 days, after which the same was used as inoculum to the test vessel. The microbial count evaluated for the surface water on the day of exposure to experiment bottles was found to bearound 107CFU

The microbial interaction with the test item in test suspension was positive, supported by the data generated on carbon dioxide evolution for 28 days. The test item degraded by the microorganisms was 10% between 4thand 5thday. This was followed by degradation of test item to around 60% within 13 day i.e. within 7 to 8 days after 10% of test item degradation. This substantiates that the test item was readily biodegradable.

The validity of the study was supported by the reference suspension behavior in which the bio-degradation was achieved greater than 60% within 14 days window period.

Based on the above facts the study was concluded as, test item was readily biodegradable.  

Description of key information

Based on the test results the substance is considered as readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:

Additional information