Monalazone disodium

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:

26.10.2018 - 07.11.2019

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)

GLP compliance:

yes

Specific details on test material used for the study:

Test Item : Monalazone Disodium

Physical appearance : White or off white powder or crystals

Lot No. : 72617S

% Total Active Ingredient : >99% excluding water

Molecular formula : C7H4ClNNa2O4S

Molecular weight : 387 g/mol

Manufactured date : 26 July 2017

Expiry date : 01 February 2019

Recommended storage condition : Ambient (+18 to +36 °C)

pH : 8-10



Notes: a) Date of receipt of test item at test facility: 22 September 2018
b) Test Item code by test facility: H017-01
c) Theoretical molecular weight of the anhyrdous form of the test item is 279.602 g/mol which exist above 60 C

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

The inoculum was a secondary effluent, collected from a treatment plant receiving predominantly domestic sewage. This effluent was used as test system as it is recommended in the guideline. A fresh sample of secondary effluent was collected from the treatment plant and was kept aerobic during transport.
The bacterial population in the inoculum was 6.0 x 10^7 CFU/L

This effluent was allowed to settle for one hour, decanted and the decanted effluent was used in the test.

Source of the Inoculum

Sewage Treatment Plant
Eurofins Advinus Limited
Bengaluru – 560 058
India

Determination of Bacterial Population in the Inoculum

The bacterial population in the inoculum was determined as colony forming units (CFU/mL) by diluting the inoculum to an appropriate dilution and then plating on nutrient agar plates.

Preconditioning of the Inoculum

The decanted effluent was preconditioned by aerating for 5 days at 22 to 24C.

Duration of test (contact time):

29 d

Initial conc.:

>= 10 - <= 20 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

A measured volume of the inoculated mineral medium, containing a known concentration of the test item [10-20 mg total organic carbon (TOC) per litre] as the nominal sole source of organic carbon was aerated by the passage of carbon dioxide-free air at a controlled rate in the dark. Degradation was followed over 28 days by determining the carbon dioxide produced. The carbon dioxide was trapped in barium hydroxide and was measured by titration of the residual hydroxide or as inorganic carbon. The amount of carbon dioxide produced from the test item (corrected for that derived from the blank inoculum) was expressed as a percentage of the theoretical carbon dioxide (ThCO2).
6.2.2 Test Item Solubility
The test item was tested for its solubility in mineral media at 500 mg/L.
6.2.3 Preparation of Test Medium
The stock solutions and the test medium were prepared as per the compositions of the chemicals given in Appendix 2. High quality ultra-pure water delivered by a Milli-Q system was used to prepare the stock solutions and the mineral medium.
6.2.4 Performance of the Test
Test System Identification

Test flasks were labeled to indicate the study number and the flask numbers.

Preparation of Test Flasks

Flask No. Contents
1 & 2 Test suspension – test item and inoculum
3 & 4 Inoculum blank – only inoculum
5 Procedure control – reference item and inoculum
6 Toxicity control – test item, reference item and inoculum

To each 5 L flask, 2400 mL of mineral medium was added and mixed with 300 mL of the pre-conditioned inoculum. A separate 3000 mL of mineral medium was also prepared in a flask to use it for further dilutions.

A sample of the mineral medium was checked for the inorganic carbon content.

These flasks were aerated with CO2 free air at 43 to 50 mL/minute, overnight to purge the system of carbon dioxide.

Inoculum Blanks

To the test flasks numbered 3 and 4 (inoculum blanks), 300 mL each of mineral medium (previously aerated with CO2-free air) was added to make the total suspension volume to 3000 mL in each flask.

Addition of Test Item and Reference Item

The total organic carbon (TOC) in the test as well as the reference item was determined using the formula:

% TOC = Carbon content of the test/reference item x 100
Molecular weight of the test/reference item
Molecular formula of test item – C7H4ClNNa2O4S
Molecular formula of reference item (Sodium benzoate) – C7H5NaO2

Based on the TOC, 150 mg of test item was weighed and made up to 300 mL using mineral medium (previously aerated with CO2-free air) and added to each test flasks 1 and 2 separately. Similarly, 78 mg of reference item was made up to 300 mL with mineral medium (previously aerated with CO2-free air) and added to test flask 5. A quantity of 75 mg test item and 39 mg of the reference item was mixed and made up to 300 mL using mineral medium (previously aerated with CO2-free air) and added to test flask 6. Final volume in the test flask was 3000 mL.

Exposure to Treatment

The outlet of each test flask was connected to the inlet of a gas absorption bottle containing 100 mL of 0.0125 M barium hydroxide solution in a series of 3 keeping the outlet of the last absorption bottle open.

Before each use, the strength of barium hydroxide was determined by titrating against potassium hydrogen phthalate (PHP). For this, 10 mL of 0.025 M PHP was transferred into a conical flask, 2 to 3 drops of phenolphtalein indicator was added and this was titrated against 0.0125 M barium hydroxide solution until the colour of PHP just turned pink. The molarity of barium hydroxide was calculated using the formula –

Molarity of Barium hydroxide = Volume of PHP taken x Molarity of PHP
2 x Titre value

The test was carried out by bubbling carbon dioxide free air through the suspension at a rate of 100 mL/minute for 28 days. Once a week, the flow rate of carbon dioxide free air was checked using the bubble flow meter.
On the 28th day, pH of the test suspension was recorded and 1 mL of concentrated hydrochloric acid was added to each flask and the bubbling of carbon dioxide free air was continued.

On the 29th day, the bubbling was stopped. All the barium hydroxide absorption bottles were disconnected and were titrated for the determination of carbon dioxide production.

The temperature of the room was maintained at 22 to 24ºC during the treatment period.
6.2.5 Observations
6.2.5.1 Determination of CO2
To identify the 10-day window period, during the first 10 days, carbon dioxide analysis was made every second-third day and then at least every fourth day until the 29th day.
6.2.5.2 Data Presentation
The amount of CO2 produced was calculated from the amount of base remaining in the absorption bottle. The amount of CO2 remaining is assessed by titrating 0.0125 M Ba(OH)2 with 0.05 M HCl [Thus 50 mL HCl would be needed to titrate 100 mL Ba(OH)2].

Since, 1 mmol of CO2 is produced for every mmol of Ba(OH)2 reacted to BaCl2 and 2 mmol of HCl are needed for the titration of the remaining Ba(OH)2 and given that the molecular weight of CO2 is 44 g, the weight of CO2 produced (mg) was calculated by:

CO2 produced (mg) = Molarity of HCl x (50 – mL HCl titrated) x 44
2

The weights of CO2 produced from the inoculum alone and from the inoculum plus test item was calculated using the respective titration values; the difference is the weight of CO2 produced from the test item alone.

The percentage biodegradation is calculated from:

% degradation = mg CO2 produced x 100
mg TOC added in test x 3.67

where 3.67 was the conversion factor (44/12) for carbon to carbon dioxide.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

13.29

Sampling time:

3 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

56.91

Sampling time:

13 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

73.17

Sampling time:

29 d

Details on results:

Test item solubility
The test item formed a solution in mineral medium at 500 mg/L.

Bacterial Population
The bacterial population in the inoculum was 6.0 x 10^7 CFU/L.

Production of Carbon-dioxide during the Treatment Period
The carbon dioxide production during the treatment period is given in Table 1. pH of the test solutions at the end of the test is given in Table 2.

The mean carbon dioxide produced from the inoculum blank (Flask No. 3 and 4) on days 3, 6, 8, 10, 13, 16, 20, 24, 27 and 29 after the treatment was 9.30, 13.92, 14.58, 13.48, 15.18, 18.54, 10.84, 11.99, 9.24 and 0 mg, respectively. The total carbon dioxide produced from the inoculum blank throughout the test period was 39.02 mg/L.

The cumulative carbon dioxide produced from the test item was 15.67, 34.09, 37.44, 52.89, 66.86, 71.97, 75.76, 79.61, 83.90 and 83.90 mg in Flask 1 on days 3, 6, 8, 10, 13, 16, 20, 24, 27 and 29 after the treatment, respectively. Simmilaröy, in Flask 2 it was 16.22, 31.45, 38.43, 57.29, 69.72, 80.22, 82.14, 85.77, 91.71 and 91.71 mg on days 3, 6, 8, 10, 13, 16, 20, 24, 27 and 29 after the treatment respectively.

The cumulative carbon dioxide produced from the refrence item (Flask No. 5) was 23.26, 47.62, 75.50, 101.40, 117.24, 121.03, 121.08, 123.83, 133.18 and 133.18 mg on days 3, 6, 8, 10, 13, 16, 20, 24, 27 and 29 after the treatment respectively.

The cumulative carbon dioxide produced from the toxicity control (Flask No 6) was 15.45, 33.87, 48.77, 57.62, 64.22, 60.53, 62.23, 64.76, 68.83 and 68.83 mg on days 3, 6, 8, 10, 13, 16, 20, 24, 27 and 29 after the treatment respectively.

The pH of the test solutions at the end of the test was 7.85, 7.86, 7.87, 7.86, 7.81 and 7.82 in Flask 1, 2, 3, 4, 5 and 6 respectively.

Percent degradation
The percent degradation of the test item, refernce item and toxicity control is presented in Table 3 and Figure 1.

The percent degradation of the test item was 69.91% and 76.42% in Flask 1 and 2, respectively, at the end of the test. The mean per cent degradation of the test itme was 73.17% at the end of the test while, the percent degradation of the refernce item was 79.58% and the toxicity control was 48.09% at the end of the test.

Based on the results, it was concluded that the test item, Monalazone disodium, is biodegradable but not readily biodegrdable as the mean degradation achieved is less than 60% pass level in a 10-day window period within the 28-day period of the test.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

Based on the results, it was concluded that the test item, Monalazone Disodium is biodegradable but not readily biodegradable as mean degradation achieved is less then 60% pass level in a 10-day window period within the 28-day period of the test.

Executive summary:

The ready biodegradability of Monalazone Disodium was t5ested using the CO2 Evolution Test. The test item was added to two test vessels at a concentration of 50 mg/L (equivalent to 15 mg of Total Organic Carbon/L). Two control treatments containing only the inoculum, one reference item treatment and one toxicity control treatment containing the test item and the reference item were also tested. All the treatments were added with an equal volume of inoculum which was collected from a secondary effluent treatment plant receiving predomnantly domestic sewage.

Treatment mixtures were aerated for 29 days with carbon dioxide (CO2) free air. The released CO2 was trapped in a series of bottles containing barium hydroxide, which were connected to the outlet of each test vessel. The residual barium hydroxide was measured on Days 3, 6, 8, 10, 13, 16, 20, 24, 27 and 29 after the initiation of the test while, the per cent degradation of reference item was 79.58% and the toxicity control was 48.09% at the end of the test.

The test fulfilled all the validity criteria.

Based on the results, it was concluded that the test item, Monalazone disodium is biodegradable but not readily biodegradable as the % mean degradation achieved is less than 60% pass level in a 10 -day window period within the 28 -day period test.

The mean per cent degradation of test item was 73.17% at the end of the test

Description of key information

The percent degradation of the test item was 69.91% and 76.42% in Flask 1 and 2, respectively, at the end of the test. The mean per cent degradation of the test itme was 73.17% at the end of the test while, the percent degradation of the refernce item was 79.58% and the toxicity control was 48.09% at the end of the test.

Based on the results, it was concluded that the test item, Monalazone disodium, is biodegradable but not readily biodegrdable as the mean degradation achieved is less than 60% pass level in a 10-day window period within the 28-day period of the test.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable but failing 10-day window

Type of water:

freshwater

Additional information

The ready biodegradability of Monalazone Disodium was t5ested using the CO2 Evolution Test. The test item was added to two test vessels at a concentration of 50 mg/L (equivalent to 15 mg of Total Organic Carbon/L). Two control treatments containing only the inoculum, one reference item treatment and one toxicity control treatment containing the test item and the reference item were also tested. All the treatments were added with an equal volume of inoculum which was collected from a secondary effluent treatment plant receiving predomnantly domestic sewage.

Treatment mixtures were aerated for 29 days with carbon dioxide (CO2) free air. The released CO2 was trapped in a series of bottles containing barium hydroxide, which were connected to the outlet of each test vessel. The residual barium hydroxide was measured on Days 3, 6, 8, 10, 13, 16, 20, 24, 27 and 29 after the initiation of the test while, the per cent degradation of reference item was 79.58% and the toxicity control was 48.09% at the end of the test.

The test fulfilled all the validity criteria.

Based on the results, it was concluded that the test item, Monalazone disodium is biodegradable but not readily biodegradable as the % mean degradation achieved is less than 60% pass level in a 10 -day window period within the 28 -day period test.

The mean per cent degradation of test item was 73.17% at the end of the test