A dried sludge product resulting from the treatment process of domestic wastewater. The exact processes followed for the production of the dried sludge are: Preliminary + secondary treatment of the wastewater stream, thickening, dehydration and drying of the excess sludge.

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:

Study: From 25 Sep 2021 to 18 Jan 2022
Treatment: From 01 Oct 2021 to 30 Oct 2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Ready Biodegradability,
Method 301B “CO2 Evolution (Modified Sturm Test)”, adopted on 17 July
1992.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

The inoculum was secondary effluent, sourced 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.
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 6 days at 22.4 to 23.8°C.

Duration of test (contact time):

28 d

Initial conc.:

ca. 2 122 mg/L

Based on:

test mat.

Remarks:

(equivalent to 15.5 mg of Total Organic Carbon/L)

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

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 on start of the test.
These flasks were aerated with CO2 free air at 100 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)/(Molecular weight of the test/reference item))x 100
Molecular formula of reference item (Aniline) – C7H5NH2
Based on the TOC, 127.32 mL of test item stock was mixed 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, 60 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 63.66 mL test item stock and 30 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
phenolphthalein indicator was added and this was titrated against 0.0125 M
barium hydroxide solution until the color 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 Titer value)

The test was carried out by bubbling carbon dioxide free air through the
suspension at a rate of 46 to 50 mL/minute and continued 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 22.0 to 23.9 ºC during the
treatment period.

Observations
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.
Data Presentation:
The amount of CO2 produced was calculated from the amount of base remaining in the absorption bottle. The amount of CO2 remaining was 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:

aniline

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 97.77

Sampling time:

28 d

Test Item Solubility: The test item formed turbid opaque grey color soluble preparation in mineral medium at 2122 mg/L.

Bacterial Population in the Inoculum:

The bacterial population in the inoculum was 6.0 x 107 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 2, 5, 7, 10, 13, 16, 19, 23, 27 and 29 after the treatment was 10.12, 9.90, 11.77, 21.07, 15.29, 11.94, 10.18, 10.56, 9.35 and 0.00 mg, respectively. The total carbon dioxide produced from the inoculum blank for throughout the test period was 36.8 mg/L.
The cumulative carbon dioxide produced from the test item was 35.86, 73.15, 103.84, 127.21, 149.76, 160.04, 165.48, 167.68, 169.55 and 169.55 mg in Flask 1 on days 2, 5, 7, 10, 13, 16, 19, 23, 27 and 29 after the treatment, respectively. Similarly, in Flask 2 it was 37.95, 73.15, 104.83, 127.65, 151.08, 158.28, 162.51, 162.73, 164.16 and 164.16 mg on days 2, 5, 7, 10, 13, 16, 19, 23, 27 and 29 after the treatment, respectively.
The cumulative carbon dioxide produced from the reference item (Flask No. 5) was 34.10, 67.54, 98.89, 119.18, 138.21, 158.72, 167.68, 168.34, 168.45 and 168.45 mg, on days 2, 5, 7, 10, 13, 16, 19, 23, 27 and 29 after the treatment, respectively.

The cumulative carbon dioxide produced from the toxicity control (Flask No. 6) was 28.93, 55.55, 79.09, 94.54, 113.79, 130.67, 146.45, 161.74, 163.83 and 163.83 mg, on days 2, 5, 7, 10, 13, 16, 19, 23, 27 and 29 after the treatment, respectively.
The pH of the test solutions at the end of the test was 8.16, 8.17, 8.40, 8.36, 8.45 and 8.43 in Flask 1, 2, 3, 4, 5 and 6 respectively.

Percent Degradation:

The percent degradation of the test item, reference item and toxicity control is
presented in Table 3 and the attached Figure.
The percent degradation of test item was 99.35 % and 96.19 % in the Flask
No. 1 and 2, respectively at the end of the test. The mean per cent
degradation of test item was 97.77 % at the end of test. The percent
degradation of reference item was 98.71 % and the toxicity control was
96.0 % at the end of the test. This shows the test item is not inhibitory to the
test system.

 

Table 1. Carbon-dioxide Production

Day	CO2 Produced (mg)							Cumulative CO2 produced (mg) Test – Blank Mean
	Test Flasks				Blank
	Flask 1	Flask 2	Flask 5	Flask 6	Flask 3	Flask 4	Mean	Flask 1	Flask 2	Flask 5	Flask 6
2	45.98	48.07	44.22	39.05	10.78	9.46	10.12	35.86	37.95	34.10	28.93
5	47.19	45.10	43.34	36.52	9.46	10.34	9.90	73.15	73.15	67.54	55.55
7	42.46	43.45	43.12	35.31	11.66	11.88	11.77	103.84	104.83	98.89	79.09
10	44.44	43.89	41.36	36.52	21.45	20.68	21.07	127.21	127.65	119.18	94.54
13	37.84	38.72	34.32	34.54	15.95	14.63	15.29	149.76	151.08	138.21	113.79
16	22.22	19.14	32.45	28.82	11.44	12.43	11.94	160.04	158.28	158.72	130.67
19	15.62	14.41	19.14	25.96	9.57	10.78	10.18	165.48	162.51	167.68	146.45
23	12.76	10.78	11.22	25.85	11.44	9.68	10.56	167.68	162.73	168.34	161.74
27	11.22	10.78	9.46	11.44	8.25	10.45	9.35	169.55	164.16	168.45	163.83
29	0.00	0.00	0.00	0.00	0.00	0.00	0.00	169.55	164.16	168.45	163.83
29	0.00	0.00	0.00	0.00	0.00	0.00	0.00	169.55	164.16	168.45	163.83
29	0.00	0.00	0.00	0.00	0.00	0.00	0.00	169.55	164.16	168.45	163.83

Total CO₂ produced in the inoculum blank during the test    = 110.18 mg/3L                                                                                                                                                                                 

                                                                                                = 36.73 mg/L

Note:  

Flask 1 and 2: Contain test item (Dried sludge from domestic wastewater) and inoculum in mineral media

Flask 3 and 4: Contain inoculum in mineral media

Flask 5: Contain reference item (Aniline) and inoculum in mineral media

Flask 6: Contain test item, reference item and inoculum in mineral media

 

Table 2. pH of Test Solutions

Flask No.	pH at the end of the test (Day 28)
Flask 1	8.16
Flask 2	8.17
Flask 3	8.40
Flask 4	8.36
Flask 5	8.45
Flask 6	8.43

Note:  

Flask 1 and 2: Contain test item (Dried sludge from domestic wastewater) and inoculum in mineral media

Flask 3 and 4: Contain inoculum in mineral media

Flask 5: Contain reference item (Aniline) and inoculum in mineral media

Flask 6: Contain test item, reference item and inoculum in mineral media

 

Table 3.Degradation of Test Item and Reference Item

Day	Test Item			Reference Item	Test Item + Reference Item
	Flask 1	Flask 2	Mean
2	21.01	22.24	21.63	19.98	16.95
5	42.86	42.86	42.86	39.58	32.55
7	60.85	61.43	61.14	57.95	46.34
10	74.54	74.80	74.67	69.84	55.40
13	87.76	88.53	88.15	80.99	66.68
16	93.78	92.75	93.27	93.01	76.57
19	96.97	95.23	96.10	98.26	85.82
23	98.26	95.36	96.81	98.64	94.78
27	99.35	96.19	97.77	98.71	96.00
29	99.35	96.19	97.77	98.71	96.00
29	99.35	96.19	97.77	98.71	96.00
29	99.35	96.19	97.77	98.71	96.00

 

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

Note:  

Flask 1 and 2: Contain test item (Dried sludge from domestic wastewater) and inoculum in mineral media

Flask 3 and 4: Contain inoculum in mineral media

Flask 5: Contain reference item (Aniline) and inoculum in mineral media

Flask 6: Contain test item, reference item and inoculum in mineral media

 

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the results, the test item was concluded as readily biodegradable
since degradation of test item reached >60% degradation in 10-day window
period within 28-day test period.

Executive summary:

The ready biodegradability of Dried sludge from domestic wastewater was tested using the CO2 Evolution Test. The test item stock was added to two test vessels at the concentration of 2122 mg/L (equivalent to 15.5 mg of Total Organic Carbon/L). Two flasks as controls containing only the inoculum, one flask as procedure control containing reference item and one flask as toxicity control containing the test item and the reference item were included in the test. All the treatments were added with equal volume of inoculum which was collected from the secondary effluent treatment plant receiving predominantly domestic sewage and volume made with mineral media. 
Treatment mixtures were aerated for 29 days with carbon dioxide (CO2) free air. The CO2 released 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 2, 5, 7, 10, 13, 16, 19, 23, 27 and 29 after the initiation of the test.
The mean per cent degradation of test item was 97.77 % at the end of test while, the percent degradation of reference item was 98.71 % and the toxicity control was 96.0 % at the end of the test. This shows the test item is not inhibitory to the test system.
The test fulfilled all the validity criteria.
Based on the results, the test item was concluded as readily biodegradable since, degradation of test item reached >60% degradation in 10-day window period and also test item is not inhibitory since, the degradation of toxicity control is >25% ThCO2 at 14th day within 28 day test period.

Description of key information

Based on the results, the test item was concluded as readily biodegradable since degradation of test item reached >60% degradation in 10-day window period within 28-day test period. 

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable