Balmywood

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:

From February 14, 2022 to August 09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspection :18 to 20 August 2020 Statement : 17 March 2021

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The sample of inoculum was collected from the secondary effluent of a treatment plant receiving predominantly domestic sewage (Aureilhan, France)
- Laboratory culture: The sample was aerated for about 6 days before use as inoculum at the test temperature to reduce endogenous respiration.

- Concentration of sludge: A concentration of inoculum of 8.3×104 cells/L was used.

Duration of test (contact time):

60 d

Initial conc.:

5 mg/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium, Reconstituted water, as prescribed by OECD Guideline No. 301 (1) (see 2.2.)

- Test temperature: Controlled environment cabinet (22°C ± 2°C); the test was performed in the dark. Measured continuously in the growth chamber.
- pH:

- Suspended solids concentration:
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: All-glass sealed with ground glass stoppers BOD (Biological Oxygen Demand) bottles of approximately 300 mL capacity. Each test vessel was uniquely identified with study code, replicate number, date of the start of the biodegradability test (=J0) and type of series.
- Number of culture flasks/concentration: - 10 bottles containing inoculum, mineral medium and glass coverslip (inoculum blank with solid support)
- 10 bottles containing inoculum and mineral medium (inoculum blank without solid support)
- 10 bottles containing test item, inoculum, mineral medium and glass coverslip (test suspension)
- 6 bottles containing reference substance, inoculum and mineral medium (procedure control)
- 6 bottles containing test item (and glass coverslip), reference substance, inoculum and mineral medium (toxicity control)

- Measuring equipment:
- Test performed in closed vessels due to significant volatility of test substance


Due to the properties of the test item (sticky and viscous substance), the administration of the test item was accomplished by addition onto a solid support and sonication. For this purpose, an appropriate amount of test item was weighed onto a glass coverslip. The test item/support combination was added to the respective test vessels that were filled with 200 mL of mineral medium and closed. Each test vessel was placed in an ultrasonic bath at ambient temperature and sonicated for 40 minutes. Then the vessels were allowed to cool to room temperature before being completely filled with the inoculated mineral medium to obtain a nominal concentration of 5 mg test item/L, and were then closed. The test was carried out without adjustment of the pH.

Determinations of the concentration of dissolved oxygen using an oxygen electrode in duplicate vessels were made on day 0, 7, 14, 21, 28, 35, 42, 49, 56 and 60 for the test suspension and the inoculum blank with solid support, on day 0, 7, 14, 21 and 28 for the inoculum blank without solid support, and only at the start of the test and after 7 and 14 days of incubation for the procedure and toxicity control. Determinations of dissolved oxygen concentration after day 28 were made from reincubated test vessels (used for O2 measurements on previous days). This was made possible by the funnel-shaped bulge in the neck of the test vessels that enabled the oxygen sensor to be submerged without overflowing the sample (i.e. the sample displaced by the sensor was collected in the neck of the test vessel and flowed back into it when the sensor was removed; then the test vessel was closed and reincubated up to a possible additional O2 measurement in the following days).

Reference substance:

benzoic acid, sodium salt

Preliminary study:

no

Key result

Parameter:

% degradation (O2 consumption)

Value:

41

Sampling time:

60 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

29

Sampling time:

28 d

Details on results:

In this study, the test item was biodegraded by 29% after 28 days (see Table 3 and Figure 1 of Appendix II). The test period was therefore extended to 60 days in order to improve the biodegradability and persistence assessments. The biodegradation percentage on day 60 was 41%. It should be noted that the test item had no inhibitory effect on the activity of the microbial inoculum, as demonstrated by the test results of the toxicity control, in agreement with OECD 301 (oxygen consumption in the toxicity control must be greater than 25% of the ThOD value within 14 days).

Therefore, ZA3155 BALMYWOOD cannot be considered as readily biodegradable under the experimental conditions, but can be considered as inherently primary (partially) biodegradable.

Results with reference substance:

The biodegradation percentage of the reference substance, sodium benzoate, was 76% at day 14, thus confirming the suitability of the inoculum used.

Moreover, in the toxicity control containing both the test item and reference item sodium benzoate, 47% biodegradation was noted at day 14 of the test. According to the test guideline, the test item can be assumed to be not inhibitory to the microbial inoculum because degradation was greater than 25% within 14 days (based on ThOD).

Thus all validity criteria of the test have been fulfilled in the present study

Table 2. Dissolved oxygen concentrations (mg/L) throughout the test. 

 

	Time (days)
	0	7	14	21	28	35	42	49	56	60
Inoculum Blank  + solid support	8.22	8.15	8.12	7.72	7.22	7.27	7.30	7.25	7.21	7.09
	8.34	8.12	8.07	7.56	7.46	7.34	7.28	7.13	7.00	7.24
Mean	8.28	8.14	8.10	7.64	7.34	7.31	7.29	7.19	7.11	7.17
Test Suspension  (5 mg test item/L)	8.07	5.34	4.90	3.52	2.69	1.56	0.89	0.86	0.72	0.62
	7.88	5.56	5.10	3.68	2.62	1.62	0.93	0.59	0.99	1.04
Mean	7.98	5.45	5.00	3.60	2.66	1.59	0.91	0.73	0.86	0.83
Inoculum Blank  (without solid support)	8.18	8.09	7.98	7.69	7.43	-	-	-	-	-
	8.18	8.15	8.04	7.73	7.44	-	-	-	-	-
Mean	8.18	8.12	8.01	7.71	7.44	-	-	-	-	-
Procedure Control (5 mg  reference substance/L)	8.20	1.55	1.09	-	-	-	-	-	-	-
	8.20	1.80	1.16	-	-	-	-	-	-	-
Mean	8.20	1.68	1.13	-	-	-	-	-	-	-
Toxicity Control  (5 mg  reference substance/L  + 5 mg test item/L)	8.06	5.39	3.71	-	-	-	-	-	-	-
	7.92	5.36	4.05	-	-	-	-	-	-	-
Mean	7.99	5.38	3.88	-	-	-	-	-	-	-

 

 

 

 

 

 

		Time (days)
		0	7	14	21	28	35	42	49	56	60
Test Suspension  (5 mg test item/L)	Replicate 1	0.00	17.65	20.38	26.69	30.31	37.78	42.25	41.77	42.15	43.24
	Replicate 2	0.00	14.85	17.71	24.30	29.49	36.08	40.68	42.32	39.01	39.08
	Mean	0	16	19	25	30	37	41	42	41	41
Procedure Control  (5mg reference substance/L)	Replicate 1	0.00	78.92	83.11	-	-	-	-	-	-	-
	Replicate 2	0.00	75.93	82.28	-	-	-	-	-	-	-
	Mean	0	77	83	-	-	-	-	-	-	-
Toxicity Control  (5 mg  reference substance/L  + 5 mg test item/L)	Mean	0	30	47	-	-	-	-	-	-	-

 

 

 

	Values for   OECD 301D	Validity criteria for the test
Oxygen depletion in the Control after 28 days	< 1.5 mg/L	OK   (Control:  1.13 mg/L)  (Solid support Control 0.75 mg/L)
Dissolved Oxygen in the bottles (test item) during the study	≥ 0.5 mg/L	OK (0.62 to 1.04 mg/L)
Variation between test item replicates at the end of the test	< 20%	OK (Cv =7%)
Biodegradadability of the reference item within 14 days	≥ 60%	OK (83% Day 14)
Biodegradadability of the toxicity control at 14 days	≥ 25% (ThOD)	OK (47%)

 

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

In this study, the test item was biodegraded by 29% after 28 days. The test period was therefore extended to 60 days in order to improve the biodegradability and persistence assessments. The biodegradation percentage on day 60 was 41%. It should be noted that the test item had no inhibitory effect on the activity of the microbial inoculum, as demonstrated by the test results of the toxicity control, in agreement with OECD 301
The test item cannot be considered as readily biodegradable under the experimental conditions, but can be considered as inherently primary (partially) biodegradable.

Executive summary:

The readily biodegradability of the tested substance was assessed according to the OECD Guideline for testing of Chemicals No. 301 D, "Ready biodegradability – Closed Bottle Test", referenced as Method C.4-E of Commission Regulation No. 440/2008 and with ISO 10634,  over a period of 60 days. BOD bottles were filled with mineral medium inoculated with a secondary effluent of a treatment plant and test item added at a nominal concentration of 5 mg/L with the help of a solid support (glass coverslip) followed by sonication. In the meantime, two series of blanks (with and without solid support) were filled with inoculated mineral medium. Furthermore, bottles containing reference substance sodium benzoate (5 mg/L) were tested in order to check the procedure. A toxicity control, containing both the test item and the reference substance, was also performed in order to check the absence of test item effect on the microbial inoculum. The concentrations of dissolved oxygen in duplicate bottles from each group incubated at 22°C ± 2°C in darkness were measured at the start of the test and thereafter once a week until the end of the test (except for the procedure and toxicity control, where measurements were made until day 14).

The mean biodegradation of the test item at day 28 was 29% (ThOD), therefore the study was prolonged till day 60. The mean biodegradation at test end after 60 days was 41% (ThOD). Therefore, test item is considered to be not readily biodegradable based on ThOD  after 28 days but reached degradation rate of 41% (ThODNH4) after 60 days.

The test item cannot be considered as readily biodegradable under the experimental conditions, but can be considered as inherently primary (partially) biodegradable.

Description of key information

OECD Guideline 301D, GLP, key study, validity 1:

29% after 28 days, 41% after 60 days.

The test item cannot be considered as readily biodegradable under the experimental conditions, but can be considered as inherently primary (partially) biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

The readily biodegradability of the tested substance was assessed according to the OECD Guideline for testing of Chemicals No. 301 D, "Ready biodegradability – Closed Bottle Test", referenced as Method C.4-E of Commission Regulation No. 440/2008 and with ISO 10634,  over a period of 60 days. BOD bottles were filled with mineral medium inoculated with a secondary effluent of a treatment plant and test item added at a nominal concentration of 5 mg/L with the help of a solid support (glass coverslip) followed by sonication. In the meantime, two series of blanks (with and without solid support) were filled with inoculated mineral medium. Furthermore, bottles containing reference substance sodium benzoate (5 mg/L) were tested in order to check the procedure. A toxicity control, containing both the test item and the reference substance, was also performed in order to check the absence of test item effect on the microbial inoculum. The concentrations of dissolved oxygen in duplicate bottles from each group incubated at 22°C ± 2°C in darkness were measured at the start of the test and thereafter once a week until the end of the test (except for the procedure and toxicity control, where measurements were made until day 14).

The mean biodegradation of the test item at day 28 was 29% (ThOD), therefore the study was prolonged till day 60. The mean biodegradation at test end after 60 days was 41% (ThOD). Therefore, test item is considered to be not readily biodegradable based on ThOD  after 28 days but reached degradation rate of 41% (ThODNH4) after 60 days.

The test item cannot be considered as readily biodegradable under the experimental conditions, but can be considered as inherently primary (partially) biodegradable.