4,5-bis(hydroxymethyl)-2-phenyl-1H-imidazole

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 15 October 2021 to 03 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance OECD 301F guideline without deviation. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

July 17, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 18-20 August 2020, Date on certificate: 04 March 2021

Specific details on test material used for the study:

- Theoretical Oxygen Demad was determined to be 1.80 mg O2/mg

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge was collected (15 October 2021) from a wastewater treatment plant (Aureilhan, France) receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The inoculum derived from the activated sludge was aerated for about 5 days before use at the test temperature to reduce endogenous respiration.
- Pretreatment: Upon arrival in the test facility, the sludge was sieved (using a 1 mm2 mesh sieve) to remove coarse particles, washed twice with mineral medium and re-suspended in mineral medium. The dry weight of the sludge suspension was determined, then the sludge was diluted in mineral medium to obtain the required sludge solids concentration of approximately 3 g/L. An aliquot was used before the start of the test for the determination of the concentration of suspended solids.

Duration of test (contact time):

39 d

Initial conc.:

100 mg/L

Based on:

test mat.

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. The mineral medium was prepared by adding 10 mL of solution (a) and one mL of each of the other stock solutions (b to d) reported in "Any other information on materials and methods incl. tables" to each litre (final volume) of deionised water (containing no more than 10% of the organic carbon content introduced by the test material (checked by DOC analysis)).
- Administration of the test and reference substances: The mixing vessels were cylindrical glass bottles sealed with a screw cap and fitted with a drain port near the bottom for drawing off the stock solution. The volume of the mixing vessels were approximately 1 L. A magnetic stirring bar was placed in the vessel and mineral medium was added. Then 150.11 mg test substance were weighed on a weighing boat that afterwards was placed above the mixing vessel and rinsed with mineral medium. The mixing vessel was then carefully filled with the remaining volume of mineral medium to obtain 1 L of stock solution and closed. Mixing was initiated with the vortex in the centre extending at least to 20 % of the vessel depth from the top to the bottom of the vessel. After approx. 23 hours of stirring in the dark at approx. 80 °C (set temperature of the magnetic stirrer with hot plate, corresponding to an actual temperature of the stock solution around 60 °C), the contents of the vessel were allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port. Samples were taken from the filtered stock solution and chemically analysed. Then the filtered stock solution (through 11μm cellulose filter paper) was diluted with mineral medium (based on the analytically confirmed concentration of the stock solution (157 mg/L)) as necessary to achieve the required final test concentration of 100 mg/L in the test vessels after the addition of inoculum. The test was carried out without adjustment of the pH.
The reference substance sodium benzoate was used at a nominal concentration of 100 mg/L. A concentrated stock solution (1000 mg/L) was prepared in mineral medium. Then it was diluted with mineral medium and a fixed amount of inoculum to obtain the required reference concentration in the test vessels.
- Test temperature: Controlled environment cabinet (22 °C ± 2 °C). The temperature was measured continuously in the incubator.
- pH: Measured at the start (before inoculum addition) and the end of the test in all treatments.
- pH adjusted: no
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: All-glass brown bottles of approximately 510 mL capacity, containing a magnetic stirring rod, topped with a rubber sleeve (CO2-absorber compartment with sodium hydroxide) and tightly sealed with a manometric pressure indicator cap (measuring head).
- Number of culture flasks/concentration: 3
- Measuring equipment: O2 uptake was measured by a closed OxiTop® -IDS respirometer. The measured values were recorded by means of a pressure measurement via piezo-resistive electronic pressure sensors. One measurement per day were taken in each test vessel until the end of the test. Daily checks were made (on working days) to ensure that the correct temperature and adequate stirring were maintained.
- Test performed in closed vessels: yes
- Other: Since the test substance is a N-containing substance, samples from the test vessels (test suspension and inoculum blank) were prepared for analysis of nitrite and nitrate concentrations at the start and the end of the test. These analyses were not performed in compliance with the OECD GLP principles but in accordance with ISO 17025.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes (3 flasks containing only inoculum).
- Abiotic sterile control: Yes (1 flask containing only test substance, without inoculum).
- Toxicity control: Yes (1 flask containing test substance, reference substance and inoculum).
- Procedure control: Yes (2 flasks containing reference substance and inoculum).

Reference substance:

benzoic acid, sodium salt

Remarks:

(batch no.: 19F274116; purity: 100%; ThOD = 1.67 mg O2/mg)

Test performance:

As the test substance failed to reach the pass level for the ready biodegradability at 28 days, the test period was prolonged in order to assess if it was possible to improve biodegradability and persistence profile. As the biodegradation level was not satisfactory (i.e. minimal further biodegradation) during the prolonged test, the test was stopped on day 39.

Key result

Parameter:

% degradation (O2 consumption)

Value:

7.4

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

11

Sampling time:

39 d

Details on results:

See tables in "Any other information on results incl. tables" and illustration for the biodegradation curves.

- Toxicity control: In the toxicity control containing both the test substance and reference item sodium benzoate, 46 % of biodegradation was noted at day 14 of the test. According to the test guideline, the test substance can be assumed to be not inhibitory to the microbial inoculum because degradation was greater than 25 % within 14 days (based on ThOD; actual 46 %). In addition, the abiotic control did not reveal any particular abiotic degradation process.

- Inoculum blank: The oxygen uptake of the inoculum blank was 15.6 mg/L (mean) after 28 days and thus did not exceed 60 mg/L.

- Replicate values: The difference of extremes of replicate values with the test substance at the end of the test (day 39) was less than 20 % (actual ~10 %).

- pH: The pH values of all test vessels were within the range 6.0-8.5 (actual: 7.5 - 8.3).

- Temperature: Temperatures were situated between 21.5 and 22.0 °C throughout the test (average value: 21.7 °C), and complied with the requirements as laid down in the study plan (22 °C ± 2 °C, constant within 1 °C).

- Nitrate/nitrite analysis: Analysis of nitrite and nitrate concentrations in the inoculum blank and the test suspension at the start and the end of the test revealed there was no nitrification process from the test substance. Indeed, the values obtained with inoculum blanks and test suspensions were in the same order of magnitude (close to or below the LOQ of the analytical methods). Thus, a correction for the oxygen consumed by nitrification was not relevant for the present study.

Results with reference substance:

The biodegradation percentage of the reference substance, sodium benzoate, was 94.4% at day 14, confirming the suitability of the inoculum used.

Table 5.2.1/2: Oxygen uptake (mg/L) throughout the test

		Time (days)*
		1	3	5	7	9	11	14	15	17	19	21	23	25	27	28	29	31	33	35	37	39
Inoculum Blank	Rep.1	2.4	7.0	8.9	9.6	10.3	10.5	11.7	12.2	13.1	13.4	14.8	15.4	16.0	15.8	16.2	16.3	16.8	16.9	17.7	18.2	18.8
	Rep.2	2.7	7.2	9.1	9.5	10.3	10.6	11.7	12.2	12.9	12.9	14.2	14.9	15.6	15.7	15.7	15.9	16.3	16.4	17.3	17.7	17.8
	Rep.3	2.7	6.9	8.4	8.8	9.7	9.9	10.9	11.5	11.9	12.3	13.3	14.0	14.5	14.8	14.8	15.2	15.5	15.7	16.5	17.1	17.3
	Mean	2.6	7.0	8.8	9.3	10.1	10.3	11.4	12.0	12.6	12.9	14.1	14.8	15.4	15.4	15.6	15.8	16.2	16.3	17.2	17.7	18.0
Test Suspension (100 mg test item/L)	Rep.1	2.1	7.2	9.0	9.4	10.5	11.4	13.6	14.6	18.4	22.1	26.5	28.9	31.6	33.9	34.8	35.8	36.9	37.6	39.4	40.0	40.5
	Rep.2	3.2	7.8	9.0	9.8	10.6	10.9	12.3	12.3	13.8	15.9	19.2	22.0	24.4	25.1	25.8	26.6	28.5	30.5	32.7	34.2	34.9
	Rep.3	2.6	7.4	8.6	9.1	9.6	10.3	11.2	11.2	12.8	15.1	18.3	20.9	23.6	25.4	26.3	27.6	30.0	32.4	34.9	36.7	37.7
	Mean	2.6	7.5	8.9	9.4	10.2	10.9	12.4	12.7	15.0	17.7	21.3	23.9	26.5	28.1	29.0	30.0	31.8	33.5	35.7	37.0	37.7
Procedure Control (100 mg reference substance/L)	Rep.1	59.4	126.0	145.9	153.4	160.3	165.2	170.2	171.6	173.9	175.3	177.5	178.7	180.0	180.2	180.9	181.2	182.4	182.9	184.3	185.0	185.8
	Rep.2	58.3	125.8	146.5	153.6	160.3	164.2	167.8	169.0	170.7	172.4	174.3	175.4	176.6	176.8	177.0	177.5	177.8	178.3	179.3	180.1	180.2
	Mean	58.9	125.9	146.2	153.5	160.3	164.7	169.0	170.3	172.3	173.9	175.9	177.1	178.3	178.5	179.0	179.4	180.1	180.6	181.8	182.6	183.0
Toxicity Control (100 mg reference substance/L + 100 mg test item/L)		59.0	119.0	140.2	148.3	154.7	161.8	171.0	173.4	178.8	183.5	188.0	191.2	194.0	196.4	197.3	198.2	199.6	200.6	201.9	202.7	202.9
Abiotic Control (100 mg test item/L; sterile)		0.8	2.0	2.9	3.3	2.9	3.2	3.2	3.1	3.1	3.4	3.7	3.8	3.7	3.7	3.5	3.7	3.1	4.0	4.3	4.1	3.1

*Only results from odd-numbered days, day 14 and day 28 are shown for display purposes.

 

Table 5.2.2/3: % biodegradation of the test substance, the reference substance, the toxicity and the abiotic controls throughout the test

		Time (days)
		1	3	5	7	9	11	14	15	17	19	21	23	25	27	28	29	31	33	35	37	39
Test Suspension	Rep.1	-0.3	0.1	0.1	0.1	0.2	0.6	1.2	1.5	3.2	5.1	6.9	7.9	9.0	10.3	10.7	11.1	11.5	11.8	12.4	12.4	12.5
	Rep.2	0.3	0.4	0.1	0.3	0.3	0.3	0.5	0.2	0.6	1.7	2.8	4.0	5.0	5.4	5.7	6.0	6.8	7.9	8.6	9.2	9.4
	Rep.3	0.0	0.2	-0.1	-0.1	-0.3	0.0	-0.1	-0.4	0.1	1.2	2.3	3.4	4.6	5.5	6.0	6.6	7.7	8.9	9.9	10.6	11.0
	Mean	0.0	0.2	0.0	0.1	0.1	0.3	0.5	0.4	1.3	2.7	4.0	5.1	6.2	7.1	7.4	7.9	8.7	9.5	10.3	10.7	11.0
Procedure Control	Rep.1	34.0	71.2	82.1	86.3	89.9	92.7	95.1	95.6	96.6	97.3	97.8	98.2	98.6	98.7	99.0	99.0	99.5	99.7	100.1	100.2	100.5
	Rep.2	33.4	71.1	82.5	86.4	89.9	92.1	93.6	94.0	94.7	95.5	95.9	98.2	96.5	96.6	96.7	96.8	96.8	97.0	97.1	97.3	97.1
	Mean	33.7	71.2	82.3	86.3	89.9	92.4	94.4	94.8	95.6	96.4	96.9	97.2	97.6	97.6	97.8	97.9	98.1	98.4	98.6	98.7	98.8
Toxicity Control		16.3	32.3	37.9	40.1	41.7	43.7	46.0	46.5	47.9	49.2	50.1	50.8	51.5	52.2	52.4	52.6	52.9	53.1	53.2	53.3	53.3
Abiotic Control		0.4	1.1	1.6	1.8	1.6	1.8	1.8	1.7	1.7	1.9	2.1	2.1	2.1	2.1	1.9	2.1	1.7	2.2	2.4	2.3	1.7

 

Table 5.2.1/4: pH-values during the test.

	Start (day 0)	End (day 39)
Inoculum Blank	7.602	7.483
		7.502
		7.517
Test Suspension	7.576	7.290
		7.338
		7.287
Procedure Control	7.603	8.254
		8.297
Toxicity Control	-	7.724
Abiotic Control	-	7.700

pH measurements were taken on additional unincubated flasks (destructive sampling) at the beginning of the test and in all incubated replicates at the end of the test.

 

Table 5.2.1/5: Analytical monitoring of nitrate & nitrite concentrations in inoculum blanks and test suspensions at the start and the end of the test.

		Start (day 0)	End  (day 39)*
Inoculum Blank	Concentration of nitrate (mg N/L)	0.894	9.17
	Concentration of nitrite (mg N/L)	<0.020	<0.020
Test Suspension (100 mg test item/L)	Concentration of nitrate (mg N/L)	0.849	5.02
	Concentration of nitrite (mg N/L)	<0.020	<0.020

LOQ_Nitrate: 0.1 mg NO₃/L; LOQ_Nitrite: 0.02 mg NO₂/L

*Average of the 3 replicates.

Validity criteria fulfilled:

yes

Remarks:

The validity of the test was demonstrated by an endogenous respiration of the inoculum blank < 60 mg/L and by a biodegradation of the reference substance of 94.4 % of its ThOD after 14 days of incubation. Moreover, the difference of extremes of replicate

Interpretation of results:

not readily biodegradable

Conclusions:

The test substance is not readily biodegradable with 7.4 % biodegradation after 28 days.

Executive summary:

The study was performed to assess the biotic degradation of the test substance by performing a ready biodegradability test, according to OECD Test Guideline 301F and EU Method C.4-D, with GLP compliance. 

Test vessels were filled with the test substance at a nominal concentration of 100 mg/L and inoculated with pre-conditioned activated sludge at a concentration of suspended solids of 30 mg/L. In the meantime, a series of blanks were filled with inoculated mineral medium.  Test vessels containing the reference substance sodium benzoate (100 mg/L) were tested in order to check the procedure. A toxicity control, containing both the test substance and the reference substance, was also performed in order to check the absence of test substance toxic effect on the microbial inoculum. The oxygen uptake in test vessels from each group incubated at 22 °C ± 2 °C in darkness was continuously recorded until the end of the test.

All validity criteria were fulfilled. The test substance was biodegraded by 7.4 % after 28 days and the toxicity control showed that the test substance had no inhibitory effect on the activity of the microbial inoculum. 

In conclusion, the test substance cannot be considered as readily biodegradable under the experimental conditions.

As the test substance failed to reach the pass level for the ready biodegradability, the test period was prolonged in order to see if the biodegradability and persistence profiles could be improved. The biodegradation percentage on day 39 was 11 %. As this biodegradation level was not satisfactory to lead to an indication of biodegradability, the test was stopped on day 39.

Description of key information

7.4% biodegradation after 28 days; OECD TG 301F and EU Method C.4-D; N. DELPIT (2022).

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Additional information

One key study for the assessment of ready biodegradability is available for the test item. The study was in conducted in accordancwe with OECD (1992) 301F, "Ready Biodegradability; Manometric Respirometry Test" referenced as Method C.4-D of Commission Regulation (EC) No. 440/2008.

Test vessels were filled with the test substance at a nominal concentration of 100 mg/L and inoculated with pre-conditioned activated sludge at a concentration of suspended solids of 30 mg/L. In the meantime, a series of blanks were filled with inoculated mineral medium.  Test vessels containing the reference substance sodium benzoate were tested in order to check the procedure (positive control). A toxicity control, containing both the test substance and the reference substance, was also performed in order to check the absence of test substance toxic effect on the microbial inoculum. The oxygen uptake in test vessels from each group incubated at 22 °C ± 2 °C in darkness was continuously recorded until the end of the test.

All validity criteria were fulfilled. The test substance was biodegraded by 7.4 % after 28 days and the toxicity control showed that the test substance had no inhibitory effect on the activity of the microbial inoculum. 

In conclusion, the test substance cannot be considered as readily biodegradable under the experimental conditions.

As the test substance failed to reach the pass level for the ready biodegradability the test period was prolonged in order to further assess biodegradability and persistence. The biodegradation percentage on day 39 was 11 %. The test was stopped on day 39 as the persistence profile was not imporved during prolongation.