Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental dates: 23 September 2021 to 03 November 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

No. 440/2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

(Paragraph (q))

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol

Molecular formula:

not applicable

IUPAC Name:

Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol

Test material form:

solid: particulate/powder

Details on test material:

Appearance: beige powder

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at measured temperatures of approximately 20 °C prior to use.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Mineral Medium
The mineral medium used in this study was that recommended in the OECD Guidelines.

Toxicity Control
A toxicity control, containing the test item and aniline, was prepared in order to assess any toxic effects of the test item on the sewage treatment micro-organisms used in the test.
A nominal amount of test item (50 mg) was dispersed in mineral medium (350 mL) and subjected to ultrasonication for 15 minutes prior to the addition of an aliquot (50 mL) of the 1000 mg/L aniline stock solution and measurement of the pH value. The inoculum (5 mL) was then added prior to adjusting to a final volume of 500 mL with mineral medium to give the test concentration of 100 mg test item/L and 100 mg aniline/L.

The Biological Oxygen Demand (BOD) values for the inoculum control, test item, procedure control and the toxicity control were measured daily. The temperature of the water bath was recorded daily. In order to confirm whether the pH of the test preparations changed, the pH was measured on Days 0 and 28.

Preparation of Test System
The following test preparations were prepared and inoculated in 500 mL clear glass bottles:
a) Three replicate bottles containing inoculated mineral medium to act as the inoculum control
b) Two replicate bottles containing inoculated mineral medium and the reference item, aniline, at a concentration of 100 mg/L to act as the procedure control
c) Three replicate bottles containing the test item at a concentration of 100 mg/L
d) Two replicate bottles containing at a concentration of 100 mg/L and the test item at a concentration of 100 mg/L in inoculated mineral medium plus the reference item, aniline, at a concentration of 100 mg/L to act as toxicity control vessels
All vessels were inoculated with the prepared inoculum at a rate of 1% v/v.
On Day 0, the test and reference items were added to the mineral medium. The pH of all vessels was measured using a Hach HQ30d Flexi handheld meter prior to the addition of the inoculum and the volume in all the vessels being adjusted to 500 mL by the addition of mineral medium.
In order to confirm that the aniline stock solution was prepared correctly, a diluted 100 mg/L stock solution (in reverse osmosis water) was also sampled for TOC analysis.
All remaining inoculum control, test item, procedure control and toxicity control vessels were placed in a CES Multi-Channel Aerobic Respirometer.
The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. The samples were stirred for the duration of the test with a magnetically coupled stirrer.
As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into the ethanolamine solution (50% v/v) causing a net reduction in gas pressure within the sample flask. The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolyzing current and the duration of the current is proportional to the amount of oxygen supplied to the micro-organisms. The data generated from the respirometer’s own battery backed memory was collected four times a day on the hard disk drive of a non-dedicated computer.
The test was conducted in diffuse light at a temperatures of between approximately 21 and 22 °C.
On Day 28, two inoculum controls, one procedure control, two test item and one toxicity control vessel were sampled for pH analysis.
The first required vessels in each series were sampled with the exception of Inoculum Control A which was considered to be an erroneous/outlier result which was confirmed using Dixons Q test.
The remaining vessels which were not sampled were not reported. Additional replicate vessels were prepared and incubated in order that in the event of a leak in the test system a replicate vessel could be discarded without jeopardizing the integrity of the test.

STATISTICAL METHODS:
Statistical analysis of the Day 28 BOD values of the three replicate inoculum control vessels was performed using Dixons Q-test, to determine if any of the replicate vessels was considered to be an outlier (J.C. Miller and J.N. Miller, (1988)).

Reference substance

Reference substance:

aniline

Results and discussion

Preliminary study:

From preliminary solubility work and following the recommendations of the International Standards Organisation (ISO 10634, 2018) and Handley et al (2002), it was concluded that the best testable dispersion was found to be obtained when using the ultrasonication method of preparation.

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

St. dev.:

0

Sampling time:

28 d

Details on results:

The test item attained 0% biodegradation after 28 days, calculated from the oxygen consumption values, and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

BOD5 / COD results

Results with reference substance:

Aniline (procedure control) attained 72% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 79% biodegradation was attained.

Any other information on results incl. tables

Validation Criteria and Biodegradation

The mean BOD of the inoculated mineral medium (control) was 28.68 mg O2/L after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The pH of the inoculated test item vessels on Day 28 were 7.5 and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between extremes of replicate BOD values at the end of the test was less than 20% and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The toxicity control attained 42% biodegradation after 14 days and 45% biodegradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.
Aniline (procedure control) attained 72% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window. After 28 days 79% biodegradation was attained thereby confirming the suitability of the inoculum and test conditions.

Total Organic Carbon Confirmation
Total Organic Carbon of the diluted aniline stock solution confirmed that it had been prepared correctly

 

Table 1             Biological Oxygen Demand Values

 

Day	BOD (mg O2/L)
	Inoculum Control		Procedure Control	Test Item		Toxicity Control
	R1	R2		R1	R2
0	0.00	0.00	0.00	0.00	0.00	0.00
1	0.16	0.04	0.00	0.12	0.08	0.12
2	1.80	1.04	3.66	0.74	0.92	3.42
3	2.96	1.74	61.18	1.34	1.70	63.14
4	4.54	2.92	143.02	2.30	2.74	148.10
5	6.38	4.58	186.14	3.70	4.28	185.18
6	7.34	5.42	198.84	4.20	5.00	199.42
7	8.50	6.46	208.84	4.84	5.70	211.08
8	9.46	7.24	214.84	5.38	6.38	217.00
9	10.54	8.46	220.00	6.08	7.38	221.42
10	11.88	9.66	225.24	7.12	8.46	226.96
11	12.50	10.12	229.40	7.16	8.74	231.36
12	13.62	11.16	232.86	7.74	9.70	235.36
13	14.32	11.58	235.12	7.84	10.04	238.12
14	15.66	12.70	238.08	8.58	11.12	241.52
15	17.90	15.00	241.78	10.62	13.24	245.70
16	19.00	16.08	244.36	11.58	14.24	248.32
17	19.82	16.50	246.66	11.88	14.62	250.06
18	20.90	17.46	249.28	12.54	15.42	252.02
19	21.78	18.20	251.60	12.82	15.92	253.74
20	23.04	19.32	254.44	13.74	17.00	255.98
21	23.82	19.86	256.52	13.86	17.24	257.56
22	24.86	21.04	259.06	14.50	18.16	259.56
23	25.58	21.86	261.06	14.96	18.82	261.36
24	26.82	23.04	263.56	16.16	20.04	263.64
25	27.78	24.16	265.72	17.12	21.08	265.64
26	28.58	25.12	267.76	17.90	21.94	267.44
27	29.44	26.12	269.90	18.70	22.82	269.18
28	30.24	27.12	272.18	19.36	23.62	271.02

 R = Replicate

Table 2             Percentage Biodegradation Values

 

Day	Biodegradation (%)
	Procedure Control	Test Item			Toxicity Control
		R1	R2	Mean
0	0	0	0	0	0
1	0	0	0	0	0
2	1	0	0	0	0
3	19	0	0	0	11
4	45	0	0	0	27
5	58	0	0	0	34
6	62	0	0	0	36
7	65	0	0	0	38
8	67	0	0	0	39
9	68	0	0	0	40
10	69	0	0	0	40
11	71	0	0	0	41
12	71	0	0	0	42
13	72	0	0	0	42
14	72	0	0	0	42
15	73	0	0	0	43
16	73	0	0	0	43
17	74	0	0	0	43
18	74	0	0	0	43
19	75	0	0	0	44
20	75	0	0	0	44
21	76	0	0	0	44
22	76	0	0	0	44
23	77	0	0	0	44
24	77	0	0	0	45
25	78	0	0	0	45
26	78	0	0	0	45
27	78	0	0	0	45
28	79	0	0	0	45

 R = Replicate

Negative biodegradation values are reported as 0% biodegradation

Table 3      pH Values of the Test Preparations on Days 0 and 28

 

Test Vessel	pH
	Day 0	Day 28
Inoculum Control R1	7.4	7.5
Inoculum Control R2	7.4	7.5
Procedure Control	7.4	8.1
Test Item R1	7.4	7.5
Test Item R2	7.4	7.5
Toxicity Control	7.4	8.1

R =   Replicate

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

In a biodegradatio study performed according to OECD guideline No. 301F, BTMR attained 0% biodegradation after 28 days and therefore cannot be considered as readily biodegradable.

Executive summary:

The study was performed to assess the ready biodegradability of BTMR in an aerobic aqueous media according to OECD Guideline 301F.

The test item at a concentration of 100 mg/L was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at temperatures of between 21 and 22 ºC for 28 days.
The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values for 28 days. Control solutions with inoculum and the reference item, aniline, and a toxicity control were used for validation purposes.

The test item attained 0% biodegradation after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.
Aniline (procedure control) attained 72% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 79% biodegradation was attained.