2-Amino-5-Chloro-N,3-Dimethylbenzamide

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:

21 January 2020 to 02 March 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item: 2-amino-5-chloro-N,3-dimethylbenzamide (M198)
Batch number: 628-042-00
Date of receipt: November 07, 2019
Packaging: Plastic tube
Internal number: 1906385-001
Production date: October 16, 2019
Expiry date: ctober 17, 2021
Purity: 983.7 ± 2.8 g/kg
Storage: At room temperature without particular attention to light.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Aerobic activated sludge from BRIANZACQUE civil wastewater treatment plant.
- Collected on January 23, 2020.
- A homogenized aliquot of the wet sludge was centrifuged, the supernatant was removed while the solid phase was weighed and dried
- The ratio of wet to dry weight was calculated to be equal to 7.2%.
- Based on this ratio, calculated amounts of wet sludge was centrifuged, the supernatant was removed and the solid phase was suspended in the test medium to get a concentration equivalent to 3 g dry material per litre.
- The prepared sludge inoculum was pre-conditioned to the experimental conditions by aerating it in the dark.
- The concentrated suspension was used as inoculum to give a final concentration of 15 mg dry material per litre into test flasks.

Duration of test (contact time):

28 d

Initial conc.:

50 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The test medium was prepared according to the OECD Guidelines No. 301 A, 1992: salts of analytical grade were dissolved in purified water to obtain the following stock solutions (up to 1000 mL):
a) KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4 x 2H2O 33.40 g/L
NH4Cl 0.50 g/L
The pH of this solution was 7.57
b) CaCl2 x 2H2O 36.40 g/L
or
CaCl2 27.50 g/L
c) MgSO4 x 7H2O 22.50 g/L
d) FeCl3 x 6 H2O 0.25 g/L
- In order to allow the storage of solution, one drop of concentrated HCl was added.
10 mL of stock solution a) and 1 mL of stock solutions b), c) and d) were added to 800 mL of purified water, then made up to 1000 mL with purified water. The pH of test medium was 7.58 (mean value between two blank bottles), so adjustment with a diluted hydrochloric acid or sodium hydroxide solution, before making up to volume, was not needed.

- Test solution: Since the test item was poor soluble in water, the test flask was prepared by weighing proper amount of test item directly in mineral medium (0.0125 g into 250 mL and 0.0075 g into 150 mL).
The volume of mineral medium was dosed in the test flasks to obtain the final concentrations of 50 mg/L of test item.
Moreover, stock solutions (10 g/L) of reference item Sodium Benzoate and sterilizing agent HgCl2 were prepared and an appropriate aliquot of the stock solution was added to the test medium (final volume: 250 mL or 150 mL ) in the test flasks to obtain a final concentration of 100 mg/L.
Activated sludge was added to each flask (with the exception of the abiotic control) before incubation.
Finally, the test flasks, containing stir bars covered with teflon, were kept under continuous stirring by the means of a magnetic stirrer for the 28-days test period.

- Test temperature: The incubation temperature was continuously monitored during the course of the study by means of a data logger.
It was in the range 21.1 – 21.4 °C with a mean value of 21.3 °C and a standard deviation of 0.05 °C.
- pH of test flask: 7.36 – 7.65 at the test start. 7.34 – 8.15 at the test end.
The pH of test item mixtures at the end of test period was 7.53 (mean value), within the provided range by guideline of 6.0 - 8.5.

Determination of BOD: The sensor on the top of the test bottle detects the pressure decrease inside the bottles and gives the measure as BOD, expressed in mg O2/L. The BOD value is registered daily except for not working days.
Determination of ThOD: It was calculated on the basis of the chemical formula and molecular weight of reference item.
Temperature: Test temperature was recorded by the means of a data logger.
pH: At the start of the test, pH values of test solutions were measured taking a small aliquot of solutions from the flasks before the addition of the activated sludge (inoculum). At the end of the incubation period, pH was measured in all test flasks

Light conditions: Darkness
Stirring: Continuous
Test duration: 28 days
Continuous darkness: yes


SAMPLING
The assay on test item was carried out in parallel with the following bottles:
- a known ready biodegradable reference item (one replicate), Sodium Benzoate, at 100 mg/L;
- two inoculum control replicates (blank);
- two test item replicates at 50 mg/L concentration;
- one abiotic control containing the test item at 50 mg/L and a sterilizing agent (HgCl2) at 100 mg/L (without inoculum);
- one toxicity control, containing the test item at 50 mg/L and reference item at 100 mg/L.
BOD flasks of 500 mL capacity, equipped with pressure sensors fitted on the bottle mouth, were used as test units.


NITRIFACATIONS
Nitrate and nitrite concentration were measured at the start and at the test end.
At the start of test, nitrate and nitrite were not observed.
At the end of test (28 days) nitrite was not observed.
The quantity of nitrate in the inoculum control flasks (as mean value) was subtracted from the mean nitrate concentration determined in the test item flasks.
The oxygen consumed in the formation of nitrate was calculated multiplying the increase of nitrate-N concentration by a factor of 4.57, which is the oxygen consumed per gram of nitrogen, as the oxygen taken up by 14 g of nitrogen is 64 g (OECD Guideline No. 301, 1992).

Reference substance:

other: Sodium Benzoate

Remarks:

EC No.: 208-534-8 , CAS No.: 532-32-1

Test performance:

CONDITIONS FOR THE VALIDITY OF THE TEST

The test complies with the validity criteria because:
- The mean oxygen uptake in the inoculum control at the end of test period (28 days) was 27 mg O2/L, not exceeding the maximum value provided by OECD guideline 301F of 60 mg O2/L.
- The difference between duplicate values of test item removal was 5%; this value was within the maximum value provided by OECD guideline 301F of 20 %.
- The reference item exceeded the biodegradation pass level (60%) in 14 days (79.6%), within the guideline fixed range by OECD guideline 301F.
- The toxicity control exceeded the 25 % value (31.2 %), within the first 14 days period as provided by OECD Guideline 301F, showing that no inhibitory effect was due to test item.
- The pH of test item mixtures at the end of test period was 7.53 (mean value), within the provided range by guideline of 6.0 – 8.5.

Key result

Parameter:

% degradation (O2 consumption)

Value:

-7

Sampling time:

28 d

Details on results:

A study to evaluate the ready biodegradability of the test item 2-amino-5-chloro-N,3-dimethylbenzamide, following the “Manometric Respirometry” method, was performed.
Test item concentration should be 100 mg/L or a concentration giving at least 50-100 ThOD or COD/L.
The tested concentration was 50 mg/L (corresponding to 93.5 mg ThOD/L).
All reported results are related to the nominal concentration of test item, since the test concentrations were not measured during the test.
The biodegradation was followed by measuring the oxygen uptake (mg/L) of the test item under defined conditions by the means of a manometric method and calculating from these values the BOD (Biochemical Oxygen Demand).
The ThODNH3 value (Theoretical Oxygen Demand without nitrification process) was calculated to be equal to 1.67 mg O2/mg for reference item, Sodium Benzoate. while ThODNO3 value (Theoretical Oxygen Demand with nitrification process) was assessed to be 1.87 mg O2/mg for the test item.
These values, 1.67 mg O2/mg for reference item and 1.87 mg O2/mg for the test item, were used to calculate per % biodegradation.
Nitrate and nitrite concentration were measured at the start and at the test end.
At the start and test end no nitrite were present.
At the test end nitrate concentration in the blank bottles (10.00 mg/L as mean value) was slightly higher than nitrate concentration found in test item bottles (9.15 mg/L as mean value), therefore the found negative value (-0.219 mg/L) was assumed as equal to zero, so Nitrification wasn’t occurred during test period.
At the end of the test the biodegradation was -7.0% at the end of the test (mean percentage values between two replicates).
Therefore, the test item, was found to be not ready and completely biodegradable under the conditions applied in a manometric respirometry test, since the 10-day window wasn’t start and a maximum biodegradation of -7.0% was found at the end of the test, (mean percentage values between two replicates).
No significant degradation was observed in the abiotic control (equal to 0.0% at the test end).
The toxicity control clearly showed that the test item does not cause inhibitory effect on the microorganisms activity at the tested concentration. Actually, the toxicity control exceeded the 25 % pass level within the 14 days of exposure (31.2%).
The reference item exceeded (79.6%) the biodegradation pass level (60%) within the range fixed by OECD Guideline 301F (first 14 days).

BOD value in the test flasks (mg/L):

DATE	TIME (DAYS)	BOD mg O2/L (letture testine)
		BLANK 1	BLANK 2	REF. ITEM	TEST ITEM 1	TEST ITEM 2	TOX. CONTROL	ABIOTIC CONTROL
31/01/2020	1	1	0	14	0	1	29	0
01/02/2020	2	4	3	70	3	4	70	0
02/02/2020	3	9	7	98	4	7	67	0
03/02/2020	4	10	9	104	6	7	99	0
04/02/2020	5	14	12	115	9	10	109	0
05/02/2020	6	14	15	125	9	12	122	0
06/02/2020	7	15	17	131	10	14	125	0
07/02/2020	8	17	17	137	10	15	132	0
08/02/2020	9	18	18	140	12	15	135	0
09/02/2020	10	19	20	145	13	14	138	0
10/02/2020	11	20	21	148	14	17	140	0
11/02/2020	12	20	23	150	14	17	145	0
12/02/2020	13	20	24	152	14	16	145	0
13/02/2020	14	20	24	155	15	18	146	0
14/02/2020	15	21	25	156	15	18	147	0
15/02/2020	16	23	25	157	15	18	148	0
16/02/2020	17	23	25	160	15	18	151	0
17/02/2020	18	23	26	162	15	18	151	0
18/02/2020	19	23	26	162	15	18	151	0
19/02/2020	20	23	27	163	15	19	151	0
20/02/2020	21	23	28	164	15	20	151	0
21/02/2020	22	23	28	164	15	20	154	0
22/02/2020	23	23	28	165	17	20	154	0
23/02/2020	24	23	28	165	17	20	154	0
24/02/2020	25	25	29	167	17	20	154	0
25/02/2020	26	25	29	167	18	20	154	0
26/02/2020	27	25	29	167	18	21	154	0
27/02/2020	28	25	29	167	20	21	154	0

*Mean oxygen uptake in the inoculum control at the end of test period (28 days) was equal to 27 mg/L,

according to the maximum value provided by OECD Guideline of 60 mg O2/L.

BOD mg O₂/mg of test item:

DATE	BOD mg02/mg test item
	REF. ITEM	TEST ITEM 1	TEST ITEM 2	TEST ITEM MEAN	TOXiCITY CONTROL	ABIOTIC CONTROL
31/01/2020	0.1350	-0.0100	0.0100	0.0000	0.2817	0.000
01/02/2020	0.6650	-0.0100	0.0100	0.0000	0.6117	0.000
02/02/2020	0.9000	-0.0800	-0.0200	-0.0500	0.8567	0.000
03/02/2020	0.9450	-0.0700	-0.0500	-0.0600	0.9317	0.000
04/02/2020	1.0200	-0.0800	-0.0600	-0.0700	1.0033	0.000
05/02/2020	1.1050	-0.1100	-0.0500	-0.0800	1.0083	0.000
06/02/2020	1.1500	-0.1200	-0.0400	-0.0800	1.0533	0.000
07/02/2020	1.2000	-0.1400	-0.0400	-0.0900	1.0667	0.000
08/02/2020	1.2200	-0.1200	-0.0600	-0.0900	1.0800	0.000
09/02/2020	1.2550	-0.1300	-0.1100	-0.1200	1.0750	0.000
10/02/2020	1.2750	-0.1300	-0.0700	-0.1000	1.1083	0.000
11/02/2020	1.2850	-0.1500	-0.0900	-0.1200	1.0917	0.000
12/02/2020	1.3000	-0.1600	-0.0800	-0.1200	1.0933	0.000
13/02/2020	1.3300	-0.1400	-0.0800	-0.1100	1.1033	0.000
14/02/2020	1.3300	-0.1600	-0.1000	-0.1300	1.0967	0.000
15/02/2020	1.3300	-0.1800	-0.1200	-0.1500	1.0800	0.000
16/02/2020	1.3600	-0.1800	-0.1200	-0.1500	1.1100	0.000
17/02/2020	1.3750	-0.1900	-0.1300	-0.1600	1.1017	0.000
18/02/2020	1.3750	-0.1900	-0.1300	-0.1600	1.1017	0.000
19/02/2020	1.3800	-0.2000	-0.1200	-0.1600	1.0933	0.000
20/02/2020	1.3850	-0.2100	-0.1100	-0.1600	1.0850	0.000
21/02/2020	1.3850	-0.1700	-0.1100	-0.1400	1.1150	0.000
22/02/2020	1.3950	-0.1700	-0.1100	-0.1400	1.1150	0.000
23/02/2020	1.3950	-0.1700	-0.1100	-0.1400	1.1150	0.000
24/02/2020	1.4000	-0.2000	-0.1400	-0.1700	1.0900	0.000
25/02/2020	1.4000	-0.1800	-0.1400	-0.1600	1.0900	0.000
26/02/2020	1.4000	-0.1800	-0.1200	-0.1500	1.0900	0.000
27/02/2020	1.4000	-0.1400	-0.1200	-0.1300	1.0900	0.000

Oxygen uptake (mg):

DATE	mg UPTAKE 02
	BLANK 1	BLANK 2	BLANK MEAN	REF. ITEM	TEST ITEM 1	TEST ITEM 2	TOX. CONTROL	ABIOTIC CONTROL
31/01/2020	0.25	0.00	0.13	3.50	0.00	0.25	4.35	0.00
01/02/2020	1.00	0.75	0.88	17.50	0.75	1.00	10.05	0.00
02/02/2020	2.25	1.75	2.00	24.50	1.00	1.75	14.85	0.00
03/02/2020	2.50	2.25	2.38	26.00	1.50	1.75	16.35	0.00
04/02/2020	3.50	3.00	3.25	28.75	2.25	2.50	17.40	0.00
05/02/2020	3.50	3.75	3.63	31.25	2.25	3.00	18.30	0.00
06/02/2020	3.75	4.25	4.00	32.75	2.50	3.50	18.75	0.00
07/02/2020	4.25	4.25	4.25	34.25	2.50	3.75	19.80	0.00
08/02/2020	4.50	4.50	4.50	35.00	3.00	3.75	20.25	0.00
09/02/2020	4.75	5.00	4.88	36.25	3.25	3.50	20.70	0.00
10/02/2020	5.00	5.25	5.13	37.00	3.50	4.25	21.00	0.00
11/02/2020	5.00	5.75	5.38	37.50	3.50	4.25	21.75	0.00
12/02/2020	5.00	6.00	5.50	38.00	3.50	4.50	21.75	0.00
13/02/2020	5.00	6.00	5.50	38.75	3.75	4.50	21.90	0.00
14/02/2020	5.25	6.25	5.75	39.00	3.75	4.50	22.05	0.00
15/02/2020	5.75	6.25	6.00	39.25	3.75	4.50	22.20	0.00
16/02/2020	5.75	6.25	6.00	40.00	3.75	4.50	22.20	0.00
17/02/2020	5.75	6.50	6.13	40.50	3.75	4.50	22.65	0.00
18/02/2020	5.75	6.50	6.13	40.50	3.75	4.50	22.65	0.00
19/02/2020	5.75	6.75	6.25	40.75	3.75	4.75	22.65	0.00
20/02/2020	5.75	7.00	6.38	41.00	3.75	5.00	22.65	0.00
21/02/2020	5.75	7.00	6.38	41.00	4.25	5.00	23.10	0.00
22/02/2020	5.75	7.00	6.38	41.25	4.25	5.00	23.10	0.00
23/02/2020	5.75	7.00	6.38	41.25	4.25	5.00	23.10	0.00
24/02/2020	6.25	7.25	6.75	41.75	4.25	5.00	23.10	0.00
25/02/2020	6.25	7.25	6.75	41.75	4.50	5.00	23.10	0.00
26/02/2020	6.25	7.25	6.75	41.75	4.50	5.25	23.10	0.00
27/02/2020	6.25	7.25	6.75	41.75	5.00	5.25	23.10	0.00

Percentage of biodegradation in test item, reference item, toxicity control and abiotic flask:

DATE	% BIODEGRADATION
	REF. ITEM	TEST ITEM Replicate 1	TEST ITEM Replicate 2	TEST ITEM MEAN	TOXICITY CONTROL	ABIOTIC CONTROL
31/01/2020	8.1	-0.5	0.5	0.0	8.0	0.0
01/02/2020	39.8	-0.5	0.5	0.0	17.3	0.0
02/02/2020	53.9	-4.3	-1.1	-2.7	24.2	0.0
03/02/2020	56.6	-3.7	-2.7	-3.2	26.3	0.0
04/02/2020	61.1	-4.3	-3.2	-3.7	28.3	0.0
05/02/2020	66.2	-5.9	-2.7	-4.3	28.5	0.0
06/02/2020	68.9	-6.4	-2.1	-4.3	29.8	0.0
07/02/2020	71.9	-7.5	-2.1	-4.8	30.1	0.0
08/02/2020	73.1	-6.4	-3.2	-4.8	30.5	0.0
09/02/2020	75.1	-7.0	-5.9	-6.4	30.4	0.0
10/02/2020	76.3	-7.0	-3.7	-5.3	31.3	0.0
11/02/2020	76.9	-8.0	-4.8	-6.4	30.8	0.0
12/02/2020	77.8	-8.6	-4.3	-6.4	30.9	0.0
13/02/2020	79.6	-7.5	-4.3	-5.9	31.2	0.0
14/02/2020	79.6	-8.6	-5.3	-7.0	31.0	0.0
15/02/2020	79.6	-9.6	-6.4	-8.0	30.5	0.0
16/02/2020	81.4	-9.6	-6.4	-8.0	31.4	0.0
17/02/2020	82.3	-10.2	-7.0	-8.6	31.1	0.0
18/02/2020	82.3	-10.2	-7.0	-8.6	31.1	0.0
19/02/2020	82.6	-10.7	-6.4	-8.6	30.9	0.0
20/02/2020	82.9	-11.2	-5.9	-8.6	30.6	0.0
21/02/2020	82.9	-9.1	-5.9	-7.5	31.5	0.0
22/02/2020	83.5	-9.1	-5.9	-7.5	31.5	0.0
23/02/2020	83.5	-9.1	-5.9	-7.5	31.5	0.0
24/02/2020	83.8	-10.7	-7.5	-9.1	30.8	0.0
25/02/2020	83.8	-9.6	-7.5	-8.6	30.8	0.0
26/02/2020	83.8	-9.6	-6.4	-8.0	30.8	0.0
27/02/2020	83.8	-7.5	-6.4	-7.0	30.8	0.0

The difference between duplicate values of test item removal at the end of the test was 5 %; this value

was within the maximum value provided by OECD Guideline of 20 %.

Value of biodegradation at the end of the test is -7.0%

 

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item, 2-amino-5-chloro-N,3-dimethylbenzamide, was found to be not ready and completely biodegradable under the conditions applied in a manometric respirometry test, having showed a maximum biodegradation of -7.0% at the end of the test (mean percentage values between two replicates).
No significant degradation was observed in the abiotic control.
The test item showed no inhibitory effects on the microorganism activity at the tested concentration, as demonstrated by the biodegradation percentage in the toxicity control exceeding the 25% pass level within 14 days of exposure (31.2%).

Executive summary:

For 2 -amino-5 -chloro-N,3 -dimethylbenzamide a 28 days biodegradation test according to OECD 301F (1992) was performed. The study lead to no significant biodagradation measured by oxygen demand.

No significant degradation was observed in the abiotic control (equal to 0.0% at the test end). The toxicity control clearly showed that the test item does not cause inhibitory effect on the microorganisms activity at the tested concentration. Actually, the toxicity control exceeded the 25 % pass level within the 14 days of exposure (31.2%).

The reference item exceeded (79.6%) the biodegradation pass level (60%) within the range fixed by OECD Guideline 301F (first 14 days).

Description of key information

The test item, 2-amino-5-chloro-N,3-dimethylbenzamide, was found to be not ready and completely biodegradable under the conditions applied in a manometric respirometry test, having shown a maximum biodegradation of -7.0% at the end of the test (mean percentage values between two replicates).

Biological results: Maximum biodegradation at the end of the 10-day window: the 10-day window was not started.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater