2-Naphthalenol, heptyl-1-[2-[3-methyl-4-[2-(3-methylphenyl)diazenyl]phenyl]diazenyl]-

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:

from July 7 to September 29, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 3-Methylaniline with heptyl naphthalen-2-ol

Cas Number:

1619917-05-3

Molecular formula:

Not applicable (UVCB Substance)

IUPAC Name:

Reaction products of 3-Methylaniline with heptyl naphthalen-2-ol

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: 78-231-15
- Expiration date of the lot/batch: December 01, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

The study was performed using aerobic activated sludge from BRIANZACQUE civil wastewater treatment plant. The sludge was collected on June 30, 2017. A homogenized aliquot of the wet sludge was centrifuged, the supernatant was removed while the solid phase was weighed, dried and the ratio of wet to dry weight was calculated to be equal to 10.59 %.
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 test substance concentration

Initial conc.:

50 mg/L

Based on:

COD

Remarks:

50 mg test item/L corresponding to 67.5 mg COD/L

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

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.

The method is based on an electro-chemical analyses process. The biodegradation process consumes the dissolved oxygen in liquid and generates CO2. Evolved carbon dioxide is adsorbed by potassium hydroxide and the total pressure decreases in the test flasks. The pressure decrease is detected and converted into an electrical signal by means of an electrode type manometer.
The test medium was prepared according to the OECD Guidelines No. 301F, 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 7.41

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 7.56 (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.

Since the test item is not totally soluble in test medium, the test item was directly weighed into the test flasks to obtain the final concentrations of 50 mg/L of test item. The test flasks were made up to a final volume of 250 mL or 150 mL (based on setting scale) with test medium (see Table 1).

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.

In Table 1 (see below), number of replicates, weighed test item amount, volume added and concentration of sterilizing agent and test/reference item is shown.

Temperature:

The incubation temperature was continuously monitored during the course of the study by means of a data logger. It was in the range 20.7 – 21.5 °C with a mean value of 21.3 °C and a standard deviation of 0.1 °C. The temperature range recommended by OECD guideline is 22 ± 2 °C, this range was corrected according to the data logger precision, therefore the range to be considered for the test was 20.6 – 23.5 °C while the recorded temperature values were corrected according to the correction factor.
The recorded temperature values during the test period were within the recommended range
and the maximum deviation was lower than 1 °C, as recommended by the guideline.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

2.2

Sampling time:

28 d

Details on results:

All reported results are related to the nominal concentration of test item, since the test concentrations were not measured during the test.
The test item, RED 2596, was found to be not ready and completely biodegradable under the conditions applied in a manometric respirometry test, having showed a maximum biodegradation of 2.2% at the end of the test (mean percentage values between two replicates).

The oxygen concentrations measured in the inoculum control (blank), test item, reference item and toxicity control bottles are reported in Table 4.
The oxygen uptake (mg) of the inoculum control (blank) bottles are reported in Table 5.

The BOD values and percentage biodegradation of test item are reported in Table 6.
The BOD values and percentage biodegradation of reference item and toxicity control bottles are reported in Table 7 and 8, respectively.
The BOD values and percentage biodegradation of abiotic control bottle are reported in Table 9.

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 COD value (Chemical Oxygen Demand) was assessed to be 1.35 mg O2/mg for the test item.

These values, 1.67 mg O2/mg for reference item and 1.35 mg O2/mg for the test item, were used to calculate per % biodegradation.
Nitrate and nitrite concentration was measured at the start and at the test end.
At the start no nitrite nor nitrate were present. At test end no nitrite formation was observed, while nitrate concentration was increased.

At test end nitrate concentration in the blank bottles (7.54 mg/L as mean value) was slightly higher than nitrate concentration found in test item (7.33 mg/L as mean value), therefore is assumed that this negative nitrification value (-0.054 mg/L) is equal to zero.

No significant degradation was observed in the abiotic control (equal to 13.3 % 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 after four days of exposure (31.0%) (Table 8).

BOD5 / COD results

Results with reference substance:

The reference item exceeded the biodegradation pass level (60%) on the five day (63.8 %), within the range fixed by OECD Guideline (first 14 days)

Any other information on results incl. tables

Table4BOD in the test flasks (mg/L).

 

Day of measurement	BOD (mg/L)
	Blank 1	Blank 2	Reference item	Test item 1	Test item 2		Tox control		Abiotic control
1	7	6	10	7	9	19		9
2	7	7	57	9	10	67		10
3	9	7	78	9	10	87		7
4	12	10	109	12	12	112		9
5	13	10	118	13	14	119		9
6	15	11	124	13	15	127		9
7	15	12	135	14	17	138		9
8	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.		n.d.
9	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.		n.d.
10	17	15	146	15	18	148		10
11	17	15	150	17	18	151		10
12	17	15	153	17	18	156		9
13	17	15	154	17	18	157		9
14	17	15	156	17	18	157		9
15	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.		n.d.
16	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.		n.d.
17	18	15	162	18	20	164		9
18	18	17	164	18	20	164		9
19	20	17	165	18	20	164		10
20	20	17	167	18	20	167		9
21	20	17	167	18	20	167		9
22	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.		n.d.
23	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.		n.d.
24	20	17	168	20	21	167		9
25	21	17	170	20	21	167		9
26	21	17	170	20	21	170		9
27	21	17	170	20	21	170		9
28	21*	17*	170	20	21	170		9

n.d. not determined being not working days.

*mean oxygen uptake in the inoculum control at the end of test period (28 days) was equal to 19 mg/L, according to the maximum value provided by OECD Guideline of 60 mg O₂/L.

  Table5Oxygen uptake in inoculum control flasks (mg).

 

Day of measurement		Inoculum control
		Oxygen uptake (mg)
		Repl. 1		Repl. 2		Mean
1	1.75		1.50		1.63
2	1.75		1.75		1.75
3	2.25		1.75		2.00
4	3.00		2.50		2.75
5	3.25		2.50		2.88
6	3.75		2.75		3.25
7	3.75		3.00		3.38
8	n.d.		n.d.		n.d.
9	n.d.		n.d.		n.d.
10	4.25		3.75		4.00
11	4.25		3.75		4.00
12	4.25		3.75		4.00
13	4.25		3.75		4.00
14	4.25		3.75		4.00
15	n.d.		n.d.		n.d.
16	n.d.		n.d.		n.d.
17	4.50		3.75		4.13
18	4.50		4.25		4.38
19	5.00		4.25		4.63
20	5.00		4.25		4.63
21	5.00		4.25		4.63
22	n.d.		n.d.		n.d.
23	n.d.		n.d.		n.d.
24	5.00		4.25		4.63
25	5.25		4.25		4.75
26	5.25		4.25		4.75
27	5.25		4.25		4.75
28	5.25		4.25		4.75

n.d. not determined being not working days.

 

Table6Oxygen uptake, BOD and percentage biodegradation in test item flasks.

 

Day of measurement	Test item
	Oxygen uptake (mg)		BOD			% biodegradation
	Repl. 1	Repl. 2	Repl. 1		Repl. 2	Repl. 1		Repl. 2		Mean
1	1.75	2.25	0.0100	0.0500		0.7	3.7		2.2
2	2.25	2.50	0.0400	0.0600		3.0	4.4		3.7
3	2.25	2.50	0.0200	0.0400		1.5	3.0		2.2
4	3.00	3.00	0.0200	0.0200		1.5	1.5		1.5
5	3.25	3.50	0.0300	0.0500		2.2	3.7		3.0
6	3.25	3.75	0.0000	0.0400		0.0	3.0		1.5
7	3.50	4.25	0.0100	0.0700		0.7	5.2		3.0
8	n.d.	n.d.	n.d.	n.d.		n.d.	n.d.		n.d.
9	n.d.	n.d.	n.d.	n.d.		n.d.	n.d.		n.d.
10	3.75	4.50	-0.0200	0.0400		-1.5	3.0		0.7
11	4.25	4.50	0.0200	0.0400		1.5	3.0		2.2
12	4.25	4.50	0.0200	0.0400		1.5	3.0		2.2
13	4.25	4.50	0.0200	0.0400		1.5	3.0		2.2
14	4.25	4.50	0.0200	0.0400		1.5	3.0		2.2
15	n.d.	n.d.	n.d.	n.d.		n.d.	n.d.		n.d.
16	n.d.	n.d.	n.d.	n.d.		n.d.	n.d.		n.d.
17	4.50	5.00	0.0300	0.0700		2.2	5.2		3.7
18	4.50	5.00	0.0100	0.0500		0.7	3.7		2.2
19	4.50	5.00	-0.0100	0.0300		-0.7	2.2		0.7
20	4.50	5.00	-0.0100	0.0300		-0.7	2.2		0.7
21	4.50	5.00	-0.0100	0.0300		-0.7	2.2		0.7
22	n.d.	n.d.	n.d.	n.d.		n.d.	n.d.		n.d.
23	n.d.	n.d.	n.d.	n.d.		n.d.	n.d.		n.d.
24	5.00	5.25	0.0300	0.0500		2.2	3.7		3.0
25	5.00	5.25	0.0200	0.0400		1.5	3.0		2.2
26	5.00	5.25	0.0200	0.0400		1.5	3.0		2.2
27	5.00	5.25	0.0200	0.0400		1.5	3.0		2.2
28	5.00**	5.25**	0.0200	0.0400		1.5	3.0		2.2***

n.d. not determined being not working days.

**the difference between duplicate values of test item removal was 5 %; this value was within the maximum value provided by OECD Guideline of 20 %.

***Value of biodegradation at the end of the test.

Table7Oxygen uptake, BOD and percentage biodegradation in reference item flask.

 

Day of measurement	Reference item
	Oxygen uptake (mg)	BOD	% biodegradation
1	2.50	0.0350	2.1
2	14.25	0.5000	29.9
3	19.50	0.7000	41.9
4	27.25	0.9800	58.7
5	29.50	1.0650	63.8*
6	31.00	1.1100	66.5
7	33.75	1.2150	72.8
8	n.d.	n.d.	n.d.
9	n.d.	n.d.	n.d.
10	36.50	1.3000	77.8
11	37.50	1.3400	80.2
12	38.25	1.3700	82.0
13	38.50	1.3800	82.6
14	39.00	1.4000	83.8
15	n.d.	n.d.	n.d.
16	n.d.	n.d.	n.d.
17	40.50	1.4550	87.1
18	41.00	1.4650	87.7
19	41.25	1.4650	87.7
20	41.75	1.4850	88.9
21	41.75	1.4850	88.9
22	n.d.	n.d.	n.d.
23	n.d.	n.d.	n.d.
24	42.00	1.4950	89.5
25	42.50	1.5100	90.4
26	42.50	1.5100	90.4
27	42.50	1.5100	90.4
28	42.50	1.5100	90.4

n.d. not determined being not working days

*the reference item exceeded the biodegradation pass level (60%) after five days (63.8 %), within the guideline fixed range by OECD Guideline (first 14 days).

 

 

 

 

 

 

Table8Oxygen uptake, BOD and percentage biodegradation in toxicity control flask.

 

Day of measurement	Toxicity control
	Oxygen uptake (mg)	BOD	% biodegradation
1	2.85	0.0817	2.7
2	10.05	0.5533	18.3
3	13.05	0.7367	24.4
4	16.80	0.9367	31.0*
5	17.85	0.9983	33.1
6	19.05	1.0533	34.9
7	20.70	1.1550	38.2
8	n.d.	n.d.	n.d.
9	n.d.	n.d.	n.d.
10	22.20	1.2133	40.2
11	22.65	1.2433	41.2
12	23.40	1.2933	42.8
13	23.55	1.3033	43.2
14	23.55	1.3033	43.2
15	n.d.	n.d.	n.d.
16	n.d.	n.d.	n.d.
17	24.60	1.3650	45.2
18	24.60	1.3483	44.6
19	24.60	1.3317	44.1
20	25.05	1.3617	45.1
21	25.05	1.3617	45.1
22	n.d.	n.d.	n.d.
23	n.d.	n.d.	n.d.
24	25.05	1.3617	45.1
25	25.05	1.3533	44.8
26	25.50	1.3833	45.8
27	25.50	1.3833	45.8
28	25.50	1.3833	45.8

n.d. not determined being not working days

*the toxicity control exceeded the biodegradation pass level (25%) after four days (31.0 %), within the guideline fixed range by OECD Guideline (first 14 days).

 

 

Table9Oxygen uptake, BOD and percentage biodegradation in abiotic control flask.

 

Day of measurement	Abiotic control
	Oxygen uptake (mg)	BOD	% biodegradation
1	2.25	0.1800	13.3
2	2.50	0.2000	14.8
3	1.75	0.1400	10.4
4	2.25	0.1800	13.3
5	2.25	0.1800	13.3
6	2.25	0.1800	13.3
7	2.25	0.1800	13.3
8	n.d.	n.d.	n.d.
9	n.d.	n.d.	n.d.
10	2.50	0.2000	14.8
11	2.50	0.2000	14.8
12	2.25	0.1800	13.3
13	2.25	0.1800	13.3
14	2.25	0.1800	13.3
15	n.d.	n.d.	n.d.
16	n.d.	n.d.	n.d.
17	2.25	0.1800	13.3
18	2.25	0.1800	13.3
19	2.50	0.2000	14.8
20	2.25	0.1800	13.3
21	2.25	0.1800	13.3
22	n.d.	n.d.	n.d.
23	n.d.	n.d.	n.d.
24	2.25	0.1800	13.3
25	2.25	0.1800	13.3
26	2.25	0.1800	13.3
27	2.25	0.1800	13.3
28	2.25	0.1800	13.3

n.d. not determined being not working days.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item RED2596 was found to be not ready and completely biodegradable under the conditions applied in a manometric respirometry test, having showed a maximum biodegradation of 2.2% at the end 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 after four days of exposure (31.0%).