Sebacohydrazide

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:

05 Jun 2018 - 05 Jul 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 10634. "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium"

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sebacohydrazide

EC Number:

213-126-8

EC Name:

Sebacohydrazide

Cas Number:

925-83-7

Molecular formula:

C10H22N4O2

IUPAC Name:

sebacohydrazide

Test material form:

solid: particulate/powder

Details on test material:

Test item name (as stated in report): SDH
Test item storage: at room temperature protected from light

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium.
- Concentration of suspended solids (SS): After pretreatment, SS concentration was determined to be 3.5 g/L in the concentrated sludge as used for the test. The magnetically stirred sludge was used as inoculum at the amount of 3 mL per litre of mineral medium, leading to a SS concentration of 10.5 mg/L.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

23 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

12 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium, according to OECD 201
- Test temperature: 22 - 23°C
- pH: at start: 7.6; on day 14: 7.7; on day 28: 7.6
- pH adjusted: no
- Continuous darkness: yes
- Continuous aeration and stirring: yes
- Pre-incubation medium: The day before the start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

TEST SYSTEM
- Culturing apparatus: 2 litre brown coloured glass bottles
- Number of culture flasks/concentration: 2 for the test suspension, 2 for inoculum blank, 1 for procedure control, 1 for toxicity control.
- Method used to create aerobic conditions: aeration with synthetic air (mixture of oxygen (ca. 20%) and nitrogen (ca. 80%), CO2 < 1 ppm)

PREPARATION OF TEST SOLUTIONS
- Since SDH was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2 litre test bottles containing medium with microbial organisms and mineral components (test item bottle A: 46.01 mg; test item bottle B: 46.22 mg and toxicity control bottle: 45.97 mg). To this end, 10 mL of Milli- RO water was added to each weighing bottle containing the test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test, to ensure optimal contact between test item and test organisms. Furthermore, with the exception of Day 1, the test medium was swirled around daily, since the test item tended to float on the water surface.

SAMPLING
- Sampling frequency: Titrations were made on day 2, 5, 8, 12, 15, 19, 23 and 29, for the inoculum blank and test item. Titrations for the procedure and toxicity control were made on day 2, 5, 8, 12 and 15.
- Sampling method: titration of Ba(OH)2 in the gas scrubbing bottles

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes; containing only inoculum
- Procedure control: yes; containing reference item and inoculum
- Toxicity control: yes; containing test item, reference item and inoculum

Reference substance

Reference substance:

acetic acid, sodium salt

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

86

Sampling time:

28 d

Remarks on result:

other: Mean of two replicate bottles.

Details on results:

- The relative biodegradation values calculated from the measurements performed during the test period revealed 91% and 81% biodegradation of the test item, for vessel A and B, respectively (based on ThCO2).
- Average biodegradation in vessel A and B reached ≥60% within a 10- day window.
- In the toxicity control, more than 25% biodegradation occurred within 14 days (30%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
- The ThCO2 of SDH was calculated to be 1.91 mg CO2/mg.
- The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

BOD5 / COD results

Results with reference substance:

The reference substance showed a normal biodegradation curve and reached >60% (i.e. 81%) biodegradation within 14 days.

Any other information on results incl. tables

Table 1: CO₂Production and Percentage Biodegradation of the Test Item (Bottle A)

Day	HCl (0.05 N) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^(1) (%)
	Blank (mean)	Bottle A
2	47.21	47.43	0.00	0.0	0.0	0
5	45.78	45.38	0.39	0.4	0.4	0
8	44.32	43.19	1.13	1.2	1.7	2
12	44.27	41.35	2.92	3.2	4.9	6
15	46.21	27.50	18.71	20.6	25.5	29
19	45.81	25.44	20.37	22.4	47.9	54
23	46.30	33.73	12.57	13.8	61.7	70
29^(2)	44.99	30.88	14.11	15.5	77.2	88
29^(2)	47.86	45.67	2.19	2.4	79.6	91
29^(2)	46.46	48.01	0.00	0.0	79.6	91

⁽¹⁾: Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of the test item: 87.9 mg CO₂/2L.

⁽²⁾: CO₂measured on day 29 is actually part of CO₂production of day 28, since microbial activity was ended on day 28 by addition of HCl

Table 2: CO₂Production and Percentage Biodegradation of the Test Item (Bottle B)

Day	HCl (0.05 N) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^(1) (%)
	Blank (mean)	Bottle B
2	47.21	48.12	0.00	0.0	0.0	0
5	45.78	46.15	0.00	0.0	0.0	0
8	44.32	44.17	0.14	0.2	0.2	0
12	44.27	39.20	5.07	5.6	5.7	6
15	46.21	24.38	21.83	24.0	29.7	34
19	45.81	28.49	17.32	19.1	48.8	55
23	46.30	37.17	9.13	10.0	58.8	67
29^(2)	44.99	35.30	9.69	10.7	69.5	79
29^(2)	47.86	46.40	1.46	1.6	71.1	81
29^(2)	46.46	48.00	0.00	0.0	71.1	81

⁽¹⁾: Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of the test item: 88.3 mg CO₂/2L.

⁽²⁾: CO₂measured on day 29 is actually part of CO₂production of day 28, since microbial activity was ended on day 28 by addition of HCl.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria

Interpretation of results:

readily biodegradable

Conclusions:

The relative biodegradation values calculated from the measurements performed during the test period revealed 91% and 81% biodegradation of the test substance in two replicate bottles.The test substance was readily biodegradable under the conditions of the modified Sturm test.

Executive summary:

The 'ready' biodegradability of the test substance was investigated using the Carbon Dioxide (CO2) Evolution Test (Modified Sturm Test), according to OECD Guideline 301 B and ISO standard 10634. A single test concentration of 23 mg/L test substance, corresponding to 12 mg TOC/L, was tested during 28 days. Based on the results, biodegradation was 91% and 81% in two replicates (mean 86%), surpassing the pass level of 60% within a 10 -day window set by OECD 301 B. The test substance is considered readily biodegradable. The test met all criteria for acceptability and is considered reliable without restriction.