Isophthalohydrazide

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:

20 September 2017 till 20 October 2017.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Remarks:

Exception: The concentration of suspended solids in the sludge was determined by the sewage treatment plant, their facilities are not GLP compliant.

Test material

Test material information

Constituent 1

Reference substance name:

Isophthalohydrazide

EC Number:

220-425-7

EC Name:

Isophthalohydrazide

Cas Number:

2760-98-7

Molecular formula:

C8H10N4O2

IUPAC Name:

isophthalohydrazide

Test material form:

solid: particulate/powder

Details on test material:

Appearance: White crystal powder
Test item storage: At room temperature

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage. The freshly obtained sludge was used immediately. The concentration of suspended solids was determined to be 4.9 g/L. Before use, the sludge was allowed to settle (approximately 30 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

24.5 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 Concentration and Preparation of Test Solutions: Since IDH was easily soluble in water, the test media were prepared using a stock solution of 1 g/L in Milli- RO water. A weighed amount of 500.01 mg of IDH was dissolved in Milli- RO water and made up to 500 mL. After stirring for 110 minutes, the final stock solution (pH 5.9) was clear and colourless. Aliquots of 49 mL of the stock solution were added to the test item bottles A and B and to the toxicity control. These test bottles contained medium with microbial organisms (final volume: 2 litres). The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms.
Stock solutions of mineral components: A)8.50 g KH2PO4, 21.75 g K2HPO4, 67.20 g Na2HPO4.12H2O, 0.50 g NH4Cl, dissolved in Milli- RO water and made up to 1 litre, pH 7.4 ± 0.2; B)22.50 g MgSO4.7H2O dissolved in Milli- RO water and made up to 1 litre; C)36.40 g CaCl2.2H2O dissolved in Milli- RO water and made up to 1 litre; D)0.25 g FeCl3.6H2O dissolved in Milli- RO water and made up to 1 litre.
Mineral medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli- RO water.
Test duration: 28 days for the inoculum blank and test item (last CO2 measurement on day 29); 14 days for the positive and toxicity control (last CO2 measurement on day 15). During the test period, the test media were aerated and stirred continuously.
Test vessels: 2 litre brown coloured glass bottles.
Type and number of bottles: Test suspension: containing test item and inoculum (2 bottles); Inoculum blank: containing only inoculum (2 bottles); Positive control: containing reference item and inoculum (1 bottle); Toxicity control: containing test item, reference item and inoculum (1 bottle).
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 (1% of final volume) were added to each bottle; this mixture was aerated with synthetic air overnight to purge the system of CO2.
Preparation: At the start of the test (day 0), test and reference item were added to the bottles containing the microbial organisms and mineral components. The volumes of suspensions were made up to 2 litres with Milli- RO water, resulting in the mineral medium described before. Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.
Suspended solids concentration: 49 mg/L (10 mL/L of the supernatant of a suspension with 4.9 g/L suspended solids).
Measurements: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test item. Titrations for the positive and toxicity control were made over a period of at least 14 days. On the penultimate day, the pH of respective test suspensions was measured and 1 mL of concentrated HCl was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 15 (positive and toxicity control) and on day 29 (remaining vessels).
Theoretical CO2 production: The theoretical CO2 production was calculated from the molecular formula. The ThCO2 of IDH was calculated to be 1.81 mg CO2/mg. The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.
pH: Measured at the start of the test (day 0) and on the penultimate day (day 14 for the positive and toxicity control and day 28 for the inoculum blanks and test item), before addition of concentrated HCl.
Temperature of medium: Measured continuously in a vessel with Milli- RO water in the same room.

Reference substance

Reference substance:

acetic acid, sodium salt

Remarks:

Tested at 40 mg/L (12 mg TOC/L)

Results and discussion

Preliminary study:

Not applicable.

Test performance:

The validity criteria of OECD 301B were satisfied:
1. The positive control item was biodegraded by at least 60% (81%) within 14 days.
2. The difference of duplicate values for %-degradation of the test item was always less than 20 (≤ 3%).
3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (48.8 mg CO2 per 2 litres of medium, corresponding to 24.4 mg CO2/L).
4. The Inorganic Carbon content (IC) of the test item (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis, IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

5

St. dev.:

0

Sampling time:

28 d

Details on results:

The relative biodegradation values calculated from the measurements performed during the test period revealed no biologically significant biodegradation of IDH (5% in both test vessels, based on ThCO2).
In the toxicity control, more than 25% biodegradation occurred within 14 days (31%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
The positive control item was biodegraded by 81% within 14 days.
The temperature recorded in a vessel with water in the same room varied between 22.2 and 23.5°C. The pH was 7.6 in all test bottles at the start, 7.8 in the toxicity control and the positive control on day 14, and 7.7 in control and IDH treated bottles on day 28.

BOD5 / COD results

Results with reference substance:

The positive control item was biodegraded by 81% within 14 days.

Any other information on results incl. tables

Please refer to atached background material for a graph showing the curves for biodegradation of the two bottles with IDH, the positive control and the toxicity control.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

IDH was not readily biodegradable under the conditions of the modified Sturm test.

Executive summary:

The objective of the study was to evaluate the non-volatile test item IDH for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with the supernatant of activated sludge; Carbon dioxide (CO2) evolution test (modified Sturm test). The study procedures described in this report were in compliance with the OECD guideline No. 301 B, 1992.

IDH was a white crystal powder with a purity of 99.4%. The test item was tested in duplicate at a concentration of 24.5 mg/L, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula. The Theoretical CO2production (ThCO2) of IDH was calculated to be 1.81 mg CO2/mg. The study consisted of six bottles:

2 inoculum blanks (no test item),

2 test bottles (IDH),

1 positive control (sodium acetate) and

1 toxicity control (IDH plus sodium acetate).

Since IDH was easily soluble in water the test media were prepared using a stock solution of1 g/L in Milli- RO water. Aliquots of 49 mL of the clear and colourless stock solution were added to the test item bottles A and B and to the toxicity control. These test bottles contained medium with microbial organisms. The volumes of suspensions were made up to 2 litres with Milli- RO water. The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms. Test duration was 28 days for the inoculum blank and test item (last CO2 measurement on day 29) and 14 days for the positive and toxicity control (last CO2 measurement on day 15).

The relative biodegradation values calculated from the measurements performed during the test period revealed no biologically significant biodegradation of IDH (5% in both test vessels, based on ThCO2).

In the toxicity control, IDH was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, IDH was designated as not readily biodegradable.