3,4-dihydroxybenzonitrile

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:

March - April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

Adopted July 17, 1992

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3,4-dihydroxybenzonitrile

EC Number:

241-367-9

EC Name:

3,4-dihydroxybenzonitrile

Cas Number:

17345-61-8

Molecular formula:

C7H5NO2

IUPAC Name:

3,4-dihydroxybenzonitrile

Test material form:

solid: particulate/powder

Remarks:

white to brownish

Details on test material:

Batch 151222

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, 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 3.5 g/L in the concentrated sludge. Before use, the sludge was allowed to settle (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 conc.:

19.5 mg/L

Based on:

TOC

Remarks:

The concentration was corresponding to 12 mg TOC/L

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water.
Stock solutions
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.
- Test temperature: 22.1 - 23.3 °C
- pH: 7.6 - 7.8
- pH adjusted: no
- Suspended solids concentration: 3.5 g/L
- Continuous darkness: yes

TEST SYSTEM
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min)
- Details of trap for CO2 and volatile organics if used: A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).


SAMPLING
- Sampling frequency: 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 suspension. Titrations for the positive and toxicity control were made over a period of at least 14 days.
- Sampling method: Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.
- Sample storage before analysis: no

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 bottles
- Toxicity control: 1 bottle
- Other: 1 positive control: reference item (sodium acetate) + inoculum

:

Reference substance

Reference substance:

other: sodium acetate

Remarks:

final concentration of 40 mg sodium acetate per litre (12 mg TOC/L)

Results and discussion

Test performance:

In the toxicity control, more than 25% biodegradation occurred within 14 days (85%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve
Furthermore, biodegradation of CH02906 of at least 60% was reached within a 10-day window in both bottles.

% Degradation

% Degradation 1

Parameter:

% degradation (CO2 evolution)

Remarks:

Bottle A

Value:

87

Sampling time:

28 d

% Degradation 2

Parameter:

% degradation (CO2 evolution)

Remarks:

Bottle B

Value:

100

Sampling time:

28 d

Details on results:

The ThCO2 of CH02906 was calculated to be 2.28 mg CO2/mg.
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

Any other information on results incl. tables

Comparison of Biodegradation of the Test Item in Bottles A and B

Day	Biodegradation (%)
	Bottle A	Bottle B	Mean A and B	∆ A-B^1)
1	0	0	0	0
4	0	0	0	0
6	3	1	2	2
8	5	1	3	4
11	23	1	12	22
13	41	4	23	37
15	55	8	32	47
18	66	18	42	48
20	72	66	69	6
25	78	92	85	14
29	86	100	93	14
29	87	100	94	13
29	87	100	94	13

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

CH02906 was readily biodegradable under the conditions of the modified Sturm test presently performed.

Executive summary:

The objective of the study was to evaluate the non-volatile test item CH02906 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.

CH02906 was a white to brownish powder with a purity of 99.9% (HPLC). The test item was tested in duplicate at a concentration of 19.5 mg/L, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula. The Theoretical CO2production (ThCO2) of CH02906 was calculated to be 2.28 mg CO2/mg.

The study consisted of six bottles:

• 2 inoculum blanks (no test item),
• 2 test bottles (CH02906),
• 1 positive control (sodium acetate)
• 1 toxicity control (CH02906 plus sodium acetate

0. Since CH02906 was easily soluble in water the test media were prepared using a stock solution of 1 g/L in Milli-RO water. After stirring for approximately 1 hour, aliquots of
   39 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 (last CO2measurement on day 29).

   The relative biodegradation values calculated from the measurements performed during the test period revealed 87% and 100% biodegradation of CH02906, for A and B, respectively (based on ThCO2). Biodegradation in bottle B was maximized at 100% since this is the theoretical maximum attainable.

   Furthermore, biodegradation of CH02906 of at least 60% was reached within a 10-day window in both bottles.

   In the toxicity control, CH02906 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, CH02906 was designated as readily biodegradable.