2[2'-Hydroxy-5'-(phenyl)ureylenphenyl]benzothiazole

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:

23 Jan 18 - 22 Feb 18

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

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

Test material form:

solid: particulate/powder

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
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.

- Treatment
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.
The concentration of suspended solids (SS) was determined to be 3.0 g/L in the concentrated sludge as used for the test.
The magnetically stirred sludge was used as inoculum at the amount of 7.5 mL per litre of mineral medium, leading to a SS concentration of 22 mg/L.

- Reason for selection
The test has been accepted internationally for determining the 'ready' biodegradability of test items under aerobic conditions.

Duration of test (contact time):

28 d

Initial test substance concentration

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 MATERIAL PREPARATION:
Test item was a slight yellow powder with a purity of 98%. The test item was tested in duplicate at a target concentration of 18 mg/L, corresponding to 12 mg TOC/L.The organic carbon content was based on the molecular formula.
Since test item 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-litres test bottles containing medium with microbial organisms and mineral components (test item ottle A: 35.82 mg; test item bottle B: 36.12 mg and toxicity control bottle: 35.82 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 the test item and the test organisms. Furthermore, the test medium was daily swirled around, since the test item tended to float on the water surface.

TEST CONDITIONS
Test duration 28 days for the inoculum blank and test item (last CO2 measurement on day 29). 14 days for the procedure 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. Milli- RO water Tap-water purified by reverse osmosis (Milli- RO) and subsequently passed over activated carbon.

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.

Barium hydroxide: 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.

Synthetic air (CO2 < 1 ppm): 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 synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).

Illumination : The test media were excluded from light.

Preparation of Bottles
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.

Type and number of bottles:
- Test suspension: containing test item and inoculum (2 bottles).
- Inoculum blank: containing only inoculum (2 bottles)
- Procedure control: containing reference item and inoculum (1 bottle).
- Toxicity control: containing test item, reference item and inoculum (1 bottle).

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.

Determination of CO2:
Experimental CO2 production:
- 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).

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 procedure and toxicity control were made over a period of at least 14 days.
- Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers were 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.
- On the penultimate day, the pH of respective test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) 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 (procedure and toxicity control) and on day 29 (remaining vessels).

Theoretical CO2:
production The theoretical CO2 production was calculated from the molecular formula.

Measurements and Recordings
pH: At the start of the test (day 0) and on the penultimate day (day 14 for the procedure and toxicity control and day 28 for the inoculum blanks and test item), before addition of concentrated HCl.
Temperature of medium: Continuously in a vessel with Milli- RO water in the same room.

Reference substance

Reference substance:

acetic acid, sodium salt

Remarks:

sodium acetate

Results and discussion

Test performance:

The test satisfied the validation criterion given in the OCDE test guideline
1. The reference item was biodegraded by at least 60% (83%) within 14 days.
2. The difference of duplicate values for %-degradation of the test item was always less than 20 (≤ 2%).
3. The total CO2 release in the blank at the end of the test slightly exceeds 40 mg/L (81.1 mg CO2 per 2 litres of medium, corresponding to 40.6 mg CO2/L). Since the CO2 release in the blank was still well below 70 mg/L this does not constitute a breach of the validity criterion.
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 (Milli- RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).
Since all criteria for acceptability of the test were met, this study was considered to be valid.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

4

Sampling time:

28 d

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 & B	Δ A-B1)
2	2	1	2	1
5	5	3	4	2
8	5	3	4	2
12	5	3	4	2
15	5	3	4	2
19	5	3	4	2
23	5	3	4	2
29 2)	5	3	4	2
29 2)	5	3	4	2
29 2)	5	3	4	2

1): Absolute difference in biodegradation between bottles A and B

2): Biodegradation is ended on day 28 by addition of HCl. Therefore, differences observed on day 29 are actually differences of day 28.

CO2 Production and Percentage Biodegradation of the Reference Item

Day	HCl (0.05 N) titrated (mL)		Produced	Produced	Cumulative	Biodegradation
	Blank	Procedure	CO2	CO2	CO2	(%)1)
	(mean)	control	(mL HCl)	(mg)	(mg)
2	48.06	30.63	17.43	19.2	19.2	22
5	44.66	24.62	20.04	22.0	41.2	48
8	42.11	29.97	12.14	13.4	54.6	64
12	41.85	39.41	2.44	2.7	57.2	67
152)	44.42	32.08	12.34	13.6	70.8	83

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of sodium acetate: 85.6 mg CO2/2L.

2): CO2 measured on day 15 is actually part of CO2 production of day 14, since microbial activity was ended on day 14 by addition of HCl

CO2 Production and Percentage Biodegradation of the Toxicity Control

Day	HCl (0.05 N) titrated (mL)		Produced	Produced	Cumulative	Biodegradation
	Blank	Procedure	CO2	CO2	CO2	(%)1)
	(mean)	control	(mL HCl)	(mg)	(mg)
2	48.06	36.41	11.65	12.8	12.8	7
5	44.66	42.06	2.60	2.9	15.7	9
8	42.11	24.45	17.66	19.4	35.1	20
12	41.85	25.90	15.95	17.5	52.6	30
152)	44.42	30.85	13.57	14.9	67.6	39

1): Calculated as the ratio between CO2 produced (cumulative) and the sum of the ThCO2 of the test item and reference item: 172.7 mg CO2/2L (ThCO2 test item: 87.0 mg CO2/2L + ThCO2 sodium acetate: 85.6 mg CO2/2L).

2): CO2 measured on day 15 is actually part of CO2 production of day 14, since microbial activity was ended on day 14 by addition of HCl.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

In conclusion, the test item was not readily biodegradable under theconditions of the modified Sturm test presently performed.

Executive summary:

The objective of the study was to evaluate the test item for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge.

The study procedures described in this report were in compliance with the OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the ISO standard 10634, 1995.

the testitem was a slight yellow powder with a purity of 98%. The test item was tested in duplicate at a target concentration of 18 mg/L, corresponding to 12 mg TOC/L.

The organic carbon content was based on the molecular formula. The Theoretical CO2 production (ThCO2) of test item was calculated to be 2.43 mg CO2/mg.

The study consisted of six bottles:

* 2 inoculum blanks (no test item),

* 2 test bottles (test item),

* 1 procedure control (sodium acetate) and

* 1 toxicity control (test item plus sodium acetate).

Since the test item 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-litres test bottles containing medium with microbial organisms and mineral components. 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 the test item and test organisms. Furthermore, the test medium was daily swirled around, since the test item tended to float on the water surface. Test duration was 28 days for the inoculum blank and test item (last CO2 measurement on day 29) and 14 days for the procedure and toxicity control (last CO2 measurement on day 15).

The relative biodegradation values calculated from the measurements performed during the test period revealed 5% and 3% biodegradation of test item (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control, the test item 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, the test item was designated as not readily biodegradable.