4-amino-5-(ethylsulphonyl)-o-anisic acid

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 December 2014 - 30 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): Acilian
- batch No.of test material:GS00012
- Expiration date of the lot/batch:February 28, 2015
- Purity test date:March 13, 2014
- Purity: 98.0 – 101.5% (nominal)
100.9% (analytical), according to certificate of analysis

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:At room temperature (20 ± 5 °C), in the dark and dry conditions
- Stability under test conditions: yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge :Microorganisms from a domestic waste water treatment plant was supplied by the sewage plant Rossdorf, Germany
- Preparation of inoculum for exposure: The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was resuspended in tap water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated overnight. This suspension was used for the experiment.
- Concentration of sludge: Stock suspension of 3.5 g/L on dry matter base (final sludge concentration in test flasks: 28.7 mg sludge/L)

Duration of test (contact time):

ca. 28 d

Initial conc.:

ca. 102.7 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium:
Fifty mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of 5000 mL with deionised water.
Stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with deionised water to 1000 mL volume; The pH-value was 7.4.
b) 11.25 g MgSO4 x 7 H2O filled up with deionised water to 500 mL volume
c) 18.2 g CaCl2 x 2 H2O filled up with deionised water to 500 mL volume
d) 0.125 g FeCl3 x 6 H2O filled up with deionised water to 500 mL volume
In order to avoid precipitation of iron hydroxide in the stock solution d) after storage and before use, one drop of concentrated HCl per litre was added.

- Test temperature: 22°C ± 1°C.
- pH: 7.5 to 7.6 (measured at the start of the test)
7.3 to 7.9 (measured at the end of the test)
- pH adjusted: NO
- Suspended solids concentration: 28.7 mg sludge/L
- Continuous darkness: yes
- Water:deionised water

TEST SYSTEM
- Number of culture flasks/concentration: 2 flask - Test item: flask 1and 2 (Total - 7 flasks, see part CONTROL AND BLANK SYSTEM below)
- Method used to create aerobic conditions: Stirring
- Measuring equipment: automated Software AMR Wincontrol - to measure Temperature
pH-electrode WTW pH 340i. - to measure pH
Measurement of oxygen:(BSB/BODSensor - System, Aqualytic Dortmund, Germany)
- Test performed in closed vessels due to significant volatility of test substance:yes
- Test performed in open system: No
- Details of trap for CO2 :It was absorbed in an aqueous solution (45%) of potassium hydroxide.
- Measuremnt of oxygen: The consumption of oxygen was determined by measuring the change of pressure in the flasks. The change of pressure in the test flasks was measured by means of a manometric method each day.

SAMPLING
- Sampling frequency: every day, 28 days

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes - FLASK 3 and 4
- Abiotic sterile control:Yes - FLASK 6 (Poisoned with 5 mL HgCl2 solution in test water (stock solution of 1 g/L))
- Toxicity control:Yes - FLASK 7
- Procedure control:Yes - FLASK 5

Reference substance:

benzoic acid, sodium salt

Remarks:

SLBK0666V, 99.9%

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 0

Sampling time:

28 d

Details on results:

The oxygen demand of the inoculum control (medium and inoculum) was 30 mg O2/L, of the test item was 25 mg O2/L and for toxicity control was 185 mg O2/L at the day 28.The difference of duplicate values for the degradation of the test item at the plateau, at the end of the test and at the end of the 10-day window was less than 20%. The difference of duplicate values at day 28 differed by 0%. At the end of the test at day 28, the degradation of test item was 0% and for the toxicity control the biodegradation was 49% after 28 days of incubation. Test item did not reach 60% of biodegradation during the incubation period of 28 days and therefore the 10 window could not be determined.
In the toxicity test, containing both the test substance and a reference compound more than 25% biodegradation (based on total ThOD) occurred within 14 days, therefore the test substance can be assumed to be not inhibitory.

ABIOTIC CONTROL: as the oxygen demand was 0 mg/L during the test, there was no need to use correction of degradation of the test item and toxicity control

Results with reference substance:

The reference item sodium benzoate was degraded to more than 60% after 3 days of incubation. It was sufficiently degraded to 81% after 14 days and to 82% after 28 days of incubation. The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Nitrogen is part of the molecular structure of the test item; therefore nitrification (ThODNO3) has to be considered for the evaluation of results.

Table No.2: Cumulative Biochemical Oxygen Demand (mg O2/L) in Test Flasks during the Test

Period of 28 Days

Time days	FLASK No.
	1	2	3	4	5	6	7
1	0	0	0	0	60	0	60
2	0	0	0	0	85	0	85
3	0	0	0	0	110	0	110
4	5	0	10	10	120	0	120
5	5	0	10	10	125	0	130
6	5	5	10	10	135	0	135
7	5	5	10	10	140	0	140
8	10	10	15	15	145	0	145
9	10	10	15	15	145	0	145
10	10	10	15	15	150	0	150
11	10	10	15	15	155	0	155
12	10	10	15	15	155	0	155
13	15	15	20	15	155	0	160
14	15	15	20	15	155	0	160
15	15	15	20	15	160	0	165
16	20	20	20	20	160	0	170
17	20	20	25	20	160	0	170
18	20	20	25	20	160	0	170
19	20	20	25	20	160	0	175
20	20	20	25	20	160	0	175
21	20	20	25	20	160	0	175
22	20	20	25	20	160	0	175
23	20	20	25	20	160	0	175
24	20	20	25	25	165	0	180
25	20	20	25	25	165	0	180
26	20	20	25	25	165	0	180
27	20	20	25	25	165	0	180
28	25	25	30	30	170	0	185

Flasks 1 and 2: Acilian

Flasks 3 and 4: inoculum control

Flask 5: reference (procedure control)

Flask 6: abiotic control

Flask 7: toxicity control

Table No.3: Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity

Control based on ThODNH4

Time days	FLASK No.
	1[%]	2[%]	5[%]	7[%]
1	0	0	35	19
2	0	0	50	27
3	0	0	63	34
4	0	0	68	36
5	0	0	71	39
6	0	0	75	40
7	0	0	78	42
8	0	0	79	42
9	0	0	79
10	0	0	81	42
11	0	0	81	43
12	0	0	84	45
13	0	0	81	45
14	0	0	81	45
15	0	0	81	46
16	0	0	82	47
17	0	0	81	46
18	0	0	81	46
19	0	0	81	48
20	0	0	81	48
21	0	0	81	48
22	0	0	81	48
23	0	0	79	47
24	0	0	82	49
25	0	0	82	49
26	0	0	82	49
27	0	0	82	49
28	0	0	82	49

1 ThODNH4 of Acilian: 1.425 mg O2/mg test item

2 ThODNH4 of sodium benzoate: 1.666 mg O2/mg reference item

Table No.4: Percentage Biodegradation of Test Item and of the Toxicity Control based on ThODNO3, and of Sodium Benzoate based on ThODNH4

Time days	FLASK No.
	1[%]	2[%]	5[%]	7[%]
1	0	0	35	17
2	0	0	50	25
3	0	0	63	31
4	0	0	68	33
5	0	0	71	36
6	0	0	75	37
7	0	0	78	39
8	0	0	79	39
9	0	0	79	39
10	0	0	81	40
11	0	0	81	41
12	0	0	84	41
13	0	0	81	41
14	0	0	81	41
15	0	0	81	43
16	0	0	82	44
17	0	0	81	43
18	0	0	81	43
19	0	0	81	44
20	0	0	81	44
21	0	0	81	44
22	0	0	81	44
23	0	0	79	44
24	0	0	82	45
25	0	0	82	45
26	0	0	82	45
27	0	0	82	45
28	0	0	82	45

Table No.5: pH values at the End of the Test

Flask No.	Treatment	pH-value
1	Anicilian	7.3
2	Anicilian	7.3
3	Inoculum control	7.6
4	Inoculum control	7.6
5	Reference item (procedure control)	7.9

Validity criteria fulfilled:

yes

Remarks:

Differences of replicates < 20%.The reference item reached the pass level >=60%on day 3 .The oxygen uptake of the inoculum blank was 30 mg O2/l in 28 days.The pH values of flasks was inside the range 6.0 - 8.5,Oxygen consumption by the test item is < 60%

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test substance is considered to be not readily biodegradable under aerobic aqueous conditions.

Executive summary:

The study of the 28 day ready aerobic biodegradability of test item, was carried out according GLP principles and OECD 301F/EC C.4 – D manometric respirometry methods, at the temperature 22 °C. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The test item contains nitrogen; therefore the evaluation of biodegradation has to be based ThODNH4 and ThONO3. The 102.7 mg/L of test item was inoculated with activated sludge (28.7 mg/L SS) and incubated under aerobic conditions in a closed respirometer flask at the constant temperature. A reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. A blank test, toxicity test and Abiotic sterile control test were run in parallel. The reference item was sufficiently degraded to 81% after 14 days and to 82% after 28 days of incubation. In the toxicity test, containing both the test item and a reference item, on the 14th test day the biodegradation (based on ThODNH4 ) attained 45 % and after 28 days of incubtaion 49% ( ThODNH4 ) - test item can be asumed to be not inhibitory.

At the 28th day of the test the measured aerobic biodegradation of the test item equals 0 %. According to OECD criteria the pass level for ready biodegradability for respirometric methods are 60% of ThOD.

Thus the test item cannot be deemed to be readily biodegradable.

Description of key information

Key study: OECD guideline 301F, GLP study. The test substance is considered to be not readily biodegradable under aerobic aqueous conditions.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

Key study : The study of the 28 day ready aerobic biodegradability of test item, was carried out according GLP principles and according to OECD 301F/EC C.4 – D manometric respirometry methods, at the temperature 22 °C.The biodegradation was followed by the oxygen uptake of the microorganisms during exposure.The consumption of oxygen was determined by measuring the change of pressure in the flasks.

The evaluation of biodegradation has to be based ThODNH4 and ThODNO3 because test item contains nitrogen. Test item in concentration 102,7 mg/L was inoculated with activated sludge (28.7 mg/L SS) in closed flask at the constant temperature. A reference item was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. A blank test, toxicity test and Abiotic sterile control test were run in parallel. Reference item reach the level of biodegradation within 14 days,the oxygen demand of the blank control was 30 mg O2/L and according the toxicity control the test item can be assumed to be not inhibitory. At the end of the test, at day 28, the degradation of Acilian was 0% (ThODNH4, ThODNO3). Thus the test item cannot be deemed to be readily biodegradable.