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Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 01 February 2010; Experiment completion date - 04 March 2010; Study completion date - 08 April 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 40851/A TE
Batch Number: TZ 5891 / BOP 02-09
Purity: 69.9 % all coloured components
Appearance: Orange powder
Expiry Date: July 31, 2014
Storage Conditions: At room temperature at about 20 °C

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed three times with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.

- Storage conditions: Based on the ratio of wet to dry weight, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10 %) dry material per liter. During the holding period prior to use, the sludge was aerated at room temperature.

- Storage length: 3 days

- Preparation of inoculum for exposure: Prior to use, the sludge was first thoroughly mixed and then diluted with test water to a concentration of 1 g per liter (dry weight basis). Based on the determined dry weight of this diluted activated sludge, defined amounts were added to test water to obtain the final test concentration.

- Pretreatment: The sludge was washed three times with tap water by centrifugation and the supernatant liquid phase was decanted.

- Concentration of sludge: 30 mg/L (dry weight basis).

- Water filtered: no

Duration of test (contact time):

28 d

Initial conc.:

111 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Test water prepared according to the test guideline
- Test temperature: 22 °C, maintained with a built-in thermostat and checked once per week
- pH: 7.4 - 7.7 Prior to test start, the pH was measured in each test flask before the addition of the activated sludge inoculum. At the end of incubation, the pH was measured again in each test flask.
- pH adjusted: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: The test flasks (500 mL reaction vessels, labeled with the necessary information to ensure unmistakable identification) were incubated under continuous stirring in a SAPROMAT D12 (Voith GmbH, Heidenheim, Germany). Oxygen consumption was recorded manually by taking a daily reading at least on each working day
- Principle: Electro-chemical analysis process; The biodegradation process consumes the dissolved oxygen in the test medium and generates CO2. The CO2 is adsorbed by soda lime, which results in a decrease of the total pressure in the airtight test flasks. The pressure drop is detected and converted into an electrical signal by means of an electrode type manometer.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: The consumed oxygen is replaced by electrolytically generated oxygen from a copper sulfate solution
- Measuring equipment: SAPROMAT D12

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: yes
- Other: reference substance (sodium benzoate) as positive control, i.e. known readily biodegradable substance, called the procedure control thereafter.

Reference substance:

benzoic acid, sodium salt

Remarks:

Purity: 99.8 %

Parameter:

% degradation (O2 consumption)

Value:

11

St. dev.:

2

Sampling time:

28 d

Details on results:

Biodegradation of the Test Item:
The percent biodegradation of the test item was calculated based on the chemical oxygen demand (COD) of 0.68 mg O2/mg test item. The biochemical oxygen demand (BOD) of test substance in the test media slightly increased from about Exposure Day 5 until test termination after 28 days. At the end of the 28-day exposure period, the mean biodegradation of test substance amounted to 11 %. Consequently, test substance was found to be not biodegradable under the test conditions within 28 days.

Biochemical Oxygen Demand After 5 Days (BOD5)
The BOD5 (biochemical oxygen demand after 5 days) was recorded (EU Commission Directive 92/69 EEC, C.5, Degradation: Biochemical Oxygen Demand). After the test duration of five days, a low oxygen consumption in the normal range found for the inoculum controls was determined in the two flasks containing the test item test substance. Thus, the BOD5 (biochemical oxygen demand after 5 days) can be stated as follows:
BOD5 = 0 mg O2/mg test item

Chemical Oxygen Demand (COD) of the Test Item:
The chemical oxygen demand (COD) of the test item test substance was determined according to the EU Commission Directive 92/69/EEC, Part C.6 following DIN 38414-S9. The duplicate values were determined to be 0.684 and 0.684 mg O2/mg test item. The mean COD was calculated to be 0.68 mg O2/mg test item.

Parameter:

BOD5

Value:

0 g O2/g test mat.

Parameter:

COD

Value:

680 g O2/g test mat.

Results with reference substance:

Biodegradation in the Toxicity Control:
The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the COD of the test item and the ThOD of the reference item. In the toxicity control, the course of biodegradation over the 28-day exposure period was similar to the two procedure controls, containing only the reference item. Within 14 days of exposure, biodegradation amounted to 65 %. Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 111 mg/L because biodegradation in the toxicity control was >25 % within 14 days.

Biodegradation of the Reference Item (Procedure control):
The percent biodegradation of the reference item sodium benzoate was calculated based on the theoretical oxygen demand of 1.67 mg O2/mg (ThOD). In the procedure controls, the reference item was degraded by an average of 91% by Exposure Day 14, thus confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by an average of 95 %.

The tabulated values represent rounded results obtained by calculation using the exact raw data.

Oxygen Consumption in the Test Flasks

 

Time [days]	Cumulative oxygen consumption [mg/L]
	Test item Replicate No.		Inoculum control Replicate No.		Procedure control Replicate No.		Toxicity control Replicate No.
	1	2	1	2	1	2	1
0	0	0	0	0	0	0	0
1	0	0	0	0	38	36	52
2	--	--	--	--	--	--	--
3	--	--	--	--	--	--	--
4	0	0	0	2	128	127	130
5	6	7	2	2	136	135	138
6	6	7	2	3	140	139	142
7	8	7	4	4	144	143	146
8	8	7	4	4	149	147	148
9	--	--	--	--	--	--	--
10	--	--	--	--	--	--	--
11	8	7	4	4	154	153	157
12	8	10	4	5	157	155	159
13	12	10	4	6	157	157	161
14	12	10	4	6	157	157	162
15	12	10	4	6	159	157	163
16	--	--	--	--	--	--	--
17	--	--	--	--	--	--	--
18	12	10	6	6	163	161	167
19	14	15	6	8	163	163	169
20	14	15	6	8	165	163	169
21	14	15	6	8	165	163	169
22	14	15	6	8	165	163	169
23	--	--	--	--	--	--	--
24	--	--	--	--	--	--	--
25	14	15	6	8	167	165	173
26	14	15	9	8	167	165	173
27	14	15	9	8	167	165	175
28	16	18	9	9	169	167	175

-- No reading taken

Biodegradation in the Test Flasks

Time [days]	Test item		Percentage Biodegradation^1(Procedure control)		Toxicity control
	Replicate 1	Replicate 2	Replicate 1	Replicate 2	Replicate1
0	0	0	0	0	0
1	0	0	23	22	21
2	--	--	--	--	--
3	--	--	--	--	--
4	-1	-1	76	75	53
5	5	7	80	80	56
6	5	6	82	82	57
7	5	4	84	83	59
8	5	4	87	86	59
9	--	--	--	--	--
10	--	--	--	--	--
11	5	4	90	89	63
12	5	7	91	90	64
13	9	7	91	91	64
14	9	7	91	91	65
15	9	7	92	91	65
16	--	--	--	--	--
17	--	--	--	--	--
18	8	5	94	93	66
19	9	11	93	93	67
20	9	11	95	93	67
21	9	11	95	93	67
22	9	11	95	93	67
23	--	--	--	--	--
24	--	--	--	--	--
25	9	11	96	95	68
26	7	9	95	94	68
27	7	9	95	94	69
28	9	12	96	95	68
Mean (Day 28)	11		95		not applicabe

 

¹ Corrected for the mean oxygen uptake of the inoculum controls
-- Not determined

pH Values at the Start and at the End of the Test

Replicate	Identification	pH Start	pH End
1	Test item	7.4	7.4
2	Test item	7.4	7.4
1	Inoculum control	7.4	7.4
2	Inoculum control	7.4	7.4
1	Procedure control	7.4	7.7
2	Procedure control	7.4	7.7
1	Toxicity control	7.4	7

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test substance was not readily biodegradable under the test conditions but showed some potential for biodegradation within 28 days. It exhibited not toxicity to microorganisms in a concentration of 111 mg/L.

Executive summary:

The test item test substance was investigated under GLP (except the supplementary COD measurement) for its ready biodegradability in a manometric respirometry test over 28 days according to the EU Commission Directive 92/69 EEC, C.4-D, Commission Regulation (EC) No 440/2008, C.4-D and the OECD Guideline for Testing of Chemicals No. 301 F. Activated domestic non-adapted sludge micro-organisms were exposed to the 111 mg/L of the test item during 28 days. The oxygen consumption was recorded, the BOD5 and the COD were determined..

 

Biodegradation of test substance:

The percent biodegradation of the test item was calculated based on the chemical oxygen demand (COD) of 0.68 mg O₂/mg test item. The biochemical oxygen demand (BOD) of test substance in the test media slightly increased from about Exposure Day 5 until test termination after 28 days. At the end of the 28-day exposure period, the mean biodegradation of test substance amounted to 11 %. Consequently, test substance was found to be not biodegradable under the test conditions within 28 days. In the toxicity control, containing both test substance and the reference item sodium benzoate, test substance had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 111 mg/L. In the procedure controls, the reference item sodium benzoate was degraded by an average of 91 % by Exposure Day 14, and reached an average biodegradation of 95% by the end of the test (Day 28), thus confirming suitability of the activated sludge. 

 

BOD₅ (biochemical oxygen demand after 5 days) of test substance:

The BOD₅ (biochemical oxygen demand after 5 days) was recorded (EU Commission Directive 92/69 EEC, C.5, Degradation: Biochemical Oxygen Demand). After the test duration of five days, a low oxygen consumption in the normal range found for the inoculum controls was determined in the two flasks containing the test item test substance. Thus, the BOD₅(biochemical oxygen demand after 5 days) was found to be 0 mg O₂/mg test item.

 

The chemical oxygen demand (COD) of the test item test substance was determined according to the EU Commission Directive 92/69 EEC, C.6 following DIN 38414-S9 was found to be 0.68 mg O₂/mg test item. The BOD:COD ratio was found to be 0 indicating no biodegradation is expected. In conclusion, test substance was not readily biodegradable under the test conditions but showed some potential for biodegradation within 28 days as 9 and 12 % (mean 11 %) biodegradation were observed. The test item exhibited not toxicity to aquatic sludge mirco-organisms in a concentration of 111 mg/L.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 01 February 2010; Experiment completion date - 02 March 2010; Study completion date - 08 April 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.9 (Biodegradation: Zahn-Wellens Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 40851/A TE
Batch Number: TZ 5891 / BOP 02-09
Purity: 69.9 % all coloured components
Appearance: Orange powder
Expiry Date: July 31, 2014
Storage Conditions: At room temperature at about 20 °C

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater.

- Preparation of inoculum for exposure: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted.

-Pretreatment: During the holding period of one day prior to use, the sludge was aerated at room temperature.

- Concentration of sludge: A: A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10 %) dry material per liter. before use, the dry weight of this diluted activated sludge was determined again and defined amounts were added to test water to obtain a final concentration of 300 mg dry material per liter. The ratio between inoculum and test item (based on the mean DOC measured) was 3 : 1.

- Water The test water was prepared according to the testing guidelines. Analytical grade salts were dissolved in purified water to obtain the following stock solutions:
1) KH2PO4 8.50 g/L K2HPO4 21.75 g/L Na2HPO4 × 2H2O 33.40 g/L NH4Cl 0.50 g/L. The pH of this solution was 7.4.
2) MgSO4 × 7H2O 22.50 g/L
3) CaCl2 × 2H2O 36.40 g/L
4) FeCl3 × 6H2O 0.25 g/L, stabilized with one drop of concentrated HCl per liter
To obtain the final test water, 10 mL of stock solution 1) and 1 mL each of stock solutions 2) - 4) were combined and made up to 1000 mL with purified water. The pH of the final test water was adjusted from 7.8 to 7.4 with a diluted hydrochloric acid solution.

Duration of test (contact time):

28 d

Initial conc.:

382.9 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

Test Conditions:
Test vessels:
2000-mL Erlenmeyer flasks, cleaned with alcoholic hydrochloric acid, rinsed with deionized water and dried. The final volume was 1000 mL per flask. Each flask was loosely covered with aluminum foil to allow the exchange of air between the flask and the surrounding atmosphere. The test media were continuously stirred by magnetic stirrers.

Identification: The test vessels were labeled with the necessary information to ensure unmistakable identification.

Test duration: 28 days.

Test temperature: 21 - 24 °C. The inoculated flasks were incubated in a temperature-controlled room. The temperature was checked on each sampling date in an inoculum blank. Additionally, the room temperature was continuously recorded.

pH: 6.6 – 7.6 (before adjustment). Prior to test start (after the addition of activated sludge inoculum) and before each sampling, the pH was measured in each test flask. If necessary, the pH was adjusted to 7.5 ± 0.5 with a diluted sodium hydroxide solution.

Light conditions: The test flasks were incubated in the dark.

Oxygen concentration: 7.1 – 8.3 mg O2/L. The oxygen concentration was measured at the start of the test and before each sampling

Sampling:
Per sampling interval, one sample of about 10 mL was taken and analyzed for DOC. Prior to sampling, water evaporation losses were compensated by adding purified water up to the mark. Deposits on the test vessels were resuspended in the test vessels.

Reference substance:

diethylene glycol

Parameter:

% degradation (DOC removal)

Value:

0

Sampling time:

28 d

Details on results:

Degradation of the test item:
In the test flasks containing the test item and activated sludge (inoculum) the mean concentration of DOC (dissolved organic carbon) varied between 90 and 95 mg/L over the exposure period of 28 days and were not significantly different from the initial mean DOC concentration of 92 mg/L measured on Day 0 (starting value after 3 hours of exposure). Expressed as percentage DOC removal, mean values in the range from -4 to 2 % were noted. Therefore, FAT 40851/A was not biodegradable under the test conditions. No significant DOC removal was observed during the first three hours of exposure which is an indication that the test item did not adsorb on activated sludge.

Results with reference substance:

Degradation of the reference item:
In the procedure controls, containing the reference item diethylene glycol and activated sludge (inoculum), the reference item was completely degraded within the first seven days of exposure, thus confirming suitability of the activated sludge.

Degradation in the Toxicity Control

In the toxicity control, containing the test item (corresponding to 45 % of total DOC), the reference item (corresponding to 55 % of total DOC) and activated sludge (inoculum), the initial DOC concentration of 182 mg/L measured on Day 0 (3 hours) rapidly decreased by 50 % within the first 14 days of exposure. Thus, according to the test guidelines the test item was not inhibitory to activated sludge at the tested concentration of 383 mg/L because degradation was >35 % within 14 days of incubation.

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

FAT 40851/A was not biodegradable.

Executive summary:

The test item FAT 40851/A was investigated for its potential (inherent) ultimate biodegradability in a Zahn-Wellens / EMPA test over 28 days, based on the OECD Guideline for Testing of Chemicals, No. 302 B (1992) and EU Commission Directive 88/302/EEC, Part C.9. In the test flasks, containing the test item FAT 40851/A TE and activated sludge (inoculum), the mean concentrations of dissolved organic carbon (DOC) were not significantly different from the initial mean DOC concentration measured on Day 0. Therefore, FAT 40851/A was not biodegradable under the test conditions. The reference item diethylene glycol was ultimately and completely degraded by 100% within the first 14 days of exposure, thus confirming suitability of the activated sludge. In the toxicity control, containing the test item, the reference item diethylene glycol and activated sludge (inoculum), the initial DOC decreased by 50% within 14 days of exposure. Thus, according to the test guidelines the test item was not inhibitory to activated sludge at the tested concentration of 383 mg/L because degradation was >35 % within 14 days.

Description of key information

The substance is neither readily nor inherently biodegradable in tests.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

The ready as well as the inherent biodegradability of the test item FAT 40851/A were tested in two valid GLP studies according to EU Methods C.4-D and C.9, respectively. The substance did not fulfil the criteria for a readily biodegradable substance, but showed a certain potential for biodegradation under the test conditions since about 10 % of the substance was found to be degraded after a period of 28 days. The potential for biodegradation could not be confirmed in the Zahn-Wellens test for inherent biodegradability as no biodegradation is observed. Based on the results of these studies, it can be concluded that FAT 40851/A is not biodegradable.