[Name confidential or not available]

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

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:

yes

Remarks:

(the nominal ThOD based test material concentration was 37 mg O2/L instead of > 50 mg O2/L as recommended by the testing guideline. This deviation did not adversely impact the results of this study.)

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

(the nominal ThOD based test material concentration was 37 mg O2/L instead of > 50 mg O2/L as recommended by the testing guideline. This deviation did not adversely impact the results of this study.)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Worlingworth sewage treatment works (Suffolk, UK)
- Method of cultivation / Preparation of inoculum for exposure: At the time of collection, the sludge was sieved (1 mm²) then transported to the laboratory and left stand for approximately 30 minutes to allow the sewage solids to settle. A portion of the supernatant was removed and the sludge aerated until required.

Duration of test (contact time):

28 d

Initial conc.:

37 mg/L

Based on:

ThOD/L

Initial conc.:

14 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: mineral salts medium (MSM) according to guideline, prepared using tap-water that had been softened and treated by reverse osmosis and then purified; nominal resistivity ≥ 18 MegOhm.cm. The pH wasadjusted to 7.4 ± 0.2 with 5M HCl. This water complies with the relevant standards (British Standard (BS) 3978, 1987; American Society for Testing and Materials (ASTM) D 1193-06) for classification as Grade 1 or Type 1 water for laboratory use.
- Test temperature: 20 to 22°C.
- pH: 7.5 to 7.6 at the start of the test and 7.4 to 7.6 at the end
- pH adjusted: not necessary
- Suspended solids concentration: 30 mg/L
- Continuous darkness: not reported
- Other: The inoculum was added to the culture bottles 1 day before test initiation to allow a period of ageing.

TEST SYSTEM
- Culturing apparatus: glass culture bottles in a temperature controlled water bath
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: internal oxygen generating process
- Measuring equipment: Co-ordinated Environmental Services (CES) Ltd automated respirometer

SAMPLING
- Sampling frequency: continuous monitoring of the cumulative amount of oxygen consumed; the cumulative oxygen demand made by each cell was printed at, typically, hourly intervals

CONTROL AND BLANK SYSTEM
- Inoculum blank (2 flasks): inoculated mineral salts medium (MSM)
- Reference (1 flask): inoculated MSM + sodium benzoate (50 mg O₂/L)
- Toxicity control (1 flask): inoculated MSM + test material (50 mg O₂/L) + sodium benzoate (50 mg O₂/L)
- Nitrification test (1 flask): inoculated MSM + test material (50 mg O₂/L) + Allylthiourea (ATU, 11.6 mg/L)
- Nitrification control (1 flask): inoculated MSM + ATU (11.6 mg/L)

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

13

Sampling time:

3 d

Remarks on result:

other: based on ThOD

Key result

Parameter:

% degradation (O2 consumption)

Value:

64

Sampling time:

28 d

Remarks on result:

other: based on ThOD

Details on results:

Mean oxygen consumption in biotic mixtures containing the test material was equivalent to 10% of the ThOD value after approximately 3 days, 50% after 13 days and 64% at the end of the test (Table 1). Since the test material is an UVCB the 10-day window condition (i.e. at least 60% degradation within 10 days after 10% degradation have been reached) does not apply (Commission Regulation (EU) No 286/2011).
In the nitrification controls the degradation of the test material was similar to that in uninhibited cultures (Table 1)
In the presence of test material the degradation of sodium benzoate achieved 67% after 3 days indicating that the test substance was not inhibitory to the microbial inoculum (Table 1)

Results with reference substance:

The reference substance sodium benzoate had achieved 60% of the ThOD after 4 days of incubation and 73% by Day 28 (Table 1).

Table 1: Biodegradation of the test and reference substances

 

% Biodegradation
Day	Test material			Test material in the presence of ATU	Sodium Benzoate	Sodium Benzoate in the inhibition mixture
	Test 1	Test 2	Mean
1	0	0	0	1	34	21
2	0	0	0	9	47	58
3	14	13	13	24	54	67
4	23	22	23	34	60	-
5	28	28	28	38	63	-
6	32	33	32	41	65	-
7	35	37	36	44	67	-
8	38	40	39	45	68	-
9	41	43	42	46	68	-
10	43	46	44	48	69	-
11	45	48	46	49	70	-
12	46	49	48	50	70	-
13	48	51	49	50	71	-
14	49	53	51	51	71	-
15	50	54	52	52	71	-
16	52	56	54	52	71	-
17	53	57	55	53	71	-
18	54	58	56	53	71	-
19	55	59	57	54	72	-
20	56	60	58	54	72	-
21	57	61	59	55	72	-
22	58	62	60	55	72	-
23	59	63	61	55	72	-
24	60	64	62	55	73	-
25	61	65	63	55	73	-
26	61	66	63	56	73	-
27	62	66	64	55	73	-
28	62	66	64	56	73	-

 

Validity criteria fulfilled:

yes

Remarks:

The results obtained for the rate of degradation of sodium benzoate (60% of its ThOD after 4 days) and for the cumulative amount of oxygen consumed by the control mixtures (28 and 37 mg O₂/L) fulfill the validity criteria for this test.

Interpretation of results:

other: readily biodegradable, but failing 10-day window. For UVCB-substances the 10-day window condition does not apply (Commission Regulation (EU) No 286/2011).

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

unsuitable test system

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)

GLP compliance:

yes

Remarks:

Quality Assurance Programme/GLP standards not specified

Oxygen conditions:

aerobic

Inoculum or test system:

other: secondary effluent, domestic

Details on inoculum:

The inoculum was derived from the secondary effluent of a treatment plant receiving predominantly domestic sewage. Inoculum was collected and filtered through a folded filter. The sample was aerated for 6 days at room temperature (pre-conditioning), filtered again and used for test preparation.

Duration of test (contact time):

28 d

Initial conc.:

< 10 mg/L

Based on:

other: DOC (measured in test material solution before addition of inoculum)

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: mineral medium as in test guideline (containing yeast extract as source for vitamines)
- Additional substrate: none
- Solubilising agent (type and concentration if used): none
- Test temperature: 22 °C ± 2 °C
- pH: 7.4 ± 0.1
- pH adjusted: no
- Suspended solids concentration: not determined
- Continuous darkness: no (diffuse light)

PREPARATION OF TEST MATERIAL SOLUTION
Approximately 2 g of test material was each weighed into two 1L glass-bottles. The bottles were filled up to 1 L with mineral medium. The flasks were shaken overnight on a laboratory shaker. Additional filtration through a folded filter resulted in a clear solution. The DOC of the saturated solutions was below 10 mg DOC/L. The pH value of the test substance solution was determined to 7.4 ± 0.1. The prepared saturated solutions were used for the test without dilution. The sample was inoculated by addition of 0.5 mL of inoculum.

TEST SYSTEM
- Culturing apparatus: 1 L glass-bottles covered with aluminium foil to allow free exchange of air
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: samples were incubated on a laboratory shaker
- Measuring equipment: DOC analyser
- Test performed in open system: yes

SAMPLING
- Sampling frequency: Day 0 (immediately after preparation), 1, 5, 9, 16, 26, and 28
- Sampling method: A volume of about 10 ml of each test suspension was taken at sampling. Before sampling any evaporation losses from the flasks were made good by adding water in the required amount if necessary. The culture medium was thoroughly shaken before withdrawing a sample and it was ensured that no material was adhering to the walls of the vessels. The samples were membrane filtered (0.45 µm) immediately after they had been taken.
- Sample storage before analysis: filtered samples were stored at -18 C until sample analysis.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Positive control: sodium acetate (2x); sodium benzoate (2x)

Reference substance:

benzoic acid, sodium salt

Remarks:

42.8 mg/l

Reference substance:

acetic acid, sodium salt

Remarks:

141.7 mg/l

Parameter:

% degradation (DOC removal)

Value:

100

Sampling time:

28 d

Results with reference substance:

Both reference samples, sodium benzoate and sodium acetate, reached a degradation level of more than 70% within 10 days after the substance degradation had reached a l0% threshold degradation level.

Table 1: DOC values measured in test material flasks

Day	DOC¹			Degradation in %³
	Blank²	Flask 1	Flask 2	Flask 1	Flask 2
0	3.95	11.0	10.4	0	0
1	4.05	5.0	3.5	87	109
5	4.8	4.9	4.3	99	108
9	6.3	6.0	5.1	104	119
16	6.05	6.6	6.1	92	99
26	4.8	4.2	4.2	109	109
28	5.5	5.0	5.7	107	97

¹: Flasks 1 and 2 corrected for blank

² Mean of two replicates

³: The DOC of the test material solutions before addition of inoculum was <10 mg DOC/L.

Validity criteria fulfilled:

not applicable

Remarks:

the chosen test method is not suitable

Interpretation of results:

other: decrease of DOC fulfills the criteria for ready biodegradability. However, the decrease of DOC is most probably due to adsorption.

Conclusions:

The results obtained in this study are disregarded due to the following reasons:
- According to OECD Guideline for Testing of Chemicals the used test method (OECD 301E) is not suitable for poorly soluble substances. Poorly soluble substances are such with a solubility limit <100 mg/L (OECD Monograph 23 (2000) Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures). The solubility in water for the test material has been determined to be < 1 mg/L.
- The initial DOC concentration in the test material solutions was below 10 mg DOC/L. According to OECD 301E 10-40 mg DOC/L should be tested.
- The DOC originating from the test material in the flasks was only little above the background (DOC content in blanks was about 4 mg/L).
- The DOC decreased very fast (90-100% within the first day). This could indicate adsorption/desorption processes.

None of these items was addressed in the study report.

Description of key information

Biodegradation in water: screening tests: biodegradation 64% (ThOD) in 28 days (OECD 301F, EU C.4-D)

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable but failing 10-day window

Type of water:

freshwater

Additional information

According to Commission Regulation (EU) No 286/2011, the 10-day window condition does not apply for UVCB substances.