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Environmental fate & pathways

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:
26 Feb - 27 Mar 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
adopted July 17, 1992
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: 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.
- Storage conditions: The freshly obtained sludge was kept under continuous aeration until further treatment.
- Preparation of inoculum for exposure: 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. At the start of the test (day 0), test and reference substance 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.
- Concentration of sludge: The concentration of suspended solids was determined to be 4.6 g/L (suspended solids) in the concentrated sludge. Before use, the sludge was allowed to settle (79 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 concentrationopen allclose all
Initial conc.:
21 mg/L
Based on:
test mat.
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 CONDITIONS
- Composition of medium: according to guideline
- Test temperature: 21.7 - 22.3 °C. Temperature was measured continuously in a vessel with Milli-RO water in the same room.
- pH: 7.5 - 7.8. pH was measured at the start of the test (day 0) and on day 28, before addition of concentrated HCl.
- Suspended solids concentration: 4.6 g/L.
- Continuous darkness: yes

TEST SYSTEM
- Preparation of test solutions: Weighed amounts of the test substance were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (test substance bottle A: 41.78 mg; test substance bottle B: 41.88 mg and toxicity control bottle: 41.82 mg). To this end, 10 ml of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous shaking (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 substance and the test organisms.
- Culturing apparatus: 2 litre glass brown coloured bottles
- 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, 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 sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).
- Measuring equipment: 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.
- Details of trap for CO2 and volatile organics if used: 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.

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: The amount of CO2 produced in the process of biodegradation was determined by titration. 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.
On day 28, the pH of all 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 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 2 bottles
- Toxicity control: yes, 1 bottle
- Reference control: yes, 1 bottle
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
7
Sampling time:
28 d
Details on results:
The substance achieved a degradation of 7% after 28 d. The substance is therefore not readily biodegradable.
The toxicity control achieved 36% degradation after 14 d and therefore exceeded the pass level (35% after 14 d). The substance is therefore not considered inhibitory to microorganisms.

BOD5 / COD results

Results with reference substance:
The reference control achieved 78% degradation after 14 d. The test system is therefore considered valid.

Any other information on results incl. tables

Table 1: Degradation values [%]

Sampling Day

Test substance

Toxicity Control

Reference Control

Bottle A

Bottle B

2

0

0

1

12

5

0

0

16

42

7

1

0

25

59

9

4

3

31

69

14

4

3

36

78

19

5

4

-

-

23

5

5

-

-

27

5

6

-

-

29

5

7

-

-

29

6

7

-

-

29

7

7

-

-

Table 2: Validity criteria

Criterion from the guideline

Outcome

Validity criterion fulfilled

Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.

 < 20%

 yes

Percentage degradation of the reference compound has reached the pass levels by day 14.

 78% after 14 d

 yes

The toxicity control should degrade to at least 35% (based on DOC) or at least 25% (based on ThOD or ThCO2) within 14 d.

 36%

 yes

The IC content of the test substance suspension in the mineral medium at the beginning of the test must be less than 5% of the TC.

 < 5%

 yes

The total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/L medium.

 22 mg/L

 yes

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Interpretation of results:
not readily biodegradable
Conclusions:
The substance is not readily biodegradable according to OECD criteria (7% degradation after 28 d).