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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 1991 to December 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study without deviations, performed under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Secondary activated sludge was obtained from the RWZI Horstermeer in Nederhorst den Berg, The Netherlands
- Storage conditions: Not reported
- Storage length: Not reported
- Preparation of inoculum for exposure: The sludge was preconditioned to reduce the endogenous respiration rates. This was done by aerating the sludge (200 mg dry weight/litre) for a period of one week.
- Concentration of sludge: 200 mg dry weight/litre
- Initial cell/biomass concentration: The sludge was diluted to a concentration in the bottles of 2 mg dry weight/litre.
- Water filtered: no
Duration of test (contact time):
56 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:
Add 1 mL of solution a, b, d and c to distilled water and make up to 1000 mL.
a. Solution A contains per 1000 ml distilled water:
KH2PO4: 8.5 g
K2HPO4: 21.75 g
Na2HP04 · 2H2O: 33.3 g
b. Solution B contains 22.5 g MgSO4 · 7H20 per 1000 mL distilled water.
c. Solution C contains 27.5 g CaCl2 per 1000 mL distilled water.
d. Solution D contains 0.14 g FeCl3 per 1000 ml distilled water.
- Additional substrate: mineral salt solution
- Solubilising agent (type and concentration if used): dichloromethane (1 gram/litre)
- Test temperature: 20 ± 1°C
- pH:
day 0: 7.0 - 7.1
day 28: 6.8 - 7.5
- pH adjusted: no
- Aeration of dilution water: no data
- Suspended solids concentration: 2 mg dry weight/litre
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 280 mL BOD-bottles (Wertheim, cat. no. 270202).
- Number of culture flasks/concentration: 7 (2 flasks analysed per timepoint)
- Method used to create aerobic conditions: not applicable
- Measuring equipment: DO probe
- Test performed in closed vessels: yes
- Test performed in open system: no

SAMPLING
- Sampling frequency:
day 0: oxygen content measured in one bottle of every test condition
day 5,15 and 28: oxygen content measured in two bottles of every test condition
day 56: oxygen of the bottles used at 28 days measured
day 0 and 28: pH measured
- Sampling method: not reported
- Sample storage before analysis: not reported

CONTROL AND BLANK SYSTEM
- Inoculum blank:
7 bottles with mineral salt solution and inoculum (2.8 mL of the preconditioned sludge)
7 bottles with mineral salt solution, inoculum and silicagel
- Abiotic control: 7 bottles with mineral salt solution (not sterile)
- Toxicity control: No
- Other:
Method control: 7 bottles with mineral salt solution, inoculum and with bis(2-ethylhexyl)phthalate coated silicagel
Reference substance:
acetic acid, sodium salt
Reference substance:
other: bis(2-ethylhexyl)phthalate: poorly soluble in water (comparable with C2AQ), biodegradability is known (method control)
Parameter:
% degradation (O2 consumption)
Value:
11
Sampling time:
5 d
Parameter:
% degradation (O2 consumption)
Value:
13
Sampling time:
15 d
Parameter:
% degradation (O2 consumption)
Value:
81
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
77
Sampling time:
56 d
Details on results:
Only biodegradation observed with 28 days testing time is relevant for assessing ready biodegradability. The study however was prolonged until 56 days to see if degradation of the test compounds succeeds.
Results with reference substance:
About 60% of the sodium acetate is degraded within 5 days. This is a degradation that could be reasonably expected because some use of the carbon in anabolic processes is normal under these conditions. In the guidelines it is stated that the biodegradation of sodium acetate must be more than 60% within 28 days. Our results (57-59%) are not significantly different from 60%.
The reference compound bis(2-ethylhexyl)phthalate degraded 42% after 28 days and 60% after 56 days. It can be seen that after a lag period of 15 days the substance begins to degrade. These results are in good correspondence with the results found in literature.

Endogenous Oxygen Depletion

In the OECD guidelines it was stated that the endogenous oxygen depletion should not exceed 0.6 mg O2/L. In this study the endogenous oxygen depletion at day 28 is 0.95. To the opinion of the study director this phenomena does not influence the test results. Also in the update of the EEC guidelines it is stated that the oxygen depletion should not exceed 1.5 mg O2/L after 28 days.

Oxygen Depletion

The results are mean values of duplicate bottles.

O2 depletion (mg O2/L)

Time

(days)

endogenous

silica

gel

sodium

acetate

Bis(2-ethylhexy1)phthalate

C2AQ

 

5

0.35

0.70

3.10

0.15

0.55

15

0.90

1.15

3.05

-0.30

0.65

28

0.80

0.70

3.00

2.15

3.95

56

0.55

0.90

3.20

3.10

3.75

Percentage biodegradation

 

%Biodegradation

Sampling time (days)

5 days

15 days

28 days

56 days

sodium acetate

59

58

57

61

Bis(2-ethylhexyl)phthalate

2.9

-5.8

42

60

C2AQ

11

13

81

77

Measured values of the oxygen content in the test bottles, expressed as mg O2/L

Sampling

time

(days)

mineral

salt

solution

mineral

salt

solution+inoculum

Mineral                                    salt

solution

+inoculum

+sodiumacetate

Mineral salt solution+inoculum

+silicagel

mineral

salt

solution+inoculum+silicagel+bis(2-ethylhexyl)phthalate

 

mineral

salt

solution

+inoculum+silicagel

+ C2AQ

0

9.4

9.1

9.1

9.4

9.5

9.7

5

8.8

8.4

5.3

7.7

7.2

7.1

8.7

8.4

5.3

7.7

7.9

7.2

15

8.3

7.4

4.4

6.7

6.5

5.7

8.3

7.4

4.3

5.8

6.6

5.5

28

7.7

6.9

3.8

6.0

3.8

2.9

7.6

6.8

3.9

6.3

4.2

1.5

56

7.4

6.9

3.7

5.8

2.8

3.1

7.4

6.8

3.6

6.1

2.9

1.3

Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
The test substance can be classified as well biodegradable (>80% in 28 days), but fails the 10-day window.
Executive summary:

The biodegradability of 2-ethylanthraquinone was determined by the method described in OECD TG 301D, adapted for poorly soluble substances.

A test with the reference compound sodium acetate showed that the activity of the inoculum was sufficient. The compound bis(2-ethylhexyl)phthalate (BEF) was used in this study as a reference compound which is poorly soluble in water, comparable with the 2-ethylanthraquinone and of which the biodegradability is known. The biodegradability of BEF found in this study (42% in 28 days) is not in contradiction with literature.

2-ethylanthraquinone was applied coated on silicagel. More than 80% is degraded within 28 days and it can be concluded that 2-ethylanthraquinone can be classified as biodegradable. In the guidelines it is stated that a compound may be classified as readily biodegradable if the level of 60% degradation is reached within 10 days after the biodegradation reached 10%. According to this criterion the compounds may not be classified as readily biodegradable. The substance can therefore be classified as well biodegradable but failing the 10-day window.

Description of key information

The test substance 2-ethylanthraquinone is regarded to be biodegradable but failing the 10-day window.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable but failing 10-day window

Additional information

Two studies on the ready biodegradability in water and one study on the inherent biodegradability in water are available. In the ready biodegradability study conducted by Thus and van der Laan-Straathof (1991), the test substance was coated on silicagel. Under these conditions 81% biodegradation with no lag-phase was observed after 28 days, but the test item failed the 10-day window criterion. Matla and Blom (1991) performed a Sturm test and reported that 2-ethylanthraquinone was not biodegradable under the test conditions. On the website of the National Institute of Technology and Evaluation (NITE), the result of an inherent biodegradation test is published which indicates that no biodegradation was observed within 2 weeks.
The difference in the results of the biodegradation tests is probably caused by the way the test compound was applied. Due to the relatively low water solubility of the test item (0.245 mg/L) the mode of application is likely to influence the resulting its bioavailability to the microorganisms. 

Thus and van der Laan-Straathof performed the only study with application of the test item coated on silicagel, in this way making the substance in the best possible way available to the microorganisms. This eventually leads to a fair estimate of the biodegradability of the test item, and thus to reliable study results.

In contrast, Matla and Blom weighed the powdered compound directly into the incubation bottles and then added the inoculum (no stirring of the mixture). As 10 and 20 mg/L test substance was directly applied to the flasks, i.e. at levels way above the water solubility, only a fraction of the test item was getting dissolved. As only the dissolved part of the test item can get subject to biodegradation, and the by far biggest portion of the test item remained undissolved in the system, the measured biodegradability is expected to get underestimated by far, eventually making the study unreliable.

On the NITE website the method of application is not reported, however it seems appropriate to assume that the test item has directly been applied (30 mg/L), eventually making the study unreliable for the same reasons as mentioned above for the Matla and Blom study.

Taking the above considerations into account, the study performed by Thus and van der Laan-Straathof is regarded as the most reliable one. On this basis the substance is considered to be biodegradable but failing the 10-day window.

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