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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:
12 Jan. - 16 Feb. 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study was conducted similar to OECD 301 C, is well documented and is acceptable for assessment. The main deviations from OECD 301C concern the inoculum and the buffered medium used; both were prepared according to the Dutch Standard NPR 6513 (2); in addition, the laboratory provided controls to test inoculum activity, toxicity and suitability of the test substance as nitrogen source.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
yes
Remarks:
A test medium with higher buffer capacity than specified in the guideline was used. In addition to the standard controls (inoculum viability and toxicity control), a control testing the suitability of the test substance as a nitrogen source was included.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Oxidation ditch situated on the premises of TNO, Delft, The Netherlands; the oxidation ditch is used to treat domestic sewage
- Concentration of original sludge: Approximately 5 g/L of solid substance per litre (organic content: 79 %)
- Preparation of inoculum for exposure: The original sludge was stirred vigorously and 0.8 ml was used to inoculate one litre of medium (see "any other information on materials and methods incl. tables"); the inoculum was added to the test medium to give a target concentration of 3 mg organic matter/L.
Duration of test (contact time):
ca. 34 d
Initial conc.:
10 mg/L
Based on:
test mat.
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: See "any other information on materials and methods incl. tables"
- Test temperature: 20 °C
- pH: 7.53 (initial) to 9.34 (test end)
- pH adjusted: no

TEST SYSTEM
- Culturing apparatus:
* Sapromat flasks equipped with a carbon dioxide trap filled with soda lime pellets with indicator (Merck)
* 200 mL of inoculated medium was placed in each of ten flasks; the remaining two flasks received inoculated medium from which the nitrogen source (NH4Cl) was omitted.
* two test concentrations (10 and 30 mg thiourea per litre) were prepared by adding 1 mL or 0.33 mL of a stock solution (0.2296 g in 50 mL water) to 200 mL medium in the flasks
- Number of culture flasks: 12 flasks altogether

CONTROL AND BLANK SYSTEM
- Inoculum activity control: Yes (one control flask containing 100 mg/L sodium acetate only as substrate)
- Toxicity control: Yes (one flask with 100 mg/L sodium acetate and 10 mg/L thiourea; two flasks with 100 mg/L sodium acetate and 30 mg/L thiourea)
- In order to determine the ability of the test substance to serve as nitrogen source, sodium acetate only and sodium acetate plus 10 mg/L of thiourea were added to the flasks containing inoculated medium without nitrogen source.
Reference substance:
acetic acid, sodium salt
Test performance:
Oxygen consumption during the whole test period was recorded in one of the flasks containing 10 mg/L thiourea; to check the validity of this observation, the dissolved organic carbon (DOC) content of the medium in all flasks was determined at the end of the test. The results (see Table 2 and Table D3) indicate that the DOC content in the two 10 mg/l thiorurea flasks was equivalent (corresponding to 96 % of the nominal test concentration). It is therefore concluded that the observed oxygen consumption in one of the flasks was spurious and probably caused by malfunction of the recording system.
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
34 d
Details on results:
INOCULUM ACTIVITY, TOXICITY AND NUTRIENT DEFICIENCY
Biodegradation of acetate in the control vessels in absence of the test substance was used as a measure for the microbial activity of the inoculum. A reduction of the biodegradation of acetate in the presence of the test substance would indicate a toxic effect on the inoculum activity.
In the control replicates the pH increased from an initial value of 7.53 to a maximum value of 9.34 during the test. The results, as given in Table D1, show that acetate was completely degraded (13 mg per flask = 96 %) within one week, as expected.

Addition of 10 mg thiourea per litre (flask no. 8) did not significantly affect the initial degradation of acetate (10 mg per flask: 74 % after ten days which is regarded as a complete mineralisation), but inhibited the following phase in which oxygen consumption probably resulted from nitrification and oxidation of inoculum material. The former process is known to be particularly sensitive to thiourea. The presence of 30 mg/L thiourea (flasks no. 9 and 10) also partly inhibited the initial rapid acetate degradation. The control with thiourea and without additional NH4Cl (flask no. 11) showed no oxygen consumption at all. However, when acetate was added to a flask containing thiourea as the only nitrogen source (flask no. 12) acetate was readily degraded, although at a lower rate than with NH4Cl as a nitrogen source. At the end of the test total oxygen consumption was similar to that in the flasks containing acetate as the only substrate. This result clearly indicates that thiourea was able to serve as a nitrogen source for microorganisms degrading acetate.

In order to check this result the dissolved organic carbon (DOC) remaining in the medium at the end of the test was determined (Table 1 and Table D3). The results indicate that all the acetate added had been degraded in all flasks. The remaining carbon corresponds well with the amount of introduced thiourea (see Table 1). However, there was also some carbon left in the flask (no. 12) where only thiourea served as nitrogen source. Although the remaining carbon probably is not thiourea, this observation weakens the the conclusion that thiourea served as nitrogen source for the acetate degradation.

BIODEGRADABILITY
The pH of the medium remained constant during the test.

The results of the determination of the biodegradability of thiourea are given in Table D2, showing that generally oxygen consumption was low and in the same range as the background of the inoculated medium.
One of the flasks containing 10 mg/L thiourea, however, shoed oxygen consumption during the whole test; to check the validity of this result, the dissolved organic carbon (DOC) of the medium in all flasks was determined at the end of the test. The results (see Table 2 and Table D3) indicate that an identical amount of DOC (corresponding to 96 % of the nominal value introduced) remained in the two flasks. It is therefore concluded that the oxygen consumption in one of the flasks was spurious and probably caused by malfunction of the registration system.
The DOC remaining in the flasks with 30 mg/L thiourea added, also indicates that no degradation had taken place.
It was concluded that thiourea is not readily biodegradable in this test.
Results with reference substance:
Acetate was significantly degraded (13 mg per flask = 96 %) within one week.

Table 1: Results of the determination of DOC in the control tests after 34 days of incubation

Flask no.

Thiourea [mg/L]

DOC added as thiourea

[mg/L]a)

Sodium acetate

DOC measured [mg/L]

7

0

0

+

0.3

8

10

1.6

+

2.2

9

30

4.8

+

4.7

10

30

4.8

+

5.2

11b)

10

1.6

-

1.3

12b)

10

1.6

+

2.4

a)Carbon content of thiourea = 0.16 mg C/ mg

b)NH4Cl has been deleted from the medium

Table 2: Results of the determination of DOC in the medium of the biodegradation test with thiourea after 34 days of incubation.

Flask no.

Thiourea [mg/L]

DOC added [mg/L]

DOC measured as thiourea [mg/L]

% of added

1

0

-

0.1

-

2

0

-

0.2

-

3

10

1.6

1.5

96

4

10

1.6

1.5

96

5

30

4.8

4.9

103

6

30

4.8

4.5

95

Table 2: Results of the determination of DOC in the medium of the biodegradation test with thiourea after 34 days of incubation.

Flask no.

Thiourea [mg/L]

DOC added [mg/L]

DOC measured as thiourea [mg/L]

% of added

1

0

-

0.1

-

2

0

-

0.2

-

3

10

1.6

1.5

96

4

10

1.6

1.5

96

5

30

4.8

4.9

103

6

30

4.8

4.5

95

Table D2: Results of the biodegradability test of thiourea

Thiourea [mg/L]

Oxygen consumption (mg per flask, cumulative)

0

10

30

Flask no.

1

2

3

4

5

6

Days

 

0

0

0

0

0

0

0

1

0

0

3

0

3

1

2

0

0

4

0

3

1

3

0

0

5

0

3

1

4

0

0

6

0

3

1

5

0

0

6

0

3

1

6

0

0

6

0

3

1

7

0

0

8

0

3

1

8

0

0

8

0

3

1

9

1

0

9

0

3

1

10

1

0

10

0

3

1

11

1

0

10

0

3

1

12

1

0

10

0

3

1

13

1

0

10

0

3

1

14

1

0

10

0

3

1

15

1

0

10

0

3

1

16

2

0

10

0

3

1

17

2

0

11

0

3

1

18

2

0

12

0

3

1

19

2

0

13

0

3

1

20

2

0

13

0

3

1

21

2

0

13

0

3

1

22

2

0

13

0

3

1

23

3

0

14

0

3

1

24

3

0

15

0

3

1

25

3

0

15

0

3

1

26

3

0

16

0

3

1

27

3

0

18

0

3

1

28

3

0

18

0

3

1

29

4

0

19

0

3

1

30

4

0

19

0

3

1

31

4

0

19

0

3

1

32

4

0

19

0

3

1

33

4

0

20

0

3

1

34

4

0

22

0

3

1

 

Table D3: Results of the DOC determinations

Flask no.

Integration values (x 10-1)

DOC [mg/L]

1

92

113

95

-

0.1

2

87

109

220

-

0.2

3

454

426

422

-

1.5

4

439

432

467

-

1.5

5

961

935

929

-

4.9

6

930

858

877

-

4.5

7

153

186

172

-

0.3

8

704

531

547

479

2.2

9

914

915

-

-

4.7

10

982

948

1004

-

5.2

11

414

460

348

-

1.3

12

599

599

568

-

2.4

 

Validity criteria fulfilled:
yes
Remarks:
but only for test with 30 mg/L thiourea; test with 10 mg/L is considered unreliable due to large difference in O2 consumption between replicates.
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Thiourea is not readily biodegradable.
Executive summary:

The biodegradability of the substance thiourea was determined using a method derived from the OECD guideline 301C (ready biodegradability: modified MITI test) using oxygen consumption as test parameter. Degradation of thiourea was tested at concentrations of 10 mg/L and 30 mg/L. This test was specifically designed to study the biodegradability of test compounds with relatively low carbon and relatively high oxidisable nitrogen content. The test was carried out using an automatic system (Sapromat) for recording oxygen consumption. The test medium had a higher buffer capacity than required by the guideline. In addition to the standard controls (inoculum activity, toxicity control) a control on the suitability of the test substance as nitrogen source was conducted. The controls showed that the activity of the inoculum was sufficient. Toxic effects of the test substance on inoculum activity were observed; degradation of the reference substance acetate was only weakly affected, but strong inhibitory effects were noted with respect to nitrification. Thiourea was not degraded in this test and is therefore considered not to be readily biodegradable. However, when thiourea served as sole nitrogen source, acetate was rapidly degraded, indicating that thiourea was degraded under these circumstances. Determination of the dissolved organic carbon (DOC) remaining in the medium at the end of the test confirmed that thiourea was not fully degraded in the biodegradation test and did not definitely confirm its use as a nitrogen source.

Description of key information

Based on the available screening level tests thiourea cannot be considered biodegradable.


Complete biodegradation could only be observed under the conditions of the SCAS tests which provide a high potential for adaption of the micro-organisms to the test substance thiourea.
Based on the available screening level tests thiourea cannot be considered readily biodegradable. The available test results on inherent biodegradability do not allow for a persistence assessment of the substance. Therefore, persistence is assessed based on higher tier biodegradation studies in surface water and sediment as well as in soil (please refer to the respective sections in IUCLID/the CSR).
Complete biodegradation could only be observed under the conditions of the SCAS tests which provide a high potential for adaption of the micro-organisms to the test substance thiourea. Thus, thiourea is considered inherently biodegradable under the conditions of the SCAS Test.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria

Additional information

One key study (Klimisch 2) for the assessment of the ready biodegradability of thiourea is available. In this study conducted by Hanstveit (1990) the biodegradability of thiourea was determined by a method derived from the OECD guideline 301C (ready biodegradability: modified MITI test), using oxygen consumption as test parameter. Thiourea was tested at concentrations of 10 and 30 mg/L. This test was specifically designed to study the biodegradability of test compounds with a relatively low carbon content and a relatively high content of oxidisable nitrogen. The test was carried out using an automatic system (Sapromat) for recording oxygen consumption and involving a test medium with higher buffer capacity than specified by the guideline. In addition to the normal controls for inoculum activity and toxicity, a control on the suitability of the test substance as a nitrogen source was included. The controls showed that the activity of the inoculum was sufficient and that the test substance exerted a toxic effect to a limited degree; the toxic effect was only slight on acetate degradation itself, but strong on other processes such as nitrification. Thiourea was not degraded in this test and is therefore considered not to be readily biodegradable. However, when thiourea served as sole nitrogen source, acetate was rapidly degraded, indicating that thiourea was also degraded under these circumstances. Determination of the dissolved organic carbon (DOC) remaining in the medium at the end of the test confirmed that thiourea was not fully degraded in the biodegradation test, but did not completely confirm its use as a nitrogen source.

On the other hand, urea is readily biodegradable. The main mode of degradation is enzymatic mineralization. The degrading microorganisms are expected to be generally capable of degrading these chemical structures even in case the oxygen atom (in urea) is replaced by sulphur (in thiourea). Therefore, biodegradation of thiourea should be generally possible.

In addition to the information on the ready biodegradability of thiourea several studies determining the inherent biodegradability of the test substance are available:

Friesel et al. (1984) investigated the biodegradation of thiourea in a SCAS Test (similar to OECD 302 A) in two test series for 84 and 29 days, respectively. The method involves exposure of the chemical to relatively high concentrations of micro-organisms over a long time period. The viability of the micro-organisms is maintained over this period by daily addition of a settled sewage feed. The conditions provided by the test are highly favourable to the selection and/or adaptation of micro-organisms capable of degrading the test compound. Thiourea was applied at 20 mg/L DOC in the test vessels. The test system consisted of 6 SCAS units (test vessels). Two vessels were used for the test substance, two were used for the blank controls, and another two vessels were used for the reference substances diethylene glycol and p-chloroaniline that were used in the first and second test series, respectively. Under the conditions of the SCAS test thiourea was biodegradable by 90-100 % after an adaption phase of six weeks (first test series). In the second test series 70-85 % biodegradation could be observed after 29 days (possibility of inoculation of activated sludge with adapted micro-organisms via ambient air).

Broecker et al. (1984) also determined the inherent biodegradability of thiourea in a test according to OECD 302A (SCAS Test). The substance (initial concentration: 13 mg DOC/L) was exposed to activated sludge of a municipal sewage treatment plant under aerobic conditions. Diethylene glycol served as positive control. Based on the data of DOC determinations 96 ± 20 % biodegradation of thiourea was observed (test duration not reported). Based on these results for thiourea that were obtained in the SCAS test, the substance is considered to be inherently biodegradable under the conditions of this test method.

It should be noted that this test method provides a high potential for adaption of the micro-organisms to the test substance. Therefore, this test could potentially overestimate biological degradation.

In contrast to the above results, NITE (2012) cites results from a test on the inherent biodegradability of thiourea according to OECD guideline 302C (MITI-II). The substance was incubated at an initial concentration of 30 ppm with activated sludge (100 ppm) for two weeks. Biodegradation of the test substance was followed by indirect measurement via determination of the biological oxygen demand (BOD) as well as by direct measurement of the test substance concentration (TOC, HPLC, and UV-VIS). After two weeks the following results were obtained:

- BOD: 2.6 % degradation

- TOC: 7.1 % degradation

- HPLC: 10.0 % degradation

- UV-VIS: 13.5 % degradation

Therefore, thiourea was assessed to be non-biodegradable based on these screening-level tests.

In addition to the tests mentioned above, Rott et al. (1982) studied the biodegradation of thiourea in the GSF-test using 14C-labelled test substance at an initial concentration of 50 µg/L. Activated sludge from laboratory culture at a concentration of 1 g solids/L served as inoculum. Under the conditions of the GSF-test thiourea degraded by 17 % in 5 days.

Conclusion:

The screening test results suggest that thiourea should not be considered as readily biodegradable.

According to ECHA Guideline R.7b, there are three tests that can be used to determine the inherent biodegradability of organic: Modified SCAS test (OECD 302 A), Zahn-Wellens/EMPA test (OECD 302 B) and modified MITI test (II) (OECD 302 C). The results for biodegradation of thiourea obtained in the two available SCAS tests (Friesel et al., 1984 and Broecker et al., 1984) were above 70 % of theoretical (DOC removal). Therefore, thiourea may be regarded as inherently and ultimately biodegradable under the conditions of the SCAS test.

In cases were DOC removal is used care must be taken to ensure that elimination did not occur through adsorption. No adsorption controls were included in both these experiments, but as the substance has a log Kow of -0.92, is non-ionizing, has a low (experimental) Koc value of 36.49 for adsorption to soil (Korte & Freitag, 1984), and a Kd value of 90 for adsorption to sludge (Freitag et al., 1985), adsorption is expected to be of limited influence on the results in the SCAS Test.

However, according to the ECHA Guidance R.7b only test results from inherent biodegradability studies according to OECD 302C (MITI II study) conducted with non pre-adapted bacteria can be used to conclude that the substance is not persistent where full mineralisation occurs (> 70 % degradation). Thus, persistence of thoiurea cannot be assessed based on basis of SCAS Test results as this test provides a high potential for adaption of the micro-organisms to the test substance.

The available MITI II test resulted in only minor degradation within 14 days (NITE, 2012). Therefore, a definitive conclusion on persistence cannot be drawn based on the available screening level results