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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start 12 February 2013 - Completion 22 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study without deviations affecting integrity of the results.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
no
Principles of method if other than guideline:
The (modified) study procedures described in this report were modified and originally based on the Organization for Economic Co-operation and Development (OECD), OECD guidelines for Testing of Chemicals, Section 3, Degradation and Accumulation, guideline No. 310: "Ready Biodegradability: CO2 in sealed vessels (Headspace Test)" adopted March 23, 2006.

In addition, the modified procedures were originally designed to meet the test methods prescribed by the following guideline:
• ISO Standard 14593 (1999) : Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test).
• OPPTS 835.3215 Inherent Biodegradability – Concawe test.
• Proposal for a new OECD guideline 302D – Inherent biodegradability – Concawe test (draft document October 2001).

The major modifications on the procedure were:
-Pre adaptation of sludge during a 13-day period, before the start of the test.
-Prolongation of the test period up to 56 days.
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewage.
Duration of test (contact time):
ca. 56 d
Initial conc.:
ca. 20 mg/L
Based on:
ThIC
Parameter followed for biodegradation estimation:
CO2 evolution
Reference substance:
other: 1-octanol
Preliminary study:
A 28-day biodegradation study was conducted prior to this test (see Biodegradation in water: screening tests.001)
Test performance:
The test proceded normally without significant deviations
Parameter:
% degradation (CO2 evolution)
Value:
ca. 0
Sampling time:
56 d
Results with reference substance:
1-octanol showed 72.9 degradation by exposure day 14.

Percentages biodegradation

Nominal day

% Biodegradation

Reference

substance

di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA)

1

1.3

1.4

7

n.d.

1.7

14

72.9

3.2

21

n.d.

 0

28

n.d.

3.0

35

n.d.

2.4

42

n.d.

 0

49

n.d.

0.0

56

n.d.

 0

n.d.: not determined

Negative values are expressed as 0.

 

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Not inherently biodegradable under the conditions of the test.
Executive summary:

An inherent biodegradation study was conducted with di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) in sealed vessels with headspace test analysis of C02. The study procedures were modified and originally based onthe OECD guideline No. 310, 2006. In addition, the modified procedures were originally designed to meet the test methods of theISO 14593 (1999), OPPTS 835.3215 and the proposal for a new OECD guideline 302D (2001). The major modifications on the procedure were the re adaptation of sludge during a 13-day period, before the start of the test and prolongation of the test period up to 56 days.

 

The batch of di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) tested was a brown highly viscous liquid and a UVCB substance (treated as 100% pure). di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) as supplied contained 68% TOC.

Since, di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) was poorly soluble in water, 3.2 mg test substance,corresponding toa final organic carbon concentration of 20 mg C/l, was added directly to the test medium (final volume bottle 107 ml). The substance was incubated in a buffer-mineral salts medium inoculated with a mixed population of micro-organisms. Note that the test substance was added immediately before closing the bottles. The test was performed in sealed bottles with a headspace of air.

The test consisted of 3 groups:

1.        Blank control: bottles containing inoculated medium;

2.        Procedure control: bottles each containing inoculated medium and 1-Octanol at 20 mg C/l;

3.        Test substance: bottles each containing inoculated medium and di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) at 20 mg C/l;

 

The CO2evolution resulting from the aerobic biodegradation of the test substance was determined by measuring the inorganic carbon (IC) produced in the test bottles in excess of that produced in blank vessels containing inoculated medium only. The extent of biodegradation was expressed as a percentage of the theoretical maximum IC production, based on the quantity of test substance (as organic carbon) added initially.

The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA). Since all criteria for acceptability of the test were met, this study was considered to be valid.

 

In conclusion, di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) is designated as not inherently biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 6, 2012 to May 22, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP OECD guideline study without deficiencies that affected study validity
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
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.
Duration of test (contact time):
>= 28 d
Initial conc.:
>= 19.5 mg/L
Based on:
ThIC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The study consisted of six bottles:
- 2 inoculum blanks (no test substance),
- 2 test bottles (di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA)),
- 1 positive control (sodium acetate) and
- 1 toxicity control (di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) plus sodium acetate).
Preliminary study:
No preliminary test was conducted.
Test performance:
1. The positive control substance was biodegraded by at least 60% (73%) within 14 days.
2. The difference of duplicate values for %-degradation of the test substance was always less than 20.
3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/l (46 mg CO2 per 2 litres of medium, corresponding to 23 mg CO2/l).
4. The Inorganic Carbon content (IC) of the test substance (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli-RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test substance, 13 mg TOC/l).

Since all criteria for acceptability of the test were met, this study was considered to be valid.

Parameter:
% degradation (CO2 evolution)
Value:
>= 14 - <= 17
Sampling time:
29 d
Details on results:
See results table
Results with reference substance:
The relative biodegradation values calculated from the measurements performed during the test period revealed 14 and 17% biodegradation of di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control more than 25% biodegradation occurred within 14 days (33%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.

Table 1    CO2 production and percentage biodegradation of the test substance (bottle A).

 

 

Day

HCl (0.05 N) titrated (ml)

Produced CO2

(ml HCl)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)

(%)

 

Blank (mean)

bottle A

 

2

47.89

47.09

0.79

0.9

0.9

1

 

5

47.04

45.71

1.33

1.5

2.3

3

 

7

47.15

45.91

1.24

1.4

3.7

4

 

9

46.78

45.83

0.95

1.0

4.7

5

 

14

46.06

45.29

0.77

0.8

5.6

6

 

19

44.81

43.71

1.10

1.2

6.8

8

 

23

44.98

43.41

1.57

1.7

8.5

10

 

27

45.32

44.23

0.00

0.0

8.5

10

 

29

45.93

44.93

1.00

1.1

9.6

11

 

29

48.63

47.12

1.51

1.7

11.3

13

 

29

49.53

48.46

1.07

1.2

12.5

14

 

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test substance: 87.3 mg CO2/2l

 

 

Table 2    CO2 production and percentage biodegradation of the test substance (bottle B).

 

 

Day

HCl (0.05 N) titrated (ml)

Produced CO2

(ml HCl)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)

(%)

 

Blank (mean)

bottle B

 

2

47.89

47.49

0.39

0.4

0.4

0

 

5

47.04

46.21

0.82

0.9

1.3

2

 

7

47.15

45.75

1.40

1.5

2.9

3

 

9

46.78

46.10

0.68

0.7

3.6

4

 

14

46.06

44.78

1.28

1.4

5.0

6

 

19

44.81

43.15

1.66

1.8

6.9

8

 

23

44.98

43.35

1.63

1.8

8.6

10

 

27

45.32

43.11

2.21

2.4

11.1

13

 

29

45.93

44.60

1.33

1.5

12.5

14

 

29

48.63

46.86

1.77

1.9

14.5

16

 

29

49.53

48.76

0.77

0.8

15.3

17

 

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test substance:88.2 mg CO2/2l

Validity criteria fulfilled:
yes
Interpretation of results:
other: not readily biodegradable
Conclusions:
Under the conditions of the test, di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) was not readily biodegradable.
Executive summary:

A ‘ready’ biodegradability (modified Sturm test) was conducted with di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA). The study procedures described in this report were based on the OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No. 440/2008 of 30 May 2008, Publication No.L142, Part C.4-C and the ISO International Standard 9439, 1999 and ISO Standard 10634, 1995.

 

di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) was a brown highly viscous liquidwith a purity of provisional 84%. The test substance was tested in duplicate at 19.5 mg/l, corresponding to 13 mg TOC/l. The organic carbon content was based on the molecular formula and the purity of the testsubstance. The Theoretical CO2production (ThCO2) of di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) was calculated to be 2.49 mg CO2/mg.

 

The study consisted of six bottles:

-         2 inoculum blanks (no test substance),

-         2 test bottles (di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA)),

-         1 positive control (sodium acetate) and

-         1 toxicity control (di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) plus sodium acetate).

 

Since di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/l, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms. Test duration was 28 days (last CO2-measurement on the 29thday).

 

The relative biodegradation values calculated from the measurements performed during the test period revealed 14 and 17% biodegradation of di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met. In the toxicity control, di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) was found not to inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid.

 

In conclusion, di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) was designated as not readily biodegradable.

Description of key information

A ‘ready’ biodegradability (modified Sturm test) was conducted with di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA). Since di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/l, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. The relative biodegradation values calculated from the measurements performed during the test period revealed 14 and 17% biodegradation of di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA), for the duplicate bottles tested. In conclusion, di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) was designated as not readily biodegradable.

An inherent biodegradation study was conducted withdi C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) in sealed vessels with headspace test analysis of C02. Since, di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) was poorly soluble in water, 3.2 mg test substance,corresponding toa final organic carbon concentration of 20 mg C/l, was added directly to the test medium (final volume bottle 107 ml). The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA).

In conclusion, di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) is designated as not inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information