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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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study was performed according to an OECD guideline under GLP with three noted deviations. The test was prolonged because the pass level was not reached at day 28. The purity of the test substance was reported to be as received from Akzo Nobel Fibres, Amhem. Since the test substance may be toxic to microorganisms, it was also tested in the presence of silica gel to reduce the concentration in the water phase.
Justification for type of information:
Additional documentation, provided within the IUCLID Assessment Reports (Section 13), supports the read-across approach.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
The test was prolonged because the pass level was not reached at day 28. Since the test substance may be toxic to microorganisms, it was also tested in the presence of silica gel to reduce the concentration in the water phase.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): WWTP Nieuwgraaf in Duiven, The Netherlands. The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic waste water. The activated sludge was preconditioned to reduce the endogenous respiration rates by aerating for 1 week.
- Concentration of sludge: 2 mL DW/L
- Preparation of inoculum for exposure: The dry weight (DW) of the inoculum was determined by filtrating 100 mL of the activated sludge over a preweighed filter. The filter was dried for 1.5 hours at 104ºC and weighed after cooling. DW was calculated by subtracting the weighed filters.

Preparation of Mineral Medium: 8.5 g of KH2PO4, 21.75 g of K2HPO4, 33.3 g of Na2HPO4 2H20, 22.5 mg MgSo4 7H20, 27.5 mg CaCl2, 0.25 mg FeCl3 6H20 were dissolved in 1 L of deionised water. Ammonium chloride was omitted to prevent nitrification. Due to this omission the pH of the medium decreased slightly. The test compound was added to the bottles using a stock solution of 1.0 g/L.
Duration of test (contact time):
0 - 28 d
Initial conc.:
2 mg/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Use was made of 3 bottles containing only inoculum, and 3 bottles containing test substance and inoculum. The concentration of the test substance in the bottles was 2.0 mg/L. The inoculum was diluted to 2 mg DW/L in the closed bottles. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. Subsequently all bottles were closed and incubated at 21±1°C in the dark. The oxygen concentration in the bottles was measured in the bottles using a special funnel that fit exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode, the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle. Dissolved oxygen concentrations were measured at day 7, 14, 21, 28, 42, 56, and 84.

One extension from the protocol of the closed Bottle test was introduced. Since the test substance may be toxic to microorganisms, the test substance was tested in the presence of silica gel to reduce the concentration in the water phase. During the test period, the test substance should be released slowly from the silica gel (1 g/bottle). Although no additional oxygen consumption was expected, controls with silica gel were carried out as well (10 bottles containing test substance, inoculum, and silica gel).

The pH of the media was 6.9 at the start of the test and was 6.5-6.9 at day 28. Temperatures ranged from 20-22°C. ThOD and BOD were calculated, and biodegradation was calculated as the ratio of BOD to ThOD.
Reference substance:
not required
Remarks:
Inhibition of the degradation of a well-degradable compound by the test substance was not determined because possible toxicity of the test substance to microorganisms degrading acetate is not relevant.
Parameter:
other: % Biodegradation (BOD/ThOD)
Value:
30
Sampling time:
28 d
Remarks on result:
other: without silica gel
Parameter:
other: % Biodegradation (BOD/ThOD)
Value:
30
Sampling time:
84 d
Remarks on result:
other: without silica gel
Parameter:
other: % Biodegradation (BOD/ThOD)
Value:
28
Sampling time:
28 d
Remarks on result:
other: with silical gel
Parameter:
other: % Biodegradation (BOD/ThOD)
Value:
28
Sampling time:
84 d
Remarks on result:
other: with silica gel

Oxygen Consumption and Percent Biodegradation

Time (days)

Oxygen Consumption (mg/L)

Biodegradation (%)

Without Silica Gel

With Silica Gel

Without Silica Gel

With Silica Gel

7

0.7

0.6

18

15

14

1.1

1.1

28

28

21

1.3

1.1

33

28

28

1.2

1.1

30

28

42

1.4

1.1

35

28

56

1.3

1

33

25

84

1.2

1.1

30

28

Validity criteria fulfilled:
yes
Interpretation of results:
other: the test substance is not ready biodegradable
Executive summary:

The ready biodegradability of the test substance was determined in the Closed Bottle test performed according to slightly modified OECD guidelines. The test was prolonged because the pass level was not reached at day 28 and the test substance was used as received from Akzo Nobel Fibres, Amhem. Validity of the test was confirmed by endogenous respiration of 1.5 mg/L, oxygen concentrations > 0.5 mg/L in all bottles during the test period, and less than 20% differences between replicates. The test substance caused no reduction in the endogenous respiration. Therefore, the test substance is considered to be non-inhibitory to the inoculum. The test substance was biodegraded approximately 30% at day 28 in the Closed Bottle test. After 84 d, the biodegradation percentage did not increase. Therefore, the test substance is not readily biodegradable. The biodegradation percentages found were comparable in the presence and absence of silica gel, suggesting that the test substance is not toxic to activated sludge at the tested concentration.

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The substance and other analogue substances are all not readily biodegradable. p-Phenylenediamine had 30% biological oxygen demand in an OECD 301D study after 28 days at 2 mg/L. No additional degradation was observed when the test was extended to 84 days. In an OECD 301C study at 100 mg/L, 2% biological oxygen demand was observed for the substance after 28 days. Biodegradation is not expected to be a primary route of degradation for this substance.