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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:
08-04-2010 - 08-06-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test performed under GLP according guidelines with (minor) acceptable deviations, meeting all validity criteria

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
acceptable deviations
Principles of method if other than guideline:
One minor deviation from the guidelines of the Closed Bottle test was introduced;
a) ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does
not result in nitrogen limitation as shown by the biodegradation of the reference compound).
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Nitrilotriacetonitrile
EC Number:
230-804-9
EC Name:
Nitrilotriacetonitrile
Cas Number:
7327-60-8
Molecular formula:
C6H6N4
IUPAC Name:
2-[bis(cyanomethyl)amino]acetonitrile
Details on test material:
A sample of nitrilotriacetonitrile was received from AkzoNobel Functional Chemicals on 08-03-2010.
The following test substance data were submitted by the sponsor, who accepted full responsibility for the validity thereof.
- chemical name (see annex 2) nitrilotriacetonitrile
- CAS reg. No. 7327-60-8
- purity (see annex 2) 99.6 +/- 1.5
- Batch/lot no. CFC 9161
- appearance white crystals
- stability not relevant in test; stable under storage conditions
- solubility in water soluble
- storage at room temperature in the dark
The concentrations cited in this report refer to the as-received sample of nitrilotriacetonitrile.

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Secondary activated sludge (02-04-2010) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an
activated sludge plant treating predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration
rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the BOD bottles (van Ginkel and Stroo, 1992).
Duration of test (contact time):
60 d
Initial test substance concentration
Initial conc.:
4 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

Nutrients and stock solutions
Deionized water used in the Closed Bottle test contained per liter of water with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4.2H2O, 22.5 mg MgSO4.7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3.6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. Sodium acetate and
the test substance were added to the bottles using stock solutions of 1.0 g/L.

Test procedures
The Closed Bottle test was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of
10 bottles containing only inoculum, 10 bottles containing inoculum and 16.0 mg/L humic acid, 10 bottles containing inoculum, test substance and
humic acid (16.0 mg/L), and 6 bottles containing sodium acetate and inoculum. The concentrations of the test substance and sodium acetate in the
bottles were 1.0 and 6.7 mg/L, respectively. 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 zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The
remaining bottles were closed and incubated in the dark. The bottles with the test substance and the bottles with humic acid were placed on
magnetic stirrer plates (600 rpm). The bottles contained magnetic bars in the bottles. Two duplicate bottles of all series were withdrawn for analyses
of the dissolved oxygen concentration at day 7, 14, 21, and 28. One extension from the protocol of the Closed Bottle test was introduced. The Closed Bottle test was prolonged by measuring the course of the oxygen decrease in the bottles of day 28 using a special funnel. This funnel fitted 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 (van Ginkel and Stroo 1992)

Calculation of the results

Calculation of endogenous respiration
The endogenous respiration (oxygen depletion in the control) was calculated as follows;
Oxygen depletion (endogenous respiration) (mg/L) = Mc (day 0) - Mc (day 28)
Mc is the mean oxygen level in the control bottle inoculated with activated sludge.

Calculation of the theoretical oxygen demand (ThOD)
The ThODs of nitrilotriacetonitrile,and sodium acetate were calculated from their molecular formulae and molecular weights
The calculated theoretical oxygen demand of nitrilotriacetonitrile, is 1.1 mg/mg.
The theoretical oxygen demand of sodium acetate is 0.8 mg/mg.

Calculation of the biochemical oxygen demand (BOD)
Provided that the oxygen concentrations in all bottles at the start of the test were equal, the amounts of oxygen consumed in test and reference
compound bottles were calculated as follows:
Oxygen consumption (mg/L) by test substance = Mch - Mt
Oxygen consumption (mg/L) by reference compound = Mc - Ma
Mc or ch = the mean oxygen level in the control bottles inoculated with activated sludge n days after the start of the test (C). The control of
the test substance contained humic acid (CH).
Mt or a = the mean oxygen concentration in the bottles containing the test compound (t) or the reference compound, sodium acetate (a),
and inoculated with activated sludge n-days after the start of the test.
The biological oxygen demand (BOD) mg/mg of the test compound and sodium acetate was calculated by dividing the oxygen consumption by the
concentration of the test substance and sodium acetate in the closed bottle, respectively.

Calculation of the biodegradation percentages
The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD).

Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

% Degradationopen allclose all
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
60 d
Details on results:
Toxicity
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test compound in the Closed Bottle test was not determined because possible toxicity of nitrilotriacetonitrile to microorganisms degrading acetate is not relevant. A slight inhibition of the endogenous
respiration of the inoculum by the test substance was detected. The test substance may therefore be slightly inhibitory to the inoculum.

Test conditions
The pH of the media was 7.0 at the start of the test. The pH of the medium at day 28 was 7.0 (control and test).
Temperatures were within the prescribed temperature range of 22 to 24°C

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
endogenous respiration of <1.5 mg/L; differences between replicates at day 28 < 20%; sodium acetate was degraded 74% after 14 days.; oxygen concentrations >0.5 mg/L in all bottles during the test period.
Interpretation of results:
inherently biodegradable
Conclusions:
Test performed under GLP according guidelines with (minor) acceptable deviations, meeting all validity criteria
Nitrilotriacetonitrile is not biodegraded in the prolonged Closed Bottle test. Nitrilotriacetonitrile should therefore not be classified as readily
biodegradable. The lack of biodegradation in the Closed Bottle test does not mean that nitrilotriacetonitrile is persistent in nature because the
stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.
Executive summary:

In order to assess the biotic degradation, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice. Nitrilotriacetonitrile did cause a slight reduction in the endogenous respiration. The test substance may therefore be slightly inhibitory to the inoculum. Nitrilotriacetonitrile was not biodegraded in the Closed Bottle test after 28 and 60 days (prolonged) and should therefore not be classified as readily biodegradable. The lack of biodegradation in the Closed Bottle test does not mean that nitrilotriacetonitrile is persistent in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test. The test is valid as shown by an endogenous respiration of 0.9 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 74% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

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