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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No GLP but performed under identical conditions, described in detail, enough details to characterize the substance but no certificate of Analysis
Justification for type of information:
Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
ISO guidlines also followed
Principles of method if other than guideline:
This Test has been performed according to slightly modified EU, OECD and IS0 Test Guidelines.To reduce the toxicity of the test substance 2.5 mg/L humic acid was added to the bottles.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
other: Secondary activated sludge
Details on inoculum:
Secondary activated sludge was obtained from the wastewater treatment plant Nieuwgraaf in Duiven,
The Netherlands. This plant is an activated sludge plant treating predominantly domestic waste water.
The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 200 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted to a
concentration of 2 mg DW/L in the BOD bottles (van Ginkel and Stroo, 1992).
Duration of test (contact time):
112 d
Initial conc.:
1.75 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Reference substance:
other: no data
Preliminary study:
Previous studies conducted measured no biodegredation. Method adapted and re-tested.
Test performance:
Inhibition of the endogenous respiration of the inoculum by the test substance in the Presence of humic
acid was not detected. here fore, no inhibition of the biodegradation due to' the "high" initial
concentration of the test compound is expected. The pH of the media was 7.0 at the start of the test.
Temperatures ranged from 19 to 21 "C. The validity of the test is demonstrated by oxygen concentrations
>0.5 mg/L in all bottles during the test period..
Key result
Parameter:
% degradation (O2 consumption)
Value:
43
Sampling time:
28 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
60
Sampling time:
112 d
Details on results:
Substance should be classified inherently biodegradable.
Results with reference substance:
Not Reported

The dissolved oxygen concentrations were determined electrochemicalty using an oxygen electrode (WTW Trioxmatic EO 200) and meter (WTW OX1 530) (Retsch, Ochten, The Netherlands). The pH was measured using a Consort pH meter (Salm en Kipp BV, Breukelen, The Netherlands). The temperature was measured and recorded with a thermo couple connected to a data logger. The dry weight (DW) of the inoculum was determined by filtrating 50 mL of the activated sludge over a preweighed 12 pm Schleicher and Schull filter. This filter was dried for 1.5 hours at 104°C and weighed after cooling. DW was calculated by subtracting the weighed filters and by dividing this difference by the filtered volume. The COD of the test compound was determined by oxidation with an acid-dichromate mixture in which cr6' was reduced to cr3'. The assay used high range COD reaction. The reaction vials were sealed and placed in a heating block and the contents heated at a temperature of 148°C for two hours. The vials were cooled to room temperature and the increase in cr3' was determined spectrophotometrically at 620 nm.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the study conditions, the test substance is considered to be not readily biodegradable.
Executive summary:

A study was conducted to determine the ready biodegradability of the read across substance, N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) C18 unsat.-alkyl-1,3-propanediammonium chloride, according to OECD Guideline 301D. The calculated theoretical oxygen demand of the test substance is 2.2 mg/mg. The chemical oxygen demand of test substance is 2.0 mg/mg. The chemical oxygen demand is used to calculate the biodegradation percentages. Humic acid (2.5 mg/L) has been added to reduce the concentration in the water phase thus preventing toxic effects of the test substance. Test substance is biodegraded 43% by Day 28 in the closed bottle test. Since in the prolonged Closed Bottle test the test substance is biodegraded up to 60% by Day 112, the test substance should be classified as inherently biodegradable. The test substance is a mixture of isopropanol and a surfactant (N,N,'N,'N,'N-pentamethyl-N-tallow-l,3 - propanediammonium chloride). Surfactants include at least two distinct components in their molecular structure. A hydrocarbon part which is poorly soluble in water and a water-soluble moiety. Degradation of the two moieties of the surfactants are degraded by consortia of at least two microorganisms. In the Closed Bottle test isopropanol is biodegraded within 7 d. Biodegradation of isopropanol will account for 20 to 30% (depending on percentage of isopropanoi build in new biomass). The biodegradation of N,N, 'N, 'N, 'N-pentamethyl-N-tallow-l,3 -propanediammonium chloride is probably initiated by a C-alkyl-N cleavage. This cleavage probably results in the production of alkanals and N,N,N',N',N1-pentamethyl-1 ,3-diaminopropane (name uncertain). The tallow alkyl-chain also represents 20 to 30% of the biodegradation measured in a Closed Bottle test. The 43% biodegradation reached at Day 28 is probably caused by the oxidation of isopropanol and the alkyl chain. The biodegradation percentage of 60 at the end of the test strongly indicates that test substance is ultimately biodegradable (mineralised to carbon dioxide, water and ammonium or nitrate). The hydrophilic moiety is probably difficult to degrade. Test substance is anticipated to be environmentally safe with respect to the biodegradation potential because the alkyl chain is readily biodegraded and the non-toxic water-soluble N,N,NJ,N',N'-pentamethyl-l,3 - diaminopropane is slowly degraded (not proven). Under the study conditions, the test substance is considered to be not readily biodegradable (Ginkel, 2001). Based on the results of the read across study, similar biodegrdation levels can be expected for the substance, Quaternary ammonium compounds, N,N,N'-tris(hydroxyethyl)-N,N'-dimethyl-N'-C16 -18 (even numbered) and C18 -unsatd., alkyltrimethylenedi-, bis(Me sulfates) (salts).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From April 08, 2010 to June 08, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 as RA study
Justification for type of information:
Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
Ammonium chloride was omitted to prevent additional oxygen consumption from nitrification
GLP compliance:
yes (incl. certificate)
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 conc.:
2 mg/L
Based on:
test mat.
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
The water used in the Closed Bottle test was spiked per liter of water with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2 HPO4·2H2O, 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
nitrilotriacetonitrile 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 test substance and humic acid, 10 bottles containing only humic acid, and 6 bottles containing sodium acetate and inoculum. The concentration of the test substance, humic acid and sodium acetate in the bottles were 2.0, 16 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).
Reference substance:
acetic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
28
Sampling time:
28 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
30
Sampling time:
60 d
Details on results:
Theoretical oxygen demand (ThOD)
The calculated theoretical oxygen demand of the test substance is 2.5 mg/mg.
The theoretical oxygen demand of sodium acetate is 0.8 mg/mg.

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 N,N,N’,N’,N’-pentamethyl-N-tallowalkyl-1,3- propanediammonium chloride to microorganisms degrading acetate is not relevant. Humic acid was added to the bottles with N,N,N’,N’,N’-pentamethyl-Ntallowalkyl- 1,3-propanediammonium chloride because this substance is toxic to the competent bacteria. Inhibition of the endogenous respiration of the inoculum by the test substance in the presence of humic acid was not observed. Therefore, no inhibition of the biodegradation due to the "high" initial concentration of the test compound is expected.
Results with reference substance:
The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 74.

Biodegradation N,N,N’,N’,N’-Pentamethyl-N-tallowalkyl-1,3-propanediammonium chloride is biodegraded 28% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test this substance was biodegraded 32% at day 60. N,N,N’,N’,N’-Pentamethyl-N-tallowalkyl-1,3-propanediammonium chloride should therefore not be classified as readily biodegradable. The biodegradation percentage strongly indicates that part of the molecule of N,N,N’,N’,N’- Pentamethyl-N-tallowalkyl-1,3-propanediammonium chloride is degraded. N,N,N’,N’,N’-Pentamethyl-N-tallowalkyl-1,3-propanediammonium chloride is a surfactant. Surfactants are chemicals in which a hydrophilic group is linked to a hydrophobic moiety. Biodegradation of both moieties of surfactants requires the concerted action of at least two microorganisms as a single organism usually lacks the full complement of enzymatic capabilities (van Ginkel, 1996). Degradation of a surfactant is only possible when both moieties can be degraded by microorganisms. Partial degradation of N,N,N’,N’,N’-pentamethyl-N tallowalkyl- 1,3-propanediammonium chloride has to be attributed to the inability of microorganisms to degrade the hydrophilic moiety. The linear alkyl chains of fatty amine derivatives are always degraded through 􀇃-oxidation. The inability to degrade the hydrophilic moiety may be concluded from the recalcitrance of N,N’–bis(2-carboxymethyl)-N,N,N’,N’-tetramethyl-1,2-ethaneammonium dibromide and N,N’-bis(2-hydroxyethyl)-N,N,N’,N’-tetramethyl-1,3- propanediammonium dibromide in the Closed Bottle test (Tehrani-Bagha et al 2007). The 30% biodegradation should therefore be attributed to the oxidation of the alkyl chain. The 28% biodegradation is already reached after 28 days demonstrating that the alkyl chain is readily biodegradable.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the study conditions, the substance was considered to be not readily biodegradable (28% in 28 d and 32% in 60 d) under aerobic conditions.
Executive summary:

A study was conducted to determine the ready biodegradability of the read-across substance,N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) C18 unsat.-alkyl-1,3-propanediammonium chloride (94.5%), in water according to OECD Guideline 301D, in compliance with GLP. The test was 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 by 74% of its theoretical oxygen demand after 14 d. Finally, the most important criterion was met with the oxygen concentrations being > 0.5 mg/L in all bottles during the test period. Biodegradability was determined to be 28% within 28 d, in the closed bottle test. In the prolonged closed bottle test substance was biodegraded 32% by Day 60, therefore the substance can be considered not readily biodegradable. Furthermore, test substance did not cause a reduction in the endogenous respiration in the presence of humic acid. The test substance reacted with humic acid and is therefore considered to be non-inhibitory to the inoculum. Under the study conditions, the substance was considered to be not readily biodegradable (Akzo nobel, 2010).

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

Study 1:

A study was conducted to determine the ready biodegradability of the read-across substance,N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) C18 unsat.-alkyl-1,3-propanediammonium chloride (94.5%), in water according to OECD Guideline 301D, in compliance with GLP. The test was 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 by 74% of its theoretical oxygen demand after 14 d. Finally, the most important criterion was met with the oxygen concentrations being > 0.5 mg/L in all bottles during the test period. Biodegradability was determined to be 28% within 28 d, in the closed bottle test. In the prolonged closed bottle test substance was biodegraded 32% by Day 60, therefore the substance can be considered not readily biodegradable. Furthermore, test substance did not cause a reduction in the endogenous respiration in the presence of humic acid. The test substance reacted with humic acid and is therefore considered to be non-inhibitory to the inoculum. Under the study conditions, the substance was considered to be not readily biodegradable (Akzo nobel, 2010).

 

Study 2:

A study was conducted to determine the ready biodegradability of the read across substance, N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) C18 unsat.-alkyl-1,3-propanediammonium chloride, according to OECD Guideline 301D. The calculated theoretical oxygen demand of the test substance is 2.2 mg/mg. The chemical oxygen demand of test substance is 2.0 mg/mg. The chemical oxygen demand is used to calculate the biodegradation percentages. Humic acid (2.5 mg/L) has been added to reduce the concentration in the water phase thus preventing toxic effects of the test substance. Test substance is biodegraded 43% by Day 28 in the closed bottle test. Since in the prolonged Closed Bottle test the test substance is biodegraded up to 60% by Day 112, the test substance should be classified as inherently biodegradable. The test substance is a mixture of isopropanol and a surfactant (N,N,'N,'N,'N-pentamethyl-N-tallow-l,3 - propanediammonium chloride). Surfactants include at least two distinct components in their molecular structure. A hydrocarbon part which is poorly soluble in water and a water-soluble moiety. Degradation of the two moieties of the surfactants are degraded by consortia of at least two microorganisms. In the Closed Bottle test isopropanol is biodegraded within 7 d. Biodegradation of isopropanol will account for 20 to 30% (depending on percentage of isopropanoi build in new biomass). The biodegradation of N,N, 'N, 'N, 'N-pentamethyl-N-tallow-l,3 -propanediammonium chloride is probably initiated by a C-alkyl-N cleavage. This cleavage probably results in the production of alkanals and N,N,N',N',N1-pentamethyl-1 ,3-diaminopropane (name uncertain). The tallow alkyl-chain also represents 20 to 30% of the biodegradation measured in a Closed Bottle test. The 43% biodegradation reached at Day 28 is probably caused by the oxidation of isopropanol and the alkyl chain. The biodegradation percentage of 60 at the end of the test strongly indicates that test substance is ultimately biodegradable (mineralised to carbon dioxide, water and ammonium or nitrate). The hydrophilic moiety is probably difficult to degrade. Test substance is anticipated to be environmentally safe with respect to the biodegradation potential because the alkyl chain is readily biodegraded and the non-toxic water-soluble N,N,NJ,N',N'-pentamethyl-l,3 - diaminopropane is slowly degraded (not proven). Under the study conditions, the test substance is considered as inherently biodegradable (Ginkel, 2001). Based on the results of the read across study, similar biodegrdation levels can be expected for the substance, Quaternary ammonium compounds, N,N,N'-tris(hydroxyethyl)-N,N'-dimethyl-N'-C16 -18 (even numbered) and C18 -unsatd., alkyltrimethylenedi-, bis(Me sulfates) (salts).