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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
The ready biodegradation of bis(2-hydroxyethyl)oleylmethylammonium chloride, CAS number 18448-65-2, EC Number 242 -332 -0, was estimated using BIOWIN v4.10, [EPI Suite version 4.11].
Qualifier:
according to guideline
Guideline:
other: QSAR
Version / remarks:
The ready biodegradation of bis(2-hydroxyethyl)oleylmethylammonium chloride, CAS number 18448-65-2, EC Number 242 -332 -0, was estimated using BIOWIN v4.10, [EPI Suite version 4.11].
Principles of method if other than guideline:
The ready biodegradation of bis(2-hydroxyethyl)oleylmethylammonium chloride, CAS number 18448-65-2, EC Number 242 -332 -0, was estimated using BIOWIN v4.10, [EPI Suite version 4.11].
GLP compliance:
no
Key result
Parameter:
other: QSAR
Remarks:
The ready biodegradation of bis(2-hydroxyethyl)oleylmethylammonium chloride, CAS number 18448-65-2, EC Number 242 -332 -0, was estimated using BIOWIN v4.10, [EPI Suite version 4.11].
Value:
>= 60 - <= 70
Remarks on result:
other: The ready biodegradation of bis(2-hydroxyethyl)oleylmethylammonium chloride, CAS number 18448-65-2, EC Number 242 -332 -0, was estimated using BIOWIN v4.10, [EPI Suite version 4.11].
Details on results:
Probability of Rapid Biodegradation (BIOWIN v4.10):
Biowin1 (Linear Model) : 0.9801
Biowin2 (Non-Linear Model) : 0.7894
Expert Survey Biodegradation Results:
Biowin3 (Ultimate Survey Model): 2.9200 (weeks )
Biowin4 (Primary Survey Model) : 3.7898 (days )
MITI Biodegradation Probability:
Biowin5 (MITI Linear Model) : 0.7691
Biowin6 (MITI Non-Linear Model): 0.7895
Anaerobic Biodegradation Probability:
Biowin7 (Anaerobic Linear Model): 0.5338
Ready Biodegradability Prediction: YES
Interpretation of results:
readily biodegradable
Conclusions:
Bis(2-hydroxyethyl)oleylmethylammonium chloride, CAS number 18448-65-2, EC Number 242 -332 -0, was estimated by BIOWIN v4.10, [EPI Suite version 4.11] to be readily biodegradable.
Executive summary:

The ready biodegradation of the test substance, bis(2-hydroxyethyl)oleylmethylammonium chloride, CAS number 18448-65-2, EC Number 242 -332 -0, was estimated using BIOWIN v4.10, [EPI Suite version 4.11].

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The output results are shown below :

Probability of Rapid Biodegradation (BIOWIN v4.10):

Biowin1 (Linear Model)         :   0.9801

Biowin2 (Non-Linear Model)     :   0.7894

Expert Survey Biodegradation Results:

Biowin3 (Ultimate Survey Model):   2.9200  (weeks       )

Biowin4 (Primary Survey Model) :   3.7898  (days        )

MITI Biodegradation Probability:

Biowin5 (MITI Linear Model)    :   0.7691

Biowin6 (MITI Non-Linear Model):   0.7895

Anaerobic Biodegradation Probability:

Biowin7 (Anaerobic Linear Model):  0.5338

Ready Biodegradability Prediction:   YES

Therefore, Bis(2-hydroxyethyl)oleylmethylammonium chloride, CAS number 18448-65-2, EC Number 242 -332 -0, was estimated by BIOWIN v4.10, [EPI Suite version 4.11] to be readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test performed under GLP, according guidelines with acceptable (minor) deviations, meeting all the validty criteria
Justification for type of information:
Read-across data is presented from quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808-53-2, EC Number 276-038-9. This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.
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:
Two minor deviations from the guidelines of the Closed Bottle test were 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), b) river water was used as inoculum
GLP compliance:
yes
Specific details on test material used for the study:
A sample of cocobis(2-hydroxyethyl)methylammonium chloride was received from Akzo Nobel Surface Chemistry on 31-03-2009. The following test
substance data were submitted by the sponsor, who accepted full responsibility for the validity thereof.
• chemical name (active ingredient) cocobis(2-hydroxyethyl)methylammonium chloride
• CAS reg. No. (active ingredient) 70750-47-9
• composition.(see annex 2) cocobis(2-hydroxyethyl)methylammonium chloride (77%)
2-propanol (20%)
Water (3%)
• product name Ethoquad C/12
•batch/Lot no S001314
•appearance yellow liquid
•stability not relevant
•solubility soluble at test concentrations
•storage at ambient temperature in the dark under nitrogen
The concentrations cited in this report refer to the as-received sample of cocobis(2-hydroxyethyl)methylammonium chloride.
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Details on inoculum:
River water was sampled from the Rhine near Heveadorp, The Netherlands (02-04-2009). The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992). River water without particles was used as inoculum. The particles were removed by sedimentation.
Duration of test (contact time):
28 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 river 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 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 solution and suspension 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 river water (inoculum and medium), 10 bottles containing river water and silica gel (2 g/bottle), 10 bottles containing
river water, the test substance and silica gel, and 6 bottles containing sodium acetate and river water The concentrations of the test substance
and sodium acetate in the bottles were 2.0 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. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.

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 river water.

Calculation of the theoretical oxygen demand (ThOD)
The ThODs of the test substance, and sodium acetate were calculated from their molecular formulae and molecular weights
The calculated theoretical oxygen demand of the test substance is 2.3 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 consumptionn (mg/L) by test substance = Mch - Mt
Oxygen consumptionn (mg/L) by reference compound = Mc - Ma
Mc or ch = the mean oxygen level in the control bottles inoculated with river water, n days after the start of the test (c) or the control
bottles with humic acid (ch).
Mt or a = the mean oxygen concentration in the bottles containing the test substance (t) or the reference compound, sodium acetate (a),
and inoculated with river water, n-days after the start of the test.
The biological oxygen demand (BOD) mg/mg of the test substanceand 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:
acetic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
70
Sampling time:
28 d
Remarks on result:
other: tested in the presence of silica gel
Details on results:
Toxicity
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined
because possible toxicity of cocobis(2-hydroxyethyl) methylammonium chloride to microorganisms degrading acetate is not considered to be relevant.
Inhibition of the endogenous respiration of the inoculum by the test substance was not detected. Inhibition is usually detected prior to the onset of the biodegradation of the test substance through suppression of the endogenous respiration (lower oxygen consumption in the presence of a test substance as compared to the control). Therefore, no inhibition of the biodegradation due to the presence of the test substance is expected.

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 7.3(control) and 7.2 (control with silica gel and test).
Temperatures were within the prescribed temperature range of 22 to 24°C.
Validity criteria fulfilled:
yes
Remarks:
Endogenous respiration of 1.4 mg/L at day 28 •Differences between the replicate values at day 28 less than 20% • 70 % biodegradation of the reference compound, sodium acetate, at day 14 •Oxygen concentrations >0.5 mg/L in all bottles during the test
Interpretation of results:
readily biodegradable
Conclusions:
The test substance, cocobis(2-hydroxyethyl)methylammonium chloride was biodegraded by 70% at day 28 in the Closed Bottle test, and can therefore be classified as readily biodegradable.
Test performed under GLP, according guidelines with acceptable (minor) deviations, meeting all the validity criteria
The test substance did not meet the ten day window validation criteria as mentioned in the OECD guideline 301D, but there are scientific arguments to demonstrate that the 10-day window is not a valid method to quantify biodegradation rates of fatty amine derivatives (see attached doccument).
Executive summary:

The biodegradation in water : ready biodegradability of the test substance, Quaternary ammonium compounds, coco alkylbis(hydroxyethy​l)​methyl, chlorides, CAS Number 70750 -47 -9, EC Number 274 -846 -6 (Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808 -53 -2, EC Number 276 -038 -9) was investigated in a study conducted according to OECD Guideline 301D (Ready Biodegradability: Closed Bottle Test).

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

Under the conditions of the study 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. Cocobis(2-hydroxyethyl)methylammonium chloride did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be noninhibitory to the inoculum. Cocobis(2-hydroxyethyl)methylammonium chloride was biodegraded 70% at day 28 in the Closed Bottle test. Hence this compound should be classified as readily biodegradable. The test is valid as shown by an endogenous respiration of 1.4 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 70% 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.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test performed under GLP, according guidelines with acceptable (minor) deviations, meeting all the validty criteria
Justification for type of information:
Read-across data is presented from quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808-53-2, EC Number 276-038-9. This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.
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:
Two minor deviations from the guidelines of the Closed Bottle test were 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), b) river water was used as inoculum
GLP compliance:
yes
Specific details on test material used for the study:
A sample of cocobis(2-hydroxyethyl)methylammonium chloride was received from Akzo Nobel Surface Chemistry on 31-03-2009. The following test
substance data were submitted by the sponsor, who accepted full responsibility for the validity thereof.
• chemical name (active ingredient) cocobis(2-hydroxyethyl)methylammonium chloride
• CAS reg. No. (active ingredient) 70750-47-9
• composition.(see annex 2) cocobis(2-hydroxyethyl)methylammonium chloride (67%)
diethyleneglycol (32%)
Water (1%)
• product name Agrisol A260H
•batch/Lot no 5101652
•appearance Light brown liquid
•stability not relevant
•solubility soluble at test concentrations
•storage at ambient temperature in the dark under nitrogen
The concentrations cited in this report refer to the as-received sample of cocobis(2-hydroxyethyl)methylammonium chloride.
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Details on inoculum:
River water was sampled from the Rhine near Heveadorp, The Netherlands (02-04-2009). The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992). River water without particles was used as inoculum. The particles were removed by sedimentation.
Duration of test (contact time):
28 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 river 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 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 solution and suspension 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 river water (inoculum and medium), 10 bottles containing river water and silica gel (2 g/bottle), 10 bottles containing
river water, the test substance and silica gel, and 6 bottles containing sodium acetate and river water The concentrations of the test substance
and sodium acetate in the bottles were 2.0 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. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.


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 river water.

Calculation of the theoretical oxygen demand (ThOD)
The ThODs of the test substance, and sodium acetate were calculated from their molecular formulae and molecular weights
The calculated theoretical oxygen demand of the test substance is 2.1 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 consumptionn (mg/L) by test substance = Mch - Mt
Oxygen consumptionn (mg/L) by reference compound = Mc - Ma
Mc or ch = the mean oxygen level in the control bottles inoculated with river water, n days after the start of the test (c) or the control
bottles with humic acid (ch).
Mt or a = the mean oxygen concentration in the bottles containing the test substance (t) or the reference compound, sodium acetate (a),
and inoculated with river water, n-days after the start of the test.
The biological oxygen demand (BOD) mg/mg of the test substanceand 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:
acetic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
64
Sampling time:
28 d
Remarks on result:
other: tested in the presence of silica gel
Details on results:
Toxicity
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined
because possible toxicity of cocobis(2-hydroxyethyl) methylammonium chloride to microorganisms degrading acetate is not considered to be relevant.
Inhibition of the endogenous respiration of the inoculum by the test substance was not detected. Inhibition is usually detected prior to the onset of the biodegradation of the test substance through suppression of the endogenous respiration (lower oxygen consumption in the presence of a test substance as compared to the control). Therefore, no inhibition of the biodegradation due to the presence of the test substance is expected.

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 7.3(control) and 7.2 (control with silica gel and test).
Temperatures were within the prescribed temperature range of 22 to 24°C.
Validity criteria fulfilled:
yes
Remarks:
Endogenous respiration of 1.4 mg/L at day 28 •Differences between the replicate values at day 28 less than 20% • 70 % biodegradation of the reference compound, sodium acetate, at day 14 •Oxygen concentrations >0.5 mg/L in all bottles during the test
Interpretation of results:
readily biodegradable
Conclusions:
The test substance, cocobis(2-hydroxyethyl)methylammonium chloride was biodegraded by 64% at day 28 in the Closed Bottle test, and can therefore be classified as readily biodegradable.
Test performed under GLP, according guidelines with acceptable (minor) deviations, meeting all the validity criteria
The test substance did not meet the ten day window validation criteria as mentioned in the OECD guideline 301D, but there are scientific arguments to demonstrate that the 10-day window is not a valid method to quantify biodegradation rates of fatty amine derivatives (see attached doccument).
Executive summary:

The biodegradation in water : ready biodegradability of the test substance, Quaternary ammonium compounds, coco alkylbis(hydroxyethy​l)​methyl, chlorides, CAS Number 70750 -47 -9, EC Number 274 -846 -6 (Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808 -53 -2, EC Number 276 -038 -9) was investigated in a study conducted according to OECD Guideline 301D (Ready Biodegradability: Closed Bottle Test).

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

Under the conditions of the study 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. Cocobis(2 -hydroxyethyl)methylammonium chloride did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be noninhibitory to the inoculum.Cocobis(2 -hydroxyethyl)methylammonium chloride was biodegraded 64% at day 28 in the Closed Bottle test. Hence this compound should be classified as readily biodegradable.The test is valid as shown by an endogenous respiration of 1.4 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 70% 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.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: See "Remark"
Remarks:
Combination of semi-continous activated sludge test with closed bottle test screening (to determine the "true" biodegradability). Tests performed according guidelines with acceptable minor devaiations, non-GLP study. Data can be used as supporting evidence. Optimal conditions found in this screening work were used in GLP test (T09020C and T09021C)
Justification for type of information:
Read-across data is presented from quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808-53-2, EC Number 276-038-9. This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
minor acceptable deviatons
Principles of method if other than guideline:
Several types of Inocula weer tested:
Unadpted secondary activated sludge
Adapted sludge from the SCAS unit was used upon sampling.
River water was sampled from the Rhine near Heveadorp, The Netherlands.

The Closed Bottle tests were performed according to OECD Test Guidelines (1992). Use was made of bottles containing only inoculum, and bottles containing Ethoquad C/12 (1.0 or 2.0 mg/L). In some cases, Ethoquad C/12 was reacted with humic acid and lignosulphonic acid, respectively, both at a concentration of 2.0 mg/L in the bottles. Possible toxic effects of Ethoquad C/12 were also counteracted through addition of 2 g of silica gel in the bottles. During the test period, the test substance should be released slowly from the silica gel. Although no additional oxygen consumption was expected, controls with silica gel, humic acid and lignosulphonic acid were carried out as well.
GLP compliance:
no
Specific details on test material used for the study:
A sample of Ethoquad C/12 was received from AkzoNobel Surface Chemistry.
The composition of the sample was 76% coco-alkylbis-(2-hydroxyethyl)methylammonium chloride, 21% 2-propanol and 3% water.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Inoculum and settled sewage
Secondary activated sludge and primary settled sewage were obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands.
This plant is an activated sludge plant treating predominantly domestic wastewater. The primary settled sewage was collected weekly and stored at
-20°C until required. 150 ML of secondary activated sludge containing approximately 2 g DW/L of suspended solids was used as an inoculum for
each unit.
The activated sludge for the Closed Bottle test 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). Adapted sludge from the SCAS unit was used upon sampling. River water was sampled from the Rhine near Heveadorp, The Netherlands. River water was aerated for 5 to 7 days before use. River water with particles (slightly turbid) and without particles was used as inoculum. The particles were removed by sedimentation.
Duration of test (contact time):
>= 28 - <= 60 d
Initial conc.:
>= 1 - <= 2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
SCAS test
The SCAS test was performed according to Test Guidelines (OECD, 1981). The test was performed in 150 ml SCAS units. At the start each SCAS unit was
filled with 150 ml of activated sludge and the aeration was started. After 23 hours the aeration was stopped and the sludge was allowed to settle for 45 minutes. After settling 100 ml of the supernatant liquor was withdrawn from the tap. Subsequently, 95 mL of primary settled sewage and 5 ml deionized water were added to the control unit, and 95 ml of primary settled sewage and 5 ml of the test substance stock solution were added to the test unit. Ethoquad C/12 was added to the test unit using a stock solution of 2.0 g/L. Aeration was started again and continued for 23 hours. The above fill and draw procedure was repeated 6 times per week throughout the test.
Supernatant drawn off was analyzed for non-purgeable organic carbon (NPOC). The NPOC values were used to follow the removal of the test substance. The percentage removal in the SCAS unit was calculated by the following equation:

% removal = 100 (CT-(Ct-Cc)) / CT

CT is the concentration of the test compound as NPOC added to the settled sewage at the start of the aeration period.
Ct is the concentration of NPOC found in the supernatant liquor of the test at the end of the aeration period.
Cc is the concentration of NPOC found in the supernatant liquor of the control.

Closed Bottle tests
The Closed Bottle tests were performed according to OECD Test Guidelines (1992). The test was performed in 0.3 L BOD (biological oxygen demand) bottles with glass stoppers. Use was made of bottles containing only inoculum, and bottles containing Ethoquad C/12 (1.0 or 2.0 mg/L). In some cases, Ethoquad C/12 was reacted with humic acid and lignosulphonic acid, respectively, both at a concentration of 2.0 mg/L in the bottles. Possible toxic effects of Ethoquad C/12 were also counteracted through addition of 2 g of silica gel in the bottles. During the test period, the test substance should be released slowly from the silica gel. Although no additional oxygen consumption was expected, controls with silica gel, humic acid and lignosulphonic acid were carried out as well. The inoculum was diluted to 2 mg DW/L in the closed bottles. The turbid river water was diluted with the mineral salts medium at a ratio of 1:1 (river water: medium). The clear river water was used undiluted. 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 closed and incubated at 23±1°C in the dark. The biodegradation was measured by following the course of the oxygen decrease in the bottles with a spe¬cial funnel. This funnel fitted exactly in the BOD bottle (van Ginkel and Stroo 1992).
The ThOD of Ethoquad C/12 was calculated from its molecular formula and molecular weight
The biological oxygen demand (BOD) mg/mg of the test compound was cal¬culated by dividing the oxygen consumption by the concentration of the test
substance in the closed bottle. The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD).
Key result
Parameter:
% degradation (O2 consumption)
Value:
63
Sampling time:
28 d
Remarks on result:
other: tested with silica gel for detoxification and clear river water as inoculum
Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
An NPOC removal of >90% and 80% removal of the organic nitrogen in the scas test shows the ability of micro-organisms to completely biodegrade coco-alkylbis-(2-hydroxyethyl)methylammonium chloride. This result is supported by the closed bottle test result with adapted sludge which resulted in 65% biodegradation after 28 days.
Executive summary:

The biodegradation in water : ready biodegradability of the test substance, Quaternary ammonium compounds, coco alkylbis(hydroxyethy​l)​methyl, chlorides, CAS Number 70750 -47 -9, EC Number 274 -846 -6 (Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808 -53 -2, EC Number 276 -038 -9) was investigated in a study conducted according to OECD Guideline 301D (Ready Biodegradability: Closed Bottle Test)R.

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

Under the conditions of the study in the closed bottle test result with adapted sludge resulted in 65% biodegradation after 28 days.

Description of key information

There is one experimental OECD 301B study available for the registered substance (Bis(2-hydroxyethyl)methyloleylammonium chloride), three OECD 301D studies for a structurally analagous substance (quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides) and a QSAR prediction using BIOWIN v4.10 [EPI Suite v4.11]

1. The biodegradation in water : ready biodegradability of the registered substance (Bis(2-hydroxyethyl)methyloleylammonium chloride) was investigated in a study conducted according to OECD Guideline 301B (CO2 Evolution Test).

The reference compound sodium benzoate reached the pass level for ready biodegradability within 4 days (71% degradation after 4 days)

The extent of degradation in the toxicity control was 32.9% within 14 days which only just meets the criteria within the OECD 301 test guidelines for the test substance to be considered as having no inhibitory effect on the inoculum (>25% based on total ThCO2 within 14 days). However, the mean degradation of the test item alone at the end of the test showed a negative value (-15.3%), indicating a slight inhibitory effect to the inoculum. Furthermore, the concentrations of test item used in this OECD 301B study (~44 mg/L) are an order of magnitude higher than those used in the OECD 301D studies for the read-across analogue quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides (~2 mg/L). Based on the 3h EC50 value of 29 mg/L determined for the activated sludge respiration inhibition of the read-across analogue (quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides), the dosing concentration of 44 mg/L for the registered substance in the OECD 301B test is very likely to have been toxic to the mircoorganisms comprising the inoculum, despite the results of the toxicity control employed in the study itself.

Since no degradation was observed within the test duration (35 d after acidification) the test item is considered to be not readily biodegradable under the conditions of the test. However, for the reasons specified above, inhibitory effects cannot be ruled out for the test item and this result may be considered as being a false negative.

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

2. The biodegradation in water : ready biodegradability of the test substance, Quaternary ammonium compounds, coco alkylbis(hydroxyethy​l)​methyl, chlorides, CAS Number 70750 -47 -9, EC Number 274 -846 -6 (Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808 -53 -2, EC Number 276 -038 -9) was investigated in a study conducted according to OECD Guideline 301D (Ready Biodegradability: Closed Bottle Test).

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

Under the conditions of the study 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. Cocobis(2-hydroxyethyl)methylammonium chloride did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be noninhibitory to the inoculum.Cocobis(2-hydroxyethyl)methylammonium chloride was biodegraded 70% at day 28 in the Closed Bottle test. Hence this compound should be classified as readily biodegradable. The test is valid as shown by an endogenous respiration of 1.4 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 70% 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.

3. The biodegradation in water : ready biodegradability of the test substance, Quaternary ammonium compounds, coco alkylbis(hydroxyethy​l)​methyl, chlorides, CAS Number 70750 -47 -9, EC Number 274 -846 -6 (Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808 -53 -2, EC Number 276 -038 -9) was investigated in a study conducted according to OECD Guideline 301D (Ready Biodegradability: Closed Bottle Test).

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

Under the conditions of the study 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. Cocobis(2 -hydroxyethyl)methylammonium chloride did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be noninhibitory to the inoculum.Cocobis(2 -hydroxyethyl)methylammonium chloride was biodegraded 64% at day 28 in the Closed Bottle test. Hence this compound should be classified as readily biodegradable.The test is valid as shown by an endogenous respiration of 1.4 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 70% 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.

4. The biodegradation in water : ready biodegradability of the test substance, Quaternary ammonium compounds, coco alkylbis(hydroxyethy​l)​methyl, chlorides, CAS Number 70750 -47 -9, EC Number 274 -846 -6 (Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808 -53 -2, EC Number 276 -038 -9) was investigated in a study conducted according to OECD Guideline 301D (Ready Biodegradability: Closed Bottle Test).

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

Under the conditions of the study in the closed bottle test result with adapted sludge resulted in 65% biodegradation after 28 days.

5. The ready biodegradation of the test substance, bis(2-hydroxyethyl)oleylmethylammonium chloride, CAS number 18448-65-2, EC Number 242 -332 -0, was estimated using BIOWIN v4.10, [EPI Suite version 4.11].

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The output results are shown below :

Probability of Rapid Biodegradation (BIOWIN v4.10):

Biowin1 (Linear Model)         :   0.9801

Biowin2 (Non-Linear Model)     :   0.7894

Expert Survey Biodegradation Results:

Biowin3 (Ultimate Survey Model):   2.9200  (weeks       )

Biowin4 (Primary Survey Model) :   3.7898  (days        )

MITI Biodegradation Probability:

Biowin5 (MITI Linear Model)    :   0.7691

Biowin6 (MITI Non-Linear Model):   0.7895

Anaerobic Biodegradation Probability:

Biowin7 (Anaerobic Linear Model):  0.5338

Ready Biodegradability Prediction:   YES

Therefore, Bis(2-hydroxyethyl)oleylmethylammonium chloride, CAS number 18448-65-2, EC Number 242 -332 -0, was estimated by BIOWIN v4.10, [EPI Suite version 4.11] to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Since no degradation was observed within the test duration (35 d after acidification) the test item is considered to be not readily biodegradable under the conditions of the test. However, for the reasons specified within the endpoint summary record, inhibitory effects cannot be ruled out for the test item and this result may be considered as being a false negative.

On this basis it is considered appropriate to make use of the read-across OECD 301D data for Quaternary ammonium compounds, coco alkylbis(hydroxyethy​l)​methyl, chlorides to draw conclusions on the biodegradability of the registered substance. The substance is therefore considered to be readily biodegradable on the basis of read-across.

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