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Environmental fate & pathways

Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose for cross-reference:
data waiving: supporting information
Reason / purpose for cross-reference:
data waiving: supporting information
Transformation products:
not specified
Remarks:
Refer to the endpoint summary
Endpoint:
biodegradation in water: sediment simulation testing
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose for cross-reference:
data waiving: supporting information
Reason / purpose for cross-reference:
data waiving: supporting information
Reason / purpose for cross-reference:
data waiving: supporting information
Transformation products:
not specified
Remarks:
Refer to the endpoint summary
Endpoint:
biodegradation in water: sewage treatment simulation testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From February 3, 2010 to May 12, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.10 (Biodegradation: Activated Sludge Simulation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ISO 11733: Activated sludge simulation test
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on source and properties of surface water:
Not applicable
Details on source and properties of sediment:
No data
Details on inoculum:
- Details on collection (e.g. location, sampling depth, contamination history, procedure): Wastewater treatment plant (WWTP) Nieuwgraaf in Duiven, The Netherlands
- Storage conditions: Frozen
- Storage length: 1 Week
- Other:
- 0.35 L of secondary activated sludge containing approximately 3 g/L dry weight was used as an inoculum for each CAS unit
Duration of test (contact time):
ca. 42 d
Initial conc.:
5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
other: Analysing the non-purgeable organic carbon (NPOC) content
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 1 L
- Composition of stock solution: 7.3 g of test substance to 1.0 L of deionized water and mixing for a few hours on a magnetic stirrer. This stock suspension was subsequently diluted 10 times to a concentration of 0.73 g/L. The flow rate of the syringe pump was 9.6 mL/day giving a nominal concentration of the test substance in the influent of the unit of 5.0 mg/L
- pH adjusted: yes
- Aeration of dilution water:

TEST SYSTEM
- Culturing apparatus: Hussmann-type units constructed of glass
- Method used to create aerobic conditions: Aeration was achieved through a capillary on the bottom of the aeration section at a rate of 7-8 L/h of air
- Test performed in closed vessels due to significant volatility of test substance: Yes
- Test performed in open system: No

SAMPLING
- Sampling frequency: Day 1, 5, 8, 12, 15, 19, 20, 22, 26, 27, 29, 33, 34, 36, 40, 41, 42

Analyses
Before the determination of the non-purgeable organic carbon (NPOC) content in the effluents of the CAS units, the effluents were filtered using Schleicher and Schüll (cellulose nitrate) filters with pores of 0.8 um to remove sludge particles. To ensure that the membrane filters used did not release carbon nor adsorb the test substance, the NPOC of a diluted stock solution was determined before and after filtration. Filtered samples were acidified prior to injection in a TOC apparatus (Shimadzus Hertogenbosch, The Netherlands).

The chemical oxygen demand (COD) of the influent and effluent was determined by oxidation with an acid-dichromate mixture in which Cr6+ was reduced to Cr3+ using Hach Lange test kits (LCK 114 and 314). The reaction vials were sealed and placed in a heating block and the contents heated at a temperature of 148°C for two hours. Ammonium and nitrite were measured with LCK 303 and LCK 342 Dr Lange test kits, respectively. The spectrophotometer (Xion 500) and heating block used were obtained from Hach Lange, Duesseldorf, Germany.

The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode (WTW Trioxmatic EO 200) and meter (WTW OXI 530) (Retsch, Ochten, The Netherlands). The pH was measured using a Knick 765 calimatic pH meter (Elektronische Messgerate GmbH, Berlin, Germany). The temperature was measured with a Tegam thermometer Model 820 (Applikon, Schiedam, The Netherlands). The dry weight (DW) of the inoculum was determined by filtering the activated sludge over a pre weighed 12 µm Schleicher and Schüll filter. This filter was dried for 1.5 hours at 104°C and weighed after cooling. DW was calculated by subtracting the weight of the filters and by dividing this difference by the filtered volume.

Collected influent and effluent samples were centrifuged at 8.000 g for 15 minutes. Effluent and sludge samples from the control unit collected in the same period were spiked with the test substance. These samples were used to assess the recovery and storage stability of the test substance. Subsequently the samples were acidified with HCl to approximately pH 2. The samples were stored in the refrigerator until analysis.

Procedures of the CAS test
The CAS test was performed according to ISO (1995), EC (1988) and OECD (1981) test guidelines. The test and control unit were not coupled. The units were started with activated sludge. The aeration was achieved by operating an air-lift. The aeration rate was regulated so that the activated sludge was kept in suspension and the dissolved oxygen concentration was at least 2 mg/L. This oxygen concentration in the aeration vessel was measured at least two times a week. The domestic sewage supply was supplied at a rate of approximately 1.4 L/day to give a hydraulic retention time of 6 hours. The flow was checked by measuring the total volume of effluent over a 24-hour period. After brushing, 35 mL of sludge was daily removed from the aeration tank to maintain a sludge retention time of 10 days. The effluent samples (50 mL) were taken from the settler. The NPOC values were primarily used to assess the performance of biological treatment system fed with quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides containing wastewater and to preliminary follow the removal of the test substance during the test period.

NPOC values of the last period of the test were used to calculate the mean removal percentage. The daily removal percentages were calculated by the following equation: 100 x (CT-(Ct-Cc)) / CT. Where CT is the carbon of the test compound measured as NPOC added to the settled sewage, Ct is the carbon found as NPOC in the effluent of the CAS unit spiked with the test substance and Cc is the carbon found as NPOC in the effluent of the control CAS unit.

The analysis values in the test and control unit were treated as paired observations. Outliers of the mean difference (Xd) series were eliminated according to the Dixon test at a 95% probability level. From the set of 'n' paired observations the mean difference (Xd) and the standard deviation (Sd) were calculated. The Sd is calculated with the following formula Sd = ✓(x -X)/n-1. The statistical significance of the observed difference was then assessed from the t-statistics given by the following equation: Xd x ✓n/Sd. The critical value of t at the required confidence level was obtained from statistical tables for a one tailed test with n-1 degrees of freedom. The percentage biodegradation/removal was given by; (SL-Xd)/SL x 100 where Xd the mean difference and SL is the spiking level, both values being expressed in mg/L carbon. The 95% confidence interval was calculated as follows: tn x Sd /✓n where tn is the t statistic for a two-tailed test, n-1 degrees of freedom, P = 0.05.

Specific analyses of quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides were used to determine the primary removal of the test substance. The removal percentage of quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides was determined with the following equation;
(Is-Es)/Is x 100,
where Is is the nominal test substance concentration in the influent and
Es is the mean of the measured test substance concentrations in the effluent.

The concentration of the test substance on the activated sludge (Csludge) and the theoretical maximum concentration on sludge are used to assess the removal of the test substance by adsorption. Provided biodegradation nor evaporation of the test substance occurs in the system, the theoretical maximum concentration of quaternary ammonium compounds, di- C12-18-alkyldimethyl, chlorides adsorbed onto the sludge is;
Cmax adsorption = Is x SRT/HRT,
where SRT is the sludge retention time,
HRT is the hydraulic retention time (both expressed in days) and
Is is the nominal test substance concentration in the influent.

The % removal of quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides by adsorption is
100 x Csludge/Cmax adsorption.

STATISTICAL METHODS:
t-test for the statistical significance of the observed difference

Deviation and amendments
Guidelines
A few minor deviations to the guidelines were introduced. The primary settled sewage was collected weekly and stored in the refrigerator until required instead of a daily collection of wastewater. The units consisted of aeration vessels capable of holding only 0.35 L from which the liquor was then passed continuously to settler of 0.30 liter capacities.

Study plan
There are three amendments to the study plan; 1) An additional test unit fed with 5.0 mg/L of test substance was included after three weeks because the unit fed with 50 mg/L was still turbid after this period. Turbidity of the effluent indicates that the test substance is toxic to microorganisms at the load used. 2) The test unit fed with 50 mg/L of test substance was still turbid after 6 weeks of operation. An influent concentration of 50 mg/L was therefore considered not representative although the removal of organic carbon from the waste water was still high. Operation of the unit fed with 50 mg/L was therefore stopped. 3) A method to determine the concentrations of the test substance, in aqueous and sludge samples was issued. 4) The information on the test substance has been changed.
Reference substance:
other: Ammonium chloride,
Reference substance:
other: Potassium hydrogen phthalate
Reference substance:
other: Sodium nitrite
Test performance:
The incubation temperature of both CAS units ranged from 19 to 21°C. The pH of the effluent of both CAS units varied from 7.0 to 7.3. The oxygen concentrations measured in both units were always ≥3.7 mg/L. These test conditions are believed to allow biodegradation by micro-organisms present in activated sludge
Key result
% Degr.:
> 99.9
Parameter:
other: based on LC-MS/MS analysis
Remarks:
using C12 DAQ as representative
Sampling time:
42 d
Remarks on result:
other: (Total mean removal)
Key result
% Degr.:
4
Parameter:
other: based on LC-MS/MS analysis
Remarks:
using C12 DAQ as representative
Sampling time:
42 d
Remarks on result:
other: (via sorption)
Key result
% Degr.:
96
Parameter:
other: based on LC-MS/MS analysis
Remarks:
using C12 DAQ as representative
Sampling time:
42 d
Remarks on result:
other: (degraded)
Transformation products:
not measured
Details on transformation products:
Not applicable
Evaporation of parent compound:
not measured
Volatile metabolites:
not measured
Residues:
not measured
Details on results:
Test conditions and validity of the test
The incubation temperature of both CAS units ranged from 19 to 21°C. The pH of the effluent of both CAS units varied from 7.0 to 7.3. The oxygen concentrations measured in both units were always >=3.7 mg/L. These test conditions are believed to allow biodegradation by micro-organisms present in activated sludge. The CAS test was started with a high concentration of aerobic micro-organisms (3.0 g/L dry weight) maintained by the daily addition of primary settled sewage and sludge from a full-scale treatment plant (Duiven). The daily removal of 35 mL of activated sludge from the aeration vessel resulted in a sludge retention time of 10 days. The dry weight in the CAS units ranged from 2.7 to 3.0 g/L. The performance of the control unit was checked by measuring the COD removal at Day 14 and at day 42 and the concentrations of ammonium and nitrite in the effluent (Day 14). At Day 14 the COD contents (means of two measurements) in the influent and effluent were 516 and 35 mg/L, respectively. At day 42, the COD levels in the influent and effluent were 472 and 34 mg/L, respectively. COD removal percentages at both days were 93. The ammonium-N and nitrite-N concentrations in the effluent at Day 14 were 0.7 and <0.6 mg/L. These results demonstrate that the test is valid.

Biodegradation in the CAS test
At day 0 the control unit had been in operation for 28 days. The test unit was preconditioned for 3 days before day 0. At day 0 administration of the test substance at a concentration of 5.0 mg/L was started (first and second amendment to the study plan). The test substance was introduced with a syringe pump. The sludge wasting resulted in a sludge retention time of 10 days. The calculated carbon content of test substance in the influent of the reactor was 3.8 mg/L. The measured concentration of the non purgeable organic carbon (NPOC) in an aqueous solution with 5.0 mg/L of test substance was 3.4 mg/L. After filtration through a 0.45 µm filter the NPOC was 3.3 mg/L. 3.8 mg/L (CT) was used to calculate the carbon removal percentages. After the introduction of the test substance at day 0, high removal percentages of NPOC i.e. 74 were immediately accomplished. These high removal percentages have to be ascribed to the adsorption and/or biodegradation of test substance. The biodegradation percentages were high during the entire test period except for days 15 and 42. From Day 25 to 42, samples were taken to assess a mean of the removal percentage with NPOC contents. No outliers were identified using the Dixon test. Subsequently, the data obtained from day 25 to 42 were used in a t-statistic. The mean difference between the NPOC in the effluents of control and test unit was -1.7 ± 0.6 mg/L (95 per cent confidence interval). The mean removal percentage calculated with this mean difference was 145 ± 11 (95% confidence). The t-statistic (n = 15) did exceed the critical value and the mean difference is therefore statistically significant. The results demonstrate that the continuous activated sludge system treating domestic wastewater spiked with test substance removes >100% of the organic carbon of the test substance. These results strongly indicate that test substance are degraded by micro-organisms. The removal percentages demonstrate that formation of recalcitrant water-soluble substances is unlikely during the biodegradation process. The biodegradation percentage in excess of 100% shows that the removal of organic compounds present in the domestic wastewater are removed better when quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides are present in the influent. Removal percentages calculated with NPOC values are not very accurate. An accurate assessment of the removal of quaternary ammonium compounds, di-C12-18- alkyldimethyl, chlorides was therefore established with specific analyses.

The concentrations of quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides in the effluent of the test unit using didodecyldimethylammonium as representative component were 3 µg/L (day 38), 4 µg/L (day 39), 3 µg/L (day 40), 4 µg/L (day 41) and 5 µg/L (day 42) µg/L. These concentrations correspond with 99.9 % removal. The mean quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides concentrations in the sludge of the reactor monitored on days 41 and 42 were 6.0 and 9.7 mg/L, respectively. This was also determined with didodecyldimethylammonium as representative component. Using 7.9 mg/L (mean of day 41 and 42), 4.0% removal through adsorption onto sludge of the quaternary ammonium compounds, di-C12-18- alkyldimethyl, chlorides present in the influent of the test unit was calculated. These results demonstrate that quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides biodegrades 96% in properly operating conventional biological wastewater treatment plants.
Results with reference substance:
- At Day 14 the COD contents (means of two measurements) in the influent and effluent were 516 and 35 mg/L, respectively. At day 42, the COD levels in the influent and effluent were 472 and 34 mg/L, respectively.
- COD removal percentages at both days were 93. The ammonium-N and nitrite-N concentrations in the effluent at Day 14 were 0.7 and <0.6 mg/L.

Results

Table 1. Oxygen concentrations, pH values in the effluent, and dry weight of the activated sludge in the control and test unit.

Time

(days)

Oxygen (mg/L)

Dry weight (g/L)

pH

 

Control

Test

Control

Test

Control

Test

1

3.7

3.7

 

 

7.2

7.2

5

4.1

3.8

 

 

7.3

7.2

6

 

 

2.9

3.0

 

 

8

4.2

4.1

 

 

7.2

7.0

12

3.7

3.9

 

 

7.0

7.0

13

 

 

2.8

2.9

 

 

15

4.2

4.7

 

 

7.0

7.2

19

4.2

4.9

 

 

7.1

7.1

20

 

 

2.7

3.0

 

 

22

4.5

4.5

 

 

7.4

7.1

26

5.1

4.1

 

 

7.3

7.2

27

 

 

2.8

2.9

 

 

29

4.8

4.9

 

 

7.2

7.0

33

4.3

4.1

 

 

7.1

7.1

34

 

 

2.9

2.9

 

 

36

4.8

4.8

 

 

7.2

7.3

40

3.9

5.0

 

 

7.0

7.1

41

 

 

2.8

2.9

 

 

42

4.5

5.0

 

 

7.2

7.0

Table 2. NPOC concentrations in the effluent of the control and test unit and the calculated removal percentages of test substance

Time (days)

NPOC (mg/L)

Removal (%)

Control

Test

1

11.8

12.8

74

5

11.5

9.9

142

8

12.6

9.3

187

12

11.7

9.6

155

15

12

14.5

35

19

9.8

9.3

118

21

12.1

13

76

22

13.1

11

155

25

13.1

11.7

136

26

10.6

9.3

134

27

11.5

9.7

147

28

10.8

10.4

111

29

12.5

10.3

156

32

11.4

8.8

168

33

11.3

9.3

153

34

12.7

11.2

139

35

11.9

10.5

137

36

13.4

9.3

208

38

9.1

8.6

113

39

12.5

10.1

163

40

12.3

10.3

153

41

12.1

11.3

121

42

13.2

15

53

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the test substance was almost completely removed from wastewater in conventional biological wastewater treatment plants primarily by biodegradation.
Executive summary:

A study was conducted to determine the biodegradation of the test substance, C12-18 DAQ (99.6% active) using a continuous activated sludge (CAS) test, according to OECD guideline 303 A, EU method C10 and ISO guideline 11733, in compliance with GLP. The test substance spiked at a nominal influent concentration of 5.0 mg/L (3.8 mg/L carbon; calculated) was exposed to micro-organisms maintained by addition of domestic waste water for a period of 42 d and included a control fed with domestic wastewater only. Based on non-purgeable organic carbon (NPOC)), the mean organic carbon removal between Day 25 and 42 (14 measurements) was 145 ± 11% (95% confidence interval). The biodegradation percentage in excess of 100% shows that the removal of organic compounds present in the domestic wastewater are removed better when the test substance is present in the influent. However, as removal percentages calculated with NPOC values are not very accurate, an accurate assessment of the removal of the test substance was therefore established with specific analyses using LC-MS/MS. The LC-MS/MS method for the determination of test substance was satisfactory with regard to the linearity, repeatability of the injections, limit of quantification (LOQ), precision and specificity. Based on specific analysis of the test compound, the mean removal percentage from Day 38 to 42 was 99.9%. Test substance in the mixed liquid suspended solids of the reactor sampled on Day 41 and 42 were 6.0 and 9.7 mg/L, respectively. The mean removal of test substance from the influent through adsorption onto sludge assessed with two samples was therefore 4.0% demonstrating that, it is primarily removed by biodegradation. In the study, the test substance biodegraded 96% in properly operating conventional biological wastewater treatment plants. Under the study conditions, the test substance was almost completely removed from wastewater in conventional biological wastewater treatment plants primarily by biodegradation (Van Ginkel, 2010).

Endpoint:
biodegradation in water and sediment: simulation testing, other
Remarks:
(river water and water spiked with sediment)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Published study not performed according to standard guidelines or GLP
Remarks:
(study also referred in the EU RAR report available on DODMAC (CAS 107-64-2)
Qualifier:
no guideline followed
Principles of method if other than guideline:
The kinetics of ultimate biodegradation (mineralization of CO2) of the test substance was studied in freshwater/sediment systems.
GLP compliance:
no
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water / sediment: freshwater
Details on source and properties of surface water:
River water and sediment samples were collected form two rivers (Rapid Creek, Rapid City, SD; Ohio River, Cincinnati, OH) at locations 0.5 to 1.0 mile below the discharge of municipal wastewater treatment plants. Rapid Creek is a slow-flowing mountainn stream with a low wastewater dilution factor whereas the Ohio River is a rapidly flowing major river with high wastewater dilution factor.
Details on source and properties of sediment:
River water and sediment samples were collected form two rivers (Rapid Creek, Rapid City, SD; Ohio River, Cincinnati, OH) at locations 0.5 to 1.0 mile below the discharge of municipal wastewater treatment plants. Rapid Creek is a slow-flowing mountainn stream with a low wastewater dilution factor whereas the Ohio River is a rapidly flowing major river with high wastewater dilution factor.

Sediment samples were analyzed for cation exchange capacity (CEC), pH, particle distribution, organic carbon, and suspended solids levels by standard procedures. They were tested on a dry wt basis.
Duration of test (contact time):
>= 28 - ca. 63 d
Initial conc.:
> 1 - < 100 µg/L
Based on:
other: 14CO2
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on study design:
Low concentrations of test material (1-100 µg/L), which approximate actual environmental levels, were added in duplicate or triplicate to 2-liter Erlenmeyer flasks containing 1 liter (final volume) of water or water/sediment suspensions. Flasks were incubated at 24 ± 2°C with agitation and aerated with CO2-free air in closed, flow-through shake-flask system. Off-gases were bubbled through appropriate CO2 sorbents to remove 14CO2 produced during biodegradation. At various intervals, samples also were taken from the aqueous phase and acidified (
Key result
% Degr.:
8
Parameter:
radiochem. meas.
Remarks:
14CO2
Sampling time:
28 d
Remarks on result:
other: of 0.05 mg/LC18 DAQ in river water alone
Key result
% Degr.:
19
Parameter:
radiochem. meas.
Remarks:
14CO2
Sampling time:
28 d
Remarks on result:
other: of 0.5 mg/L C18 DAQ in river water alone
Key result
% Degr.:
11
Parameter:
radiochem. meas.
Remarks:
14CO2
Sampling time:
63 d
Remarks on result:
other: of 0.05 mg/L C18 DAQ in river water alone
Key result
% Degr.:
22
Parameter:
radiochem. meas.
Remarks:
14CO2
Sampling time:
63 d
Remarks on result:
other: of 0.5 mg/L C18 DAQ in river water alone
Key result
% Degr.:
43
Parameter:
radiochem. meas.
Remarks:
14CO2
Sampling time:
28 d
Remarks on result:
other: of 0.05 mg/L C18 DAQ in river water + 5g/L adapted sediment
Key result
% Degr.:
65
Parameter:
radiochem. meas.
Sampling time:
63 d
Remarks on result:
other: of 0.05 mg/L C18 DAQ in river water + 5g/L adapted sediment
Transformation products:
not measured
Volatile metabolites:
no
Residues:
no
Details on results:
Degradation of 14C-C18 DAQ was slow in river water containing low levels of suspended solids (<25mg/L). However, in the presence of sediments (5g/L), degradation was significantly higher and again followed apparent first-order kinetics over the concentration range tested (50 and 500 µg/L).
Degradation rates for C18 DAQ did not decrease at the level of sediment present, even though 99% of the DSDMAC initially added was bound to particulate matter. After an S-shaped lag phase, degradation was directly proportional to C18 DAQ concentration with estimated half-lives for mineralization of ~5d. The lag phase observed presumably represents a period of microbial growth during which time C18 DAQ degradation rates are a function of both C18 DAQ concentration and biomass levels.
Validity criteria fulfilled:
not applicable
Conclusions:
In river water alone (50 mg/L suspended solids, < 25 mg/L sediment), degradation was low (8% of 0.05 mg/L and 19% of 0.5 mg/L in 28 days). After 63 days the degradation results were not much higher (11% and 22% respectively), and the degradation curve ends in a plateau, suggesting that degradation will not continue. However, in the presence of sediments (5g/L), degradation was significantly higher (43% of 0.05 mg/L after 28 days and 65% after 63 days)
Executive summary:

A study was conducted to determine the kinetics of ultimate biodegradation (mineralization of CO2) of the test substance, C18 DAQ in freshwater/sediment systems. Low concentrations of test substance (1-100 µg/L), in duplicate or triplicate with river water or river water/sediment suspensions, were used. Low concentrations of test material (1-100 µg/L), which approximate actual environmental levels, were added in duplicate or triplicate to 2-liter Erlenmeyer flasks containing 1 liter (final volume) of water or water/sediment suspensions. Flasks were incubated at 24 ± 2°C with agitation and aerated with CO2-free air in closed, flow-through shake-flask system. Off-gases were bubbled through appropriate CO2 sorbents to remove 14CO2 produced during biodegradation. At various intervals, samples also were taken from the aqueous phase and acidified (<pH2) to release 14CO2 trapped as carbonates and bicarbonates. The amount of 14CO2 present in the various sorbents was determined by LSC and converted to a cumulative percentage of the total amount of 14C-activity initially added, based on LSC of zero-time samples. Sterile controls were included to normalize for 14C-activity arising from nonbiological sources. In river water alone (50 mg/L suspended solids, < 25 mg/L sediment), degradation was low (8% of 0.05 mg/L and 19% of 0.5 mg/L in 28 days). After 63 days the degradation results were not much higher (11% and 22% respectively), and the degradation curve ends in a plateau, suggesting that degradation will not continue. However, in the presence of sediments (5g/L), degradation was significantly higher (43% of 0.05 mg/L after 28 days and 65% after 63 days) (Larson, 1983).

Endpoint:
biodegradation in water and sediment: simulation testing, other
Remarks:
(river water and water spiked with sediment)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Published study not performed according to standard guidelines or GLP
Remarks:
(study also referred in the EU RAR report available on DODMAC (CAS 107-64-2)
Justification for type of information:
Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.
Reason / purpose for cross-reference:
read-across source
Qualifier:
no guideline followed
Principles of method if other than guideline:
The kinetics of ultimate biodegradation (mineralization of CO2) of the test substance was studied in freshwater/sediment systems.
GLP compliance:
no
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water / sediment: freshwater
Details on source and properties of surface water:
River water and sediment samples were collected form two rivers (Rapid Creek, Rapid City, SD; Ohio River, Cincinnati, OH) at locations 0.5 to 1.0 mile below the discharge of municipal wastewater treatment plants. Rapid Creek is a slow-flowing mountainn stream with a low wastewater dilution factor whereas the Ohio River is a rapidly flowing major river with high wastewater dilution factor.
Details on source and properties of sediment:
River water and sediment samples were collected form two rivers (Rapid Creek, Rapid City, SD; Ohio River, Cincinnati, OH) at locations 0.5 to 1.0 mile below the discharge of municipal wastewater treatment plants. Rapid Creek is a slow-flowing mountainn stream with a low wastewater dilution factor whereas the Ohio River is a rapidly flowing major river with high wastewater dilution factor.

Sediment samples were analyzed for cation exchange capacity (CEC), pH, particle distribution, organic carbon, and suspended solids levels by standard procedures. They were tested on a dry wt basis.
Duration of test (contact time):
>= 28 - ca. 63 d
Initial conc.:
> 1 - < 100 µg/L
Based on:
other: 14CO2
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on study design:
Low concentrations of test material (1-100 µg/L), which approximate actual environmental levels, were added in duplicate or triplicate to 2-liter Erlenmeyer flasks containing 1 liter (final volume) of water or water/sediment suspensions. Flasks were incubated at 24 ± 2°C with agitation and aerated with CO2-free air in closed, flow-through shake-flask system. Off-gases were bubbled through appropriate CO2 sorbents to remove 14CO2 produced during biodegradation. At various intervals, samples also were taken from the aqueous phase and acidified (
Key result
% Degr.:
8
Parameter:
radiochem. meas.
Remarks:
14CO2
Sampling time:
28 d
Remarks on result:
other: of 0.05 mg/LC18 DAQ in river water alone
Key result
% Degr.:
19
Parameter:
radiochem. meas.
Remarks:
14CO2
Sampling time:
28 d
Remarks on result:
other: of 0.5 mg/L C18 DAQ in river water alone
Key result
% Degr.:
11
Parameter:
radiochem. meas.
Remarks:
14CO2
Sampling time:
63 d
Remarks on result:
other: of 0.05 mg/L C18 DAQ in river water alone
Key result
% Degr.:
22
Parameter:
radiochem. meas.
Remarks:
14CO2
Sampling time:
63 d
Remarks on result:
other: of 0.5 mg/L C18 DAQ in river water alone
Key result
% Degr.:
43
Parameter:
radiochem. meas.
Remarks:
14CO2
Sampling time:
28 d
Remarks on result:
other: of 0.05 mg/L C18 DAQ in river water + 5g/L adapted sediment
Key result
% Degr.:
65
Parameter:
radiochem. meas.
Sampling time:
63 d
Remarks on result:
other: of 0.05 mg/L C18 DAQ in river water + 5g/L adapted sediment
Transformation products:
not measured
Volatile metabolites:
no
Residues:
no
Details on results:
Degradation of 14C-C18 DAQ was slow in river water containing low levels of suspended solids (<25mg/L). However, in the presence of sediments (5g/L), degradation was significantly higher and again followed apparent first-order kinetics over the concentration range tested (50 and 500 µg/L).
Degradation rates for C18 DAQ did not decrease at the level of sediment present, even though 99% of the DSDMAC initially added was bound to particulate matter. After an S-shaped lag phase, degradation was directly proportional to C18 DAQ concentration with estimated half-lives for mineralization of ~5d. The lag phase observed presumably represents a period of microbial growth during which time C18 DAQ degradation rates are a function of both C18 DAQ concentration and biomass levels.
Validity criteria fulfilled:
not applicable
Conclusions:
In river water alone (50 mg/L suspended solids, < 25 mg/L sediment), degradation was low (8% of 0.05 mg/L and 19% of 0.5 mg/L in 28 days). After 63 days the degradation results were not much higher (11% and 22% respectively), and the degradation curve ends in a plateau, suggesting that degradation will not continue. However, in the presence of sediments (5g/L), degradation was significantly higher (43% of 0.05 mg/L after 28 days and 65% after 63 days)
Executive summary:

A study was conducted to determine the kinetics of ultimate biodegradation (mineralization of CO2) of the read across substance, C18 DAQ in freshwater/sediment systems. Low concentrations of test substance (1-100 µg/L), in duplicate or triplicate with river water or river water/sediment suspensions, were used. Low concentrations of test material (1-100 µg/L), which approximate actual environmental levels, were added in duplicate or triplicate to 2-liter Erlenmeyer flasks containing 1 liter (final volume) of water or water/sediment suspensions. Flasks were incubated at 24 ± 2°C with agitation and aerated with CO2-free air in closed, flow-through shake-flask system. Off-gases were bubbled through appropriate CO2 sorbents to remove 14CO2 produced during biodegradation. At various intervals, samples also were taken from the aqueous phase and acidified (<pH2) to release 14CO2 trapped as carbonates and bicarbonates. The amount of 14CO2 present in the various sorbents was determined by LSC and converted to a cumulative percentage of the total amount of 14C-activity initially added, based on LSC of zero-time samples. Sterile controls were included to normalize for 14C-activity arising from nonbiological sources. In river water alone (50 mg/L suspended solids, < 25 mg/L sediment), degradation was low (8% of 0.05 mg/L and 19% of 0.5 mg/L in 28 days). After 63 days the degradation results were not much higher (11% and 22% respectively), and the degradation curve ends in a plateau, suggesting that degradation will not continue. However, in the presence of sediments (5g/L), degradation was significantly higher (43% of 0.05 mg/L after 28 days and 65% after 63 days) (Larson, 1983).

Description of key information

Based on the available information, the test substance is expected to undergo complete removal (99.9%) in sewage treatment plants, where about 4% will removed by adsorption to the sludge and the remaining 96% is considered to be biodegraded. The biodegradation in the sediment on the other hand is expected to be slow. In the absence of good quality studies, the half-life has been extrapolated at 5000 days, considering the soil half-life of 500 days and a factor 10 as per the ECHA R.16 guidance.

Key value for chemical safety assessment

Half-life in freshwater sediment:
5 000 d

Additional information

No simulation biodegradation study in water or sediment could be located on C12-18 DAQ. Therefore, the endpoint has been assessed based on available weight of evidence from sewage treatment simulation testing and biodegradation screening test results with C12-18 DAQ together with the literature evidence on read across substance C18 DAQ, which has been exhaustively reviewed in the frame of an European Risk Assessment (RAR) published in 2002 by German Authorities. Both the test and read across substances are di-alkyl dimethyl ammonium chloride compounds. C18 DAQ is structurally the same but only differs in containing a longer average alkyl chain length. Longer alkyl chains are expected to be less bioavailable which likely results in lower biodegradation rates. Using the available biodegradation and half-life results available for C16-18 (and C18) DAQ for read-across to C12-18 DAQ is therefore considered as a worst case approach (Refer to the read across justification for details).

Sewage treatment simulation testing:

A study was conducted to determine the biodegradation of the test substance, C12-18 DAQ (99.6% active) using a continuous activated sludge (CAS) test, according to OECD guideline 303 A, EU method C10 and ISO guideline 11733, in compliance with GLP. The test substance spiked at a nominal influent concentration of 5.0 mg/L (3.8 mg/L carbon; calculated) was exposed to micro-organisms maintained by addition of domestic wastewater for a period of 42 d and included a control fed with domestic wastewater only. Based on non-purgeable organic carbon (NPOC)), the mean organic carbon removal between Day 25 and 42 (14 measurements) was 145 ± 11% (95% confidence interval). The biodegradation percentage in excess of 100% shows that the removal of organic compounds present in the domestic wastewater are removed better when the test substance is present in the influent. However, as removal percentages calculated with NPOC values are not very accurate, an accurate assessment of the removal of the test substance was therefore established with specific analyses using LC-MS/MS. The LC-MS/MS method for the determination of the test substance was satisfactory regarding the linearity, repeatability of the injections, limit of quantification (LOQ), precision and specificity. Based on specific analysis of the test compound, the mean removal percentage from Day 38 to 42 was 99.9%. Test substance in the mixed liquid suspended solids of the reactor sampled on Day 41 and 42 were 6.0 and 9.7 mg/L, respectively. The mean removal of the test substance from the influent through adsorption onto sludge assessed with two samples was therefore 4.0% demonstrating that, it is primarily removed by biodegradation. In the study, the test substance biodegraded 96% in properly operating conventional biological wastewater treatment plants. Under the study conditions, the test substance was almost completely removed from wastewater in conventional biological wastewater treatment plants primarily by biodegradation (Van Ginkel, 2010).  

Surface water/sediment simulation testing:

A study was conducted to determine the kinetics of ultimate biodegradation (mineralization to CO2, H2O and NH3) of the read across substance,C18 DAQin freshwater/sediment systems. Low concentrations of test substance (1-100 µg/L), in duplicate or triplicate with river water or river water/sediment suspensions, were used. Low concentrations of test material (1-100 µg/L), which approximate actual environmental levels, were added in duplicate or triplicate to 2-liter Erlenmeyer flasks containing 1 liter (final volume) of water or water/sediment suspensions. Flasks were incubated at 24 ± 2°C with agitation and aerated with CO2-free air in closed, flow-through shake-flask system. Off-gases were bubbled through appropriate CO2 sorbents to remove 14CO2produced during biodegradation. At various intervals, samples were taken from the aqueous phase and acidified (<pH2) to release 14CO2trapped as carbonates and bicarbonates. The amount of 14CO2present in the various sorbents was determined by LSC and converted to a cumulative percentage of the total amount of 14C-activity initially added, based on LSC of zero-time samples. Sterile controls were included to normalize for 14C-activity arising from nonbiological sources. In river water alone (50 mg/L suspended solids, < 25 mg/L sediment), degradation was low (8% of 0.05 mg/L and 19% of 0.5 mg/L in 28 days). After 63 days the degradation results were not much higher (11% and 22% respectively), and the degradation curve ends in a plateau, suggesting that degradation will not continue. However, in the presence of sediments (5 g/L), degradation was significantly higher (43% of 0.05 mg/L after 28 days and 65% after 63 days) (Larson, 1983).

Further, the available monitoring data with the read across substances reveal that biodegradation in environmental sediments is lower. Hellmann (1995) found an increase of the C16-18 DAQ concentration at high river flows. As the causes whirling of sediments and rinsing of agricultural soil during strong rainfalls are stated. These results indicate that C16-18 DAQ adsorbed onto sediments is not or very slowly degraded. A degradation rate could not be derived from the monitoring data. Therefore, analogously to the degradation in soil, a half-life of 500 days (k = 1.4 . 10-3/d) was extrapolated for the aerobic sediment layer. There is no information available that the test substance or read across substances can be degraded under anaerobic conditions.

Based on the available information, the below conclusions could be drawn for the test substance:

  •  The water-soluble fraction is readily biodegradable and completely mineralized when not hampered by toxicity to the inoculum
  • The biodegradation rate is due to the lowered bioavailability of the longer alkyl chain constituents reduced.
  • The fraction sorbed is slowly released and immediately degraded, the substance is thus either sorbed or biodegraded.

Taking into consideration the above information, the test substance is considered to be inherently biodegradable, where the water-soluble fraction (ca. 80%) is considered to be degraded or not persistent and the poorly water soluble fraction (ca. 20%) is sorbed and hence likely to persist as indicated by the evidence available on C18 DAQ. However, in the absence of experimental data due to underlying technical challenges with OECD 308 test, asame classification for C12-18DAQ is applied as for C18DAQ, which is ‘Persistent (P) or very Persistent (vP)’ persistent in sediment compartment (see section 2.3 of IUCLID or section 8 of the CSR).

For the purpose of risk assessment, the half-life in sediment as a worst-case has been derived analogously to soil at 5000 days, considering the soil half-life of 500 days and using a factor 10 as per the ECHA R.16 guidance.