D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C18-C18 (unsaturated) acyl] derivs.

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: sewage treatment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-17 to 2015-07-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was carried out according GLP and has no defiencies

Qualifier:

according to guideline

Guideline:

OECD Guideline 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units)

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Test System
Non-adapted activated sludge

Reasons for the selection of the study system
Activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly industrial chemical waste.

Source
Municipal sewage treatment plant, 31137 Hildesheim, Germany

Pretreatment
The activated sludge was washed once with tap water and maintained in an aerobic condition by aeration at room temperature until use. Based on the dry matter, the starting concentration was adjusted to 2.5 ± 0.5 g/L DW (nominal value).

Duration of test (contact time):

28 d

Initial conc.:

895 other: kBq/L (test start until day 15)

Based on:

test mat.

Initial conc.:

384.3 other: µg/L (test start until day 15)

Based on:

test mat.

Initial conc.:

440 other: KBq/L (day 15 until test end)

Based on:

test mat.

Initial conc.:

192.1 other: µg/L (day 15 until test end)

Based on:

test mat.

Details on study design:

Duration
After an acclimatisation period of 3 days the test item dosage was started. The study was terminated after 28 days corresponding to 25 days of test item application.
Test design
The study was performed in a closed gas flow-through system connected to a series of base traps to capture evolved 14CO2. The test item was continuously pumped through the system, and the headspace of the test vessel was continuously purged with air at a rate sufficient to maintain the activated sludge in an aerobic condition. The evolved 14CO2 was measured directly in the base traps. The activated sludge settled in the setting tank was pumped back into the aeration vessel by an airlift pump.
Set-up
The system was started with an activated sludge content of 3.2 g DW/L (test item unit). The test volume in the aeration vessel was 372 mL, the total volume of activated sludge in the aeration vessel and settling tank was 550 mL. The complete system, consisting of influent dosage system, aeration vessel, settling vessel, effluent vessel and 14CO2 traps, was sealed. All exhausted gas was collected in sodium hydroxide traps (2 mol/L), which were connected to the effluent vessel. To avoid any losses of 14CO2, the system was not opened for re-suspension of activated sludge, samplings and emptying of the effluent vessel. Activated sludge and effluent samples were withdrawn from sampling ports with a valve. The effluent vessel was emptied by suction of the effluent from the closed system. 4mL H3PO4 (conc.) were given into the effluent vessel at the start of each 24 h period. To strip off the 14CO2, the acidified effluent was purged continuously with the exit gas from the aeration chamber and airlift pump.
To re-suspend activated sludge adhering to the walls of the aeration vessel, a magnetic bar was placed inside the aeration vessel permanently. For re-suspension, the magnetic bar was swiped over the glass walls with a second magnetic bar from outside the vessel.
Incubation
Incubation took place in an activated sludge unit at 20 - 25°C. The aeration vessel was aerated continuously. Airflow was adjusted to maintain an oxygen level of at least 2 mg/L and ensured by strong bubbling in the aeration vessel.
Sludge retention time (SRT)
8 d, 1/8 of the volume of the activated sludge in the aeration vessel was removed daily. The complete volume of about 46.5 mL was removed once per day directly from the aeration vessel.
Steady-state phase
After a period, in which the system had stabilized and the removal of DOC of the organic medium was efficient (> 80 %), the test item solution was dosed with a syringe pump into the aeration vessel of the test item unit.
Throughout the study, the sludge adhering to the walls of the activated sludge units was re-suspended at least daily. Regularly all tubes and tubing were checked and cleaned to prevent a growth of biofilm as far as possible.
The rate of ultimate biodegradation of the organic medium was checked by determination of the DOC in the influents and effluents in regular time intervals during this phase of the test. The elimination and mineralisation of the test item was determined by LSC and LC-FSA.
Completion of the test
The study was completed after reaching a sufficient degree of elimination / mineralisation and the plateau.
Replicates
One replicate
Water
Tap water for the preparation of organic medium
Deionised water for the preparation of the synthetic sewage stock solutions
Organic medium
Domestic sewage with a DOC of approx. 100 mg/L was used as an organic medium. If necessary, the DOC was adjusted with tap water or synthetic sewage to 100 mg C/L. The organic medium was prepared freshly at least every 7 days. The DOC of each new batch of organic medium was determined.
Domestic sewage
Domestic sewage was collected from the overflow channel of the primary sedimentation tank, free of coarse particles. The DOC and pH value were measured from each batch. Each batch was stored at about 4°C to reduce microbial activity and used for a maximum of 7 days. Domestic sewage of the same source as for activated sludge was used.
Synthetic sewage
Synthetic sewage was prepared according to OECD 303 A and used for DOC adjustment of domestic sewage if necessary. The composition of the synthetic sewage was as follows:
Test temperature 20 - 25°C
pH value 7.5 ± 0.5
Initial dry weight content 3.2 g/L
Mean hydraulic retention time
6 h; The total influent flow of 62 mL/h was maintained by dosing the test item stock solution and organic medium in appropriate amounts. Both solutions were pumped directly into the aeration vessels.
Influent flow
Organic medium: 62.5 mL/h
Test item solution: 50 µL/h (until day 15)
25 µL/h (from day 15)
Hydraulic parameters
Hydraulic Retention Time
[h] Influent Rate

[mL/h] Sludge Retention Time
[d] Wastage Sludge Rate
[mL/d]
6 62 8 46.5

Application
The test item was applied continuously to the test item unit by a syringe pump. As the test item stock solution was added directly to the activated sludge unit, no biodegradation may have occurred before it had reached the aeration vessel.

Equipment
Test vessels
Activated sludge units consisting of an aeration vessel (approx. 500 mL) and a separator (approx. 250 mL)
Temperature
Temperature data logger with sensor device 147 T, TESTO
pH-value
HQ 40d multi, HACHLANGE and Multi 350i, WTW
Pumps
Precision syringe pump, KD SCIENTIFIC and Peristaltic pump, ISMATEC
Aeration
Medo Kompressor, REBIE
TOC
Photometer DR3900, HACHLANGE and TOC Analyser Multi N/C 3100, ANALYTIK JENA
Balances
Balance, KERN and Analytical Balance, SARTORIUS
Dry weight
Drying oven T6, THERMO SCIENTIFIC
Pipette
Multipette Xstream, EPPENDORF
14CO2 Trap
99.7 % NaOH, Sodium hydroxide, VWR
Acidification of effluent
85 % H3PO4, ROTH
Ultrapure water
Ultra pure water system, BARNSTEAD

Type and Frequency of Measurements
(exclusive test item analysis)
Parameter Frequency
Influent volumes : At least twice per week
DOC of the effluents: At least 3 x per week
DOC of the organic media (influent): Every new batch
Temperature, pH-value in the aeration vessel (activated sludge): At least weekly, depended on the stability of the system, measured in the surplus sludge
Suspended solids (activated sludge): Weekdays

LSC and LC-FSA Analysis
Matrix Frequency
LSC LC-FSA
Influent
(Test item
stock solution) Every new batch -—
Effluent Daily At least weekdays
Activated
sludge Daily —
NaOH traps Daily —
— not measured
 
Sampling Procedure
To avoid any losses of CO2 from the headspace as far as possible, all samplings were done without opening the respective vessels.
Effluent
Composite sampling (24h) of the effluent was carried out. The complete effluent was purged from the effluent vessel without opening the vessel. Aliquots of the effluent were measured by LSC. Furthermore, 1L effluent was enriched and measured via LC-FSA.
Activated sludge
Analysis of the activated sludge was done from the surplus sludge. The complete volume of the surplus sludge (46.5 mL) was removed once per day by a syringe.
NaOH-Traps
The first NaOH Trap was removed daily, and a new trap was connected to the last one.

Test performance:

as given in the Guideline

% Degr.:

99

Parameter:

radiochem. meas.

Sampling time:

25 d

Remarks on result:

other: Elimination

Mineralization rate (in CO2):

36 other: %

Transformation products:

no

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

no

Details on results:

Temperature, pH-Value and Suspended Solids
The operational parameters pH-value, temperature and suspended solids in the activated sludge unit are given below.
The pH-value in the activated sludge was in the required range of 7.5 ± 0.5, except on one day the pH was slightly below 7.0. The decrease of the pH value was associated with the volume of synthetic sewage needed to adjust the organic medium.
The temperature in the aeration vessels was in the range of 20 - 25°C throughout the study.
Activated sludge with a dry weight of 3.20 g/L was used for the start of the activated sludge unit. Within the first week of the study the dry weight settled at 2.8 /L and remained in the range 2.9 – 2.3 g/L until the end of the study.

pH-value and Temperature in the sampled activated sludge
Study day Test Item Unit
Date pH temperature [°C]
-2 2015-06-17 7.47 21.9
5 2015-06-24 7.22 22.2
11 2015-06-30 7.19 22.4
19 2015-07-08 6.86 22.4
21 2015-07-10 7.08 21.8
25 2015-07-14 7.16 21.9

Suspended Solids
Study Day of Suspended solids
Day test item Date
application [g/L]
2 0 2015-06-19 3.2
5 3 2015-06-22 3.1
6 4 2015-06-23 2.8
7 5 2015-06-24 2.8
8 6 2015-06-25 2.7
9 7 2015-06-26 2.5
12 10 2015-06-29 2.7
13 11 2015-06-30 2.3
14 12 2015-07-01 2.8
15 13 2015-07-02 2.9
16 14 2015-07-03 2.7
19 17 2015-07-06 2.5
20 18 2015-07-07 2.9
21 19 2015-07-08 2.6
22 20 2015-07-09 2.5
23 21 2015-07-10 2.5
26 24 2015-07-13 2.3
27 25 2015-07-14 2.3

Biodegradation of Organic Medium (DOC)
The biodegradation of the organic medium was calculated from the difference of influent and effluent DOC. DOC values of the effluent were determined from composite effluent samples taken over a period of 24 h. Therefore, influent DOC and effluent DOC of the following 24 h were used for the calculation of elimination.
During acclimatisation, the activated sludge units had stabilized and biodegradation of the DOC of the organic medium became efficient (> 80 %), (Table 11).
Biodegradation > 80 % was already achieved after two days. Then it decreased slightly and with the exception of day 6 oscillated around 80 % (77 – 84 %). This was probably due to variations in the quality and composition of the organic medium. The decrease had no influence on the biodegradation efficiency, the unit worked stable and efficiently throughout the test (Figure 2).

DOC Concentration and Degradation
Study DOC
Day Date [mg C/L] Elimination
influent effluent [%]
0 2015-06-16 109.9 - -
1 2015-06-17 109.9 12.8 88.39
3 2015-06-19 109.9 15.7 85.71
6 2015-06-22 90.5 30.0 72.70
8 2015-06-24 94.9 19.9 78.02
10 2015-06-26 94.9 15.4 83.77
13 2015-06-29 94.9 21.4 77.44
14 2015-06-30 107.9 21.3 77.55
17 2015-07-03 107.9 25.7 76.18
20 2015-07-06 107.9 23.4 78.31
22 2015-07-08 101.9 22.8 78.87
24 2015-07-10 101.9 18.0 82.33
27 2015-07-13 101.9 21.3 79.09
 
Elimination, Mineralisation and Biodegradation of the Test Item
Stability of the Test Item (Radiochemical Purity)
The radiochemical purity of the test item was determined directly after preparation of the working solution and prior to the start of the study by HPLC-FSA method. The radiochemical purity was 99.5 % prior to the start of the study.

Stability and Concentration of the Test Item Stock Solution (non-GLP)
Before the start of exposure, losses of the test item stock solution to the application equipment (syringe, tubing) were assessed over a period of 7 days. No significant loss of activity was observed.

Test Item Stock Solution Stability during Application
Percentage of Day 0 Value
Day
[%]
1 97.2
2 87.5
3 100.5
7 99.5

Concentration of the Test Item Stock Solution (Definitive Study)
A new stock solution was prepared at the start of exposure and after 12 days. The concentration of the new batch of the stock solution was analysed by LSC. The recovery of the test item in the stock solution was 97.9 %. This value was used for the calculation of the influent concentration.

Test Item Concentrations in the Stock Solutions
Day of Nominal Value Stock Solution Activity Percentage of Nominal Value
test item Date
application [kBq/mL] [µg/mL] [kBq/mL] [µg/mL] [%]
0 2015-06-19 1110 476.4 1087.1 466.6 97.9
12 2015-07-01 1086.2 466.2 97.9


Influent Concentration
The initial influent concentration of 879.1 kBq/L (377 µg/L, actual measured values) was chosen to facilitate the determination of ≤ 0.15 % N-[1-14C]Oleoyl-N-methylglucamine (corresponding to 1.3 kBq/L) in the effluent by LC-FSA (LOQ = 1.02 kBq/L, 0.44 µg/L). On the other hand the estimated maximum influent concentration in treatment plants is about 50 µg/L.
As the actual measured effluent concentration was distinctly above the LOQ and increased within the duration of the study, the high influent concentration was not essential to analyse N-[1-14C]Oleoyl-N-methylglucamine in the effluent reliably.
Therefore, the influent concentration was reduced to approach the estimated maximum influent concentration in treatment plants and to investigate the influence of the influent concentration on the degree of elimination and biodegradation.
The application was started with an influent concentration of 879 kBq/L (377 µg/L) corresponding to a daily influent load of 1308 kBq/d. At day 15 of test item application the influent concentration was reduced to 440 kBq/L (188.5 µg/L), corresponding to a daily influent load of 654 kBq/d.

Elimination of N-[1-14C]Oleoyl-N-methylglucamine
After the activated sludge units had stabilized, the dosage of the test item was started (June 19, 2015). Influent (stock solution) and effluent samples were analysed by LSC and LC-FSA after enrichment.
The elimination of N-[1-14C]Oleoyl-N-methylglucamine and total 14C-elimination was calculated based on influent and effluent activities.
Until day 12 of test item application, the elimination was in the range 98.8 – 99.9 %. Between day 12 and day 14 the elimination decreased slightly but increased immediately after reduction of the influent concentration and was in the range 98.1 – 99.2 % until the end of the study. The influence of the influent concentration on the degree of elimination was negligible.
A elimination of N-[1-14C]Oleoyl-N-methylglucamine of 99 % was calculated for the tes item application of 25 days.
The total 14C-elimination was in the range 53.1 – 83.3 (mean 75.9 %).

Effluent Concentration and Elimination of Total 14C Activity and N-[1-14C]Oleoyl-N-methylglucamine

Influent Concentration Effluent Concentration Elimination
Day of Test Item Appli-cation Date N-[1-14C] Oleoyl-N-methyl-glucamine

SI Total 14C Activity

SE N-[1-14C] Oleoyl-N-methyl-glucamine

SET Total 14C Activity

ETA N-[1-14C] Oleoyl-N-methyl-glucamine

ETI
[kBq/L] [kBq/L] [kBq/L] [%] [%]
2 21.06.2015 879 154.40 — 82.44 —
3 22.06.2015 154.39 0.85 82.44 99.90
4 23.06.2015 246.96 2.07 71.91 99.76
5 24.06.2015 162.31 1.43 81.54 99.84
6 25.06.2015 161.00 1.57 81.69 99.82
7 26.06.2015 159.11 2.35 81.90 99.73
8 27.06.2015 198.93 — 77.37 —
9 28.06.2015 178.96 — 79.64 —
10 29.06.2015 252.18 7.40 71.32 99.16
11 30.06.2015 250.68 7.82 71.49 99.11
12 01.07.2015 224.26 10.17 74.49 98.84
13 02.07.2015 412.69 20.10 53.06 97.71
14 03.07.2015 334.05 16.43 62.00 98.13
15 04.07.2015 440 266.91 — 69.64 —
16 05.07.2015 174.29 — 60.35 —
17 06.07.2015 88.62 5.98 79.84 98.64
18 07.07.2015 86.71 3.44 80.27 99.22
19 08.07.2015 106.59 7.20 75.75 98.36
20 09.07.2015 86.50 4.60 80.32 98.95
21 10.07.2015 81.30 6.87 81.50 98.44
22 11.07.2015 78.49 5.41 82.15 98.77
23 12.07.2015 73.58 5.33 83.26 98.79
24 13.07.2015 77.62 7.14 82.34 98.38
25 14.07.2015 112.14 8.48 74.49 98.07
— not measured italic values not included in statistical evaluation

N-[1-14C]Oleoyl-N-methylglucamine in the Activated Sludge Phase
Samples of the activated sludge from the aeration vessel were taken each day. The percentage load of 14C activity in the activated sludge phase was calculated based on the total influent load of N-[1-14C]Oleoyl-N-methylglucamine and the total 14C load in the activated sludge.

N-[1-14C]Oleoyl-N-methylglucamine in the Activated Sludge Phase
Day of
test item application Date Total Influent Load

TLI 14C Activity on Sludge

SA Total Sludge Load1)

LA Percentage of Total Influent Load
A
[kBq] [kBq/mL] [kBq] [%]
2 21.06.2015 1308.2 0.43 216.7 16.6
3 22.06.2015 2616.3 0.53 265.3 10.1
4 23.06.2015 3924.5 0.78 389.5 9.9
5 24.06.2015 5123.62) 0.79 392.9 7.7
6 25.06.2015 6431.8 0.97 484.2 7.5
7 26.06.2015 7740.0 1.07 535.5 6.9
8 27.06.2015 9048.1 1.11 553.9 6.1
9 28.06.2015 10356.3 1.09 545.6 5.3
10 29.06.2015 11664.5 1.03 517.4 4.4
11 30.06.2015 12972.6 0.89 444.0 3.4
12 01.07.2015 14280.8 0.75 373.7 2.6
13 02.07.2015 15588.9 1.41 704.5 4.5
14 03.07.2015 16897.1 1.15 572.8 3.4
15 04.07.2015 18205.3 0.90 451.9 2.5
16 05.07.2015 18859.4 0.89 444.2 2.4
17 06.07.2015 19513.4 0.62 308.7 1.6
18 07.07.2015 20167.5 0.81 406.7 2.0
19 08.07.2015 20821.6 0.80 399.4 1.9
20 09.07.2015 21475.7 0.71 356.7 1.7
21 10.07.2015 22129.8 0.74 370.5 1.7
22 11.07.2015 22783.8 0.66 329.7 1.4
23 12.07.2015 23437.9 0.46 228.0 1.0
24 13.07.2015 24092.0 0.36 180.9 0.8
25 14.07.2015 24746.1 0.37 184.2 0.7
1) calculated with an activated sludge volume of 500 mL
2) only 22 h influent flow

Mineralisation and Ultimate Biodegradation of N-[1-14C]Oleoyl-N-methylglucamine
The mineralisation was calculated based on the influent load and the total load of the NaOH traps.
The 14CO2 activity of the NaOH traps was determined daily.
The influent load was corrected by the daily removed 14C load of the surplus sludge solution, as this fraction was not accessible for mineralisation.
The mineralisation was in the range 28.6 % – 38.3 % and the mean value was 36.1 %.

Mineralisation of N-[1-14C]Oleoyl-N-methylglucamine during the Plateau Phase
Day of
test item application Date Sum Influent Load
TLI Sum Surplus Load
TLS Load NaOH Traps
LDT Sum Traps Load
TLT Mineralisation

M
[kBq] [kBq] [kBq/d] [kBq] [%]
2 21.06.2015 1308.2 30.3 374 374 28.6
3 22.06.2015 2616.3 63.6 465 839 32.4
4 23.06.2015 3924.5 113.0 522 1361 35.3
5 24.06.2015 5123.6 158.4 472 1833 36.6
6 25.06.2015 6431.8 211.6 425 2258 36.0
7 26.06.2015 7740.0 271.9 530 2788 37.0
8 27.06.2015 9048.1 335.4 577 3366 38.3
9 28.06.2015 10356.3 399.9 444 3810 38.0
10 29.06.2015 11664.5 463.2 357 4167 37.0
11 30.06.2015 12972.6 517.5 372 4539 36.3
12 01.07.2015 14280.8 565.4 302 4841 35.2
13 02.07.2015 15588.9 655.2 501 5342 35.6
14 03.07.2015 16897.1 722.4 497 5839 36.0
15 04.07.2015 18205.3 777.3 473 6312 36.1
16 05.07.2015 18859.4 824.2 406 6718 37.2
17 06.07.2015 19513.4 856.7 252 6970 37.3
18 07.07.2015 20167.5 898.6 223 7193 37.2
19 08.07.2015 20821.6 942.5 256 7449 37.4
20 09.07.2015 21475.7 978.9 193 7642 37.2
21 10.07.2015 22129.8 1017.3 177 7819 37.0
22 11.07.2015 22783.8 1052.1 194 8014 36.8
23 12.07.2015 23437.9 1077.5 156 8170 36.5
24 13.07.2015 24092.0 1099.0 131 8300 36.1
25 14.07.2015 24746.1 1120.6 167 8468 35.8

The biodegradation was evaluated based on the composition of the 14C-activity in the effluent.
About 24 % of the applied radioactivity were determined in the effluent. A maximum of 9.2 % (mean 4.6 %) of the 14C-activity in the effluent were N-[1-14C]Oleoyl-N-methylglucamine and a further mean of 1.8 % (0.8 % – 4.4 %) of the 14C-activity were extractable from the effluent.
This 1.8 % might be associated with degradation products. However, the respective LC-FSA chromatograms show no further distinct peaks (no detection of transformation products) but an increased background activity.
The remaining 14C-activty (≥ 88 % of the 14C-activity in the effluent) was not extractable from the effluent.

From the absence of distinct degradation products and the low amount of extractable 14C-activty it can be concluded, that the degradation of N-[1-14C]Oleoyl-N-methylglucamine is almost ultimate and that the main amount of 14C activity in the effluent is 14C associated with biomass, fragments of bacteria biomass and small molecules excreted by the bacteria.

Consequently the ultimate biodegradation was calculated from the influent concentration and the extractable 14C-activity in the effluent measured by LC-FSA. Furthermore is was assumed that the composition of 14C-activity in the activated sludge and activated sludge liquid phase is comparable to the 14C-activity in the effluent.

The ultimate degradation was in the range 97.2 – 99.6 %, the mean value was 98.5 %.

Composition of 14C Activity in the Effluent
Effluent Concentration % of Total 14C Activity in Effluent
Day of Test Item Application Date Total 14C Activity

SE N-[1-14C] Oleoyl-N-methylgluca mine
Activity
SET Total Extractable 14C-Activity

SETE N-[1-14C] Oleoyl-N-methylglucamine*

TI Total Extractable 14C-Activity*

TA
[kBq/L] kBq/L kBq/L % %
3 22.06.2015 154.39 0.85 3.49 0.55 2.26
4 23.06.2015 246.96 2.07 3.92 0.84 1.59
5 24.06.2015 162.31 1.43 3.87 0.88 2.38
6 25.06.2015 161.00 1.57 4.05 0.97 2.52
7 26.06.2015 159.11 2.35 4.06 1.48 2.55
10 29.06.2015 252.18 7.40 10.90 2.93 4.32
11 30.06.2015 250.68 7.82 11.12 3.12 4.43
12 01.07.2015 224.26 10.17 13.34 4.53 5.95
13 02.07.2015 412.69 20.10 24.69 4.87 5.98
14 03.07.2015 334.05 16.43 21.34 4.92 6.39
17 06.07.2015 88.62 5.98 9.09 6.75 10.26
18 07.07.2015 86.71 3.44 5.08 3.97 5.86
19 08.07.2015 106.59 7.20 9.79 6.75 9.18
20 09.07.2015 86.50 4.60 6.78 5.32 7.84
21 10.07.2015 81.30 6.87 10.42 8.45 12.81
22 11.07.2015 78.49 5.41 6.71 6.89 8.55
23 12.07.2015 73.58 5.33 6.50 7.25 8.83
24 13.07.2015 77.62 7.14 8.12 9.19 10.46
25 14.07.2015 112.14 8.48 10.53 7.56 9.39
* total 14C activity in effluent = 100 %

Ultimate Degradation of N-[1-14C]Oleoyl-N-methylglucamine
Influent Concentration Effluent Concentration Ultimate Degradation
Day of Test Item Application Date N-[1-14C] Oleoyl-N-methyl-glucamine

SI Total Extractable 14C-Activity

SEX N-[1-14C] Oleoyl-N-methyl-glucamine

U
kBq/L kBq/L %
3 22.06.2015 879 3.49 99.6
4 23.06.2015 3.92 99.6
5 24.06.2015 3.87 99.6
6 25.06.2015 4.05 99.5
7 26.06.2015 4.06 99.5
10 29.06.2015 10.90 98.8
11 30.06.2015 11.12 98.7
12 01.07.2015 13.34 98.5
13 02.07.2015 24.69 97.2
14 03.07.2015 21.34 97.6
17 06.07.2015 440 9.09 97.9
18 07.07.2015 5.08 98.8
19 08.07.2015 9.79 97.8
20 09.07.2015 6.78 98.5
21 10.07.2015 10.42 97.6
22 11.07.2015 6.71 98.5
23 12.07.2015 6.50 98.5
24 13.07.2015 8.12 98.2
25 14.07.2015 10.53 97.6

Mass Balance of N-[1-14C]Oleoyl-N-methylglucamine
Based on the data obtained for N-[1-14C]Oleoyl-N-methylglucamine in the influent, total 14C activity in the effluent, 14C activity in activated sludge and 14CO2 in the NaOH traps, a mass balance was calculated for the total 14C activity. Throughout the test, a mass balance slightly below 70 % was achieved.
During the preliminary investigations and set-up of the test design, is was determined that N-[1-14C]Oleoyl-N-methylglucamine has a high tendency to adsorb on the surfaces of the test equipment. Therefore it is assumed that parts of the test item were lost by adsorption on the inner walls and tubing system of the activated sludge unit. This assumption was confirmed by the high 14C-activity determined in the washing water after cleaning of the activated sludge unit (non-GLP).

Mass Balance of N-[1-14C]Oleoyl-N-methylglucamine
Day of
test item application Date Total Influent Load
TLI Total Effluent Load
TLEU Sludge Load1)

LA + LL Surplus Sludge Load1)
TLS NaOH Traps Load
TLT Mass Balance

MB
[kBq] [kBq] [kBq] [kBq] [kBq] [%]
2 21.06.2015 1308.2 229.7 244.9 30.3 374 71.9
3 22.06.2015 2616.3 459.5 337.0 63.6 839 64.8
4 23.06.2015 3924.5 827.0 366.7 113.0 1361 71.3
5 24.06.2015 5123.6 1068.5 546.2 158.4 1833 68.5
6 25.06.2015 6431.8 1308.0 496.8 211.6 2258 67.0
7 26.06.2015 7740.0 1544.8 581.5 271.9 2788 67.2
8 27.06.2015 9048.1 1840.8 659.8 335.4 3366 68.2
9 28.06.2015 10356.3 2107.1 695.2 399.9 3810 67.2
10 29.06.2015 11664.5 2482.3 708.5 463.2 4167 66.4
11 30.06.2015 12972.6 2855.4 696.7 517.5 4539 65.2
12 01.07.2015 14280.8 3189.0 598.7 565.4 4841 63.6
13 02.07.2015 15588.9 3803.1 528.9 655.2 5342 68.7
14 03.07.2015 16897.1 4300.2 991.9 722.4 5839 68.2
15 04.07.2015 18205.3 4697.4 737.0 777.3 6312 67.8
16 05.07.2015 18205.3 4956.7 604.8 799.1 6718 67.8
17 06.07.2015 18859.4 5088.6 509.7 845.9 6970 68.7
18 07.07.2015 19513.4 5217.6 454.9 920.3 7193 67.8
19 08.07.2015 20167.5 5376.2 480.0 964.3 7449 68.0
20 09.07.2015 20821.6 5504.9 394.1 1000.6 7642 68.2
21 10.07.2015 21475.7 5625.9 417.7 1039.1 7819 67.4
22 11.07.2015 22129.8 5742.7 377.9 1073.8 8014 67.1
23 12.07.2015 22783.8 5852.2 278.6 1099.3 8170 66.5
24 13.07.2015 23437.9 5967.6 236.0 1120.8 8300 65.5
25 14.07.2015 24092.0 6134.5 237.2 1142.4 8468 64.7
1) Activated sludge solids and liquid phase

Statistical Evaluation of the Elimination, Mineralisation, Ultimate Degradation and Mass Balance of N-[1-14C]Oleoyl-N-methylglucamine

N-[1-14C]Oleoyl-N-methylglucamine
Total 14C Elimination Test Item Elimination Mineralisation Ultimate Degradation Recovery Rate /
Mass Balance
[%] [%] [%] [%] [%]
Range measured 53.1 - 83.3 98.1 - 99.9 28.6 - 38.3 97.2 – 99.6 63.6 - 71.9
Median 79.7 99.0 36.6 98.5 67.3
Mean 75.9 99.0 36.1 98.5 67.3
95 % CI 72.5 - 79.3 99.3 - 98.7 35.3 - 36.9 98.1 – 98.9 66.5 - 68.1
Number of case n 24 17 24 20 24
CI = Confidence Interval

Validity criteria fulfilled:

yes

Conclusions:

Based on the analysis of N-[1-14C]Oleoyl-N-methylglucamine in the effluent the mean elimination rate of the total influent concentration was calculated to be 99.0 % in a continuously operating activated sludge unit.
The mineralisation had reached a mean value of 36.1 %; the mass balance was determined to be 67.3 % on average.

Statistical Evaluation of the Elimination, Mineralisation, Ultimate Degradation
and Mass Balance of N-[1-14C]Oleoyl-N-methylglucamine

N-[1-14C]Oleoyl-N-methylglucamine

Total 14C Elimination Test Item Elimination Mineralisation Ultimate Degradation Recovery Rate /
Mass Balance
[%] [%] [%] [%] [%]
Range measured 53.1 - 83.3 98.1 - 99.9 28.6 - 38.3 97.2 – 99.6 63.6 - 71.9
Median 79.7 99.0 36.6 98.5 67.3
Mean 75.9 99.0 36.1 98.5 67.3
95 % CI 72.5 - 79.3 99.3 - 98.7 35.3 - 36.9 98.1 – 98.9 66.5 - 68.1
Number of case n 24 17 24 20 24
CI = Confidence Interval

Background Information on Data Evaluation
14C-Elimination
The mean 14C-Elimination is 75.9% in comparison to an N-[1-14C]Oleoyl-N-methylglucamine elimination of 99% and an ultimate biodegradation of 98.5%. It has to be taken into account that during biodegradation test item fragments are incorporated into biomass present in the effluent. Furthermore fragments of bacteria biomass containing 14C and organic microbial biosynthesis products are present in the effluent.
This explains why the 14C-Elimination is lower than the elimination and ultimate biodegradation of N-[1-14C]Oleoyl-N-methylglucamine.

Mineralisation
The mean mineralisation of the test item is 36.1% and much lower than ultimate biodegradation of 98.5%. One explanation is that test item fragments have been incorporated in the biomass of the microorganisms, which therefore are not available for mineralisation. A quantification of the amount of test item fragments incorporated in biomass is not feasible within the scope of the study. In addition, due to the complex construction of the activated sludge unit it is also very difficult to capture the evolved CO2 completely and on-line.

Ultimate Biodegradation
The ultimate biodegradation was evaluated based on the composition of the 14C-activity in the effluent.
About 24 % of the applied radioactivity were determined in the effluent. A maximum of 9.2 % (mean 4.6 %) of the 14C-activity in the effluent were N-[1-14C]Oleoyl-N-methylglucamine and a further mean of 1.8 % (0.8 % – 4.4 %) of the 14C-activity were extractable from the effluent.
This 1.8 % might be associated with degradation products. However, the respective LC-FSA chromatograms show no further distinct peaks (no detection of transformation products) but an increased background activity. The remaining 14C-activty (≥ 88 % of the 14C-activity in the effluent) was not extractable from the effluent.
From the absence of distinct degradation products and the low amount of extractable 14C-activty it can be concluded, that the degradation of N-[1-14C]Oleoyl-N-methylglucamine is almost ultimate and that the main amount of 14C-activity in the effluent is 14C associated with biomass, fragments of bacteria biomass and small molecules excreted by the bacteria (organic microbial biosynthesis products). Consequently the ultimate biodegradation was calculated from the influent concentration and the extractable 14C-activity in the effluent measured by LC-FSA.
Furthermore it was assumed that the composition of 14C-activity in the activated sludge and activated sludge liquid phase is comparable to the 14C-activity in the effluent. This results in a mean ultimate degradation was of 98.5 %.

Executive summary:

The elimination and mineralisation of the test itemN-[1-14C]Oleoyl-N-methylglucaminein an activated sludge unit was determined over a test period of 28 days according to OECD guideline 303 A. The study was conducted from2015-06-17 to 2015 -07 -14 at the Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany. The definite exposure phase was from2015-06-19 to 2015-07-14.

Principle of the Study

The elimination and mineralisation of N-[1-14C]Oleoyl-N-methylglucamineby aerobic microorganisms were determined in a continuously operating laboratory activated sludge unit simulating the activated sludge process. The degree of N-[1-14C]Oleoyl-N-methylglucamineelimination,¹⁴C-elimination, ultimate degradation, mineralisation and the total mass balance were assessed.

The study was performed in a closed gas flow-through activated sludge unit connected to a series of base traps to capture evolved¹⁴CO₂. The system was started with an activated sludge content of 3.2 g/L DW. After a short acclimatization period of the activated sludge (DOC elimination > 80 %), the application of N-[1-14C]Oleoyl-N-methylglucamine was initiated. Samples of activated sludge and effluent were taken in regular intervals. Emerging¹⁴CO₂was captured in NaOH traps. LSC and LC-FSA analysis was performed on the samples and traps to determine the elimination and mineralisation ofN-[1-14C]Oleoyl-N-methylglucamine.

Test Procedure and Results

During acclimatization, the activated sludge unit was inoculated with activated sludge. The DOC elimination of the organic medium reached a degradation rate > 80 % already after 1 day. The dosage of the test item was started after an acclimatization period of 3 days.

The nominal concentration of the test item stock solution was 1110 kBq/mL. The stock solution was dosed at a constant rate of 50 µl/h into the activated sludge unit until day 15, resulting in a nominal influent concentration of 895 kBq/L. Afterwards, the flow was decreased to 25 µl/h corresponding to 444 kBq/L.

The elimination ofN-[1-14C]Oleoyl-N-methylglucaminewas calculated based on the influent and effluent concentration ofN-[1-14C]Oleoyl-N-methylglucamine. Furthermore the total¹⁴C elimination was calculated based on the total activity ofN-[1-14C]Oleoyl-N-methyl-glucamine in the influent and the total¹⁴C-activity the effluent.

The mineralisation was calculated based on the total influent load and the total load of¹⁴C activity in NaOH traps.

The ultimate biodegradation was calculated from the influent concentration and the extractable¹⁴C-activity in the effluent measured by LC-FSA.

The mean elimination rate forN-[1-14C]Oleoyl-N-methylglucamine was calculated to be 99.0 %, whereas the total¹⁴C-elimination was 75.9 %. The mineralisation had a mean value of 36.1 %, the mean ultimate degradation was 98.5 % and the mass balance was determined to be 67.3 % on average.

Statistical Evaluation of the Elimination, Mineralisation, Ultimate Degradation and Mass Balance ofN-[1-14C]Oleoyl-N-methylglucamine

	N-[1-14C]Oleoyl-N-methylglucamine
	Total^14C Elimination	Test Item Elimination	Mineralisation	Ultimate Degradation	Recovery Rate / Mass Balance
	[%]	[%]	[%]	[%]	[%]
Range measured	53.1 - 83.3	98.1 - 99.9	28.6 - 38.3	97.2 – 99.6	63.6 - 71.9
Median	79.7	99.0	36.6	98.5	67.3
Mean	75.9	99.0	36.1	98.5	67.3
95 % CI	72.5 - 79.3	99.3 - 98.7	35.3 - 36.9	98.1 – 98.9	66.5 - 68.1
Number of case n	24	17	24	20	24

CI = Confidence Interval

Reference 2

Endpoint:

biodegradation in water: sewage treatment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-03-01 to 2016-05-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 314 C (Simulation Tests to Assess the Biodegradability of Chemicals in Wastewater. C: Mineralization and Transformation in Anaerobic Digester Sludge)

Version / remarks:

October 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Oxygen conditions:

anaerobic

Inoculum or test system:

digested sludge

Details on inoculum:

Test system
Digested sludge, not adapted

Source
Anaerobic digester of the municipal sewage treatment plant of the city of 31137 Hildesheim, Germany.

Date of receipt
2016-02-25

Pretreatment
After sampling the sludge was sieved through a 2 mm screen and the dry weight was determined. The dry weight was 24.8 g/L, adjustment of the dry weight was not necessary.

Duration of test (contact time):

62 d

Initial conc.:

2.33 other: kBq/Replicate

Based on:

test mat.

Initial conc.:

1 000 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CH4 evolution

CO2 evolution

radiochem. meas.

Details on study design:

Test Procedures
Application The digester sludge was filled in the replicates. Afterwards 735 µL of test item solution were pipetted directly in each replicate. The test solution was swiveled carefully to ensure a homogeneous distribution of the test item.
Afterwards, the replicates were purged with Nitrogen (≥ 15 s) to ensure anaerobic conditions.

Frequency of application The application was carried out once at test start.

Test duration 62 days (Abiotic control 63 days)

Test vessels 125 mL sealed gastight headspace flasks

Digester sludge amount 20 mL
per replicate

Dry sludge weight 23.25 g/L

Replicates As 14CO2 and 14CH4 were strongly associated with the biomass and were only completely released after acidification separate test replicates were prepared for each sampling.

The following number of replicates was prepared in total:
40 test item replicates
20 abiotic control replicates
20 control replicates

Volatile traps Crimped headspace bottles containing 30 mL 1 mol/L aqueous sodium hydroxide solution were used for 14CO2.
14C-methane was combusted to 14CO2 and trapped in further aqueous sodium hydroxide traps.
Two 14CO2 traps and two 14CH4 traps were used for each replicate.

Combustion system The combustion system for 14C-methane consisted of a tube furnace for oxidation of methane to CO2. Inside of the furnace, a glass tube filled with copper oxide as catalyst was placed which was connected on one side to the outlet of the last volatile trap for 14CO2 and on the other side to crimped bottles containing 30 mL 1 mol/L aqueous sodium hydroxide solution were used for trapping the 14CO2 coming from the furnace.
 
Incubation All replicates were incubated at 35 ± 3 °C in the dark. Anaerobic conditions were established and maintained by flushing the sealed gastight test vessels with moistened N2 after application.

Type and Frequency of Measurements and Observations
Sampling 2 test item replicates were sacrificed at each sampling time. Sampling for determination of mineralization (LSC analysis of NaOH traps after sampling of 14CO2 and 14CH4) was carried out on day 1, 2, 3, 6, 7, 10, 14, 20, 28, 37 and 62.

Evaluation
The amounts of residual 14C-activity and mineralisation products were given as % of applied initial concentration for each sampling time.

A mass balance was calculated in percentage of the initial applied radioactivity for each sampling time.

The amount of mineralised carbon was related to the applied amount of carbon at the beginning of the test.


Sampling of the abiotic replicates was done on day 63.

14CO2 / 14CH4 At sampling, the respective replicates were connected to the gas trapping system and were acidified with 1 mL phosphoric acid (85 %) to a pH < 2. Furthermore, 20 mL HPLC water were added to fluidize the digester sludge and to improve the release of 4CO2 and 14CH4.
Afterwards, the headspace gas was purged through the gas trapping system for at least 4 hours. Produced 14C-methane was combusted to 14CO2. The newly formed 14CO2 was trapped and analysed. 14CO2 in the traps was determined by LSC.

14C-Activity in test solution The residual 14C-activity in the digester sludge was determined by LSC. Therefore, the liquid phase was separated by centrifugation.

The residual 14-C activity associated with the sludge particles was determined by LSC after combustion.

Incubation temperature The incubation temperature was documented continuously.

Test performance:

Stability of the Test Item (Working Solution)
HPLC-FSA analysis of the working solution prior to experimental starting confirmed the stability of the test item (100 % radiochemical purity).

Dry weight of the Digester Sludge
The dry weight of the digested sludge was determined at test start prior to the preparation of the test replicates from the total sludge batch. Measurements during the study were done from the control replicates.

Dry Weight of the Digester Sludge
Exposure Dry weight
Day g/L
Test start 23.25
6 19.75
After test end (day 73) 19.46

Visual inspection of the control vessels revealed that parts of the digested sludge remained stuck to the inner vessel walls and were not captured for the dry weight determination. Therefore, the calculation of the total 14C-activity associated with the sludge particles was done with the determined sludge dry weight at test start.
The dry weight determination after the end of the incubation phase indicated that the dry weight remained constant during the incubation phase.

Compartment:

anaerobic sludge

% Recovery:

82.6

Remarks on result:

other:

Remarks:

The determination of the mass balance was affected by the high adsorption potential of N-[1-14C]Oleoyl-N-methylglucamine and the difficulties to transfer the digested sludge particles completely from the test vessels to the centrifuge tubes. Visual inspection of the test vessels revealed that a biofilm remained on the inner surface of the test vessels and the associated 14C-activity was therefore not captured for LSC measurements. Furthermore, N-[1-14C]Oleoyl-N-methylglucamine adsorbed on the test vessel walls was not measurable.

Key result

% Degr.:

54

Parameter:

CO2 evolution

Sampling time:

62 d

Key result

% Degr.:

5.5

Parameter:

CH4 evolution

Sampling time:

62 d

Transformation products:

not measured

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Details on results:

The adsorption of N-[1-14C]Oleoyl-N-methylglucamine on the sludge particles was high, on day 3 a maximum of 72.1 % of the applied radioactivity was determined on the sludge particles. Within the course of the study the amount of 14C-activity associated with the sludge particles decreased to 21 % on day 37 and remained at that range until test end (day 62).

The 14C-activity in the liquid phase remained < 10 % throughout the study duration and reached a maximum of 8.0 % on day 3.


14C-Activity in Digested Sludge
Exposure Day Liquid Phase Sludge Particles
% of AR % of AR
Repl. 1 Repl. 2 mv Repl. 1 Repl. 2 mv
1 4.2 4.4 4.3 66.4 60.0 63.2
2 6.4 6.3 6.3 64.5 65.5 65.0
3 7.3 8.7 8.0 70.8 73.5 72.1
6 5.0 4.4 4.7 49.5 46.5 48.0
7 5.1 4.7 4.9 42.6 56.6 49.6
10 3.9 4.4 4.2 45.2 41.0 43.1
14 3.5 3.2 3.4 51.9 49.4 50.6
20 3.5 3.2 3.4 41.3 35.4 38.4
28 3.0 3.0 3.0 31.5 29.2 30.3
37 2.1 2.2 2.2 21.4 20.5 21.0
62 2.0 1.6 1.8 22.2 19.3 20.7
63 AC 3.5 2.0 2.8 70.5 66.2 68.3
AR = Applied Radioactivity mv = mean values AC = Abiotic control

Validity criteria fulfilled:

yes

Conclusions:

The anaerobic mineralization of the test item N-[1-14C]Oleoyl-N-methylglucamine was 60 % within a test duration of 62 days.

At the plateau phase of the study, approx. 20 % of the remaining 14C-activity were associated with the sludge particles.

Based on the position of the 14C-label, the course of mineralization and the amount of 14C-activity associated with sludge particles within the study duration, it can be concluded that the anaerobic degradation of N-[1-14C]Oleoyl-N-methylglucamine is almost ultimate and that the major part of the remaining 14C-activity is incorporated in the bacteria biomass.

Executive summary:

The anaerobic mineralization of N-[1-14C]Oleoyl-N-methylglucamine, (batch no. not specified) in digester sludgewas determinedover a test period of 62 days according to OECD guideline 314 C. The study was conducted from2016-03-01to2016-05-13 at the test facility.

Sludge samples were incubated in the dark under anaerobic conditions for 62 days under controlled laboratory conditions.

The anaerobic mineralization was determined by trapping and analysis of the evolved¹⁴CO₂and¹⁴CH₄ in 1 mol/L NaOH. Furthermore¹⁴C-activity associated with sludge particles and in the liquid phase was determined. The radioactivity of the NaOH, sludge samples and liquid phase was determined by LSC.

 

Mineralization ofN-[1-14C]Oleoyl-N-methylglucamine

Mineralisation of N-[1-14C]Oleoyl-N-methylglucamine started directly after application and proceeded steadily until day 28. At that point of time the mineralisation was 55 % and the plateau was reached. At test end (day 62) the mineralisation was 60 %.

 

Mineralisation ofN-[1-14C]Oleoyl-N-methylglucamine

Exposure Day	Total Mineralization
	% of AR
1	5
2	8
3	14
6	19
7	26
10	33
14	38
20	47
28	55
37	57
62	60

                        AR = Applied Radioactivity

Based on the position of the¹⁴C-label, the course of mineralization and the amount of¹⁴C-activity associated with sludge particles within the study duration, it can be concluded that the anaerobic degradation o fN-[1-14C]Oleoyl-N-methylglucamine is almost ultimate and that the major part of the remaining¹⁴C-activity is incorporated in the bacteria biomass.

 

Distribution of¹⁴C-Activity and Mass Balance

The adsorption ofN-[1-14C]Oleoyl-N-methylglucamineon the sludge particles was high, on day 3 a maximum of 72.1 % of the applied radioactivity was determined on the sludge particles. Within the course of the study the amount of¹⁴C-activity associated with the sludge particles decreased to 21 % on day 37 and remained at that range until test end (day 62). The¹⁴C-activity in the liquid phase remained < 10 % throughout the study duration and reached a maximum of 8.0 % on day 3.

 

Throughout the study the mass balance was in the range 71.2 – 94.1 % (mean 82.6 %).

 

Distribution of¹⁴C-Activity and Mass Balance

Exposure Day	Mineralization	Liquid Phase	Sludge Particles	Mass Balance
	% of AR			% of AR
	mv	mv	mv
1	4.6	4.3	63.2	72.1
2	7.7	6.3	65.0	79.1
3	14.0	8.0	72.1	94.1
6	18.5	4.7	48.0	71.2
7	26.1	4.9	49.6	80.6
10	32.9	4.2	43.1	80.2
14	37.8	3.4	50.6	91.8
20	46.6	3.4	38.4	88.3
28	55.2	3.0	30.3	88.5
37	57.3	2.2	21.0	80.4
62	59.5	1.8	20.7	82.0
63 AC	11.5	2.8	68.3	82.6

AR = Applied Radioactivityv = mean values           AC = Abiotic control

Description of key information

Two guideline studies ar available to describe the biodegradation of the test item in water and sediment. One test was conducted according OECD Guideline 303 A, the second study was conducted according OECD Guideline 314 C.

OECD 303 A

Based on the analysis of N-[1-14C]Oleoyl-N-methylglucamine in the effluent the mean elimination rate of the total influent concentration was calculated to be 99.0 % in a continuously operating activated sludge unit.

The mineralisation had reached a mean value of 36.1 %; the mass balance was determined to be 67.3 % on average.

 

Statistical Evaluation of the Elimination, Mineralisation, Ultimate Degradation

and Mass Balance of N-[1-14C]Oleoyl-N-methylglucamine

               

N-[1-14C]Oleoyl-N-methylglucamine

	Total 14C Elimination [%]	Test Item Elimination [%]	Mineralisation [%]	Ultimate Degradation [%]	Recovery Rate / Mass Balance [%]
Range measured	53.1 -83.3	98.1 -99.9	28.6 -38.3	97.2 -99.6	63.6 -71.9
Median	79.7	99.0	36.6	98.5	67.3
Mean	75.9	99.0	36.1	98.5	67.3
95 % CI	72.5 -79.3	99.3 -98.7	35.3 -36.9	98.1 -98.9	66.5 -68.1
Number of case n	24	17	24	20	24

 

CI = Confidence Interval

 

Background Information on Data Evaluation

14C-Elimination

The mean 14C-Elimination is 75.9% in comparison to an N-[1-14C]Oleoyl-N-methylglucamine elimination of 99% and an ultimate biodegradation of 98.5%. It has to be taken into account that during biodegradation test item fragments are incorporated into biomass present in the effluent. Furthermore fragments of bacteria biomass containing 14C and organic microbial biosynthesis products are present in the effluent.

This explains why the 14C-Elimination is lower than the elimination and ultimate biodegradation of N-[1-14C]Oleoyl-N-methylglucamine.

 

Mineralisation

The mean mineralisation of the test item is 36.1% and much lower than ultimate biodegradation of 98.5%. One explanation is that test item fragments have been incorporated in the biomass of the microorganisms, which therefore are not available for mineralisation. A quantification of the amount of test item fragments incorporated in biomass is not feasible within the scope of the study. In addition, due to the complex construction of the activated sludge unit it is also very difficult to capture the evolved CO2 completely and on-line.

 

Ultimate Biodegradation

The ultimate biodegradation was evaluated based on the composition of the 14C-activity in the effluent.

About 24 % of the applied radioactivity were determined in the effluent. A maximum of 9.2 % (mean 4.6 %) of the 14C-activity in the effluent were N-[1-14C]Oleoyl-N-methylglucamine and a further mean of 1.8 % (0.8 % – 4.4 %) of the 14C-activity were extractable from the effluent.

This 1.8 % might be associated with degradation products. However, the respective LC-FSA chromatograms show no further distinct peaks (no detection of transformation products) but an increased background activity. The remaining 14C-activty (≥ 88 % of the 14C-activity in the effluent) was not extractable from the effluent.

From the absence of distinct degradation products and the low amount of extractable 14C-activty it can be concluded, that the degradation of N-[1-14C]Oleoyl-N-methylglucamine is almost ultimate and that the main amount of 14C-activity in the effluent is 14C associated with biomass, fragments of bacteria biomass and small molecules excreted by the bacteria (organic microbial biosynthesis products). Consequently the ultimate biodegradation was calculated from the influent concentration and the extractable 14C-activity in the effluent measured by LC-FSA.

Furthermore it was assumed that the composition of 14C-activity in the activated sludge and activated sludge liquid phase is comparable to the 14C-activity in the effluent.This results in a mean ultimate degradation was of 98.5 %.

OECD 314 C

The anaerobic mineralization of the test item N-[1-14C]Oleoyl-N-methylglucamine was 60 % within a test duration of 62 days.

 

At the plateau phase of the study, approx. 20 % of the remaining 14C-activity were associated with the sludge particles.

 

Based on the position of the 14C-label, the course of mineralization and the amount of 14C-activity associated with sludge particles within the study duration, it can be concluded that the anaerobic degradation of N-[1-14C]Oleoyl-N-methylglucamine is almost ultimate and that the major part of the remaining 14C-activity is incorporated in the bacteria biomass.