Fatty acid chlorides, C8-14 (even numbered), reaction products with glycine

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: sewage treatment simulation testing

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2014-10-23 to 2014-11-28

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

GLP compliance:

yes

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
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, D-31137 Hildesheim

Pretreatment
The activated sludge was washed twice 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.

Duration of test (contact time):

32 d

Initial conc.:

564 other: kBq/L

Based on:

other:

Initial conc.:

70 µg/L

Based on:

test mat.

Details on study design:

Duration
After an acclimatisation period of 4 days the test item dosage was started. The adaptation phase lasted 10 days and was followed by the plateau phase, which lasted 21 days. The test was terminated after 36 days corresponding to 32 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 head space 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 air lift pump.
Set-up
The system was started with an activated sludge content of 2.5 ± 0.5 g DW/L (test item unit). Test volume in the aeration vessel was 372 mL, the total volume of activated sludge in the aeration vessel and settling tank was 500 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. 1 mL H3PO4 (conc.) (2 mL on day 9/10) was 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 (46.5 mL) in the aeration vessel was removed once daily 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 test, 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 mineralization of the test item was determined by LSC.
Completion of the test
The study was completed after reaching a sufficient degree of elimination / mineralization and the plateau. The plateau had lasted three weeks and had about 21 valid values.
Replicates
One replicate
Water
Tap water for the preparation of organic medium
Deionized 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 4 days. The DOC of each new batch of organic medium was determined. As the adjustment with a high amount synthetic sewage can influence the degradation capability of the activated sludge, the use of synthetic sewage was decided by the study director on a case-by-case basis.
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.
Test temperature 20 - 25 °C
pH value 7.5 ± 0.5
Initial dry weight content 2.5 ± 0.5 g/L, the activated sludge was adjusted with organic medium at test start.
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. All solutions were pumped directly into the aeration vessels.
Influent flow Organic medium: 62 mL/h
Test item solution: 250 µL/h

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 continuously applied 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 and Reagents
Test vessels Activated sludge units consisting of an aeration vessel (approx. 500 mL) and a separator (approx. 250 mL)
Pumps Precision syringe pump, KD SCIENTIFIC
Peristaltic pump, ISMATEC
Aeration Medo Kompressor, REBIE
Temperature Temperature Data Loggers with Sensor Device, TESTO
Balances KERN, SARTORIUS
Dry weight Drying oven T6, THERMO SCIENTIFIC
pH-value HQ 40d multi, HACH
TOC Photometer DR3900, HACHLANGE
TOC Analyser Multi N/C 3100, ANALYTIK JENA
14CO2 Trap 99.7 % NaOH, Sodium hydroxide, VWR
Acidification of effluent 85 % H3PO4, ROTH

Type and Frequency of Measurements

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) At least weekdays

LSC Analysis
Matrix Frequency
Adaptation Plateau phase
Influent
(Test item
stock solution) Every new batch
Effluent Daily Daily
Activated
sludge Daily Daily
NaOH traps Daily Daily

Sampling Procedure
To avoid any losses of 14CO2 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.
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.

Compartment:

other: water / sediment, material (mass) balance

Remarks on result:

other: See results

% Degr.:

> 99

Parameter:

radiochem. meas.

Remarks:

14 CO2

Sampling time:

32 d

Remarks on result:

other: 2.2% of 14C was found in the effluent but only 37% could be adsorbed in a reversed phase cartouche, 67% of 14C is most likely incorporated in biological material not sorbing to the cartouche. This means elimination is >99%.

Mineralization rate (in CO2):

87 other: %

Transformation products:

not specified

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Residues:

not measured

Details on results:

Temperature, pH-Value and Suspended Solids
The operational parameters pH-value, temperature and suspended solids in the activated sludge units 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 2.76 g/L was used for the start of the activated sludge unit. During acclimatisation, the activated sludge accumulated in the aeration vessel and increased to 4.2 g/L dry weight. Subsequently during the adaptation phase, the suspended solids in the aeration vessel levelled off to 3.0 g/L and stabilized in the range 2.0 – 2.6 g/L during the plateau phase.

pH-value and Temperature in the sampled Activated Sludge
Study day Test Item Unit
Date pH temperature [°C]
0 2014-10-27 7.76 20.7
8 2014-11-04 6.95 23.1
15 2014-11-11 7.45 22.1
22 2014-11-18 7.16 22.0
29 2014-11-25 7.32 21.9

Suspended Solids
Study Day of Suspended solids
Day test item Date
application [g/L]
5 1 2014-10-28 4.2
6 2 2014-10-29 3.8
7 3 2014-10-30 3.7
8 4 2014-10-31 3.7
9 5 2014-11-01 3.8
10 6 2014-11-03 3.1
12 8 2014-11-04 3.0
13 9 2014-11-05 3.3
14 10 2014-11-06 3.0
15 11 2014-11-07 2.5
18 14 2014-11-10 2.6
19 15 2014-11-11 2.4
20 16 2014-11-12 2.2
21 17 2014-11-13 2.1
22 18 2014-11-14 2.4
25 21 2014-11-17 2.5
26 22 2014-11-18 2.3
27 23 2014-11-19 2.4
28 24 2014-11-20 2.0
29 25 2014-11-21 2.1
33 28 2014-11-24 2.2
34 29 2014-11-25 2.4
35 30 2014-11-26 2.3
36 31 2014-11-27 2.4
37 32 2014-11-28 2.4

 
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 efficiently (> 80 %).
Biodegradation > 80 % was already achieved after one day and remained > 80 % until day 14. From day 14 until test end the biodegradation of the DOC decreased slightly and oscillated around 80 % (75 – 80 %). It is assumed that this decrease is associated with the increased DOC of the domestic sewage and reduced volume of synthetic sewage needed for the adjustment of the organic medium. The decrease had no influence on the biodegradation efficiency, the unit worked efficiently throughout the test.

DOC Concentration and Degradation (Elimination)
Study DOC
Day Date [mgC/L] Elimination
influent effluent [%]
0 2014-10-23 97.5 -
1 2014-10-24 97.5 9.5 90.29
2 2014-10-25 97.6 - -
4 2014-10-27 97.6 10.8 88.98
5 2014-10-28 97.6 9.8 89.98
6 2014-10-29 105.4 - -
7 2014-10-30 105.4 8.2 92.22
11 2014-11-03 61.9 9.4 91.11
12 2014-11-04 97.5 - -
13 2014-11-05 97.5 10.9 88.82
15 2014-11-07 96.4 13.9 85.74
18 2014-11-10 96.4 19.3 79.98
20 2014-11-12 96.4 20.3 78.94
21 2014-11-13 105.5 - -
22 2014-11-14 105.5 20.8 80.28
25 2014-11-17 105.5 21.5 79.62
27 2014-11-19 110.9 22.7 78.48
29 2014-11-21 110.9 21.1 80.97
32 2014-11-24 93.4 19.9 82.06
34 2014-11-26 93.4 21.2 77.30
36 2014-11-28 93.4 22.9 75.48

Elimination and Mineralization 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 experimental starting via LC-RFD (radio flow detection) method. The radio chemical purity was 95.7 % prior to experimental starting.

Stability and Concentration of the Test Item Stock Solution (non-GLP)
Before experimental starting, losses to the application equipment and the stability of the test item stock solution were assessed for the intended application interval of 7 days. When prepared in sterilized water and equipment, no significant loss of activity was observed and no additional peaks appeared during the 7 day application interval. Therefore, the stability of the test item in a set-up with sterilized compounds was considered suitable.

Test Item Stock Solution Stability during the Intended Application Interval
Study Stock Solution Acitivity Percentage of
Day 0 Value
Day
[kBq/mL] [%]
0 93.3 -
1 92.4 99.0
2 92.0 98.7
3 92.1 98.7
7 89.9 96.3
8 93.9 100.7

After the start of exposure, a new stock solution was prepared approx. every 7 days. The concentration of each new batch of the stock solution was analysed by LSC. The recovery of the test item in the stock solution was in the range of 99 - 103 %.

Test Item Concentrations in the Stock Solutions
Study Day of Nominal Value Stock solution Acitivity Percentage of Nominal Value
Day test item Date
application [kBq/mL] [kBq/mL] [%]
4 0 2014-10-27 140 141 100
11 7 2014-11-03 144 103
18 14 2014-11-10 140 100
25 21 2014-11-17 139 99
32 28 2014-11-24 142 101

Elimination of N-Dodecanoyl[1-14C]glycine, sodium salt
After the activated sludge unit had stabilized, the dosage of the test item was started (October 27, 2014). Influent (stock solution) and effluent samples were analysed by LSC.

The elimination was calculated based on the total activity of N-Dodecanoyl[1-14C]glycine, sodium salt in the influent and total 14C activity in the effluent samples. The mean 14C elimination during the test item application was calculated based on 32 values obtained during the test item application.

The total 14C activity of the effluent was analysed daily. As 14CO2 was stripped off continuously and 14C associated with biomass was removed by centrifugation (until day 16) and filtration (from day 17 until test end), respectively, it is assumed, that the remaining 14C activity in the effluent samples is associated with N-Dodecanoyl[1-14C]glycine, sodium salt or its degradation products.

The mean 14C elimination during the 32 days of test item application was determined to be 97.77 %.
 
Effluent Concentration and Elimination of N-Dodecanoyl[1-14C]glycine, sodium salt
Daily influent concentration: 564520 Bq/L N-Dodecanoyl[1-14C]glycine, sodium salt
Day of
test item application Day of plateau Phase Date 14C Activity Effluent
SET 14C- Elimination
E
[kBq/L] [%]
1 28.10.2014 0.93 99.83
2 29.10.2014 2.70 99.52
3 30.10.2014 3.56 99.37
4 31.10.2014 4.37 99.23
5 01.11.2014 6.32 98.88
6 02.11.2014 7.32 98.70
7 03.11.2014 8.85 98.43
8 04.11.2014 8.84 98.43
9 05.11.2014 11.16 98.02
10 06.11.2014 12.43 97.80
11 1 07.11.2014 15.75 97.21
12 2 08.11.2014 19.63 96.52
13 3 09.11.2014 22.02 96.10
14 4 10.11.2014 20.73 96.33
15 5 11.11.2014 22.75 95.97
16 6 12.11.2014 23.02 95.92
17 7 13.11.2014 14.58 97.42
18 8 14.11.2014 13.67 97.58
19 9 15.11.2014 13.14 97.67
20 10 16.11.2014 14.07 97.51
21 11 17.11.2014 14.52 97.43
22 12 18.11.2014 15.33 97.28
23 13 19.11.2014 13.80 97.56
24 14 20.11.2014 13.35 97.64
25 15 21.11.2014 13.68 97.58
26 16 22.11.2014 14.40 97.45
27 17 23.11.2014 14.15 97.49
28 18 24.11.2014 12.92 97.71
29 19 25.11.2014 11.81 97.91
30 20 26.11.2014 10.70 98.10
31 21 27.11.2014 10.08 98.21
32 22 28.11.2014 11.58 97.95

Examination of 14C-Activity in the Effluent
As the course of 14C activity in the effluent showed an unusual progression additional examinations were done at test end to characterize the 14C activity in the effluent and to prove if N-Dodecanoyl[1-14C]glycine, sodium salt is still present in the effluent.

As a high mineralization (mean 86.6 %) was reached during the plateau phase, it is assumed that ultimate degradation of the test item took place and that the main amount of the remaining 14C activity in the filtered and acidified effluent samples is not N-Dodecanoyl[1-14C]glycine, sodium salt but 14C associated with fragments of bacteria biomass and small molecules excreted by the bacteria.

The effluent of day 32 (test end) in comparison with HPLC water acidified with H3PO4 and spiked with 25 kBq/L N-Dodecanoyl[1-14C]glycine, sodium salt were used for the following investigations. Filtered samples of 10 mL (0.45 µm and 0.2 µm filter in series) were used for extractions with cartridges with different polarity. The following cartridges were used1):

1. Cation exchanger cartridge: Chromabond PS H+
2. Non-polar SPE cartridge: HF Bond Elut C18 – Agilent
3. Polar SPE cartridge: C2Bond Elut -Agilent
4. Polar SPE cartridge: Chromabond NH2
1) Prior to extraction all cartridges were conditioned with 5 mL methanol and 5 mL HPLC water.

The 14C activity in the extracts which passed the cartridges was determined by LSC.

Non-Retained 14C Activity in the Effluent
Effluent Spiked HPLC Water
Cartridge Bq/ 10 mL % Bq/ 10 mL %
None1) 163.9 100.0 207.7 100.0
Cationic 61.9 37.8 6.1 2.9
Non-Polar 41.5 25.3 5.1 2.4
Polar C2 93.1 56.8 5.5 2.7
Polar NH2 102.5 62.6 4.5 2.2
1) filtered and acidified sample


The extractions with cartridges with different polarity showed, that N-Dodecanoyl[1-14C]glycine, sodium salt was nearly completely retained by all 4 types of cartridges. In contrast varying amounts of 14C activity of the filtered effluent samples passed the cartridges, e.g. about 63 % of the 14C activity in the effluent sample passed the polar SPE NH2 cartridge.

These results clearly confirm, that a large fraction of the remaining 14C activity in the effluent is not associated with N-Dodecanoyl[1-14C]glycine, sodium salt. Furthermore the elimination of N-Dodecanoyl[1-14C]glycine, sodium salt is even higher than the calculated elimination of 97.77 % based on the total 14C activity in the effluent.

 
N-Dodecanoyl[1-14C]glycine, sodium salt 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-Dodecanoyl[1-14C]glycine, sodium salt and the total 14C load in the activated sludge. The results are given in Table 16.

N-Dodecanoyl[1-14C]glycine, sodium salt in the Activated Sludge Phase
Daily influent load: 840 kBq/d N-Dodecanoyl[1-14C]glycine, sodium salt
Day of
test item application Date Total Influent Load
TLI 14C Activity on Sludge
SA Total Sludge Load1)
LA Percentage of Total Influent Load
[kBq] [kBq/mL] [kBq] [%]
1 28.10.2014 665.02) 0.77 382.5 57.5
2 29.10.2014 1505.0 1.35 675.3 44.9
3 30.10.2014 2345.0 1.55 776.5 33.1
4 31.10.2014 3185.0 2.09 1045.7 32.8
5 01.11.2014 4025.0 2.32 1159.0 28.8
6 02.11.2014 4865.0 2.20 1101.8 22.6
7 03.11.2014 5705.0 1.94 970.4 17.0
8 04.11.2014 6545.0 2.25 1126.6 17.2
9 05.11.2014 7385.0 2.24 1118.1 15.1
10 06.11.2014 8225.0 2.06 1028.2 12.5
11 07.11.2014 9065.0 2.29 1144.7 12.6
12 08.11.2014 9905.0 2.16 1081.4 10.9
13 09.11.2014 10745.0 1.97 985.6 9.2
14 10.11.2014 11585.0 2.21 1104.9 9.5
15 11.11.2014 12425.0 2.13 1066.6 8.6
16 12.11.2014 13265.0 1.99 996.5 7.5
17 13.11.2014 14105.0 2.04 1018.5 7.2
18 14.11.2014 14945.0 1.97 986.9 6.6
19 15.11.2014 15785.0 2.25 1124.7 7.1
20 16.11.2014 16625.0 1.78 891.1 5.4
21 17.11.2014 17465.0 1.86 929.8 5.3
22 18.11.2014 18305.0 2.01 1003.8 5.5
23 19.11.2014 19145.0 1.74 868.7 4.5
24 20.11.2014 19985.0 1.32 659.2 3.3
25 21.11.2014 20825.0 1.07 532.5 2.6
26 22.11.2014 21665.0 1.21 606.8 2.8
27 23.11.2014 22505.0 1.08 538.4 2.4
28 24.11.2014 23345.0 1.14 568.6 2.4
29 25.11.2014 24158.82) 1.19 593.6 2.5
30 26.11.2014 24998.8 1.08 541.7 2.2
31 27.11.2014 25838.8 1.22 608.6 2.4
32 28.11.2014 26678.8 1.18 587.6 2.2
1) calculated with an activated sludge volume of 500 mL
2) influent flow paused for 45 min
 
Mineralization of N-Dodecanoyl[1-14C]glycine, sodium salt during the Plateau Phase
The 14CO2 activity of the NaOH traps was determined daily. The mineralization during the plateau phase was calculated based on the influent load and the total load of the NaOH traps from day 10 until test end (day 32).
The influent load was corrected by the daily removed 14C load of the surplus sludge, as this fraction was not accessible for mineralization.

During the plateau phase the mineralization was in the range 83.4 % – 88.9 % and the mean value was 86.6 %.

Mineralization of N-Dodecanoyl[1-14C]glycine, sodium salt during the Plateau Phase
Daily influent load: 840 kBq/d N-Dodecanoyl[1-14C]glycine, sodium salt
Day of
test item application Date Sum Influent Load
TLI Sum Surplus Load
TLS Load NaOH Traps
LDT Sum Traps Load
TLT Mineralization


M
[kBq] [kBq] [kBq/d] [kBq] [%]
10 06.11.2014 840.0 201.7 654 654 88.9
11 07.11.2014 1680.0 309.4 599 1253 84.8
12 08.11.2014 2520.0 412.5 656 1909 86.4
13 09.11.2014 3360.0 507.2 593 2502 84.9
14 10.11.2014 4200.0 613.4 594 3096 83.8
15 11.11.2014 5040.0 715.8 596 3692 83.4
16 12.11.2014 5880.0 811.6 657 4349 84.2
17 13.11.2014 6720.0 909.9 667 5016 84.9
18 14.11.2014 7560.0 1004.2 693 5709 85.8
19 15.11.2014 8400.0 1111.1 684 6393 86.4
20 16.11.2014 9240.0 1197.1 684 7077 87.1
21 17.11.2014 10080.0 1286.7 647 7725 87.0
22 18.11.2014 10920.0 1383.5 618 8342 86.6
23 19.11.2014 11760.0 1468.4 690 9033 87.1
24 20.11.2014 12600.0 1533.0 747 9780 87.9
25 21.11.2014 13440.0 1586.7 668 10448 87.7
26 22.11.2014 14280.0 1647.9 743 11191 88.2
27 23.11.2014 15120.0 1702.0 700 11891 88.3
28 24.11.2014 15960.0 1757.2 702 12592 88.3
29 25.11.2014 16773.81) 1815.4 547 13139 87.5
30 26.11.2014 17613.8 1868.6 668 13807 87.4
31 27.11.2014 18453.8 1928.0 663 14470 87.2
32 28.11.2014 19293.8 1985.3 693 15163 87.3
1) influent flow paused for 45 min

Mass Balance of N-Dodecanoyl[1-14C]glycine, sodium salt
Based on the data obtained for N-Dodecanoyl[1-14C]glycine, sodium salt in the influent, total 14C activity in the effluent, 14C activity in activated sludge and 14CO2 in the NaOH trap samples, a mass balance was calculated for the total 14C activity. Throughout the test, a mass balance > 90 % was achieved, except on day 3 the mass balance decreased to 85.1 %.

Mass Balance of N-Dodecanoyl[1-14C]glycine, sodium salt
Daily influent load: 840 kBq/d N-Dodecanoyl[1-14C]glycine, sodium salt
Day of
test item application Day of plateau Phase 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] [%]
1 28.10.2014 665.0 1.39 395.4 36.7 213 91.6
2 29.10.2014 1505.0 5.40 697.2 101.3 628 90.6
3 30.10.2014 2345.0 10.69 795.0 175.1 1106 85.1
4 31.10.2014 3185.0 17.19 1086.5 275.8 1755 94.9
5 01.11.2014 4025.0 26.59 1191.0 386.3 2469 98.3
6 02.11.2014 4865.0 37.48 1124.9 490.7 3177 96.9
7 03.11.2014 5705.0 50.65 997.4 583.2 3821 93.3
8 04.11.2014 6545.0 63.79 1142.8 689.4 4540 96.4
9 05.11.2014 7385.0 80.39 1127.9 794.2 4970 92.2
10 06.11.2014 8225.0 98.89 1043.3 891.1 5624 90.9
11 1 07.11.2014 9065.0 152.66 1159.0 998.8 6223 92.0
12 2 08.11.2014 9905.0 211.82 1111.8 1101.9 6880 91.9
13 3 09.11.2014 10745.0 273.79 1021.0 1196.5 7472 90.6
14 4 10.11.2014 11585.0 321.78 1146.4 1302.8 8066 91.5
15 5 11.11.2014 12425.0 375.18 1103.8 1405.1 8662 90.9
16 6 12.11.2014 13265.0 430.72 1033.7 1501.0 9319 90.6
17 7 13.11.2014 14105.0 479.03 1061.4 1599.3 9986 91.1
18 8 14.11.2014 14945.0 524.32 1015.8 1693.5 10679 91.2
19 9 15.11.2014 15785.0 565.78 1152.2 1800.5 11363 92.5
20 10 16.11.2014 16625.0 613.40 928.2 1886.5 12048 91.3
21 11 17.11.2014 17465.0 665.70 967.0 1976.1 12695 91.6
22 12 18.11.2014 18305.0 719.56 1043.9 2072.8 13313 92.0
23 13 19.11.2014 19145.0 759.44 917.8 2157.8 14003 91.5
24 14 20.11.2014 19985.0 802.31 698.0 2222.4 14750 90.7
25 15 21.11.2014 20825.0 842.86 582.6 2276.1 15418 90.1
26 16 22.11.2014 21665.0 886.15 662.2 2337.2 16161 90.9
27 17 23.11.2014 22505.0 928.45 586.3 2391.4 16861 90.7
28 18 24.11.2014 23345.0 968.24 595.9 2446.5 17563 90.9
29 19 25.11.2014 24158.8 1004.27 629.1 2504.8 18109 90.5
30 20 26.11.2014 24998.8 1036.25 575.2 2558.0 18777 90.2
31 21 27.11.2014 25838.8 1071.48 642.1 2617.4 19441 90.4
32 22 28.11.2014 26678.8 1109.16 618.4 2674.6 20134 90.4
1) Activated sludge solids and liquid phase
 

Statistical Evaluation of the Elimination, Mineralization and Mass Balance of N-Dodecanoyl[1-14C]glycine, sodium salt

N-Dodecanoyl[1-14C]glycine, sodium salt

14C Elimination Mineralization
(Plateau Phase) Recovery Rate /
Mass Balance
[%] [%] [%]
Range measured 95.92 - 99.83 83.4 - 88.9 85.1 - 98.3
Median 97.65 87.1 91.2
Mean 97.77 86.6 91.7
95 % CI 97.42 - 98.12 85.9 - 87.2 90.8 - 92.5
Number of case n 32 23 32
CI = Confidence Interval

Validity criteria fulfilled:

yes

Conclusions:

Based on the LSC analysis of total 14C activity in the effluent the mean elimination rate of the total influent concentration of N-Dodecanoyl[1-14C]glycine, sodium salt was calculated to be 97.77 % in a continuously operating activated sludge unit. 2.2% of 14C was found in the effluent but only 37% could be adsorbed in a reversed phase cartouche, 67% of 14C is most likely incorporated in biological material not sorbing to the cartouche. This means that only 37% of the 2.2% 14C in the effluent is as worst case related to the test substance itself (=0.8%) and thereforie the elimination in the sewage treatment plant is >99%. For exposure estimations 99% primary degradation in the sewage treatment plant is used.
The mineralization reached a mean value of 86.6 %; the mass balance was determined to be 91.7 % on average.

Executive summary:

The elimination and mineralization of the test item N-Dodecanoyl[1-14C]glycine, sodium saltin an activated sludge unit was determined over a test period of 32 days according to OECD guideline 303 A. The study was conducted from 2014-10-23 to 2014-11-28 at the Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany. The definite exposure phase was from 2014-10-27 to 2014-11-28.

  

Principle of the Study

The elimination and mineralization of N-Dodecanoyl[1-14C]glycine, sodium saltby aerobic microorganisms were determined in a continuously operating laboratory activated sludge unit simulating the activated sludge process. The degree of¹⁴C-elimination, the mineralization 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 2.5 ± 0.5 g/L DW. After acclimatization of the system, the application of N-Dodecanoyl[1-14C]glycine, sodium salt was started. Samples of activated sludge and effluent were taken in regular intervals. Emerging¹⁴CO₂was captured in NaOH traps. LSC analysis was performed on the samples and traps to determine the elimination and mineralization of N-Dodecanoyl[1-14C]glycine, sodium salt.

  

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 4 days.

 

The nominal concentration of the test item stock solution was 140 kBq/mL. The stock solution was dosed at a constant rate of 250 µL/h into the activated sludge unit, resulting in a nominal influent concentration of 564 kBq/L, corresponding to 70 µg/L.

The elimination was calculated based on the total activity of N-Dodecanoyl[1-14C]glycine, sodium saltin the influent and total¹⁴C activity in the effluent.

The mineralization during the plateau phase (day 10 to day 32) was calculated based on the total influent load and the total load of¹⁴C activity in NaOH traps.

The amounts of N-Dodecanoyl[1-14C]glycine, sodium salteliminated from the aqueous phase as well as the mineralization and the total recovery rate are summarized in the Table below.

 

The mean elimination rate for N-Dodecanoyl[1-14C]glycine, sodium saltof the influent concentration and total¹⁴C-activity in the effluent was calculated to be 97.77 %. 2.2% of 14C was found in the effluent but only 37% could be adsorbed in a reversed phase cartouche, 67% of 14C is most likely incorporated in biological material not sorbing to the cartouche. This means that only 37% of the 2.2% 14C in the effluent is as worst case related to the test substance itself (=0.8%) and thereforie the elimination in the sewage treatment plant is >99%. For exposure estimations 99% primary degradation in the sewage treatment plant is used. The mineralization had a mean value of 86.6 %, the mass balance was determined to be 91.7 % on average.

 

Statistical Evaluation of the Elimination, Mineralization and Mass Balance of N-Dodecanoyl[1-14C]glycine, sodium salt

	N-Dodecanoyl[1-14C]glycine, sodium salt
	^14C Elimination	Mineralization (Plateau Phase)	Recovery Rate / Mass Balance
	[%]	[%]	[%]
Range measured	95.92 - 99.83	83.4 - 88.9	85.1 - 98.3
Median	97.65	87.1	91.2
Mean	97.77	86.6	91.7
95 % CI	97.42 - 98.12	85.9 - 87.2	90.8 - 92.5
Number of case n	32	23	32

 

CI = Confidence Interval

Description of key information

Based on the LSC analysis of total 14C activity in the effluent the mean elimination rate of the total influent concentration of N-Dodecanoyl[1-14C]glycine, sodium salt was calculated to be 97.77 % in a continuously operating activated sludge unit. 2.2% of 14C was found in the effluent but only 37% could be adsorbed in a reversed phase cartouche, 67% of 14C is most likely incorporated in biological material not sorbing to the cartouche. This means that only 37% of the 2.2% 14C in the effluent is as worst case related to the test substance itself (=0.8%) and thereforie the elimination in the sewage treatment plant is >99%. For exposure estimations 99% primary degradation in the sewage treatment plant is used.

The mineralization reached a mean value of 86.6 %; the mass balance was determined to be 91.7 % on average.