1,3,6-Naphthalenetrisulfonic acid, 7-[2-[2-[(aminocarbonyl)amino]-4-[[4-chloro-6-[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]phenyl]diazenyl]-, sodium salt

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-06-15 to 2021-08-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Facility Actions
Receipt: 2020-06-17
Identification parameter: Name, batch number, state and color
Retention of test item : At least 1 g has been retained on 2020-07-06 and
stored at 6 ± 2 °C.
Storage conditions: 18 - 25 °C, dark, in tightly closed original container
 

Radiolabelling:

no

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on source and properties of surface water:

Parameter
Sampling site location: Am Anger, 30982 Pattensen, Germany
Coordinates: 52˚11’39.419'' N 9˚47’7.49'' E
Sampling date & time: 2021-06-09 /10:38 h
Weather conditions: Sunny, dry
Depth of collection: Surface (upper 0 – 20 cm)
Appearance : Clear, yellowish-brownish
Temperature [°C]: 18.1
pH value: 7.07
O2 concentration [mg O2/L]: 8.37
DOC [mg/L]: 5.57
TOC [mg/L]: 5.73
TIC [mg/L]: 41.90
NO2-N [mg/L]: 0.04
NO3-N [mg/L]: 3.09
NH3-N [mg/L]: 0.14
Total N [mg/L]: 3.78
Ortho-P [mg/L]: 0.119
Total P [mg/L]: 0.151
Suspended matter*: 10.2 mg/L#
# determined with membrane filter with pore size 0.45 µm

Test System: Natural surface water (field fresh sampled, without sediment) of the river ‘LEINE’. The sampling site was selected with respect to the regional biological and chemical water quality maps (interactive online version) of the Lower Saxony Water Management, Coastal Defence and Nature Conservation Agency (German: NLWKN).
Reasons for the selection: The surface water meets the recommendations of the guideline.
Collection / Sampling: The water was collected from the surface of the river (approx. upper 20 cm water) at an undisturbed recess. Furthermore, temperature, pH value and O2 concentration of the water were measured at field sampling.
Handling: The water was yellowish and clear. Small fishes were removed by filtration.
Acclimation: Until test start the surface water was acclimated for three days under test conditions at 12 ± 2 °C in the dark with aeration.

Duration of test (contact time):

62 d

Initial conc.:

30 µg/L

Based on:

test mat.

Initial conc.:

100 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

Experimental Procedure
Nominal test item 30 µg/L and 100 µg/L
concentration: 10 mg/L for Co-Chromatography/Metabolite Identification

Application solution: 25 mg/L (30 µg/L and 100 µg/L)
2 g/L (10 mg/L)
Solvent: Ultrapure Water
Pretreatment: None
Blank control: Surface water (test system) without test and/or reference
item.
Sterile control: Sterilized surface water with Reactive Yellow 176 at test
concentration.
For sterilization, the water was autoclaved prior to
application.
Sterile Blank Control: Sterilized surface water (test system) without test and/or
reference item.

Preparation of Calibration Standards
Ten matrix matched calibration standards in the range from 2 to 150 µg/L were prepared. A stock solution of 1000 mg/L of the test item in ultrapure water was prepared for this purpose. This stock solution was diluted in two steps with surface water to a concentration of 1 mg/L for the dilution solution (DS1). Dilution solution DS2 was prepared from DS 1 by a factor 100 dilution with surface water. The preparation of the calibration standards is summarized below.
 

Preparation of Calibration Standards
Concentration Volume DS1 Volume DS2 Volume Surface Water
[µg/L] [mL, 1000 µg/L] [mL, 10 µg/L] [mL]
150 0.15 - 0.85
100 0.1 - 0.90
80 0.08 - 0.92
40 0.04 - 0.96
20 0.02 - 0.98
10 0.05 - 4.95
5 - 0.5 0.5
4 - 0.4 0.6
3 - 0.3 0.7
2 - 0.2 0.8

Preparation of Samples
Surface water samples were measured directly.

Reference control
Reference item: Aniline sulfate, [14C(U)]-
Test facility code: 60007
Batch number: 373-002-110-A-20141024-PVA
Radiochemical purity: 99.2%
Specific activity: 110 mCi/mmol
Expiry date: Not specified, stability and radiochemical purity was
confirmed via analysis by the supplier prior to shipment.
Replicates: Duplicates
Test concentration: 10 µg/L

Water samples: Samples with a volume of 1 mL were mixed with Ultima
Gold XR and the residual 14C-activity was determined by
LSC.

Dissolved 14CO2: Samples with a volume of 1 mL were acidified with 20 µL
H3PO4 (conc.) and shaken vigorously. Afterwards the
sample was mixed with Ultima Gold XR and the residual
14C-activity was determined by LSC.

Test Procedure
Duration: 62 days
Application: At application, appropriate volumes of the test item application
solution were pipetted directly into the test medium of each
replicate.
Test vessels: 2.0 L glass bottles for test item replicates, blank controls and
sterile controls controls (transformation).
1.0 L glass bottles for test item replicates (metabolite
identification).
150 mL gastight bottles for reference control replicates and sterile
blank controls.
Test medium: Natural surface water of the river ‘Leine’.
Volume of the test medium: 1.3 L (test item replicates, blank controls and sterile
controls)
650 mL (test item replicates metabolite identification)
100 mL (reference control and sterile blank control)

Replicates: Duplicates per concentration and treatment

Incubation: Nominal: 12 ± 2 °C in the dark
Actual: 10.5 – 14.0 °C in the dark
Agitation: Continuous stirring at approx. 120 rpm to maintain particles and
microorganisms in suspension and to facilitate oxygen transfer
from the headspace to the liquid.

Types and frequency of measurements
Temperature: The incubation temperature was recorded continuously
throughout the test.

pH and Oxygen: Measurements of pH and oxygen concentration in the test
system (Blank control) were done on day 0, 1, 14, 29 and 55.

Transformation: Sampling for determination of the transformation was carried
out directly after application and at 10 additional sampling
points.

The amount of test item and the degradation product (RRT of
1.45 in the preliminary test) in the test solution and sterile test
item solution were determined by LC-DAD.

The sampling points were chosen to enable the establishment
of the pattern of decline of the test item. Sampling was done
after application (day 0) and at day 1, 2, 3, 4, 6, 14, 37 and day 62.

Sterile controls were analysed at test start and test end. Blank
controls and sterile blank controls were analysed at test start to
verify the absence of the test item in the used surface water.

For the reference control sampling for mineralization was
stopped once degradation was finished. Residual activity in the
test solution and dissolved 14CO2 (after acidification of test
solution) were determined by LSC.

Analysis of dissolved 14CO2 and residual 14C-activity in the test
solution was carried out via radiometric analysis by LSC. The
total radioactivity in the water samples and water samples after
acidification was measured.

Reference substance:

aniline

Remarks:

Reference item:Aniline sulfate, [14C(U)]- Test facility code:60007 Batch number:373-002-110-A-20141024-PVA Radiochemical purity:99.2% Specific activity :110 mCi/mmol Expiry date:Not specified, stability and radiochemical purity was confirmed via analysis

Compartment:

abiotic control measured at end of test

Sampling date:

2021

% Recovery:

1.5

Remarks on result:

other: 100µg/L

Compartment:

abiotic control measured at end of test

Sampling date:

2021

% Recovery:

0

Remarks on result:

other: 30 µg/L

Compartment:

biologically active treatment at end of test

Sampling date:

2021

% Recovery:

3.7

Remarks on result:

other: 100 µg/L

Compartment:

biologically active treatment at end of test

Sampling date:

2021

% Recovery:

1.7

Remarks on result:

other: 30 µg/L

Parent/product:

parent

Compartment:

water

% Degr.:

96.3

Parameter:

test mat. analysis

Sampling date:

2021

Sampling time:

62 d

Remarks on result:

other: 100 µg/L

Parent/product:

parent

Compartment:

water

% Degr.:

98.3

Parameter:

test mat. analysis

Sampling date:

2021

Sampling time:

62 d

Remarks on result:

other: 30 µg/L

Compartment:

natural water: freshwater

DT50:

2.05 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: 100 µg/L

Compartment:

natural water: freshwater

DT50:

1.91 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: 30 µg/L

Transformation products:

yes

Remarks:

Molecular Formula: C28H22ClN9O12S4

No.:

#1

Details on transformation products:

Transformation of Reactive Yellow 176 started directly after application. About 55 % transformation was reached within 3 days, after 14 days the detected concentration was < 10 % of the applied concentration (Table 11 and Table 12).
The test item was degraded to one metabolite with a relative retention time (RRT) of 1.45 (UPLC-DAD; 1.3 in the preliminary study with another chromatographic method) to the test item. The molecular formula of this metabolite was determined by LC-HRMS analysis of the samples prepared for the metabolite identification (day 0 versus day 62).
The UPLC-DAD system used for the quantitative analysis and the LC-HRMS system used the same chromatographic set-up. The LC-HRMS analysis yielded a peak with a RRT of 1.45 to the test item peak. A molecular formula of C28H22ClN9O12S4 was obtained for this peak. The experimentally obtained accurate mass was within a range of ± 5 ppm of the theoretical mass as proposed as quality indicator for a positive confirmation of a molecular formula by the guidance document SANTE/12682/2019.
This metabolite was formed by an elimination of the terminal alkyl sulfate moiety. This metabolite was also formed in the abiotic controls at the end of the study in equal quantities. This result indicates that the degradation is driven by an abiotic process.

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Residues:

not measured

Details on results:

30 µg/L Reactive Yellow 176
% Applied Reactive Yellow 176

Day 0 1 2 3 6 14
Repl. 1 124.7 80.7 55.7 40.9 16.9 3.1
2 124.8 81.7 59.4 43.5 19.1 2.5
Mean 124.7 81.2 57.5 42.2 18.0 2.8

Day 37 62
Repl. 1 2.9 3.2
2 0 4.2
Mean 1.4 3.7

% M1 of Applied Reactive Yellow 176

Day 0 1 2 3 6 14
Repl. 1 16.5 53.1 68.9 84.7 107.7 131.6
2 18.5 55.6 74.4 90.3 110.3 138.6
Mean 17.5 54.4 71.6 87.5 109.0 135.1

Day 37 62
Repl. 1 127.7 112.9
2 134.0 115.3
Mean 130.8 114.1

100 µg/L Reactive Yellow 176
% Applied Reactive Yellow 176

Day 0 1 2 3 6 14
Repl. 1 121.1 81.4 57.0 43.6 19.7 3.2
2 121.4 80.2 58.7 46.1 18.9 2.6
Mean 121.3 80.8 57.9 44.8 19.3 2.9

Day 37 62
Repl. 1 1.5 2.0
2 1.6 1.5
Mean 1.6 1.7

% M1 of Applied Reactive Yellow 176

Day 0 1 2 3 6 14
Repl. 1 24.6 58.4 79.0 97.0 120.7 141.2
2 26.1 59.9 78.6 91.5 118.4 141.6
Mean 25.4 59.2 78.8 94.3 119.6 141.4

Day 37 62
Repl. 1 139.6 127.9
2 138.8 126.4
Mean 139.2 127.2

Sterile Controls
% Applied Reactive Yellow 176

30 µg/L 100 µg/L
Day
Repl. 0 62 0 62
Reactive Red 239 1 120.4 < LCL 118.8 1.3
2 123.9 < LCL 116.3 1.7
Mean 122.1 - 117.6 1.5
M1 1 15.5 89.8 28.9 111.5
2 18.7 88.3 31.7 115.7
Mean 17.1 89.0 30.3 113.6

Results with reference substance:

Mineralisation of the reference item Aniline sulphate, [14C(U)]- in the reference control was already > 50% after 8 days The high mineralization indicates that the surface water contained an active microbial population..

Mineralization of Aniline Sulphate, [14C(U)]- in the Reference Control and Solvent Control
Exposure Day Reference Control
% Mineralisation
Repl. 1 Repl. 1
3 48.8 51.2
6 62.9 61.5
14 68.8 70.5

Validity criteria:

Biodegradation Reference Substance Aniline

Observed value:

> 50 % Mineralisation within 14 days

Validity criteria fulfilled:

yes

Conclusions:

not P/vP

Executive summary:

The aerobic mineralization of Reactive Yellow 176 (batch no. LK2003715) in surface water was determined in a simulation biodegradation test over a test period of 60 days according to OECD guideline 309. The study (experimental phase) was conducted from 2021-06-15 to 2021-08-16.

Transformation of Reactive Yellow 176 started directly after application. About 55 % transformation was reached within 3 days, after 14 days the detected concentration was < 10 % of the applied concentration.The test item was degraded to one metabolite with a relative retention time (RRT) of 1.45 (UPLC-DAD) to the test item.
A molecular formula of C28H22ClN9O12S4 was obtained for this peak. The experimentally obtained accurate mass was within a range of ± 5 ppm of the theoretical mass as proposed as quality indicator for a positive confirmation of a molecular formula by the guidance document SANTE/12682/2019.
This metabolite was formed by an elimination of the terminal alkyl sulfate moiety. This metabolite was also formed in the abiotic controls at the end of the study in equal quantities. This result indicates that the degradation is driven by an abiotic process.

DTx values for Reactive Yellow 176 were calculated with single first order kinetics (SFO).

30 µg/L

DT50 1.91 days
DT90 6.34 days

100 µg/L

DT50 2.05 days
DT90 6.82 days

 

Description of key information

The test substance demonstrated to be rapidly degraded in water but driven by an abiotic process.

Key value for chemical safety assessment

Half-life in freshwater:

2 d

at the temperature of:

12 °C