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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 April - 17 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was carried out in accordance with OECD guideline no. 301F and Good Laboratory Practice.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
(1992)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
(2S)-2-(1,3-DIOXO-2,3-DIHYDRO-1H-ISOINDOL-2-YL)PENTANEDIOIC ACID
EC Number:
608-945-6
Cas Number:
340-90-9
Molecular formula:
C13H11NO6
IUPAC Name:
(2S)-2-(1,3-DIOXO-2,3-DIHYDRO-1H-ISOINDOL-2-YL)PENTANEDIOIC ACID
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): Phthalyl-L-Glu
- Molecular formula: C13H11NO6
- Molecular weight: 277.23 g/mol
- CAS number: 340-90-9
- Appearance: White solid
- Theoretical Oxygen Demand: ThOD(NH4): 1.39 mg oxygen per mg test item, ThOD(NO3): 1.62 mg oxygen per mg test item

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (sewage plant Rossdorf, Germany) was used.
The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was resuspended in tap water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated overnight. This suspension was used for the experiment.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
103 mg/L
Based on:
test mat.
Initial conc.:
143 mg/L
Based on:
other: THOD(NH4)
Initial conc.:
167 mg/L
Based on:
other: ThOD(NO3)
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral medium according to OECD301F
- Test temperature: 22 ± 1 °C
- pH: 7.2 - 7.6
- pH adjusted: no
- Suspended solids concentration: 29 mg/L

TEST SYSTEM
- Culturing apparatus: Manometric Test System with 500 mL flasks
- Number of culture flasks/treatment: 2
- Measuring equipment: BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany
- Test performed in closed vessels: Yes, the test flasks were closed gas-tight by a measuring head
- Details of trap for CO2: 45% Potassium hydroxide solution (this trap was only for adsorbing, it was not analysed)

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 flasks
- Abiotic sterile control: Yes
- Toxicity control: Yes
- Procedural control (with reference substance): Yes

PREPARATION OF THE FLASKS
Test substance and/or reference substance samples (~25 mg) were weighed and added directly to the test flasks.
In the abiotic sterile control flask 5 mL of a 1 g/L HgCl solution was added
Then all test flasks, except for the abiotic sterile control flask were filled with test medium inoculated with 2 mL of a 3.5 g/L suspended sludge solution to a total volume of 244 mL. The abiotic sterile contriol flask was was filled with test medium to a total volume of 244 mL.

The closed test flasks were incubated in a climatised room under continuous stirring.

MEASUREMENTS

Oxygen:
The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel, which was measured daily with the BSB/BOD sensor.

Temperature:
The temperature was recorded by means of the automated software AMR Wincontrol©.

pH-Value:
pH-values were measured in procedure control, a separately prepared test flask with test item (to prevent loss of test item in the test flasks) and a separately prepared test flask without test item (control) at test start and in all flasks at the end of the test, except in the abiotic and toxicity control, using a pH-electrode WTW pH 340i.

Determination of nitrification:
For N-containing test items as Phthalyl-L-Glu, a correction for a potential uptake of oxygen by nitrification was made. For this reason, on exposure day 0 a sufficient aliquot was withdrawn from the bottles containing the test item and inoculum, from the inoculum control (after measurement of
oxygen concentration) for analysis of nitrate and nitrite using Continuous Flow Analysis. At day 28 an aliquot from both inoculum controls, and both test item flasks was taken for analysis. Due to an increase in the concentration of nitrate and nitrite in the test bottles, the oxygen uptake by nitrification was calculated as described in Annex V of the test guidelines.
Samples were taken of a control and a test item treated flask at test start and stored deep frozen (=-10°C) until nitrate determination was done. At test end, samples were taken of a control and test item treated flask including toxicity control and the nitrification was determined.
The nitrogen determination was done using a AA3 Continuous Flow Analyzer and equipment.
Reference substance
Reference substance:
benzoic acid, sodium salt
Remarks:
ThOD(NH4): 1.67 mg oxygen per mg sodium benzoate (calculated)

Results and discussion

% Degradation
Key result
Parameter:
% degradation (O2 consumption)
Value:
85
Sampling time:
28 d
Remarks on result:
other: corrected for nitrification
Details on results:
The test item Phthalyl-L-Glu contains nitrogen; therefore the evaluation of biodegradation has to be based on ThOD(NH4) and ThOD(NO3).
The mean biodegradation of at least 10% of Phthalyl-L-Glu was reached at day 9 (start of the 10-day window), when the mean degradation was 14% and 12%
based on ThOD(NH4) and on ThOD(NO3), respectively. At the end of the 10-day window at day 19, the degradation of Phthalyl-L-Glu was 86% based on ThOD(NH4) and 73% based on ThOD(NO3) and therefore the 10 day window criterion was passed.
The mean biodegradation at test end after 28 days was 96% based on ThOD(NH4) and 83% based on ThOD(NO3).
The nitrate concentration in the controls after 28 days of incubation was 2.3 mg/L Nitrate-N (mean). The nitrate concentration in the test item treated
vessels after 28 days was 6.3 mg/L Nitrate-N (mean). Therefore, nitrification occurred and results were corrected for nitrication in accordance with Annex V of the test guidelines.
The degradation of Phthalyl-L-Glu corrected for nitrification was 85% after 28 days

BOD5 / COD results

Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to 83% after 14 days and to 89% after 28 days of incubation.: the activity of the inoculum was thus verified (validity criterion).

Any other information on results incl. tables

The oxygen demand in the abiotic control was 0 mg/L during the test duration. There was no use to correct the degradation of the test item and toxicity control.

The validity criteria were fulfilled as follows:

Inoculum Control:

The oxygen demand of the inoculum control (medium and inoculum) was 15 mg O2/L and thus not greater than 60 mg

O2/L within 28 days as required by the test guideline.

pH-Value:

The pH-value of the test item flasks at the end of the test was 7.5 and therefore within the range of pH 6.0 to 8.5 as

required by the test guideline.

Reference Item:

The percentage degradation of the reference item should reach the level for ready biodegradability (>60%) within 14

days as required by the test guideline. The reference item sodium benzoate was degraded to more than 60% after 4 days of incubation.

Test Item:

The difference of duplicate values for the degradation of the test item at the plateau, and at the end of the 10-day window

was less than 20%. The difference of duplicate values at the end of the 10-day window and at day 28 was 0%

and 2.6%, respectively.

Toxicity Control:

If in a toxicity test, containing both the test substance and a reference compound less than 25% biodegradation (based

on total ThOD) occurred within 14 days, the test substance can be assumed to be inhibitory. The biodegradation was

52% (ThOD(NH4)) at day 14; the test item was not toxic.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See above
Interpretation of results:
readily biodegradable
Conclusions:
The test substance is ready biodegradable.
Executive summary:

The ready biodegradability of the test substance was investigated according to OECD guideline no. 301F and Good Laboratory Practice.

The test item Phthalyl-L-Glu contains nitrogen; therefore the evaluation of biodegradation has to be based on ThOD(NH4) and ThOD(NO3).

The mean biodegradation of at least 10% of Phthalyl-L-Glu was reached at day 9 (start of the 10-day window), when the mean degradation was 14% and 12% based on ThOD(NH4) and on ThOD(NO3), respectively. At the end of the 10-day window at day 19, the degradation of Phthalyl-L-Glu was 86% based on ThOD(NH4) and 73% based on ThOD(NO3) and therefore the 10 day window criterion was passed.

The mean biodegradation at test end after 28 days was 96% based on ThOD(NH4) and 83% based on ThOD(NO3).

The nitrate concentration in the controls after 28 days of incubation was 2.3 mg/L Nitrate-N (mean). The nitrate concentration in the test item treated vessels after 28 days was 6.3 mg/L Nitrate-N (mean). Therefore, nitrification occurred and results were corrected for nitrication in accordance with Annex V of the test guidelines.

The degradation of Phthalyl-L-Glu corrected for nitrification was 85% after 28 days

Based on these results it can be concluded that the test substance is ready biodegradable.

The test substance was not inhibitory to the inoculum.

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