Mycophenolic acid

Biodegradation in water and sediment: simulation tests

Administrative data

Endpoint:

biodegradation in water: sewage treatment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 April 2017 - 22 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 314 B (Simulation Tests to Assess the Biodegradability of Chemicals in Wastewater. B: Biodegradation in Activated Sludge)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Mycophenolic acid

EC Number:

246-119-3

EC Name:

Mycophenolic acid

Cas Number:

24280-93-1

Molecular formula:

C17H20O6

IUPAC Name:

6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-2-benzofuran-5-yl)-4-methylhex-4-enoic acid

Test material form:

other: Dissolved in ethanol

Details on test material:

- Lot No.: ELN024694-MM-195
- Molecular Formula: C17H20O6
- Specific activity: 47.7 mCi/mmol
- Molecular weight: 321.87 @ 47.7 mCi/mmol
- Storage requirements: Freezer (–20 °C or below, recommended)
- Stability under correct storage conditions (expiry date): August 01, 2017; assigned by the study director

Specific details on test material used for the study:

- Storage Conditions: Freezer (–20 °C or below, recommended)

Radiolabelling:

yes

Remarks:

14C

Study design

Oxygen conditions:

aerobic

Remarks:

CO2-free air

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Details on collection: The activated sludge was sampled from a well-mixed region of the aeration basin of the sewage treatment plant (STP) in Flörsheim/Main into a collecting vessel. During transport and storage the activated sludge was continuously aerated.
- Storage conditions: Date of samling / date of arrival: April 24, 2017. Storage temperature 20.5-44.5°C (number of measurements: 18)
- Storage length: 1 day
- Temperature (°C) at time of collection: 13.5°C
- pH at time of collection: 7.0 (based on information obtained from personnel of the sewage treatment plant in Flörsheim/Main)

- Preparation of inoculum for exposure:
* Biotic activated sludge:The entire activated sludge sample was sieved through a 2 mm sieve prior to use. Based on information obtained from personnel of the sewage treatment works in Flörsheim/Main, the actual total suspended solids (TSS) concentration in the sampled activated sludge was 3480 mg/l. Thus, the TSS concentration was adjusted to a default level of 4000±500 mg/l. The solids were allowed to settle for 5 minutes and a volume of 1200 ml of the supernatant was removed. The adjusted TSS concentration of the activated sludge was then determined
to be 3965 mg/l. The pH of the activated sludge sample was determined to be 7.42 (day 0 measurement).
* Abiotic activated sludge: Abiotic (sterilised) sludge samples were prepared by autoclaving three times for at least 45 minutes at 121 °C. After autoclaving, the pH of the abiotic sludge was determined to be 7.86.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

20 µg/L

Based on:

other: nominal conc.

Initial test substance concentration 2

Initial conc.:

6 580 other: dpm/ml

Based on:

other: nominal radioactive concentration

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Details on study design:

TEST CONDITIONS
Test system: Activated sludge
(MLSS; mixed-liquor suspended solids)
Test setup: Flow-through
Biological parameters: Biodegradation (primary and ultimate)
Test duration: 28 d
Temperature: 20–25 °C, controlled at ±2 °C
Amount of MLSS per test vessel (biotic): 1518 ml (nominal)
Amount of MLSS per test vessel (abiotic): 212.6 ml (nominal)
Aeration of the test vessels: CO2-free air
Test units: 2000 ml glass bottles
Test concentrations (nominal): Solvent control; 20 µg/l, corresponding to 6580 dpm/ml
Number of replicates: 2

TEST SYSTEM
- Culturing apparatus: glass bottles closed with a screw-cap, equipped with connections for influent and effluent gas lines plus a sampling port for removing MLSS samples
- Number of culture flasks/concentration: 2 for biotic, 6 (plus 1 reserve vessel) for abiotic, 1 for solvent control
- Method used to create aerobic conditions: CO2-free air
- Test performed in open system: no, flow-through batch system
- Details of trap for CO2 and volatile organics if used: CO2: 1.5 M NaOH / volatile fatty acids (VFA): BaCl2 precipitation of the radioactivity was conducted. The radioactivity in the trapping solutions was determined by LSC. Thereafter, 20 ml 0.25 M BaCl2 solution was added to 10 ml aliquots of trapping solution (1.5 M NaOH) each, followed by precipitation of Ba14CO2.

Biotic treatments
Glass bottles (2000 ml volume) closed with a screw cap and equipped with connections for influent and effluent gas lines plus a sampling port for removing MLSS samples were used as test vessels. Based upon the number and volume of the samples needed for the assessment, 1518 ml of MLSS was filled into each test vessel and the solvent control vessel. The MLSS was stirred continuously (500 rpm) to keep it well mixed and in suspension and to ensure aerobic conditions.
The test was performed using a closed flow-through batch system. The test vessels were connected to a series of traps to capture evolved 14CO2 (containing 1.5 N NaOH) and other volatile transformation products (containing ethylene glycol, EG). The headspace of the test vessels was continuously purged with CO2-free air. CO2-free air was obtained by conducting ambient air through a washing bottle containing soda lime pellets. Empty safety traps were included (e.g. behind the test vessels) as a precaution against back-flow or condensation.

Abiotic treatments
Glass bottles (250 ml volume) closed with a screw cap with an air-tight septum made of butylrubber were used as test vessels. A nominal volume of 212.6 ml MLSS was filled into each test vessel. Abiotic (sterilised) sludge samples were prepared by autoclaving three times for at least 45 minutes at =121 °C (for details see Annex III). The test was performed using a closed static batch system. The MLSS was stirred continuously (250 rpm) to keep it well mixed and in suspension.

SAMPLING
- Sampling frequency: biotic test vessels: 8x in 28 days (0, 0.25, 2, 3, 7, 10, 14 and 28); Abiotic test vessels: 3x in 28d (0, 7 and 28/29)
- Sampling method used per analysis type: Distribution of radioactivity between liquor (aqueous phase extractable and non-extractable residues), solids (sludge extractable and non-extractable residues) and trapping solutions was determined. A 14C mass balance was established by summing up the total radioactive residues (TRR) from all samples.

Calculation of DT50/DT90 values
For each sampling the calculation of the actual amount of 14C-parent was based on the total radioactivity, which was detected in the extracts considering the respective results of the TLC analysis, i.e. the contribution of the relative peak area of the test item to the total peak area. For each sampling, the actual amount of test item and known and unknown transformation products was expressed in % of the total applied radioactivity [%aR] and of initially recovered radioactivity [%iRR]. Based on the achieved datasets the DegT50/DegT90 values of Mycophenolic acid including 95% confidence limits, chi²-values and prob>t values were calculated by means of the “CAKE” program [Tessella Ltd. 2015]. The best fit model was selected on basis of chi²-values, prob>t values and a visual check of the obtained graphs.

STATISTICAL METHODS:
Mass balance and plot of data
For each sampling time point a total 14C mass balance is reported. In addition, the percentage of dosed radioactivity recovered as parent in liquor extracts is reported. These data are plotted against time for both the biotic and abiotic treatments.
Kinetic analyses
The results of the test were fitted to kinetic models (decay models for the parent compound and production models for mineralization).
Primary degradation (parent compound)
Liquor extract 1 was quantitatively analyzed by TLC. Characterization was done by cochromatography of 14C-MPA taken from stock solution and of unlabelled transformation product MPAG. By doing so, it was possible to annotate MPA and MPAG, whereas other
peaks were not annotated to one of these compounds. They are named as unknown transformation products 1 and 2 (unknown TP 1 and TP 2).
The degradation rate (DegT50,primary) of MPA in activated sludge was determined by means of the “CAKE” program [Tessella Ltd. 2015]. First, kinetic analyses were performed using all available kinetic models, namely single first order (SFO), first order multi compartment (FOMC), hockey stick (HS) and double first order in parallel (DFOP).
Ultimate degradation (mineralization)
In addition, the mineralization of the test item as a function of time was fitted to single firstorder kinetics (SFO) and double first-order in parallel kinetics (DFOP) using the data analysis software STATISTICA, Version 13 (Dell Inc., 2017). Both kinetic models are described and recommended in the test guideline (OECD, 2008) and in FOCUS Guidance (FOCUS, 2006).
Two replicate data points for each sampling interval were used. The kinetic model with the best fit was identified in consideration of the recommendations of the FOCUS Guidance (FOCUS, 2006):
a) Visual assessment: Examination of the visual fit by plotting measured against fitted data and by plotting calculated minus measured data (residual plot).
b) Chi-square test: According to FOCUS Guidance (2006), the chi2 error value of the best-fit model should be below 15%.

Reference substance

Reference substance 1

Reference substance:

aniline

Reference substance 2

Reference substance:

benzoic acid, sodium salt

Results and discussion

Test performance:

Based on the results the study is assessed as valid.

Mean total recovery

Mean total recovery 1

Compartment:

biologically active treatment at end of test

% Recovery:

96.2

St. dev.:

3.5

Remarks on result:

other: based on initially recovered radioactivity (iRR)

Mean total recovery 2

Compartment:

abiotic control measured at end of test

% Recovery:

99

St. dev.:

4.9

Remarks on result:

other: based on the total radioactivity measured in the MLSS

% Degradation

Key result

% Degr.:

82.2

St. dev.:

5.3

Parameter:

CO2 evolution

Sampling time:

28 d

Half-life of parent compound / 50% disappearance time (DT50)

Half-life of parent compound / 50% disappearance time (DT50) 1

Key result

Compartment:

biologically active treatment at end of test

DT50:

1.7 d

Type:

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

Temp.:

22.3 °C

Remarks on result:

other: primary degradation

Half-life of parent compound / 50% disappearance time (DT50) 2

Key result

Compartment:

biologically active treatment at end of test

DT50:

2.9 d

Type:

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

Temp.:

22.3 °C

Remarks on result:

other: ultimate degradation

Transformation products:

yes

Identity of transformation products

No.:

#1

Reference

Reference substance name:

Unnamed

IUPAC name:

Mycophenolic acid-ß-D-glucuronide

CAS number:

31528-44-6

Molecular formula:

C23H28O12

Molecular weight:

496.46

Details on transformation products:

Besides mineralization to CO2, parent MPA was transformed to Mycophenolic acid-ß-Dglucuronide (max. value 0.5% iRR at day 2) and the unknown, transitory transformation products TP1 (max. value 2.8% iRR at day 3) and TP 2 (max. value 1.8% iRR at day 2). Evaluation of the sterile samples showed that the transformation of MPA is a biotic process.

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

yes

Details on results:

TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Yes/No
- Anomalies or problems encountered (if yes):

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
- Range of maximum concentrations in % of the applied amount at end of study period:
on the - the and -th day of incubation, respectively. At the end of the study period, the corresponding concentrations were - and -- % of the applied amount, respectively.

MINOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
- Range of maximum concentrations in % of the applied amount at end of study period:

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:

EXTRACTABLE RESIDUES
- % of applied amount at day 0:
- % of applied amount at end of study period:

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0:
- % of applied amount at end of study period:

MINERALISATION
- % of applied radioactivity present as CO2 at end of study:

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study:

STERILE TREATMENTS (if used)
- Transformation of the parent compound:
- Formation of transformation products:
- Formation of extractable and non-extractable residues:
- Volatilization:

RESULTS OF SUPPLEMENTARY EXPERIMENT (if any):

Results with reference substance:

The test had to be stopped after 8.09 days since the climate cabinet malfunctioned resulting in a rapid increase in temperature (>30°C).
Both compounds were rapidly mineralized within the test period of 8 days. Biodegradation was determined using the respirometric OxiTop® system (WTW, Weilheim, Germany). Mineralization kinetics were determined using the 5-parameter logistic regression model and the data analysis software STATISTICA (Dell Inc., 2016). The mean mineralization at the plateau of the curve after 8.09 days was 67.7%ThOD for aniline (n = 3, chi2 error = 4.91) and 83.5%ThOD for sodium benzoate (n = 3, chi2 error = 5.49). Based on the results, both reference compounds would be rated as readily biodegradable. Thus, the activated sludge used in the test proved to be biologically active at the start of the test.

Any other information on results incl. tables

Validity of the test

The following validity criteria are specified in the test guideline and were used by the study director to assess the validity of the study.

-Criterion: The mass balance from the abiotic treatment is used to confirm the recovery of parent from the test system. Target recoveries from the test matrix should be 85–100%. If parent recoveries from initial samples taken from the abiotic control are within this range, the sample preparation procedures are suitable for the recovery of the parent

compound from the test matrix. However, this range should not be used as a criterion for acceptance of the test.

Results: The recovery of the parent from the test matrix in the abiotic control at test start was 67.9% iRR. This can be explained by the distribution in the test system according to the 14C-mass balance, where only 67.9% iRR could be detected in the analysed liquor extract 1. However, the entire radioactivity in the liquor extracts from the abiotic control

could be attributed to parent MPA and the recovery of radioactivity at the start of the test was was 93.4% iRR. Thus, the sample preparation procedures are considered to be suitable.

-Criterion: Total recovery of radioactivity in both biotic and abiotic samples should normally range from 75% to 115% in each individual sample and average total recovery for all samples within a treatment should normally range from 85% to 110%. However, this range should not be used as a criterion for acceptance of the test.

Results:

Biotic samples

The total recovery of radioactivity based on initially recovered radioactivity (iRR) was in the range between 89.1% and 103.4% in individual samples and average total recovery for all samples was 96.2±3.5%.

Based on nominally applied radioactivity (aR), the total recovery of radioactivity was in the range between 100.4% and 117.2% in individual samples and average total recovery for all samples was 108.7±4.0%.

Abiotic samples

The total recovery of radioactivity based on the total radioactivity measured in the MLSS at the respective sampling time point was in the range between 90.1% and 103.8% in individual samples and average total recovery for all samples was 99.0±4.9%. Based on nominally applied radioactivity (aR), the total recovery of radioactivity was in

the range between 104.2% and 119.4% in individual samples and average total recovery for all samples was 113.9±5.4%.

-Criterion: If possible, the limit of detection (LOD) of the analytical method for the test substance should be =1% of the initial test concentration added to the test system. If possible, the limit of quantification (LOQ) should be =3% of the added concentration.

Results: The LOD was determined to be 0.01 [%aR] and 0.01 [%iRR], respectively. The LOQ was determined to be 0.03 [%aR] and 0.03 [%iRR], respectively.

-Criterion: When a reference substance is included, the results for the reference substance should approximate those anticipated based upon the reasons for its selection.

Results: As expected, both reference substances were rapidly mineralized in a non-GLP ready biodegradability test on the basis of OECD Guideline 301F. The mean mineralization a after 8 days was 67.7% ThOD for aniline (n = 3, chi2 error = 4.9) and 83.5%ThOD for sodium benzoate (n = 3, chi2 error = 5.5).

Based on the results the study is assessed as valid.

Applicant's summary and conclusion

Validity criteria

Validity criteria fulfilled:

yes

Conclusions:

The test item 14C-Mycophenolic acid (MPA) was rapidly degraded in biotic activated sludge within a test period of 28 days. Besides mineralization to CO2 (82.2 ± 5.3% iRR at the end of the test), parent MPA was transformed to Mycophenolic acid-ß-D-glucuronide (MPAG; max. value 0.5% iRR at day 2) and the unknown, transitory transformation products TP1 (max. value 2.8% iRR at day 3) and TP 2 (max. value 1.8% iRR at day 2). Evaluation of the sterile samples showed that the transformation of MPA is a biotic process. The calculated DegT50,primary value is 1.7 days, the calculated DegT90,primary value is 5.5 days for both, [%aR] and [%iRR] by SFO kinetics. The calculated DegT50,ultimate value is 2.9 days based on [%iRR] (DFOP kinetics).

Executive summary:

Report: Thomas Junker, Monika Herrchen: "Mycophenolic acid, [carboxyl-14C]: A Study on the Biodegradation in Activated Sludge "

Source: ECT Oekotoxikologie GmbH, unpublished report No.: 17AZ2BW

December 22, 2017

Guideline: OECD Guideline 314B, "Simulation tests to assess the biodegradability

of chemicals discharged in wastewater – Biodegradation in Activated

sludge ", October 03, 2008

GLP: Yes (certified laboratory)

Dates of experimental work (biological phase):

Experimental Starting Date: April 24, 2017

Experimental Completion Date (sampling sterile day 28): June 01, 2017

Experimental Completion Date (latest LSC measurements): August 16, 2017

Test item:

- Name of the test item: Mycophenolic acid, [carboxyl-14C]

- Chemical name: 6-(1,3-Dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoic acid

- Molecular Formula: C17H20O6

- Sponsor’s number: RO1005797-901-002

- Lot No.: ELN024694-MM-195

- Radiochemical purity: >98%

- Specific activity: 47.7 mCi/mmol

- Molecular weight: 321.87 @ 47.7 mCi/mmol

Material and methods:

Test system: Activated sludge (MLSS; mixed-liquor suspended solids)

Test setup: Flow-through

Biological parameters: Biodegradation (primary and ultimate)

Test duration: 28 d

Temperature: 20–25 °C, controlled at ±2 °C

Amount of MLSS per test vessel (biotic): 1518 ml (nominal)

Amount of MLSS per test vessel (abiotic): 212.6 ml (nominal)

Aeration of the test vessels: CO2-free air

Test units: 2000 ml glass bottles

Test concentrations (nominal): Solvent control; 20 µg/l, corresponding to 6580 dpm/ml

Number of replicates per concentration (biotic): 2

Number of replicates per concentration (abiotic): 6 (plus one reserve vessel)

Number of replicates in the solvent control: 1

Quantification of radioactivity associated with parent compound: Thin-layer chromatography (TLC)

In accordance with GLP regulations the biodegradation of the test item in activated sludge (MLSS, mixed-liquor suspended solids) was assessed within a test period of 28 days. The study was performed according to the OECD Guideline 314B, "Simulation tests to assess the biodegradability of chemicals discharged in wastewater – Biodegradation in Activated sludge". In addition, the level of parent compound and potential degradation products was determined in liquor extracts using chromatographic separation (thin-layer chromatography) and radioanalytical detection methods.

Findings:

Biotic activated sludge

The radioactivity measured at day 0 in the test vessels was 7412.5 dpm/ml (112.7% of nominal; replicate A) and 7457.0 dpm/ml (113.3% of nominal; replicate B), corresponding to a test item concentration of 22.5 µg/l and 22.7 µg/l, respectively.

Based on initially recovered radioactivity (iRR), the mean recovery per sampling time point was between 92.6%iRR (day 28) and 101.3%iRR (day 2). The overall mean recovery for all sampling time points was 96.2±3.5%iRR.

Based on nominally applied radioactivity (aR), the mean recovery per sampling time point was between 104.6%aR (day 28) and 114.5%aR (day 2). The overall mean recovery for all sampling time points was 108.7±4.0%aR. The test item was rapidly mineralized. Mineralization of the test item started directly after application and increased rapidly at the beginning and more slowly afterwards up to 82.2%iRR at day 28. Mineralization comprises evolved CO2 (detected in the traps) as well as CO2 dissolved in the MLSS. The latter was highest at day 2 and decreased afterwards, whereas the amount of evolved CO2 increased continuously until the end of the study.

At the start of the test (day 0), 82.5%iRR was detected in the liquor extracts. Subsequently, the radioactivity in the liquor extracts decreased to 1.2% iRR at the end of the test. At no time point could a noteworthy amount of radioactivity (>2% iRR throughout the test period) be detected in the liquor after extraction. The amount of radioactivity in the solid extract was

highest at day zero (5.5% iRR) and decreased afterward to 0.3% iRR after 28 days. The amount of NER increased at the beginning of the study up to 10.2% iRR at day 2 and did not change substantially until day 14 (range between 11.1% iRR and 13.1% iRR). At the

end of the test period (day 28) the amount of NER was 7.3%iRR.

Abiotic activated sludge

For the abiotic controls, duplicate test vessels were sacrificed at each sampling time point. The radioactivity measured at the sampling time points in the duplicate test vessels was between 7491 dpm/ml (113.8% of nominal) and 7673 dpm/ml (116.6% of nominal), corresponding to 22.8 µg test item/l and 23.3 µg test item/l, respectively. Based on the total radioactivity measured in the MLSS at the respective sampling time point, the mean recovery was between 93.4% and 102.6%. Based on nominally applied

radioactivity, the recovery measured at the sampling time points was in the range between 107.6%aR and 117.4%aR (mean of duplicates). The mean total recovery for all sampling time points (mean of duplicates) was 99.0±4.9% based on measured radioactivity and 113.9±5.4%aR based on nominally applied radioactivity.

Primary degradation (parent compound)

Besides mineralization to CO2, parent MPA was transformed to Mycophenolic acid-ß-Dglucuronide (max. value 0.5% iRR at day 2) and the unknown, transitory transformation products TP1 (max. value 2.8% iRR at day 3) and TP 2 (max. value 1.8% iRR at day 2).

Evaluation of the sterile samples showed that the transformation of MPA is a biotic process. The degradation rate (DegT50,primary) of MPA in activated sludge was determined by means of the “CAKE” program. The disappearance of MPA in activated sludge could be described with a Chi-square (¿2) value of 5.82 (for basis [%aR]) and 5.85 (for basis [%iRR]) using SFO

kinetics. The calculated DegT50,primary value is 1.7 days, the calculated DegT90,primary value is 5.5 days for both, [%aR] and [%iRR] basis.

Ultimate degradation (mineralization)

The best fit for mineralization data was obtained for the DFOP model. The data are well described by the DFOP kinetic and the curve fits closely to the measured data. The residuals are small and they are randomly scattered around the zero line. Chi2 error is 2.09 (non-GLP). The calculated DegT50,ultimate value is 2.9 days.

Conclusions

The test item 14C-Mycophenolic acid (MPA) was rapidly degraded in biotic activated sludge within a test period of 28 days. Besides mineralization to CO2 (82.2 ± 5.3% iRR at the end of the test), parent MPA was transformed to Mycophenolic acid-ß-D-glucuronide (MPAG; max. value 0.5% iRR at day 2) and the unknown, transitory transformation products TP1 (max. value 2.8% iRR at day 3) and TP 2 (max. value 1.8% iRR at day 2). Evaluation of the sterile samples showed that the transformation of MPA is a biotic process. The calculated DegT50,primary value is 1.7 days, the calculated DegT90,primary value is 5.5 days for both, [%aR] and [%iRR] by SFO kinetics. The calculated DegT50,ultimate value is 2.9 days based on [%iRR] (DFOP kinetics).

Validity of the Test

The recovery of the parent from the test matrix in the abiotic control at test start was 67.9% iRR. This can be explained by the distribution in the test system according to the 14C-mass balance, where only 67.9% iRR could be detected in the analysed liquor extract 1. However, the entire radioactivity in the liquor extracts from the abiotic control could be attributed to parent MPA and the recovery of radioactivity at the start of the test was was 93.4% iRR. Thus, the sample preparation procedures are considered to be suitable.

In biotic activated sludge the total recovery of radioactivity based on initially recovered radioactivity (iRR) was in the range between 89.1% and 103.4% in individual samples and average total recovery for all samples was 96.2±3.5%. Based on nominally applied radioactivity, the total recovery of radioactivity was in the range between 100.4% and 117.2% in individual samples and average total recovery for all samples was 108.7±4.0%. In abiotic activated sludge the total recovery of radioactivity based on the total radioactivity measured in the MLSS at the respective sampling time point was in the range between 90.1% and 103.5% in individual samples and average total recovery for all samples was 99.0±4.9%. Based on nominally applied radioactivity, the total recovery of radioactivity was in the range between 104.2% and 119.1% in individual samples and average total recovery for all samples was 113.9±5.4%.

The LOD was determined to be 0.01 [%aR] and 0.01 [% iRR], respectively. The LOQ was determined to be 0.03 [%aR] and 0.03 [% iRR], respectively.

As expected, both reference substances were rapidly mineralized in a non-GLP ready biodegradability test on the basis of OECD Guideline 301F. The mean mineralization a after 8 days was 67.7%ThOD for aniline (n = 3, chi2 error = 4.9) and 83.5%ThOD for sodium benzoate (n = 3, chi2 error = 5.5).

Based on the results the study is assessed as valid.