Phenoxymethylpenicillin potassium

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Item: Pen V Potassium
Lot No.: B519322
Appearance: White, solid powder
Expiry date: 30 April 2024
Storage: Room temperature, protected from light

Oxygen conditions:

anaerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Species: Activated sludge, microorganisms from a domestic waste water treatment plant.

Origin: The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 27 September 2019 (seven days before the test). The prepared activated sludge was continuously aerated (2L/minute) at the test temperature of 22±2oC, for about 7 days until use.

Preparation of Activated Sludge Inoculum:
The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 5 g dry material per litre was mixed with mineral medium (see Section 5.4) and then aerated under test conditions until use. The pH of the activated sludge inoculumafter preparation was: 7.43, just before use: 7.09. A pH adjustment of activated sludge inoculum was not performed.

Pre-conditioning of Activated Sludge Inoculum:
Pre-conditioning consisted of aerating (2L/minute) activated sludge (in mineral medium1) for 7 days at the test temperature. During the aeration the cell count of inoculum was checked as follows: the viability of the cultured sludge was determined by plating 0.1 mL of the different, usually 10^-1, 10^-2, 10^-3 and 10^-4 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by these plating experiments by manual colony counting. The approximately cell count of aerated inoculum fell in the range of ~10^10 cells/L; therefore, on the day of the test this inoculum was diluted 1000000 x with mineral medium to reach the necessary ~10^4-10^5cells/L cell concentration. After preparation the sludge was filtered through cotton wool. Pre-conditioning improves the precision of the method. The inoculum was not pre-adapted to the test chemical.
The approximately cell count of applied, aerated inoculum was 9.17 x 10^5/L, fell in the range of 10^4-10^6/L.

Duration of test (contact time):

28 d

Initial conc.:

3.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Environmental Conditions
The test was carried out in a controlled environment room (during the formulation and oxygen measuring) at a temperature of 22 ± 2°C according to the guideline. The test bottleswere incubated in an incubator at 22 ± 2°C, in the dark. Temperature was measured continuously using min/max thermometer (in controlled environment room) or built-in thermometer (in incubator) and recorded at least once a day.During pre-conditioning of activated sludge inoculum t he temperature was 20.0-21.1oC; during the preparation, aeration and incubation of the mineral medium, preparation of test bottles (units), the temperature was 20.1-20.8oC. During the incubation (28 days) of the test units the temperature range was the following: 20.1-21.0oC. The oxygen concentration of test water (mineral medium) was in the range of 8-9 mg/L. It was measured at the start of the test and found to be 8.39 mg/L.The pH was checked prior study start and found to be 7.43. A pH adjustment was considered as not necessary. The test conditions were measured with suitable instruments and documented in the raw data.

Preparation of the Test Solutions
For the preparation of test item test solutions, at first the suitable amount (175 mg) of Pen V Potassium was dissolved in the respective volume (500 mL) of aqueous test medium (mineral medium) to prepare a 350 mg/L stock solution. The test item stock solution was continuously stirred until use to ensure a good dispersion and homogeneity (extra care was taken to avoid air bubbles in the stirred solution). During the incubation period the test solutions were stirred. The stock solution was adequately diluted in the test item containing test groups. The test solutions were freshly prepared in the testing laboratory at the beginning of the experiment. Reference item stock solution: In the present study 500 mL stock solution with a concentration of 300 mg/L was prepared: 0.15 g of sodium benzoate was dissolved in 500 mL of mineral medium.

The Test Groups
1.) Test Item (flasks 1a and 1b):

- 60 mL test item stock solution (350 mg/L, according to the calculated theoretical oxygen demand ThODNH3 assuming that no nitrification occurs of 1.48 mg O2/mg test item) [ In 3.5 mg/L test item concentration, respectively the ThODNH3 corresponds to about 3.5 x 1.48 = 5.18 mg O2]
- 12 mL activated sludge inoculum, ad. 6000 mL mineral medium. 4.5 mg/L test item concentration corresponded to about 4.5 x 1.24 = 5.58 mg O2/L.

2.) Procedure Control, Sodium benzoate (flasks 2a and 2b):
- 60 mL reference item stock solution (300 mg/L), (ThODNH3 of reference item: 1.67 mg O2 per mg) [ In 3.0 mg/L reference item concentration, respectively the ThODNH3 corresponded to about 3.0 x 1.67 = 5.01 mg O2/L]
- 12 mL activated sludge inoculum (final concentration: 2 mL/L),
- ad. 6000 mL mineral medium;

3.) Inoculum Control (flasks 3a and 3b):
- 12 mL activated sludge inoculum (final concentration: 2 mL/L),
- ad. 6000 mL mineral medium;

4.) Toxicity Control (flasks 4a and 4b):
- 60 mLtest item stock solution (350 mg/L,
- 60 mL reference item stock solution (300 mg/L)
- 12 mL activated sludge inoculum
- ad. 6000 mL mineral medium;

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

4.9

Sampling time:

28 d

Details on results:

Biodegradation of the Test Item:
Under the test conditions the percentage biodegradation of the test item reached a mean of 32.1 % after 28 days based on its ThODNH3. Therefore the test item can be considered to be not ready biodegradable.
See also the attachment.

Results with reference substance:

The reference item Sodium benzoate was sufficiently degraded to a mean of 80.3 % after 14 days, and to a mean of 85.6 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum

Correction for Oxygen Uptake for Interference with Nitrification:

Errors due to not considering nitrification in the assessment by oxygen uptake of the biodegradability of test substances not containing N are marginal (not greater than 5%), even if oxidation of the ammonium N in the medium occurs erratically as between test and blank vessels. However, for test substances containing N, serious errors can arise if the observed oxygen uptake is not corrected for the amount of oxygen used in oxidising ammonium to nitrite and nitrate. For that reason, at this N-containing test item, the oxidised nitrogen (nitrate and nitrite) concentrations were determined following each oxygen measurement with photometric method using nitrite and nitrate cell tests. The LOQ (Limit Of Quantification) of the measurements was 0.03 mg NO2/L and 0.4 mg NO3/L, respectively. The nitrate concentration of the samples was less than 0.4 mg/L in all measurement occasions, throughout the study. The nitrite concentration in the 7-, 14-, 21-, and 28-day samples (test item, inoculum control and toxicity control) was below the LOQ. At the start (0-day) measurements 0.04 mg/L nitrite concentrations were measuered in the test item samples, in average 0.05 mg/L in the inoculum control samples and in average 0.06 mg/L in the toxicity control samples. According to the referred OECD 301 guideline (Annex V) the oxygen consumed in nitrate formation approximates 4.57 x increase of nitrate-N concentration, and the oxygen consumed in nitrite formation is 3.43 x increase of nitrite-N concentration. In this study any relation between the change of the measured dissolved oxygen concentrations in the inoculum control, test item and procedure control bottles and the measured slightly higher nitrite concentrations (that corresponds to consumed oxygen of ammonium oxidation processes) in the start samples could not be established. In this study the measured nitrite concentrations were in the evaluable range exclusively in the start (0-day) samples and increasing tendencies in nitrite and/or nitrate concentrations were not established. The oxygen uptake resulting from a possible ammonium oxidation did not influence the amount of oxygen taken up by microbial population. Therefore, any correction of the measured dissolved oxygen concentrations was considered as not possible. The measured relatively higher nitrite concentration values at the start samples were caused likely by a technical effect (possible discoloration of the solutions and/or turbidity).

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The biodegradation of the test item reached 4.9 % after 28 days.

Executive summary:

The test item was considered to be not readily biodegradable (4.9 % biodegradation on day 28). According to the test guidelines the pass level for ready biodegradability is 60 % of ThODNH3. The relationship between oxygen uptake resulting from a possible ammonium oxidation (measured exclusively in the start (0-day) test item, inoculum control and toxicity control samples) and oxygen uptake of applied microbial population was equivocal, any correction of the measured dissolved oxygen concentrations was considered as not possible. Most likely technical effects (possible discoloration of the solutions and/or turbidity) influenced the nitrite concentration determinations.According to the test guidelines the test item can be assumed as not inhibitory on the activated sludge microorganisms because the degradation in the toxicity control group was 29.4 % within 14 days, and therefore higher than 25 %. The percentage biodegradation of the reference item (80.3 % after 14 days) confirmsthe suitability of the used activated sludge inoculum.

Description of key information

The biodegradation of the test item reached 4.9 % after 28 days.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

[Type of water: freshwater]