Phenoxymethylpenicillin potassium

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1 October 2019 - 26 November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

2010

Deviations:

yes

Remarks:

Only the preliminary test is reported in this record.

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

2016

Deviations:

yes

Remarks:

Only the preliminary test is reported in this record.

Qualifier:

according to guideline

Guideline:

other: OCSPP 850.330 Modified Activated Sludge, Respiration Inhibition Test

Version / remarks:

2012

Deviations:

yes

Remarks:

Only the preliminary test is reported in this record.

Principles of method if other than guideline:

A preliminary tests were performed showing a significant inhibition of oxygen consumption by the test substance. The preliminary test was used to estimate the range of concentrations of the test item needed in a possible definite test. A definitive test will be performed later and will be reported in an own record.

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

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Defined amounts of the test item (1 x 3; 1 x 30 mg and 3 x 300 mg test item that corresponded to the investigated 10, 100 and 1000 mg/L concentrations) were directly weighed (administered) into each test flask. The subsequent calculations refer to the initial weighed nominal concentration. Concentrations in excess of nominal 1000 mg test item/L were not tested.

Test organisms (species):

activated sludge, domestic

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 02 October 2019 (one day before the test).
Preparation:
The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed centrifuged and the supernatant liquid phase was d ecanted. The solid material was re-suspended in isotonic saline solution with shaking and the wash-water was removed by centrifuging again. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed (5.985 g wet weight), dried and the ratio of wet sludge to dry weight (0.5531 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (24 g dry weight that was equivalent to 259.7 g wet sludge) was suspended in isotonic saline solution (ad. 8 L) to yield a concentration equivalent to about 3 g per litre (on dry weight basis). (In the test containers (300 mL final volume) the final concentration of suspended solids, containing 150 mL inoculum was 1.5 g per litre on dry weight basis.) The activated sludge was not used on the day of the collection but continuously aerated(2 L/minute) at the test temperature for about 24 hours (1 day) and was fed once with 50 mLsynthetic sewage/L activated sludge. The pH of the activated sludge inoculum was checked after preparation (pH: 7.46), pH adjustment of the inoculum was considered not necessary.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

no

Hardness:

Not determined.

Test temperature:

20 ± 2 °C

pH:

pH 7 - 8

Dissolved oxygen:

Aeration with compresed air at 0.5 L/min

Salinity:

/

Conductivity:

/

Nominal and measured concentrations:

0 - 10 - 100 - 1000 mg/L nominal.

Details on test conditions:

Blank Control (CB): In this preliminary test eight replicates of blank control group (water, synthetic sewage and inoculum, but without addition of the test or reference item) were tested concurrently, four at the start and four at the end of the test series.
Abiotic Control (CA):Abiotic controls (water, synthetic sewage and the test item at the concentration of 1000 mg/L, but without inoculum) were prepared to measure the abiotic oxygen consumption. In this preliminary test the abiotic control was tested at the highest test item concentration (for that reason 3 x 300 mg test item was directly weighed into assigned containers) in three parallels.
Reference Control (R):Concurrent to the test item, the reference item 3,5-Dichlorophenol was tested at three concentrations (the nominal test concentrations of 2, 7 and 24.5 mg/L) under otherwise identical test conditions.
Nitrification Control (CN):In order to decide whether the sludge nitrifies and at what rate, mixtures in two parallels (same as the blank controls, however, containing 11.6 mg/L N-allylthiourea) were included in the preliminary experiment.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol. Nitrification inhibitor: N-allythiourea (ATU).

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

ca. 1 597.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: The EC50 (based on the inhibition of the oxygen consumption rate) was estimated Probit analysis, IBM® SPSS® Statisticssoftware.

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

ca. 5.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: The EC10 was calculated by means of Probit analysis, IBM® SPSS® Statistics software.

Duration:

3 h

Dose descriptor:

NOEC

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

not determinable

Remarks:

Due to the statistically significant differences (2-Sample T-test (α=0.05)) in the comparison with the blank control and 1000 mg/L concentration values exact NOEC value could not bedetermined.

Details on results:

Nitrification Controls: Additional nitrification control was examined in the test with two parallels to check the possible nitrification potential of the applied activated sludge batch. With the applying of the nitrification control the differentiation between the total, heterotrophic and nitrification respiration was possible. The total respiration (RT) was 66.40 mg/Lh, the heterotrophic respiration (RH) was 66.80 mg/Lh, the nitrification respiration (RN): -0.40 mg/Lh was calculated according to the equation: RN= RT-RH. The obtained -0.40 mg/Lh was considered as not significant difference, being within a biological variability range of the applied test system, that lower than the 5 % of RT (3.32 mg/Lh) in blank controls. According to the above calculations and based on the OECD 209 guideline it was assumed that the heterotrophic oxygen uptake equals the total uptake.

Test substance:
The observed oxygen consumption rates consequently the specific respiration rates were inhibited by 10 % at the lowest examined concentration of 10 mg/L, 33 % inhibition was obtained at the concentration of 100 mg/L and 42 % at the highest test item concentration of 1000 mg/L. Based on measured oxygen consumption values and calculated specific respiration rates definite test item effect was established.

Results with reference substance (positive control):

Positive reference control:
The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 15.66 mg/L, (95 % confidence limits: 12.37-21.12 mg/L).

This test was performed without pH adjustment. The measured pH values in the prepared solutions: 3,5-Dichlorophenol reference control stock solution, synthetic sewage, activated sludge inoculum, prepared test mixtures etc. were in the acceptable pH 7 - 8 range at the start of the test. The mixtures were aerated at 0.5 L/min in the temperature range of 20 ± 2 oC. The temperature in the test mixtures during the measurements in average: 21.4 oC, the measured minimum: 20.6 °C, maximum: 22.0 °C.

Validity criteria fulfilled:

yes

Conclusions:

Based on the measured oxygen consumption values and the calculated specific respiration rates, unequivocal, dose-related effect of the test item was established.

Executive summary:

The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions, according to the OECD guideline 209.

During the performance of the pre-experiment strong test item effect on the pH was realized at the test item concentration of 1000 mg/L. Due to the statistically significant differences (2-Sample T-test (α=0.05)) in comparison with the blank control and 1000 mg/L concentration values an exact NOEC value could not be determined. The 3-hour EC10 value of the test item is < 1000 mg/L (EC10 within the range: 10 mg/L to 100 mg/L); and the 3-hour EC50 and EC80 values are higher than 1000 mg/L. In conclusion, this preliminary test results demonstrate the significant inhibition of oxygen consumption by the test item at the highest concentration of 1000 mg/L examined; therefore, in line with OECD 209 Guideline a definite test was considered as necessary. The definite test will be performed under a new study code; the concentration levels to be examined in the definite test will be based on the results of the present preliminary study.

Reference 2

Endpoint:

activated sludge respiration inhibition testing

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

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OCSPP 850.330 Modified Activated Sludge, Respiration Inhibition Test

Version / remarks:

2012

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.

Analytical monitoring:

no

Remarks:

In this test analytical concentration measurements did not follow the actual test item concentrations, because of the test item direct addition.

Vehicle:

no

Details on test solutions:

In the main test six concentrations in a geometric series with five replicates were tested. The examined concentrations were: 3.2; 10; 32; 100; 320 and 1000 mg/L. Concentrations in excess of nominal 1000 mg test item/L were not tested.Defined amounts of the test item (5 x 1 mg; 5 x 3 mg; 5 x 9.6 mg; 5 x 30 mg; 5 x 96 mg and 5 x 300 mg) were directly weighed (administered) into each test flask and the subsequent calculations refer to the initial weighed nominal concentrations.

Test organisms (species):

activated sludge, domestic

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 09 December 2019 (one day before the test).
Preparation of Activated Sludge Inoculum:
The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed centrifuged and the supernatant liquid phase was d ecanted. The solid material was re-suspended in isotonic saline solution with shaking and the wash-water was removed by centrifuging again. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed (5.228 g wet weight), dried and the ratio of wet sludge to dry weight (0.5158 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (30 g dry weight that was equivalent to 304 g wet sludge) was suspended in isotonic saline solution (ad. 10 L) to yield a concentration equivalent to about 3 g per litre (on dry weight basis). (In the test containers (300 mL final volume) the final concentration of suspended solids, containing 150 mL inoculum was 1.5 g per litre on dry weight basis.) The activated sludge was not used on the day of the collection but continuously aerated(2 L/minute) at the test temperature for about 24 hours (1 day) and was fed once with 50 mLsynthetic sewage/L activated sludge. The pH of the activated sludge inoculum was checked after preparation (pH: 7.48), pH adjustment of the inoculum was considered not necessary.
Foaming:
In this test the occurring foaming was not significant, controlling was not necessary during the incubation.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

no

Hardness:

Not determined.

Test temperature:

The test was carried out in a controlled environment room (during the incubation, during the formulation and oxygen concentration measuring) at a temperature of 20 ± 2 °C according to the guideline. The recorded temperatures in the environmental room varied between: 20.3 - 21.1 °C. The temperature was measured in the controlled environment room with a min/max thermometer during the incubation period. The water temperature was recorded during the oxygen measurement in all test bottles.

pH:

The pH and the oxygen concentrations were determined in all test vessels before the inoculum addition, at the start (just after the inoculation) and at the end of the incubation period in all test concentrations, reference item concentrations and controls. The results of the pH and dissolved oxygen concentration measurements are summarized in the attached file [Tables 3 and 4].

Dissolved oxygen:

The test flasks were aerated with compressed air (0.5 L/minute).

Salinity:

/

Conductivity:

/

Nominal and measured concentrations:

0 - 3.2 - 10 - 32 - 100 - 320 - 1000 mg/L nominal. Five replicates were tested. Concentrations in excess of nominal 1000 mg test item/L were not tested.

Details on test conditions:

The test item Pen V Potassium was investigated in a preliminary test (Study number: 880-209-4611). The preliminary test was performed to estimate the range of concentrations of the test item needed in a possible main test for determining the inhibition of oxygen consumption. The test item was investigated in the preliminary test at the nominal concentrations of 10; 100 and 1000 mg/L. The nitrification respiration was not significant in the preliminary test and it can be assumed that the heterotrophic oxygen uptake equals the total uptake and no significant nitrification was occurring. In the preliminary test no abiotic oxygen consumption was noticed. The observed oxygen consumption rates consequently the specific respiration rates were inhibited by 10 % at the lowest examined concentration of 10 mg/L, 33 % inhibition was obtained at the concentration of 100 mg/L and 42 % at the highest test item concentration of 1000 mg/L. The preliminary experiment was performed without pH adjustment. The test item did not have any effect on the pH within the test system and additional neutralization step of test item containing mixtures before inoculum addition was not necessary. The preliminary test results demonstrate a statistically significant inhibition of oxygen consumption by the test item at the highest examined concentration of 1000 mg/L; therefore, in line with OECD 209 Guideline the performance of the main test was considered as necessary.

Controls Blank Control (CB): In the main test eight controls (according to the number of the available O2 electrodes) four at the start and four at the end of the test series were investigated.
Abiotic Control (CA):The abiotic control was not repeated because no oxygen uptake was observed in the abiotic control group in the preliminary test.
Reference Control (R):The reference item 3,5-Dichlorophenol was tested at three concentrations with threeparallels (at the nominal test concentrations of 2, 7 and 24.5 mg/L) under otherwise identical test conditions; as the test mixtures.
Nitrification Control (CN):The nitrification respiration was not significant in the preliminary test and it was assumed that the heterotrophic oxygen uptake equals the total uptake and no significant nitrification was occurring. The main test was performed in one set of test vessels, without ATU addition, but for informative reason a nitrification control group (containing N-all ylthiourea) was investigated with three parallels.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol. Nitrification inhibitor: N-allythiourea (ATU).

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

ca. 6.37 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: (95 % conf. limits: 1.82–13.71 mg/L)

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

ca. 2 411.59 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

119 018.04 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Based on the calculated oxygen consumption rates, slight, in average 3.40 % inhibition (well within the biological variability range of the applied test system) was noticed already at the lowest examined concentration of 3.2 mg/L. The observed-calculated oxygen consumption rates and corresponding inhibitory tendencies showed a dose-related tendency up to the highest concentration of 1000 mg/L (where 39.04 % inhibition was noticed).The degree of inhibition of oxygen consumption rates (3.40 % at 3.2 mg/L, 14.11 % at 10 mg/L, 20.11 % at 32 mg/L, 28.55 % at 100 mg/L, 32.21 % at 320 mg/L and 39.04 % at 1000 mg/L) obtained allowed the calculation of the 3-hour exact EC10 value and estimation of EC50 and EC80 values. Based on the available data the 3-hour EC10, EC50 and EC80 values and their 95 % confidence limits were calculated (in the case of EC50 and EC80 estimated) by Probit analysis using IBM® SPSS® Statistics software.
The specific respiration rates were compared with the blank control values using Dunnett’s t- test (2-sided, α = 0.05). The specific respiration rates did not differ statistically significantly from the blank control at the concentration of 3.2 mg/L (Dunnett’st- test, α = 0.05). The specific respiration rates differ statistically significantly from the blank control at the concentration range of 10-1000 mg/L (Dunnett’s t-test, α = 0.05). Consequently, the NOEC can be statistically and biologically determined as 3.2 mg/L.
An additional nitrification control was examined in the test with three parallels to check the possible nitrification potential of the applied activated sludge batch. By applying the nitrification control the differentiation between the total, heterotrophic and nitrification respiration was possible. The total respiration (RT) was 52.70 mg/Lh and the heterotrophic respiration (RH) was 51.17 mg/Lh. The nitrification respiration (RN) of 1.53 mg/Lh was calculated according to the following equation: RN= RT-RH. The obtained nitrification respiration of 1.53 mg/Lh was considered to be within the biological variability range of the applied test system, since it is lower than 5 % of the average RT in the blank controls (2.63 mg/Lh). According to the above calculations it was assumed that the heterotrophic oxygen uptake equals the total uptake.

Results with reference substance (positive control):

Positive reference control:
The following concentrations of the positive reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 2, 7 and 24.5 mg/L. In comparison to the blank controls the oxygen consumption rate of the activated sludge was inhibited by 9.13 % at the lowest concentration of 2 mg/L and at the nominal concentrations of 7 and 24.5 mg/L, the oxygen consumption rate was inhibited by 16.73 % and 71.10 %, respectively. The 3-hour EC50 of 3,5-Dichlorophenol was calculated (Probit analysis, IBM® SPSS®Statistics software) t o be 14.85 mg/L.

This test was performed without pH adjustment. The measured pH values in the prepared solutions: 3,5-Dichlorophenol reference control stock solution, synthetic sewage, activated sludge inoculum, prepared test mixtures (controls, test item treatments) etc. were in the acceptable pH 7-8 range at the start of the test. The measured pH values and pH changes are summarized in Appendix I, Tables 3 and 4. The mixtures were aerated at 0.5 L/min in the temperature range of 20 ± 2 oC. The temperature in the test mixtures during the measurements in average: 21.6 oC, the measured minimum: 20.7 °C, maximum: 22.0 °C.

Validity criteria fulfilled:

yes

Conclusions:

Based on the statistical and biological evaluation in this test the NOEC was determined as 3.2 mg/L.

Executive summary:

The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions, according to the OECD guideline 209. Based on the results of the earlier performed preliminary test (statistically significant inhibition of oxygen consumption by the test substance at the concentration of 1000 mg/L) a main test was performed. In the present main test, based on measured oxygen consumption values and calculated specific respiration rates the 3-hour EC10 value of the test item was calculated as 6.37 mg/L (within the range of 1 mg/L to 10 mg/L), the 3-hour EC50 and EC80 values of the test item are higher than 1000 mg/L.

Description of key information

Based on measured oxygen consumption values and calculated specific respiration rates the 3-hour EC10 value of the test item was calculated as 6.37 mg/L (within the range of 1 mg/L to 10 mg/L), the 3-hour EC50 and EC80 values of the test item are higher than 1000 mg/L.

Based on the statistical and biological evaluation in this test the NOEC was determined as 3.2 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

2 411.59 mg/L

EC10 or NOEC for microorganisms:

3.2 mg/L

Additional information

The EC10 was calculated to 3.2 mg/L