3-aminophenol

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 8 August, 2013 to 16 April, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed according to the standard guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Principles of method if other than guideline:

Study was conducted to determine the rate of toxicological effect of test chemical on the microorganisms. The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions according to the OECD Guideline for Testing of Chemicals, No. 209: "Activated Sludge, Respiration Inhibition Test".

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Appropriate amounts of test item were directly weight into the test vessels and test water was added. The composition was stirred intensively.
A stock solution of 3,5-dichlorophenol was prepared according to the OECD Guideline No. 209: 0.5 g of 3,5-dichlorophenol was dissolved in 500 mL deionised water. Warm water was used to accelerate the dissolution. The solution was filled up to volume when it had cooled to room temperature. The final pH was 7.3 and therefore in the range of 7 to 8. 1N NaOH was used for the adjustment of the pH.
In parallel a nitrification inhibitor N-allylthiourea (ATU) was tested. A stock solution of N-allylthiourea was prepared according to the OECD Guideline No. 209: a stock solution of 2.32 g/L Nallylthiourea was prepared. 2.5 mL of this stock solution were added to an incubation mixture of final volume of 500 mL. This resulted in a final concentration of 11.6 mg ATU/L.

Preparation of Test Flasks:
For each replicate a test solution with a final volume of 500 mL was tested per treatment in a glass flask. 16 mL synthetic sewage and an adequate amount of the test item or an adequate volume of the stock solution of the reference item was filled up with tap water to 300 mL before the start of the test.
At the start of the test 200 mL activated sludge inoculum with a sludge concentration of 3.0 g/L suspended solids was added, first to two controls, then in time intervals of approximately 15 minutes (an arbitrary but convenient interval) to the test solutions of the reference item, to further controls, to the test item and finally to additional controls. During 3 hour aeration period the flasks were stirred on a magnetic stirrer to maintain sludge flocs in suspension.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Species / Origin:
Activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the municipal sewage treatment plant Bensheim, Germany.

Conditioning:
The activated sludge used for this study was used as collected, but coarse particles were removed by settling for a short period (10 minutes) and then the upper layer decanted. During holding prior to use the sludge was fed with 50 mL synthetic sewage per litre and kept aerated at room temperature overnight. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on the sludge dry matter, calculated amounts of wet sludge were suspended in tap water to yield a concentration equivalent to about 3.0 g/L on dry weight basis. This level gives a concentration of 1.2 g/L suspended solids in the test medium. The pH of the activated sludge inoculum was 6.9 and therefore no adjustment necessary.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 - 22 °C during pre-incubation (3 hours)
18 - 23 °C during evaluation period

pH:

From 7.3 to 8.5

Nominal and measured concentrations:

10, 32, 100, 320 and 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel, Material, size, headspace, fill volume: Glass flasks of approximately 1 litre volume and Karlsruher flasks of 250 mL volume
- Type (delete if not applicable): open
- Aeration: Compressed air (1.026 litre per minute)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): Six controls (tap water, synthetic sewage and inoculum, but without addition of the test item) were tested in parallel.
In parallel to the study with the test item, the reference item 3,5-dichlorophenol was tested at the nominal test concentrations of 3.2, 10 and 32 mg/L (five replicates each test concentration) under otherwise identical test conditions.
In parallel a nitrification inhibitor N-allylthiourea (ATU) was tested in the same way with six separate controls and at the identical nominal concentrations of the test item and also the reference item 3,5-dichlorophenol under otherwise identical test conditions.
- No. of vessels per vehicle control (replicates): n.a.
- Biomass loading rate: 200 mL activated sludge inoculum with a sludge concentration of 3.0 g/L suspended solids was added per flask.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: per flask, 16 mL synthetic sewage and 300 mL tap water.
Synthetic Sewage: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2 · 2H2O, 0.2 g MgSO4 · 7H2O, 2.8 g K2HPO4 filled up to 1 litre with deionised water; pH: 7.1
- Intervals of water quality measurement: The oxygen concentrations were determined at the start and at the end of the incubation period in one replicate of each test concentration and control. The pH-value was determined at the start and at the end in one replicate of the test concentrations and controls. The water temperature was measured in one control medium at the start and the end of the incubation period.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: /
- Light intensity: /

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
For the measurement of the respiration rate a well-mixed sample of each test medium was poured into a Karlsruher flask after exactly 3 hours incubation time. The oxygen concentration was then measured with an oxygen electrode and recorded for about ten minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption (in mg O2 L-1 minute-1) was determined from the most linear part of the respiration curve in the range between approximately 9.0 - 2.0 mg O2/L. If necessary shorter time intervals were also used.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.125-3.2
- Justification for using less concentrations than requested by guideline: n.a.
- Range finding study: yes, the pre-experiment was not performed in compliance with the GLP-Regulations and is excluded from the Statement of Compliance in the final report.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (3.2, 10 and 32 mg/L)

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

961.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Total respiration

Remarks on result:

other: Other details not known

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

406.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Heterotrophic Respiration without Nitrification

Remarks on result:

other: 239.5-552.0

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

1 145.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Heterotrophic Respiration without Nitrification

Remarks on result:

other: 840.2-1982.9

Details on results:

Total respiration:
In comparison to the inoculum controls the respiration rates of the activated sludge were moderately inhibited by 27.3% and 41.3% at the test concentrations of 10 and 32 mg/L. For the concentrations of 100 mg/L and 320 mg/L the inhibition rates were 46.5% and 42.8%. The highest test concentration of 1000 mg/L resulted in an inhibition of 48.7%. Concentrations exceeding 1000 mg/L nominal were not tested.
EC10 and EC20 were not determined due to mathematical reasons.
Inhibition of Respiration Rate (Heterotrophic Respiration without Nitrification):
In comparison to the inoculum controls the respiration was not inhibited at concentrations of 10 mg/L and 32 mg/L. For the test item concentration of 100 mg/L and 320 mg/L the inhibition was 4.7% and 7.3%. At the concentration of 1000 mg/L the respiration rate was moderately inhibited by 18.7%.
EC50 was not determined due to mathematical reasons.
3-hour EC10, EC20 and 3-hour EC50 of Oxygen Uptake due to Nitrification: Based on measured inhibition rates, the 3-hour EC10, EC20 and EC50 and their 95% confidence limits could not be determined due to mathematical reasons.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels:
Inhibition of Respiration Rate of Total Respiration: The inhibition of the activated sludge treated with the reference item was in the range of 40% to 85% for the test concentrations from 3.2 until 32 mg/L.
EC50=6.0 mg/L and therefore is in the range of 2 to 25 mg/L for the used activated sludge batch.
Inhibition of Respiration Rate of Heterotrophic Respiration without Nitrification: The inhibition of the activated sludge treated with the reference item was in the range of 8% to 75% for the test concentrations from 3.2 until 32 mg/L.
EC50=17.7 mg/L and therefore also in the range of 5 to 40 mg/L for the used activated sludge batch.

3-hour EC50 of 3,5-Dichlorophenol of Oxygen Uptake due to Nitrification: EC50=1.4 mg/L and therefore is in the range of 0.1 to 10 mg/L for the used activated sludge batch.

Validity criteria fulfilled:

yes

Remarks:

The O2 uptake in the blank controls was 27.1 mg/g/h ie > 20 mg oxygen/g of AS (dw of SS) in 1H. The respiration rates of the 6 controls differed not more than 30%. The value was 11.1% for the total respiration and 11.0% for the heterotrophic respiration.

Conclusions:

The 3-hour EC50 for total respiration was calculated to be 961.9 mg/L. For heterotrophic respiration the 3-hour EC10 was 406.4 mg/L. For the oxygen uptake due to nitrification the 3-hour EC50 could not be calculated. The results indicate the test item has a moderate effect on the total respiration and a low effect on the heterotrophic respiration. This indicates that the test item has a strong effect on nitrifying bacteria. The inhibition on the nitrification is determined by subtracting the heterotrophic respiration from the total respiration. Due to the high standard deviation in the treatment groups of the nitrification the EC50 could not be calculated.

Executive summary:

The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions according to the OECD Guideline for Testing of Chemicals, No. 209: "Activated Sludge, Respiration Inhibition Test", adopted July 22, 2010. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations of the test item after an incubation period of 3 hours. Concentrations of 10, 32, 100, 320 and 1000 mg test item/L and concentrations of 3.2, 10 and 32 mg 3,5-Dichlorophenol/L and six inoculum controls were used to determine the inhibition on total respiration and heterotrophic respiration (without nitrification) in a dose-response study.

In comparison to the inoculum controls the total respiration rates of the activated sludge were moderately inhibited by 27.3% and 41.3% at the test concentrations of 10 and 32 mg/L. For the concentrations of 100 mg/L and 320 mg/L the inhibition rates were 46.5% and 42.8%. The highest test concentration of 1000 mg/L resulted in an inhibition of 48.7%. Concentrations exceeding 1000 mg/L nominal were not tested.

In comparison to the inoculum controls the heterotrophic respiration (respiration without nitrification) was not inhibited at concentrations of 10 mg/L and 32 mg/L. For the test item concentration of 100 mg/L and 320 mg/L the inhibition was 4.7% and 7.3%. At the concentration of 1000 mg/L the respiration rate was moderately inhibited by 18.7%. Test item has a strong inhibiting effect on nitrifying bacteria. At a concentration of 10 mg/L the inhibition was 65.3%. For the concentrations of 32 and 100 mg/L the inhibition rates were 97.5% and 98.3%. For 320 and 1000 mg/L the inhibition rates were 86.8% and 86.0%. The 3-hour EC50 for total respiration was calculated to be 961.9 mg/L. For heterotrophic respiration the 3-hour EC10 was 406.4 mg/L. For the oxygen uptake due to nitrification the 3-hour EC50 could not be calculated. The positive control 3,5-Dichlorophenol was tested in the same way as the test item. The 3-hour EC50 for total respiration, heterotrophic respiration and for the oxygen uptake due to nitrification was in the range recommended by the test guidelines and confirms the suitability of the activated sludge used.

Description of key information

The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions according to the OECD Guideline for Testing of Chemicals, No. 209: "Activated Sludge, Respiration Inhibition Test", adopted July 22, 2010. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations of the test item after an incubation period of 3 hours. Concentrations of 10, 32, 100, 320 and 1000 mg test item/L and concentrations of 3.2, 10 and 32 mg 3,5-Dichlorophenol/L and six inoculum controls were used to determine the inhibition on total respiration and heterotrophic respiration (without nitrification) in a dose-response study. In comparison to the inoculum controls the total respiration rates of the activated sludge were moderately inhibited by 27.3% and 41.3% at the test concentrations of 10 and 32 mg/L. For the concentrations of 100 mg/L and 320 mg/L the inhibition rates were 46.5% and 42.8%. The highest test concentration of 1000 mg/L resulted in an inhibition of 48.7%. Concentrations exceeding 1000 mg/L nominal were not tested. In comparison to the inoculum controls the heterotrophic respiration (respiration without nitrification) was not inhibited at concentrations of 10 mg/L and 32 mg/L. For the test item concentration of 100 mg/L and 320 mg/L the inhibition was 4.7% and 7.3%. At the concentration of 1000 mg/L the respiration rate was moderately inhibited by 18.7%. Test item has a strong inhibiting effect on nitrifying bacteria. At a concentration of 10 mg/L the inhibition was 65.3%. For the concentrations of 32 and 100 mg/L the inhibition rates were 97.5% and 98.3%. For 320 and 1000 mg/L the inhibition rates were 86.8% and 86.0%. The 3-hour EC50 for total respiration was calculated to be 961.9 mg/L. For heterotrophic respiration the 3-hour EC10 was 406.4 mg/L. For the oxygen uptake due to nitrification the 3-hour EC50 could not be calculated. The positive control 3,5-Dichlorophenol was tested in the same way as the test item. The 3-hour EC50 for total respiration, heterotrophic respiration and for the oxygen uptake due to nitrification was in the range recommended by the test guidelines and confirms the suitability of the activated sludge used.

Key value for chemical safety assessment

EC50 for microorganisms:

961.9 mg/L

Additional information

Based on the experimental data from various database and peer reviewed journals for the target chemical study have been reviewed to determine the mode of action of test chemical 3-aminophenol on the growth rate, and mortality of microorganisms. The studies are as mentioned below:

 

In the first key study from experimental report 2015. The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions according to the OECD Guideline for Testing of Chemicals, No. 209: "Activated Sludge, Respiration Inhibition Test", adopted July 22, 2010. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations of the test item after an incubation period of 3 hours. Concentrations of 10, 32, 100, 320 and 1000 mg test item/L and concentrations of 3.2, 10 and 32 mg 3,5-Dichlorophenol/L and six inoculum controls were used to determine the inhibition on total respiration and heterotrophic respiration (without nitrification) in a dose-response study. In comparison to the inoculum controls the total respiration rates of the activated sludge were moderately inhibited by 27.3% and 41.3% at the test concentrations of 10 and 32 mg/L. For the concentrations of 100 mg/L and 320 mg/L the inhibition rates were 46.5% and 42.8%. The highest test concentration of 1000 mg/L resulted in an inhibition of 48.7%. Concentrations exceeding 1000 mg/L nominal were not tested. In comparison to the inoculum controls the heterotrophic respiration (respiration without nitrification) was not inhibited at concentrations of 10 mg/L and 32 mg/L. For the test item concentration of 100 mg/L and 320 mg/L the inhibition was 4.7% and 7.3%. At the concentration of 1000 mg/L the respiration rate was moderately inhibited by 18.7%. Test item has a strong inhibiting effect on nitrifying bacteria. At a concentration of 10 mg/L the inhibition was 65.3%. For the concentrations of 32 and 100 mg/L the inhibition rates were 97.5% and 98.3%. For 320 and 1000 mg/L the inhibition rates were 86.8% and 86.0%. The 3-hour EC50 for total respiration was calculated to be 961.9 mg/L. For heterotrophic respiration the 3-hour EC10 was 406.4 mg/L. For the oxygen uptake due to nitrification the 3-hour EC50 could not be calculated. The positive control 3,5-Dichlorophenol was tested in the same way as the test item. The 3-hour EC50 for total respiration, heterotrophic respiration and for the oxygen uptake due to nitrification was in the range recommended by the test guidelines and confirms the suitability of the activated sludge used.

 

 

Similarly study from peer reviewed journal 1994, toxicity study was conducted to determine the effect of test chemical on the growth of microorganisms. Test conducted under static system in which Escherichia coli exposed for 12 hrs with the test chemical. After the 12 hhrs of exposure the Effective concentration (EC50) value of test chemical to Escherichia coli was determine to be 4054.5 mg/l on the basis of growth rate inhibition.

 

Similar study was conducted to determine the rate of toxicological effect of test chemical on the strain of microorganisms E.coli ATCC 11775 for 45 minutes. The strain of E.coli used was purchased from the American Type Culture Collection. Stock cultures were maintained on agar slants at room temperature. For culture broth dextrose-salts-casaminoacids-(vitamin free)-medium was used. From agar cultures the culture broth was inoculated and allowed to grow for 12 to 16 h at 37 °C. Test cultures were prepared by dilution with culture medium (104 cells/ml) and 150 ml portions were transferred to 1 liter Erlenmeyer culture flasks. After 0.5 h the toxicant was added. Samples of the incubated cultures (37 °C) were taken every 45 minutes and the number of bacteria was determined with a Coulter Counter. Therefor samples were diluted with particle free saline-(0.85%)-formaldehyde-(2%)-solutions to obtain counts of 1000 to 10000 organisms. The Coulter Counter Model ZB was equipped with a 0.030 mm orifice obtaining counts for 0.050 ml. After the exposure of test chemical for 45 minutes effect on the bacterial population were count. Based on the effect the I50 was determine to be 4070.176 mg/l.

Above key study was supported by the second study from authoritative database 2018. Toxicological study was conducted to determine the nature and effect of test chemical when comes in contact with the Tetrahymena Thermophile. Test performed for the total exposure period of 48 hrs. Based on the effect observed on the Population growth rate inhibition of Tetrahymena thermophile (Ciliate Protozoa) during the exposure period of 48 hrs, the EC50 was determine to be 2.55 mg/l. Based on the effect observed on the microorganisms, chemical was consider as toxic.

 

Similarly in the fifth study from authoritative database 2018 toxicity was measured. Toxicological study was conducted to determine the nature and effect of test chemical when comes in contact with the Tetrahymena Thermophile. Test performed for the total exposure period of 48 hrs. Based on the effect observed on the Population growth rate inhibition of Tetrahymena thermophile (Ciliate Protozoa) during the exposure period of 48 hrs, the EC50 was determine to be 2.9 mg/l. Based on the effect observed on the microorganisms, chemical was consider as toxic.

 

Sixth study from secondary sources supports the previous 2 and 3^rd study. Aim of this study was to investigate the effect of test chemical on the growth and population effect of Tetrahymena Thermophile by providing the incubation period of 2 days. Based on the effect observed on the Population growth rate inhibition of Tetrahymena thermophile (Ciliate Protozoa) during the exposure period of 48 hrs, the EC10 was determine to be 0.7 mg/l and EC20 was observed in the range of 1.25mg/l – 1.5 mg/l. Based on the effect observed on the microorganisms, chemical was consider as toxic.