L-4-hydroxyproline

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:

2017-05-08 to 2017-07-21

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 (Carbon and Ammonium Oxidation))

Version / remarks:

July 22, 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Version / remarks:

2007

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Thai Hakko Biotechnologies Co., Ltd. / batch 160006
- Expiration date of the lot/batch: retest date 2018-02-22
- Purity test date: 2016-03-08

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 10 - 25 °C in a tightly closed container, stored in a dry and well ventilated place
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: highly soluble
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: n.a.

Analytical monitoring:

yes

Remarks:

Analytical monitoring in pre-study revealed nominal reccovery rate at test initiation and end of test.

Details on sampling:

During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption (in mg O2 L-1 minute-1) was determined over a period up to 10 minutes in the range between approximately 8.2 – 2.0 mg O2/L.

Vehicle:

yes

Details on test solutions:

The activated sludge used for this study was used as collected, but coarse particles were removed by settling for a short period (30 minutes) and then the upper layer decanted. During holding prior to use the sludge was fed daily with 50 mL synthetic sewage (see below) 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 pure water to yield a concentration equivalent to 3 g/L on dry weight basis. This level gave a concentration of 1.5 g/L suspended solids in the test medium. The pH of the activated sludge was 6.1 and therefore no adjustment was necessary.

Appropriate amounts (about 50 mg) of test item were directly weight into the test vessels and test water was added. The composition was stirred intensively

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Laboratory culture:
- Name and location of sewage treatment plant where inoculum was collected: Bensheim, Germany
The activated sludge used for this study was used as collected, but coarse particles were removed by settling for a short period (30 minutes) and then the upper layer decanted. During holding prior to use the sludge was fed daily with 50 mL synthetic sewage (see below) 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 pure water to yield a concentration equivalent to 3 g/L on dry weight basis. This level gave a concentration of 1.5 g/L suspended solids in the test medium. The pH of the activated sludge was 6.1 and therefore no adjustment was necessary

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20 °C +/- 2°C

pH:

6.1

Dissolved oxygen:

measured

Nominal and measured concentrations:

Nominal 100 mg test item/L (three replicates)

Details on test conditions:

TEST SYSTEM
- Test vessel: Karlsruher flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL
- Aeration: Compressed air (1.017 litre per minute)
- No. of vessels per concentration (replicates): 3
- No. of vessels per positive control (replicates): 3
- No. of vessels per vehicle control (replicates): 6
- Nutrients provided for bacteria: synthetic seage

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water

OTHER TEST CONDITIONS
- Adjustment of pH: not required

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : respiration rate
TEST CONCENTRATIONS
- Test concentrations: 100 mg/L

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

The mean respiration rates of the activated sludge treated with the test item at a concentration of 100 mg/L differed by less than 10% from control.

At a nominal test item concentration of 100 mg test item/L less than 10% inhibition (mean of three replicates) was observed after three hours of incubation. The mean deviation in the respiration rate between L-4-HYDROXYPROLINE (n=3) and the control (n=6) was 1.9%.

The respiration rate of the activated sludge with L-4-HYDROXYPROLINE at a test item concentration of 100 mg/L was not statistically significant reduced when compared to the control (Student t-test).

L-4-HYDROXYPROLINE at the rate of 100 mg/L had no toxic effects on respiration activity of activated sludge.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC10, EC20, EC50

Reported statistics and error estimates:

Student t-test

Validity criteria fulfilled:

yes

Conclusions:

L-4-HYDROXYPROLINE at the rate of 100 mg/L had no toxic effects on respiration activity of activated sludge. Therefore it can be assumed that the NOEC is above 100 mg test item/L.

Executive summary:

The influence of the test item L-4-HYDROXYPROLINE on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions according to OECD 209. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of one concentration of the test item after an incubation period of 3 hours.

This limit test was performed in compliance with the test guidelines to demonstrate that the test item had no toxic effect on activated sludge up to at least this concentration.

Nominal test concentration was 100 mg/L.

L-4-HYDROXYPROLINE at the rate of 100 mg/L had no toxic effects on respiration activity of activated sludge. Therefore it can be assumed that the NOEC is above 100 mg/L

Description of key information

NOEC > 100 mg/L

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

100 mg/L