L-(+)-2,5-diaminopentanoic acid

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 2017-09-06

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:

2010-07-22

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

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

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: Kyowa Hakko Bio Co., Ltd. / lot 160060
- Purity test date: 2016-12-01

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 20 +/- 5 °C, in the dark.
- Solubility and stability of the test substance in the solvent/vehicle: highly soluble

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Appropriate amounts of test item were directly weight into the test vessels and test water was added. The composition was stirred intensively.
- Controls:
negative: pure water, synthetic sewage and inoculum
positive for total respiration: 3,5-dichlorphenol
positive for nitification inhibition: N-allylthiourea (ATU)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: municipal sewage treatment plant Bensheim, Germany
- Method of cultivation:
The activated sludge used for this study was used as collected, but coarse particles were removed by settling for a short period (15 minutes) and then the upper layer decanted. During holding prior to use the sludge was fed 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 3.0 g/L on dry weight basis. This level gives a concentration of 1.5 g/L suspended solids in the test medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 °C +/- 2°C during pre-incubation (at least 3 hours) and evaluation period

pH:

7.0

Nominal and measured concentrations:

Nominal 10, 32, 100, 320 and 1000 mg test item/L (5 replicates each)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass flasks of approximately 1 litre volume and Karlsruher flasks of 250 mL volume
- Material, size, headspace, fill volume: see above
- Aeration: Compressed air (1.017 litre per minute)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per positive control (replicates) 3,5-dichlorophenol: 5 per test concentration, except for 4 mg/L where 4 replicates were evaluated
- No. of vessels per positive control (replicates) ATU: 6 per test concentration
- Sludge concentration (weight of dry solids per volume): 3.0 g/L dry weight basis, 1.5 g/L suspended solids
- Nutrients provided for bacteria: sythetic sewage
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea (ATU)
- Biomass loading rate: 1.5 g/L suspended solids
OTHER TEST CONDITIONS
- Adjustment of pH: not required
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Respiration rate: oxygen consumtion
TEST CONCENTRATIONS
- Test concentrations: 10, 32, 100, 320 and 1000 mg/L

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

7.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Adsorption (e.g. of test material to the walls of the test container): no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: 3,5-Dichlorphenol: 3 -hour EC10, EC20, EC50

Reported statistics and error estimates:

A non-linear, 3-parametric normal Cumulative Distribution Function (CDF) was chosen to compute the Effective Concentrations (3-hour ECx and their 95 %-confidence limits), followed by an analysis of variance and test for lack of fit for the 3-param. normal CDF.
For determination of NOEC concentrations, the Shapiro-Wilk`s Test was used for check of normal distribution, the variance homogeneity was tested by the Leven`s Test. Subsequently, the multiple sequentially-rejective Welsh-t-test (test item, total respiration and nitrification respiration, reference item total respiration) or the Williams Multiple Sequential t-test Procedure (test item, heterotroph respiration and reference item, heterotroph respiration and nitrification respiration; α= 0.05, one-sided smaller) with Levenberg-Marquardt optimization was used to determine LOEC and NOEC values, if possible.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ® ToxRat Solutions GmbH.

Inhibition of Respiration Rate (Total Respiration):

A dose-response inhibition of total respiration rate could be observed for test item concentrations up to 320 mg/L. The highest tested test item concentration of 1000 mg/L resulted in a reduced inhibition, comparable to the lowest tested concentrations.

The 3-hour EC_10, EC₂₀ and EC₅₀ for total respiration could not be established; the NOEC was determined to be at a test item concentration of 32 mg/L.

Inhibition of Respiration Rate (Heterotrophic Respiration without Nitrification):

In comparison to the inoculum controls, the heterotrophic respiration was not inhibited at any test concentration.

The 3-hour EC_x values for heterotrophic respiration could not be established, since no dose-response inhibition was measured; the NOEC was determined to be ≥ 1000 mg/L.

Inhibition of Respiration Rate based on Nitrification:

The effects on nitrification respiration were above 45% for all test item concentrations and showed no clear dose-response relationship.

For the oxygen uptake due to nitrification the 3-hour EC₅₀ was established to be 7.8 mg/L; the NOEC was determined to be at a test item concentration of <10 mg/L. 

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item L-(+)-2,5-diaminopentanoic acid (L-Orn-HCl) on heterotrophic oxygen uptake (heterotrophic respiration) from the oxidation of organic carbon is negligible; the NOEC could be established to be above 1000 mg/L. In contrast, strong effects on the oxidation of ammonium (nitrification respiration) could be determined. It can be concluded, that the effects on total oxygen uptake (total respiration) are caused by the nitrification respiration.
The test item L-(+)-2,5-diaminopentanoic acid (L-Orn-HCl) has no inhibiting effect on the heterotrophic respiration of activated sludge.
However, the test item L-(+)-2,5-diaminopentanoic acid (L-Orn-HCl) has an influence on the oxygen uptake due to nitrification.

Executive summary:

The influence of the test item L-(+)-2,5-diaminopentanoic acid (L-Orn-HCl) on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions. 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 at least 3 hours.

Total respiration:

A dose-response inhibition of total respiration rate could be observed for test item concentrations up to 320 mg/L. The highest tested test item concentration of 1000 mg/L resulted in a reduced inhibition, comparable to the lowest tested concentrations.

The 3-hour EC_10,EC₂₀ and EC₅₀ for total respiration could not be established; the NOEC was determined to be at a test item concentration of 32 mg/L.

Respiration without nitrification (Heterotrophic Respiration):

In comparison to the inoculum controls, the heterotrophic respiration was not inhibited at any test concentration.

The 3-hour EC_x values for heterotrophic respiration could not be established, since no dose-response inhibition was measured; the NOEC was determined to be ≥ 1000 mg/L.

Respiration based on nitrification:

In comparison to the inoculum controls, the respiration rates of the activated sludge based on nitrification were strongly inhibited at test item concentrations of 100, 320 and 1000 mg/L (72.6%, 74.0% and 65.6% inhibition, respectively). For all other test item concentrations there was moderate inhibition on nitrifying activated sludge bacteria (45.3% and 46.1%). For the oxygen uptake due to nitrification the 3-hour EC₅₀ was established to be 7.8 mg/L; the NOEC was determined to be at a test item concentration < 10 mg/L. 

Description of key information

L-(+)-2,5-diaminopentanoic acid (L-Orn-HCl) has no inhibiting effect on the heterotrophic respiration of activated sludge.

However, L-(+)-2,5-diaminopentanoic acid (L-Orn-HCl) has an influence on the oxygen uptake due to nitrification.

Key value for chemical safety assessment

EC50 for microorganisms:

7.8 mg/L

Additional information

The EC50 of 7.8 mg/L refers to inhibition of oxygen uptake due to nitrification.

A NOEC of 32 mg/L applies for total respiration.

A NOEC of > 1000 mg/L applies for heterotrophic respiration