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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Lysine hydrochloride
EC Number:
211-519-9
EC Name:
Lysine hydrochloride
Cas Number:
657-27-2
Molecular formula:
C6H14N2O2.ClH
IUPAC Name:
lysine hydrochloride
Details on test material:
Identification : L-Lysine Monohydrochloride
Molecular formula : C6H14N2O2HCl
Molecular weight : 182.65
CAS Number : 657-27-2
Description : Beige powder with lumps (determined at NOTOX)
Batch : 0148
Purity : 99.72%
Test substance storage : At room temperature in the dark
Stability under storage conditions : Stable
Expiry date : 28 May 2012

Stability in water : At least 48 hours
Solubility in water : Not indicated

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
The batch of L-Lysine Monohydrochloride tested was a beige powder with lumps and a purity of 99.72%. A stock solution of 0.5 g/l was prepared by dissolving 0.1251 g test substance in 250 ml of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). An amount of synthetic sewage feed (16 ml), activated sludge (200 ml) and an adequate amount of the test substance stock were mixed and made up to 500 ml with Milli-RO water in a 1-litre bottle. A concentration of 100 mg/l was tested in duplicate.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The sludge was coarsely sieved, washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.6 g/l of sludge, as used for the test). The pH was 8.2 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic sewage feed was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
Oxygen consumption was measured and recorded for approximately 10 minutes after the 3 hours exposure period.

Test conditions

Test temperature:
Between 18.7 and 19.2°C.
pH:
8.6 - 8.7
Dissolved oxygen:
Oxygen consumption:
Controls: 38-39 mg O2/l/h
L-Lysine Monohydrochloride: 36-41 mg O2/l/h
The oxygen concentration at the start of measurements was at least 7.0 mg O2/l
Nominal and measured concentrations:
Nominal concentration: 100 mg/l, in duplicate.
Details on test conditions:
TEST SYSTEM
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: All glass, approximately 300 ml oxygen bottles (measuring) and
1 litre test bottles(exposure)
- Aeration:During exposure with clean, oil-free air
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2 / series
- Biomass loading rate: 1.4 g/l suspended solids in final test mixture

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The respiration rate from each vessel, in mg O2/l/hr, was calculated from the linear part of the respiration curve, which was generally between 2.5 and 6.5 mg O2/l.

TEST CONCENTRATIONS
- Test concentrations: limit study at 100 mg/l
Reference substance (positive control):
yes
Remarks:
(3,5-dichlorophenol)

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
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
Remarks:
respiration rate
Details on results:
No (significant) inhibition of respiration rate of the sludge was recorded at 100 mg L-Lysine Monohydrochloride per litre. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed. Hence, the EC50 of L-Lysine Monohydrochloride exceeded 100 mg/l (based on nominal concentrations).
Results with reference substance (positive control):
Results with reference substance valid: Yes
The inhibitory effect of 3,5-dichlorophenol on aerobic waste water (activated sludge) bacteria increased with increasing concentration , ranging from a 28 % inhibition at 5.0 mg/l to 73% at 30 mg/l. Relevant effects levels: The EC50 of 3,5-dichlorophenol was: 10.8 mg/l
Reported statistics and error estimates:
For the reference substance the percentage inhibition was plotted against the logarithm of the concentrations and the EC50 was determined using linear regression analysis.
For L-Lysine Monohydrochloride no EC50 could be calculated because the test substance proved to be non-toxic (EC50 > 100 mg/l).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
L-Lysine Monohydrochloride was not toxic to waste water (activated sludge) bacteria at a nominal concentration of 100 mg/l (EC50 > 100 mg/l).