(6R-trans)-7-amino-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 January 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 tot GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

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

Test material information

Constituent 1

Reference substance name:

(6R-trans)-7-amino-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

EC Number:

244-870-1

EC Name:

(6R-trans)-7-amino-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Cas Number:

22252-43-3

Molecular formula:

C8H10N2O3S

IUPAC Name:

7-amino-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Details on test material:

- Name of test material (as cited in study report): 7-ADCA
- Substance type: White powder
- Physical state: Solid
- Analytical purity: 99.0%
- Lot/batch No.: D212013
- Expiration date of the lot/batch: 28 February 2011 (retest date)
- Stability under storage conditions: Stable
- Storage condition of test material: In refrigerator (2-8°C) in the dark
- Stability in water Not indicated
- Solubility in water 0.1% (20°C)

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

Since 7-ADCA was hardly soluble in water test concentrations were prepared separately in
Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA).
Two 1 litre test bottles were filled with 250 ml of test substance mixtures with initial loading rates of 200 mg/l. These mixtures were treated with ultrasonic waves for 5 minutes and stirred in closed brown glass bottles for 24 hours. Subsequently, 16 ml synthetic sewage feed, 200 ml sludge and Milli-RO water up to 500 ml were added resulting in a loading rate of 100 mg/l . This loading rate was tested in duplicate. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring during the 3 hour exposure period.

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 (4.2 g/l of sludge, as used for the test). The pH was 7.8 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 18.9 and 19.5°C.

pH:

7.9 - 8.0

Dissolved oxygen:

Oxygen consumption:
Controls for 7-ADCA:60-62 mg O2/l/h
7-ADCA:59-60 mg O2/l/h
The oxygen concentration at the start of the measurements was at least 6.5 mg O2/l

Nominal and measured concentrations:

Loading rate at 100 mg/l

Details on test conditions:

TEST SYSTEM
- Type (delete if not applicable): open
- Material, size, headspace, fill volume:All glass, approximately 300 ml oxygen bottles and 1 litre test bottles.
- Aeration:During exposure with clean, oil free air
- No. of vessels per concentration (replicates):2
- No. of vessels per control (replicates):2
- Biomass loading rate: 1.7 g/l suspended solids in the final 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
limt at a loading rate of 100 mg/l

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

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

Effect concentrations 2

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 7-ADCA per litre. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed. Hence, the EC50 of 7-ADCA exceeded a loading rate of 100 mg/l.

Results with reference substance (positive control):

The EC50 of 3,5-dichlorophenol was in the accepted range of 5-30 mg/l (11 mg/l)

Reported statistics and error estimates:

The percentage inhibition was plotted against the logarithm of the concentrations. The EC50 for the reference substance, was determined using linear regression analysis.
For 7-ADCA 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:

Under the conditions of this present test, 7-ADCA was not toxic to waste water (activated sludge) bacteria at a loading rate of 100 mg/l (EC50 > a loading rate of 100 mg/l).