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Toxicity to microorganisms

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toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data based on the inhibition control of a ready biodegradability study. This approach is in accordance with the Guidance on information requirements and chemical safety assessment (Chapter R.7b: Endpoint specific guidance, ECHA 2017)).
Reason / purpose for cross-reference:
reference to same study
according to guideline
other: OECD 301B Ready Biodegradation
Principles of method if other than guideline:
Microbial toxicity was derived from the results of a biodegradation test.
GLP compliance:
yes (incl. QA statement)
Test organisms (species):
other: activated sludge, domestic, adapted
Details on inoculum:
The source of test organism was activated sludge freshly obtained from a municipal sewage treatment plant: Waterschap de Maaskant, s-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage. The freshly obtained sludge wask kept under continuous aeration until further treatment. The concentration of suspended solids was 4.4 g/l in the concentrated slkudge (infomration obtained from the municipal sewage treatment plant) Before use, the sludge was allowed to settle (83 minutes) and the liquid was decanted for use as inoculum at the amount of 10 ml/l of mineral medium.
Test type:
Water media type:
Limit test:
Total exposure duration:
14 d
Details on test conditions:
Pre-incubation medium: Mineral components, Milli-RO water (ca. 80% total volume) and inoculum (1% final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
Type and number of bottles: Test suspension: containing test substance and inoculum (2 bottles). Inoculum blank: containing only inoculum (2 bottles). Positive control: containing reference substance and inoculum (1 bottle). Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
Preparation: The test substance and positive control were added to the bottles containing the microbial organisms and mineral components (ca. 80% of total volume). The volumes of suspensions were made up to 2 litres with MiIIi-RO water, resulting in the mineral medium described before. Three CO2-absorbers (bottles filled with 100 ml 0.0125 M Ba(OH), were connected in series to the exit air line of each test bottle.
Experimental CO2 production: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH), with 0.05 M standardized HCI (1 :20 dilution from 1 M HCI (Titrisol® ampul), Merck, Darmstadt, Germany).
Measurements: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day. Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1 % solution in ethanol, Merck, Darmstadt, Germany) was used as pH-indicator. On the 28th day, the pH of the test suspensions was measured and 1 ml of concentrated HCI (37%, Merck, Darmstadt, Germany) was added to each bottle. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.
Theoretical CO, production: The theoretical CO2 production was calculated from the molecular formula.
Measurements and recordings: pH- at the start of the test and on the 28th day. Temperature of medium: Continuously in a vessel with Milli-RO water in the same room. Electronic data capture: Observations/measurements in the study were recorded electronically using the following programme:
REES version 1.5 (REES scientific, Trenton, NJ, USA): Temperature.
Data evaluation: Relative degradation values were calculated from the cumulative CO2 production relative to the total expected CO2 production based on the total carbon content of the amount of test material present in the test bottles. A figure of more than 10% degradation was considered as significant. The relative degradation values were plotted versus time together with the relative degradation of the positive control. If applicable, the number of days is calculated from the attainment of 10% biodegradation until 60% biodegradation. Should this period be 10 days (10-day window), then the test substance is designated as readily biodegradable. Toxicity control: if less than 25% degradation (based on ThC02) occurred within 14 days, the test substance was assumed to be inhibitory. The total CO2 evolution in the inoculum blank was determined by the cumulative difference (in ml of titrant) between the blank Ba(OH)2 traps and fresh Ba(OH)2.
14 d
Dose descriptor:
Effect conc.:
20 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: CO2 evolution
result from toxicity control form ready biodegradability test
Details on results:
The ThCO2 of 2-Pentanone oxime (Methyl propylketoxime) was calculated to be 2.17 mg CO2/mg.
The ThCO2 of Sodium Acetate was calculated to be 1.07 mg CO2/mg.
The relative degradation values calculated from the measurements performed during the test period revealed 4 and 12% degradation of 2-Pentanone oxime (Methyl propylketoxime), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60%biodegradation within a 10-day window) was not met.
In the toxicity control more than 25% degradation occurred within 14 days (33%, based on ThCO2). Therefore, the test substance was assumed not toinhibit microbial activity.

Results with reference substance (positive control):
The ThCO2 of Sodium Acetate was calculated to be 1.07 mg CO2/mg.
Positive control substance was degraded by 69% within 14d, therefore reference substance considered to be valid.
Validity criteria fulfilled:
Executive summary:

Microbial toxicity of 2 -PO was derived from available Ready Biodegradation test. In this test, ready biodegradability was tested by monitoring CO2 evolution. Both a positive control and toxicity control were included in the test set up. In the toxicity control, 2 -PO was added at a concentration of 40 mg/L. Validity criteria of the test were fulfilled. In the toxicity control more than 25% degradation occurred within 14 days (33%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.

Description of key information

NOEC (14 d): 20 mg/L (nominal, inhibition control of OECD 301B).

Key value for chemical safety assessment

Additional information

The assessment of the toxicity of the substance to microorganisms is based on the results of toxicity control included in the available ready biodegradability test according to OECD 301B. If a compound degrades well in a ready biodegradability test, or does not inhibit the degradation of a toxicity control at a certain concentration, this concentration can be used as a NOEC value (Chapter R.7b: Endpoint specific guidance, ECHA 2017). A substance can be assumed to be not inhibitory to aquatic microorganisms, if in the toxicity control of a ready biodegradation test more than 25% degradation based on oxygen demand (BOD/ThOD) occurred within 14 days (OECD guideline 301). The available ready biodegradability test includes an inhibition control containing 20 mg/L test substance and 40 mg/L sodium acetate. The inhibition control reached 33% degradation within 14 days (based on ThCO2). Therefore, the substance is assumed not to inhibit microbial activity and the test item concentration of 20 mg/L can be used as NOEC.