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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
Justification for Read Across is detailed in the report attached to the IUCLID section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
1981
GLP compliance:
no
Vehicle:
yes
Details on test solutions:
Stock solution (0.5 to 5.0 g/l) of the test chemical was prepared in deionized water prior to inhibition testing. When necessary, the stock solution was adjusted to pH 7.5 ± 0.5 by the addition of 1N H2SO4 or 1 N NaOH as required.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: local municipal wastewater treatment plant.
- Collection: the day prior to the first day of inhibition test.
- Preparation of inoculum for exposure: The solids were allowed to settle and the waste liquor was discarded. The solids were then transferred into a 9-liter laboratory-scale, semi continuous activated sludge cylinder and diluted to approximately 8.5 liters with deionized water. The system was initially mixed by aerating at a rate of approximately 0.5 l/min, and the concentration of mixed liquor suspended solids was determined by gravimetric analysis. The solids were then allowed to settle in the cylinder and the upper layer of waste liquor was discarded. The activated sludge was washed three times with appropriate volumes of deionized water. After washing, the system was adjusted to contain 4000 ± 400 mg of mixed liquor suspended solids (dry weight) per liter. The system was then aerated continuously at a rate of 0.5 l/min and incubated at ambient temperature (21°C). The system was supplemented daily with 50 ml of a synthetic sewage stock solution per liter of activated sludge. The synthetic sewage stock solution was composed of (per liter): Bacto-Peptone, 16.0 g; Bacto-Beef extract, 11.0 g; urea, 3.0 g; K2HPO4 , 28.0 g; MgSO4.7H2O, 0.2 g; CaCI2.2H2O , 0.4 g; and NaCl, 0.7 g. Final pH of the stock solution was adjusted to pH 7.0 with H3PO4. The synthetic sewage stock solution was similar to that described by the OECD with the exception that the level of K2HPO4 was increased to 28.0 g for increased buffering capacity. Fresh stock solution was prepared as required and stored for not more than two days at 5 °C.
- Pre-Treatment: prior to inhibition testing, the activated sludge system was adjusted to pH 7.5 ± 0.5 by the addition of H3PO4.
Test type:
static
Water media type:
freshwater
Total exposure duration:
3 h
Test temperature:
approximately 21 °C
pH:
7.4 - 8.0 (following analysis of the oxygen consumption rate)
Details on test conditions:
TEST SYSTEM
- Test vessel: 1-litre bottle.
- Aeration: the reaction mixture was aerated at a rate between 0.5 and 1.0 l/min to ensure complete mixing using a Pasteur pipette aeration device.
- Sludge: 200 ml
- Weight of dry solids: 800 mg of suspended solids.
- Nutrients provided for bacteria: 16 ml of synthetic sewage stock solution.

TEST MEDIUM / WATER PARAMETERS
- Dilution water: up tp a volume of 300 ml with deionised water.

EFFECT PARAMETERS MEASURED
Following the 3 - hour incubation period, a portion of a reaction mixture was transferred to a standard BOD dilution bottle. The rate of oxygen consumption in the sample was immediately analyzed using an Orion model 97-08 polarographic oxygen electrode and an Orion model 701 Ionanalyzer. An additional magnetic stirrer was added to the BOD bottle to ensure proper mixing of the sample, since the captive stirrer supplied with the oxygen electrode did not provide adequate mixing. Output of the Ionanalyzer was connected to a Perkin-Elmer Model 56 chart recorder, and the change in dissolved oxygen concentration in the sample was recorded over a 5 minute period. The respiration rate of the sample, defined as mg of O2/l consumed per minute, was calculated from the slope of the recorder trace between approximately 6.0 mg O2/l and 2.0 mg O2/l.

CONTROL
- Blank: control reaction mixtures were prepared at the beginning and end of the study in which the test chemical was omitted.

POSITIVE CONTROL
- Stock solution: prepared by dissolving 0.5 g of 3,5-dichlorophenol in 10 ml of 1N NaOH and diluting to 30 ml with deionized water. The solution was adjusted to pH 7.5 ± 0.5 by the addition of 8 ml of 1N H2SO4, and then diluted to a total volume of 1liter with deionized water.
- Concentrations: at least 3 concentrations of the reference substance were tested.

Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
IC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Validity criteria requirements were readily met during the course of the study.
Results with reference substance (positive control):
3,5-Dichlorophenol IC50 (3 h): 12.5 (mg/l), based on graphic analysis.

REPRODUCIBILITY OF INHIBITION TESTING

3,5-Dichlorophenol Municipal IC50 (3 h): 12.2 ± 2.2 (mg/l), based on 24 determinations showing a variance of 18 %.

3,5-Dichlorophenol Industrial IC50 (3 h): 11.4 ± 1.5 (mg/l), based on 3 determinations showing a variance of 13 %.

Mercury Dichloride Municipal IC50 (3 h): 2.3 ± 0.5 (mg/l), based on 3 determinations showing a variance of 22 %.

Mercury Dichloride Industrial IC50 (3 h): 0.9 ± 0.2 (mg/l), based on 3 determinations showing a variance of 22 %.

Mercury Dichloride IC50 (3 h): 2.2 (mg/l), based on graphic analysis.

Phenol Municipal IC50 (3 h): 798.9 ± 97.1 (mg/l), based on 3 determinations showing a variance of 12 %.

Phenol Industrial IC50 (3 h): 766.4 ± 96.6 (mg/l), based on 3 determinations showing a variance of 13 %.

Phenol IC50 (3 h): 800 (mg/l), based on graphic analysis.

Validity criteria fulfilled:
yes
Conclusions:
Acetophnone IC50 (3 h) > 1000 mg/l.
Executive summary:

The OECD Activated Sludge, Respiration Inhibition Test was evaluated as a method for assessing the potential impact of chemicals on wastewater treatment systems. Reproducibility of the test method was examined with respect to variation in oxygen consumption rates measured for a series of reactions set under identical conditions, and the variability in IC50 values estimated for three reference compounds. Several statistical procedures were also evaluated for analyzing activated sludge inhibition data. The test method was subsequently used to examine the effects of a wide variety of inorganic and organic chemicals, including Acetophenone, on activated sludge respiration rates.

Acetophnone IC50 (3 h) > 1000 mg/l.

Description of key information

Not expected as harmful/toxic for microorganisms.

Key value for chemical safety assessment

Additional information

There are no information about propiophenone, thus the available data on the structural analogous acetophenone have been taken into account.

The respiration inhibition potential of acetophenone was assayed using activated sludge. Reproducibility of the test method was examined with respect to variation in oxygen consumption rates measured for a series of reactions set under identical conditions, and the variability in IC50 values estimated for three reference compounds. The IC50 value resulted to be greater than 1000 mg/l, after an exposure period of 3 hours (Klecka and Landi, 1985).

A further EC50 acetophenone value of 15.5 mg/l (no further details) can be retrieved from secondary sources (Nendza and Wenzel, 2006).

Furthermore, data on Spirotox test performed with acetophenone are available. Spirotox test is a method for estimating the toxicity of volatile compounds; the test was carried out in the disposable polystyrene multiwells. After the organisms, protozoa Spirostomum ambiguum, were added to the wells, microplate was tightly closed using silicone grease and polyethylene film. No control mortality was observed. Transparent PE film enabled good observation of test response. The EC50 (48h), i.e. concentration producing different deformations of 50 % of the test organisms, resulted to be 1.91 ±. 0.31 mmol/l (ca 229 mg/l); the LC50 (48h), i.e. the concentration producing lethal response of 50 % of the test organisms, (48 h) has been indicated at 6.18 ± 1.14 mmol/l (ca 742 mg/l) (Nalecz-Jawecki and Sawicki, 1999).

The Read Across approach can be considered appropriate for the assessement of toxicity to microorganisms. Details can be found in the Read Across justification document attached in section 13 of IUCLID.

REFERENCE

Nalecz-Jawecki G., Sawicki J., 1999. Spirotox - a new tool for testing the toxicity of volatile compounds. ChemosphereV.ol . 38, No. 14, pp. 3211.3218, 1999

Nendza, M. and Wenzel, A. 2006. Discriminating Toxicant Classes by Mode of Action 1. (Eco)toxicity Profiles. Environ Sci & Pollut Res 2006: 1 – 12.