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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13. Jan. 2006 - 13. Jan. 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
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
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The batch of FORMAMIDOPROPYLBETAINE consisted of FORMAMIDOPROPYLBETAINE (40.1%), water (56.1%) and sodium chloride (3.8%).
As a worse case approach a correction for the water content and sodium chloride was madel . A stock solution of 1.0832 g total test substance/I was prepared in Milli-RO water, corresponding with 0.434 g pure FORMAMIDOPROPYLBETAINE/1. Thorough mixing (15 minutes) was used to accelerate dissolving and to ensure homogeneity. Volumes of the stock solution corresponding to the test concentration were then added to the test media. The pH of the clear and colourless stock solution was 7.2. A concentration of 87 mg pure FORMAMIDOPROPYLBETAINE/I, corresponding with 216.5 mg
total test substance/I, was tested in duplicate.
1: Based on information given by the sponsor. This information was amended later on. Therefore, the pure FORMAMIDOPROPYLBETAINE was 87 mg/l instead of 100 mg/L The total test concentration is still above 100 mg/l.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Test System : Micro-organisms in activated sludge.
Source : Municipal sewage treatment plant: 'Waterschap de Maaskant', 's-Hertogenbosch, the Netherlands,receiving predominantly domestic sewage.
Number of micro-organisms : Number of micro-organisms was determined as the amount of Mixed Liquor Suspended Solids (MLSS) per litre test medium.
Preparation of the sludge : The sludge was coarsely sieved, washed and diluted with tap-water. A small amount of the sludge was weighed and dried at ca. 105°C to determine the amount of suspended solids (2.7 g/l of sludge, as used for the test). The pH was 7.5 on the day of sampling and 7.9 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.
Rationale : Recognized by international guidelines as the recommended test system.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
30 min
Test temperature:
18.8 - 19.9 °C
pH:
8.1 - 8.4
Dissolved oxygen:
8.3 - 8.5 mg/L at study start
Nominal and measured concentrations:
Nominal concentration: 216.5 mg/L = 87 mg/L pure substance
Details on test conditions:
Contact time : 30 minutes, during which aeration and stirring took place.
Vessels : All glass, approximately 300 ml oxygen bottles and 1 litre test bottles.
Milli-RO / Milli-Q water : Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ionexchange cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).
Synthetic sewage feed : 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCI, 0.4 g CaCI2.2H20, 0.2 g MgS04.7H20, 2.8 g K2HP04. Dissolved in Milli-Q water, made up t01 litre and filtered. The pH was 6.7 and adjusted with 1 M NaOH to 7.5. All chemicals were obtained from Merck, Darmstadt, Germany.
Air supply : Clean, oil-free air.
Oxygen meter : WTW inolab Oxi Level 2 supplied with a WTW CeliOx 325 oxygen electrode, electrolyte type ELY/G.
Recorder : Flatbed recorder SE 102 (Kipp & Zonen).
Performance of the test : The synthetic sewage feed (16 ml) and an adequate amount of the test substance solution were mixed and made up to 300 ml with Milli-RO water. Activated sludge (200 ml) was added to provide a final volume of 500 ml. The mixture was then aerated in a 1 litre bottle during the contact time, using a pipette as an aeration device. Then a well mixed sample of the contents was poured into a 300 ml oxygen bottle, and the flask was sealed with an oxygen electrode connected to a recorder, forcing the air out of the vessel. Oxygen consumption was measured and recorded for approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH and temperature were determined in the remaining part of the reaction mixture. This procedure was repeated for the duplicate concentration. Two controls without test substance were tested in each test series, one at the start and one at the end.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 87 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
EC 50 (30 min) for 3,5-dichlorophenol = 16.6 mg/L
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test, FORMAMIDOPROPYLBETAINE was not toxic to waste water (activated sludge) bacteria at a nominal concentration of 87 mg pure FORMAMIDOPROPYLBETAINE/I, corresponding with 216.5 mg total test substance/I. As a worse case approach a correction for the water content and sodium chloride was made 1.
1: Based on information given by the sponsor. This information was amended later on. Therefore, the pure FORMAMIDOPROPYLBETAINE was 87 mg/I instead of 100 mg/I. The total test concentration is still above 100 mg/I.
Executive summary:

The influence of FORMAMIDOPROPYLBETAINE on the respiration rate of activated sludge was investigated after a contact time of 30 minutes. The study procedure was based on OECD Guideline No. 209, adopted April 4, 1984, EEC Directive 67/548 amended November 18, 1987 (87/302), Part C, Publication No. L 133, adopted May 30, 1988 and ISO Standard 8192 (1986).

The batch of FORMAMIDOPROPYLBETAINE consisted of FORMAMIDOPROPYLBETAINE (40.1 %), water (56.1 %) and sodium chloride (3.8%). As a worse case approach a correction for the water content and sodium chloride was made 1. A stock solution of 1.0832 g total test substance/L was prepared in Milli-RO water, corresponding with 0.434 g pure FORMAMIDOPROPYLBETAINE/L. Thorough mixing (15 minutes) was used to accelerate dissolving and to ensure homogeneity. Volumes of the stock solution corresponding to the test concentration were then added to the test media. The pH of the clear and colourless stock solution was 7.2. A concentration of 87 mg pure FORMAMIDOPROPYLBETAINE/L, corresponding with 216.5 mg

total test substance/I, was tested in duplicate. No inhibition of respiration rate of the sludge was recorded at the tested concentration. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed. Hence, the EC50 of

FORMAMIDOPROPYLBETAINE exceeded 87 mg pure FORMAMIDOPROPYLBETAINE/L, corresponding with 216.5 mg total test substance/I (based on nominal concentrations). Since all criteria for acceptability of the test were met, this study was considered to be valid. In conclusion, under the conditions of this present test, FORMAMIDOPROPYLBETAINE was not toxic to waste water (activated sludge) bacteria at a nominal concentration of 87 mg pure FORMAMIDOPROPYLBETAINE/L, corresponding with 216.5 mg total test substance/L. Based on information given by the sponsor. This information was amended later on. Therefore, the pure

FORMAMIDOPROPYLBETAINE was 87 mg/L instead of 100 mg/L. The total test concentration is still above 100 mg/L.

1: Based on information given by the sponsor. This information was amended later on. Therefore, the pure

FORMAMIDOPROPYLBETAINE was 87 mg/L instead of 100 mg/L. The total test concentration is still above 100 mg/L.

Description of key information

Under the conditions of this present test, FORMAMIDOPROPYLBETAINE was not toxic to waste water (activated sludge) bacteria at a nominal concentration of 87 mg pure FORMAMIDOPROPYLBETAINE/L, corresponding with 216.5 mg total test substance/L.

Key value for chemical safety assessment

Additional information

The influence of FORMAMIDOPROPYLBETAINE on the respiration rate of activated sludge was investigated after a contact time of 30 minutes. The study procedure was based on OECD Guideline No. 209, adopted April 4, 1984, EEC Directive 67/548 amended November 18, 1987 (87/302), Part C, Publication No. L 133, adopted May 30, 1988 and ISO Standard 8192 (1986).

The batch of FORMAMIDOPROPYLBETAINE consisted of FORMAMIDOPROPYLBETAINE (40.1 %), water (56.1 %) and sodium chloride (3.8%). As a worse case approach a correction for the water content and sodium chloride was made 1. A stock solution of 1.0832 g total test substance/I was prepared in Milli-RO water, corresponding with 0.434 g pure FORMAMIDOPROPYLBETAINE/L. Thorough mixing (15 minutes) was used to accelerate dissolving and to ensure homogeneity. Volumes of the stock solution corresponding to the test concentration were then added to the test media. The pH of the clear and colourless stock solution was 7.2. A concentration of 87 mg pure FORMAMIDOPROPYLBET AINE/I, corresponding with 216.5 mg

total test substance/I, was tested in duplicate. No inhibition of respiration rate of the sludge was recorded at the tested concentration. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed. Hence, the EC50 of FORMAMIDOPROPYLBETAINE exceeded 87 mg pure FORMAMIDOPROPYLBETAINE/L, corresponding with 216.5 mg total test substance/L (based on nominal concentrations). Since all criteria for acceptability of the test were met, this study was considered to be valid. In conclusion, under the conditions of this present test, FORMAMIDOPROPYLBETAINE was not toxic to waste water (activated sludge) bacteria at a nominal concentration of 87 mg pure FORMAMIDOPROPYLBETAINE/L, corresponding with 216.5 mg total test substance/L. Based on information given by the sponsor. This information was amended later on. Therefore, the pure FORMAMIDOPROPYLBETAINE was 87 mg/L instead of 100 mg/L. The total test concentration is still above 100 mg/L.

1: Based on information given by the sponsor. This information was amended later on. Therefore, the pure FORMAMIDOPROPYLBETAINE was 87 mg/L instead of 100 mg/L. The total test concentration is still above 100 mg/L.