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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:
13/3/2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
In the range-finding test activiated sewage sludge micro-organisms were exposed to a series of nominal test concentration of 10, 100 and 1000 mg/l. The test item was dissolved directly in water. In order to aid weighing and homogenise the test item it was first warmed to approximately 80 °C prior to weighing.

A nominal amount of test item (2500 mg) was dissolved in water with the aid of ultrasonication for approximately 20 minutes and the volume adjusted to 1 litre to give a 2500 mg/l stock solution from which dilutions were made to give 250 and 25 mg/l stock solutions. An aliquot (200 ml) of the 25 mg/l stock solution was dispersed with synthetic sewage (16 ml), activated sewage sludge (250 ml) and water, to a final volume of 500 ml, to give the required concentration of 10 mg/l. Similarly, aliquots (200 ml) of thew 250 mg/l and 2500 mg/l stock solutions were used to prepare the test concentrations of 100 and 1000 mg/l. The 1000 mg/l test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogenity.

The pH of the test item stock solutions were measured using a Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 and pH 8.0.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C and was used on the day of collection. The pH of the sample was 7.5, measured using a Hach HQ40d Flexi handheld meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper (rinsed three times with 20 ml of deionised reverse osmosis water prior to drying in an oven) using a Buchner funnel which was then rinsed 3 times with 10 ml of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 °C for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/l.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
Temperatures ranged between 18 to 20 °C
pH:
The pH ranged from 7.0 to 7.8 in the test preparations
Dissolved oxygen:
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70% of the dissolved oxygen saturation level of 8.9 mg O2/L.
Nominal and measured concentrations:
Nominal concentrations of 10, 100 and 1000 mg/L
Details on test conditions:
At time '0' 16 ml of synthetic sewage was diluted to 250 ml with water and 250 ml of inoculum added in a 500 ml conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tunes at a rate of 0.5 to 1.0 litres per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. The test item vessels were prepared as previously detailed ('Details on test solutions'). Finally, two further control vessels were prepared.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 - <= 1 000 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
The reference item gave a 3-Hour EC50 value of 7.6 mg/L, 95% confidence limits 5.9 – 9.9 mg/L

Oxygen consumption rates and percentage inhibition values for the control, test and reference item after 3 hours contact time are given in the following table:

 Nominal Concentration (mg/L)  Initial O2 Reading (mg O2/L) Measurement Period (minutes)   Final O2 Reading (mg O2/L)  O2 Consumption Rates (mg O2/L/hour) % Inhibition 
 Control 1 5.8   7  2.0  32.57  -
 Control 2  5.3  6  2.0  33.00  -
 Control 3  5.8  7  2.1  31.71  -
 Control 4  5.1  5  2.3  33.60  -
 Test Item 10 mg/L  5.3  6  1.9  34.00  [4]
 Test Item 100 mg/L  5.3  6  1.9  34.00  [4]
 Test Item 1000 mg/L  5.8  7  2.3  30.00  8
 Test Item 1000 mg/L  6.6  10  2.1  27.00  17
 Test Item 1000 mg/L  5.8  8  2.1  27.75  15
 3,5-dichlorophenol 3.2 mg/L  6.6  10  2.5  24.60  25
 3,5-dichlorophenol 10 mg/L  7.7  10  5.7  12.00  63
 3,5-dichlorophenol 32 mg/L  8.4  10  7.5  5.40  83

No statistically significant toxic effects were shown at the test concentrations of 10 and 100 mg/L, however statistically significant toxic effects were shown at the test concentration of 1000 mg/L. It was considered as unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
The 3 hour EC50 value is > 1000 mg/L. The NOEC after 3 hours of exposure was between 100 and 1000 mg/L.
Executive summary:

Activated sewage sludge was exposed to an aqueous solution of the test item at concentration of 10, 1000 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at temperatures between 18 and 20 degrees centigrade with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

The effect of the test item on the respiration of activated sewage sludge gave a 3 hour EC 50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was between 100 and 1000 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

The reference item gave a 3 hour EC50 value of 7.6 mg/L, 95% confidence limits 5.9 to 9.9 mg/L.

Description of key information

A reliable study (Klimisch 1) is available. The study was conducted according to GLP and to internationally accepted test guidelines.

The 3h EC50 was determined to be >1000 mg/L and the NOEC was in the range 100 - 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information