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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The test was conducted on 31 January 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
1984
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Preparation of stock solutions:
- reference substance : on the day of the test, a stock solution of 1.0 g 3,5-dichlorophenol/L was prepared by dissolving 25.0 mg 3,5-dichlorophenol in 25 mL isotonic medium. The stock solution was ultrasonicated for about 30 minutes and after this period the solution did not contain visible amounts of undissolved test substance.
- test item : a stock solution was prepared at test iniation by dissolving 250.4 mg F1EC in 500 mL isotonic medium resulting in a concentration of 500 mg/L. The stock solution was clear and no undissolved test substance was visible.

Preparation of test solutions:
The study was carried out on one day on which both 3,5-dichlorophenol and F1EC were tested. The isotonic medium, the 3,5-dichlorophenol or F1EC stock solution and the synthetic waste water were added to a 1-litre glass beaker.
Incubation started when the activated sludge and the test substance or reference substance were added to the glass beaker. Every 15 minutes the incubation of one glass beaker was started. The sequence was 0 mg/L (control replicate A); 5.0 mg/L (ref); 10 mg/L (ref); 20 mg/L (ref); 5.0 mg/L (F1EC); 10 mg/L (F1EC); 20 mg/L (F1EC), 40 mg/L (F1EC); 80 mg/L (F1EC), 160 mg/L (F1EC) and 0 mg/L ( control replicate B). Each test solution was aerated immediately and vigorously using filtered air (via carbon filter) after the addition of activated sludge. The dry weight content of the activated sludge in the test solutions was 4.0 x 200/500 = 1.6 g/L.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Synthetic waste water:
1L synthetic waste water was prepared to provide the activated sludge with "feed" during the test. The synthetic waste water was prepared on 30 January 2008 which was one day before the start of the test. The pH of this solution was 7.3 which is in agreement with the criterion of 7.5 ± 0.5 °C.

Activated sludge solution:
About 5L secondary (biological) activated sludge was obtained from the R WZI Horstermeer in Nederhorst den Berg, The Netherlands on 31 January 2008. The R WZI Horstermeer is an activated sludge plant treating 160,000 wastewater (about 80% domestic wastewater). The sludge was obtained from the second tank (from the building) on the day of test initiation and was used directly. After receipt at the laboratory tubes with 200 mL of the activated sludge were centrifuged at 4000 rpm for 20 minutes. After centrifugation, one tube was used to determine the dry weight of the activated sludge. The activated sludge was dried for 2 hours at 105°C. The dry weight of the activated sludge appeared to be 1.2 gram per 200 mL. The supernatant of the tubes was decanted and isotonic medium was added to a dry weight of 4.0 g/L. The minimum volume needed for the study was 2200 mL. The contents of 10 vessels (total dry weight of 12 g) was resuspended in 3000 mL isotonic medium resulting in a dry weight content of 4 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
Not specified
Test temperature:
between 20.5 and 21.1°C
pH:
no data
Nominal and measured concentrations:
nominal concentrations were : 5, 10, 20, 40, 80 and 160 mg/L
Details on test conditions:
Test conditions:
The glass beakers were incubated in a climate chamber with a temperature set point of 20°C. The temperature in the climate chamber was recorded once every hour during the test period and ranged between 20.5 and 21 .1°C.

Oxygen measurement:
Thirty minutes after the start of the incubation phase, the measurement phase was started. A 250-mL Erlenmeyer flask was completely filled with the test solution and stoppered without a headspace. Each minute the oxygen content was recorded (Hach HQ 10 oxygen meter). After 11 minutes, the test solution was poured back into the glass beaker and incubation (with vigorous aeration) was continued. After three hours of incubation the oxygen content was measured again during an 11-minutes period.

Calculations:
For each glass beaker the respiration rate was determined for bath measurement periods. The respiration rate is defined as the slope of the linear part of the curve and is expressed as mg oxygen per litre per minute. For each concentration the inhibition percentage of the respiration rate will be expressed as a percentage of the mean of the two control respiration rates. Linear interpolation, with concentration on a logarithmic scale (base = 10), was used to determine the E(I)C50 after 30 minutes and after 3 hours.

Validity criteria:
- The difference in respiration rate of the two control replicates should be less than 15%;
- After an incubation period of three hours the E(I)C50 of 3,5-dichlorophenol should be in the range of 5 to 30 mg/L.
Reference substance (positive control):
yes
Remarks:
3.5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
IC50
Effect conc.:
> 160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Results of the controls
The results of the oxygen measurements show a stable decline of the oxygen content during the 11 minutes period, which is equivalent with a constant respiration rate during the measurement period.
After 30 minutes of incubation the difference between the two control respiration rates (replicate A and B) was 7.8%. After 3 hours of incubation the difference between the two contral respiration rates was 14.7%. In both cases the difference between the two controls was less than 15% which is in agreement with the first validity criterion.

Results of F1EC
The respiration rates and the inhibition percentages of the respiration rate are presented in Table 1. The inhibition percentages are based on the mean respiration rate of controls A and B.
At the highest test concentration of 160 mg/L the respiration inhibition was 24 and 19%, respectively after 30 minutes and 3 hours incubation. The E(I)C50 value after 30 minutes and 3 hours of incubation was > 160 mg/L.
Results with reference substance (positive control):
The inhibition percentages of 3.5-dichlorophenol are based on the mean respiration rate of controls A and B.
After 30 minutes of incubation the E(I)C50 value was estimated to be 5.5 mg/l. After 3 hours of incubation the E(I)C50 value was also 5.5 mg/l which is in agreement with the second validity criterion. According to this validity criterion the E(I)C50 value after 3 hours of incubation should be in the range of 5 to 30 mg/l. For this reason it can be concluded that the sensitivity of the activated sludge was sufficient.

  Table 1 : Respiration rates and inhibition at various F1EC concentrations

 Nominal concentrations

(mg/L)

 Respiration rate at 30 min incubation

(mgO2/L/min)

 Respiration rate at 3h incubation

(mgO2/L/min)

 Inhibition at 30 min incubation

(%)

 Inhibition at 3h incubation

(%)

 0 A  0.54  0.47  -  -
 5.0  0.57  0.51  0  -0.8
 10  0.53  0.52  5.9  -3.0
 20  0.56  0.54  2.0  -7.0
 40  0.60  0.61  -5.9  -20.8
 80  0.52  0.67  8.8  -32.4
 160  0.43  0.41  23.5  18.9
 0 B  0.59  0.54  -  -
Validity criteria fulfilled:
yes
Remarks:
The difference in respiration rate of the two control replicates was < 15%; The 3h E(I)C50 of 3,5-dichlorophenol was in the range of 5 to 30 mg/L.
Conclusions:
The 30 min and 3 h IC50 of F1EC to activated sludge were determined to be above 160 mg/L.
Executive summary:

The objective of this study was to determine the effect of F1EC (MONOFLUOROETHYLENE CARBONATE) on activated sludge by measuring the respiration rate under defined conditions in the presence of different concentrations of the test substance.

3,5-Dichlorophenol was used as reference substance to evaluate the sensitivity of the activated sludge. The test was conducted according to OECD Guideline 209 (1984) and according to the OECD GLP guidelines (1998).

The respiration rate of an activated sludge suspension (4.0 g dry weight/L) fed with a standard amount of synthetic wastewater was measured after a contact time with the test substance of 30 minutes and 3 hours. The respiration rate is defined as the oxygen consumption of a given concentration of activated sludge expressed as mg oxygen per liter per minute. The study was carried out on one day on which both 3,5-dichlorophenol and F1EC were tested.

Two controls (replicate A and B) were included. As the difference between the two controls was < 15%, the test can be considered as valid.

The reference test was conducted at concentrations of 5.0, 10 and 20 mg 3,5-dichlorophenol /L. The E(I)C50 value was estimated to be 5.5 mg/l after 30 min and 3h of incubation. As the E(I)C50 value after 3 hours is in the range of 5 to 30 mg/l it can be concluded that the sensitivity of the activated sludge was sufficient.

The test with F1EC was conducted at concentrations of 5.0; 10; 20; 40; 80 and 160 mg/l. At the highest test concentration of 160 mg/L the respiration inhibition was 24 and 19%, respectively after 30 minutes and 3 hours incubation. The E(I)C50 value after 30 minutes and 3 hours of incubation was > 160 mg/L.

Description of key information

The 3 h IC50 of the substance to activated sludge were determined to be above 160 mg/L and the NOEC 80 mg/L. The NOEC is used as the key value for chemical safety assessment as the EC50 was determined to be greater than the highest test concentration.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
80 mg/L

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