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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 05 November 2015 to 11 December 2015
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 (Carbon and Ammonium Oxidation))
Version / remarks:
(2010)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Version / remarks:
(2007)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTIONS
A stock solution of the test substance of 10 g/L was prepared for the combined limit/range-finding and final test by adding 5.0 g test item to 500 mL of Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Vigorous stirring was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear and colourless stock solution (pH 2.5) corresponding to the test concentration were then added to the test media (16 mL synthetic medium and made up to 250 mL with Milli-RO). Thereafter, 250 mL of activated sludge was added resulting in the required concentrations. Optimal contact between the test item and test organisms was ensured applying continuous aeration and stirring.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands
- Preparation: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.6 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (=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.
- The amount of suspended solids in the final test mixture was 1.5 g/L (250 mL of 3.0 g/L in 500 mL)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
- Combined limit/range-finding test: 20.1 - 22.0°C
- Final test: 20.2 - 21.3°C
pH:
- Combined limit/range-finding test:
-- Before addition of sludge: 7.0 - 7.5
-- After the 3 hour exposure period: 6.9 - 8.4
- Final test:
-- Before addition of sludge: 7.0 - 7.6
-- After the 3 hour exposure period: 7.3 - 8.2
Dissolved oxygen:
The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation
Nominal and measured concentrations:
- Combined limit/range-finding test: 10, 100 and 1000 mg/L
- Final test: 10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass open bottles/vessels
- Total volume: 500 mL
- Aeration: yes, during the exposure period
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per reference (replicates): 3
- Sludge concentration: 250 mL/500 mL
- Synthetic medium concentration: 16 mL/500 mL
- Nitrification inhibitor used: No (based on the results of the combined limit/range finding study, see below)
MEDIUMS
- ISO medium: adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, The Netherlands) with the following composition:
CaCl2.2H2O: 211.5 mg/L
MgSO4.7H2O: 88.8 mg/L
NaHCO3: 46.7 mg/L
KCL: 4.2 mg/L
- Synthetic medium (=sewage feed): according to OECD 209 (2010)
OTHER TEST CONDITIONS
- Adjustment of pH: no
EFFECT PARAMETERS MEASURED:
- Calculation of EC50 value based on probit analysis using linear maximum likelihood regression with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
COMBINED LIMIT/RANGE FINDING STUDY:
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study:
- 1000 mg/L test substance + nitrification inhibitor (11.6 mg/L N-allylthiourea) (3 replicates): heterotrophic inhibition comparable to the total inhibition
- 10 mg/L test substance: no inhibition of total resp. (1 replicate)
- 100 mg/L test substance: 37% inhibition of total resp. (1 replicate)
- 1000 mg/L test substance: 74% inhibition of total resp. (average of 3 replicates)
- Abiotic control: no oxygen uptake from abiotic processes (1 replicate)
- Based on the results of the total and heterotrophic respiration, nitrification inhibition was calculated to be 71%
PERFORMANCE OF THE TEST
The synthetic medium (=sewage feed,16 mL) and an appropriate amount of the test item stock were mixed and made up to 250 mL with Milli-RO water (tap-water purified by reverse osmosis (Millipore Corp., USA)). The pH was determined. Thereafter 250 mL activated sludge was added. This was the start of the test. After the 3-hour contact time the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls. The medium temperature was recorded continuously in a temperature control vessels. The temperature control vessels were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.
OXYGEN RECORDING
Determination of oxygen was performed with multiple oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.
Reference substance (positive control):
yes
Remarks:
(3,5-dichlorophenol)
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
270 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% confidence interval: 160 - 540 mg/L
Details on results:
- At all concentrations, statistically significant inhibition of the respiration rate of the sludge was recorded (see the table below)
- A NOEC or EC10 could not be determined as 23% inhibition was observed at the lowest tested concentration of 10 mg/L
Results with reference substance (positive control):
- Combined limit/range-finding test: EC50: 5.6 mg/L
- Final test: EC50: 4.1 mg/L

Treatment

Concentration

pH

 

Mean respiration rate

 

%

Inhibition (mean value)

 

(mg/L)

Start

End

(mg O2/L h)

(mg O2/g h)

 

Blank control

 

7.5-7.6

7.3-7.7

44

29

 

T1

10

7.5

7.6-7.7

33

22

23

T2

32

7.5

7.5-7.6

32

22

26

T3

100

7.4-7.5

7.5-7.6

26

17

41

T4

320

7.3-7.4

7.4-7.6

22

15

49

T5

1000

7.0

7.4-7.5

15

10

65

 

Validity criteria fulfilled:
yes
Conclusions:
In a study in accordance with OECD 209 (2010) and according to GLP principles, an EC50 value of 270 mg/L was determined for the inhibitory effect of the substance on activated sludge bacteria.
Executive summary:

In accordance with OECD 209 (2010) and according to GLP principles, the influence of the substance on the respiration rate of activated sludge was investigated after a contact time of 3 hours. The final test was performed based on the result of a preceding combined limit/range-finding test. Five concentrations, ranging from 10 to 1000 mg/L, increasing with a factor 3.2 were tested. Five replicates per concentration and six replicates for an untreated control group were tested. In the combined limit/range-finding test it was determined that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition. The inhibitory effect of the substance on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 23% inhibition of the respiration rate (total) at the lowest tested concentration of 10 mg/L to 65% at 1000 mg/L. Statistically significant inhibition of the respiration rate of the sludge was recorded at all concentrations tested. The study met the acceptability criteria. Based on the results of the study, an EC50 value of 270 mg/L was determined for the inhibitory effect of the substance on activated sludge bacteria. No EC10 or NOEC could be derived.

Description of key information

In accordance with OECD 209 (2010) and according to GLP principles, the influence of the substance on the respiration rate of activated sludge was investigated after a contact time of 3 hours.The final test was performed based on the result of a preceding combined limit/range-finding test. Five concentrations, ranging from 10 to 1000 mg/L, increasing with a factor 3.2 were tested. Five replicates per concentration and six replicates for an untreated control group were tested. In the combined limit/range-finding test it was determined that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition. The inhibitory effect of the substance on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 23% inhibition of the respiration rate (total) at the lowest tested concentration of 10 mg/L to 65% at 1000 mg/L. Statistically significant inhibition of the respiration rate of the sludge was recorded at all concentrations tested.The study met the acceptability criteria. Based on the results of the study, an EC50 value of 270 mg/L was determined for the inhibitory effect of the substance on activated sludge bacteria. No EC10 or NOEC could be derived.

Key value for chemical safety assessment

EC50 for microorganisms:
270 mg/L

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