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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
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:
yes
Remarks:
see Any other information on materials and methods incl. tables
GLP compliance:
yes (incl. QA statement)
Remarks:
Health Care Inspectorate, Ministry of Health, Welfare and Sport, The Netherlands
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was not completely soluble in test medium at the loading rates initially prepared. Therefore, test substance mixtures in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate were stirred in closed dark brown bottles for approximately 25 hours, except for the abiotic control which was stirred for 28.5 hours. Subsequently, to 200 mL of the prepared solutions, 16 mL synthetic medium, 250 mL sludge and Milli-RO water up to 500 mL were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm), washed and diluted with ISO-medium. 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 8.0 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Concentration of suspended solids in the test mixture: 1.5 g/L in the final test mixture.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
Combined limit/range-finding test
Test temperature:
18.2 - 20.1°C
pH:
before addition of sludge: 7.3 - 7.4
after the 3 hour exposure period: 7.2 - 8.5.
Dissolved oxygen:
at the start above 60-70%
Nominal and measured concentrations:
Loading rates: 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: all glass bottles/vessels
- Aeration: clean, oil-free air, constantly
- No. of vessels per concentration (replicates): The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate.
- No. of vessels per control (replicates): 6
- Biomass loading rate: 1.5 g/L in the final test mixture

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis
- Intervals of water quality measurement: Temperature and pH were measured at the start and at the end of the study.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After the 3-hour contact time, the oxygen consumption was recorded for a period of 10-15 minutes.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: The test was performed as a limit test at 1000 mg/L. Additional lower concentrations were tested to be able to use the study as a range finding study, in case effects would have occurred.
Reference substance (positive control):
yes
Remarks:
1, 3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: carbon and ammonium oxidation
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: carbon and ammonium oxidation
Details on results:
- The combined limit/range-finding test showed no inhibition, 18% and an average of 5% inhibition of the respiration rate at a loading rate of 10, 100 and 1000 mg/l, respectively. The inhibition observed at a loading rate of 1000 mg/l was not statistically significant.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
- Effect concentrations exceeding solubility of substance in test medium: not reported
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (3h) = 14 mg/L
Reported statistics and error estimates:
Two sample t-test: alpha= 0.05

Table: Study results

Loading rate (mg/L)

Respiration rate (mg O2/L h)

Respiration rate (mg O2/g h) (1)

% Inhibition of respiration relative to the control (mean)

0 (Blank control, mean)

39

26

-

0 (Nitrification control)

32

21

19

1000 (Abiotic control)

0

0

0

1000 (Test substance with N-allylthiourea)

32

21

1 (2)

10

41

27

-4

100

32

21

18

1000 (mean)

38

25

5

(1) The amount of suspended solids in the final test mixture was 1.5 g/L

(2) Relative to Nitrification control

Executive summary:

One study is available on the toxicity of 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl 2-chloropropenoic acid ester (CAS 96383-55-0) to microorganisms. The study was conducted according to OECD guideline 209 and GLP (Desmares-Koopmans, 2013). The test was designed as a combined limit- and range finding study. The limit test loading 1000 mg/L was tested in triplicate. Additionally, single replicates of 10 and 100 mg/L loadings were tested, to be used for range finding, incase effects would occur. Activated sludge form a municipal sewage treatment plant, receiving predominantly domestic sewage, was exposed to the test substance for three hours, and the inhibition of respiration was analysed. The test showed no inhibition, 18% inhibition and an average of 5% inhibition of the respiration rate at a loading rates of 10, 100 and 1000 mg/L, respectively. The inhibition observed at a loading rate of 1000 mg/L was not statistically significant. Hence, the EC50 and NOEC can considered to be > 1000 mg/L and 1000 mg/L, respectively.

Description of key information

EC50 (3h) > 1000 mg/L (OECD 209; activated sludge)

NOEC 1000 mg/L (OECD 209; activated sludge)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

One study is available on the toxicity of 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl 2-chloropropenoic acid ester (CAS 96383-55-0) to microorganisms. The study was conducted according to OECD guideline 209 and GLP (Desmares-Koopmans, 2013). The test was designed as a combined limit- and range finding study. The limit test loading 1000 mg/L was tested in triplicate. Additionally, single replicates of 10 and 100 mg/L loadings were tested, to be used for range finding, incase effects would occur. Activated sludge form a municipal sewage treatment plant, receiving predominantly domestic sewage, was exposed to the test substance for three hours, and the inhibition of respiration was analysed. The test showed no inhibition, 18% inhibition and an average of 5% inhibition of the respiration rate at a loading rates of 10, 100 and 1000 mg/L, respectively. The inhibition observed at a loading rate of 1000 mg/L was not statistically significant. Hence, the EC50 and NOEC can considered to be > 1000 mg/L and 1000 mg/L, respectively.