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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Aug 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Umwelt und Naturschutz, Landwirtschaft und Verbraucherschutz des Landes Nordrhein-Westfalen
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct weighting
- Differential loading: yes
- Controls: blank control
- Eluate: no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: no
- Name and location of sewage treatment plant where inoculum was collected: STP, Cologne-Stammheim, Germany (aeration tank)
- Method of cultivation: Aeration of the activated sludge at 20 °C ± 2 °C, daily feed with synthetic medium.
- Preparation of inoculum for exposure: First, the sludge is settled and supernatant is decanted. After centrifuging (15 min at 4000 rpm and 20°C) the supernatant is decanted again. 1 g of wet sludge is dried in order to calculate the amount of wet sludge to achieve a concentration of activated sludge of 3 g/L (dry wet) suspended solids. The calculated amount of sludge is first dissolved in synthetic medium and then filled up to a defined end volume with de-ionized water.
- Pretreatment: none
- Concentration of Inoculum: 3 g/L (dry weight).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.7 - 20.9 °C
pH:
7.7 - 7.9
Dissolved oxygen:
See "Any other information on results incl. tables"
Nominal and measured concentrations:
nominal: control, 10, 18, 32, 56 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL glass Erlenmeyer flasks
- Aeration: yes
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 800 mg/L suspended solids

TEST MEDIUM / WATER PARAMETERS
- Preparation of test medium: according to guideline

OTHER TEST CONDITIONS
- Adjustment of pH: yes, adjusted to pH 7±0.5 using HCl

EFFECT PARAMETERS MEASURED: respiration inhibition after 3 h of incubation

TEST CONCENTRATIONS
- Range-finding test : 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: The range-finding test revealed inhibition rates of 20.1%, 74.2% and 100% for test substances concentrations of 10, 10 and 100 mg/L, respectively.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
5.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: respiration rate
Remarks:
95% CL: 1.6 - 10.2 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
41.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: respiration rate
Remarks:
95% CL: 30.8 - 58.7 mg/L
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: no
- Blank controls oxygen uptake rate: no physico-chemical oxygen consumption was observed
Results with reference substance (positive control):
- Results with reference substance valid? yes, the EC50 of 3,5-dichlorophenol is in the accepted range 5 to 30 mg/L
- EC50 (3 h): 7.2 mg/L (95% CI: 3.8 - 13.2 mg/L)
Reported statistics and error estimates:
Inhibitory effect of the test item: Percentage of the mean of the respiration rates of two controls. EC50 values: Determined by probit analysis of the respiration rates at different test substance concentrations.

Table 1: Main test (test substance CMK): Oxygen content, temperature and pH values during exposure phase

 

Test item concen-tration [mg/L]

O2 start [mg O2/L]

O2 end [mg O2/L]

Time (start-end) [min.]

Temp.

[°C]

pH

Test item

10

5.7

2.6

7

20.6

7.7

18

5.5

2.8

7

20.5

7.7

32

5.8

2.7

9

20.7

7.7

56

5.9

3.9

8

20.8

7.6

100

6.2

5.0

9

20.9

7.5

Control 1

 

5.7

2.8

5

21.1

7.7

Control 2

 

5.4

2.7

5

20.6

7.7

Physico-chemical oxygen consumption control *

 

7.5

7.5

9

20.8

7.3

  Comments: * Data obtained in the range finding test. The test item was added to about 130 mL deionized water and stirred overnight before testing (equilibration phase).

Table 2: Results of test item

Test item concen-tration [mg/L]

Respiratory ratetest item [mg/L h]

Phys.-chem. O2 consumption [mg/Lh]

Respiratory rate - phys.-chem. O2 consumption [mg/Lh]

Inhibition [%]

10

26.6

0.0*

26.6

20.9

18

23.1

0.0*

23.1

31.1

32

20.7

0.0*

20.7

38.5

56

15.0

0.0*

15.0

55.4

100

8.0

0.0

8.0

76.2

Control. Mean

33.6

 

Control 1

34.8

 

Control 2

32.4

 

* The physico-chemical oxygen consumption has been determined at 1000 mg/L test item concentration. As no physico-chemical oxygen consumption was observed at that test item concentration this observation also holds true for the lower test item concentrations.

Table 3: Validity criteria

Criterion from the guideline

Outcome

Validity criterion fulfilled

The two control respiration rates are within 15 per cent of each other.

Respiratory rates of the 2 controls differ less than 15 %

yes

The EC 50 (3 hours) of 3,5-dichlorophenol is in the accepted range 5 to 30 mg/L.

EC50 of the reference compound 3.5-Dichlorophenol is in the range 5 – 30 mg/L

yes

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Conclusions:
EC 50 (3 h) = 41.4 mg/L
EC 10 (3 h) = 5.7 mg/L
Executive summary:

The effect of p-chloro-m-cresol on aquatic micro-organism was investigated by Neuhahn (2008) in accordance with the OECD guideline 209 and EC Method C.11. P-chloro-m-cresol showed inhibitory effects on activated sludge. 50% inhibition of microbial activity was determined at 41.4 mg/L. The EC10 was determimed at 5.7 mg/L.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to analogue justification document provided in IUCLID section 13.
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
5.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: respiration rate
Remarks:
95% CL: 1.6 - 10.2 mg/L
Remarks on result:
other:
Remarks:
source, CAS 59-50-7, key rel 1, Neuhahn 2008
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
41.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: respiration rate
Remarks:
95% CL: 30.8 - 58.7 mg/L
Remarks on result:
other:
Remarks:
source, CAS 59-50-7, key rel 1, Neuhahn 2008

Description of key information

EC50 (3 h) = 41.4 mg/L (nominal, activated sludge microorganisms), read-across

EC10 (3 h) = 5.7 mg/L (nominal, activated sludge microorganisms), read-across

Key value for chemical safety assessment

EC50 for microorganisms:
41.4 mg/L
EC10 or NOEC for microorganisms:
5.7 mg/L

Additional information

No study investigating the toxicity of sodium p-chloro-m-cresolate (CAS 15733-22-9) to aquatic microorganisms is available. Therefore, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read-across to the source substance (p-chloro-m-cresol, CAS 59-50-7) which is the main transformation product of the target substance sodium p-chloro-m-cresolate (CAS 15733-22-9) is applied.

The source substance p-chloro-m-cresol is the common compound in this analogue approach and is solely responsible for the (absence of) effects. Thus, the source substance is considered a suitable representative for the evaluation of the toxicity of the target substance to aquatic organisms. The read-across approach is justified in detail within the analogue justification in IUCLID section 13.

One experimental study is available investigating the effect of p-chloro-m-cresol on aquatic microorganism (Neuhahn, 2008). The test was conducted according to OECD guideline 209 and EC Method C.11. Activated sludge microorganisms were exposed for 3 hours to nominal concentrations of 10, 18, 32, 56 and 100 mg/L. The inhibition of respiration was measured at the end of the study. P-chloro-m-cresol showed inhibitory effects on activated sludge microorganisms. 50% inhibition of microbial activity was determined at 41.4 mg/L. Therefore the EC50 (3 h) was 41.4 mg/L. The EC10 (3 h) was determined to be 5.7 mg/L.

Based on the available results from a structurally similar source substance (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) which is the main transformation product of the target substance and is characterized by a similar ecotoxicological profile and comparable structure, it can be concluded that sodium p-chloro-m-cresolate has effects in the same range as the target substance.