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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:
2012-08-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
adopted 22 July 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Municipal sewage treatment plant of 31137 Hildesheim, Germany (predominantly municipal sewage and hardly any industrial chemical waste)
- Pretreatment: The sludge was washed twice with autoclaved tap water. Adjustment to a dry sludge concentration of 3.0 g/L ± 10 % was not necessary. The sludge was used within 24 h after sampling.
- Initial biomass concentration: Dry sludge concentration 2.90 g/L, corresponding to 1.45 g/L in the test vessel
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.8°C
pH:
activated slugde: 7.83
synthetic waste water: 7.37
Nominal and measured concentrations:
nominal: 10, 32, 100, 320, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flasks
- Aeration: Shaking of flasks at 150 rpm to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes suspension.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per reference item (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic waste water according to OECD Guideline 209


OTHER TEST CONDITIONS
- Adjustment of pH: not necessary


EFFECT PARAMETERS MEASURED:
O2 consumption

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 1, 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: 14-16% inhibition at 100 mg/L, 52% inhibition at 1000 mg/L
Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
72.8 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 62.6-84.7 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
557 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 495-634 mg/L
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 = 91.8 mg/L, 95% c.i = 83.2 - 102 mg/L, this is in the recommended range of validity of 53 - 155 mg/L

Validity criteria:

The mean specific oxygen uptake rate of the control replicates was 19.6 mg O2/g×h (validity criterion: should be >/=20 mg O2/g h). This is only slightly below the recommended value and is therefore considered to have no impact on the quality and integrity of the study.

The coefficient of variation of the oxygen uptake rates in the control replicates was 4.1% (validity criterion: < 30%).

The EC50 of the reference item was in the range of 53 - 155 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
The NOEC of ETMA based on respiration inhibition of activated sludge is 32 mg/L. The EC10 is 72.8 mg/L. The EC50 is 557 mg/L.
Executive summary:

In a 3 h Respiration Inhibition Test according to OECD Guideline 209, adopted 22 July 2010, activated sludge obtained from a municipal treatment plant was exposed to ETMA at nominal concentrations of 10, 32, 100, 320 and 1000 mg/L under static conditions. In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item.

The reference item produced the appropriate response.

The NOEC of ETMA based on respiration inhibition of activated sludge is 32 mg/L. The EC10 is 72.8 mg/L. The EC50 is 557 mg/L.

 

Results Synopsis

 

Test Organism: activated sludge

Test Type: Static

 

3 hr EC10: 72.8mg/L  95% C.I.:  62.6 - 84.7 mg/L

3 hr EC50: 557mg/L   95% C.I.:  495 - 634 mg/L

3 hr NOEC:  32 mg/L

Endpoint(s) Effected:respiration inhibition

Description of key information

EC10 = 72.8 mg/L; OECD Guideline 209; GLP; activated sludge respiration inhibition (static)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
72.8 mg/L

Additional information

For the assessment of toxicity to microorganisms of ETMA two reliable (RL=1 - 2), relevant and adequate studies are available:

In a 3 h Respiration Inhibition Test according to OECD Guideline 209, adopted 22 July 2010, activated sludge obtained from a municipal treatment plant was exposed to ETMA at nominal concentrations of 10, 32, 100, 320 and 1000 mg/L under static conditions. In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item. The reference item produced the appropriate response.

The NOEC of ETMA based on respiration inhibition of activated sludge is 32 mg/L. The EC10 is 72.8 mg/L. The EC50 is 557 mg/L.

 

This study is supported by the results of a test on ready biodegradation which included a toxicity control:

The ready biodegradation of ETMA was investigated in a study conducted according to OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test), adopted 17 July 1992 and EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test), over a period of 28 days. The type of inoculum was not specified in the study report. The biodegradation rate was determined by measurement of O2 consumption. Inoculum blank and procedural/functional control with the reference substance sodium benzoate were performed.

The functional control reached the pass level >60% after 14 d. The biodegradation of the test substance remained below 5% throughout the study period of 28 d (1.1 – 4.3%). No biodegradation was observed under the conditions of the present study.

The biodegradation in the toxicity control ranged between 41.0 and 42.4%. According to the criteria of the OECD guideline 301 D a substance can be assumed to be inhibitory, if less than 25% (based on total ThOD) degradation occurred within 14 days. Thus, ETMA did not inhibit bacterial activity at a concentration of 1.65 mg/L