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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 February 1999 - 17 May 1999
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 AND APPLICATION OF TEST SOLUTION
- Method:
Test substance: a stock dispersion of the test substance was prepared by dispering 0.2010 g of Ziram in 1000 mL of BOD dilution water
Reference substance: a stock solution of the reference substance was prepared according to the OECD guideline by dissolving 0.1251 g in 5 mL NaOH solution, adjusting the pH to 7.7 with HCl solution, and the making up to 250 mL with Nanopure water
- Controls: yes
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: oxidation ditch treating domestic sewage, Berkel en Rodenrijs, The Netherlands
- Preparation of inoculum for exposure: the sample was centrifugated and the supernatant discarded. Activated sludge was washed three times by centrifugation (10 min at 2000 rpm = max. 1218 g) and resuspension in tap water twice and in dilution water once. The dry weight of the sludge suspension was determined and the suspension diluted to approximately 4 g/L of mixed liquor suspended solids (MLSS) by addition of dilution water. Then, it was aerated vigorously and kept at approximately 20°C in the dark.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20°C
pH:
7.9
Nominal and measured concentrations:
Nominal concentrations: Negative control, 0.10, 0.32, 1, 3.2, 10, 32 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 1 L glass beakers containing 16 mL of synthetic sewage feed, 284 mL of control (dilution water) or treatment solutions and 200 mL of activated sludge
- Aeration: 0.5 to 1 L/min
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Suspended solids: 4.5 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the dilution water was prepared according to NEN standard No. 6634.
- Synthetic sewage feed was prepared according to OECD guideline

EFFECT PARAMETERS MEASURED: respiration inhibition

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
19.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
The EC50 for 3,5-dichlorophenol was determined to be 13.2 mg/L, value within the validity range of the OECD guideline (5 to 30 mg/L)

Table 1: Validity criteria for OECD 209

Criterion from the guideline

Outcome

Validity criterion fulfilled

The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.

Respiration rates of the two controls were 20.2 and 21.7 mg O2/g/h

yes

The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.

Variation was 7%

yes

Validity criteria fulfilled:
yes
Remarks:
For details please refer to field "any other information on results incl. tables"
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
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
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
2008
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
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Nominal and measured concentrations:
Nominal: Control (tap water, synthetic sewage and inoculum), 10, 32, 100, 320, 1000 mg/L
Reference substance (positive control):
yes
Remarks:
3.2, 10 and 32 mg 3,5-Dichlorophenol/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
58.8 mg/L
95% CI:
>= 29.7 - <= 106.9
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
187.6 mg/L
95% CI:
>= 100 - <= 371.5
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks:
without nitrification
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
19.8 mg/L
95% CI:
>= 2 - <= 47
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks:
without nitrification
Results with reference substance (positive control):
- Results with reference substance valid: yes
- Relevant effect levels: EC50 (3 h) = 4 mg/L (total respiration), EC50 (3 h) = 15.5 mg/L (heterotrophic respiration without nitrification)

The oxygen uptake in the blank controls was 28.5 mg/g/h and therefore clearly above 20 mg oxygen/g of activated sludge (dry weight of suspended solids) in an hour. The respiration rates of the six controls differed not more than 30 %. The value was 10.0 % for the total respiration and 9.9 % for the heterotrophic respiration. These validity criterions were fulfilled.


Validity criteria fulfilled:
yes
Remarks:
For details please refer to field "any other information on results incl. tables"

Description of key information

EC50 (3 h) = 19.2 mg/L (nominal, activated sludge, OECD 209)

NOEC (3 h) = 0.32 mg/L (nominal, activated sludge, OECD 209)

Key value for chemical safety assessment

EC50 for microorganisms:
19.2 mg/L
EC10 or NOEC for microorganisms:
0.32 mg/L

Additional information

Two studies are available assessing the toxicity of zinc bis dimethyldithiocarbamate (CAS No. 137-30-4) towards aquatic microorganisms.

In the key study (1999) the effect of the substance on aquatic microorganisms activity was investigated according to the OECD Guideline No. 209 and GLP. Activated sludge of a predominantly domestic sewage was exposed for 3 h to the test item concentrations of 0.10, 0.32, 1, 3.2, 10, 32 and 100 mg/L and a negative control. The inhibition of total respiration rate was measured, resulting in an EC50 (3 h) value of 19.2 mg/L (nominal). This study was chosen as key study due to its methodological reliability and due to the most sensitive effect values.

In the supporting study (2014) the effects of the substance on sewage sludge microorganisms were assessed according to the OECD Guideline No. 209 and GLP. The activated sludge of a predominantly domestic sewage was exposure for 3 h to 10, 32, 100, 320 and 1000 mg test item/L and the respiration rate (oxygen consumption) was measured. The 3-hour EC50 was 58.8 mg/L (nominal, inhibition of total respiration). For heterotrophic respiration without nitrification the 3-hour EC50 was 187.6 mg/L (nominal).