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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:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Qualifier:
according to guideline
Guideline:
other: Amtsblatt der EG L 133 Teil C: Biologische Abbaubarkeit: Prüfung der Atmungshemmung
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
not applicable
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
-Type: activated sludge (mixed population of different microorganisms)
-Origin: laboratory scale sewage treatment unit treating predominantly municipal sewage. (Location: Southern Wupperverband/Germany)
-no pretreatment
-pH: 7.2
-biomass concentration: 320.0 mg/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Hardness:
no data
Test temperature:
20 ± 2 °C
pH:
7.3-7.6
Dissolved oxygen:
O2-Start: 6.0-7.6 mg/L
O2-End: 3.9-6.9 mg/L
Salinity:
n.a.
Conductivity:
no data
Nominal and measured concentrations:
Nominal concentrations: 5.6, 10, 18, 32, 56 mg/L
Details on test conditions:
-Test item concentration: 5.6, 10, 18, 32, 56 mg/L
-Test item concentration in physico-chemical oxygen consumption control: 10000 mg/L
-Method of application: direct weighing
-Test concentration of the activated sludge: 320 mg/L
-Test temperature: 20 ± 2°C
-Incubation time: 30 minutes with permanent aeration
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorphenol (Purity 97 %)
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The concentrations of the test item and reference compound were not confirmed by analytical methods, but instead based on nominal concentrations.

To measure the oxygen consumption, 250 mL of sludge with test item (or control or reference compound) was incubated for 30 min.
Results with reference substance (positive control):
For 3,5-Dichlorophenol an EC50 of 8.6 mg/L has been determined.
The EC50 value was calculated, using Probit–analysis, from the inhibition of the respiration rates at different concentrations.

Results:

 Test conc. [mg/L]  Respiratory rate [mg/L*h]  Inhibition [%]    Reference substance [mg/L]   Respiratory rate [mg/L*h]  Inhibition [%]
 5.6  21.0  25.5    2.5  24.0  14.9
 10  18.0  36.2    5  22.5  20.2
 18  15.6  44.7    10  10.0  64.5
 32  12.0  57.4    20  5.1  81.9
 56  7.0  75.2    40  3.0  89.4
Validity criteria fulfilled:
yes
Remarks:
on the basic of graphic evaluation: the EC50 for 3,5-DCP was 8.6 mg/L and between the prescribed limits of 2 and 25 mg/L
Conclusions:
The test item showed  75.2 % respiration inhibition of activated sludge at the highest test item concentration (56 mg/L) and an EC50 value of 20 mg/L was determined. Test concentrations are given as nominal concentrations.
Executive summary:

A study with activated sludge was performed according to the German standard method "Amtsblatt der EG L 133 Teil C: Biologische Abbaubarkeit: Prüfung der Atmungshemmung", which is equivalent to the OECD Guideline 209. Two controls without test item were included in the test design. Each batch of activated sludge was checked using 3,5-Dichlorophenol as a reference compound. The physico-chemical oxygen consumption control should be carried out, since some substances may consume oxygen by chemical reactivity. In order to be able to differentiate between physico-chemical oxygen consumption and biological oxygen consumption (respiration), at least the maximum concentration of the test item was additionally tested without activated sludge. To measure the oxygen consumption, 250 mL of sludge with test item (or control or reference compound) was incubated for 30 min. The respiration rate for each concentration was determined graphically from the linear part of the curve of O2-content versus time. The inhibitory effect of the test item at a particular concentration is expressed as a percentage of the mean of the respiration rates of two controls. The sludge was exposed to five different nominal concentrations (5.6, 10, 18, 32, 56 mg/L) of the test item. Because of the strong oxygen consumption of the activated sludge, only 0.32 g/L was used in all batches. The respiration rate of each mixture was determined after aeration period of 30 min. The test item showed 75.2 % respiration inhibition of activated sludge at the highest test item concentration of 56 mg/L. An EC50 value of 20 mg/L was determined. Test concentrations are given as nominal concentrations and were not confirmed by analytical methods.

Description of key information

The test item showed  75.2 % respiration inhibition of activated sludge at the highest test item concentration (56 mg/L) and an EC50 value of 20 mg/L was determined. Test concentrations are given as nominal concentrations.

Key value for chemical safety assessment

EC50 for microorganisms:
20 mg/L

Additional information

A study with activated sludge was performed according to the German standard method "Amtsblatt der EG L 133 Teil C: Biologische Abbaubarkeit: Prüfung der Atmungshemmung", which is equivalent to the OECD Guideline 209. Two controls without test item were included in the test design. Each batch of activated sludge was checked using 3,5-Dichlorophenol as a reference compound. The physico-chemical oxygen consumption control should be carried out, since some substances may consume oxygen by chemical reactivity. In order to be able to differentiate between physico-chemical oxygen consumption and biological oxygen consumption (respiration), at least the maximum concentration of the test item was additionally tested without activated sludge. To measure the oxygen consumption, 250 mL of sludge with test item (or control or reference compound) was incubated for 30 min. The respiration rate for each concentration was determined graphically from the linear part of the curve of O2-content versus time. The inhibitory effect of the test item at a particular concentration is expressed as a percentage of the mean of the respiration rates of two controls. The sludge was exposed to five different nominal concentrations (5.6, 10, 18, 32, 56 mg/L) of the test item. Because of the strong oxygen consumption of the activated sludge, only 0.32 g/L was used in all batches. The respiration rate of each mixture was determined after aeration period of 30 min. The test item showed 75.2 % respiration inhibition of activated sludge at the highest test item concentration of 56 mg/L. An EC50 value of 20 mg/L was determined. Test concentrations are given as nominal concentrations and were not confirmed by analytical methods. This toxicity study is classified as acceptable and satisfies the guideline requirements for the toxicity study to microorganisms.