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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:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
The method followed that described in the OECD Guidelines for
Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", EEC
Commission Directive 87/302/EEC and US EPA Draft Ecological Effects Test Guidelines OPPTS
850.6800.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements of Directives 87/18/EEC (as amended by Directive 1999/11/EC) and 881320/EEC (as amended by Directive 1999/12/EC).
Test material information:
Composition 1
Specific details on test material used for the study:
For the purposes of the test a reference material, 3,5-dichloropbenol (Sigma-Aldrich Batch
No. 10330 H102611ES) was used. Two stock solutions of 50 and 160 mg/l were prepared by
dissolving the reference material directly in water with the aid of ultrasonic disruption. Aliquots
(10 and 100 ml) of the 160 mg/I stock solution were removed and dispersed with activated sewage
sludge, synthetic sewage and water to give the final concentrations of 3.2 and 32 mg/I. Similarly,
a 100 ml aliquot of the 50 mg/I stock solution was used to prepare the 10 mg/I concentration.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:

- Preparation of inoculum for exposure:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a
temperature of 21 °C and was used on the day of collection. The pH of the sample was 7.5 and the
suspended solids equal to 4.0 g/l prior to use.
Test type:
not specified
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
Observations were made on the test preparations throughout the test period, and the pH of the control, reference material and test material preparations was measured at the end of the exposure period prior to measurement of the oxygen consumption rate.
Post exposure observation period:
n/a
Hardness:
100mg/L Calcium carbonate
Test temperature:
21C
pH:
7.5
Dissolved oxygen:
6.9mg/L
Salinity:
n/a
Conductivity:
n/a
Nominal and measured concentrations:
3.2 - 32mg/L
Details on test conditions:
For the purpose of the definitive test, the test material was dissolved directly in water.
An amount oft.est material (100 mg) was dissolved in water with the aid of ultrasonic disruption
(approximately 60 minutes) and high shear mixing (7500 rpm, 10 minutes) and the volume
adjusted to 1 OOO ml to give a 100 mg/I stock solution from which a dilution was made to give a
10 mg/I stock solution. Aliquots (9, 28 and 90 ml) of the 10 mg/I stock solution were separately
dispersed with synthetic sewage (16 ml), activated sewage sludge (200 ml) and water, to a final
volume of 500 ml, to give the required concentrations of0.18, 0.56 and 1.8 mg/I. Similarly
aliquots (28, 90 and 280 ml) of the 100 mg/I stock solution were used to give the required
concentrations of 5.6, 18 and 56 mg/I.
The volumetric flasks containing the stock solutions were inverted several times to ensure
homogeneity of the stock solutions.
Analysis of the concentration, homogeneity and stability of the test material in the test
preparations was not appropriate to the Test Guideline.
Reference substance (positive control):
yes
Remarks:
activated and synthetic sewage plus water
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
31 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other:
Details on results:
The effect of the test material on the respiration of activated sewage sludge gave a
3-Hom ECso of 31 mg/I. The No Observed Effect Concentration (NOEC) after 3 hours exposure
was 0.56 mg/I.
Results with reference substance (positive control):
Oxygen consumption rates and percentage inhibition values for the control, test and reference
materials in the definitive test are given in Tables 2 and 3. The pH values of the test preparations
at the end of the exposure period are given in Table 4, and observations made on the test
preparations throughout the study are given in Table 5.
Percentage inhibition is plotted against concentration for the test and reference materials (Figures
1to4).
The following results were derived:
PCMX ECso (30 minutes): 52 mg/I
3,5-dichlorophenol ECso (30 minutes): 18 mg/I
Variation in respiration rates of controls 1 and 2 after 30 minutes: ::1:: 2%.
PCMX ECso (3 homs): 31 mg/I
3,5-dichlorophenol ECso (3 hours): 9.7 mgll
Variation in respiration rates of conttols 1 and 2 after 3 hours: ::!:: 6%.
The No Observed Effect Concentration (NOEC) after 3 hours was 0.56 mgll.
The validation criteria for the con1rol respiration rates and reference material ECso values have
been satisfied.
Reported statistics and error estimates:
n/a
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the respiration of activated sewage sludge micro-organisms gave
a 3-Hour EC50 of 31 mg/I. The No Observed Effect Concentration (NOEC) after 3 hours
exposme was 0.56 mg/I.

Description of key information

The effect of the test material on the respiration of activated sewage sludge micro-organisms gave

a 3-Hour EC50 of 31 mg/I. The No Observed Effect Concentration (NOEC) after 3 hours

exposme was 0.56 mg/I.

Key value for chemical safety assessment

EC50 or LC50 for microorganisms:
31 mg/L
EC10, LC10 or NOEC for microorganisms:
0.56 mg/L

Additional information

see full study report