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Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Cell Multiplication Inhibition Assay acc. to Bringmann
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
no data available
Analytical monitoring:
not specified
Details on sampling:
no data available
Vehicle:
not specified
Details on test solutions:
Pollutant solution was prepared by adding the test material in sterile double-distilled water. From this pollutant solution four parallel dilution series were made in 300 mL Erlenmeyer flasks, stoppered with cotton-lined plastic caps.
Dilution series were done as follows: the first flasks contain 160 mL of pollutant solution. Starting from this flask the subsequent solution steps at a constant dilution ratio by consistently mixing 80 mL of preliminary pollutant dilution and 80 mL double distilled water. At the end of the dilution series each flasks were made up to 100 mL by adding 5 mL solution I, 5 mL solution II and 10 mL of the prepared bacterial solution.
Not inoculated flasks were prepared by adding 10 mL saline instead of bacterial suspension.
Stock solution I: 20 g glucose, 4.24 g sodium nitrate, 2.4 g dipotassium hydrogen phosphate, 1.2 g potassium dihydrogen phosphate
30 mL trace elements solution. Glucose and nutrient salts were dissolved seperatly in 500 mL double-distilled water each ad united when cooled
Stock solution II: 0.2 g ferrous sulphate, 4.0 g magnesium sulphate in 1000 mL sterile double-distilled water
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Method of cultivation: stock culture of Pseudomonas putida are kept in agar slant tubes, each stock culture were renewed in a one week interval
- Preparation of inoculum for exposure: inoculum were prepared from the stock culture on nutrient medium in agar slant tubes and incubate at 25 °C for 24 h. Then the cells material were washed with sterile saline and the extinction of the monochromatic radiation at 436 nm for a 10 mm layer of the bacterial suspension by photoelectric measurement were determined.
- Initial biomass concentration: final turbidity value of bacterial suspension corresponded to the extinction value of a Formazin standard suspension of TE/F/436 nm = 10.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
16 h
Post exposure observation period:
no data available
Hardness:
no data available
Test temperature:
25 °C
pH:
pH 7
Dissolved oxygen:
no data available
Salinity:
no data available
Nominal and measured concentrations:
no data available
Details on test conditions:
See details on test solution.
Reference substance (positive control):
not specified
Duration:
16 h
Dose descriptor:
other: TT (toxicity thresholds)
Effect conc.:
135 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Results with reference substance (positive control):
no data available
Reported statistics and error estimates:
For the evaluation the mean values of impulses / mL in the test cultures non-stimulated were calculated and compared to the mean value of of counts performed in test cultures of the lowest toxic pollutant concentration. The findings based on pollutant concentrations at which the onset of inhibitory action was observed.
Validity criteria fulfilled:
not applicable
Conclusions:
In a cell multiplication inhibition test the toxicity threshold for the model organism Pseudomonas putida for 1,2-dichloroethane was determined to be 135 mg/L
Executive summary:

This study was designed to determine the effect of 1,2 -dichloroethane on growth of Peudomonas putida in liquid medium. The toxicity threshold (NOEC) is observed for 135 mg/L.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
not specified
Principles of method if other than guideline:
in comparison with a closed serum bottle test according to Blum (1989)
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
no data available
Analytical monitoring:
no
Details on sampling:
No detailed information given. Concentration of 1,2-dichlorethane was selected to cover a broad range. Four to five concentrations were used.
Vehicle:
no
Details on test solutions:
no data available
Test organisms (species):
activated sludge
Details on inoculum:
Local activated sludge was used as source and was fed onm a daily basis with Isomil and other inorganic nutrients. The sludge retention time was maintenend at five days. After two weeks of feeding the mixed liquor suspended solids (MLSS) reached a steady average of 1.650 mg/L.
The laboratory activated sludge, which was concentrated by sedimentation to about 4.0 mg/L MLSS (mixed liquor suspend solids) was used as test media.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
no data available
Hardness:
no data available
Test temperature:
no data available
pH:
no data available
Dissolved oxygen:
no data available
Salinity:
no data available
Nominal and measured concentrations:
no information given
Details on test conditions:
OECD design: a total of 18 Erlenmeyer flasks of 500 mL was set up for an experiment. Volume of test culture was 200 mL and 16 mL synthetic sewage feed was used, water was added to make a mixture of 500 mL. The first, the middle and last flask were used as control, in which no toxic chemical was added. Air flow rate was at about 0.9 L/min for each flask.
The time to set up each flask was staggered 10 minutes apart. After exactly 3 hours of aeration, the contents of each flask were poured into a biochemical oxygen demand bottle. A Yellow Springs dissolved oxygen probe equipped with a mixing device was inserted into each BOD bottle. The dissolved oxygen concentration changed with time was plotted on a strip chart recorder.
The percent inhibition was plotted against concentration. After gathering enough data, the best fit curve was drawn by hand. The experiment was repeated at least 3 times to obtain the IC50 value.

Serum bottle design: test procedure was described in D.J.W. Blum's PhD thesis in 1989. The feed solution is composed of 150 mL of AS is mixed with a nutritive solution (250 mL synthetic sewage + 600 ml water). For each serum bottle 25 mL is added. a small flash with soda solution is inserted in the bottle, then the bottles are sealed. For one experiment 36 bottles are used, of which 4 as controls. The chemical is added through a syringe (some µL, according to concentration range) then 50mL of pure oxygen. Bottles are placed on a shaker at 25°C. At the end of the experiment (24h) the oxygen remaining in the bottle is measured by volumetry (depression). IC50 corresponds to 50% less oxygen consumption than in the control.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
IC50
Effect conc.:
35 500 mg/L
Nominal / measured:
nominal
Conc. based on:
not specified
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: regular OECD 209 design
Duration:
24 h
Dose descriptor:
IC50
Effect conc.:
2 780 mg/L
Nominal / measured:
nominal
Conc. based on:
not specified
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: closed serum bottle
Results with reference substance (positive control):
The IC50 for 3,5-dichlorophenol was 19 mg/L
Reported statistics and error estimates:
no data available
Validity criteria fulfilled:
yes
Conclusions:
Based on the results of this study the IC50 of 1,2 -dichloroethane on activated sludge is 2780 mg/L.
Executive summary:

This study was designed to determine the effects of 1,2-dichloroethane on sewage sludge micro-organisms by measuring the respiration rate after exposure to the test substance after a 3 h contact time OECD 209. In parallel a design in serum bottle preventing losses of volatile substances is described. For non volatile substances the results of both design show a comparable sensitivity, thus validating results obtained by this procedure.

Based on the results of this study the IC50 -24h of 1,2-dichloroethane on activated sludge is 2780 mg/L, using a design that prevents losses by volatilisation.

Description of key information

Based on OECD 209 study from Tang (1990) on DCE:

EC50 (24h)=2.78 g/L

Key value for chemical safety assessment

EC50 for microorganisms:
2.78 g/L

Additional information

No experimental study was conducted on the multiconstituent substance (Flux1). Instead, a constituent-based, Weigh-of-Evidence approach, was performed.

The three major constituents were targeted, representing ca 95% of a typical composition (carbon tetrachloride(CAS n° 56-23-5), 1,2-Dichloroethane (CAS n°107 -06 -2) and chloroform (CAS n° 67-66 -3)). Numerous published data were available, and a single consensus value was selected for each, from a reliable source.

No average key value was derived for the multiconstituent substance: as the Risk Assessment is conducted per constituent, individual values are the key data.

One study was designed to determine the effects of 1,2-dichloroethane on sewage sludge micro-organisms by measuring the respiration rate after exposure to the test substance after a 3 h contact time OECD 209. In parallel a design in serum bottle preventing losses of volatile substances is described. For non-volatile substances the results of both design show a comparable sensitivity, thus validating results obtained by this procedure.

Based on the results of this study the IC50 -24h of 1,2-dichloroethane on activated sludge is 2780 mg/L, using a design that prevents losses by volatilisation.