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Toxicity to microorganisms

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Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
justification for analogue approach: see IUCLID section 13 "Read-across justification"
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
Sampling method: a portion of a reaction mixture was transferred to a standard BOD dilution bottle.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions (0.5 to 5.0 g/L) of the test chemical were prepared in deionized water. When necessary, the stock solutions were adjusted to pH 7.5 ± 0.5 by the addition of 1N H2SO4 or 1N NaOH as required
Test organisms (species):
other: activated sludge from domestic and industrial sewage treatment plants
Details on inoculum:
Activated sludge was obtained on the day prior to the first day of each series of inhibition tests. On return to the laboratory, the solids were allowed to settle and the waste liquor was discarded. The solids were then transferred into a 9-liter laboratory-scale, semi-continuous activated sludge cylinder and diluted to approximately 8.5 liters with deionized water. The system was initially mixed by aerating at a rate of approximately 0.5 L/min, and the concentration of mixed liquor suspended solids was determined by gravimetric analysis. The solids were then allowed to settle in the cylinder and the upper layer of waste liquor was discarded. The activated sludge was washed three times with appropriate volumes of deionized water. After washing, the system was adjusted to contain 4000 ± 400 mg of mixed liquor suspended solids (dry weight) per liter. The system was then aerated continuously at a rate of 0.5 L/min and incubated at ambient temperature (21°C).
The system was supplemented daily with 50 mL of a synthetic sewage stock solution per litter of activated sludge. The synthetic sewage stock solution was composed of, per liter: Bacto-Peptone, 16.0 g; Bacto-Beef extract, 11.0 g; urea 3.0 g; K2HPO4, 28 g; MgSO4 • 7H2O, 0.2 g; CaCl2 • 2H2O, 0.4 g; and NaCl, 0.7 g. Final pH of the stock solution was adjusted to pH 7.0 with H3PO4. Fresh stock solution was prepared as required and stored for not more than two days at 5°C.
When the same source of inoculum was to be used for testing on a subsequent day (maximum of 7 days). an additional 50 mL of synthetic sewage stock solution was added per liter of activated sludge. The system was incubated overnight.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
21°C
pH:
7.4 - 8.0
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 1L-bottle, final volume 500 mL
- Aeration: 0.5 - 1.0 L/min

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water

OTHER TEST CONDITIONS
- Adjustment of pH: yes, 7.5 ± 0.5
Reference substance (positive control):
yes
Remarks:
3,5-dichlorphenol
Key result
Duration:
3 h
Dose descriptor:
IC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
During a number of preliminary inhibition studies, it was observed that the pH of a test reaction often changed during the 3-hour incubation period. Thus, decreases in the respiration rate may have been due to a combination of both the effects of pH and toxicity of the test chemical. As a consequence, the buffering capacity of the synthetic sewage nutrient medium was increased to prevent changes in pH of reaction mixtures.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: IC50 for 3,5-dichlorphenol: 12.2 ± 2.2 mg/L (18% variance) (municipal sewage), 11.4 ± 1.5 mg/L (13% variance) (industrial sewage)
Reported statistics and error estimates:
Inhibition data were analyzed using Thompson's method of moving averages to estimate IC50 values. The experimental data were also analyzed using a probit-transformation model similar to that described by Larson and Schaeffer (1982). A nonlinear curve-fitting program (Procedure NLIN of SAS) was used to estimate IC50 values and associated 95% confidence intervals.
Endpoint:
toxicity to microorganisms
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, non guideline study, published in peer reviewed literature, limitations in reporting but otherwise adequate for assessment
Justification for type of information:
justification for analogue approach: see IUCLID section 13 "Read-across justification"
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
The effect of the test substance on the inhibition of the multiplication of bacterial cells of the genus Pseudomonas was investigated. Cultures of the test organisms were exposed to a series of test concentrations of the test substance for 16 hours and the effects on cell multiplication measured using a turbidimetric technique. Results are reported relative to the blank controls.
GLP compliance:
no
Remarks:
predates GLP
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No data reported
Analytical monitoring:
no
Details on sampling:
The concentration of the bacterial suspension was measured turbidimetrically at 0 and 16 hours
Vehicle:
no
Details on test solutions:
Test solutions prepared from a stock solution, with each dilution containing 1 part v/v test substance solution in 2^0 to 2^14 v/v double distilled water. Nutrient medium, trace element solution and vitamin solution were added to each test vessel together with 10ml of bacterial suspension.
Test organisms (species):
Pseudomonas putida
Details on inoculum:
Stock cultures of the test strain kept on the nutrient for stock and preliminary cultures in agar slant tubes. New stock cultures prepared at 1 week intervals. The inoculated stock culture incubated at 25°C for 24h
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
16 h
Post exposure observation period:
None
Hardness:
No data reported
Test temperature:
25°C
pH:
authors state that the solution of test substance was neutralised by the addition of a minimal volume of acid or alkaline solution
Dissolved oxygen:
No data reported
Salinity:
Not applicable
Nominal and measured concentrations:
No data reported
Details on test conditions:
Four parallel dilution series in 300ml Erlenmeyer flasks, stoppered with cotton-lined plastic caps. Both inoculated and non-inoculated dilution series left at 25°C for 16h
Reference substance (positive control):
no
Remarks:
No data reported
Key result
Duration:
16 h
Dose descriptor:
NOEC
Effect conc.:
2 850 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
After 16 hours the extinction of the monochromatic radiation at 436nm in a 10mm layer in the inoculated dilution series was measured. The lowest concentration having a mean extinction value ≥3 below the mean for non-toxic dilutions is reported as the EC3.
Results with reference substance (positive control):
No data reported
Reported statistics and error estimates:
No data reported
Validity criteria fulfilled:
no
Remarks:
non-standard test
Conclusions:
An 16h EC3 of 2850 mg/l has been reported for Pseudomonas putida . This can be considered to be the NOEC.
Executive summary:

An 16h EC3 of 2850 mg/l has been reported for Pseudomonas putida. This can be considered to be the NOEC. Although this study is non-GLP and is non guideline, it is considered suitable for use as a key study. The study investigates the effects of the test substance on a standard test organism. A series of exposure concentrations were used and results reported as the inhibition of culture growth relative to controls.

Description of key information

Trimethyl orthoacetate is rapidly hydrolysed to methanol (CAS no. 67 -56 -1) and methyl acetate, which further hydrolyses to acetic acid (CAS no. 64 -19 -7) as final reaction product in the presence of water (< 2.4 h at pH 4, 7 and 9 at 50°C). Under neutral (pH 7) and acedic (pH 4) conditions, the half-life was < 1 h. The measured degradation of TMOA at pH 7 and 50°C correspond to a half-life time well below one day under normal outdoor conditions. Accordingly, reliable data of the hydrolysis products methanol and acetic acid are used to address the endpoint, which is entirely appropriate to draw conclusions on the toxicity of Trimethyl orthoacetate to microorganisms.

1. Methanol

- Toxicity to microorganism: IC50 (3 h) > 1000 mg/L for activated sludge based on growth inhibition (OECD TG 209)

2. Acetic Acid

- Toxicity to microorganism: EC3 (16 h) = 2850 mg/L (= NOEC) for Pseudomonas putida based on growth inhibition (non-standard test, predates GLP)

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

Concerning study data for acetic acid:

An 16h EC3 of 2850 mg/l has been reported for Pseudomonas putida. This can be considered to be the NOEC. Although this study is non-GLP and is non guideline, it is considered suitable for use as a key study. The study investigates the effects of the test substance on a standard test organism. A series of exposure concentrations were used and results reported as the inhibition of culture growth relative to controls.