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Toxicity to microorganisms

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Endpoint:
toxicity to microorganisms
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Refer to the section 13 for details on the category justification.
Qualifier:
according to guideline
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
Deviations:
yes
Remarks:
Highest concentration tested was 10,00 g/L upon request of Study Sponsor; due to nature of the material, test temperature was 25 +/- 1°C instead of 21 +/- 1°C, cultures plated on full agar plates instead of Erlenmeyers and duration 72 h instead of 16 h
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Maximal soluble amount of test material (saturation concentration) diluted in doubly distilled water. Test concentrations prepared by serial dilution of initial saturated solution.
Test organisms (species):
Pseudomonas putida
Details on inoculum:
Pseudomonas putida MIGULA, strain Berlin 33/2, from the German Microorganism culture Collection (D-3300 Braunschweig)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
16 h
Post exposure observation period:
None
Test temperature:
25 +/- 1°C
Nominal and measured concentrations:
- Nominal: 0.01, 0.05, 0.10, 0.50, 1.00, 2.50, 5.00 and 10.00 g/L
Details on test conditions:
- Temperature: 25 +/- 1°C
Reference substance (positive control):
no
Key result
Duration:
16 h
Dose descriptor:
EC10
Effect conc.:
ca. 830 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Key result
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
ca. 6 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition

None.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the nominal 16 h EC50 and EC10 were 6.0 and 0.83 g/L (i.e. 6000 and 830 mg/L), respectively.
Executive summary:

A study was conducted to assess the toxicity of the read across substance, C8-18 and C18-unsatd. DEA, to the bacteria Pseudomonas putida according to DIN Guideline 38412 L8, in compliance with GLP. Pseudomonas putida colonies, in triplicate, were exposed to the test substance (directly diluted in test medium) at nominal concentrations of 0.01, 0.05, 0.10, 0.50, 1.00, 2.50, 5.00 and 10.00 g/L. Inhibition of cell growth compared to controls was recorded after 16 h. Under the conditions of the study, the nominal 16 h EC50 and EC10 were 6.0 and 0.83 g/L (i.e. 6000 and 830 mg/L), respectively (Poth, 1992). Based on the results of the read across study, the test substance is also expected to have similar toxicity to microorganisms.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
From May 10, 1999 to May 11, 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the section 13 for details on the category justification.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 87/302/EEC, Part C, Activated Sludge Respiration Inhibition Test, 1988.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
- The nominal final test substance concentrations tested were 10, 32, 100, 320, and 1000 mg/L.
For the preparation of test media containing the test substance, a stock dispersion of the test substance with a nominal concentration of 5 g/L was prepared. The test substance was warmed up by means of a waterbath to about 40°C for 5 minutes to increase the fluidity and to improve the spreading properties. Then 5025 mg of test substance was dispersed in 1000 mL of deionized water by ultrasonic treatment and intense stirring. In this way a turbid but homogenous dispersion was obtained. Aliquots of this stock dispersion were mixed with synthetic wastewater and deionized water in the respective test flasks to obtain the desired concentrations. Immediately prior to the start of the test (incubation) activated sludge was added. Concentrations in excess of 1000 mg test item per liter were not tested.
Additionally, two controls (deionized water, synthetic wastewater and inoculum, without test substance) and the reference substance 3,5-dichlorophenol (positive control) at nominal concentrations of 5, 16 and 50 mg/l were tested in parallel under otherwise identical test conditions.
For test media containing the reference substance, a stock solution of 3,5-dichlorophenol was prepared according to the test guidelines: 0.5g of 3,5-dichlorophenol was dissolved in 10 mL 1 M NaOH and diluted to about 30 mL with purified water. Excess of NaOH was neutralized with 0.5 mol/L H2S04 to the point of incipient precipitation. Thereafter, the mixture was made up to one liter with purified water. The pH was determined to be pH 7.8 and the final concentration amounted to 500 mg/L. Aliquots of this 3,5-dichlorophenol stock solution were mixed with synthetic wastewater and deionized water in the respective test flasks to obtain the desired concentrations. Immediately prior to the start of the test (incubation) activated sludge was added.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz 1, Sissach, Switzerland) treating predominantly domestic wastewater. The sludge was washed by centrifugation, the supernatant liquid phase was decanted and the solid material resuspended in tap water. This procedure was repeated twice. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to a mixed liquor suspended solids (MLSS) level of 3g per liter (on dry weight basis). During the holding of one day prior to use, the sludge was fed daily with 50 mL synthetic wastewater per liter and was kept at room temperature under continuous aeration until use. Immediately before use, the dry weight of the activated sludge was measured again in the inoculum used in the test. The pH of the activated sludge inoculum was pH 6.5.
Test type:
other: aerobic
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20-21°C
pH:
7.1-7.9
Dissolved oxygen:
> or = to 2.5 mg/L
Nominal and measured concentrations:
0, 10, 32, 100, 320 and 1000 mg/L (nominal)
Details on test conditions:
Experimental Conditions
The inhibitory effect of the test substance on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3h respiration inhibition test. The test was performed in 1000-mL glass flasks. At the start of the test 200 mL activated sludge inoculum with a sludge concentration of 3.1 g/L dry weight (corresponding to a final MLSS level of about 1.2 g/L) was added. The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) first to a control, then to the test solutions of the reference substance, then to the test solutions of the test substance, and finally to the second control. During the incubation period of exactly 3h all test media and the controls were continuously aerated with compressed air at a flow of approximately one liter per minute. The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period, and just before the measurements of the respiration rates the oxygen concentrations were at least 7.5 mg/L. The temperature in the test media, measured in one control, was 21°C at the start and 20°C at the end of the incubation period. The following nominal concentrations of test substance: 10, 32, 100, 320, and 1000 mg/L. In addition, two controls and three different concentrations of the reference substance 3,5-dichlorophenol (5, 16 and 50 mg/L) were tested in parallel.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
oxygen consumption
Key result
Duration:
3 h
Dose descriptor:
other: EC20 and EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
oxygen consumption
Details on results:
Up to and including the concentration of nominal 1000 mg/L, the test substance had no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours. The respiration rates were in the range of -9.2% to -17.8% compared to the controls (negative values: enhanced respiration rates compared to the controls). Thus, the 3h EC20, EC50 and EC80 of the test substance to activated sludge microorganisms could not be calculated, but were clearly higher than 1000 mg/L.
Results with reference substance (positive control):
The 3h EC50 of the reference substance 3,5-dichlorophenol (positive control) was calculated to be 7.0 mg/L (95% confidence limits were not calculable). Thus, the EC50 of the reference substance was in the accepted range of 5-30 mg/L and confirmed the suitability of the used activated sludge.

None.

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the 3 h EC50 of the read across substance to microorganisms was determined to be >1000 mg/L (nominal).
Executive summary:

A study was conducted to determine the toxicity of the read across substance, isoC18 MIPA (100% active), to microorganisms according to OECD Guideline 209 and Commission Directive 87/302/EEC, Part C, in compliance with GLP. Activated sludge was exposed to the test substance at nominal concentrations of 0, 10, 32, 100, 320 and 1000 mg/L for 3 h. In addition, two controls and three different concentrations of the reference substance 3,5 -dichlorophenol (5, 16 and 50 mg/L) were tested. The respiration rate (oxygen consumption) of the aerobic activated sludge fed with standard amounts of synthetic wastewater was measured. The inhibitory effect of the test substance was expressed as a percentage of the mean respiration rate of the controls. Up to and including the nominal concentration of 1000 mg/L, the test substance had no inhibitory effect on the respiration rate of activated sludge after the incubation period of 3 h. The respiration rates were in the range of -9.2 to ‑17.8% compared to the controls. The 3 h EC50 of the reference substance 3,5 -dichlorophenol (positive control) was calculated to be 7.0 mg/L and therefore was in the accepted range of 5 -30 mg/L, confirming the suitability of the used activated sludge. Under the study conditions, the 3 h EC50 of the test substance to microorganisms was determined to be >1000 mg/L (nominal) (Batscher, 1999). Based on the results of the read across study, the test substance is also expected to have similar toxicity to microorganisms.

Description of key information

Key value for chemical safety assessment

EC50 for microorganisms:
6 000 mg/L
EC10 or NOEC for microorganisms:
830 mg/L

Additional information

A study was conducted to assess the toxicity of the read across substance, C8-18 and C18-unsatd. DEA, to the bacteria Pseudomonas putida according to DIN Guideline 38412 L8, in compliance with GLP. Pseudomonas putida colonies, in triplicate, were exposed to the test substance (directly diluted in test medium) at nominal concentrations of 0.01, 0.05, 0.10, 0.50, 1.00, 2.50, 5.00 and 10.00 g/L. Inhibition of cell growth compared to controls was recorded after 16 h. Under the conditions of the study, the nominal 16 h EC50 and EC10 were 6.0 and 0.83 g/L (i.e. 6000 and 830 mg/L), respectively (Poth, 1992).

A study was conducted to determine the toxicity of the read across substance, isoC18 MIPA (100% active), to microorganisms according to OECD Guideline 209 and Commission Directive 87/302/EEC, Part C, in compliance with GLP. Activated sludge was exposed to the test substance at nominal concentrations of 0, 10, 32, 100, 320 and 1000 mg/L for 3 h. In addition, two controls and three different concentrations of the reference substance 3,5 -dichlorophenol (5, 16 and 50 mg/L) were tested. The respiration rate (oxygen consumption) of the aerobic activated sludge fed with standard amounts of synthetic wastewater was measured. The inhibitory effect of the test substance was expressed as a percentage of the mean respiration rate of the controls. Up to and including the nominal concentration of 1000 mg/L, the test substance had no inhibitory effect on the respiration rate of activated sludge after the incubation period of 3 h. The respiration rates were in the range of -9.2 to ‑17.8% compared to the controls. The 3 h EC50 of the reference substance 3,5 -dichlorophenol (positive control) was calculated to be 7.0 mg/L and therefore was in the accepted range of 5 -30 mg/L, confirming the suitability of the used activated sludge. Under the study conditions, the 3 h EC50 of the test substance to microorganisms was determined to be >1000 mg/L (nominal) (Batscher, 1999).