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Endpoint:
toxicity to microorganisms, other
Remarks:
Pseudomonas putida growth inhibition test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6-9-1996 to 7-9-1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study done under an accepted guideline and GLP, by read-across on an analogue substance. The study is valid and compliant to the guideline. However data is missing on phys/chem properties of the test substance (like solubility and stability) and therefore the actual exposure is uncertain, this is the main restriction.
Qualifier:
according to guideline
Guideline:
other: ISO 10712
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Monitoring Authority, Dep. Helth UK, 1996
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylsulphoxide
- Method: For the purpose of the definitive study the test material was prepared using a preliminary slurry in dimethylsulphoxide. An amount of test material (20 mg) was mixed with 200 uL of dimethylsulphoxide and 15 mL sterile reverse osmosis water to form a slurry prior to dispersal in sterile reverse osmosis water with the aid of ultrasonic disruption.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s) The volume was then adjusted to 2 L to give a 10 mg/L stock solution. To an aliquot (80 mL) of the 10 mg/L stock solution, nutrient stock solutions and bacterial suspension were added to give the required test concentration of 8.0 mg/L.
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Strain: Pseudomonas putida strain NCIB 8248
- Laboratory culture: Freeze dried cultures of Pseudomonas putida were obtained from the National Collection of Industrial Bacteria, Aberdeen, Scotland
- Method of cultivation: maintained in the laboratory by routine sub-culturing onto fresh agar slopes, approximately once per week. The cultures were maintained in the laboratory at a temperature of 25 C.
- Preparation of inoculum for exposure: Approximately 17 hours prior to commencing the test, an aqueous suspension of Pseudomonas putida was produced by adding pre-culture medium to a stock culture of the bacterium and gently shaking in order to wash the bacterial cells off the solid medium. The resultant suspension was dispersed into a sterile flask plugged with sterile non-absorbent cotton wool and incubated at 25· C. After the initial incubation period of approximately 17 hours, the bacterial suspension was diluted using pre-culture medium to give a turbidity of approximately 100 Formazine Turbidity Units (FTU) An aliquot (50 mL) of the 100 FTU bacterial suspension was added to 450 mL of pre-culture medium and incubated at 25 C.
After incubation at 25 C for 6 hours the bacterial suspension had a turbidity of approximately 50 FTU.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
16 h
Test temperature:
25 °C
Nominal and measured concentrations:
Range-finding study: nominal: 0.8 and 8.0 mg/L
Definitive study: 8.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mlL glass conical flasks
- Type (delete if not applicable): closed
- Fill volume: 100 mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 10


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): bacterial growth by measurement of the absorbance at 436 nm

TEST CONCENTRATIONS
- Range finding study: 0.8 and 8 mg/L
- Test concentrations: 6 mg/L
- Results used to determine the conditions for the definitive study: no effect on bacterial growth
Reference substance (positive control):
no
Key result
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
> 8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Key result
Duration:
16 h
Dose descriptor:
EC10
Effect conc.:
> 8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Key result
Duration:
16 h
Dose descriptor:
NOEC
Effect conc.:
8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
Statistical analysis of the absorbance values was carried out for the solvent control and 8.0 mg/L test group using a Students t-test. There were no statistically significant differences (P ~0.05), between the solvent control and 8.0 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) is given as ~ 8.0 mg/L
The test concentration of 8.0 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and the limitations imposed for the addition of nutrient solutions and bacterial suspension to the test material stock solution. Other recognised auxiliary solvents were used during the preliminary solubility work performed, however, dimethylsulphoxide was found to give the best testable dispersion of the test material in water.
Reported statistics and error estimates:
A Students t-test was carried out on the absorbance values after 16 hours exposure for the solvent control and 8.0 mg/L test concentration to determine any statistically significant differences between the test and solvent control groups.
Validity criteria fulfilled:
yes
Conclusions:
The effect of on the growth of Pseudomonas putida has been investigated and gave EClO and ECso values of greater than 8.0 mg/l. The EClO value of greater than 8.0 mg/L corresponds to an evaluation number (Bewertungszahl, BWZ) for the German Water Hazard Classification Scheme of less than 5.1. The study is valid and compliant to the guideline. However data is missing on phys/chem properties of the test substance (like solubility and stability) and therefore the actual exposure is uncertain.
Executive summary:

Methods

A study was performed to assess the effect of the test material on the growth of the bacteriaPseudomonas putida. The method followed that described in the German Water Hazard Classification Scheme (Bewertung Wassergefahrdender Stoffe LTWS - Nr 10) and ISO 10712 "Determination of the inhibitory effect of water constituents on bacteria (Pseudomonascell multiplication inhibition test)".

Procedure:

Following a preliminary range-finding study,Pseudomonas putidawas exposed to an aqueous dispersion of the test material at a concentration of 8.0 mg/L (six replicate flasks) for approximately 16 hours at a temperature of 25 C. Samples of the bacterial populations were removed after approximately 16 hours and absorbance values determined for each control and treatment group.

Results

Exposure ofPseudomonas putidato the test material gave an EC10 and an EC50 values of greater than 8.0 mg/l. The EC10 value of greater than 8.0 mg/I corresponds to an evaluation number (Bewertungszahl, BWZ) for the German Water Hazard Classification Scheme of less than 5.1. The test concentration of 8.0 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and the limitations imposed by the addition of nutrient solutions and bacterial suspension to the test material stock solution.

 

 

Endpoint:
toxicity to microorganisms, other
Remarks:
Pseudomonas putida growth inhibition test
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
6-9-1996 to 7-9-1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study done under an accepted guideline and GLP, by read-across based on a chemical category. The study is valid and compliant to the guideline. However data is missing on phys/chem properties of the test substance (like solubility and stability) and therefore the actual exposure is uncertain, this is the main restriction.
Justification for type of information:
This study was conducted on 2,5-furandione, dihydro-, mono-C15- 20-alkenyl derivatives (CAS 68784-12-3), an analogue substance used as the source of information for the assessment of the target substance through read-across. Therefore, this study is informative for evaluation of the environmental fate and toxicity of the target substance, Reaction products of furan-2,5-dione and octadec-1-ene (known here as n-ODSA EC 701-338-8; no CASRN available), and it is adequate for classification and risk assessment.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: ISO 10712
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Monitoring Authority, Dep. Helth UK, 1996
Specific details on test material used for the study:
As a result of increasingly rigorous criteria being applied to the analysis of commercial material used in physical property/toxicity testing, the identity of the material has been modified to reveal a more accurate and precise depiction of the commercial substance. This enhancement is reflected in changes in chemical identifiers such as EC and/or CAS numbers from those noted in earlier versions of data records or in study reports.

Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylsulphoxide
- Method: For the purpose of the definitive study the test material was prepared using a preliminary slurry in dimethylsulphoxide. An amount of test material (20 mg) was mixed with 200 uL of dimethylsulphoxide and 15 mL sterile reverse osmosis water to form a slurry prior to dispersal in sterile reverse osmosis water with the aid of ultrasonic disruption.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s) The volume was then adjusted to 2 L to give a 10 mg/L stock solution. To an aliquot (80 mL) of the 10 mg/L stock solution, nutrient stock solutions and bacterial suspension were added to give the required test concentration of 8.0 mg/L.
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Strain: Pseudomonas putida strain NCIB 8248
- Laboratory culture: Freeze dried cultures of Pseudomonas putida were obtained from the National Collection of Industrial Bacteria, Aberdeen, Scotland
- Method of cultivation: maintained in the laboratory by routine sub-culturing onto fresh agar slopes, approximately once per week. The cultures were maintained in the laboratory at a temperature of 25 C.
- Preparation of inoculum for exposure: Approximately 17 hours prior to commencing the test, an aqueous suspension of Pseudomonas putida was produced by adding pre-culture medium to a stock culture of the bacterium and gently shaking in order to wash the bacterial cells off the solid medium. The resultant suspension was dispersed into a sterile flask plugged with sterile non-absorbent cotton wool and incubated at 25· C. After the initial incubation period of approximately 17 hours, the bacterial suspension was diluted using pre-culture medium to give a turbidity of approximately 100 Formazine Turbidity Units (FTU) An aliquot (50 mL) of the 100 FTU bacterial suspension was added to 450 mL of pre-culture medium and incubated at 25 C.
After incubation at 25 C for 6 hours the bacterial suspension had a turbidity of approximately 50 FTU.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
16 h
Test temperature:
25 °C
Nominal and measured concentrations:
Range-finding study: nominal: 0.8 and 8.0 mg/L
Definitive study: 8.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mlL glass conical flasks
- Type (delete if not applicable): closed
- Fill volume: 100 mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 10


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): bacterial growth by measurement of the absorbance at 436 nm

TEST CONCENTRATIONS
- Range finding study: 0.8 and 8 mg/L
- Test concentrations: 6 mg/L
- Results used to determine the conditions for the definitive study: no effect on bacterial growth
Reference substance (positive control):
no
Key result
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
> 8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Key result
Duration:
16 h
Dose descriptor:
EC10
Effect conc.:
> 8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Key result
Duration:
16 h
Dose descriptor:
NOEC
Effect conc.:
8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
Statistical analysis of the absorbance values was carried out for the solvent control and 8.0 mg/L test group using a Students t-test. There were no statistically significant differences (P ~0.05), between the solvent control and 8.0 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) is given as ~ 8.0 mg/L
The test concentration of 8.0 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and the limitations imposed for the addition of nutrient solutions and bacterial suspension to the test material stock solution. Other recognised auxiliary solvents were used during the preliminary solubility work performed, however, dimethylsulphoxide was found to give the best testable dispersion of the test material in water.
Reported statistics and error estimates:
A Students t-test was carried out on the absorbance values after 16 hours exposure for the solvent control and 8.0 mg/L test concentration to determine any statistically significant differences between the test and solvent control groups.
Validity criteria fulfilled:
yes
Conclusions:
The effect of on the growth of Pseudomonas putida has been investigated and gave EClO and ECso values of greater than 8.0 mg/l. The EClO value of greater than 8.0 mg/L corresponds to an evaluation number (Bewertungszahl, BWZ) for the German Water Hazard Classification Scheme of less than 5.1. The study is valid and compliant to the guideline. However data is missing on phys/chem properties of the test substance (like solubility and stability) and therefore the actual exposure is uncertain.
Executive summary:

Methods

A study was performed to assess the effect of the test material on the growth of the bacteriaPseudomonas putida. The method followed that described in the German Water Hazard Classification Scheme (Bewertung Wassergefahrdender Stoffe LTWS - Nr 10) and ISO 10712 "Determination of the inhibitory effect of water constituents on bacteria (Pseudomonascell multiplication inhibition test)".

Procedure:

Following a preliminary range-finding study,Pseudomonas putidawas exposed to an aqueous dispersion of the test material at a concentration of 8.0 mg/L (six replicate flasks) for approximately 16 hours at a temperature of 25 C. Samples of the bacterial populations were removed after approximately 16 hours and absorbance values determined for each control and treatment group.

Results

Exposure ofPseudomonas putidato the test material gave an EC10 and an EC50 values of greater than 8.0 mg/l. The EC10 value of greater than 8.0 mg/I corresponds to an evaluation number (Bewertungszahl, BWZ) for the German Water Hazard Classification Scheme of less than 5.1. The test concentration of 8.0 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and the limitations imposed by the addition of nutrient solutions and bacterial suspension to the test material stock solution.

 

 

Description of key information

The key study was an acute bacterial toxicity test (Pseudomonas putida cell multiplication inhibition test) by read-across from an analogue substance, conducted in accordance with an established guideline.   

Key value for chemical safety assessment

EC50 for microorganisms:
8 mg/L
EC10 or NOEC for microorganisms:
8 mg/L

Additional information

The inhibition of cell multiplication of Pseudomonas putida, by read-across from an analogue substance, was determined in a limit test from the key study. The 16-h NOEC was reported as ≥ 8.0 mg/L; the 16-h EC10 and 16-h EC50 were each reported as > 8.0 mg/L.

This information is from the substance 2,5-furandione, dihydro-,mono-C15-20-alkenylderivatives (CAS 68784-12-3, a mixture of a hexadecenyl- and octadecenyl succinic anhydrides, and also known as PentasizeTM68, AS 1100TMand AS 1000TM), an analogue used for the assessment of several endpoints through read-across. The hypothesis for read-across between the substance being registered (Reaction products of furan-2,5-dione and octadec-1-ene; known here as n-ODSA EC 701-338-8; no CASRN available), and the analogue substance is a common functional group: a 2,5-furandione, dihydro- structure, also known as a succinic anhydride, to which is attached a long-chain monounsaturated olefin. In the environment, the anhydride moiety is quickly hydrolysed to form a dioic acid.  When the substance to be registered and the analogue substance are compared, changes in the purity of the starting olefin stock, or small differences in the length (between sixteen and twenty) or arrangement (linear or branched) of the carbon chain are not anticipated to significantly affect the environmental fate properties or the toxicity of the substances. For each endpoint study based upon read-across, the analogue approach is substantiated by an evaluation provided in the Analogue Approach Report Format (AARF) attached to the endpoint study summary file. The AARF allows the read-across information to fulfil the information requirements of the REACH Annexes VII-X, to be the basis for classification and labelling decisions, and for risk assessment.