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Toxicity to microorganisms

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Reference
Endpoint:
toxicity to microorganisms, other
Remarks:
toxicity to microorganisms
Type of information:
experimental study
Remarks:
read-across to similar substance
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 GLP and with accepted guideline with the test substance CAS 28777-98-2. 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
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
> 8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Duration:
16 h
Dose descriptor:
EC10
Effect conc.:
> 8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
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 bacteria Pseudomonas 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 (Pseudomonas cell multiplication inhibition test)".

Procedure:

Following a preliminary range-finding study, Pseudomonas putida was 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 of Pseudomonas putida to 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 sol ution.

Description of key information

NOEC = 8 mg/L; EC50 and EC10 (16 h) > 8 mg/L (CAS 28777-98-2)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
8 mg/L

Additional information

Because no test with 2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs on acute toxicity in fish is available, a test with the main constituent C18 ASA is assessed.

In a limit test performed in accordance with ISO 10712, no effect on the growth of Pseudomonas putida strain NCIB 8248 has been noted after exposure for 16 hto the nominal concentration of 8 mg/L in DMSO. Thus, the NOEC was 8 mg/L and the EC50 > 8.0 mg/L, respectively.

Since the tested substance C18 ASA is a main constituent of 2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs, the same outcome of the tests would be expected for 2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs.