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Ecotoxicological information

Toxicity to microorganisms

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Reference
Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 19th to June 21st 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study was performed according to ISO Guideline 10712 (1995) without GLP statement as this study was originally conducted by R&D department to increase knowledge on the fate of the substance, not related to registration purposes. Minor deviation was observed but all validity criteria were fullfiled and this study is well documented.
Qualifier:
according to
Guideline:
other: ISO 10712 (1995)
Deviations:
yes
Remarks:
. Maximum temperature during 16 hour's incubation was 24.4°C which is 0.4°C higher than the target temperature range of 23 +/- 1°C.
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Remarks:
This study was originally conducted by R&D department to increase knowledge on the fate of the substance, not related to registration purposes.
Specific details on test material used for the study:
No additional information
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item is of low water solubility. Therefore, water accommodated fractions (WAF) were performed. The following loading rates were prepared with bi-distilled water: 10, 2.5, 1, 0.5, 0.1 and 0.05 mg/L. The two highest loading rates (10 and 2.5 mg/L) were prepared by direct addition of the test item to bi-distilled water: for the loading rate of 10 mg/L, 10 mg of the test item was diluted to 1 L of bi-distilled water; for the loading rate of 2.5 mg/L, 5 mg of the test item was diluted to 2 L of bi-distilled water. For the preparation of the four lower loading rates (1, 0.5, 0.1 and 0.05 mg/L), the WAF with the loading rate of 2.5 mg/L was diluted because it was technically not possible weight lower amounts of the test item than 4 mg and to stir much higher volumes of bi-distilled water. The WAFs were stirred for 24 hours followed by a sedimentation period of 30 minutes. The test solution was taken out from the middle of the liquid with a pipette.
- Eluate: bi-distilled water
- Controls: The controls were prepared by adding 20 mL bi-distilled water, 2.5 mL 10-fold concentrated exposure medium and 2.5 mL of the diluted pre-culture bacteria suspension. In addition, blanks have been prepared without bacteria for the loading rates 10, 2.5 and 1 mg/L. Instead of the bacterial inoculumsuspension growth medium was added.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The loading rate of 10 mg/L was slightly whitely and turbid. At the end of the sedimentation period, very small white flakes could be observed on the ground of the bottle and an oily film remained on the surface of the liquid. With the loading rate of 2.5 mg/L, the liquid was uncoloured and clear.
Test organisms (species):
Pseudomonas putida
Details on inoculum:
Pseudomonas putida is a non pathogenic gram-negative, saprophytic, free living bacterium in soil or water where it plays an important role in decomposition, biodegradation, and the carbon and nitrogen cycles. The optimum growth temperature is between 25 and 30°C.
Pseudomonas putida, MIGULA strain Berlin 33/2 (SSM 50026) was obtained from the German Collection of Microorganisms (DSMZ - Deutsche Stammsammlung für Mikroorganismen und Zellkulturen GmbH, Braunschweig) on April 4th, 2006. A working culture was prepared at May 3rd, 2006, which was kept at -80°C deep-frozen till test performance.
Preparation of the pre-culture and the inoculum: about 5 hours before starting the test a pre-culture with a starting concentration of 50 FNU (Formazine Nephelometric Units) was prepared with the permanent working suspension and incubated at 23.5-24.4°C in a tempered water-bath shaker in the dark. At the end of the pre-culture period an optical density of the pre-culture of 0.594 corresponding to 258 FNU at 436 nm was measured. The pre-culture was diluted to obtain an optical density corresponding to 50 FNU.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
16 h
Remarks on exposure duration:
none
Post exposure observation period:
none
Hardness:
No data
Test temperature:
23.9-24.4°C
pH:
At the start of the experiment, pH of the test and control solutions is 7.0. With the higher loading rates pH increased up to 8.0.
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
- Nominal concentrations: loading rates 10, 2.5, 1, 0.5, 0.1 and 0.05 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: each test vessel and the control vessels contained 25 mL of the test solution.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable

TEST MEDIUM / WATER PARAMETERS
See "stock solutions" in "Any other information on materials and methods incl. tables".
The preculture medium is made up adding 25 mL of each of solution 1 and 3 and 50 mL of solution 4 to 900 mL sterilized water.
The exposure medium consists of 25 mL of each of solutions 2 and 3 and 50 mL of solution 4 in 900 mL sterilized water.

OTHER TEST CONDITIONS
The vessels were kept for 16 h +/- 1 h in a water-bath shaker at 23.9-24.4°C in the dark. At the end of the incubation period the optical density of the cell suspension was measured photometric at 436 nm vs. growth medium. The measured optical density was converted into FNU. The pH was determined at the end of the experiment.

The photometer used was calibrated with formazine solution according to ISO 7027 for converting the E 436 nm to FNU. Only E 436 < 0.4 were used for correlation. The following correlation was determined: FNU = E 436 / 0.0023 with r² = 0.999.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : For each test vessel, the inhibition of cell growth in comparison to the control is calculated according to the following formula:
Inhibition (sample) [%] = [(FNU control at 16h - FNU sample at 16h) / (FNU control at 16h - FNU control at 0h)] * 100
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
16 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
None of the loading rates tested between 10 mg/L and 0.05 mg/L resulted in an inhibition of bacterial growth.
No turbidity occurred and no increased extinction with the blanks could be measured.
Maximum growth promotion was 10.4% with the middle concentratio of 1 mg/L.
See table 6.1.7/1 in "Any other information on results incl. tables".
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 16h-EC50 = 29.7 mg/L. The target range should be between 10 and 30 mg/L.
Reported statistics and error estimates:
None

Table 6.1.7/1: FNU (Formazine Nephelometric Units) for the controls and the replicates of the six different test concentrations

Concentrations (mg/L)

Repl.

Ext. 436 nm

FNU

Inhibition (%)

Mean Inhibition (%)

Relative standard deviation (%)

Control, 0h

0.01

4

-

-

-

Control, 16h

1

2

3

4

1.102

1.071

1.234

1.040

479

466

537

452

-

-

-

0.05

1

2

3

1.109

1.113

1.091

482

484

474

0.2

-0.1

1.9

0.7

158.0

0.1

1

2

3

1.215

1.190

1.215

528

517

528

-9.4

-7.1

-9.4

-8.6

-15.2

0.5

1

2

3

1.131

1.184

1.189

492

515

517

-1.7

-6.6

-7.0

-5.1

-57.1

1.0

1

2

3

1.231

1.224

1.225

535

532

533

-10.8

-10.2

-10.3

-10.4

-3.3

2.5

1

2

3

1.121

1.114

1.051

487

484

457

-0.8

-0.2

5.5

1.5

234.8

10

1

2

3

1.150

1.111

1.097

500

483

477

-3.5

0.1

1.3

-0.7

-362.2

Validity criteria fulfilled:
yes
Conclusions:
16h-NOEC = 10 mg/L. None of the loading rates tested between 10 mg/L and 0.05 mg/L resulted in an inhibition of bacterial growth.
Executive summary:

This study was performed according to ISO Guideline 10712 (1995) without GLP statement, to determine the inhibition of the growth of the bacterium Pseudomonas putida during a period of 16 hours of exposure of the test item HYD006. This study was originally conducted by R&D department to increase knowledge on the fate of the substance, not related to registration purposes.

Due to the low water solubility of the test item, water accommodated fractions (WAF) were performed with stirring times of 24 hours and a subsequent sedimentation period of 30 minutes. The following loading rates were prepared with bi-distilled water: 10, 2.5, 1, 0.5, 0.1 and 0.05 mg/L. These loading rates were inclubated with a defined starting concentration of the bacteria measured photometric as optical density at 436 nm of the suspension. One control was performed without test item and other controls were performed without bacteria for the loading rates 10, 2.5 and 1 mg/L. The decrease of optical density compared to a control without test item is calculated as %-inhibition of cell growth.

The loading rate of 10 mg/L was slightly whitely and turbid. At the end of the sedimentation period, very small white flakes could be observed on the ground of the bottle and an oily film remained on the surface of the liquid. With the loading rate of 2.5 mg/L, the liquid was uncoloured and clear.

None of the loading rates tested between 10 mg/L and 0.05 mg/L resulted in an inhibition of bacterial growth. Maximum growth promotion was 10.4% with the middle concentration of 1 mg/L.

In conclusion, it seems that the 16h-NOEC is equivalent to 10 mg/L of the test item.

Description of key information

ISO Guideline 10712, non GLP, key study, validity 2:

16h-NOEC (Pseudomonas putida) = 10 mg/L;

The test substance is non-toxic for the bacterium Pseudomonas putida up to 10 mg/L.

OECD Guideline 301D, non GLP, Supporting study, validity 2:

Toxic effect observed in activated sludge micro-organisms up to 84 days at the tested concentration of 2 mg/L.

The estimated NOEC can be expected at 1.0 mg/L (by dividing the toxic concentration by 2). To consider a worst case scenario, this NOEC is used for chemical safety assessment.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 mg/L

Additional information

One key study is available to assess the toxicity of the registered substance to micro-organisms. This study was performed, according to ISO Guideline 10712 (1995) without GLP statement, to determine the inhibition of the growth of the bacterium Pseudomonas putida during a period of 16 hours of exposure of the registered substance. This study was originally conducted by R&D department to increase knowledge on the fate of the substance, not related to registration purposes. Due to the low water solubility of the test substance, water accommodated fractions (WAF) were performed with stirring times of 24 hours and a subsequent sedimentation period of 30 minutes. The following concentrations were prepared with bi-distilled water: 10, 2.5, 1, 0.5, 0.1 and 0.05 mg/L. These concentrations were incubated with a defined starting concentration of the bacteria measured photometrically as optical density of the suspension at a wavelength of 436 nm. One control was performed without test item and other controls were performed without bacteria for the concentrartions 10, 2.5 and 1 mg/L. The decrease of optical density compared to a control without test item is calculated as %-inhibition of cell growth. The concentration of 10 mg/L was observed to be slightly white and turbid. At the end of the sedimentation period, very small white flakes could be observed at the bottom of the bottle and an oily film remained on the surface of the test solution. With the concentration of 2.5 mg/L, the liquid was colourless and clear. None of the concentrations tested between 10 mg/L and 0.05 mg/L resulted in an inhibition of bacterial growth. Maximum growth promotion was 10.4% with the middle concentration of 1 mg/L. In conclusion, it seems that the 16h-NOEC is equivalent to 10 mg/L of the test item, the highest concentration tested.

However, considering the toxicity effect observed in the biodegradation studies mentioned in the present dossier, the toxic effect observed in activated sludge micro-organisms up to 84 days at the tested concentration of 2 mg/L (AkzoNobel (2012) OECD 301D study) is considered relevant for chemical safety assessment. Therefore, the estimated NOEC for the toxicity to STP micro-organisms can be expected at 1.0 mg/L (by dividing the toxic concentration by 2). This worst case value is used for the chemical safety assessment.