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Ecotoxicological information

Toxicity to microorganisms

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Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 - 15 Apr 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study with acceptable restrictions. Analytical purity not stated.
Qualifier:
according to guideline
Guideline:
other: NEN-EN-ISO 10712 adopted in 1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solutions were prepared applying magnetic stirring for approximately 50 hours to achieve the maximum soluble fraction of the test substance in the test medium. Subsequently, this stock solution was transferred to a separating funnel and left stabilising for ~ 24 hours. The water phase in the middle of the separating funnel was used in this test. Final test solutions (310, 625, 1250, 2500, 5000 and 10000 mg/L) were prepared from 390, 780, 1560, 3130, 6250 and 12500 mg/L stock solutions.
- Evidence of undissolved material: All test solutions were turbid emulsions.
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Laboratory culture: Strain Migula, Berlin 33/2 (DSM 50026) from BGA, Berlin, Germany
- Method of cultivation: Cultivated in nutrient medium: 18 g agar dissolved in water, plus 50 mL of solution I (10 g NaNO3 + 2.4 g K2HPO4 + 1.2 g KH2PO4 + 1 g yeast extract dissolved in 500 mL dilution water), 125 mL of solution III (40 glucose monohydrate dissolved in 500 mL dilution water) and 100 mL of solution IV (0.01 g Fe(III) citrate + 4 g MgSO4x7H2O dissolved in 1 L dilution water), made up to one litre in water.
- Preparation of inoculum for exposure: A small amount of bacteria from a maximally 7d old stock culture was inoculated in pre-culture medium (25 mL of solution I and III plus 50 mL of solution IV, made up to one L with water) in Erlenmeyer flasks. After incubation under continuous shaking in the dark, the final turbidity value of the bacterial suspension was adjusted up to FNU (Formazine Nephelometric Units)/436 nm = 50.
- Pretreatment: The bacteria were incubated for 6 h under test conditions prior to exposure.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
16 h
Hardness:
not stated
Test temperature:
23 ± 1 °C
pH:
7.6 - 8.6
Nominal and measured concentrations:
Nominal: 310, 625, 1250, 2500, 5000 and 10000 mg/L siphoned test solutions
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask covered with aluminium caps
- Material: glass; Size: 100 mL; Fill volume: 40 mL
- No. of vessels per concentration: 3
- No. of vessels per control: 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water
- Culture medium different from test medium: no, according to ISO 10712

EFFECT PARAMETERS MEASURED: The cell concentration was measured after 16 h exposure to the test substance by measuring the turbidity value of all cell suspensions using an UV/VIS spectrophotometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: A range finding test was combined with the final test as no inhibitory effects on Pseudomonas were expected. However, the test substance was insoluble in water according to the information supplied by the sponsor. Therefore, a pretest was performed to examine the solubility of the test substance in Milli-Q water.
- Results used to determine the conditions for the definitive study: Since test substance was floating on the surface in a stock solution prepared at 12.5 g/L, it was decided to test toxicity of siphoned test solutions.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: siphoned test solutions
Basis for effect:
growth inhibition
Duration:
16 h
Dose descriptor:
EC10
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: siphoned test solutions
Basis for effect:
growth inhibition
Results with reference substance (positive control):
The EC50 value of 3,5-Dichlorophenol as calculated by linear regression was 15 mg/L. Thus, the EC50 is within the accepted range of 10-30 mg/L
for the EC50 of the reference substance.

Table 1: Extinction values of the inoculated dilution series (I-III), the non-inoculated dilution series t (turbidity control) and the control flasks B

Nominal concentration of test substance [mg/L]*

Extinction in FNU 436 nm

series

Mean      

 

Corrected mean

Inhibition [%]

I

II

III

I, II, III

t

310

2.18

2.23

2.04

2.15

0.06

2.09

-1.8

625

2.17

2.16

2.20

2.18

0.03

2.15

-4.6

1250

2.09

2.01

2.17

2.09

0.04

2.06

-0.1

2500

2.11

1.99

2.12

2.07

0.12

1.95

4.9

5000

2.14

2.11

2.09

2.11

0.04

2.07

-1.0

10000

2.19

2.12

2.10

2.14

0.03

2.10

-2.5

 

 

 

 

 

 

 

 

Control

2.07, 2.08, 1.96, 1.89, 2.27, 2.20, 2.25, 1.74, 1.98, 2.09

Mean

2.05

B0

0.02

*: final nominal concentration of the treatments

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Dec 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP Guideline study with acceptable restrictions.
Qualifier:
according to guideline
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solution was prepared according to the national standard procedure (DEV S4): 20 g of the test substance was added to deionised water and shacked overhead for 24 h. This solution was filtered with a folded filter. The filtrate was used for the test. It was added directly to the test vessels and diluted with deionised water to the desired concentration.
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Laboratory culture: A preculture was incubated for 7 h before test initiation at 21 °C.
- Preparation of inoculum for exposure: The preculture was diluted with sterile growth medium to obtain a turbidity of 50 TE/F436. 10 mL was inoculated to 50 mL eluate of the test substance and 10 mL of growth medium. This mixture was filled up with deionised water to a total volume of 100 mL.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
16 h
Remarks on exposure duration:
± 1h
Test temperature:
21 ± 1 °C
Nominal and measured concentrations:
Nominal concentration: 10000 mg/L filtered test solution
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flasks
- size, fill volume: 300 mL, 100 mL
- Aeration: incubated on a shaker (Braun Certomat R)
- No. of vessels per concentration: 3
- No. of vessels per control: 3

EFFECT PARAMETERS MEASURED: Growth inhibition was measured after 16 h test duration.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: Because the test substance is poorly water soluble the filtrate from a stock solution of 10 g/L was used to analyse if there are any effects in the range of water solubility.
Reference substance (positive control):
no
Duration:
16 h
Dose descriptor:
EC10
Effect conc.:
> 10 g/L
Nominal / measured:
nominal
Conc. based on:
other: filtered test solution
Basis for effect:
growth inhibition
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
> 10 g/L
Nominal / measured:
nominal
Conc. based on:
other: filtered test solution
Basis for effect:
growth inhibition

No effects of the test substance in the range of water solubility were observed testing a saturated test solution which was subsequently filtered. Thus, an LC50 (16h) greater then the highest tested concentration of 10 g/L is determined.

Description of key information

Pseudomonas putida were exposed to C9-11 branched alcohols, C10 rich diesters with nonanedioic acid during 16 hours at 10 g/L (a saturated test solution which was subsequently filtered). It can be concluded that no effects of the test substance in the range of water solubility were observed (growth measured by examination with a spectrophotometer) Thus the EC50 (16h) is greater than the water solubility (Hansonis 1993).

In another study bis(2-ethylhexyl) azelate (concentrations up to 10 g/L (siphoned)) was shown not to influence the growth rate of Pseudomonas putida at any of the concentrations tested. The NOEC in this study is 10 g/L (Bertens 1993)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
10 g/L

Additional information

The results of the test with both analogue substance are indicative for very low toxicity towards micro-organisms. As both substances are considered close analogues to diesters of alcohols, C7-9-iso-, C8-rich, 2-ethylhexyl and nonanedioic acid, no toxicity to micro-organisms is expected for this substance. Therefore the NOEC is set at 10 g/L (see also document on read-across rationale)