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Toxicity to microorganisms

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Endpoint:
toxicity to microorganisms
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Data obtained from peer-reviewed secondary source. Read-across justification: The substance is hydrolytically unstable. When it comes in contact with water or moisture complete hydrolysis will take place with no significant reaction products other than the particular alcohol and hydrated titanium dioxide. This rapid hydrolysis (hydrolysis half-life < 3 minutes to < 2 hours) is the driving force for the fate and pathways of the substance. The aquatic toxicity testing is considered scientifically unjustified as the substance degrades immediately releasing alcohol and hydrated insoluble titanium dioxides in water. The testing conducted with analogue substances of the category proves that the aquatic toxicity in daphnia and algae studies is similar to the aquatic toxicity of alcohol released to the test water, and the insoluble hydrated titanium oxides precipitated on the bottom of the test vessels. The identification of degradation products from the hydrolysis study conducted for the target substance verifies that there are no impurities in the alcohol released from the target substance, which might change the aquatic toxicity of the target substance compared to the toxicity of the pure alcohol. As there is a mechanistic reasoning to the read-across, the read-across from the degradation product (relevant alcohol) is used to evaluate the aquatic toxicity and the fate and pathways of the target substance in the environment.
Justification for type of information:
The substance is hydrolytically unstable. When it comes in contact with water or moisture, a complete hydrolysis will take place with no significant reaction products other than the particular alcohol and hydrated titanium dioxide. This rapid hydrolysis (hydrolysis half-life < 3 minutes to < 2 hours) is the driving force for the fate and pathways of the substance. The aquatic toxicity testing is considered scientifically unjustified as the substance degrades immediately releasing the particular alcohol and hydrated insoluble titanium dioxides in water.
The testing conducted with analogue substance of the category justifies that the aquatic toxicity in daphnia and algae studies is similar to the aquatic toxicity of the alcohol released to test water as the insoluble hydrated titanium oxide, precipitated on the bottom of the test vessels; lacking bioavailability. The identification of the degradation products from the hydrolysis study conducted for the target substance verifies that there are no impurities in the alcohol released from the target substance which might change the aquatic toxicity of the target substance compared to the toxicity of the pure alcohol.
As there is a mechanistic reasoning to the read-across, the read-across from the degradation product (relevant alcohol) is used to evaluate the aquatic toxicity and the fate and pathways of the target substance in the environment.
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
Principles of method if other than guideline:
The methodology follows the standarized method of P.putida cell multiplication test. The concentration of the bacterial suspension is measured turbidimetrically. It is expressed by the extinction of the primary light of the monochromatic radiation at 436 nm for a layer of 10 mm thickness. The concentration at which the inhibition effect of a pollutant is first observed is determined to have an extinction value at the end of the test period; that is ≥ 3% below the mean value of extinction for non-toxic dilutions of the test cultures.
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Before the test cultures were prepared the test material solution was neutralized by having a known content in sterile double-distilled water and by using theminimum volume of acid or alkaline solution. The initial concentration of the pollutant solution was not reported.
- Eluate:double distilled water.
- Differential loading:
From the test material solution, four parallel dilutions with varying volume ratios were prepared in 300 ml Erlenmeyer flasks, stoppered with cotton-lined plastic caps using sterile double-distilled water. These dilutions each contained 1 part v/v of test material solution in 2E0 to 2E14 parts v/v mixture.

The dilution series were prepared as follows: the first flask of each dilution series contained 160 ml of test material solution at the start. Starting from this flask, subsequent dilutions were prepared using a constant dilution ratio of 80 ml preliminary test material dilution + 80 ml double-distilled water. Consequently, each flask contained 80 ml of culture liquid at the start. Each flask of the three dilution series was made up to 100 ml by adding 5 ml each of stock solution I, 5 ml of stock solution II and 10 ml each of the prepared bacterial suspension from the preliminary culture having a known adjusted extinction value.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):not used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not reported
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Laboratory culture: P. putida strains kept on the nutrient for stock and preliminary cultures in agar slant tubes
- Method of cultivation: new stock cultures prepared at intervals of 1 week each.
- Preparation of inoculum for exposure: Incubated at 25 deg. C for 24 h and kept in stock. Cell material was washed out with sterile saline. Extinction of the monochromatic radiation at 436 nm for a 10 layer was measured photometrically from the bacteria suspension. The final turbidity was adjusted with sterile saline to correspond the extinction value of formazin standard suspension TE/436 nm of 10, by taking into a consideration the dilution ratio of the bacteria suspension in the inital test solutions (1:9).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
16 h
Test temperature:
25 deg. C
pH:
not reported.
Nominal and measured concentrations:
Nominal from 2E0 to 2E14 parts v/v mixture.
Details on test conditions:
TEST SYSTEM
- Test vessel: sterilized Erlenmeyer flask stopped with cotton-lined plastic caps
- Material, size, headspace, fill volume: glass, 300 ml, 100 ml
- Aeration: no
- No. of vessels per concentration (replicates): three
- No. of vessels per control (replicates): three
- Biomass loading rate: Following inoculation, the extinction value of the monochromatic radiation at 436 nm for a 10-mm layer of the bacterial suspension of the test cultures correspond to the extinction value of the Formazin standard suspension TE/F/436 nm = 10.

Leave both inoculated and non-inoculated dilution series at 25ºC for 16 hours. After termination of the test period measure the extinction of the monochromatic radiation at 436 nm in a 10-mm layer in the inoculated dilution series.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: not reported. double distilled water was used. No measured data on the dilution water.
Culture medium different from test medium: no

Nutrient medium (for stock and preliminary cultures)
Dissolved in 1000 ml double -distilled water:
1.060 g sodium nitrate, NaNO3
0.600 g dipotassium hydrogen phosphate, K2HPO4, anhydrous
0.300 g potassium dihydrogen phosphate, KH2PO4
0.200 g magnesium sulphate, MgSO4.7 H2O
10.000 g D(+) glucose
18.00 g Difco Bacto agar
0.010 g ferrous sulphate, FeSO4.7 H2O
1.5 ml trace elements solution.

The solution was sterilized in a steam sterilizer for 1.5 hours, after which 3 ml of vitamin solution was added.

Trace elements solution (in grams per liter of double-distilled water)
0.055 Al2(SO4)3.18 H2O
0.028 KI
0.028 KBr
0.055 TiO2
0.028 SnCl2.2 H2O
0.028 LiCl
0.389 MnCl2.4 H2O
0.614 H3BO3
0.055 ZnSO4.7 H2O
0.055 CuSO4.5 H2O
0.059 NiSO4.6 H2O
0.055 Co(NO3)2.6 H2O

Vitamin solution
0.2 mg biotin (as D+ biotin)
2.0 mg nicotinic acid
1.0 mg thiamine (as thiamine HCl)
1.0 mg p-aminobenzoic acid
0.5 mg panthothenic acid (as D-panthothenic acid, Na-salt)
5 mg pyridoxamine (as pyridoxamine dihydrochloride)
2.0 mg cyanocobalamin (vitamin B12)
100 ml double distilled water

6 ml each of the nutrient medium was put into culture tubes, the latter was sterilized in a steam sterilizer by fractionated sterilization (three times) for 30 min. Let solidify in slant position.

Stock solution I
20.000 g D(+) glucose
4.240 g sodium nitrate, NaNO3
2.400 g dipotassium hydrogen phosphate, K2HPO4 anhydrous
1.200 g potassium dihydrogen phosphate, KH2PO4
30 ml trace elements solution.

Glucose and nutrient salts were diluted separately in 500 ml double-distilled water each, sterilized in a steam sterilizer for 30 min and solutions were united when cooled.

Stock solution II
Dissolved:
0.200 g ferrous sulphate, FeSO4.7 H2O
4.000 g magnesium sulphate MgSO4.7 H2O

in 1000 ml sterile double distilled water.

Saline
Dissolved:
0.500 g sodium chloride, NaCl

in 1000 ml double-distilled water. Sterilized in a steam sterilizer for 30 min.

OTHER TEST CONDITIONS
- Adjustment of pH: no adjustment
- Photoperiod: not applicable
- Light intensity:not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : bacteria cell growth was measured as turbidity after 16 h incubation at 25 deg. C.
The concentration of the bacterial suspension is measured turbidimetrically; it is expressed by the extinction of the primary light of the monochromatic radiation at 436 nm for a layer of 10 mm thickness. The concentration at which the inhibitory action of a pollutant starts will be present in that step of a dilution series of the pollutant having an extinction value at the end of the test period that is ≥ 3% below the mean value of extinction for non-toxic dilutions of the test cultures.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: not reported
- Range finding study: not reported
- Test concentrations: From the test material solution, four parallel dilutions with varying volume ratios were prepared in 300 ml Erlenmeyer flasks, stoppered with cotton-lined plastic caps using sterile double-distilled water. These dilutions each contained 1 part v/v of test material solution in 2E0 to 2E14 parts v/v mixture.
- Results used to determine the conditions for the definitive study:

For evalution of the toxicological findings at the end of the test period, the mean value (A) of the extinction is calculated for all test cultures that are free from both toxic influence and stimulation of growth except for those having extinction values outside a standard deviation of <3% and also, the mean value (B) of the extinction for those test cultures having the lowest toxic pollutant concentration within the dilution series.

For mathematical evaluation, (a) (highest non-toxic pollutant concentration) is plotted against (A) and (b) (lowest toxic pollutant concentration) against (B) as coordinates. After having entered (A - 3%), the pollutant concentration at which the inhibitory action (c) begins may be obtained from the regression line between (a;A) and (b;B) if a negative deviation of the mean extinction by a 3% difference against the mean extinction value of all test cultures having a non-toxic and non-stimulating pollutant concentration is used as an indicator of the beginning of inhibitory action.










Duration:
16 h
Dose descriptor:
other: toxicity threshold TT
Effect conc.:
110 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: mean extinction value
Results with reference substance (positive control):
not reported
For evalution of the toxicological findings at the end of the test period, the mean value (A) of the extinction is calculated for all test cultures that are free from both toxic influence and stimulation of growth except for those having extinction values outside a standard deviation of <3% and also, the mean value (B) of the extinction for those test cultures having the lowest toxic pollutant concentration within the dilution series.

For mathematical evaluation, (a) (highest non-toxic pollutant concentration) is plotted against (A) and (b) (lowest toxic pollutant concentration) against (B) as coordinates. After having entered (A - 3%), the pollutant concentration at which the inhibitory action (c) begins may be obtained from the regression line between (a;A) and (b;B) if a negative deviation of the mean extinction by a 3% difference against the mean extinction value of all test cultures having a non-toxic and non-stimulating pollutant concentration is used as an indicator of the beginning of inhibitory action.
Validity criteria fulfilled:
not specified
Conclusions:
Based on the literature, the 16-h toxicity threshold of 2-ethylhexanol to Pseudomonas putida (cell multiplication) is 1100 mg/l based on the nominal concentrations.
Executive summary:
As the target substance hydrolyses immediately (half-life < 10 minutes) the intrinsic properties are related to this main organic degradation product (ethylhexanol) of the target substance.This information is used as a key value in CSA.

According to the ECHA Guidance on information requirements and chemical safety assessment, in absence of activated respiration test, toxicity results to microbial bacteria can be used for evaluate the inhibitory effects of chemicals to STP micro-organisms. Therefore, the results of the P.putida cell multiplication inhibition test of the organic hydrolysing product were used to calculate the PNEC STP.

Endpoint:
activated sludge respiration inhibition testing
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:

Description of key information

Testing is not scientifically justified as this substance is hydrolytically unstable and the decomposition products possess low aquatic toxicity. The read-across result of the 16-h toxicity threshold of 2-ethylhexanol to Pseudomonas putida (cell multiplication); 110 mg/l, based on the nominal concentrations, is used as key value in CSA. le and the decomposition products possess low aquatic toxicity. The read-across result of the 16-h toxicity threshold of structuraly similair decomposition product, 2 -ethylhexanol, to Pseudomonas putida (cell multiplication); 110 mg/l, based on the nominal concentrations, is used as key value in CSA.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
110 mg/L

Additional information

As the target substance hydrolyses immediately (half-life < 10 minutes) the toxicity to microorganisms is related to organic degradation product (EHD) of the target substance.

The activated sludge respiration study was considered unnecessary for the unstable target substance as structurally similair degadation product (2 -ethylhexanol) is found to be readily biodegradable. In MITI-test, 2 -ethylhexanol was not found to have any inhibitory effetcs to activated sludge at the test concentration of 100 ppm.

Since the target substance is highly water reactive, use of water is avoided in the use applications, there is no emission to a sewage treatment plant. Discharge to STP is relevant only in one use application when the target substance is used as a catalyst in industrial esterification processes. In this use application, water is used to remove the catalyst from the process. The discharge to WWTP is related to the degradation products of this substance as the hydrolysis will take place, and 2-ethylhexanol and TiO2 are released to WWTP. In addition, the concentrations of these degradation products in effluents are expected to be lower than the test concentration in MITI-test (100 ppm).

According to the ECHA Guidance on information requirements and chemical safety assessment, in absence of activated respiration test, toxicity results to microbial bacteria can be used for evaluate the inhibitory effects of chemicals to STP micro-organisms. Therefore, the result of the P.putida cell multiplication inhibition test of the organic hydrolysing product is used to calculate the PNEC STP.