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EC number: 276-014-8 | CAS number: 71786-60-2
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Short-term toxicity to fish
Administrative data
Link to relevant study record(s)
- Endpoint:
- short-term toxicity to fish
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- from 2022-05-14 to 2022-06-03 with the definitive exposure periods from 2022-05-16 to 2022-05-20 (first definitive study) and 2022-05-30 to 2022-06-03 (second definitive study)
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 203 (Fish, Acute Toxicity Test)
- Version / remarks:
- (2019)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- Test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
Batch number 20201158
CAS No. 71786-60-2
Purity (certified) 100% UVCB
Chemical name Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs
Density 910 kg/m3 at 20 °C
Appearance Light yellow liquid
Water Solubility Dispersible / insoluble
Stability under
test conditions Not specified
Expiry date 2023-02-10
Recommended storage
Store container tightly closed in a dry, well-ventilated place. Avoid elevated temperatures. - Analytical monitoring:
- yes
- Remarks:
- Samples were analyzed with a LC-MS/MS method.
- Details on sampling:
- The test loadings and the controls were analytically verified daily in the fresh media (after 0, 24, 48, 72 hours) and in the 24-hours old media (after 24, 48, 72 and 96 hours).
Peak distribution (Finger print)
The peak distribution (fingerprint) of the WAF was analyzed in freshly prepared medium in the highest loading rate of the test item once during the test. The signal distribution was compared with an analytical standard prepared in solvent and a control. - Vehicle:
- no
- Details on test solutions:
- Water Accommodated Fraction
Water accommodated fractions (WAF) were prepared because the test item is a poorly soluble UVCB substance with compounds of different water solubility. This procedure is in accordance with the OECD guidance document No. 23 (2019).
Using this approach, aqueous media was prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.
Test loadings
10.0 – 22.0 – 48.4 – 106 – 234 – 515 – 1134 µg/L (factor 2.2), corresponding to geometric mean measured concentrations of 5.37 – 11.3 – 32.3 – 72.1 – 191 – 651 - 1498 µg/L.
The concentrations are chosen due to the results of a preliminary range finding test (see Annex II) and an Early-Life Stage Toxicity Test (Noack Study Code SO21177/FSZ19203).
In the first definitive study no EC50-values could be determined since only partial mortality (<50%) was observed A second definitive study was carried out with two additional test loadings (control, 515 & 1134 µg/L), which prolonged the concentration series of the first definitive study.
Preparation of the water accommodated fractions
Seven water accommodated fractions (WAF) were prepared separately with the nominal loading rates mentioned above.
The procedure according to ASTM D6081 (2019) as described below will be carried out. For each loading rate an appropriate amount of stock solution containing the test item with methanol was placed on a curved glass slide. The methanol was evaporated. The glass slides with the test item were inserted in glass flasks with an appropriate amount of dilution water.
The test item is a poorly soluble UVCB, therefore a prolonged slow stirring procedure was applied for 24 ± 1 hours at room temperature. The stirring time was chosen based on the preliminary investigations of a daphnia reproduction test. The magnetic stirrer bar was placed with a fish-clip® system a few centimeters above the bottom of the flask to prevent direct contact with the test item on the bottom. After a separation phase of 1 hour at room temperature, the aqueous phase of the WAF was removed by siphoning (from the approximate middle (positioned in the middle third) of the glass flask). The first 25 mL were discarded.
The WAFs were checked using a laser beam (Tyndall effect) for undissolved test item (formation of emulsion). No presence of undissolved test item during the test was observed. The Tyndall effect was negative. The resulting water accommodated fractions (WAF) were used in the test. - Test organisms (species):
- Danio rerio (previous name: Brachydanio rerio)
- Details on test organisms:
- Test organism
Danio rerio (zebrafish)
Vertebrata, Gnathostomata, Pisces, Osteichthyes, Teleostei, Cypriniformes, Cyprinidae
Reason for the selection of the test system
According to the guideline, Danio rerio is suitable for this kind of study.
Origin
All fish used in the test were gained at Noack Laboratorien GmbH from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin)
Holding
Holding was performed at the test facility at 23 ± 2 °C and diffuse light (540 – 1000 Lux, photoperiod of 12 to 16 h light / 12 to 8 h dark). The water was changed at least once per week. The dissolved oxygen concentration was more than 80% of the air saturation value.
Zebrafish with at least 9 days of acclimatization and mortality < 5% within these days prior to the start of the exposure were used in the test.
No mortality was observed during this time. No disease treatments were administered throughout holding and testing.
Water
Tap water of local origin was used for holding. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
Nominal water parameters:
Total hardness: 40 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
Oxygen saturation: ≥ 80 % of air saturation value
Acidity: 0.3 mmol/L (measurement 2022-03-22)
0.3 mmol/L (recent measurement 2022-05-30)
Alkalinity: 0.8 mmol/L (measurement 2022-03-22)
0.8 mmol/L (recent measurement 2022-05-30)
Conductivity: 172 µS/cm (measurement 2022-03-22)
168 µS/cm (recent measurement 2022-05-30)
Acclimatization
A sufficient number of fish were acclimatized to dilution water and environmental conditions equivalent to the test conditions for at least 9 days. The environmental conditions were equivalent to the test conditions.
Feeding
Food was provided 3 times per week. Food was given to satiation (4% of the fish body weight) per feeding day. The test fish were not fed 24 h before test start.
Food Sera Vipan; SERA GMBH, 52518 Heinsberg, Germany - Test type:
- semi-static
- Water media type:
- freshwater
- Remarks:
- Tap water of local origin was used for holding. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
- Limit test:
- no
- Total exposure duration:
- 96 h
- Hardness:
- For details see section "Any other information on results incl. tables" below.
- Test temperature:
- The temperature during test solution preparation in both studies ranged from 22.0 to 23.5 °C.
For details see section "Any other information on results incl. tables" below. - pH:
- For details see section "Any other information on results incl. tables" below.
- Dissolved oxygen:
- For details see section "Any other information on results incl. tables" below.
- Nominal and measured concentrations:
- 10.0 – 22.0 – 48.4 – 106 – 234 – 515 – 1134 µg/L (factor 2.2), corresponding to geometric mean measured concentrations of 5.37 – 11.3 – 32.3 – 72.1 – 191 – 651 - 1498 µg/L.
- Details on test conditions:
- Controls
7 fish in dilution water (without test item but treated in the same way as the test solution) were tested under the same test conditions as the test replicates.
Reference item
No reference item is recommended for this test according to the guidelines.
Duration of the test 96 hours
Test vessel and Pre-Treatment
Glass aquaria of 3 L were used (dimensions: 11.5/13/20 cm, depth of water: approx. 17 cm) and covered with glass plates. A coating phase (saturation of the test vessels) was carried out. The test vessels were pre-treated with the appropriate test solution for at least 12 hours under test conditions. Before the start of the exposure, the test vessels were emptied and refilled with freshly prepared test solution. The test solutions for coating were prepared as described above.
Test volume About 2.5 L per vessel
Aeration No aeration was provided.
Dilution water Same as used for holding
Acclimatization
Acclimatization was not necessary, because the quality of the dilution water and the environmental conditions were equivalent to the water used for holding.
Number of fish
7 zebrafish were used for the test loadings and the controls. For the whole study 69 fish were used.
Loading, length of fish
First definitive study:
Fish density in the tanks was 0.104 g fish per liter test solution.
Average body length at the test end: 1.89 cm
Average body weight at the test end: 0.037 g
Second definitive study:
Fish density in the tanks was 0.115 g fish per liter test solution.
Average body length at the test end: 1.94 cm
Average body weight at the test end: 0.041 g
Age of fish Juvenile
Replicates One replicate each for the limit loading and the control
Introduction of fish The fish were introduced randomly to individual replicates.
Feeding of test fish The fish were not fed during the exposure.
Water temperature (target) 21 - 25 °C, controlled at ± 1 °C
Dissolved oxygen Concentration (target)
Not less than 60% of air saturation value.
Light intensity (target) 540 – 1000 Lux
Photoperiod
A photoperiod (light / dark cycle: 16 / 8 h) was maintained during exposure.
Type and Frequency of Measurements
Biological Parameters
Observation intervals
Observations were made after 2 ± 0.5 h and 5 ± 1 h after the start of the exposure. On days 1-4 of the test, all vessels with living fish were inspected twice per day.
Criteria of effects
Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behavior, respiratory function, pigmentation, etc.).
Measurement of fish size and weight
For measurements of size and weight all control fish were used at the end of the exposure.
Physico-chemical Properties
Water quality and light intensity measurements
The pH-value, temperature and oxygen saturation were measured daily in all test vessels. Chlorine and nitrate were measured from the dilution water used in the test.
Total hardness and TOC was determined at the start of the exposure in the dilution water. During the test, the water temperature was recorded continuously (once per hour) with a data logger. The light intensity was measured at the start of the exposure at one central spot on the surface per the test aquaria.
The solution appearance and behavior were observed daily before and after renewal. - Duration:
- 96 h
- Dose descriptor:
- LC0
- Effect conc.:
- 72.1 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Remarks on result:
- other: 100% Active ingredient
- Duration:
- 96 h
- Dose descriptor:
- LL0
- Effect conc.:
- 106 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Duration:
- 96 h
- Dose descriptor:
- LC100
- Effect conc.:
- 651 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Duration:
- 96 h
- Dose descriptor:
- LL100
- Effect conc.:
- 515 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Key result
- Duration:
- 96 h
- Dose descriptor:
- LC50
- Effect conc.:
- 199 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Remarks on result:
- other: 100% Active ingredient
- Key result
- Duration:
- 96 h
- Dose descriptor:
- LL50
- Effect conc.:
- 241 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Remarks on result:
- other: 100% Active ingredient
- Key result
- Duration:
- 72 h
- Dose descriptor:
- LC50
- Effect conc.:
- 208 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Remarks on result:
- other: 100% Active ingredient
- Key result
- Duration:
- 72 h
- Dose descriptor:
- LL50
- Effect conc.:
- 248 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Remarks on result:
- other: 100% Active ingredient
- Key result
- Duration:
- 48 h
- Dose descriptor:
- LC50
- Effect conc.:
- 356 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Remarks on result:
- other: 100% Active ingredient
- Key result
- Duration:
- 48 h
- Dose descriptor:
- LL50
- Effect conc.:
- 348 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Key result
- Duration:
- 24 h
- Dose descriptor:
- LC50
- Effect conc.:
- 988 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Key result
- Duration:
- 24 h
- Dose descriptor:
- LL50
- Effect conc.:
- 764 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Details on results:
- Biological Data
All effect levels are given based on the nominal test item loading concentration. The LL0 after 96 hours was the nominal test item loading rate of 106 µg/L corresponding to an overall arithmetic mean measured concentration of 72.1 µg/L.
Physicochemical Data
The environmental conditions (pH-value, temperature and oxygen-saturation) were determined to be within the acceptable limits. Light intensity was measured at the start of the exposure and ranged from 680 to 840 (mean value 776) Lux in the first definitive test and from 680 to 790 (mean value 761) Lux in the second definitive test.
Measured Exposure Concentrations during the Definitive Test
The concentrations of the four compounds C10 Amine + 2EO, C12 Amine + 2EO, C14 Amine + 2EO and C16 Amine + 2EO of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) were analytically verified daily via LC MS/MS in the fresh media (after 0, 24, 48, 72 hours) and in the 24-hours old media (after 24, 48, 72 and 96 hours) in all test item concentrations and the control.
The measured concentrations of all compounds of the test item in the fresh media at the start of an exposure-renewal interval (0, 24, 48 and 72 hours) were between < LOQ to 169% of the nominal values.
The measured concentration of the test item in the old media at the end of an exposure-renewal interval (24, 48, 72 and 96 hours) were between < LOQ and 156% of the nominal values. All test solutions were clear and showed no turbidity during sampling because of an negative Tyndall effect.
The peak distribution (fingerprint) of the WAF was analyzed in freshly prepared medium in the limit loading of the test item after 0 hours. The signal distribution was compared with an analytical standard prepared in solvent and a control. - Reported statistics and error estimates:
- Evaluation 2. Definitive test
LL50-values and corresponding confidence intervals were calculated by standard procedures (sigmoidal dose-response regression), as revised in OECD Series on Testing and Assessment No. 54 (2006).
After 2 ± 0.5 h and 5 ± 1 h no mortality occurred.
After 24 hours only 0 and 100% mortality occurred. The LL50 was calculated as a geometric mean of the corresponding concentrations. These concentrations were chosen as lower and upper confidence limits.
After 48 h the LL50-values and the confidence limits were calculated by sigmoidal regression.
After 72 to 96 hours one partial response of mortality occurred. Therefore, the LL50-values were calculated by sigmoidal regression and the corresponding concentrations were chosen as lower and upper confidence limits.
The test loadings leading to 0 and 100% mortality (LL0 and LL100) after 96 h will be determined directly from the test results.
Geometric mean measured concentrations
The geometric mean measured concentrations were determined by the sumproduct function of excel, referring to the recoveries and purities of the four compounds C10 ethoxylated alkylamine C12 ethoxylated alkylamine, C14 ethoxylated alkylamine and C16 ethoxylated alkylamine.
Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
The following software was used for calculations:
- Excel, MICROSOFT CORPORATION - Sublethal observations / clinical signs:
Preparation of the WAFs in the First Definitive Study
Stock solution [mg/L] in MeOH 1000 Preparation volume of the stock solution [mL] 20 Application volume of test item [µL/20 mL] for the stock solution (density of the test item taken into account) 21.98 Preparation volume per WAF [L] 2 Application volume of the stock solution placed 20.0 44.0 96.8 212 468 on a curved glass slide [µL/2L] Final test item Loading rate [µg/L] 10.0 22.0 48.4 106 234 Preparation of the WAFs in the Second Definitive Study
Stock solution [mg/L] in MeOH 5000 Preparation volume of the stock solution [mL] 20 Application volume of test item [µL/20 mL] for the stock solution (density of the test item taken into account) 109.9 Preparation volume per WAF [L] 2 Application volume of the stock solution placed 206 454 on a curved glass slide [µL/2L] Final test item Loading rate [µg/L] 515 1134 Observations of Sub Lethal Effects and Normal Behavior in the Test Vessels in the First
Definitive Study
(n = 7, number of fish)Nominal Test Item loading Effect * Number of fish affected at observation time [hours] [µg/L] day 0 day 1 day 2 day 3 day 4 1.5 5 18.5 25.5 42.5 49.5 67 73.5 90.5 96 234 -1 7 7 7 7 3 1 1 0 0 0 (2.1) 0 0 0 0 0 0 0 1 0 0 (2.2) 0 0 0 0 1 1 0 0 0 0 (2.4) 0 0 0 0 0 3 4 1 3 3 (2.5) 0 0 0 0 0 2 1 3 2 2 (2.7) 0 0 0 0 0 3 4 4 3 3 (2.9) 0 0 0 0 4 0 0 0 0 0 (2.10) 0 0 0 0 0 5 0 1 1 1 (3.5) 0 0 0 0 0 5 5 6 5 5 (4.1) 0 0 0 0 0 0 1 0 0 0 (5.4) 0 0 0 0 0 1 1 3 3 3 (E) 0 0 0 0 0 1 1 1 2 2 106 -1 7 7 7 7 7 7 7 7 7 7 48.4 -1 7 7 7 7 7 7 7 7 7 7 22.0 -1 7 7 7 7 7 7 7 7 7 7 10.0 -1 7 7 7 7 7 7 7 7 7 7 Control -1 7 7 7 7 7 7 7 7 7 7 *) The numbers in brackets correspond to the following observations:
(1) = Normal behavior (2.1) = Lethargy/Immobility (2.2) = Lateral position
(2.4) = Slow escape reflex (2.5) = Missing escape reflex (2.7) = Hypoactivity
(2.9) = Arresting on the ground (2.10) = Arresting below the surface (3.5) = Clamped fins
(4.1) = Hyperventilation (5.4) = Descent of the fish (E) = Exitus lethalisObservations of Sub Lethal Effects and Normal Behavior in the Test Vessels in the
Second Definitive Study
(n = 7, number of fish)Nominal Test Item loading Effect * Number of fish affected at observation time [hours] [µg/L] day 0 day 1 day 2 day 3 day 4 2 4 21 28 45 52 69 76 93 96 1134 -1 7 4 0 100 % mortality (2.4) 0 3 0 (E) 0 0 7 515 -1 7 7 6 0 0 0 0 100 % mortality (2.4) 0 0 1 7 0 0 0 (2.5) 0 0 0 0 2 1 0 (3.5) 0 0 0 7 2 1 0 (5.4) 0 0 0 0 2 1 0 (E) 0 0 0 0 5 6 7 Control -1 7 7 7 7 7 7 7 7 7 7 *) The numbers in brackets correspond to the following observations:
(1) = Normal behavior (2.4) = Slow escape reflex (2.5) = Missing escape reflex
(3.5) = Clamped fins (5.4) = Abnormal vertical orientation (E) = Exitus lethalisCumulative Mortality % in the Test Vessels in the First Definitive Study
Nominal Test Item loading Cumulative mortality [%] at observation time [hours] [µg/L] day 0 day 1 day 2 day 3 day 4 1.5 5 18.5 25.5 42.5 49.5 67 73.5 90.5 96 234 0 0 0 0 0 14 14 14 29 29 106 0 0 0 0 0 0 0 0 0 0 48.4 0 0 0 0 0 0 0 0 0 0 22.0 0 0 0 0 0 0 0 0 0 0 10.0 0 0 0 0 0 0 0 0 0 0 Control 0 0 0 0 0 0 0 0 0 0 Cumulative Mortality % in the Test Vessels in the Second Definitive Study
Nominal Test Item loading Cumulative mortality [%] at observation time [hours] [µg/L] day 0 day 1 day 2 day 3 day 4 2 4 21 28 45 52 69 76 93 96 1134 0 0 100 100 100 100 100 100 100 100 515 0 0 0 0 71 86 100 100 100 100 Control 0 0 0 0 0 0 0 0 0 0
Environmental Conditions in the First Definitive Study
Nominal Test item loading Oxygen Saturation pH Value Temperature [µg/L] [%] [°C] new old new old new old Day 0 234 93 7.34 23.8 106 94 7.12 23.8 48.4 92 7.16 23.8 22.0 92 7.36 23.8 10.0 95 7.08 23.7 Control 99 7.60 23.6 Day 1 234 96 86 7.50 7.49 24.0 24.2 106 97 83 7.38 7.52 24.1 23.8 48.4 92 80 7.48 7.43 24.1 23.8 22.0 97 76 7.46 7.41 24.6 23.8 10.0 96 85 7.46 7.41 23.8 23.8 Control 98 72 7.44 7.18 23.2 23.8 Day 2 234 97 86 7.44 7.42 24.0 23.8 106 96 81 7.44 7.38 24.0 23.9 48.4 98 81 7.43 7.40 24.0 23.9 22.0 97 81 7.41 7.36 24.0 24.0 10.0 97 81 7.33 7.35 23.8 23.9 Control 98 73 7.18 7.17 23.3 23.7 Day 3 234 97 80 7.50 7.33 24.0 24.0 106 97 88 7.48 7.40 24.0 24.0 48.4 98 95 7.48 7.44 24.0 24.0 22.0 97 79 7.45 7.38 24.1 24.0 10.0 98 85 7.46 7.41 23.8 23.9 Control 100 74 7.37 7.28 23.7 23.8 Day 4 234 81 7.37 23.8 106 86 7.37 24.0 48.4 87 7.37 24.1 22.0 83 7.29 24.1 10.0 77 7.19 24.0 Control 87 7.29 23.9 Environmental Conditions in the Second Definitive Study
Nominal Test item loading Oxygen Saturation pH Value Temperature [µg/L] [%] [°C] new old new old new old Day 0 1134 97 7.38 23.6 515 97 7.39 23.4 Control 100 7.54 23.4 Day 1 1134 - 83 - 7.39 - 23.4 515 98 84 7.47 7.42 23.5 23.2 Control 100 84 7.55 7.24 23.7 23.4 Day 2 1134 - - - - - - 515 97 81 7.66 7.40 23.4 22.9 Control 99 83 7.51 7.48 23.3 23.9 Day 3 1134 - - - - - - 515 84 7.46 22.9 Control 99 84 7.44 7.24 22.9 23.0 Day 4 1134 - - - 515 Control 84 7.41 23.2 Water Temperature in the Control (Continuous Measuring) in the First and Second Definitive Test
Period of Measurements 2022-05-16 to 2022-05-20 2022-05-30 to 2022-06-03 Minimum Temperature [°C] 22.9 22.5 Maximum Temperature [°C] 24.2 23.5 Mean Temperature [°C] 23.7 23.0 Residual Chlorine from the Dilution Water in the First and Second Definitive Test
Batch For Study Days [mg/L] First Definitive Test Second Definitive Test Day 0 0 to 1 < 0.01 < 0.01 Day 1 1 to 2 < 0.01 < 0.01 Day 2 2 to 3 < 0.01 < 0.01 Day 3 3 to 4 < 0.01 < 0.01 Nitrate from the Dilution Water in the First and Second Definitive Test
Batch For Study Days [mg/L] First Definitive Test Second Definitive Test Day 0 0 to 1 2.14 1.31 Day 1 1 to 2 1.86 1.42 Day 2 2 to 3 2.08 1.86 Day 3 3 to 4 1.55 1.58 TOC and Total Hardness from the Dilution Water
Parameter Study Days [mg/L] First Definitive Test Second Definitive Test TOC 0 to 4 < 2 < 2 Total Hardness 0 to 4 62 70 Measured Concentrations of the Test Item during the Definitive Test (C10 Amine + 2 EO)
Sampling date Fresh media Old media Fresh media Old media 0 hours 24 hours 24 hours 48 hours Nominal Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) concentration of the C10 Amine + 2 EO Test item a.s. Meas. % Meas. % Meas. % Meas. % [µg/L] [µg a.s./L] conc. conc. conc. conc. [µg a.s./L] [µg a.s./L] [µg a.s./L] [µg a.s./L] 11341) 82.4 128 155 128 156 - - - - 5151) 37.4 56.5 151 55.4 148 52.2 139 51.9 139 2342) 17.0 18.3 107 17.5 103 17.8 105 17.3 101 106 7.73 8.34 108 7.68 100 7.83 102 7.89 102 48.4 3.53 3.80 108 3.69 105 3.42 97 3.40 97 22.0 1.60 1.68 105 1.27 79 1.74 109 1.62 101 10.0 0.729 0.727 100 0.688 95 0.781 107 0.753 104 Control1) < LOQ < LOQ < LOQ < LOQ Control < LOQ < LOQ < LOQ < LOQ Sampling date Fresh media Old media Fresh media Old media 48 hours 72 hours 72 hours 96 hours Nominal Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) concentration of the C10 Amine + 2 EO Test item a.s. Meas. % Meas. % Meas. % Meas. % [µg/L] [µg a.s./L] conc. conc. conc. conc. [µg a.s./L] [µg a.s./L] [µg a.s./L] [µg a.s./L] 11341) 82.7 - - - - - - - - 5151) 37.5 56.5 151 52.2 139 - - - - 234 17.0 18.3 107 12.5 73 17.2 101 15.2 90 106 7.73 7.98 104 5.46 71 7.24 94 5.76 75 48.4 3.53 3.86 110 2.35 67 3.42 97 2.78 79 22.0 1.60 1.62 101 0.982 61 1.36 85 0.962 60 10.0 0.729 0.765 105 0.591 81 0.624 86 0.246 34 Control1) < LOQ < LOQ < LOQ - Control < LOQ < LOQ < LOQ < LOQ Meas. Conc. = Measured concentration (dilution factor taken into account)
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (2.0 µg/L of the test item, corresponding to 0.145 µg a.s./L)
a.s. = Active substance
1) = 2nd definitive study
2.) = 1st definite study
- = not determinedMeasured Concentrations of the Test Item during the Definitive Test (C12 Amine + 2 EO)
Sampling date Fresh media Old media Fresh media Old media 0 hours 24 hours 24 hours 48 hours Nominal Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) concentration of the C12 Amine + 2 EO Test item a.s. Meas. % Meas. % Meas. % Meas. % [µg/L] [µg a.s./L] conc. conc. conc. conc. [µg a.s./L] [µg a.s./L] [µg a.s./L] [µg a.s./L] 11341) 597 661 111 852 143 - - - - 5151) 271 234 86 359 132 387 143 358 132 234 123 131 107 108 88 129 105 109 88 106 55.8 59.7 107 41.4 74 58.6 105 48.8 87 48.4 25.5 28.0 110 19.9 78 24.4 96 19.6 77 22.0 11.6 12.1 105 < LOQ 12.6 109 6.17 53 10.0 5.26 5.32 101 2.77 53 5.08 97 3.57 68 Control1) < LOQ < LOQ < LOQ < LOQ Control < LOQ < LOQ < LOQ < LOQ Sampling date Fresh media Old media Fresh media Old media 48 hours 72 hours 72 hours 96 hours Nominal Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) concentration of the C12 Amine + 2 EO Test item a.s. Meas. % Meas. % Meas. % Meas. % [µg/L] [µg a.s./L] conc. conc. conc. conc. [µg a.s./L] [µg a.s./L] [µg a.s./L] [µg a.s./L] 11341) 597 - - - - - - - - 5151) 271 419 155 346 128 - - - - 234 123 127 103 63.2 51 123 100 84.6 69 106 55.8 54.7 98 21.1 38 46.6 84 13.3 24 48.4 25.5 27.7 109 5.17 20 22.3 87 9.62 38 22.0 11.6 10.7 93 1.59 14 7.46 64 < LOQ 10.0 5.26 4.84 92 2.44 46 2.09 40 < LOQ Control1) < LOQ < LOQ < LOQ - Control < LOQ < LOQ < LOQ < LOQ Meas. Conc. = Measured concentration (dilution factor taken into account)
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (2.0 µg/L of the test item, corresponding to 1.05 µg a.s./L)
a.s. = Active substance
1) = 2nd main study
- = not determinedMeasured Concentrations of the Test Item during the Definitive Test (C14 Amine + 2 EO)
Sampling date Fresh media Old media Fresh media Old media 0 hours 24 hours 24 hours 48 hours Nominal Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) concentration of the C14 Amine + 2 EO Test item a.s. Meas. % Meas. % Meas. % Meas. % [µg/L] [µg a.s./L] conc. conc. conc. conc. [µg a.s./L] [µg a.s./L] [µg a.s./L] [µg a.s./L] 11341) 211 357 169 256 121 - - - - 5151) 96 155 162 95.1 99 136 142 94.9 99 234 43.6 45.9 105 27.5 63 48.3 111 28.1 64 106 19.8 18.8 95 3.88 20 23.2 117 10.5 53 48.4 9.02 9.57 106 1.61 18 7.30 81 3.86 43 22.0 4.10 3.69 90 < LOQ 4.23 103 < LOQ 10.0 1.86 1.41 76 < LOQ 1.17 63 < LOQ Control < LOQ < LOQ < LOQ < LOQ Sampling date Fresh media Old media Fresh media Old media 48 hours 72 hours 72 hours 96 hours Nominal Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) concentration of the C14 Amine + 2 EO Test item a.s. Meas. % Meas. % Meas. % Meas. % [µg/L] [µg a.s./L] conc. conc. conc. conc. [µg a.s./L] [µg a.s./L] [µg a.s./L] [µg a.s./L] 11341) 211 - - - - - - - - 5151) 96 147 153 98.2 102 - - - - 234 43.6 40.1 92 4.57 10 40.4 93 12.3 28 106 19.8 14.2 72 0.630 3 8.61 44 < LOQ 48.4 9.02 6.17 68 < LOQ 4.78 53 < LOQ 22.0 4.10 2.30 56 < LOQ 1.49 36 < LOQ 10.0 1.86 0.813 44 < LOQ < LOQ < LOQ Control < LOQ < LOQ < LOQ < LOQ Meas. Conc. = Measured concentration (dilution factor taken into account)
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (2.0 µg/L of the test item, corresponding to 0.373 µg a.s./L)
a.s. = Active substance
1) = 2nd main study
- = not determinedMeasured Concentrations of the Test Item during the Definitive Test (C16 Amine + 2 EO)
Sampling date Fresh media Old media Fresh media Old media 0 hours 24 hours 24 hours 48 hours Nominal Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) concentration of the C16 Amine + 2 EO Test item a.s. Meas. % Meas. % Meas. % Meas. % [µg/L] [µg a.s./L] conc. conc. conc. conc. [µg a.s./L] [µg a.s./L] [µg a.s./L] [µg a.s./L] 11341) 96 133 139 87.6 91 - - - - 5151) 44 61.7 142 12.5 29 59.1 136 30.7 71 234 19.8 16.6 84 6.45 33 17.5 88 5.74 29 106 8.97 5.85 65 0.803 9 6.65 74 1.72 19 48.4 4.09 2.88 70 < LOQ 2.05 50 0.327 8 22.0 1.86 0.813 44 < LOQ 0.964 52 < LOQ 10.0 0.846 0.307 36 < LOQ 0.243 29 < LOQ Control < LOQ < LOQ < LOQ < LOQ Sampling date Fresh media Old media Fresh media Old media 48 hours 72 hours 72 hours 96 hours Nominal Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) concentration of the C16 Amine + 2 EO Test item a.s. Meas. % Meas. % Meas. % Meas. % [µg/L] [µg a.s./L] conc. conc. conc. conc. [µg a.s./L] [µg a.s./L] [µg a.s./L] [µg a.s./L] 11341) 96 - - - - - - - - 5151) 44 59.8 137 19.3 44 - - - - 234 19.8 12.1 61 0.272 1 13.8 70 0.955 5 106 8.97 2.75 31 < LOQ 1.10 12 < LOQ 48.4 4.09 0.846 21 < LOQ 1.27 31 < LOQ 22.0 1.86 0.555 30 < LOQ 0.349 19 < LOQ 10.0 0.846 0.172 20 < LOQ < LOQ < LOQ Control < LOQ < LOQ < LOQ < LOQ Meas. Conc. = Measured concentration (dilution factor taken into account)
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (2.0 µg/L of the test item, corresponding to 0.169 µg a.s./L)
a.s. = Active substance
1) = 2nd main study
- = not determinedCalculated Overall Mean Measured Test Item Concentrations of the study
Sampling day Fresh media Old media Fresh media Old media 0 hours 24 hours 24 hours 48 hours Nominal Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs concentration of the test item (CAS: 71786-60-2) [µg/L] Calculated mean measured concentration per study day [µg/L] 11341) 1472 1523 - - 5151) 583 599 730 616 234 244 184 245 183 106 106 61.8 111 78.8 48.4 50.9 29.0 42.8 31.3 22.0 21.1 2.36 22.5 9.22 10.0 8.92 4.31 8.39 5.29 Sampling day Fresh media Old media Fresh media Old media 48 hours 72 hours 72 hours 96 hours Nominal Tallow amine propoxylate (CAS: 1309955-79-0) Overall mean measured concentration of the test item [µg/L] concentration of the test item [µg/L] Calculated mean measured concentration per study day [µg/L] 11341) - - - - 1498 5151) 785 593 - - 651 234 227 91.7 224 130 191 106 91.6 31.4 73.4 22.4 72.1 48.4 44.4 8.87 36.4 14.6 32.3 22.0 17.5 3.29 12.2 2.01 11.3 10.0 7.58 3.77 3.45 1.20 5.37 1) = 2nd definitive study , all other results are from the first study
- = not determinedFingerprint of the Test Item (non-GLP)
The peak distribution of the test concentration was analyzed in freshly prepared medium in the nominal test item concentration 1134 µg/L. A solution of the analytical standard of the test item was prepared in acetonitrile and diluted to 1.00 mg/L with dilution medium. A sample of the nominal test item concentration 1134 µg/L and a control were diluted factor 2 with acetonitrile containing 2% formic acid to avoid an inhomogeneous sample. The solutions were analytically verified via high resolution MS and evaluated by the software. The detected signals of the analytical standard and the test item solution were compared. In test item and standard solutions 6 masses were observed and could be assigned to the test item. Compared to the standard in dilution medium the test item in fresh media showed a relative increase in the amounts of the compounds with 274 Da (C12), 302 Da (C14) and 358 Da (C18), whereas the relative amounts of compounds < 274 Da (C10 and C8) are decreased. In general, however, the concentrations and solubility of the test item is too low for significant MS spectra.
Percentage of the Relevant Amine
[%] C10 Amine + 2EO 7.27 C12 Amine + 2EO 52.62 C14 Amine + 2EO 18.64 C16 Amine + 2EO 8.46 Gradient Table
Time [min] A [%] B [%] 0.00 75 25 0.30 75 25 1.30 10 90 2.00 10 90 2.10 75 25 2.50 75 25 Dilution steps
Nominal Dilution Sample Final test item Factor* volume volume concentration [mL] [mL] [µg/L] 1134 200 1 10 0.1 1.0 1134 80 1 10 0.25 1.0 5151) 100 1 10 0.2 1.0 5151) 40 1 10 0.5 1.0 5152) 100 0.2 10 5152) 40 0.05 1.0 234 20 0.1 1.0 234 2 1.0 1.0 106 10 0.2 1.0 106 2 1.0 1.0 48.4 10 0.2 1.0 48.4 2 1.0 1.0 22.0 2 1.0 1.0 10.0 2 1.0 1.0 Control 2 1.0 1.0 * including factor 2
1) samples 48h and later
2) samples 0h and 24hParameter, Acceptance Criteria and Results of the Method Validation
Parameter Acceptance criteria Result Linearity ≥ 5 standard concentrations, 0.2 to 20 µg test item/L (n = 8), ü r ≥ 0.99 (R2 ≥ 0.9801 for 2nd order regression) r ≥ 0.99 Calibration range from ≤ 30% LOQ to 20% above highest concentration level after sample preparation Matrix effects Should be ≤ 20% Matrix effects: < 20% ü Lowest calibration level (LCL) S/N ≥ 9 for quantifier ion trace S/N for 0.2 µg test item/L ü S/N ≥ 3 for qualifier ion trace C10 Amine + 2 EO 187 (Quantifier), 110 (Qualifier) C12 Amine + 2 EO 937 (Quantifier), 460 (Qualifier) C14 Amine + 2 EO 493 (Quantifier), 354 (Qualifier) C16 Amine + 2 EO 185 (Quantifier), 176 (Qualifier) Limit of Quantification (LOQ) Should be at or below lowest test concentration 2.0 µg test item/L (1 x LOQ) ü 1400 µg test item/L (700 x LOQ) Accuracy1) Mean recovery rate of 70-120% C10 Amine + 2 EO ü (Fortified samples) per fortification level (2 levels) 1 x LOQ: 88% (n = 5) 700 x LOQ: 105% (n = 5) C12 Amine + 2 EO 1 x LOQ: 92% (n = 5) 700 x LOQ: 106% (n = 5) C14 Amine + 2 EO 1 x LOQ: 91% (n = 5) 700 x LOQ: 105% (n = 5) C16 Amine + 2 EO 1 x LOQ: 89% (n = 5) 700 x LOQ: 107% (n = 5) Precision1) Relative standard deviation ≤ 20% per fortification level C10 Amine + 2 EO ü 1 x LOQ: 7.9% 700 x LOQ: 2.3% C12 Amine + 2 EO 1 x LOQ: 7.8% 700 x LOQ: 2.1% C14 Amine + 2 EO 1 x LOQ: 8.2% 700 x LOQ: 2.5% C16 Amine + 2 EO 1 x LOQ: 14% 700 x LOQ: 2.2% Stability Samples: Stable if 70 – 120% of nominal concentration See section 15.1 ü Calibration standards: Stable if ±10% of freshly prepared standard Specificity: Measurement of two transitions of the same precursor ion - one quantifier (used for evaluation) and one qualifier (for confirmation of the analyte identity). C10 Amine + 2 EO ü LC-MS/(MS) quantifier [m/z]: 246.20 > 88.13 qualifier [m/z]: 246.20 > 70.17 C12 Amine + 2 EO quantifier [m/z]: 274.30 > 88.13 qualifier [m/z]: 274.30 > 70.23 C14 Amine + 2 EO quantifier [m/z]: 302.36 > 88.12 qualifier [m/z]: 302.36 > 70.29 C16 Amine + 2 EO quantifier [m/z]: 330.36 > 88.12 qualifier [m/z]: 330.36 > 70.29 Blank values < 30% of LOQ Blank values < 30% of LOQ ü Procedural recovery Procedural recoveries with experimental samples. The recovery has to be 70-120% of the nominal value. See section 15.2 ü = criterion fulfilled
- = not determinedPreparation of the fortified samples
The method was validated at the 1 x LOQ (2.0 µg test item/L) and at the 700 x LOQ 1400 µg test item/L).
Preparation of Fortified Samples
LOQ Level Control 1 700 Stock solution - 100 mg test item/L in Acetonitrile Spiking solution - 0.2 Stock solution [mg test item/L] (Dilution medium) (Medium) Replicates 2 5 5 Concentration of the LOQ - 2.0 1400 [µg test item/L] Medium for preparation Fish dilution medium Volume of spiking solution [mL] - 0.05 0.07 Volume of medium [mL] 5.0 4.95 4.93 Dilution factor - 2 100 Dilution medium Acetonitrile containing 2% formic acid 1) Dilution medium 2) Sample volume [mL] 5.01) 5.01) 5.01) 0.12) Finale volume [mL] 101) 101) 101) 5.02) Test item concentration in the final solution [µg/L] - 1.0 14.0 Dilution medium Acetonitrile : ultra-pure water (50 : 50 v/v) containing 1 % formic acid
1) First dilution step
2) Second dilution stepNominal Concentrations of the Fortified Samples of the active ingredients of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
Fortified concentrations*: 2.02 µg test item/L (1 x LOQ) and 1419 µg test item/L (700 x LOQ).Active ingredient 1 x LOQ* 700 x LOQ* [µg a.s./L] [µg a.s./L] C10 Amine + 2 EO 0.147 103 C12 Amine + 2 EO 1.06 747 C14 Amine + 2 EO 0.376 265 C16 Amine + 2 EO 0.171 120 * = weighing factor taken into account
a.s. = active substanceMeasured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
Active ingredient: C10 Amine + 2 EO and C12 Amine + 2 EOReplicate Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) C10 Amine + 2 EO 1 x LOQ 700 x LOQ Meas.conc. % Meas.conc. % [µg a.s./L] [µg a.s./L] 1 0.147 101 108 105 2 0.125 85 112 108 3 0.123 84 109 106 4 0.127 87 106 103 5 0.125 85 106 102 Mean 0.13 88 108 105 SD ± 0.01 2 CV [%] 7.9 2.3 Replicate Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) C12 Amine + 2 EO 1 x LOQ 700 x LOQ Meas. conc. % Meas. conc. % [µg a.s./L] [µg a.s./L] 1 1.11 104 814 109 2 0.956 90 797 107 3 0.941 89 782 105 4 0.922 87 776 104 5 0.948 89 773 104 Mean 0.98 92 789 106 SD ± 0.08 17 CV [%] 7.8 2.1 Meas. conc. = measured concentration of each component of the test item, dilution factor taken into account
% = percent concentration of the fortified sample
* = weighing factor taken into account
SD = standard deviation CV = coefficient of variationMeasured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
Active ingredient: C14 Amine + 2 EO and C16 Amine + 2 EOReplicate Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) C14 Amine + 2 EO 1 x LOQ 700 x LOQ Meas. conc. % Meas. conc. % [µg a.s./L] [µg a.s./L] 1 0.389 103 284 107 2 0.346 92 284 107 3 0.318 85 279 106 4 0.327 87 270 102 5 0.329 87 270 102 Mean 0.34 91 277 105 SD ± 0.03 7 CV [%] 8.2 2.5 Replicate Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) C16 Amine + 2 EO 1 x LOQ 700 x LOQ Meas. conc. % Meas. conc. % [µg a.s./L] [µg a.s./L] 1 0.187 109 131 109 2 0.149 87 131 109 3 0.135 79 130 108 4 0.138 81 126 105 5 0.151 89 126 105 Mean 0.15 89 129 107 SD ± 0.02 3 CV [%] 14 2.2 Meas. conc. = measured concentration of each component of the test item, dilution factor taken into account
% = percent concentration of the fortified sample
* = weighing factor taken into account
SD = standard deviation
CV = coefficient of variationStability
The stability of the fortified samples at the 1 x LOQ was checked after 3 days and was for C10 Amine + 2 EO, C12 Amine + 2 EO, C14 Amine + 2 EO and C16 Amine + 2 EO between 105 and 109% of the nominal concentration for each component.
The stability of the standard stock solution (1000 mg test item/L in acetonitrile) was checked after 7 days and was 103 to 106% of the nominal concentration and was found to be stable over this period.
The responses of a 3 days old calibration standard (8 µg test item/L) was compared with the responses of a freshly prepared calibration standard with the same concentration. The deviation was < 10% and was found to be stable over this period.
Procedural Recovery
A procedural recovery (Quality Control) on 1x LOQ Level was freshly prepared on each day of analysis. It was treated in parallel to the test samples.
Measured Concentrations and Percent of Nominal Concentration of the Quality Control during the Definitive Test
Sampling Fresh media Old media Fresh media Old media date 0 hours 24 hours 24 hours 48 hours Component Meas. % Meas. % Meas. % Meas. % conc. conc. conc. conc. [µg a.s./L] [µg a.s./L] [µg a.s./L] [µg a.s./L] C10 Amine + 2 EO 0.144 96 0.147 98 0.143 96 0.130 88 C12 Amine + 2 EO 1.08 100 1.09 101 1.09 101 1.02 95 C14 Amine + 2 EO 0.380 99 0.389 102 0.381 100 0.358 94 C16 Amine + 2 EO 0.186 107 0.176 101 0.170 98 0.166 95 Sampling Fresh media Old media Fresh media Old media date 48 hours 72 hours 72 hours 96 hours Component Meas. % Meas. % Meas. % Meas. % conc. conc. conc. conc. [µg a.s./L] [µg a.s./L] [µg a.s./L] [µg a.s./L] C10 Amine + 2 EO 0.136 91 0.104 70 0.157 105 0.147 99 C12 Amine + 2 EO 1.04 96 0.852 79 1.12 104 1.05 97 C14 Amine + 2 EO 0.361 94 0.299 78 0.362 95 0.364 95 C16 Amine + 2 EO 0.170 98 0.132 76 0.172 99 0.161 93 Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration
Quality Control = 2.00 µg test item/L, weighing factor taken into accountMeasured Concentrations and Percent of Nominal Concentration of the Quality Control during the second Definitive Test
Sampling Fresh media Old media Fresh media Old media date 0 hours 24 hours 24 hours 48 hours Component Meas. % Meas. % Meas. % Meas. % conc. conc. conc. conc. [µg a.s./L] [µg a.s./L] [µg a.s./L] [µg a.s./L] C10 Amine + 2 EO 0.117 79 0.113 76 0.113 76 0.140 95 0.139 94 0.139 94 0.139 94 C12 Amine + 2 EO 1.02 96 1.04 98 1.04 98 1.11 104 1.08 101 1.08 101 1.08 101 C14 Amine + 2 EO 0.358 95 0.350 93 0.350 93 0.369 98 0.366 97 0.366 97 0.366 97 C16 Amine + 2 EO 0.137 80 0.147 86 0.147 86 0.126 73 0.130 76 0.130 76 0.130 76 Sampling Fresh media Old media Fresh media Old media date 48 hours 72 hours 72 hours 96 hours Component Meas. % Meas. % Meas. % Meas. % conc. conc. conc. conc. [µg a.s./L] [µg a.s./L] [µg a.s./L] [µg a.s./L] C10 Amine + 2 EO 0.140 95 0.103 70 0.103 70 - - C12 Amine + 2 EO 1.11 104 0.890 83 0.890 83 - - C14 Amine + 2 EO 0.369 98 0.317 84 0.317 84 - - C16 Amine + 2 EO 0.126 73 0.163 95 0.163 95 - - Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration
Quality Control = 2.00 µg test item/L, weighing factor taken into account- = No measurements due to 100 % mortality
Preliminary (non-GLP)
Solubility Test
A solubility test was performed within the framework of a study on Daphnia chronic reproduction. Two water accommodated fractions were prepared. For each loading rate an appropriate amount of stock solution containing the test item with methanol was placed on a curved glass slide. The methanol was evaporated. The glass slide with the test item was inserted in a glass flask with an appropriate amount of Elendt M4 medium according to OECD 211, Annex 2. A slow stirring procedure was applied for 72 hours at room temperature. The WAFs were checked via laser beam (Tyndall effect) for undissolved test item. No presence of undissolved test item during the test was observed. For the preparation of the water accommodated fractions a stirring phase of 24 hours was found to be suitable.
Measured Concentrations of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) during the Stability Test (non-GLP)
Analytical system: LC-MSEthanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) Components Measured concentration [µg/L] Sampling date 24 hours 48 hours 72 hours Nominal test item loading rate 100 µg/L C14 Component + 2EO 66.9 68.2 81.7 C16 Component + 2EO 52.1 52.8 54.5 C18 Component + 2EO 27.7 14.3 13.4 C18:1 Component + 2EO 38.7 40.2 39.9 Nominal test item loading rate 1000 µg/L C8 Component + 2EO 768 - C10 Component + 2EO 913 C12 Component + 2EO 923 C14 Component + 2EO 849 C16 Component + 2EO 571 C18 Component + 2EO 330 C18:1 Component + 2EO 513 LCL = lowest calibration standard = 2 µg/L
Range Finding Test
A non-GLP preliminary range finding test was conducted at the test facility under semi-static conditions over a period of 96 hours.
The range finding test was carried out with the test loading of 10 µg/L. The water accommodated fraction was prepared as described above. A coating phase (saturation of the test vessels) was carried out as described above. The Tyndall effect was negative.Observations of Sub Lethal Effects and Normal Behavior in the Test Vessels
(n = 3 , number of fish)Nominal Effect * Number of fish affected at observation time Test Item Loading [µg/L] day 0 day 1 day 2 day 3 day 4 10 -1 3 3 3 3 3 Control -1 3 3 3 3 3 *) The numbers in brackets correspond to the following observations:
(1) = Normal behaviorCumulative Mortality % in the Test Vessels
Nominal Cumulative mortality [%] at observation time Test Item Loading [µg/L] day 0 day 1 day 2 day 3 day 4 10 0 0 0 0 0 Control 0 0 0 0 0 Measured Exposure Concentrations of the Test Item during the non-GLP Preliminary Range Finding Test
Determination of the test item via LC-MSSampling date Fresh media, Old media, Fresh media, Old media, Day 0 Day 1 Day 3 Day 4 Nominal test item loading rates Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) (C14 Component + 2 EO) [µg/L] [µg a.s./L] Meas. conc. % Meas. conc. % Meas. conc. % Meas. conc. % [µg a.s./L] [µg a.s./L] [µg a.s./L] [µg a.s./L] 10.0 1.86 1.72 92 0.460 25 1.24 67 < LCL Control < LCL < LCL < LCL < LCL Sampling date Fresh media, Old media, Fresh media, Old media, Day 0 Day 1 Day 3 Day 4 Nominal test item loading rates Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) (C16 Component + 2 EO) [µg/L] [µg a.s./L] Meas. conc. % Meas. conc. % Meas. conc. % Meas. conc. % [µg a.s./L] [µg a.s./L] [µg a.s./L] [µg a.s./L] 10.0 0.846 0.462 55 < LCL 0.272 32 < LCL Control < LCL < LCL < LCL < LCL % = Percent of the nominal test item concentration
a.s. = active substance
LCL = 0.5 µg test item/L (0.0932 µg C14 Component + 2 EO/L; 0.0423 µg C16 Component + 2EO))
Meas. Conc. = measured concentration of the test item, dilution factors taken into accountFingerprint – MS spectrum
The peak distribution of the test concentration was analyzed in freshly prepared medium in the nominal test item concentration 1134 µg/L. A solution of the analytical standard of the test item was prepared in acetonitrile and diluted to 1.00 mg/L with dilution medium. A sample of the nominal test item concentration 1134 µg/L and a control were diluted factor 2 with acetonitrile containing 2% formic acid to avoid an inhomogeneous sample. The solutions were analytically verified via high resolution MS and evaluated by the software. The detected signals of the analytical standard and the test item solution were compared. In test item and standard solutions 6 masses were observed and could be assigned to the test item. Compared to the standard in dilution medium the test item in fresh media showed a relative increase in the amounts of the compounds with 274 Da (C12), 302 Da (C14) and 358 Da (C18), whereas the relative amounts of compounds < 274 Da (C10 and C8) are decreased. In general, however, the concentrations and solubility of the test item is too low for significant MS spectra.
- Validity criteria fulfilled:
- yes
- Conclusions:
- The effect levels are given based on the nominal loading rates and the overall arithmetic mean measured concentrations of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2). In this study Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) was found to cause lethal effects to rainbow trout after 96 hours at the nominal test item loadings 234 – 515 – 1134 µg/L, corresponding to overall arithmetic mean measured concentrations of 191 – 651 – 1498 µg/L and higher. The LL50 after 96 hours was 241 µg test item/L (nominal loading rate), corresponding to an overall arithmetic mean measured concentration of 199 µg/L. The LL0 and LL100 after 96 hours were 106 and 515 µg test item/L (nominal loading rate), respectively, corresponding to overall arithmetic mean measured concentrations of 72.1 and 651 µg/L.
- Executive summary:
The acute toxicity of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) (batch no. 20201158) to fish (zebrafish) was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (2019) in two definitive studies from 2022-05-14 to 2022-06-03 with the definitive exposure periods from 2022-05-16 to 2022-05-20 (first definitive study) and 2022-05-30 to 2022-06-03 (second definitive study) at the test facility.
The test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) is a light yellow liquid, poorly soluble but dispersible in water. It is a UVCB substance with constituents of different water solubility. Water accommodated fractions (WAFs) were prepared 24 ± 1 hour prior to the start of exposure with the nominal loading rates of the test item of 10.0 – 22.0 – 48.4 – 106 – 234 – 515 – 1134 µg/L, corresponding to the overall arithmetic mean measured concentrations of 5.37 – 11.3 – 32.2 – 72.1 – 191 – 651 - 1498 µg/L. For the preparation of the loading rate the test item was dissolved in methanol applied onto curved glass slides. The methanol was evaporated. The glass slides with the test item were inserted in glass flasks with an appropriate amount of dilution water. A slow stirring procedure was applied for 24 ± 1 hour at room temperature. The magnetic stirrer bar was placed with a fish-clip® system a few centimeters above the bottom of the flask to prevent direct contact with the test item on the bottom. After a separation phase of 1 hour, the aqueous phase of the WAF was removed by siphoning (from the approximate middle of the glass flask). The WAFs were checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative. The resulting water accommodated fractions (WAF) were used in the test. The test vessels were pre-treated with the appropriate test solution for at least 12 hours under test conditions.
The selection of the test loadings are based on the results of preliminary range finding tests (acute, see Annex II) and Early-Life Stage Toxicity Test (Noack Study Code SO21177/FSZ19203).
In the first definitive study no EC50-values could be determined. A second definitive study was carried out with only two additional test loadings (control, 515 & 1134 µg/L), which prolonged the concentration series of the first definitive study.The studies were performed over a period of 96 hours under semi-static conditions with daily renewal of the test media. Seven test organisms were exposed to the test loadings and the controls. No significant mortality but sub-lethal effects were observed in the first definitive test. 100% mortality was observed in the test loadings 515 and 1134 µg/L. . Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits.
The measured concentrations of all compounds of the test item in the fresh media at the start of an exposure-renewal interval (0, 24, 48 and 72 hours) were between < LOQ to 169% of the nominal values.
The measured concentration of the test item in the old media at the end of an exposure-renewal interval (24, 48, 72 and 96 hours) were between < LOQ and 156% of the nominal values.All effect values given were based on the nominal loading of the test item and the overall mean measured concentration of the test item.
All validity criteria of the OECD 203 (2019) test guideline were met. The effect levels (LL0 / LL100) given in the table below are based on the nominal concentrations and the overall arithmetic mean measured concentrations of the test item.
LL-Values (96 hours) of the Test item
based on the nominal test item loading rates and the overall arithmetic mean measured concentration [µg/L]Based on nominal test item loading rates [µg/L] Based on overall arithmetic mean measured test item concentrations [µg/L] Test duration [h] LL50 Confidence interval LC50 Confidence interval 24 764 (515 – 1134) 988 (651 – 1498) 48 348 (344 – 351) 356 (345 – 368) 72 248 (234 – 515) 208 (191 – 651) 96 241 (234 – 515) 199 (191 – 651) LL100 = 515 651 Lowest test item loading with 100 % mortality after 96 h LL0 = 106 72.1 Highest test item loading with 0 % mortality after 96 h - Endpoint:
- short-term toxicity to fish
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18-12-2009 - 24-4-2009
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study, well conducted. No major restrictions.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 203 (Fish, Acute Toxicity Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Analytical monitoring:
- yes
- Details on sampling:
- Storage of samples: samples were analysed immediately after sampling without storage.
Samples treatment: samples were diluted in methanol. - Vehicle:
- no
- Details on test solutions:
- A stock solution 5 mg/L CECAJEL 210 at was prepared on the day when the test was started and then on each day the solutions were renewed, Ie. after 24, 48 and 72 hours. Solutions were prepared by mixing 10 mg of the test item in 2 litres of dilution water and diluted in order to achieve the
desired range of concentrations.
The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, colourless \
over the period of the test. No precipitation was observed at the end of the test - Test organisms (species):
- Danio rerio (previous name: Brachydanio rerio)
- Details on test organisms:
- The organisms used for the test were Dania rerio (Teleostei, Cyprinidae). Batch n° 09/Br/02/1 was supplied by Elevage de la Grande Riviere (69490 Saint-Forgeux). The fish were placed under the test conditions (medium, identical or lower biological loading, temperature, lighting, etc.) 20 days before the beginning of the test. The fish selected for the test were all from the same batch and were of the same age (date of birth stated by the supplier: 20/08/08). Their size (determined by measuring 10 randomly sampled fish) was 3.2 ± 0.18 cm. The mortality in the aquarium containing
the batch of fish used for the tests was less than 5 % (observed mortality: 0 %) over the 7 days preceding the test. - Test type:
- semi-static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 96 h
- Test temperature:
- 22.4 ± 0.5 °C
- pH:
- 7.43-8.05
- Dissolved oxygen:
- Did not fall below 77 % saturation.
- Nominal and measured concentrations:
- 0.500, 0.370, 0.274, 0.203 and 0.151 mg/L (Nominal)
- Details on test conditions:
- The study was carried out in a thermostatically controlled room. The temperature, which was measured on a continuous basis in a test flask, was 22.4 ± 0.5 °C. The room was equipped with a programmed lighting system which produced a controlled lighting sequence: intensity between 400 and 800 lux at the surface of the aquaria; photo-period of 12 hours of darkness per 24 h. Physico-chemical parameters were measured using a METTLER TOLEDO 345 pH-meter for measurement of pH and with a WTW OXI 538 oxymeter for dissolved oxygen measurement. The study was performed using aquariums with a nominal capacity of 5 Iitres, covered with Altuglas®cover plates drilled with holes to allow the test solutions to be aerated. Volume of test medium per aquarium was 2.5 L.
At the beginning of the test, appropriate volumes of the stock solution of CECAJEL 210 (nominal concentration 5 mg/L) were poured directly into the test aquarium, which were topped up with dilution water to obtain the above mentioned range of nominal concentrations (without pH-adjustment).
Number of fish :10 for each test concentration and for the control (2 replicates). The definitive test was carried out under semi-static conditions.
Five fish were added in each test aquarium which were covered and incubated. Two aquaria were prepared for each concentration. The fish were examined after 1 hour and at intervals not exceeding 24 hours. They were considered to be dead if no reaction was obtained when their caudal peduncle was stimulated and if no respiratory movement was observed. The visible anomalies were noted,
as were any sub-lethal effects such as loss of balance, pigmentation, changes in swimming behaviour, respiratory distress, etc.
Solutions were renewed after 24, 48 and 72 hours and physical parameters were measured daily. - Reference substance (positive control):
- yes
- Remarks:
- Conducted on every fish batch
- Duration:
- 24 h
- Dose descriptor:
- LC50
- Effect conc.:
- > 0.29 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Duration:
- 48 h
- Dose descriptor:
- LC50
- Effect conc.:
- 0.25 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Remarks on result:
- other: 95 %(0.24-0.28)
- Duration:
- 72 h
- Dose descriptor:
- LC50
- Effect conc.:
- 0.14 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Remarks on result:
- other: 95% (0.11-0.18)
- Duration:
- 96 h
- Dose descriptor:
- LC50
- Effect conc.:
- 0.1 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Remarks on result:
- other: ND
- Details on results:
- The test chemical displays significant toxicity to fish.
- Results with reference substance (positive control):
- The sensitivity of the test organisms was checked at least once for each new batch of fish, by determining the LC50-24h of the potassium dichromate. The results of the last test, carried out on 12/03/09, indicated a LC50-24h of 250 mglL. For reference, the former AFNOR standard NF T 90-303 states that the LC5Q-24h of thepotassium dichromate with respect to Dania rerio must be between 200 and 400 mglL. The fish batch was considered acceptable for testing.
- Reported statistics and error estimates:
- The mortality data are analyzed using an Excel sheet program. It was designed to calculate the LC50 value and the 95% confidence interval. Probit
analysis is generally used to calculate the 24-, 48-, 72 and 96-hour LC50 values. - Sublethal observations / clinical signs:
Percentage Mortality
Nominal mg/L Mean Measured mg/L 24 48 72 96 0.50 0.29 0 70 100 100 0.37 0.22 0 30 90 100 0.27 0.14 0 0 30 60 0.20 0.09 0 0 10 20 0.15 0.05 0 0 0 10 Control - 0 0 0 0 - Validity criteria fulfilled:
- yes
- Conclusions:
- Test well conducted with in general with validated analytical method ,GLP accreditation and analytical certificate. CAS number is contradictory will need clarifying. May be a toxicity overestmation for risk assesment purposes. Suitable for classification and labeling. Some minor discrepancies. Can be considered reliable without significant restrictions.
This study is considered to be reliable without restriction now the identity of the test substance has been clarified. - Executive summary:
The acute toxicity (assessed by lethality) of the test item CECAJEL 210 to Danio reria (freshwater fish) for a duration of 96 hours under semi-static conditions was assessed according to the OECO Guideline 203.
Fish were exposed to a range of concentrations of CECAJEL 210 dissolved in dilution water.
The effective concentrations killing 50% of fish at 24h, 48h, 72h, and 96h (LC50-24h, 48h, 72h and 96h) were calculated by regression analysis using the Probit/log model. The results are presented in the following table:Duration of the exposure period LC50 value, mg/L (95% CI) 24h >29 48h 0.25 (0.24 - 0.28 72h 0.14 (0.11 - 0.18 96h 0.10 (ND) CI: confidence Interval; ND: not determined
The
Referenceopen allclose all
Description of key information
There are many acute toxicity to fish studies available for the primary fatty amine ethoxylates (2EO) from which most are considered supporting studies mainly because they lack quantification of the exposure concentrations or sufficient identification of the test substance used. The available short-term toxicity test results indicate that fish are generally less sensitive to the primary fatty amine ethoxylates when compared to algae and short-term daphnia. The available acute fish data for the primary fatty amine ethoxylates which covers an alkyl chain length range from C12 to C18 reveals only a slight increase of the toxicity in relation to an increasing alkyl chain length.
Two studies however are considered adequate to address the acute fish test endpoint. Both were performed semi-statically with daily renewal of the test solutions and with quantification of the exposure concentrations. One study where the test solutions were prepared via dilution of a stock solution is performed with PFAEO-O (CAS no.: 25307-17-9) and is due to the availability of a test with the test substance, Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs. (CAS no.: 71786-60-2; Old name: Bis(2-hydroxyethyl) coco amine; abbreviation: PFAEO-C) considered supporting to the key study performed with the test substance.
The acute toxicity of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (PFAEO-C; CAS no.: 71786-60-2) to fish (zebrafish) was determined according to OECD TG 203 (2019) under GLP conditions.
The test item PFAEO-C is poorly soluble but dispersible in water. It is a UVCB substance with constituents of different water solubility. Water accommodated fractions (WAFs) were therefore prepared 24 ± 1 hour prior to the start of exposure with the nominal loading rates of the test item of 10.0 – 22.0 – 48.4 – 106 – 234 – 515 – 1134 μg/L, corresponding to the overall arithmetic mean measured concentrations of 5.37 – 11.3 – 32.2 – 72.1 – 191 – 651 - 1498 μg/L. For the preparation of the loading rate the test item was dissolved in methanol applied onto curved glass slides. The methanol was evaporated. The glass slides with the test item were inserted in glass flasks with an appropriate amount of dilution water. A slow stirring procedure was applied for 24 ± 1 hour at room temperature. The magnetic stirrer bar was placed with a fish-clip® system a few centimeters above the bottom of the flask to prevent direct contact with the test item on the bottom. After a separation phase of 1 hour, the aqueous phase of the WAF was removed by siphoning (from the approximate middle of the glass flask). The WAFs were checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative. The resulting water accommodated fractions (WAF) were used in the test. The test vessels were pre-treated with the appropriate test solution for at least 12 hours under test conditions.
The selection of the test loadings was based on the results of preliminary range finding tests and Early-Life Stage Toxicity Test (Noack Study Code SO21177/FSZ19203).
In the first definitive study no EC50-values could be determined. A second definitive study was carried out with only two additional test loadings (control, 515 & 1134 μg/L), which prolonged the concentration series of the first definitive study.
The studies were performed over a period of 96 hours under semi-static conditions with daily renewal of the test media. Seven test organisms were exposed to the test loadings and the controls. No significant mortality but sub-lethal effects were observed in the first definitive test.
100% mortality was observed in the test loadings 515 and 1134 μg/L. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits.
The measured concentrations of all compounds of the test item in the fresh media at the start of an exposure-renewal interval (0, 24, 48 and 72 hours) were between < LOQ to 169% of the nominal values.
The measured concentration of the test item in the old media at the end of an exposure-renewal interval (24, 48, 72 and 96 hours) were between < LOQ and 156% of the nominal values.
on the nominal loading of the test item and the overall mean measured concentration of the test item.
All validity criteria of the OECD 203 (2019) test guideline were met. The effect levels (LL0 / LL100) given in Table 1 are based on the nominal concentrations and the overall arithmetic mean measured concentrations of the test item.
All effect values given are based on the nominal test item loading rates (LLxx) and the overall arithmetic mean measured concentration (LCxx).
The 96h LL50/LC50 values calculated from the effects observed are: 241 and 199 µg/L
LL50 | Confidence interval | LC50 | Confidence interval | |
24 | 764 | (515 – 1134) | 988 | (651 – 1498) |
48 | 348 | (344 – 351) | 356 | (345 – 368) |
72 | 248 | (234 – 515) | 208 | (191 – 651) |
96 | 241 | (234 – 515) | 199 | (191 – 651) |
LL100 =Lowest test item loading with 100 % mortality after 96 h | 515 | 651 | ||
LL0 =Highest test item loading with 0 % mortality after 96 h | 106 | 72.1 |
Key value for chemical safety assessment
Fresh water fish
Fresh water fish
- Dose descriptor:
- LC50
- Remarks:
- Test performed according to the WAF approach. LL50 = 0.241 mg/L
- Effect concentration:
- 0.241 mg/L
Additional information
- OECD (2002). Guidance Document on the Use of the Harmonised System for the Classification of Chemicals which are Hazardous for the Aquatic Environment.
- Wheeler, J. R., D. Lyon, C. Di Paolo, A. Grosso and M. Crane (2020). "Challenges in the regulatory use of water-accommodated fractions for assessing complex substances." Environmental Sciences Europe 32(1): 1-10.
- OECD (2019): Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals. OECD series on testing and assessment no. 23 (second edition), ENV/JM/MONO(2000)6/REV1
Most of the short-term toxicity fish tests were conducted in a period when no reliable specific method of analyses was available. The concentrations were therefore not analytically verified, the reliability of the results is limited because of the poor solubility of the test substances and partial sorption onto the walls of test vessels. Hence, most of the tests were considered to be valid with restrictions. The available data reveal a comparable toxicity independent of the chain length.
Two studies are however considered adequate to address the acute fish test endpoint. Both were performed semi-statically with daily renewal of the test solutions and with quantification of the exposure concentrations. One study where the test solutions were prepared via dilution of a stock solution is performed with PFAEO-O (CAS no.: 25307-17-9) and is due to the availability of a test with the test substance, Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs. (CAS no.: 71786-60-2; PFAEO-C) considered supporting to the key study which was performed with the test substance. All effect values for this study are given based on the nominal test item loading rates (LLxx) and the overall arithmetic mean measured concentration (LCxx). The 96h LL50/LC50 values calculated from the effects observed are: 241 and 199 µg/L.
The TWA results are given despite the fact that per definition of the WAF, all terms related to concentration level should be given as loading rates (mass-to-volume ratio of the substance to the medium) because partly dissolved compounds and mixtures cannot be related to concentrations. Analytical verifications of selected components can be helpful and deliver supporting information, but they do not represent the whole test substance and therefore, toxicity results will be evaluated based on WAF loading rate (Wheeler, Lyon et al. 2020). Several guidance documents suggest to use the WAF loading rate for the environmental hazard classification of chemical substances e.g. the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (OECD 2002, OECD 2019) as well as OECD guidance documents on the classification of chemicals which are Hazardous for the Aquatic Environment.
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