Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 695-977-9 | CAS number: 1309955-79-0
- 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
Long-term toxicity to fish
Administrative data
Link to relevant study record(s)
- Endpoint:
- fish early-life stage toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- from 2022-01-13 to 2022-02-16
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
- Version / remarks:
- (2013)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- Test item Tallow amine propoxylate (CAS: 1309955-79-0)
Substance name
2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol
Batch number 20200979
CAS No. 1309955-79-0
Purity (certified) The substance is a 100% UVCB.
Density 890 kg/m3 at 20 °C
Appearance Clear to turbid liquid, amber, red-brown
Water Solubility Dispersible / insoluble
Melting point 30 – 40 °C
Stability under test concditions
Not specified
Expiry date 2023-11-20
Recommended storage
Store container tightly closed in a dry, well-ventilated place. Avoid elevated temperatures. - Analytical monitoring:
- yes
- Remarks:
- with an LC-MS/MS method
- Details on sampling:
- Determination of the test item
The samples were analyzed under GLP with an LC-MS/MS method which was implemented under non-GLP and documented finally in the GLP raw data. The method was validated.
The peak distribution (fingerprint) of the highest stock solution and highest test concentration was analyzed, once during the test. The signal distribution was compared with an analytical standard prepared in solvent.
Sampling procedure and pre-treatment
On each time of sampling one alternating replicate of the test vessels (i.e. the outlet of the test vessel) was sampled. The samples taken from the test vessels were completely clear and did not contain any particles or turbidity. On each sampling point the following constituents of the test item N-Hexadecyldiisopropanolamine, Octadecenyldiisopropanolamine and Octadecyldiisopropanolamine was analytically determined to distinguish how much test item is truly dissolved.
Organic solvent was added to the samples to stabilize the samples and limit the sorption to the sample vials.
Sampling schedule
Samples of test media including control group and solvent control group were taken from alternating test replicates (the outlet of the test vessel of these replicates) on days -1, 0 and weekly thereafter until end of exposure. The changing intervals of the stock solution were taken into account.
The highest concentrated stock solution was sampled and analyzed from one used interval during the definitive test. - Vehicle:
- yes
- Remarks:
- With regard to the limited solubility of the test item in water, methanol (VWR, 100% HPLC gradient grade, batch 21K184012) was used as a solvent. The solvent concentration was the same in all concentration levels and the solvent control (0.025 mL/L).
- Details on test solutions:
- Test concentrations
5.00 - 10.0 - 20.0 - 40.0 - 80.0 µg test item/L (factor: 2), corresponding to overall arithmetic mean measured test item concentrations of 4.25 – 6.93 – 16.5 – 36.1 – 76.4 µg test item/L.
The test concentrations were based on the results of a preliminary range finding test (non-GLP) conducted under flow-through conditions over 15 days. For results of the range finding test, see Annex II.
The test concentrations were prepared by adding the dissolved test item to dilution water as described above.
Solvent
With regard to the limited solubility of the test item in water, methanol (VWR, 100% HPLC gradient grade, batch 21K184012) was used as a solvent. The solvent concentration was the same in all concentration levels and the solvent control (0.025 mL/L).
Stock solution
A stock solution of 3200 mg/L was prepared in methanol. The test item was melted at 50°C for ca. 3 hours in an incubator until the test item was clearly dissolved. An appropriate amount of the test item was weighed out and transferred with the solvent into a glass flask. The solution was agitated until it was visually clear dissolved. Typically, this covers a time period of 20 to 120 seconds.
Further stock solutions were prepared by dilution with methanol.
The stock solutions were prepared in appropriate intervals of 7 days.
Syringes were filled with the freshly prepared stock solutions or pure methanol for the solvent control in corresponding intervals. - 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 recommended for this type of study.
Origin
All fish used in the test were reared at Noack Laboratorien GmbH from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin, Germany)
Maintenance of brood fish
A breeding stock of unexposed, mature zebrafish with an age of approx. 9-11 months was used for the egg production. Fish were free of macroscopically discernable symptoms of infection and disease. Spawners were maintained in aquaria with a loading capacity of a minimum of 1 L water per fish.
- Temperature: 25 ± 2 °C
- Dissolved oxygen concentration > 60% of air saturation value
- pH value: 6 – 8.5
- Photoperiod: 16 h light / 8 h dark cycle
(2 transition periods, 30 minutes each)
- Diffuse light (7 – 750 lux on water surface)
- Food: Artemia salina nauplii, 48 hours old, ad libitum;
Daphnia magna, juvenile and adult daphnids, ad libitum;
dry food sera vipan SERA, ad libitum.
- No disease treatments were administered.
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 of an acceptable dilution water for holding and testing:
Total hardness: 10 – 250 mg CaCO3/L
pH-value: 6.0 – 8.5
Alkalinity: 0.7 mmol/L (recent measurement: 2021-11-09)
Acidity: 0.1 mmol/L (recent measurement: 2021-11-09)
Conductivity: 171 µS/cm (recent measurement: 2021-11-09)
Spawning
15 – 35 adult zebrafish were kept in at least 3 separate aquaria. The fish were healthy with a mortality rate < 5% during the last 7 days and thus not medically treated for at least 7 days. About 15 minutes before start of artificial dawning rectangular dishes covered with a stainless-steel mesh and provided with artificial plants (plastic), were introduced into the aquaria. After 1.5 hours the glass dishes were gently removed. Eggs were checked carefully for abnormalities like fungus infections. These eggs as well as coagulated and not fertilized eggs were discarded (less than 30%). About 900 eggs were taken and washed in dilution water. Eggs originated from 4 different spawnings.
Start of exposure
The eggs that were used to start the exposure were pooled and attributed randomly (eggs were placed in alternating groups into each of the test groups) to the test groups in crystallization dishes containing test solutions (two dishes per test group, each dish loaded with at least 60 eggs, resulting in a total of about 120 eggs per test concentration).
Fertilization check
Immediately after exposing the eggs to the test solutions (start of exposure), the eggs were checked for fertilization. Eggs were fully covered with the respective test solutions. Every embryo was checked under a stereo microscope for its stage. Cleavages which form 4, 8, 16 and 32 cell blastomers can be clearly identified by the development of the blastula and were regarded to be fertilized. Eggs with only a 2 cell stage were regarded as not fertilized and discarded.
Fertilization rate
The mean fertilization rate was 88%.
Introduction of eggs
The eggs were placed in the middle of the water phase of the test vessels directly after the fertilization check at a stage before cleavage of the blastodisc commences or as close as possible to this stage. The eggs were transferred randomly into test vessels containing the respective exposure solutions. The distribution of eggs to the concentration groups was carried out indiscriminately by adding 5 eggs to the first test group, the 2nd 5 eggs to the next test group and so on, until all test groups contained the necessary number of eggs. Distribution started with the control group, followed by the solvent control group and from the lowest to the highest test concentration. - Test type:
- flow-through
- Water media type:
- freshwater
- Remarks:
- Tap water was used for testing. The water was filtered on activated charcoal to remove residual chlorine.
- Limit test:
- no
- Total exposure duration:
- 34 d
- Remarks on exposure duration:
- On study day 4, 95% of the control and 96% the solvent control larvae had hatched. Therefore, study day 4 was defined as post hatch day 0 (= PHD 0).
- Hardness:
- see section "Any other information on results incl. tables" below
- Test temperature:
- see section "Any other information on results incl. tables" below
During the exposure the water temperature was recorded continuously (once per hour) with a
data logger. The mean temperature was 26.3 °C (Table 21). The minimum temperature was
25.7 °C and the maximum temperature was 26.8 °C. - pH:
- see section "Any other information on results incl. tables" below
- Dissolved oxygen:
- see section "Any other information on results incl. tables" below
- Nominal and measured concentrations:
- 5.00 - 10.0 - 20.0 - 40.0 - 80.0 µg test item/L (factor: 2), corresponding to overall arithmetric mean measured test item concentrations of 4.25 – 6.93 – 16.5 – 36.1 – 76.4 µg test item/L.
The test concentrations were based on the results of a preliminary range finding test (non-GLP) conducted under flow-through conditions over 15 days. - Details on test conditions:
- Test design
A randomized block design with each treatment being present in each block was established. A flow-through exposure design was carried out. Membrane piston pumps provided the water flow-through via silicone tubing.
Precision syringe pumps were used for the introduction of stock solution. The stock solution and the dilution water were mixed in a mixing chamber (approx. volume 0.7 L, one mixing chamber per test replicate) by magnetic stirring at a sufficient speed to ensure a small vortex depth of about 10 % of the dispersion height in the mixing chamber before passing the test aquaria (approx. volume 7.5 L; four replicates per test concentration, control and solvent control) where the eggs/fish were exposed.
The accuracy of the water flow-through was checked prior to start of the exposure and three times per week thereafter.
Water exchange in the test aquaria was about 10 times per day (3.125 L/h).
An equilibration period of at least 8 days was carried out prior to start of the exposure. The measured concentrations were analysed four times during this period until no trend of increasing or decreasing was shown.
Equilibration period
Test solutions flowed through the test vessels for 8 days prior to the start of the exposure. The measured concentrations were analysed four times during this period until no trend of increasing or decreasing was shown.
Control Dilution water (without test item and without solvent)
Solvent control
Additionally, a solvent control with the same concentration of solvent but without test item was prepared and tested under the same conditions as the test groups.
Reference item
No reference item is recommended for this test according to the guideline.
Test duration
34 days (30 days post hatch), depending on post-hatch day 0 (study day 4).
Replicates, number of eggs
Four replicates per test concentration and control, with 20 eggs each (80 eggs per test concentration, solvent control and control) were tested.
For the whole study (including the range finding test and definitive tests) 680 healthy eggs/fish were used.
Loading
A loading rate not exceeding 0.5 g/L wet weight fish per 24 hours and not exceeding 5 g/L of solution at any time was maintained.
Test vessels
Glass aquaria of 8.7 L provided with mesh coated fittings allowing flow-through of test media (dimensions: 22/22/18 cm) were used. Test vessels were covered by glass lids. The volume of the test media was approximately 7.5 L.
Cleaning
The test vessels were siphoned as needed to remove excess fecal material and uneaten food, also to minimize microbial growth and biodegradation of the test item. Furthermore, the mesh coated fittings were cleaned once per day. Cleaning started on study day 1.
Aeration
The dilution water supply tank was aerated.
No additional aeration of the test vessels was provided.
Dilution water
Tap water was used for testing. The water was filtered on activated charcoal to remove residual chlorine.
Feeding of test fish
The feeding regime was ad libitum during the whole feeding period (study day 5 to 33).
Feeding started 3 days after the beginning of hatch on study day 5 (post-hatch day 1, where almost all non-affected larvae swum up). Larvae were fed with a starter food (ST-1 (AQUA SCHWARZ GMBH, 37081 Göttingen, Germany), as well as a suspension of the starter food ST-1 and fine milled brine shrimp nauplii (2 – 8 times daily). 1 day after start of feeding brine shrimp nauplii (24 - 48 h old) were fed until the end of the test (2 – 7 times daily).
Brine shrimp nauplii origin, breeding conditions:
Artemia salina (Brine shrimp eggs) were purchased from Kessler Zoologiegroßhandel GmbH & Co. KG, D 67122 Altrip, Germany. Fresh cultures were prepared with salt water (NaCl 20 g/L, ca. 2 g eggs to 1 L salt water, gentle aeration for 24 - 48 hours at approx. 22 °C). 24 - 48 h old brine shrimp nauplii were harvested, washed in a stainless-steel mesh and resuspended in tap water.
Feeding ad libitum was carried out.
Water temperature (target) 26 ± 1.5 °C
Dissolved oxygen
Not less than 60% of air saturation value concentration (target)
Light intensity (target) 300 ± 150 Lux
Photoperiod
A daily 16 / 8 h photoperiod (light / dark) was maintained throughout exposure.
Type and Frequency of Measurements
Biological Parameters
All biological parameters were observed daily. Dead larvae/fish and coagulated or dead eggs were removed daily, if observed as described below.
Hatching
The number of hatched larvae was determined daily until study day 5. All embryos hatched were counted as hatched, even if they had died directly afterwards. Eggs were only removed when mortality of eggs/embryos was observed as specified below.
On study day 4, 95% of the control and 96% of the solvent control larvae had hatched. Therefore, study day 4 was defined as post-hatch day 0 (= PHD 0). For evaluation of hatch, all hatched larvae (even dead ones) were counted.
The cumulative number of hatched larvae up to study day 6 was used for evaluation.
Mortality Criteria for mortality vary according to life stage:
For eggs/embryos: If fungus growth on eggs was observed, these eggs were removed and counted. Mortality as discerned by a distinct change in coloration or a marked loss of translucency and change in coloration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance and change in coloration was checked daily. Mortality caused by absence of heartbeat was checked, if applicable. Dead eggs/embryos were discarded.
For larvae and juvenile fish: Immobility and/or lack of reaction to mechanical stimulus. Dead larvae or juvenile fish were discarded.
Further effects
Abnormal appearance and behavior were also recorded daily.
Abnormalities, e.g. quiescence, hyperventilation, uncoordinated swimming, swim-up behavior, atypical quiescence and atypical feeding behavior were recorded by visually inspecting each replicate.
Measurement of fish size
At the end of exposure (post-hatch day 30) the fish were euthanized in a Benzocaine solution and the individual total length of all survivors was measured to the nearest 0.5 mm with millimeter paper. The total length (from the tip of the snout to the tip of the longer lobe of the caudal fin) was measured.
Measurement of fish wet weight
At the end of exposure (post-hatch day 30) all surviving fish were weighed on replicate basis to the nearest 0.1 mg. Fish were blotted on paper towels to remove excess moisture prior to weighing. The mean wet weight per animal was calculated from the number of surviving fish.
Physical Properties
Water quality measurements were carried out during exposure in the following intervals:
Once per hour
Temperature in the dilution water, measured in one control vessel
At least 3 times per week Determination of
- Dissolved oxygen in all replicates of each test group
- Check of flow rates of the test media (variation < 10% throughout exposure)
Weekly
- pH-value and temperature in all replicates of each test group
- TOC and Chlorine from dilution water
- Total hardness in one replicate of control, solvent control and the remaining highest test item concentration
The light intensity on the surface of the test aquaria was measured at the start of the exposure. - Reference substance (positive control):
- no
- Remarks:
- No reference item is recommended for this test according to the guideline.
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LC50
- Effect conc.:
- 16.3 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Overall survival
- Duration:
- 34 d
- Dose descriptor:
- LC50
- Effect conc.:
- 19.5 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Overall survival
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LC50
- Effect conc.:
- 16.5 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Post hatch survival
- Duration:
- 34 d
- Dose descriptor:
- LC50
- Effect conc.:
- 19.7 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Post hatch survival
- Duration:
- 34 d
- Dose descriptor:
- other: LC20
- Effect conc.:
- < 4.25 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Overall survival
- Remarks on result:
- other: determined value lower than the lowest test concentration
- Duration:
- 34 d
- Dose descriptor:
- other: LC20
- Effect conc.:
- < 5 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Overall survival
- Remarks on result:
- other: determined value lower than the lowest test item concentration
- Duration:
- 34 d
- Dose descriptor:
- other: LC20
- Effect conc.:
- < 4.25 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Post hatch survival
- Remarks on result:
- other: determined value lower than the lowest test item concentration
- Duration:
- 34 d
- Dose descriptor:
- other: LC20
- Effect conc.:
- < 5 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Post hatch survival
- Remarks on result:
- other: determined value lower than the lowest test item concentration
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LC10
- Effect conc.:
- < 4.25 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Overall survival
- Remarks on result:
- other: determined value lower than the lowest test item concentration
- Duration:
- 34 d
- Dose descriptor:
- LC10
- Effect conc.:
- < 5 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Overall survival
- Remarks on result:
- other: determined value lower than the lowest test item concentration
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LC10
- Effect conc.:
- < 4.25 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Post hatch survival
- Remarks on result:
- other: determined value lower than the lowest test item concentration
- Duration:
- 34 d
- Dose descriptor:
- LC10
- Effect conc.:
- < 5 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Post hatch survival
- Remarks on result:
- other: determined value lower than the lowest test item concentration
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 6.93 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Overall survival
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 10 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Overall survival
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 6.93 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Post hatch survival
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 10 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Post hatch survival
- Key result
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 4.25 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Overall survival
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 5 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Overall survival
- Key result
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 4.25 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Post hatch survival
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 5 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality
- Remarks:
- Post hatch survival
- Duration:
- 34 d
- Dose descriptor:
- other: EC20
- Effect conc.:
- 20.3 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- weight
- Duration:
- 34 d
- Dose descriptor:
- other: EC20
- Effect conc.:
- 25 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- weight
- Duration:
- 34 d
- Dose descriptor:
- other: EC20
- Effect conc.:
- 38.4 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- length
- Duration:
- 34 d
- Dose descriptor:
- other: EC20
- Effect conc.:
- 43.2 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- length
- Key result
- Duration:
- 34 d
- Dose descriptor:
- EC10
- Effect conc.:
- 12.1 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- weight
- Duration:
- 34 d
- Dose descriptor:
- EC10
- Effect conc.:
- 16.4 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- weight
- Key result
- Duration:
- 34 d
- Dose descriptor:
- EC10
- Effect conc.:
- 15.5 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- length
- Duration:
- 34 d
- Dose descriptor:
- EC10
- Effect conc.:
- 20.2 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- length
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 16.5 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- weight
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 20 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- weight
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 16.5 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- length
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 20 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- length
- Key result
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 6.93 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- weight
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 10 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- weight
- Key result
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 6.93 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- length
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 10 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- length
- Key result
- Duration:
- 5 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 76.4 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- time to hatch
- Duration:
- 5 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 80 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- time to hatch
- Key result
- Duration:
- 5 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 36.1 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- time to hatch
- Duration:
- 5 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 40 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- time to hatch
- Details on results:
- Results
Biological Data
Egg Fertilization Rate
The mean egg fertilization rate determined on study day 0 (start of the exposure) was 88%. Eggs were fully covered with the respective test solutions during fertilization check.
Hatch and Definition of Post Hatch Day 0
Hatch began on study day 2 in the control group and the test concentrations of 5.00, 10.0 and 80.0 µg/L. The hatch of larvae in other test concentrations and the solvent control started on study day 3. The hatch of larvae was completed until study day 5. Study day 4 was determined to be post hatch day 0 (PHD 0) with a hatching rate of 95% in the control and 96% in the solvent control.
Statistical procedures were applied for the total number of test organisms that have hatched on study days 4 and 5.
The Williams Multiple sequential t-test procedure for hatch data after 4 and 5 days was done with a significance level of 0.05. No statistically significant differences were found between the pooled controls and the nominal test concentrations of 5.00 to 80.0 µg/L for day 4. For day 5 a statistically significant difference was found between the pooled controls and the nominal test item concentrations of 80.0 µg/L.
For hatchability on day 4, the NOEC and the LOEC (based on nominal test item concentrations) for this endpoint were determined to be ≥ 80.0 and > 80.0 µg/L, respectively. The corresponding NOEC and LOEC (based on overall arithmetic mean measured dissolved test item concentrations) were ≥ 76.4 and > 76.4 µg/L, respectively. For hatchability on day 5, the NOEC and the LOEC (based on nominal test item concentrations) for this endpoint were determined to be 40.0 and 80.0 µg/L, respectively. The corresponding NOEC and LOEC (based on overall arithmetic mean measured test item concentrations) were 36.1 and 76.4 µg/L, respectively.
Swim-up
The swim-up period of the control groups and the nominal test concentrations of 5.00 to 80.0 µg/L was observed from study day 5 to 6. First swim-up of larvae was observed on study day 5 in all test groups. From study day 7 on, swim-up was reduced in the highest test concentration of 80 µg/L due to non-lethal effects or mortality. No statistical analysis of swim-up data was carried out.
Fry Survival (Post-Hatch Survival)
The post-hatch survival in the control replicates met the validity criteria of the guideline (required: ≥ 75%). The fry survival (post-hatch survival) at the end of the study was 80% in the control and 87% in the solvent control, thus fully meeting the validity criteria of the guideline. A concentration-related decrease of the post-hatch survival was detected with increasing test concentrations.
The Williams Multiple sequential t-test procedure (alpha = 0.05) was performed for statistical analysis of post-hatch survival data on study day 34 (PHD 30). Statistically significant differences were found between the pooled control groups and the nominal test item concentrations of 10.0 to 80.0 µg/L.
The NOEC and the LOEC for this endpoint were 5.00 µg/L and 10.0 µg/L (nominal concentrations), respectively. The corresponding NOEC and LOEC (based on overall arithmetic mean measured test item concentrations) were 4.25 µg/L and 6.93 µg/L, respectively.
The LC50-value (based on nominal concentrations) for post-hatch survival on study day 34 (PHD30) was 19.7 (15.6 to 24.9) µg/L, corresponding to 16.5 (12.7 to 22.4) µg/L (based on overall arithmetic mean measured dissolved test item concentrations).
Overall Survival
The cumulative mortality at the end of the exposure, related to the number of eggs introduced on day 0, was 22% per control and 14% per solvent control. A concentration-related decrease of the overall survival (increase of overall mortality) was detected in the test item concentrations.
The William’s multiple sequential t-test procedure (alpha = 0.05) was performed for statistical analysis of overall survival data on study day 34 (PHD 30). Statistically significant differences were found between the pooled control groups and the nominal test item concentrations of 10.0 to 80.0 µg/L.
The NOEC and the LOEC for this endpoint were 5.00 µg/L and 10.0 µg/L (nominal concentrations), respectively. The corresponding NOEC and LOEC (based on overall arithmetic mean measured dissolved test item concentrations) were 4.25 µg/L and 6.93 µg/L, respectively.
The LC50-value (based on nominal concentrations) for overall survival on study day 34 (PHD 30) was 19.5 (15.5 to 24.2) µg/L, corresponding to 16.3 (12.6 to 21.7) µg/L (based on overall arithmetic mean measured dissolved test item concentrations).
Morphological and Behavioral Effects
Observation of abnormal appearance and behavior of hatched larvae were carried out daily until the end of the exposure. As an isolated event a slowed escape reflex and no uptake of food of one larvae of the control group was observed on study days 24 and 25. No further morphological nor behavioral effects were observed in the control and the solvent control group throughout exposure. In the nominal test concentrations of 5.00 and 10.0 µg/L occasional non-lethal effects were observed. The nominal test concentrations of 20.0 and 40.0 µg/L showed an increase of observed non-lethal effects with ongoing exposure from study day 19 up to study day 28 and from study day 7 up to study day 31. The highest nominal test concentration of 80.0 µg/L showed non-lethal effects for a short period after hatch from study day 7 to 10 resulting in 100 % mortality.
Fry Growth
Fry growth, expressed as length and wet weight measurements, was measured on study day 34 (PHD 30) from all survivors.
The Williams Multiple sequential t-test procedure (alpha = 0.05) showed statistically significant differences for the surviving fish of the nominal test concentrations of 20.0 and 40.0 µg/L for the growth parameters fresh weight and mean total length.
Therefore, the NOEC and the LOEC (nominal test item concentrations) for the growth parameters fresh weight and total length were determined to be 10.0 and 20.0 µg/L, respectively. The corresponding NOEC and LOEC (based on overall arithmetic mean measured dissolved test item concentrations) were 6.93 and 16.5 µg/L, respectively.
Biomass Loading
The biomass-loading factor for the study was determined from the fresh weights of the control and solvent control fish at the end of the exposure.
The maximum biomass at the end of the exposure was determined in replicate 4 of the solvent control group: 507.3 mg total fish weight. The maximum biomass loading based on the 7.5 liter volume of a single growth chamber was 67.6 mg/L.
Maximum loading rate:
biomass/(volume of test solution) = (507.3 mg)/(7.5 L) =67.6 mg/L
The biomass loading rate based upon a flow of 75 liters per day through each single test aquaria was 6.76 mg per liter and day.
Maximum loading rate per day:
biomass/(volume of test solution per day) = (507.3 mg)/(75 L) = 6.76 mg/L per day
These loadings were well within the requirements to ensure adequate dissolved oxygen levels and to avoid crowding of the fish.
Physico-chemical Data
Dissolved Oxygen
The dissolved oxygen concentrations in the control, solvent control and the test item groups, expressed in percent saturation, were in the mean 95 – 98% and ranged from 80 to 100% during the exposure period
Water Temperature
During the exposure the water temperature was recorded continuously (once per hour) with a data logger. The mean temperature was 26.3 °C (Table 21). The minimum temperature was 25.7 °C and the maximum temperature was 26.8 °C.
The mean water temperature measured once per week from all replicates during the exposure period was 26.2 °C for the control. The minimum and maximum measured temperature for the control were 26.0 and 26.6 °C, respectively.
The validity criterion for the parameter temperature was fulfilled.
Total Hardness
Total hardness of the test media was measured once per week from alternating replicates of the control and the remaining highest test concentration of 80.0 µg/L (day 1 and 8), 40.0 µg/L (day 15, 22 and 29). The mean total hardness was 66 mg CaCO3/L and ranged from 63 to 68 mg CaCO3/L in the control group
Residual Chlorine
Residual chlorine, measured from the dilution water supply tank on study days 0, 7, 14, 21 and 28 was < 0.01 mg/L.
Total Organic Carbon (TOC) of the Dilution Water
The total organic carbon (TOC) sampled from the dilution water supply tank was determined on study days 0, 7, 15, 22 and 28. The mean measured TOC was 1.24 mgC/L throughout exposure (see Table 25). All measured concentrations were below the LOQ of 2 mgC/L.
Flow Rates
The mean flow rate through the mixing chambers of all test item and control groups was 3.07 ± 0.069 L/h and the individual values ranged from 2.82 to 3.36 L/h.
A precision syringe pump was used for introduction of the stock solution to the mixing chambers. At any renewal of the syringes the proper function of the pump and the applied volume was checked by the syringe volume indicator.
Light intensity
Light intensity was measured at the start of exposure on the surface of all test vessels and ranged from 170 to 315 lux (mean: 226 lux).
Specific Analysis
The concentrations of three components (N-Hexadecyldiisopropanolamine, Octadecenyldiiso-propanolamine, Octadecyldiisopropanolamine) had been measured in this study in the equilibrium phase, from the stock solutions and during the definitive test. Arithmetic mean values for all analytes were calculated.
Measured Test Concentrations during Equilibration Phase
The concentrations of the three constituents N Hexadecyldiisopropanolamine, Octadecenyldiiso-propanolamine and Octadecyldiisopropanolamine of the test item Tallow amine propoxylate (CAS: 1309955-79-0), the solvent control and the control were determined in the equilibration phase on day -1 from the test vessels of the test replicates 1 of all tested concentration levels via LC MS/MS.
To evaluate how much test item is lost due to the centrifugation step, the concentration in samples taken from the test vessels of day -1 was quantified with and without centrifugation step, before the samples were subjected to the analytical procedure as described.
The samples analysed without centrifugation showed a homogeneous distribution and no turbidity. Although the recoveries were below the nominal concentration, no precipitate could be observed. Therefore, these samples were used for the calculation of the overall mean measured dissolved concentrations. The lower recoveries of the centrifuged samples are explained by losses due to adsorption to the centrifuge tubes and are therefore not further used. This effect was also observed with similar substances in aqueous matrix.
Measured Concentrations in the Stock Solutions
Measured concentrations of the freshly prepared stock solution were 98 to 102% of the nominal values. Measured concentrations of the 7 days aged stock solution were in the range of 99 to 105% of the nominal values.
Measured Concentrations in the Test Vessels during Exposure
The concentrations of the three constituents N Hexadecyldiisopropanolamine, Octadecenyldiiso-propanolamine and Octadecyldiisopropanolamine of the test item Tallow amine propoxylate (CAS: 1309955-79-0), the solvent control and the control were determined during exposure on days 0, 7, 14, 21 and 28 from the test vessels of a defined test replicate of all tested concentration levels via LC MS/MS.
The samples taken from the test vessels were completely clear and did not contain any particles or turbidity. These samples were subjected to the analytical procedure and the results are used for the calculation of the effect values.
Fingerprint of the Test Item
The peak distribution of the highest stock solution (3200 mg test item/L), highest test item concentration (80.0 µg test item/L) and the control were analyzed. A solution of the analytical standard of the test item was prepared and diluted to 10 mg/L with acetonitrile : ultra-pure water (50 : 50). The stock solution was diluted factor 3200 with acetonitrile : ultra-pure water (50 : 50). The highest test item concentration and the control were diluted factor 2 with acetonitrile to avoid an inhomogeneous sample.
The detected signals of the analytical standards and the sample solutions were compared.
The most dominating signals in the analytical standard and stock solution spectra could be assigned to 3 constituents of the test item (see below). These constituents were used for the determination of the test item during the test phase. In the highest test item concentration, these signals could also be identified.
N Hexadecyldiisopropanolamine = 358 Da
Octadecenyldiisopropanolamine = 384 Da
Octadecyldiisopropanolamine = 386 Da
Preliminary Range Finding Test (non-GLP)
A non-GLP preliminary range finding test was conducted at the test facility as a shortened early -
life stage test over a period of 15 days under flow-through conditions as described. An
equilibration period over 27 days was carried out prior to the start of the exposure. Exposure was
started by placing fertilized eggs into the test replicates. A solvent control and two test
concentrations of the test item of 10.0 and 100 μg/L were included in the test. Two replicates per
solvent control and test concentration were tested.
The test item concentrations were analytically verified via LC-MS/MS on days -8, -3, 6 and 13.
Sampling on study day -8 was done from the test vessels, the mixing chamber supply and the
outlet of the test vessels. - Reported statistics and error estimates:
- Evaluation
All calculations are based on the nominal test item concentrations of 5.00 – 10.0 – 20.0 – 40.0 – 80.0 µg/L, corresponding to the overall arithmetric mean measured test item concentrations of 4.25 – 6.93 – 16.5 – 36.1 – 76.4 µg/L.
Replicate means were used for statistical analysis since each test vessel is the experimental unit based on the design of the test. For each parameter analyzed (hatch rate, post hatch survival, overall survival and fry growth (expressed as mean total length and fresh weight measured on PHD 30), the statistical tests given below were conducted.
Since a solvent was used, both control groups were included for statistical analysis. The two controls were compared by Fisher’s Exact Binomial Test (significance level 0.05, for the parameters hatching success, post hatch survival and overall survival) and Student-t Test (significance level 0.05, for the parameter fry growth (expressed as length and fresh weight)).
No statistically significant difference was detected between the dilution water control and the solvent control for any parameter. Therefore, the controls were pooled for statistical analysis.
The hatching rate (=hatching success) is defined as the total number of test organisms that have hatched in a replicate divided by the number of eggs introduced into the replicate at start of exposure.
The number of hatched larvae was determined daily until study day 6, when the last hatching occurred. However, not 100% of the larvae hatched successfully in all replicates.
Post-hatch survival is defined as the number of surviving fish in a replicate at the end of exposure divided by the number of test organisms in the replicate that have hatched. - Validity criteria fulfilled:
- yes
- Conclusions:
- Tallow amine propoxylate (CAS: 1309955-79-0) caused significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal concentrations of 5.00, 10.0, 20.0, 40.0 and 80.0 µg/L, corresponding to overall arithmetic mean measured dissolved test item concentrations of 4.25, 6.93, 16.5, 36.1, 76.4 µg/L.
For hatchability on day 4, the NOEC and the LOEC (based on nominal test item concentrations) for this endpoint were determined to be ≥ 80.0 and > 80.0 µg/L, respectively. The corresponding NOEC and LOEC (based on overall arithmetric mean measured dissolved test item concentrations) were ≥ 76.4 and > 76.4 µg/L, respectively. For hatchability on day 5, the NOEC and the LOEC (based on nominal test item concentrations) for this endpoint were determined to be 40.0 and 80.0 µg/L, respectively. The corresponding NOEC and LOEC (based on overall arithmetic mean measured dissolved test item concentrations) were 36.1 and 76.4 µg/L, respectively.
Therefore no EC10 value could be calculated for this parameter.
For both parameters post hatch survival and overall survival, the nominal NOECs were 5.00 µg/L. Therefore, the respective nominal LOECs were determined to be 10.0 µg/L. The corresponding NOEC and LOEC values based on overall arithmetic mean measured test item concentrations were NOEC 4.25 µg/L and LOEC 6.93 µg/L, respectively.
The EC10 values were determined to be < 5.00 µg/L (nominal test item concentrations), corresponding to < 4.25 µg/L (overall arithmetic mean measured dissolved test item concentrations).
For the parameter fry growth (expressed as length and fresh weight) the nominal NOECs were 10.0 µg/L (for both parameters). Therefore, the nominal LOECs for length and weight were determined to be 20.0 µg/L, respectively. The corresponding NOEC and LOEC values based on overall arithmetic mean measured dissolved test item concentrations were NOEC 6.93 µg/L and LOEC 16.5 µg/L, respectively.
The EC10 value for the growth parameter fresh weight was determined to be 16.4 (6.59 – 40.7) µg/L (nominal test item concentrations), corresponding to 12.1 (3.96 – 36.9) µg/L (overall arithmetic mean measured dissolved test item concentrations).
The EC10 value for the growth parameter length was determined to be 20.2 (12.1 - 33.5) µg/L (nominal test item concentrations), corresponding to 15.5 (8.81 – 27.1) µg/L (overall arithmetic mean measured dissolved test item concentrations).
All effect values are given based on the nominal and the overall arithmetic mean measured concentrations of the test item Tallow amine propoxylate. - Executive summary:
The effects of the test item Tallow amine propoxylate (CAS: 1309955-79-0) (Batch-No. 20200979) on the early-life stage of fish (Danio rerio / Zebrafish) were determined at the test facility according to OECD Guideline 210 from 2022-01-13 to 2022-02-16
The test item is an amber/red-brown clear to turbid clear liquid UVCB substance. Methanol was used as solvent with a concentration of 0.025 mL/L dilution water. Stock solutions in methanol with nominal concentrations of 200, 400, 800, 1600 and 3200 mg/L were prepared in appropriate intervals of 7 days and continuously dosed to the dilution water in a flow-through system. Based on the results of a range finding test the test was conducted as a dose-response test with the nominal test item concentrations 5.00, 10.0, 20.0, 40.0 and 80.0 µg/L, corresponding to the overall arithmetic mean measured dissolved test item concentrations of 4.25, 6.93, 16.5, 36.1 and 76.4 µg/L. The term arithmetic mean means that all measured samples (stabilized samples, from study days 0, 7, 14, 21 and 28) were taken into account for calculation of the arithmetric mean measured concentrations.
The test was started by placing fertilized eggs into the test vessels and it lasted 34 days (30 days post-hatch). 80 eggs of Danio rerio / zebrafish were exposed to each test concentration, the solvent control and the control (4 replicates with 20 eggs each).
The water quality parameters pH-value, oxygen concentration, temperature and total hardness were within the acceptable limits.
On study day 4, 95% of the control and 96% the solvent control larvae had hatched. Therefore, study day 4 was defined as post hatch day 0 (= PHD 0).
Different toxicological endpoints were determined: hatching success, fry growth (assessed via length and fresh weight measurements on PHD 30), morphological and behavioral effects, post-hatch survival and overall survival.
Specific analysis of various concentrations of Tallow amine propoxylate (CAS: 1309955-79-0) in the test media and the controls was carried out via LC-MS/MS.
The concentrations of the test item Tallow amine propoxylate (CAS: 1309955-79-0), the solvent control and the control were determined during exposure on days 0, 7, 14, 21 and 28. Three constituents of the test item (N Hexadecyldiisopropanolamine, Octadecenyldiisopropanolamine and Octadecyldiisopropanolamine) were determined via LC MS/MS.
The samples taken from the test vessels were completely clear and did not contain any particles or turbidity. These samples were subjected to the analytical procedure and the results are used for the calculation of the dose response.
The highest concentrated methanol stock solution was sampled and analyzed from the freshly prepared and corresponding 7 days aged stock solution. Measured concentrations of the freshly prepared stock solution were 98 to 102% of the nominal values. Measured concentrations of the 7 days aged stock solution were in the range of 99 to 105% of the nominal values.All effect values are given based on the nominal and the overall arithmetic mean measured dissolved concentrations of the test item Tallow amine propoxylate (CAS: 1309955-79-0).
Findings and Observations
The results of the parameters hatching success, fry growth (expressed as weight and length measurement at PHD 30), post-hatch survival and overall survival were checked for statistically significant differences.
No statistically significant difference was detected between the dilution water control and the solvent control for all parameters (hatching success, fry growth expressed as length or weight on PHD 30, post-hatch survival and overall survival). Therefore, both controls were pooled for statistical analysis.
The effect values NOEC, LOEC, ECx, LCx values were determined based on the statistical results. The results are presented in the tables below:NOEC, LOEC, ECx Values of Hatching Success and Fry Growth
Based on nominal and overall arithmetic mean measured dissolved test item concentrations [µg/L] with 95% confidence intervals in bracketsParameter Based on nominal test item concentrations [µg/L] Based on overall arithmetic mean measured test item concentrations [µg/L] Hatching success after Fry Growth Hatching success after Fry Growth 5 days1) expressed as: 5 days1) expressed as: Length Weight Length Weight NOEC 40.0 10.0 10.0 36.1 6.93 6.93 LOEC 80.0 20.0 20.0 76.4 16.5 16.5 EC10 n.d 20.2 16.4 n.d 15.5 12.1 (12.1 - 33.5) (6.59 – 40.7) (8.81 – 27.1) (3.96 – 36.9) EC20 n.d 43.2 25.0 n.d 38.4 20.3 (21.2 - > 80.0) (9.60 – 67.7) (17.7 – > 76.4) (6.29 – 68.7) EC50 n.d n.d. n.d. n.d n.d. n.d. 1) = end of hatching period
n.d. = not determinableNOEC, LOEC, LCx values of Post Hatch Survival and Overall Survival
Based on nominal and overall arithmetic mean measured test item concentrations [µg/L]
with 95% Confidence intervals in bracketsParameter Based on nominal test item concentrations [µg/L] Based on overall arithmetic mean measured test item concentrations [µg/L] Post-hatch survival Overall survival Post-hatch survival Overall survival NOEC 5.00 5.00 4.25 4.25 LOEC 10.0 10.0 6.93 6.93 LC10 < 5.00* < 5.00* < 4.25* < 4.25* LC20 < 5.00* < 5.00* < 4.25* < 4.25* LC50 19.7 19.5 16.5 16.3 (15.6 – 24.9) (15.5 – 24.2) (12.7 – 22.4) (12.6 – 21.7) * determined valued lower than the lowest test item concentration
Reference
NOEC/LOEC
The data of the parameters hatching success, post hatch survival and overall survival were arcsine transformed prior to statistical analysis.
Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01.
Levene’s test on variance was done with a significance level of 0.01.
Monotonicity was done by trend analysis by contrasts (significance level 0.05).
The Fisher´s Exact Binomial test-procedure was done with a significance level of 0.05 for the parameter hatching success on study day 4 and 5.
The Williams multiple sequential t-test procedure was done with a significance level of 0.05 for the parameters post hatch survival, overall survival, fresh weight and mean total length.
All calculations were done with ToxRat Professional and based on the nominal and overall mean measured concentrations of the test item.
ECx/LCx-calculation The ECx/LCX-values and the corresponding confidence intervals were calculated by standard procedures with GraphPad Prism (GRAPHPAD SOFTWARE, INC.) and ToxRat Professional (TOXRAT SOLUTIONS GMBH).
Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- Microsoft Excel, MICROSOFT CORPORATION
- ToxRat, TOXRAT SOLUTIONS GMBH
Arithmetic mean measured concentrations
The arithmetic mean measured concentrations were determined by the sumproduct function of excel, referring to the recoveries and purities of the three lead components of the samples. This procedure takes into account the mean measured concentrations of main constituents as well as their content in the product.
Hatch / Hatching Time (Control, Solvent Control and Test Item Concentrations)
Nominal | Rep. | PHD -2 | PHD -1 | PHD 0 | PHD 1 | PHD 2 |
test item conc. | Study day 2 | Study day 3 | Study day 4 | Study day 5 | Study day 6 | |
[µg/L] | Cumulative hatching rate [%] | |||||
Control | 1 | 0 | 45 | 85 | 95 | 95 |
2 | 0 | 85 | 100 | 100 | 100 | |
3 | 10 | 55 | 100 | 100 | 100 | |
4 | 0 | 30 | 95 | 95 | 95 | |
Mean | 3 | 54 | 95 | 98 | 98 | |
Solvent control | 1 | 0 | 80 | 100 | 100 | 100 |
2 | 0 | 60 | 95 | 100 | 100 | |
3 | 0 | 80 | 95 | 95 | 95 | |
4 | 0 | 60 | 95 | 100 | 100 | |
Mean | 0 | 70 | 96 | 99 | 99 | |
5.00 | 1 | 25 | 85 | 95 | 100 | 100 |
2 | 0 | 75 | 100 | 100 | 100 | |
3 | 0 | 70 | 95 | 95 | 95 | |
4 | 0 | 60 | 100 | 100 | 100 | |
Mean | 6 | 73 | 98(ns) | 99(ns) | 99 | |
10.0 | 1 | 0 | 55 | 85 | 95 | 95 |
2 | 5 | 70 | 95 | 95 | 95 | |
3 | 0 | 70 | 100 | 100 | 100 | |
4 | 5 | 85 | 100 | 100 | 100 | |
Mean | 3 | 70 | 95(ns) | 98(ns) | 98 | |
20.0 | 1 | 0 | 85 | 100 | 100 | 100 |
2 | 0 | 60 | 90 | 100 | 100 | |
3 | 0 | 80 | 90 | 95 | 95 | |
4 | 0 | 90 | 100 | 100 | 100 | |
Mean | 0 | 79 | 95(ns) | 99(ns) | 99 | |
40.0 | 1 | 0 | 65 | 90 | 95 | 95 |
2 | 0 | 70 | 95 | 95 | 95 | |
3 | 0 | 70 | 90 | 100 | 100 | |
4 | 0 | 65 | 95 | 95 | 95 | |
Mean | 0 | 68 | 93(ns) | 96(ns) | 96 | |
80.0 | 1 | 0 | 65 | 80 | 90 | 90 |
2 | 15 | 80 | 80 | 80 | 80 | |
3 | 0 | 45 | 90 | 90 | 90 | |
4 | 0 | 65 | 100 | 100 | 100 | |
Mean | 4 | 64 | 88(ns) | 90(s) | 90 |
(ns) / (s) = No statistically / statistically significant difference from pooled control groups
Percent Swim-up of Hatched Live Fry of the Control, Solvent Control and the Nominal Test Item Concentrations 5.00 to 80.0 µg/L
Nominal | Rep. | PHD 0 | PHD 1 | PHD 2 | PHD 3 | PHD 4 |
test item conc. | Study day 4 | Study day 5 | Study day 6 | Study day 7 | Study day 8 | |
[µg/L] | Cumulative swim up [%] | |||||
Control | 1 | 0 | 100 | 100 | 100 | 100 |
2 | 0 | 100 | 100 | 100 | 100 | |
3 | 0 | 100 | 100 | 100 | 100 | |
4 | 0 | 100 | 100 | 100 | 100 | |
Mean | 0 | 100 | 100 | 100 | 100 | |
Solvent control | 1 | 0 | 100 | 100 | 100 | 100 |
2 | 0 | 90 | 100 | 100 | 100 | |
3 | 0 | 100 | 100 | 100 | 100 | |
4 | 0 | 100 | 100 | 100 | 100 | |
Mean | 0 | 98 | 100 | 100 | 100 | |
5.00 | 1 | 0 | 95 | 95 | 90 | 100 |
2 | 0 | 100 | 100 | 100 | 100 | |
3 | 0 | 100 | 100 | 100 | 100 | |
4 | 0 | 100 | 100 | 100 | 100 | |
Mean | 0 | 99 | 99 | 98 | 100 | |
10.0 | 1 | 0 | 100 | 100 | 100 | 100 |
2 | 0 | 100 | 100 | 100 | 100 | |
3 | 0 | 100 | 100 | 100 | 94 | |
4 | 0 | 100 | 100 | 100 | 100 | |
Mean | 0 | 100 | 100 | 100 | 99 | |
20.0 | 1 | 0 | 100 | 100 | 100 | 100 |
2 | 0 | 100 | 100 | 100 | 100 | |
3 | 0 | 100 | 100 | 100 | 100 | |
4 | 0 | 100 | 100 | 100 | 100 | |
Mean | 0 | 100 | 100 | 100 | 100 | |
40.0 | 1 | 0 | 100 | 100 | 95 | 100 |
2 | 0 | 100 | 100 | 100 | 100 | |
3 | 0 | 95 | 100 | 95 | 95 | |
4 | 0 | 100 | 100 | 84 | 78 | |
Mean | 0 | 99 | 100 | 94 | 93 | |
80.0 | 1 | 0 | 89 | 100 | 75 | 50 |
2 | 0 | 100 | 100 | 88 | 0 | |
3 | 0 | 35 | 94 | 76 | 0 | |
4 | 0 | 90 | 100 | 85 | 0 | |
Mean | 0 | 79 | 99 | 81 | 13 |
Post-Hatch Survival on Study Day 34 (PHD 30) of the Control, Solvent Control and the Nominal Test Item Concentrations 5.00 and 10.0 µg/L
Nominal | Rep. | Eggs introduced on study day 0 | Cumulative number of hatched larvae | Vital Larvae on study day 34 | Post-Hatch survival |
test item conc. | (PHD 30) | on study day 34 | |||
[µg/L] | [%] | ||||
Control | 1 | 20 | 19 | 15 | 79 |
2 | 20 | 20 | 17 | 85 | |
3 | 20 | 20 | 16 | 80 | |
4 | 20 | 19 | 14 | 74 | |
Mean | 20 | 19.5 | 16 | 80 | |
Solvent control | 1 | 20 | 20 | 18 | 90 |
2 | 20 | 20 | 18 | 90 | |
3 | 20 | 19 | 16 | 84 | |
4 | 20 | 20 | 17 | 85 | |
Mean | 20 | 19.8 | 17 | 87 | |
5.00 | 1 | 20 | 20 | 16 | 80 |
2 | 20 | 20 | 15 | 75 | |
3 | 20 | 19 | 16 | 84 | |
4 | 20 | 20 | 15 | 75 | |
Mean | 20 | 19.8 | 16 | 79 (ns) | |
10.0 | 1 | 20 | 19 | 17 | 89 |
2 | 20 | 19 | 11 | 58 | |
3 | 20 | 20 | 8 | 40 | |
4 | 20 | 20 | 12 | 60 | |
Mean | 20 | 19.5 | 12 | 62 (s) |
(ns) / (s) = No statistically / statistically significant difference from pooled control groups
Post-Hatch Survival on Study Day 34 (PHD 30) of the Nominal Test Item Concentrations 20.0 to 80.0 µg/L
Nominal | Rep. | Eggs introduced on study day 0 | Cumulative number of hatched larvae | Vital Larvae on study day 34 | Post-Hatch survival |
test item conc. | (PHD 30) | on study day 34 | |||
[µg/L] | [%] | ||||
20.0 | 1 | 20 | 20 | 8 | 40 |
2 | 20 | 20 | 8 | 40 | |
3 | 20 | 19 | 13 | 68 | |
4 | 20 | 20 | 11 | 55 | |
Mean | 20 | 19.8 | 10 | 51 (s) | |
40.0 | 1 | 20 | 19 | 3 | 16 |
2 | 20 | 19 | 0 | 0 | |
3 | 20 | 20 | 0 | 0 | |
4 | 20 | 19 | 0 | 0 | |
Mean | 20 | 19.3 | 1 | 4 (s) | |
80.0 | 1 | 20 | 18 | 0 | 0 |
2 | 20 | 16 | 0 | 0 | |
3 | 20 | 17 | 0 | 0 | |
4 | 20 | 20 | 0 | 0 | |
Mean | 20 | 17.8 | 0 | 0 (s) |
(ns) / (s) = No statistically / statistically significant difference from pooled control groups
Overall Survival and Overall Mortality on Study Day 34 (PHD 30) of the Control, Solvent Control and the Nominal Test Item Concentrations 5.00 and 10.0 µg/L
Nominal | Rep. | Eggs introduced on study day 0 | Vital larvae on | Overall survival | Overall mortality |
test item conc. | study day 34 | ||||
[µg/L] | (PHD 30) | ||||
[%] | [%] | ||||
Control | 1 | 20 | 15 | 75 | 25 |
2 | 20 | 17 | 85 | 15 | |
3 | 20 | 16 | 80 | 20 | |
4 | 20 | 14 | 70 | 30 | |
Mean | 20 | 16 | 78 | 22 | |
Solvent control | 1 | 20 | 18 | 90 | 10 |
2 | 20 | 18 | 90 | 10 | |
3 | 20 | 16 | 80 | 20 | |
4 | 20 | 17 | 85 | 15 | |
Mean | 20 | 17 | 86 | 14 | |
5.00 | 1 | 20 | 16 | 80 | 20 |
2 | 20 | 15 | 75 | 25 | |
3 | 20 | 16 | 80 | 20 | |
4 | 20 | 15 | 75 | 25 | |
Mean | 20 | 16 | 78 (ns) | 22 | |
10.0 | 1 | 20 | 17 | 85 | 15 |
2 | 20 | 11 | 55 | 45 | |
3 | 20 | 8 | 40 | 60 | |
4 | 20 | 12 | 60 | 40 | |
Mean | 20 | 12 | 60 (s) | 40 |
(ns) / (s) = No statistically / statistically significant difference from pooled control groups
Overall Survival and Overall Mortality on Study Day 34 (PHD 30) of the Nominal Test Item Concentrations 20.0 to 80.0 µg/L
Nominal | Rep. | Eggs introduced on study day 0 | Vital larvae on | Overall survival | Overall mortality |
test item conc. | study day 34 | ||||
[µg/L] | (PHD 30) | ||||
[%] | [%] | ||||
20.0 | 1 | 20 | 8 | 40 | 60 |
2 | 20 | 8 | 40 | 60 | |
3 | 20 | 13 | 65 | 35 | |
4 | 20 | 11 | 55 | 45 | |
Mean | 20 | 10 | 50 (s) | 50 | |
40.0 | 1 | 20 | 3 | 15 | 85 |
2 | 20 | 0 | 0 | 100 | |
3 | 20 | 0 | 0 | 100 | |
4 | 20 | 0 | 0 | 100 | |
Mean | 20 | 1 | 4 (s) | 96 | |
80.0 | 1 | 20 | 0 | 0 | 100 |
2 | 20 | 0 | 0 | 100 | |
3 | 20 | 0 | 0 | 100 | |
4 | 20 | 0 | 0 | 100 | |
Mean | 20 | 0 | 0 (s) | 100 |
(ns) / (s) = No statistically / statistically significant difference from pooled control groups
Behavioral Effects observed in the Nominal Test Concentrations 5.00 and 10.0 µg/L (Study Day 6 – 28)
Study Day | 5.00 µg/L | 10.0 µg/L | ||||||
1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
6 | 1 Sc | - | - | - | - | - | - | - |
7 | 1Sc | - | - | - | - | - | - | - |
8 | - | - | - | - | - | - | 1 Q | - |
1 G | ||||||||
9 | - | - | - | - | - | - | 1 Q | - |
1 G | ||||||||
10 | - | - | - | - | - | - | 2 Q | - |
2 G | ||||||||
11-13 | - | - | - | - | - | - | - | - |
14-16 | - | - | - | 1 G | - | - | - | - |
17-23 | - | - | - | - | - | - | - | - |
24 | - | 1 G | - | 1 G | - | - | - | 2 G |
1 F | 1 F | 2 F | ||||||
1 E | 1 E | 2 E | ||||||
25 | 1 G | 1 G | - | 1 G | - | - | - | 1 G |
1 F | 1 F | 1 F | 1 F | |||||
1 E | 1 E | 1 E | 1 E | |||||
26 | 1 G | 1 G | - | 1 G | 1 F | 1 F | - | |
1 F | 1 F | 1 F | 1 E | 1 E | ||||
1 E | 1 E | 1 E | ||||||
27 | - | - | - | - | - | - | 1 F | - |
28 | - | - | - | - | - | - | 1F | - |
E = Slow escape reflex F = No food uptake
G = Arresting on the ground Sc = Scoliosis Q = Quiescence
Behavioral Effects observed in the Nominal Test Concentrations 20.0 and 40.0 µg/L (Study Day 7 – 28)
Study Day | 20.0 µg/L | 40.0 µg/L | ||||||
1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
7 | - | - | - | - | 1 G | - | 1 G | 3 G |
1 Q | 1 Q | 3 Q | ||||||
8 | - | - | - | - | - | - | 1 G | 4 G |
1 Q | 4 Q | |||||||
9 | - | - | - | - | - | 1 G | 1 G | 1 G |
1 Q | 1 Q | 1 Q | ||||||
10 | - | - | 1 Q | 1 Q | - | 14 G | 7 E | 8 G |
1 G | 1 G | 14 Q | 1 M | 8 M | ||||
4 G | ||||||||
7 Q | ||||||||
4 S | ||||||||
1 T | ||||||||
11 | - | - | 1 Q | - | 4 E | 7 G | 5 E | 1 G |
1 G | 4 G | 7 M | 6 G | 1 Q | ||||
2 Q | 6 Q | |||||||
2 S | 5 S | |||||||
12 | - | - | - | - | 4 E | 7 G | 1 G | 100 % Mortality |
3 G | 7 M | 1 M | ||||||
1 Q | ||||||||
2 S | ||||||||
13 | - | - | - | - | 3 E | 1 E | - | |
3 G | 1 S | |||||||
14 | - | - | - | - | 4 E | 1 E | - | |
4 G | 1 S | |||||||
15-16 | - | - | - | - | 3 E | 1 E | - | |
3 G | 1 G | |||||||
17 | - | - | - | - | 3 E | 2 E | - | |
3 G | 2 G | |||||||
18 | - | - | - | - | 2 E | 100 % Mortality | - | |
2 G | ||||||||
19 | - | 4 G | - | - | 1 E | - | ||
1 G | ||||||||
20 | - | 2 G | - | - | 1 E | - | ||
1 G | ||||||||
21 | - | - | - | 1 E | 100 % Mortality | |||
1 G | ||||||||
22-23 | - | - | - | - | - | |||
24 | - | 1 E | - | - | ||||
1 G | ||||||||
25 | 1 E | 1 E | 2 E | - | - | |||
1 F | 1 G | 2 F | ||||||
1 G | ||||||||
26 | 1 E | 1 E | 2 E | - | - | |||
1 F | 1 F | 2 F | ||||||
1 G | 1 G | |||||||
27 | 1 E | - | 2 E | - | - | |||
1 F | ||||||||
1 G | ||||||||
28 | 1 E | - | 1 E | - | - | |||
1 F | ||||||||
1 G |
E = Slow escape reflex F = No food uptake G = Arresting on the ground
M = Missing escape reflex Q = Quiescence S = Arresting at the surface
T = Tumbling
Behavioral Effects observed in the Nominal Test Concentration 80.0 µg/L
(Study Day 7 – 11)
Study Day | 80.0 µg/L | |||
1 | 2 | 3 | 4 | |
7 | 4 G | 2 G | 4 G | 3 G |
4 Q | 2 Q | 4 Q | 3 Q | |
8 | 6 F | 3 F | 8 F | 10 F |
6 G | 3 Q | 5 G | 7 G | |
6 Q | 8 Q | 10 Q | ||
9 | 2 F | 100 % Mortality | 4 F | 3 F |
2 G | 4 G | 3 G | ||
2 Q | 4 Q | 3 Q | ||
10 | 1 F | 100 % Mortality | 2 F | 2 F |
1 M | 2 M | 2 M | ||
11 | 100 % Mortality |
F = No food uptake G = Arresting on the ground
M = Missing escape reflex Q = Quiescence
Overview of Fry Growth: Length and Wet Weight on Study Day 34 (PHD 30) of the Control, Solvent Control and Nominal Test Item Concentrations 5.00 and 10.0 µg/L
Nominal | Rep. | PHD 30 (End of exposure) | |
test item conc. [µg/L] | Mean total length per fish [mm] | Mean wet weight per fish [mg] | |
Control | 1 | 14.6 | 27.0 |
2 | 14.3 | 27.5 | |
3 | 14.6 | 26.3 | |
4 | 15.1 | 29.9 | |
Mean | 14.7 | 27.7 | |
± SD | 0.287 | 1.35 | |
CV [%] | 1.95 | 4.88 | |
Solvent control | 1 | 14.6 | 27.4 |
2 | 13.8 | 25.5 | |
3 | 14.3 | 27.9 | |
4 | 15.4 | 29.8 | |
Mean | 14.5 | 27.7 | |
± SD | 0.580 | 1.53 | |
CV [%] | 4.00 | 5.53 | |
5.00 | 1 | 13.3 | 21.5 |
2 | 14.2 | 30.4 | |
3 | 13.2 | 24.5 | |
4 | 13.6 | 25.4 | |
Mean | 13.6 (ns) | 25.5 (ns) | |
± SD | 0.390 | 3.20 | |
CV [%] | 2.87 | 12.6 | |
10.0 | 1 | 13.6 | 22.2 |
2 | 13.9 | 28.4 | |
3 | 15.2 | 38.3 | |
4 | 14.8 | 30.8 | |
Mean | 14.4 (ns) | 29.9 (ns) | |
± SD | 0.650 | 5.76 | |
CV [%] | 4.51 | 19.3 |
(ns) = No statistically significant difference from pooled controls
Overview of Fry Growth: Length and Wet Weight on Study Day 34 (PHD 30) of the Nominal Test Item Concentrations 20.0 and 40.0 µg/L
Nominal | Rep. | PHD 30 (End of exposure) | |
test item conc.[µg/L] | Mean total length per fish [mm] | Mean wet weight per fish [mg] | |
20.0 | 1 | 13.3 | 19.3 |
2 | 12.5 | 21.5 | |
3 | 13.6 | 24.0 | |
4 | 12.3 | 22.3 | |
Mean | 12.9 (s) | 21.8 (s) | |
± SD | 0.540 | 1.69 | |
CV [%] | 4.19 | 7.75 | |
40.0 | 1 | 11.7 | 19.7 |
2 | - | - | |
3 | - | - | |
4 | - | - | |
Mean | (s) | -(s) | |
± SD | - | - | |
CV [%] | - | - |
(s) = Statistically significant difference from pooled controls
- = Fish died before the end of the exposure phase
Individual Length on Study Day 34 (PHD 30) of the Control and the Solvent Control
Fish No. | Control | Solvent control | ||||||
Total length of individual fish in [mm] | ||||||||
1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
1 | 14.0 | 10.0 | 7.5 | 18.5 | 14.0 | 14.0 | 18.5 | 15.0 |
2 | 16.5 | 9.0 | 15.5 | 17.0 | 13.0 | 20.5 | 16.5 | 14.0 |
3 | 18.5 | 9.0 | 16.5 | 11.5 | 12.0 | 17.0 | 10.5 | 13.0 |
4 | 13.0 | 12.5 | 17.5 | 18.5 | 9.0 | 17.0 | 10.0 | 15.0 |
5 | 11.0 | 16.5 | 16.0 | 14.5 | 10.5 | 10.5 | 20.0 | 20.5 |
6 | 19.0 | 19.5 | 14.0 | 11.0 | 18.5 | 10.0 | 20.0 | 16.5 |
7 | 15.5 | 15.0 | 11.5 | 12.0 | 15.5 | 12.0 | 17.5 | 15.0 |
8 | 15.0 | 16.0 | 17.5 | 10.0 | 16.0 | 13.0 | 17.0 | 14.0 |
9 | 14.0 | 14.0 | 10.0 | 17.5 | 17.0 | 15.5 | 16.5 | 17.0 |
10 | 17.5 | 12.0 | 19.5 | 20.0 | 16.0 | 15.5 | 10.0 | 13.0 |
11 | 14.5 | 18.0 | 17.0 | 16.0 | 16.0 | 17.5 | 11.0 | 19.0 |
12 | 12.0 | 13.0 | 15.0 | 17.0 | 18.5 | 13.5 | 15.0 | 15.0 |
13 | 18.0 | 16.0 | 15.0 | 14.0 | 15.0 | 13.0 | 8.5 | 18.0 |
14 | 8.0 | 15.0 | 10.0 | 14.0 | 10.5 | 13.0 | 10.0 | 10.0 |
15 | 12.5 | 18.0 | 17.5 | - | 15.0 | 9.0 | 18.0 | 18.0 |
16 | - | 18.0 | 13.0 | - | 17.0 | 11.0 | 13.0 | 17.0 |
17 | - | 12.0 | - | 16.0 | 12.5 | 11.0 | 11.0 | |
18 | - | - | - | - | 14.0 | 14.0 | - | - |
19 | - | - | - | - | - | - | - | - |
20 | - | - | - | - | - | - | - | - |
Mean | 14.6 | 14.3 | 14.6 | 15.1 | 14.6 | 13.8 | 14.3 | 15.4 |
± SD | 2.95 | 3.16 | 3.23 | 3.04 | 2.65 | 2.87 | 3.84 | 2.69 |
CV % | 20.2 | 22.1 | 22.1 | 20.1 | 18.1 | 20.8 | 26.9 | 17.5 |
- = Fish died before end of the study
Individual Length on Study Day 34 (PHD 30) of the Nominal Test Item Concentrations 5.00 and 10.0 µg/L
Fish No. | Nominal test item conc. [µg/L] | |||||||
5.00 | 10.0 | |||||||
Total length of individual fish in [mm] | ||||||||
1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
1 | 9.0 | 15.0 | 19.0 | 9.0 | 9.5 | 10.0 | 20.0 | 14.5 |
2 | 12.0 | 14.0 | 20.0 | 13.0 | 11.5 | 10.0 | 19.0 | 18.0 |
3 | 12.0 | 9.0 | 14.0 | 14.0 | 12.0 | 14.0 | 10.0 | 16.5 |
4 | 12.0 | 18.0 | 8.5 | 8.0 | 12.0 | 9.0 | 21.0 | 9.0 |
5 | 17.0 | 17.0 | 14.0 | 20.0 | 13.0 | 11.5 | 16.0 | 18.0 |
6 | 17.0 | 15.0 | 14.0 | 18.0 | 17.0 | 20.5 | 15.0 | 13.0 |
7 | 12.0 | 15.0 | 8.0 | 14.0 | 10.0 | 10.0 | 10.5 | 20.0 |
8 | 13.0 | 16.0 | 16.5 | 12.0 | 9.0 | 15.0 | 10.0 | 8.0 |
9 | 9.0 | 14.0 | 8.0 | 14.0 | 16.0 | 16.5 | - | 17.0 |
10 | 18.0 | 18.0 | 11.0 | 11.0 | 10.5 | 19.5 | - | 13.0 |
11 | 13.0 | 20.5 | 12.0 | 14.5 | 17.0 | 17.0 | - | 14.0 |
12 | 19.0 | 11.0 | 12.5 | 18.0 | 16.0 | - | - | 16.5 |
13 | 15.5 | 11.0 | 14.5 | 17.0 | 16.0 | - | - | - |
14 | 14.0 | 11.0 | 9.5 | 10.0 | 14.0 | - | - | - |
15 | 10.0 | 11.0 | 19.0 | 11.0 | 17.0 | - | - | - |
16 | 11.0 | 12.0 | 10.5 | - | 18.0 | - | - | - |
17 | - | - | - | - | 13.0 | - | - | - |
18 | - | - | - | - | - | - | - | - |
19 | - | - | - | - | - | - | - | - |
20 | - | - | - | - | - | - | - | - |
Mean | 13.3 | 14.2 | 13.2 | 13.6 | 13.6 | 13.9 | 15.2 | 14.8 |
± SD | 3.04 | 3.11 | 3.81 | 3.41 | 2.89 | 3.91 | 4.30 | 3.48 |
CV % | 22.8 | 21.9 | 28.8 | 25.1 | 21.3 | 28.1 | 28.3 | 23.5 |
- = Fish died before end of the study
Individual Length on Study Day 34 (PHD 30) of the Nominal Test Item Concentrations 20.0 and 40.0 µg/L
Fish No. | Nominal test item conc. [µg/L] | |||||||
20.0 | 40.0 | |||||||
Total length of individual fish in [mm] | ||||||||
1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
1 | 16.0 | 12.0 | 14.0 | 19.0 | 19.5 | - | - | - |
2 | 13.0 | 10.0 | 16.5 | 17.0 | 8.0 | - | - | - |
3 | 18.0 | 15.0 | 15.0 | 13.0 | 7.5 | - | - | - |
4 | 8.5 | 18.0 | 13.0 | 10.5 | - | - | - | - |
5 | 15.0 | 16.0 | 15.0 | 12.0 | - | - | - | - |
6 | 11.0 | 11.0 | 10.0 | 11.0 | - | - | - | - |
7 | 15.0 | 10.0 | 13.0 | 8.0 | - | - | - | - |
8 | 10.0 | 8.0 | 9.0 | 14.0 | - | - | - | - |
9 | - | - | 16.0 | 11.5 | - | - | - | - |
10 | - | - | 18.5 | 12.0 | - | - | - | - |
11 | - | - | 17.0 | 7.5 | - | - | - | - |
12 | - | - | 10.0 | - | - | - | - | - |
13 | - | - | 10.0 | - | - | - | - | - |
14 | - | - | - | - | - | - | - | - |
15 | - | - | - | - | - | - | - | - |
16 | - | - | - | - | - | - | - | - |
17 | - | - | - | - | - | - | - | - |
18 | - | - | - | - | - | - | - | - |
19 | - | - | - | - | - | - | - | - |
20 | - | - | - | - | - | - | - | - |
Mean | 13.3 | 12.5 | 13.6 | 12.3 | 11.7 | - | - | - |
± SD | 3.05 | 3.24 | 2.97 | 3.27 | 5.54 | - | - | - |
CV % | 22.9 | 25.9 | 21.8 | 26.6 | 47.4 | - | - | - |
- = Fish died before end of the study
Pooled Wet Weights on Study Day 34 (PHD 30) of the Control, Solvent Control and the Nominal Test Item Concentrations 5.00 to 40.0 µg/L
Nominal | Replicate | Number of fish alive | Pooled | Mean | Mean | ± SD | CV % |
test item conc. | on study day 34 | wet weight | wet weight | ||||
[µg/L] | per replicate | per fish per replicate | |||||
[mg] | [mg] | [mg] | |||||
Control | 1 | 15 | 405.3 | 27.0 | 27.7 | 1.353 | 4.88 |
2 | 17 | 467.1 | 27.5 | ||||
3 | 16 | 421.3 | 26.3 | ||||
4 | 14 | 418.9 | 29.9 | ||||
Solvent control | 1 | 18 | 493.3 | 27.4 | 27.7 | 1.531 | 5.53 |
2 | 18 | 458.6 | 25.5 | ||||
3 | 16 | 447.0 | 27.9 | ||||
4 | 17 | 507.3 | 29.8 | ||||
5.00 | 1 | 16 | 343.5 | 21.5 | 25.5 | 3.202 | 12.56 |
2 | 15 | 456.1 | 30.4 | ||||
3 | 16 | 392.4 | 24.5 | ||||
4 | 15 | 381.7 | 25.4 | ||||
10.0 | 1 | 17 | 377.8 | 22.2 | 29.9 | 5.764 | 19.28 |
2 | 11 | 312.7 | 28.4 | ||||
3 | 8 | 306.5 | 38.3 | ||||
4 | 12 | 369.9 | 30.8 | ||||
20.0 | 1 | 8 | 154.5 | 19.3 | 21.8 | 1.690 | 7.75 |
2 | 8 | 171.7 | 21.5 | ||||
3 | 13 | 312.0 | 24.0 | ||||
4 | 11 | 245.2 | 22.3 | ||||
40.0 | 1 | 3 | 59.0 | 19.7 | n.a. | n.a. | n.a. |
2 | 0 | - | - | ||||
3 | 0 | - | - | ||||
4 | 0 | - | - |
n.a. = Not applicable
Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 0 -15)
Study day | Replicate | Dissolved oxygen [%] | ||||||
Nominal test item concentrations [µg/L] | ||||||||
Control | Solvent control | 5.00 | 10.0 | 20.0 | 40.0 | 80.0 | ||
0 | 1 | 100 | 98 | 100 | 99 | 99 | 100 | 99 |
2 | 99 | 99 | 99 | 99 | 99 | 99 | 99 | |
3 | 99 | 98 | 100 | 99 | 99 | 99 | 99 | |
4 | 99 | 99 | 99 | 99 | 98 | 99 | 99 | |
1 | 1 | 100 | 100 | 99 | 99 | 99 | 99 | 99 |
2 | 100 | 99 | 100 | 100 | 100 | 99 | 98 | |
3 | 99 | 98 | 99 | 99 | 99 | 99 | 99 | |
4 | 99 | 99 | 100 | 99 | 98 | 99 | 91 | |
4 | 1 | 100 | 100 | 100 | 100 | 99 | 100 | 99 |
2 | 99 | 99 | 99 | 99 | 100 | 99 | 99 | |
3 | 99 | 99 | 100 | 99 | 99 | 99 | 99 | |
4 | 99 | 99 | 99 | 99 | 99 | 99 | 99 | |
6 | 1 | 100 | 100 | 99 | 98 | 98 | 98 | 98 |
2 | 99 | 98 | 98 | 98 | 99 | 99 | 98 | |
3 | 99 | 98 | 99 | 98 | 98 | 98 | 99 | |
4 | 99 | 100 | 99 | 98 | 98 | 98 | 99 | |
8 | 1 | 99 | 98 | 95 | 96 | 98 | 97 | 96 |
2 | 98 | 96 | 97 | 98 | 97 | 97 | 96 | |
3 | 97 | 95 | 98 | 97 | 97 | 96 | 96 | |
4 | 99 | 98 | 98 | 95 | 97 | 97 | 98 | |
11 | 1 | 99 | 98 | 96 | 96 | 97 | 98 | 97 |
2 | 96 | 95 | 95 | 96 | 97 | 96 | 96 | |
3 | 97 | 95 | 97 | 96 | 96 | 95 | 97 | |
4 | 98 | 97 | 96 | 96 | 96 | 95 | 97 | |
13 | 1 | 98 | 95 | 93 | 95 | 94 | 98 | - |
2 | 95 | 93 | 94 | 95 | 96 | 95 | - | |
3 | 95 | 93 | 96 | 97 | 93 | 91 | - | |
4 | 96 | 94 | 96 | 93 | 94 | - | - | |
15 | 1 | 98 | 93 | 89 | 91 | 93 | 97 | - |
2 | 95 | 92 | 95 | 94 | 96 | 94 | - | |
3 | 94 | 93 | 94 | 94 | 93 | 88 | - | |
4 | 96 | 94 | 94 | 90 | 91 | - | - |
- = No measurement, due to 100 % mortality
Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 18 - 32)
Study day | Replicate | Dissolved oxygen [%] | ||||||
Nominal test item concentrations [µg/L] | ||||||||
Control | Solvent control | 5.00 | 10.0 | 20.0 | 40.0 | 80.0 | ||
18 | 1 | 98 | 96 | 84 | 89 | 93 | 97 | - |
2 | 96 | 94 | 95 | 96 | 96 | 96 | - | |
3 | 96 | 95 | 95 | 92 | 93 | 85 | - | |
4 | 97 | 95 | 95 | 91 | 95 | - | - | |
20 | 1 | 96 | 94 | 82 | 91 | 94 | 94 | - |
2 | 95 | 93 | 95 | 93 | 96 | - | - | |
3 | 95 | 95 | 95 | 94 | 96 | 80 | - | |
4 | 95 | 93 | 95 | 91 | 95 | - | - | |
22 | 1 | 97 | 95 | 89 | 95 | 95 | 97 | - |
2 | 96 | 92 | 96 | 96 | 97 | - | - | |
3 | 94 | 95 | 96 | 95 | 96 | - | - | |
4 | 94 | 91 | 95 | 92 | 96 | - | - | |
25 | 1 | 95 | 96 | 94 | 95 | 94 | 96 | - |
2 | 96 | 95 | 96 | 95 | 96 | - | - | |
3 | 95 | 94 | 93 | 95 | 95 | - | - | |
4 | 96 | 91 | 94 | 92 | 93 | - | - | |
27 | 1 | 94 | 93 | 93 | 92 | 94 | 96 | - |
2 | 93 | 90 | 95 | 92 | 95 | - | - | |
3 | 93 | 90 | 92 | 94 | 92 | - | - | |
4 | 92 | 87 | 88 | 88 | 91 | - | - | |
29 | 1 | 95 | 92 | 93 | 93 | 94 | 95 | - |
2 | 93 | 91 | 96 | 94 | 96 | - | - | |
3 | 94 | 92 | 91 | 91 | 92 | - | - | |
4 | 92 | 88 | 93 | 89 | 91 | - | - | |
32 | 1 | 96 | 95 | 91 | 95 | 95 | 95 | - |
2 | 93 | 93 | 96 | 95 | 96 | - | - | |
3 | 96 | 95 | 92 | 94 | 95 | - | - | |
4 | 94 | 91 | 95 | 91 | 92 | - | - | |
Mean | 97 | 95 | 95 | 95 | 96 | 96 | 98 | |
± SD | 2.29 | 3.16 | 3.73 | 3.12 | 2.43 | 4.03 | 1.85 | |
Min. | 92 | 87 | 82 | 88 | 91 | 80 | 91 | |
Max. | 100 | 100 | 100 | 100 | 100 | 100 | 99 |
- = No measurement, due to 100 % mortality
Min./Max. = Minimum/Maximum measured dissolved oxygen concentration
Water Temperature (Continuous Measuring) in Replicate 2 of the Control Group
Period of measurements | 2022-01-13 to 2022-02-16 |
Minimum temperature [°C] | 25.7 |
Maximum temperature [°C] | 26.8 |
Mean temperature [°C] | 26.3 |
Water Temperature in the Test Media
Study day | Rep. | Water temperature [°C] | ||||||
Nominal test item concentrations [µg/L] | ||||||||
Control | Solvent control | 5.00 | 10.0 | 20.0 | 40.0 | 80.0 | ||
0 | 1 | 26.3 | 26.6 | 26.8 | 26.4 | 26.6 | 26.8 | 26.5 |
2 | 26.4 | 26.3 | 26.1 | 26.2 | 26.2 | 26.4 | 26.4 | |
3 | 26.1 | 26.4 | 26.2 | 26.2 | 26.2 | 26.3 | 26.0 | |
4 | 26.1 | 26.1 | 26.1 | 26.4 | 26.2 | 26.1 | 26.4 | |
7 | 1 | 26.4 | 26.7 | 26.7 | 26.3 | 26.7 | 26.4 | 26.5 |
2 | 26.6 | 26.4 | 26.3 | 26.6 | 26.4 | 26.5 | 26.5 | |
3 | 26.5 | 26.6 | 26.5 | 26.5 | 26.5 | 26.5 | 26.3 | |
4 | 26.5 | 26.6 | 26.3 | 26.7 | 26.5 | 26.8 | 26.4 | |
14 | 1 | 26.1 | 26.3 | 26.3 | 26.2 | 26.3 | 26.5 | - |
2 | 26.1 | 26.1 | 25.9 | 26.0 | 26.0 | 26.2 | - | |
3 | 26.1 | 26.1 | 26.0 | 26.0 | 25.9 | 26.0 | - | |
4 | 26.0 | 26.0 | 25.9 | 26.2 | 26.0 | - | - | |
20 | 1 | 26.1 | 26.5 | 26.5 | 26.3 | 26.2 | 26.6 | - |
2 | 26.1 | 26.3 | 26.1 | 26.2 | 26.3 | - | - | |
3 | 26.0 | 26.5 | 26.1 | 26.2 | 26.2 | 26.2 | - | |
4 | 26.1 | 26.0 | 26.0 | 26.4 | 26.2 | - | - | |
28 | 1 | 26.4 | 26.5 | 26.4 | 25.8 | 26.1 | 26.3 | - |
2 | 26.1 | 26.0 | 26.0 | 25.7 | 25.9 | - | - | |
3 | 26.0 | 26.2 | 26.0 | 25.9 | 25.7 | - | - | |
4 | 26.0 | 26.1 | 25.9 | 26.2 | 26.0 | - | - | |
Mean | 26,2 | 26.3 | 26.2 | 26.2 | 26.2 | 26.4 | 26.4 | |
± SD | 0,19 | 0.23 | 0.26 | 0.25 | 0.25 | 0.24 | 0.17 | |
Min. | 26,0 | 26.0 | 25.9 | 25.7 | 25.7 | 26.0 | 26.0 | |
Max. | 26,6 | 26.7 | 26.8 | 26.7 | 26.7 | 26.8 | 26.5 |
- = No measurement, due to 100 % mortality
Min./Max. = Minimum/Maximum measured water temperature
pH-Values in the Test Media
Study day | Rep. | pH-value | ||||||
Nominal test item concentrations [µg/L] | ||||||||
Control | Solvent control | 5.00 | 10.0 | 20.0 | 40.0 | 80.0 | ||
0 | 1 | 7.91 | 7.87 | 7.92 | 7.82 | 7.82 | 7.88 | 7.89 |
2 | 7.89 | 7.79 | 7.84 | 7.82 | 7.84 | 7.84 | 7.84 | |
3 | 7.81 | 7.82 | 7.92 | 7.81 | 7.85 | 7.81 | 7.79 | |
4 | 7.87 | 7.84 | 7.82 | 7.83 | 7.81 | 7.82 | 7.83 | |
7 | 1 | 7.99 | 8.01 | 8.02 | 8.03 | 8.12 | 8.03 | 8.02 |
2 | 8.01 | 7.98 | 7.96 | 8.06 | 8.07 | 8.03 | 8.02 | |
3 | 8.00 | 8.06 | 8.05 | 8.00 | 8.05 | 8.05 | 8.08 | |
4 | 8.07 | 8.06 | 8.01 | 8.00 | 8.02 | 8.10 | 8.04 | |
14 | 1 | 7.87 | 7.91 | 7.74 | 7.68 | 7.83 | 8.01 | - |
2 | 7.88 | 7.74 | 7.81 | 7.85 | 7.95 | 7.95 | - | |
3 | 7.83 | 7.77 | 7.94 | 7.84 | 7.86 | 7.89 | - | |
4 | 8.02 | 7.87 | 7.58 | 7.78 | 7.80 | - | - | |
20 | 1 | 7.90 | 7.95 | 7.90 | 7.94 | 7.92 | 8.00 | - |
2 | 7.89 | 7.85 | 7.99 | 7.92 | 7.98 | - | - | |
3 | 7.73 | 7.91 | 7.98 | 7.94 | 7.97 | 7.77 | - | |
4 | 7.93 | 7.86 | 7.94 | 7.84 | 7.92 | - | - | |
28 | 1 | 7.77 | 7.72 | 7.71 | 7.75 | 7.74 | 7.78 | - |
2 | 7.70 | 7.67 | 7.77 | 7.74 | 7.73 | - | - | |
3 | 7.75 | 7.75 | 7.74 | 7.72 | 7.75 | - | - | |
4 | 7.76 | 7.74 | 7.71 | 7.64 | 7.69 | - | - | |
Mean | 7,88 | 7.86 | 7.87 | 7.85 | 7.89 | 7.93 | 7.94 | |
± SD | 0,105 | 0.113 | 0.128 | 0.118 | 0.122 | 0.111 | 0.113 | |
Min. | 7,70 | 7.67 | 7.58 | 7.64 | 7.69 | 7.77 | 7.79 | |
Max. | 8,07 | 8.06 | 8.05 | 8.06 | 8.12 | 8.10 | 8.08 |
- = No measurement, due to 100 % mortality
Min./Max. = Minimum/Maximum measured pH-value
Total Hardness in the Control and the Remaining Highest Test Concentration
Study day | Replicate | Total hardness [mg CaCO3/L] | ||
Nominal test item conc. [µg/L] | ||||
Control | 40.0 | 80.0 | ||
1 | 1 | 68 | 59 | |
8 | 2 | 67 | 66 | |
15 | 3 | 65 | 64 | |
22 | 4/1 | 63 | 62 | |
29 | 1 | 67 | 65 | |
Mean | 66 | 64 | 63 | |
± SD | 2.00 | 1.53 | 4.95 | |
Min. | 63 | 62 | 59 | |
Max. | 68 | 65 | 66 |
= No measurement, due to 100 % mortality
n.a. = Not applicable
Min./Max. = Minimum/Maximum measured hardness
Total Organic Carbon (TOC) of the Dilution Water
Study day | TOC [mg/L] |
0 | 1.28 |
7 | 1.28 |
15 | 1.25 |
22 | 1.18 |
28 | 1.19 |
Mean | 1.24 |
SD | 0.048 |
Min. | 1.18 |
Max. | 1.28 |
Min./Max. = Minimum/Maximum measured TOC
Flow Rates
Flow rate | Nominal test item concentrations [µg/L] | ||||||
Control | Solvent control | 5.00 | 10.0 | 20.0 | 40.0 | 80.0 | |
Measured flow rates of the membrane piston pumps1) | |||||||
[L/h] | |||||||
Minimum | 2.88 | 2.82 | 2.94 | 2.94 | 2.82 | 2.88 | 2.88 |
Maximum | 3.12 | 3.30 | 3.24 | 3.36 | 3.24 | 3.36 | 3.12 |
Mean | 3.07 | 3.07 | 3.08 | 3.07 | 3.07 | 3.08 | 3.05 |
± Standard deviation | ± 0.060 | ± 0.076 | ± 0.068 | ± 0.068 | ± 0.064 | ± 0.079 | ± 0.064 |
Overall mean | 3.07 | ||||||
± Standard deviation | ± 0.069 |
1) Nominal flow rates of dilution water (for preparation of the test solutions): 3.125 (2.81 – 3.44 L/h)
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessel on Study Day -1 (N Hexadecyldiisopropanolamine)
Sampling day | Day -1 | |||||
Replicate | Repl. 1 | |||||
Nominal | Tallow amine propoxylate (CAS: 1309955-79-0) | |||||
concentration of the | N‑Hexadecyldiisopropanolamine | |||||
Test Vessel | Test vessel (centrifuged) | |||||
Test item | a.i. | Meas. conc. | % | Meas. conc. | % | |
[µg/L] | [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | |||
5.00 | 1.47 | 0.679 | 46 | < LOQ | ||
10.0 | 2.95 | 1.31 | 45 | 0.493 | 17 | |
20.0 | 5.89 | 6.06 | 103 | 2.32 | 39 | |
40.0 | 11.8 | 9.63 | 82 | 2.58 | 22 | |
80.0 | 23.6 | 18.2 | 77 | 4.30 | 18 | |
Solvent Control | < LOQ | < LOQ | ||||
Control | < LOQ | < LOQ |
Meas. Conc. = Measured concentration (dilution factor taken into account)
a.i. = Active ingredient
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (1.0 µg/L of the test item, corresponding to 0.295 µg a.i./L)
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessel on Study Day -1 (Octadecenyldiisopropanolamine)
Sampling day | Day -1 | |||||
Replicate | Repl. 1 | |||||
Nominal | Tallow amine propoxylate (CAS: 1309955-79-0) | |||||
concentration of the | Octadecenyldiisopropanolamine | |||||
Test Vessel | Test vessel (centrifuged) | |||||
Test item | a.i. | Meas. conc. | % | Meas. conc. | % | |
[µg/L] | [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | |||
5.00 | 2.07 | 0.930 | 45 | < LOQ | ||
10.0 | 4.14 | 1.76 | 43 | 0.592 | 14 | |
20.0 | 8.28 | 9.00 | 109 | 3.33 | 40 | |
40.0 | 16.6 | 13.9 | 84 | 3.52 | 21 | |
80.0 | 33.1 | 27.3 | 82 | 5.51 | 17 | |
Solvent Control | < LOQ | < LOQ | ||||
Control | < LOQ | < LOQ |
Meas. Conc. = Measured concentration (dilution factor taken into account)
a.i. = Active ingredient
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (1.0 µg/L of the test item, corresponding to 0.414 µg a.i./L)
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessel on Study Day -1 (Octadecyldiisopropanolamine)
Sampling day | Day -1 | |||||
Replicate | Repl. 1 | |||||
Nominal | Tallow amine propoxylate (CAS: 1309955-79-0) | |||||
concentration of the | Octadecyldiisopropanolamine | |||||
Test Vessel | Test vessel (centrifuged) | |||||
Test item | a.i. | Meas. conc. | % | Meas. conc. | % | |
[µg/L] | [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | |||
5.00 | 1.08 | 0.483 | 45 | < LOQ | ||
10.0 | 2.15 | 0.890 | 42 | 0.306 | 14 | |
20.0 | 4.30 | 5.71 | 134 | 1.99 | 46 | |
40.0 | 8.60 | 8.13 | 95 | 2.42 | 28 | |
80.0 | 17.2 | 15.7 | 4.06 | 24 | ||
Solvent Control | < LOQ | < LOQ | ||||
Control | < LOQ | < LOQ |
Meas. Conc. = Measured concentration (dilution factor taken into account)
a.i. = Active ingredient
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (1.0 µg/L of the test item, corresponding to 0.214 µg a.i./L)
Measured Concentrations in the Stock Solutions
Measured concentrations of the freshly prepared stock solution were 98 to 102% of the nominal values. Measured concentrations of the 7 days aged stock solution were in the range of 99 to 105% of the nominal values.
Measured Concentrations and Percent of Nominal Concentration in the Stock Solutions
Freshly prepared stock solutions | 7 Days aged stock solutions | |||||
Nominal | Tallow amine propoxylate (CAS: 1309955-79-0) | |||||
concentration of the | N‑Hexadecyldiisopropanolamine | |||||
Test item | a.i. | Meas. | % | Meas. | % | |
[mg/L] | [mg a.i./L] | conc. | conc. | |||
[mg a.i./L] | [mg a.i./L] | |||||
3200 | 943 | 957 | 102 | 991 | 105 | |
Freshly prepared stock solutions | 7 Days aged stock solutions | |||||
Nominal | Tallow amine propoxylate (CAS: 1309955-79-0) | |||||
concentration of the | Octadecenyldiisopropanolamine | |||||
Test item | a.i. | Meas. | % | Meas. | % | |
[mg/L] | [mg a.i./L] | conc. | conc. | |||
[mg a.i./L] | [mg a.i./L] | |||||
3200 | 1325 | 1328 | 100 | 1318 | 99 | |
Nominal | Tallow amine propoxylate (CAS: 1309955-79-0) | |||||
concentration of the | Octadecyldiisopropanolamine | |||||
Test item | a.i. | Meas. | % | Meas. | % | |
[mg/L] | [mg a.i./L] | conc. | conc. | |||
[mg a.i./L] | [mg a.i./L] | |||||
3200 | 685 | 674 | 98 | 709 | 104 |
Meas. Conc. = Measured concentration (dilution factor taken into account)
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (1.0 µg/L of the test item)
Measured Concentrations in the Test Vessels during Exposure
The concentrations of the three constituents N Hexadecyldiisopropanolamine, Octadecenyldiiso-propanolamine and Octadecyldiisopropanolamine of the test item Tallow amine propoxylate (CAS: 1309955-79-0), the solvent control and the control were determined during exposure on days 0, 7, 14, 21 and 28 from the test vessels of a defined test replicate of all tested concentration levels via LC MS/MS.
The samples taken from the test vessels were completely clear and did not contain any particles or turbidity. These samples were subjected to the analytical procedure and the results are used for the calculation of the effect values.
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day 0 to Day 28 (N Hexadecyldiisopropanolamine)
Sampling day | Day 0 | Day 7 | Day 14 | |||||||
Replicate | Repl. 2 | Repl. 3 | Repl. 4 | Rep. 1 | ||||||
Nominal | Tallow amine propoxylate (CAS: 1309955-79-0) | |||||||||
concentration of the | N‑Hexadecyldiisopropanolamine | |||||||||
Test item | a.i. | Meas. | % | Meas. | % | Meas. | % | Meas. | % | |
[µg/L] | [µg a.i./L] | conc. | conc. | conc. | conc. | |||||
[µg a.i./L] | [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | |||||||
5.00 | 1.47 | 2.03 | 138 | 1.23 | 84 | 0.945 | 64 | - | ||
10.0 | 2.95 | 2.63 | 89 | 2.31 | 782) | 1.41 | 48 | - | ||
20.0 | 5.89 | 5.97 | 101 | 4.58 | 782) | 4.36 | 74 | - | ||
40.0 | 11.8 | 11.1 | 94 | 13.8 | 117 | - 1) | 9.79 | 83 | ||
80.0 | 23.6 | 21.6 | 92 | 21.6 | 92 | - 1) | - 1) | |||
Solvent Control | < LOQ | < LOQ | < LOQ | - | ||||||
Control | < LOQ | < LOQ | < LOQ | - | ||||||
Sampling day | Day 21 | Day 28 | ||||||||
Replicate | Repl. 1 | Repl. 3 | Repl. 2 | Rep. 1 | ||||||
Nominal | Tallow amine propoxylate (CAS: 1309955-79-0) | |||||||||
concentration of the | N‑Hexadecyldiisopropanolamine | |||||||||
Test item | a.i. | Meas. | % | Meas. | % | Meas. | % | Meas. | % | |
[µg/L] | [µg a.i./L] | conc. | conc. | conc. | conc. | |||||
[µg a.i./L] | [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | |||||||
5.00 | 1.47 | 0.849 | 58 | - | 1.34 | 91 | - | |||
10.0 | 2.95 | 1.53 | 52 | - | 1.84 | 63 | - | |||
20.0 | 5.89 | 4.20 | 71 | - | 4.05 | 67 | - | |||
40.0 | 11.8 | - 1) | 7.76 | 66 | - 1) | 7.87 | 67 | |||
80.0 | 23.6 | - 1) | - 1) | - 1) | - 1) | |||||
Solvent Control | < LOQ | - | < LOQ | - | ||||||
Control | < LOQ | - | < LOQ | - |
Meas. Conc. = Measured concentration (dilution factor taken into account)
a.i. = Active ingredient
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (1.0 µg/L of the test item, corresponding to 0.295 µg a.i./L)
- = No determination of this replicate
1) = No samples due to 100% mortality
2) = Samples reanalyzed, value confirmed
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day 0 to Day 28 (Octadecenyldiisopropanolamine)
Sampling day | Day 0 | Day 7 | Day 14 | |||||||
Replicate | Repl. 2 | Repl. 3 | Repl. 4 | Rep. 1 | ||||||
Nominal | Tallow amine propoxylate (CAS: 1309955-79-0) | |||||||||
concentration of the | Octadecenyldiisopropanolamine | |||||||||
Test item | a.i. | Meas. | % | Meas. | % | Meas. | % | Meas. | % | |
[µg/L] | [µg a.i./L] | conc. | conc. | conc. | conc. | |||||
[µg a.i./L] | [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | |||||||
5.00 | 2.07 | 2.74 | 132 | 1.81 | 87 | 1.29 | 62 | - | ||
10.0 | 4.14 | 3.86 | 93 | 3.34 | 81 | 2.05 | 50 | - | ||
20.0 | 8.28 | 9.00 | 109 | 6.60 | 80 | 6.35 | 77 | - | ||
40.0 | 16.6 | 15.6 | 94 | 21.7 | 1312) | - 1) | 13.9 | 84 | ||
80.0 | 33.1 | 31.1 | 94 | 32.3 | 97 | - 1) | - 1) | |||
Solvent Control | < LOQ | < LOQ | < LOQ | - | ||||||
Control | < LOQ | < LOQ | < LOQ | - | ||||||
Sampling day | Day 21 | Day 28 | ||||||||
Replicate | Repl. 1 | Repl. 3 | Repl. 2 | Rep. 1 | ||||||
Nominal | Tallow amine propoxylate (CAS: 1309955-79-0) | |||||||||
concentration of the | Octadecenyldiisopropanolamine | |||||||||
Test item | a.i. | Meas. | % | Meas. | % | Meas. | % | Meas. | % | |
[µg/L] | [µg a.i./L] | conc. | conc. | conc. | conc. | |||||
[µg a.i./L] | [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | |||||||
5.00 | 2.07 | 1.21 | 58 | - | 1.69 | 81 | - | |||
10.0 | 4.14 | 2.40 | 58 | - | 2.39 | 58 | - | |||
20.0 | 8.28 | 6.16 | 74 | - | 5.69 | 69 | - | |||
40.0 | 16.6 | - 1) | 11.7 | 71 | -1) | 11.0 | 67 | |||
80.0 | 33.1 | - 1) | - 1) | - 1) | - 1) | |||||
Solvent Control | < LOQ | - | < LOQ | - | ||||||
Control | < LOQ | - | < LOQ | - |
Meas. Conc. = Measured concentration (dilution factor taken into account)
a.i. = Active ingredient
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (1.0 µg/L of the test item, corresponding to 0.414 µg a.i./L)
- = No determination of this replicate
1) = No samples due to 100% mortality
2) = Samples reanalyzed, value confirmed
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day 0 to Day 28 (Octadecyldiisopropanolamine)
Sampling day | Day 0 | Day 7 | Day 14 | |||||||
Replicate | Repl. 2 | Repl. 3 | Repl. 4 | Rep. 1 | ||||||
Nominal | Tallow amine propoxylate (CAS: 1309955-79-0) | |||||||||
concentration of the | Octadecyldiisopropanolamine | |||||||||
Test item | a.i. | Meas. | % | Meas. | % | Meas. | % | Meas. | % | |
[µg/L] | [µg a.i./L] | conc. | conc. | conc. | conc. | |||||
[µg a.i./L] | [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | |||||||
5.00 | 1.07 | 1.27 | 118 | 0.958 | 90 | 0.653 | 61 | - | ||
10.0 | 2.14 | 1.85 | 87 | 1.83 | 85 | 1.261 | 59 | - | ||
20.0 | 4.28 | 4.59 | 107 | 3.78 | 88 | 3.47 | 81 | - | ||
40.0 | 8.56 | 8.50 | 99 | 12.7 | 1482) | - 1) | 7.79 | 91 | ||
80.0 | 17.1 | 16.3 | 95 | 18.1 | 106 | - 1) | - 1) | |||
Solvent Control | < LOQ | < LOQ | < LOQ | - | ||||||
Control | < LOQ | < LOQ | < LOQ | - | ||||||
Sampling day | Day 21 | Day 28 | ||||||||
Replicate | Repl. 1 | Repl. 3 | Repl. 2 | Rep. 1 | ||||||
Nominal | Tallow amine propoxylate (CAS: 1309955-79-0) | |||||||||
concentration of the | Octadecyldiisopropanolamine | |||||||||
Test item | a.i. | Meas. | % | Meas. | % | Meas. | % | Meas. | % | |
[µg/L] | [µg a.i./L] | conc. | conc. | conc. | conc. | |||||
[µg a.i./L] | [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | |||||||
5.00 | 1.07 | 0.658 | 62 | - | 0.952 | 89 | - | |||
10.0 | 2.14 | 1.67 | 78 | - | 1.55 | 73 | - | |||
20.0 | 4.28 | 3.43 | 80 | - | 4.09 | 95 | - | |||
40.0 | 8.56 | - 1) | 6.32 | 74 | - 1) | 7.03 | 82 | |||
80.0 | 17.1 | - 1) | - 1) | - 1) | - | |||||
Solvent Control | < LOQ | - | < LOQ | - | ||||||
Control | < LOQ | - | < LOQ | - |
Meas. Conc. = Measured concentration (dilution factor taken into account)
a.i. = Active ingredient
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (1.0 µg/L of the test item, corresponding to 0.214 µg a.i./L)
- = No determination of this replicate
1) = No samples due to 100% mortality
2) = Samples reanalyzed, value confirmed
Overview of Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels and Calculated Overall Arithmetic Mean Measured Test Item Concentrations (Study Day 0 to 28)
Sampling day | Day 0 | Day 7 | Day 14 | Day 21 | Day 28 | |
Nominal | Tallow amine propoxylate (CAS: 1309955-79-0) | Overall arithmetric 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] | |||||
5.00 | 6.53 | 4.34 | 3.12 | 2.95 | 4.30 | 4.25 |
10.0 | 9.03 | 8.10 | 5.14 | 6.07 | 6.31 | 6.93 |
20.0 | 21.2 | 16.2 | 15.4 | 14.9 | 15.0 | 16.5 |
40.0 | 38.1 | 52.2 | 34.1 | 28.0 | 28.2 | 36.1 |
80.0 | 74.9 | 78.0 | -1) | -1) | -1) | 76.4 |
1) = No samples due to 100% mortality
Gradient Table
Time [min] | A [%] | B [%] |
0.00 | 75 | 25 |
2.00 | 5 | 95 |
3.00 | 5 | 95 |
3.10 | 75 | 25 |
3.50 | 75 | 25 |
Dilution steps (Test Vessel)
Nominal | Dilution | Sample | Final |
test item | Factor* | volume | volume |
concentration | [mL] | [mL] | |
[µg/L] | |||
80.0 | 20 | 0.1 | 1.0 |
40.0 | 10 | 0.2 | 1.0 |
20.0 | 4 | 0.5 | 1.0 |
10.0 | 2 | 1.0 | 1.0 |
5.00 | 2 | 1.0 | 1.0 |
Solvent Control | 2 | 1.0 | 1.0 |
Control | 2 | 1.0 | 1.0 |
*including factor 2
Preparation of stock solution
The stock solution was diluted with dilution medium into
samples calibration range.
Dilution steps (Stock Solution)
Nominal | Dilution | Sample | Final |
test item | Factor | volume | volume |
concentration | [mL] | [mL] | |
[mg/L] | |||
3200 | 500 000 | 0.11) | 101) |
0.12) | 102) | ||
0.13) | 5.03) |
1) first dilution step
2) second dilution step
3) third dilution step
Vital Eggs and Larvae from Study Days 0 to 34 (Control, Solvent Control and
5.00 µg/L)
Study day | Nominal test item concentration [µg/L] | |||||||||||
Control | Solvent control | 5.00 | ||||||||||
Number of vital eggs and larvae (listed eggs/larvae) by study day | ||||||||||||
1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 |
1 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 19/0 | 20/0 | 20/0 | 20/0 | 19/0 | 20/0 |
2 | 19/0 | 20/0 | 18/2 | 20/0 | 20/0 | 20/0 | 19/0 | 20/0 | 15/5 | 20/0 | 19/0 | 20/0 |
3 | 10/9 | 3/17 | 9/11 | 14/6 | 4/16 | 8/12 | 3/16 | 8/12 | 3/17 | 5/15 | 5/14 | 8/12 |
4 | 2/17 | 0/20 | 0/20 | 1/19 | 0/20 | 1/19 | 0/19 | 1/19 | 1/19 | 0/20 | 0/19 | 0/20 |
5 | 0/19 | 0/20 | 0/20 | 0/19 | 0/20 | 0/20 | 0/19 | 0/20 | 0/20 | 0/20 | 0/19 | 0/20 |
6 | 0/19 | 0/20 | 0/20 | 0/19 | 0/20 | 0/20 | 0/19 | 0/20 | 0/20 | 0/20 | 0/19 | 0/20 |
7 | 0/19 | 0/20 | 0/20 | 0/19 | 0/20 | 0/20 | 0/19 | 0/20 | 0/20 | 0/20 | 0/19 | 0/20 |
8 | 0/19 | 0/20 | 0/20 | 0/19 | 0/20 | 0/20 | 0/19 | 0/20 | 0/18 | 0/20 | 0/19 | 0/20 |
9 | 0/19 | 0/20 | 0/20 | 0/19 | 0/20 | 0/20 | 0/19 | 0/20 | 0/18 | 0/20 | 0/19 | 0/20 |
10 | 0/18 | 0/20 | 0/20 | 0/19 | 0/20 | 0/20 | 0/19 | 0/20 | 0/18 | 0/20 | 0/18 | 0/20 |
11 | 0/18 | 0/20 | 0/20 | 0/19 | 0/20 | 0/20 | 0/19 | 0/20 | 0/18 | 0/20 | 0/18 | 0/20 |
12 | 0/18 | 0/20 | 0/19 | 0/18 | 0/19 | 0/20 | 0/19 | 0/20 | 0/18 | 0/19 | 0/18 | 0/20 |
13 | 0/18 | 0/20 | 0/18 | 0/18 | 0/19 | 0/20 | 0/19 | 0/20 | 0/18 | 0/19 | 0/18 | 0/20 |
14 | 0/18 | 0/20 | 0/18 | 0/18 | 0/19 | 0/20 | 0/19 | 0/19 | 0/18 | 0/19 | 0/18 | 0/20 |
15 | 0/18 | 0/20 | 0/18 | 0/17 | 0/19 | 0/20 | 0/19 | 0/19 | 0/18 | 0/19 | 0/18 | 0/20 |
16 | 0/18 | 0/20 | 0/18 | 0/17 | 0/19 | 0/20 | 0/19 | 0/19 | 0/18 | 0/19 | 0/18 | 0/20 |
17 | 0/17 | 0/20 | 0/18 | 0/17 | 0/19 | 0/19 | 0/19 | 0/19 | 0/18 | 0/19 | 0/18 | 0/19 |
18 | 0/17 | 0/20 | 0/18 | 0/17 | 0/19 | 0/19 | 0/19 | 0/19 | 0/18 | 0/19 | 0/17 | 0/19 |
19 | 0/16 | 0/19 | 0/18 | 0/17 | 0/19 | 0/19 | 0/19 | 0/19 | 0/18 | 0/19 | 0/16 | 0/18 |
20 | 0/16 | 0/19 | 0/18 | 0/17 | 0/19 | 0/19 | 0/19 | 0/18 | 0/18 | 0/17 | 0/16 | 0/18 |
21 | 0/16 | 0/19 | 0/18 | 0/17 | 0/19 | 0/19 | 0/19 | 0/18 | 0/17 | 0/17 | 0/16 | 0/18 |
22 | 0/16 | 0/19 | 0/17 | 0/17 | 0/19 | 0/19 | 0/19 | 0/18 | 0/17 | 0/17 | 0/16 | 0/18 |
23 | 0/16 | 0/19 | 0/16 | 0/16 | 0/19 | 0/19 | 0/18 | 0/18 | 0/17 | 0/17 | 0/16 | 0/17 |
24 | 0/15 | 0/19 | 0/16 | 0/15 | 0/19 | 0/19 | 0/18 | 0/18 | 0/17 | 0/17 | 0/16 | 0/16 |
25 | 0/15 | 0/19 | 0/16 | 0/15 | 0/19 | 0/19 | 0/17 | 0/18 | 0/17 | 0/17 | 0/16 | 0/16 |
26 | 0/15 | 0/18 | 0/16 | 0/15 | 0/19 | 0/19 | 0/17 | 0/18 | 0/17 | 0/17 | 0/16 | 0/16 |
27 | 0/15 | 0/18 | 0/16 | 0/15 | 0/19 | 0/19 | 0/17 | 0/18 | 0/16 | 0/16 | 0/16 | 0/15 |
28 | 0/15 | 0/18 | 0/16 | 0/15 | 0/19 | 0/19 | 0/17 | 0/18 | 0/16 | 0/16 | 0/16 | 0/15 |
29 | 0/15 | 0/18 | 0/16 | 0/15 | 0/19 | 0/18 | 0/16 | 0/17 | 0/16 | 0/15 | 0/16 | 0/15 |
30 | 0/15 | 0/18 | 0/16 | 0/15 | 0/19 | 0/18 | 0/16 | 0/17 | 0/16 | 0/15 | 0/16 | 0/15 |
31 | 0/15 | 0/18 | 0/16 | 0/15 | 0/19 | 0/18 | 0/16 | 0/17 | 0/16 | 0/15 | 0/16 | 0/15 |
32 | 0/15 | 0/18 | 0/16 | 0/15 | 0/18 | 0/18 | 0/16 | 0/17 | 0/16 | 0/15 | 0/16 | 0/15 |
33 | 0/15 | 0/17 | 0/16 | 0/14 | 0/18 | 0/18 | 0/16 | 0/17 | 0/16 | 0/15 | 0/16 | 0/15 |
34 | 0/15 | 0/17 | 0/16 | 0/14 | 0/18 | 0/18 | 0/16 | 0/17 | 0/16 | 0/15 | 0/16 | 0/15 |
Vital Eggs and Larvae from Study Days 0 to 34 (10.0 to 40.0 µg/L)
Study day | Nominal test item concentration [µg/L] | |||||||||||
10.0 | 20.0 | 40.0 | ||||||||||
Number of vital eggs and larvae (listed eggs/larvae) by study day | ||||||||||||
1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 |
1 | 19/0 | 19/0 | 20/0 | 20/0 | 20/0 | 20/0 | 19/0 | 20/0 | 19/0 | 19/0 | 20/0 | 20/0 |
2 | 19/0 | 18/1 | 20/0 | 19/1 | 20/0 | 20/0 | 19/0 | 20/0 | 19/0 | 19/0 | 20/0 | 19/0 |
3 | 8/11 | 5/14 | 6/14 | 3/17 | 3/17 | 8/12 | 3/16 | 2/18 | 6/13 | 5/14 | 6/14 | 6/13 |
4 | 2/17 | 0/19 | 0/20 | 0/20 | 0/20 | 2/18 | 1/18 | 0/20 | 1/18 | 0/19 | 2/18 | 0/19 |
5 | 0/19 | 0/19 | 0/20 | 0/20 | 0/20 | 0/20 | 0/19 | 0/20 | 0/19 | 0/19 | 0/20 | 0/19 |
6 | 0/19 | 0/19 | 0/20 | 0/20 | 0/20 | 0/20 | 0/19 | 0/20 | 0/19 | 0/19 | 0/19 | 0/19 |
7 | 0/19 | 0/19 | 0/20 | 0/20 | 0/20 | 0/20 | 0/19 | 0/20 | 0/19 | 0/19 | 0/19 | 0/19 |
8 | 0/19 | 0/18 | 0/18 | 0/20 | 0/20 | 0/20 | 0/19 | 0/20 | 0/18 | 0/18 | 0/19 | 0/18 |
9 | 0/19 | 0/18 | 0/18 | 0/20 | 0/20 | 0/20 | 0/19 | 0/20 | 0/18 | 0/16 | 0/19 | 0/17 |
10 | 0/19 | 0/18 | 0/18 | 0/20 | 0/20 | 0/20 | 0/19 | 0/20 | 0/18 | 0/14 | 0/16 | 0/8 |
11 | 0/19 | 0/18 | 0/17 | 0/20 | 0/20 | 0/20 | 0/19 | 0/20 | 0/18 | 0/10 | 0/11 | 0/1 |
12 | 0/19 | 0/18 | 0/17 | 0/20 | 0/19 | 0/20 | 0/18 | 0/19 | 0/17 | 0/9 | 0/3 | 0/0 |
13 | 0/19 | 0/18 | 0/17 | 0/20 | 0/19 | 0/19 | 0/18 | 0/19 | 0/16 | 0/3 | 0/2 | - |
14 | 0/19 | 0/17 | 0/17 | 0/20 | 0/19 | 0/19 | 0/18 | 0/19 | 0/14 | 0/3 | 0/2 | - |
15 | 0/19 | 0/17 | 0/17 | 0/20 | 0/19 | 0/18 | 0/18 | 0/19 | 0/13 | 0/3 | 0/2 | - |
16 | 0/19 | 0/16 | 0/17 | 0/20 | 0/19 | 0/17 | 0/17 | 0/18 | 0/12 | 0/3 | 0/2 | - |
17 | 0/19 | 0/15 | 0/17 | 0/20 | 0/17 | 0/17 | 0/17 | 0/17 | 0/12 | 0/3 | 0/2 | - |
18 | 0/19 | 0/15 | 0/15 | 0/19 | 0/15 | 0/15 | 0/16 | 0/13 | 0/7 | 0/0 | 0/2 | - |
19 | 0/19 | 0/15 | 0/14 | 0/19 | 0/15 | 0/14 | 0/15 | 0/12 | 0/6 | - | 0/2 | - |
20 | 0/18 | 0/15 | 0/14 | 0/17 | 0/13 | 0/12 | 0/15 | 0/12 | 0/5 | - | 0/1 | - |
21 | 0/18 | 0/14 | 0/12 | 0/15 | 0/11 | 0/10 | 0/15 | 0/11 | 0/4 | - | 0/0 | - |
22 | 0/18 | 0/14 | 0/11 | 0/15 | 0/10 | 0/9 | 0/15 | 0/11 | 0/4 | - | - | - |
23 | 0/18 | 0/13 | 0/9 | 0/15 | 0/10 | 0/9 | 0/15 | 0/11 | 0/4 | - | - | - |
24 | 0/17 | 0/12 | 0/9 | 0/15 | 0/9 | 0/9 | 0/15 | 0/11 | 0/4 | - | - | - |
25 | 0/17 | 0/12 | 0/9 | 0/15 | 0/9 | 0/8 | 0/15 | 0/11 | 0/4 | - | - | - |
26 | 0/17 | 0/12 | 0/9 | 0/13 | 0/9 | 0/8 | 0/15 | 0/11 | 0/4 | - | - | - |
27 | 0/17 | 0/11 | 0/9 | 0/12 | 0/9 | 0/8 | 0/15 | 0/11 | 0/4 | - | - | - |
28 | 0/17 | 0/11 | 0/9 | 0/12 | 0/9 | 0/8 | 0/14 | 0/11 | 0/4 | - | - | - |
29 | 0/17 | 0/11 | 0/9 | 0/12 | 0/8 | 0/8 | 0/13 | 0/11 | 0/4 | - | - | - |
30 | 0/17 | 0/11 | 0/9 | 0/12 | 0/8 | 0/8 | 0/13 | 0/11 | 0/4 | - | - | - |
31 | 0/17 | 0/11 | 0/9 | 0/12 | 0/8 | 0/8 | 0/13 | 0/11 | 0/4 | - | - | - |
32 | 0/17 | 0/11 | 0/9 | 0/12 | 0/8 | 0/8 | 0/13 | 0/11 | 0/3 | - | - | - |
33 | 0/17 | 0/11 | 0/8 | 0/12 | 0/8 | 0/8 | 0/13 | 0/11 | 0/3 | - | - | - |
34 | 0/17 | 0/11 | 0/8 | 0/12 | 0/8 | 0/8 | 0/13 | 0/11 | 0/3 | - | - | - |
- = 100% mortality
Vital Eggs and Larvae from Study Days 0 to 11 (80.0 µg/L)
Study day | Nominal test item concentration [µg/L] | |||
80.0 | ||||
Number of vital eggs and larvae (listed eggs/larvae) by study day | ||||
1 | 2 | 3 | 4 | |
0 | 20/0 | 20/0 | 20/0 | 20/0 |
1 | 18/0 | 18/0 | 19/0 | 20/0 |
2 | 18/0 | 15/3 | 19/0 | 20/0 |
3 | 5/13 | 2/16 | 10/9 | 7/13 |
4 | 2/16 | 0/16 | 0/17 | 0/20 |
5 | 0/18 | 0/16 | 0/17 | 0/20 |
6 | 0/18 | 0/16 | 0/17 | 0/20 |
7 | 0/16 | 0/16 | 0/17 | 0/20 |
8 | 0/12 | 0/3 | 0/8 | 0/10 |
9 | 0/2 | 0/0 | 0/4 | 0/3 |
10 | 0/1 | - | 0/2 | 0/2 |
11 | 0/0 | - | 0/0 | 0/0 |
- = 100% mortality
Method Validation (non-GLP)
The analytical method was validated according to SANTE/2020/12830, Rev. 1 (2021), Guidance Document on Pesticide Analytical Methods for Risk Assessment and Post-approval Control and Monitoring Purposes using the criteria in the table below.
Parameter, Acceptance Criteria and Results of the Method Validation
Parameter | Acceptance criteria | Result | |
Linearity | ≥ 5 standard concentrations, Calibration range from ≤ 30% LOQ to 20% above highest concentration level after sample preparation | 0.1 to 10 µg test item/L (n = 8), r ≥ 0.99 | ü |
Matrix effects | Should be ≤ 20% | Matrix effects: < 20% | ü |
Lowest calibration level (LCL) | S/N ≥ 9 for quantifier ion trace S/N ≥ 3 for qualifier ion trace | S/N for 0.1 µg test item/L N-Hexadecyldiisopropanolamine 1499 (Quantifier), 700 (Qualifier) Octadecenyldiisopropanolamine 1264 (Quantifier), 1572 (Qualifier) Octadecyldiisopropanolamine 604 (Quantifier), 205 (Qualifier) | ü |
Limit of Quantification (LOQ) | Should be at or below lowest test concentration | 1.0 µg test item/L (1 x LOQ) 100 µg test item/L (100 x LOQ) | ü |
Accuracy1) | Mean recovery rate of 70-120% | N-Hexadecyldiisopropanolamine 1 x LOQ: 98% (n = 5) 100 x LOQ: 107% (n = 5) Octadecenyldiisopropanolamine 1 x LOQ: 98% (n = 5) 100 x LOQ: 106% (n = 5) Octadecyldiisopropanolamine 1 x LOQ: 96% (n = 5) 100 x LOQ: 105% (n = 5) | ü |
Precision1) | Relative standard deviation ≤ 20% per fortification level | N-Hexadecyldiisopropanolamine 1 x LOQ: 3.5% 100 x LOQ: 2.4% Octadecenyldiisopropanolamine 1 x LOQ: 2.9% 100 x LOQ: 1.8% Octadecyldiisopropanolamine 1 x LOQ: 3.0% 100 x LOQ: 1.8% | ü |
Parameter | Acceptance criteria | Result | |
Stability | Samples: Stable if 70 – 120% of freshly prepared sample Standards: Stable if ±10% of freshly prepared | See section 18.1 | ü |
Specificity: LC-MS/(MS) | Measurement of two transitions of the same precursor ion - one quantifier (used for evaluation) and one qualifier (for confirmation of the analyte identity).
| N-Hexadecyldiisopropanolamine quantifier [m/z]: 358.52 > 98.18 Octadecenyldiisopropanolamine quantifier [m/z]: 384.47 > 366.50 Octadecyldiisopropanolamine quantifier [m/z]: 386.49 > 368.52 | ü |
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 18.2 | ü |
Preparation of the The method was validated at the 1 x LOQ (1.0 µg test item/L)
fortified samples and at the 100 x LOQ ( 100 µg test item/L).
For preparation and dilution steps, please refer to table below.
Preparation of Fortified Samples
LOQ Level | Control | 1 | 100 |
Stock solution | - | 1000 mg test item/L in methanol | |
Spiking solution (Medium) | - | 0.1 (Dilution medium) | 10 (Dilution medium) |
Replicates | 2 | 5 | 5 |
Concentration of the LOQ | - | 1.0 | 100 |
Medium for preparation | Fish dilution medium | ||
Volume of spiking solution [mL] | - | 0.05 | 0.05 |
Volume of medium [mL] | 5.0 | 4.95 | 4.95 |
Dilution factor | 2 | 2 | 20 |
Dilution medium | Acetonitrile 1) Dilution medium 2) | ||
Sample volume [mL] | 5.01) | 5.01) | 5.01) 0.12) |
Finale volume [mL] | 101) | 101) | 101) 1.02) |
Test item concentration in the final solution [µg/L] | - | 0.5 | 5.0 |
Dilution medium: Acetonitrile : ultra-pure water (50 : 50 v/v)
1) First dilution step
2) Second dilution step
Nominal Concentrations of the Fortified Samples of the active ingredients of Tallow amine propoxylate (CAS: 1309955-79-0)
Fortified concentrations*: 1.07 µg test item/L (1 x LOQ) and 107 µg test item/L (100 x LOQ).
Active ingredient | 1 x LOQ [µg a.i./L] | 100 x LOQ [µg a.i./L] |
N-Hexadecyldiisopropanolamine | 0.315 | 31.5 |
Octadecenyldiisopropanolamine | 0.443 | 44.3 |
Octadecyldiisopropanolamine | 0.229 | 22.9 |
* = weighing factor taken into account
a.i. = active ingredient
Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of Tallow amine propoxylate (CAS: 1309955-79-0)
Active ingredient: N-Hexadecyldiisopropanolamine and Octadecenyldiisopropanolamine
Replicate | Tallow amine propoxylate (CAS: 1309955-79-0) | |||
N-Hexadecyldiisopropanolamine | ||||
1 x LOQ | 100 x LOQ | |||
Meas.conc. | % | Meas.conc. | % | |
1 | 0.306 | 97 | 33.1 | 105 |
2 | 0.319 | 101 | 32.7 | 104 |
3 | 0.293 | 93 | 33.8 | 107 |
4 | 0.320 | 101 | 34.8 | 110 |
5 | 0.307 | 97 | 34.0 | 108 |
Mean | 0.31 | 98 | 33.7 | 107 |
SD ± | 0.01 |
| 0.8 |
|
CV [%] | 3.5 |
| 2.4 |
|
Replicate | Tallow amine propoxylate (CAS: 1309955-79-0) | |||
Octadecenyldiisopropanolamine | ||||
1 x LOQ | 100 x LOQ | |||
Meas.conc. | % | Meas.conc. | % | |
1 | 0.416 | 94 | 47.2 | 107 |
2 | 0.436 | 98 | 45.6 | 103 |
3 | 0.437 | 99 | 47.1 | 106 |
4 | 0.448 | 101 | 47.6 | 108 |
5 | 0.423 | 96 | 47.8 | 108 |
Mean | 0.43 | 98 | 47.1 | 106 |
SD ± | 0.01 |
| 0.9 |
|
CV [%] | 2.9 |
| 1.8 |
|
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 variation
Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of Tallow amine propoxylate (CAS: 1309955-79-0)
Active ingredient: Octadecyldiisopropanolamine
Replicate | Tallow amine propoxylate (CAS: 1309955-79-0) | |||
Octadecyldiisopropanolamine | ||||
1 x LOQ | 100 x LOQ | |||
Meas. conc. | % | Meas. conc. | % | |
1 | 0.212 | 92 | 23.7 | 104 |
2 | 0.221 | 97 | 23.7 | 104 |
3 | 0.218 | 95 | 24.0 | 105 |
4 | 0.224 | 98 | 24.4 | 107 |
5 | 0.229 | 100 | 24.7 | 108 |
Mean | 0.221 | 96 | 24.1 | 105 |
SD ± | 0.007 |
| 0.4 |
|
CV [%] | 3.0 |
| 1.8 |
|
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 variation
Stability
The stability of the standard stock solution (1000 mg test item/L in acetonitrile) was checked after 21 days and was 99 to 100% of the nominal concentration and was found to be stable over this period.
The responses of an 8 days old calibration standard (30 µg test item/L) was compared with the responses of a freshly prepared calibration standard with the same concentration. The deviation was < 10% for all constituents and was found to be stable over this period.
The stability of the fortified samples at the 1xLOQ was checked after 1, 7 and after 15 days. For results, see table below.
Measured Concentrations and Percent of Nominal Concentration of the stored fortified samples
| 1 day | 7 days | 15 days | |||
Component | Meas. conc. [µg a.i./L] | % | Meas. conc. [µg a.i./L] | % | Meas. conc. [µg a.i./L] | % |
N-Hexadecyldiisopropanolamine | 0.323 | 102 | 0.297 | 94 | 0.294 | 93 |
Octadecenyldiisopropanolamine | 0.453 | 102 | 0.428 | 97 | 0.430 | 97 |
Octadecyldiisopropanolamine | 0.255 | 111 | 0.254 | 111 | 0.225 | 98 |
Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration
a.i. = active ingredient
Procedural Recovery
A procedural recovery (Quality Control) on 1 x LOQ Level was freshly prepared on each day of analysis. It was treated in parallel to the test samples. For results, see table below.
Measured Concentrations and Percent of Nominal Concentration of the Quality Control during the Definitive Test
Component | N-Hexadecyldiiso-propanolamine | Octadecenyldiiso-propanolamine | Octadecyldiiso-propanolamine | |||
Sampling date | Meas. conc. [µg a.i./L] | % | Meas. conc. [µg a.i./L] | % | Meas. conc. [µg a.i./L] | % |
Day -1 | 0.289 | 95 | 0.412 | 97 | 0.217 | 99 |
Day 0 | 0.299 | 99 | 0.435 | 102 | 0.229 | 104 |
Day 7 | 0.330 | 109 | 0.480 | 113 | 0.244 | 111 |
0.319 | 1052) | 0.464 | 1082) | 0.241 | 1092) | |
Day 121) | 0.321 | 106 | 0.453 | 106 | 0.238 | 108 |
Day 14 | 0.286 | 98 | 0.407 | 100 | 0.211 | 100 |
Day 191) | 0.293 | 101 | 0.434 | 106 | 0.212 | 101 |
Day 21 | 0.292 | 101 | 0.447 | 110 | 0.219 | 104 |
Day 28 | 0.314 | 108 | 0.429 | 105 | 0.224 | 106 |
Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration
Quality Control = 1.00 µg test item/L, weighing factor taken into account
1) = sampling date of the stock solution
2) = Quality Control of reanalyzed samples
Mortality [%] and Hatch [%] in the Range Finding Test (non-GLP)
(n = 40 per treatment group with 2 replicates and 20 eggs each under flow-through conditions)
Nominal test item concentration | Cumulative | Post hatch | Overall | Mortality [%] |
100 | 98 | 0* | 0* | 100* |
10 | 98 | 97 | 95 | 5 |
Solvent control | 100 | 98 | 98 | 2 |
* 100 % mortality of hatched larvae was already observed on study day 11
Description of key information
One valid long-term fish study is available. The study was performed according to OECD TG 210 using a flow-through test design. The study with 2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol (PFAPO T, CAS 1339955-79-0) resulted in a NOEC for overall survival and post-hatch survival of 4.25 µg/L based on mean measured test item concentrations and 5.00 µg/L based on nominal test item concentrations.
Key value for chemical safety assessment
Fresh water fish
Fresh water fish
- Dose descriptor:
- NOEC
- Effect concentration:
- 4.25 µg/L
Additional information
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.