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Ecotoxicological information

Long-term toxicity to fish

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Reference
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.

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)

























































































































































































































































































































NominalRep.PHD -2PHD -1PHD 0PHD 1PHD 2
test item conc.Study day 2Study day 3Study day 4Study day 5Study day 6
[µg/L]Cumulative hatching rate [%] 
Control1045859595
2085100100100
31055100100100
4030959595
Mean354959898
Solvent control1080100100100
206095100100
3080959595
406095100100
Mean070969999
5.001258595100100
2075100100100
3070959595
4060100100100
Mean67398(ns)99(ns)99
10.01055859595
2570959595
3070100100100
4585100100100
Mean37095(ns)98(ns)98
20.01085100100100
206090100100
3080909595
4090100100100
Mean07995(ns)99(ns)99
40.01065909595
2070959595
307090100100
4065959595
Mean06893(ns)96(ns)96
80.01065809090
21580808080
3045909090
4065100100100
Mean46488(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

























































































































































































































































































































NominalRep.PHD 0PHD 1PHD 2PHD 3PHD 4
test item conc.Study day 4Study day 5Study day 6Study day 7Study day 8
[µg/L]Cumulative swim up [%]
Control10100100100100
20100100100100
30100100100100
40100100100100
Mean0100100100100
Solvent control10100100100100
2090100100100
30100100100100
40100100100100
Mean098100100100
5.0010959590100
20100100100100
30100100100100
40100100100100
Mean0999998100
10.010100100100100
20100100100100
3010010010094
40100100100100
Mean010010010099
20.010100100100100
20100100100100
30100100100100
40100100100100
Mean0100100100100
40.01010010095100
20100100100100
30951009595
401001008478
Mean0991009493
80.010891007550
20100100880
303594760
4090100850
Mean079998113

 


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







































































































































































NominalRep.Eggs introduced on study day 0Cumulative number of hatched larvaeVital Larvae on study day 34Post-Hatch survival
test item conc.(PHD 30)on study day 34
[µg/L] [%]
Control120191579
220201785
320201680
420191474
Mean2019.51680
Solvent control120201890
220201890
320191684
420201785
Mean2019.81787
5.00120201680
220201575
320191684
420201575
Mean2019.81679 (ns)
10.0120191789
220191158
32020840
420201260
Mean2019.51262 (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



































































































































NominalRep.Eggs introduced on study day 0Cumulative number of hatched larvaeVital Larvae on study day 34Post-Hatch survival
test item conc.(PHD 30)on study day 34
[µg/L] [%]
20.012020840
22020840
320191368
420201155
Mean2019.81051 (s)
40.012019316
2201900
3202000
4201900
Mean2019.314 (s)
80.01201800
2201600
3201700
4202000
Mean2017.8 00 (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















































































































































































NominalRep.Eggs introduced on study day 0Vital larvae onOverall survivalOverall mortality
test item conc.study day 34  
[µg/L](PHD 30)  
  [%][%]
Control120157525
220178515
320168020
420147030
Mean20167822
Solvent control120189010
220189010
320168020
420178515
Mean20178614
5.00120168020
220157525
320168020
420157525
Mean201678 (ns)22
10.0120178515
220115545
32084060
420126040
Mean201260 (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











































































































































NominalRep.Eggs introduced on study day 0Vital larvae onOverall survivalOverall mortality
test item conc.study day 34  
[µg/L](PHD 30)  
  [%][%]
20.012084060
22084060
320136535
420115545
Mean201050 (s)50
40.012031585
22000100
32000100
42000100
Mean2014 (s)96
80.012000100
22000100
32000100
42000100
Mean2000 (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 Day5.00 µg/L10.0 µg/L
12341234
61 Sc -------
71Sc-------
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 F1 F2 F
1 E1 E2 E
251 G1 G-1 G---1 G
1 F1 F1 F1 F
1 E1 E1 E1 E
261 G1 G-1 G 1 F1 F-
1 F1 F1 F1 E1 E
1 E1 E1 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 Day20.0 µg/L40.0 µg/L
12341234
7----1 G-1 G3 G
1 Q1 Q3 Q
8------1 G4 G
1 Q4 Q
9-----1 G1 G1 G
1 Q1 Q1 Q
10--1 Q1 Q-14 G7 E8 G
1 G1 G14 Q1 M8 M
   4 G 
   7 Q 
   4 S 
   1 T 
11--1 Q-4 E7 G5 E1 G
1 G4 G7 M6 G1 Q
 2 Q 6 Q 
 2 S 5 S 
12----4 E7 G1 G                 100 % Mortality                    
3 G7 M1 M
1 Q  
2 S  
13----3 E1 E-
3 G1 S
14----4 E1 E-
4 G1 S
15-16----3 E1 E-
3 G1 G
17----3 E2 E-
3 G2 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
251 E1 E2 E--
1 F1 G2 F
1 G   
261 E1 E2 E--
1 F1 F2 F
1 G 1 G  
271 E-2 E--
1 F
1 G 
281 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 Day80.0 µg/L
1234
74 G2 G4 G3 G
4 Q2 Q4 Q3 Q
86 F3 F8 F10 F
6 G3 Q5 G7 G
6 Q 8 Q10 Q
92 F100 % Mortality4 F3 F
2 G4 G3 G
2 Q4 Q3 Q
101 F100 % Mortality2 F2 F
1 M2 M2 M
11100 % 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































































































































































NominalRep.PHD 30 (End of exposure)
test item conc. [µg/L]Mean total length per fish [mm]Mean wet weight per fish [mg]
Control114.627.0
214.327.5
314.626.3
415.129.9
Mean14.727.7
± SD0.2871.35
CV [%]1.954.88
Solvent control114.627.4
213.825.5
314.327.9
415.429.8
Mean14.527.7
± SD0.5801.53
CV [%]4.005.53
5.00113.321.5
214.230.4
313.224.5
413.625.4
Mean13.6 (ns)25.5 (ns)
± SD0.3903.20
CV [%]2.8712.6
10.0113.622.2
213.928.4
315.238.3
414.830.8
Mean14.4 (ns)29.9 (ns)
± SD0.6505.76
CV [%]4.5119.3

(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























































































NominalRep.PHD 30 (End of exposure)
test item conc.[µg/L]Mean total length per fish [mm]Mean wet weight per fish [mg]
20.0113.319.3
212.521.5
313.624.0
412.322.3
Mean12.9 (s)21.8 (s)
± SD0.5401.69
CV [%]4.197.75
40.0111.719.7
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.ControlSolvent control
Total length of individual fish in [mm]
12341234
114.010.07.518.514.014.018.515.0
216.59.015.517.013.020.516.514.0
318.59.016.511.512.017.010.513.0
413.012.517.518.59.017.010.015.0
511.016.516.014.510.510.520.020.5
619.019.514.011.018.510.020.016.5
715.515.011.512.015.512.017.515.0
815.016.017.510.016.013.017.014.0
914.014.010.017.517.015.516.517.0
1017.512.019.520.016.015.510.013.0
1114.518.017.016.016.017.511.019.0
1212.013.015.017.018.513.515.015.0
1318.016.015.014.015.013.08.518.0
148.015.010.014.010.513.010.010.0
1512.518.017.5 -15.09.018.018.0
1618.013.0 -17.011.013.017.0
17 -12.0  -16.012.511.011.0
18 - - -14.014.0 -
19 - - - - - - -
20 - - - - -
Mean14.614.314.615.114.613.814.315.4
± SD2.953.163.233.042.652.873.842.69
CV %20.222.122.120.118.120.826.917.5

- = 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]
5.0010.0
Total length of individual fish in [mm]
12341234
19.015.019.09.09.510.020.014.5
212.014.020.013.011.510.019.018.0
312.09.014.014.012.014.010.016.5
412.018.08.58.012.09.021.09.0
517.017.014.020.013.011.516.018.0
617.015.014.018.017.020.515.013.0
712.015.08.014.010.010.010.520.0
813.016.016.512.09.015.010.08.0
99.014.08.014.016.016.5-17.0
1018.018.011.011.010.519.5-13.0
1113.020.512.014.517.017.0-14.0
1219.011.012.518.016.0--16.5
1315.511.014.517.016.0---
1414.011.09.510.014.0---
1510.011.019.011.017.0---
1611.012.010.5-18.0---
17----13.0---
18--------
19--------
20--------
Mean13.314.213.213.613.613.915.214.8
± SD3.043.113.813.412.893.914.303.48
CV %22.821.928.825.121.328.128.323.5

- = 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]
20.040.0
Total length of individual fish in [mm]
12341234
116.012.014.019.019.5---
213.010.016.517.08.0---
318.015.015.013.07.5---
48.518.013.010.5----
515.016.015.012.0----
611.011.010.011.0----
715.010.013.08.0----
810.08.09.014.0----
9--16.011.5----
10--18.512.0----
11--17.07.5----
12--10.0-----
13--10.0-----
14--------
15--------
16--------
17--------
18--------
19--------
20--------
Mean13.312.513.612.311.7---
± SD3.053.242.973.275.54---
CV %22.925.921.826.647.4---

- = 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












































































































































































































NominalReplicateNumber of fish alivePooledMeanMean± SDCV %
test item conc.on study day 34wet weightwet weight 
[µg/L] per replicateper fish per replicate 
  [mg][mg][mg]
Control115405.327.027.71.3534.88
217467.127.5
316421.326.3
414418.929.9
Solvent control118493.327.427.71.5315.53
218458.625.5
316447.027.9
417507.329.8
5.00116343.521.525.53.20212.56
215456.130.4
316392.424.5
415381.725.4
10.0117377.822.229.95.76419.28
211312.728.4
38306.538.3
412369.930.8
20.018154.519.321.81.6907.75
28171.721.5
313312.024.0
411245.222.3
40.01359.019.7n.a.n.a.n.a.
20--
30--
40--

n.a. = Not applicable


 


Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 0 -15)






























































































































































































































































































































































Study dayReplicateDissolved oxygen [%]
Nominal test item concentrations [µg/L]
ControlSolvent control5.0010.020.040.080.0
0110098100999910099
299999999999999
3999810099999999
499999999989999
111001009999999999
2100991001001009998
399989999999999
4999910099989991
411001001001009910099
2999999991009999
3999910099999999
499999999999999
611001009998989898
299989898999998
399989998989899
4991009998989899
8199989596989796
298969798979796
397959897979696
499989895979798
11199989696979897
296959596979696
397959796969597
498979696969597
131989593959498-
2959394959695-
3959396979391-
49694969394--
151989389919397-
2959295949694-
3949394949388-
49694949091--

- = No measurement, due to 100 % mortality


 


Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 18 - 32)





























































































































































































































































































































































Study dayReplicateDissolved oxygen [%]
Nominal test item concentrations [µg/L]
ControlSolvent control5.0010.020.040.080.0
181989684899397-
2969495969696-
3969595929385-
49795959195--
201969482919494-
29593959396--
3959595949680-
49593959195--
221979589959597-
29692969697--
39495969596--
49491959296--
251959694959496-
29695969596--
39594939595--
49691949293--
271949393929496-
29390959295--
39390929492--
49287888891--
291959293939495-
29391969496--
39492919192--
49288938991--
321969591959595-
29393969596--
39695929495--
49491959192--
Mean97959595969698
± SD 2.293.163.733.122.434.031.85
Min.92878288918091
Max.10010010010010010099

- = 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 measurements2022-01-13 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 dayRep.Water temperature [°C]
Nominal test item concentrations [µg/L]
ControlSolvent control5.0010.020.040.080.0
0126.326.626.826.426.626.826.5
226.426.326.126.226.226.426.4
326.126.426.226.226.226.326.0
426.126.126.126.426.226.126.4
7126.426.726.726.326.726.426.5
226.626.426.326.626.426.526.5
326.526.626.526.526.526.526.3
426.526.626.326.726.526.826.4
14126.126.326.326.226.326.5-
226.126.125.926.026.026.2-
326.126.126.026.025.926.0-
426.026.025.926.226.0--
20126.126.526.526.326.226.6-
226.126.326.126.226.3--
326.026.526.126.226.226.2-
426.126.026.026.426.2--
28126.426.526.425.826.126.3-
226.126.026.025.725.9--
326.026.226.025.925.7--
426.026.125.926.226.0--
Mean26,226.326.226.226.226.426.4
± SD 0,190.230.260.250.250.240.17
Min.26,026.025.925.725.726.026.0
Max.26,626.726.826.726.726.826.5

- = No measurement, due to 100 % mortality
Min./Max. = Minimum/Maximum measured water temperature


 


pH-Values in the Test Media











































































































































































































































































Study dayRep.pH-value
Nominal test item concentrations [µg/L]
ControlSolvent control5.0010.020.040.080.0
017.917.877.927.827.827.887.89
27.897.797.847.827.847.847.84
37.817.827.927.817.857.817.79
47.877.847.827.837.817.827.83
717.998.018.028.038.128.038.02
28.017.987.968.068.078.038.02
38.008.068.058.008.058.058.08
48.078.068.018.008.028.108.04
1417.877.917.747.687.838.01-
27.887.747.817.857.957.95-
37.837.777.947.847.867.89-
48.027.877.587.787.80--
2017.907.957.907.947.928.00-
27.897.857.997.927.98--
37.737.917.987.947.977.77-
47.937.867.947.847.92--
2817.777.727.717.757.747.78-
27.707.677.777.747.73--
37.757.757.747.727.75--
47.767.747.717.647.69--
Mean7,887.867.877.857.897.937.94
± SD 0,1050.1130.1280.1180.1220.1110.113
Min.7,707.677.587.647.697.777.79
Max.8,078.068.058.068.128.108.08

- = 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 dayReplicateTotal hardness [mg CaCO3/L]
Nominal test item conc. [µg/L]
Control40.080.0
1168 59
8267 66
1536564 
224/16362 
2916765 
Mean666463
± SD2.001.534.95
Min.636259
Max.686566

= 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 dayTOC [mg/L]
01.28
71.28
151.25
221.18
281.19
Mean1.24
SD0.048
Min.1.18
Max.1.28

Min./Max. = Minimum/Maximum measured TOC


 


Flow Rates








































































Flow rateNominal test item concentrations [µg/L]
ControlSolvent control5.0010.020.040.080.0
Measured flow rates of the membrane piston pumps1)
[L/h]
Minimum2.882.822.942.942.822.882.88
Maximum3.123.303.243.363.243.363.12
Mean3.073.073.083.073.073.083.05
± Standard deviation ± 0.060± 0.076± 0.068± 0.068± 0.064± 0.079± 0.064
Overall mean3.07
± 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 dayDay -1 
ReplicateRepl. 1
NominalTallow amine propoxylate (CAS: 1309955-79-0)
concentration of theN‑Hexadecyldiisopropanolamine
 Test VesselTest vessel (centrifuged) 
Test itema.i.Meas. conc. %Meas. conc. %
[µg/L][µg a.i./L][µg a.i./L][µg a.i./L]
5.001.470.67946< LOQ
10.02.951.31450.49317
20.05.896.061032.3239
40.011.89.63822.5822
80.023.618.277 4.3018
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 dayDay -1
ReplicateRepl. 1
NominalTallow amine propoxylate (CAS: 1309955-79-0)
concentration of theOctadecenyldiisopropanolamine
 Test VesselTest vessel (centrifuged)
Test itema.i.Meas. conc. %Meas. conc. %
[µg/L][µg a.i./L][µg a.i./L][µg a.i./L]
5.002.070.93045< LOQ
10.04.141.76430.59214
20.08.289.001093.3340
40.016.613.9843.5221
80.033.127.382 5.5117
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 dayDay -1
ReplicateRepl. 1
NominalTallow amine propoxylate (CAS: 1309955-79-0)
concentration of theOctadecyldiisopropanolamine
 Test VesselTest vessel (centrifuged)
Test itema.i.Meas. conc. %Meas. conc. %
[µg/L][µg a.i./L][µg a.i./L][µg a.i./L]
5.001.080.48345< LOQ
10.02.150.890420.30614
20.04.305.711341.9946
40.08.608.13952.4228
80.017.215.7 4.0624
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 solutions7 Days aged stock solutions
NominalTallow amine propoxylate (CAS: 1309955-79-0)
concentration of theN‑Hexadecyldiisopropanolamine
Test itema.i.Meas.%Meas.%
[mg/L][mg a.i./L]conc. conc. 
  [mg a.i./L][mg a.i./L]
3200943957102991105
 Freshly prepared stock solutions7 Days aged stock solutions
NominalTallow amine propoxylate (CAS: 1309955-79-0)
concentration of theOctadecenyldiisopropanolamine
Test itema.i.Meas.%Meas.%
[mg/L][mg a.i./L]conc. conc. 
  [mg a.i./L][mg a.i./L]
320013251328100131899
NominalTallow amine propoxylate (CAS: 1309955-79-0)
concentration of theOctadecyldiisopropanolamine
Test itema.i.Meas.%Meas.%
[mg/L][mg a.i./L]conc. conc. 
  [mg a.i./L][mg a.i./L]
320068567498709104

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 dayDay 0Day 7Day 14
ReplicateRepl. 2Repl. 3Repl. 4Rep. 1
NominalTallow amine propoxylate (CAS: 1309955-79-0)
concentration of theN‑Hexadecyldiisopropanolamine
Test itema.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]
5.001.472.031381.23840.94564-
10.02.952.63892.31782)1.4148-
20.05.895.971014.58782)4.3674-
40.011.811.19413.81171)9.7983
80.023.621.69221.692- 1)- 1)
Solvent Control< LOQ< LOQ< LOQ-
Control< LOQ< LOQ< LOQ-
Sampling dayDay 21Day 28
ReplicateRepl. 1Repl. 3Repl. 2Rep. 1
NominalTallow amine propoxylate (CAS: 1309955-79-0)
concentration of theN‑Hexadecyldiisopropanolamine
Test itema.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]
5.001.470.84958-1.3491-
10.02.951.5352-1.8463-
20.05.894.2071-4.0567-
40.011.81)7.7666- 1)7.8767
80.023.6- 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 dayDay 0Day 7Day 14
ReplicateRepl. 2Repl. 3Repl. 4Rep. 1
NominalTallow amine propoxylate (CAS: 1309955-79-0)
concentration of theOctadecenyldiisopropanolamine
Test itema.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]
5.002.072.741321.81871.2962-
10.04.143.86933.34812.0550-
20.08.289.001096.60806.3577-
40.016.615.69421.71312)- 1)13.984
80.033.131.19432.397- 1)- 1)
Solvent Control< LOQ< LOQ< LOQ-
Control< LOQ< LOQ< LOQ-
Sampling dayDay 21Day 28
ReplicateRepl. 1Repl. 3Repl. 2Rep. 1
NominalTallow amine propoxylate (CAS: 1309955-79-0)
concentration of theOctadecenyldiisopropanolamine
Test itema.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]
5.002.071.2158-1.6981-
10.04.142.4058-2.3958-
20.08.286.1674-5.6969-
40.016.6- 1)11.771-1)11.067
80.033.1- 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 dayDay 0Day 7Day 14
ReplicateRepl. 2Repl. 3Repl. 4Rep. 1
NominalTallow amine propoxylate (CAS: 1309955-79-0)
concentration of theOctadecyldiisopropanolamine
Test itema.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]
5.001.071.271180.958900.65361-
10.02.141.85871.83851.26159-
20.04.284.591073.78883.4781-
40.08.568.509912.71482)- 1)7.7991
80.017.116.39518.1106- 1)- 1)
Solvent Control< LOQ< LOQ< LOQ-
Control< LOQ< LOQ< LOQ-
Sampling dayDay 21Day 28
ReplicateRepl. 1Repl. 3Repl. 2Rep. 1
NominalTallow amine propoxylate (CAS: 1309955-79-0)
concentration of theOctadecyldiisopropanolamine
Test itema.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]
5.001.070.65862-0.95289-
10.02.141.6778-1.5573-
20.04.283.4380-4.0995-
40.08.56- 1)6.3274- 1)7.0382
80.017.1- 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 0Day 7Day 14Day 21Day 28 
 
NominalTallow 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]
5.006.534.343.122.954.304.25
10.09.038.105.146.076.316.93
20.021.216.215.414.915.016.5
40.038.152.234.128.028.236.1
80.074.978.0-1)-1)-1)76.4

1) = No samples due to 100% mortality


 


 


 


 


 


Gradient Table



































Time [min]A [%]B [%]
0.007525
2.00595
3.00595
3.107525
3.507525

 


Dilution steps (Test Vessel)







































































NominalDilutionSampleFinal
test itemFactor*volumevolume
concentration [mL][mL]
[µg/L]   
80.0200.11.0
40.0100.21.0
20.040.51.0
10.021.01.0
5.0021.01.0
Solvent Control21.01.0
Control21.01.0

*including factor 2


Preparation of stock solution


The stock solution was diluted with dilution medium into
samples calibration range.


 


Dilution steps (Stock Solution)











































NominalDilutionSampleFinal
test itemFactorvolumevolume
concentration [mL][mL]
[mg/L]   
3200500 0000.11)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 dayNominal test item concentration [µg/L]
ControlSolvent control5.00
Number of vital eggs and larvae (listed eggs/larvae) by study day
123412341234
020/020/020/020/020/020/020/020/020/020/020/020/0
120/020/020/020/020/020/019/020/020/020/019/020/0
219/020/018/220/020/020/019/020/015/520/019/020/0
310/93/179/1114/64/168/123/168/123/175/155/148/12
42/170/200/201/190/201/190/191/191/190/200/190/20
50/190/200/200/190/200/200/190/200/200/200/190/20
60/190/200/200/190/200/200/190/200/200/200/190/20
70/190/200/200/190/200/200/190/200/200/200/190/20
80/190/200/200/190/200/200/190/200/180/200/190/20
90/190/200/200/190/200/200/190/200/180/200/190/20
100/180/200/200/190/200/200/190/200/180/200/180/20
110/180/200/200/190/200/200/190/200/180/200/180/20
120/180/200/190/180/190/200/190/200/180/190/180/20
130/180/200/180/180/190/200/190/200/180/190/180/20
140/180/200/180/180/190/200/190/190/180/190/180/20
150/180/200/180/170/190/200/190/190/180/190/180/20
160/180/200/180/170/190/200/190/190/180/190/180/20
170/170/200/180/170/190/190/190/190/180/190/180/19
180/170/200/180/170/190/190/190/190/180/190/170/19
190/160/190/180/170/190/190/190/190/180/190/160/18
200/160/190/180/170/190/190/190/180/180/170/160/18
210/160/190/180/170/190/190/190/180/170/170/160/18
220/160/190/170/170/190/190/190/180/170/170/160/18
230/160/190/160/160/190/190/180/180/170/170/160/17
240/150/190/160/150/190/190/180/180/170/170/160/16
250/150/190/160/150/190/190/170/180/170/170/160/16
260/150/180/160/150/190/190/170/180/170/170/160/16
270/150/180/160/150/190/190/170/180/160/160/160/15
280/150/180/160/150/190/190/170/180/160/160/160/15
290/150/180/160/150/190/180/160/170/160/150/160/15
300/150/180/160/150/190/180/160/170/160/150/160/15
310/150/180/160/150/190/180/160/170/160/150/160/15
320/150/180/160/150/180/180/160/170/160/150/160/15
330/150/170/160/140/180/180/160/170/160/150/160/15
340/150/170/160/140/180/180/160/170/160/150/160/15

 


Vital Eggs and Larvae from Study Days 0 to 34 (10.0 to 40.0 µg/L)












































































































































































































































































































































































































































































































































































Study dayNominal test item concentration [µg/L]
10.020.040.0
Number of vital eggs and larvae (listed eggs/larvae) by study day
123412341234
020/020/020/020/020/020/020/020/020/020/020/020/0
119/019/020/020/020/020/019/020/019/019/020/020/0
219/018/120/019/120/020/019/020/019/019/020/019/0
38/115/146/143/173/178/123/162/186/135/146/146/13
42/170/190/200/200/202/181/180/201/180/192/180/19
50/190/190/200/200/200/200/190/200/190/190/200/19
60/190/190/200/200/200/200/190/200/190/190/190/19
70/190/190/200/200/200/200/190/200/190/190/190/19
80/190/180/180/200/200/200/190/200/180/180/190/18
90/190/180/180/200/200/200/190/200/180/160/190/17
100/190/180/180/200/200/200/190/200/180/140/160/8
110/190/180/170/200/200/200/190/200/180/100/110/1
120/190/180/170/200/190/200/180/190/170/90/30/0
130/190/180/170/200/190/190/180/190/160/30/2-
140/190/170/170/200/190/190/180/190/140/30/2-
150/190/170/170/200/190/180/180/190/130/30/2-
160/190/160/170/200/190/170/170/180/120/30/2-
170/190/150/170/200/170/170/170/170/120/30/2-
180/190/150/150/190/150/150/160/130/70/00/2-
190/190/150/140/190/150/140/150/120/6-0/2-
200/180/150/140/170/130/120/150/120/5-0/1-
210/180/140/120/150/110/100/150/110/4-0/0-
220/180/140/110/150/100/90/150/110/4---
230/180/130/90/150/100/90/150/110/4---
240/170/120/90/150/90/90/150/110/4---
250/170/120/90/150/90/80/150/110/4---
260/170/120/90/130/90/80/150/110/4---
270/170/110/90/120/90/80/150/110/4---
280/170/110/90/120/90/80/140/110/4---
290/170/110/90/120/80/80/130/110/4---
300/170/110/90/120/80/80/130/110/4---
310/170/110/90/120/80/80/130/110/4---
320/170/110/90/120/80/80/130/110/3---
330/170/110/80/120/80/80/130/110/3---
340/170/110/80/120/80/80/130/110/3---

- = 100% mortality


 


Vital Eggs and Larvae from Study Days 0 to 11 (80.0 µg/L)









































































































Study dayNominal test item concentration [µg/L]
80.0
Number of vital eggs and larvae (listed eggs/larvae) by study day
1234
020/020/020/020/0
118/018/019/020/0
218/015/319/020/0
35/132/1610/97/13
42/160/160/170/20
50/180/160/170/20
60/180/160/170/20
70/160/160/170/20
80/120/30/80/10
90/20/00/40/3
100/1-0/20/2
110/0-0/00/0

- = 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, 
r ≥ 0.99 (R≥ 0.9801 for 2nd order regression)


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) 
(Fortified samples)



Mean recovery rate of 70-120% 
per fortification level (2 levels)



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
qualifier [m/z]: 358.52 > 340.52


Octadecenyldiisopropanolamine


quantifier [m/z]: 384.47 > 366.50
qualifier [m/z]: 384.47 > 98.18


Octadecyldiisopropanolamine


quantifier [m/z]: 386.49 > 368.52
qualifier [m/z]: 386.49 > 98.24



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
[mg test item/L]


(Medium)



-



0.1


(Dilution medium)



10


(Dilution medium)



Replicates



2



5



5



Concentration of the LOQ 
[µg test item/L]



-



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.
[µg a.i./L]



%



Meas.conc.
[µg a.i./L]



%



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.
[µg a.i./L]



%



Meas.conc.
[µg a.i./L]



%



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.
[µg a.i./L]



%



Meas. conc.
[µg a.i./L]



%



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 
[µg/L]



Cumulative 
hatch [%]
on study day 5



Post hatch 
survival [%] 
on study day 15



Overall 
survival [%]
on study day 15



Mortality [%] 
on study day 15



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


 


 


 


 


 


 

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 brackets























































































ParameterBased on nominal test item concentrations [µg/L]Based on overall arithmetic mean measured test item concentrations [µg/L]
Hatching success afterFry GrowthHatching success afterFry Growth
5 days1)expressed as:5 days1)expressed as:
 LengthWeight LengthWeight
NOEC40.010.010.036.16.936.93
LOEC80.020.020.076.416.516.5
EC10n.d20.216.4n.d15.512.1
(12.1 - 33.5)(6.59 – 40.7)(8.81 – 27.1)(3.96 – 36.9)
EC20 n.d43.225.0n.d38.420.3
(21.2 - > 80.0)(9.60 – 67.7)(17.7 – > 76.4)(6.29 – 68.7)
EC50n.dn.d.n.d.n.dn.d.n.d.

1) = end of hatching period
n.d. = not determinable


 


NOEC, 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 brackets

























































ParameterBased on nominal test item concentrations [µg/L]Based on overall arithmetic mean measured test item concentrations [µg/L]
Post-hatch survivalOverall survivalPost-hatch survivalOverall survival
NOEC5.005.004.254.25
LOEC10.010.06.936.93
LC10< 5.00*< 5.00*< 4.25*< 4.25*
LC20< 5.00*< 5.00*< 4.25*< 4.25*
LC5019.719.516.516.3
(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


 


 

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