Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2022-05-04 to 2022-06-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guideline
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)

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-(octadec-9-enylimino)bisethanol
EC Number:
246-807-3
EC Name:
2,2'-(octadec-9-enylimino)bisethanol
Cas Number:
25307-17-9
Molecular formula:
C22H45NO2
IUPAC Name:
2,2'-(Octadec-9-enylimino)bisethanol
Test material form:
liquid
Details on test material:
- Chemical name: Bis(2-hydroxyethyl)oleylamine
- EC number: 246-807-3

To the best of knowledge, the sample used is representative to the boundary composition shared and agreed by each registrant.
Specific details on test material used for the study:
Test item 2,2’-(Octadec-9-enylimino)bisethanol

Batch number 1950627

CAS No. 25307-17-9

Purity (certified) 100% UVCB

Density 905 kg/m3 at 20 °C

Appearance Yellow liquid

Water Solubility Dispersible / insoluble

Stability under test Not specified
conditions

Expiry date 2023-03-18

Recommended storage Keep container in a dry and well-ventilated place.
Avoid elevated temperatures.

Sampling and analysis

Analytical monitoring:
yes
Remarks:
LC-MS/MS
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 test concentration was analyzed, once during the test (under non-GLP). 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 water samples for analysis were obtained by siphoning through inert tubing. These samples did not contain any surface particulates or material dislodged from the bottom or sides of the test vessels. On each sampling point the compound C18:1 Amine + 2EO 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.
Stock solutions were sampled and analysed from freshly prepared and aged solutions of one application interval on study day 9 and 16.

Test solutions

Vehicle:
yes
Remarks:
With regard to the limited solubility of the test item in water, methanol (VWR, 100% HPLC gradient grade) 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:
A stock solution of 1600 mg/L was prepared in methanol. An appropriate volume of the test item was pipetted into a glass flask and filled up with the solvent. Density of the test item was taken into account. 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.

Equilibration period
Test solutions flowed through the test vessels for 12 days prior to the start of the exposure. The measured concentrations showed no trend of increasing or decreasing.

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.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
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. 10-13 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: 0.6 mmol/L (recent measurement: 2022-05-30)
Acidity: 0.1 mmol/L (recent measurement: 2022-05-30)
Conductivity: 153 µS/cm (recent measurement: 2022-05-30)

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 89%.

 
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.

Study design

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:
35 d
Remarks on exposure duration:
35 days (30 days post-hatch)

Test conditions

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 26.4 °C. 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 26.4 °C for the control. The minimum and maximum measured temperature for the control were 26.1 and 26.7 °C, respectively
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:
2.50 - 5.00 - 10.0 - 20.0 - 40.0 µg test item/L (factor: 2), corresponding to the overall arithmetic mean measured test item concentrations of 1.29 – 2.19 – 6.00 – 12.9 - 27.0 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 14 days.
The test concentrations were prepared by adding the dissolved test item to dilution water as described above.
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 12 days was carried out prior to start of the exposure. The measured concentrations were analysed five times during this period and showed no trend of increasing or decreasing.

Reference item
No reference item is recommended for this test according to the guideline.

Test duration
35 days (30 days post hatch), depending on post-hatch day 0 (study day 5).

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) 653 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 during exposure. Cleaning started on study day 2.

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 34).
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 – 7 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.

Reference substance (positive control):
no
Remarks:
No reference item is recommended for this test according to the guideline.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
35 d
Dose descriptor:
LC50
Effect conc.:
11.7 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Overall survival
Duration:
35 d
Dose descriptor:
LC50
Effect conc.:
18.3 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Overall survival
Duration:
35 d
Dose descriptor:
other: LC20
Effect conc.:
10 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Overall survival
Duration:
35 d
Dose descriptor:
other: LC20
Effect conc.:
15.6 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Overall survival
Key result
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
12.9 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Overall survival
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
20 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Overall survival
Key result
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
6 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Overall survival
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
10 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Overall survival
Key result
Duration:
35 d
Dose descriptor:
LC50
Effect conc.:
10.7 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Post-hatch survival
Duration:
35 d
Dose descriptor:
LC50
Effect conc.:
16.8 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Post-hatch survival
Duration:
35 d
Dose descriptor:
other: LC20
Effect conc.:
7.83 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Post-hatch survival
Duration:
35 d
Dose descriptor:
other: LC20
Effect conc.:
12.7 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Post-hatch survival
Key result
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
12.9 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Post-hatch survival
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
20 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Post-hatch survival
Key result
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
6 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Post-hatch survival
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
10 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Post-hatch survival
Duration:
35 d
Dose descriptor:
other: EC20
Effect conc.:
13.6 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
35 d
Dose descriptor:
other: EC20
Effect conc.:
> 20 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Key result
Duration:
35 d
Dose descriptor:
EC10
Effect conc.:
1.61 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
35 d
Dose descriptor:
EC10
Effect conc.:
3.54 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Key result
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
2.19 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
5 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Key result
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
1.29 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
2.5 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Key result
Duration:
35 d
Dose descriptor:
EC10
Effect conc.:
> 12.9 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
35 d
Dose descriptor:
EC10
Effect conc.:
> 20 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
2.19 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
5 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
1.29 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
2.5 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
> 27 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
time to hatch
Remarks:
hatching success
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
>= 27 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
time to hatch
Remarks:
hatching success
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
> 40 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
time to hatch
Remarks:
hatching success
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
>= 40 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
time to hatch
Remarks:
hatching success
Details on results:
Results

Biological Data

Egg Fertilization Rate
The mean egg fertilization rate determined on study day 0 (start of the exposure) was 89%. 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 goup. The hatch of larvae in other test concentrations and solvent control group started on study day 3. The hatch of larvae was completed until study day 7. Study day 5 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 5, 6 and 7.
The Dunnett`s Multiple t-test procedure for hatch data after 5 days was done with a significance level of 0.05. No statistically significant differences were found between the pooled controls and all tested concentrations up to 40.0 µg/L.
The Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm for data of hatch after 6 and 7 days was done with a significance level of 0.05. No statistically significant differences were found between the pooled controls and all tested nominal concentrations up to 40.0 µg/L.
For hatchability on study day 5 and 6, the NOEC and the LOEC (based on nominal test item concentrations) for this endpoint were determined to be ≥ 40.0 and > 40.0 µg/L, respectively. The corresponding NOEC and LOEC (based on the overall arithmetic mean measured test item concentrations) were ≥ 27.0 and > 27.0 µg/L, respectively.

Swim-up
The swim-up period of the control groups and the nominal test concentrations of 5.00 to 40.0 µg/L was observed from study day 5 to 7. 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 40 µg/L due to observed 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 88% in the control and 90% in the solvent control. 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 35 (PHD 30). Statistically significant differences were found between the pooled control groups and the nominal test item concentrations of 20.0 and 40.0 µg/L.
The NOEC and the LOEC for this endpoint were 10.0 µg/L and 20.0 µg/L (nominal concentrations), respectively. The corresponding NOEC and LOEC (based on the overall arithmetic mean measured test item concentrations) were 6.00 µg/L and 12.9 µg/L, respectively.
The LC50-value (based on nominal concentrations) for post-hatch survival on study day 35 (PHD30) was 16.8 (12.6 to 18.7) µg/L, corresponding to 10.7 (7.76 to 12.0) µg/L (based on the overall arithmetic mean measured 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 16% 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 35 (PHD 30). Statistically significant differences were found between the pooled control groups and the nominal test item concentrations of 20.0 and 40.0 µg/L.
The NOEC and the LOEC for this endpoint were 10.0 µg/L and 20.0 µg/L (nominal concentrations), respectively. The corresponding NOEC and LOEC (based on the overall arithmetic mean measured test item concentrations) were 6.00 µg/L and 12.9 µg/L, respectively.
The LC50-value (based on nominal concentrations) for overall survival on study day 35 (PHD 30) was 18.3 (10.9 to 30.8) µg/L, corresponding to 11.7 (6.60 to 21.0) µg/L (based on the overall arithmetic mean measured 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 quiescence and an unusual long arresting on the ground of one larvae of the control group was observed on study days 11 and 12. In the solvent control group one larvae showed tumbling from study day 10 to 13. Quiecscence and unusual long arresting on the ground was observed from two larvae on study day 13 and from one larvae on study day 14. No further morphological nor behavioral effects were observed in the control and solvent groups throughout exposure.
In the nominal test concentrations of 2.50 and 5.00 µg/L occasional non-lethal effects were observed. The nominal test concentration of 10.0 µg/L showed no morphological nor behavioural effects throughout exposure. The test concentration of 20.0 µg/L showed non-lethal effects with ongoing exposure from study day 11 up to study day 19. The highest nominal test concentration of 40.0 µg/L showed non-lethal effects for a short period after hatch from study day 7 to 12. This resulted in 100 % mortality.

Fry Growth
Fry growth, expressed as length and wet weight, was measured on study day 35 (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 5.00 to 20.0 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 2.50 and 5.00 µg/L, respectively. The corresponding NOEC and LOEC (based on overall arithmetic mean measured test item concentrations) were 1.29 and 2.19 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 control group: 751.5 mg total fish weight. The maximum biomass loading based on the 7.5 liter volume of a single growth chamber was 100.2 mg/L.

Maximum loading rate:
biomass/(volume of test solution) = (751.5 mg)/(7.5 L) = 100.2 mg/L

The biomass loading rate based upon a flow of 75 liters per day through each single test aquaria was 10.0 mg per liter and day.

Maximum loading rate per day:
biomass/(volume of test solution per day) = (751.5 mg)/(75 L) = 10.0 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 94 – 98% and ranged from 77 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 26.4 °C. 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 26.4 °C for the control. The minimum and maximum measured temperature for the control were 26.1 and 26.7 °C, respectively.
The validity criterion for the parameter temperature was fulfilled.

pH-Values

The mean pH-values in the control, solvent control and test item groups were between 7.75 and 7.80 and ranged from 7.58 to 8.08 during the exposure period.

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 40.0 µg/L (day 0 and 8) and 20.0 µg/L (day 15, 21 and 29). The mean total hardness was 67 mg CaCO3/L and ranged from 64 to 68 mg CaCO3/L in the control group.

Residual Chlorine

Residual chlorine, measured from the dilution water supply tank on study days 0, 8, 15, 21 and 29 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, 8, 15, 21 and 29. The mean measured TOC was 1.26 mgC/L throughout exposure. 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, control and solvent groups was 3.12 ± 0.052 L/h and the individual values ranged from 2.94 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 188 to 406 lux (mean: 275 lux).


Measured Test Concentrations during Equilibration Phase

The concentrations of the test item 2,2’-(Octadec-9-enylimino)bisethanol, the solvent control and the control were determined in the equilibration phase on day -1 from the test vessels of the test replicates 2 of all tested concentration levels via LC MS/MS.

To evaluate how much test item is lost due to the centrifiguation 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 arithmetic mean measured 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 observed with similar substances in aquaeous matrix.

Measured Concentrations in the Stock Solutions

Measured concentrations of the freshly prepared stock solution were 106 to 112% of the nominal values. Measured concentrations of the 7 days aged stock solution were in the range of 111 to 115% of the nominal values.

Measured Concentrations in the Test Vessels during Exposure
The concentrations of the test item 2,2’-(Octadec-9-enylimino)bisethanol, the solvent control and the control were determined during exposure on days 0, 7, 14, 21 and 34 from the test vessels of a defined test replicate of all tested concentration levels via LC MS/MS.

Fingerprint of the Test Item (Non-GLP)

The peak distribution of the highest test item concentration (40.0 µg test item/L) and the control was analyzed. A solution of the analytical standard of the test item was prepared and diluted to 1.00 mg/L and 20 µg/L with methanol : fish dilution water (50 : 50) containing 0.1% formic acid. The sample and the control were diluted by factor 2 with methanol containing 0.2% formic acid to avoid an inhomogeneous sample. The detected signals of the analytical standards and the sample solution were compared. In sample and standard solution the mass 356 m/z was observed and could be assigned to the test item. In general, the MS spectra of the sample solution and the analytical standard were quite similar. However, in the highest test item concentration the intensity of the signal corresponding to the test item was slightly lower compared to the 20 µg/L standard solution indicating a lower solubility of the test item in the aquaeous test medium.

Validity Criteria

The study was performed according to OECD 210 and met the validity criteria:

• Dissolved oxygen saturation was between 77 and 100% of the air saturation value
(required: > 60%).
• Water temperature was in the recommended range for the test species (i.e. 26 °C ± 1.5 °C): 26.1 – 27.1 °C. It did not differ by more than  1.5 °C between test vessels or between successive days at any time during the test.
• The cumulative hatching success was 95% in the control group and 96% in the solvent control group (required: ≥ 70%).
• Post-hatch survival was 88% in the control group and 90% in solvent control group (required: ≥ 75%).
• Concentration analysis: the concentrations of the test item in the test solutions should be maintained within 80 – 120% of the nominal values. The test item is a multi-component substance (UVCB) which is insoluble/dispersible in water. On each sampling point the compound C18:1 Amine + 2EO was analytically determined to distinguish how much of the test item is truly dissolved and dispersed in the test media. The measured concentrations of the test item (truly dissolved and dispersed fraction) were constantly below the nominal range. This effect was also observed in the preliminary test. Additionally all effect values were based on the overall arithmetic mean measured concentrations of the test item 2,2’-(Octadec-9-enylimino)bisethanol.

Stability

The stability of the standard stock solution (1000 mg test item/L in methanol) was checked after 23 days and was 92% of the nominal concentration.

The responses of a freshly prepared calibration standard (6 µg test item/L) was compared with a 19 days old calibration standard with 5 injections each. The deviation was < 10% and the calibration standards were found to be stable over this period.

The stability of the fortified samples at the 1 x LOQ was checked after 7 days. The recovery was 101% of the nominal concentration.
 
Procedural Recovery

A procedural recovery (Quality Control) on 1 x LOQ Level was freshly prepared on each day of analysis. For preparation of the quality contro. It was treated in parallel to the test samples and measured in each sample set.

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 14 days under flow-through conditions as described. An equilibration period over 12 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 5.00 and 50.0 µg/L were included in the test. Two replicates per solvent control and test concentration were tested. 20 eggs per replicate were exposed to each concentration level and the solvent control.
The test item concentrations were analytically verified via LC-MS/MS on days -1, 0, 7 and 14. The sampling was done from the test vessels and from the outlet of the test vessels. Additionally, the samples for analysis of the truly dissolved test item in the test vessels were centrifuged and analyzed.
Reported statistics and error estimates:
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 Dunnett’s multiple t-test-procedure was done with a significance level of 0.05 for the parameter hatching success on study day 5.
The multiple sequentially-rejective Welsh-t-test after Bonferroni-Holm was done with a significance level of 0.05 for the parameter hatching success on study days 6 and 7.
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 arithmetic mean measured concentrations of the test item.

Any other information on results incl. tables

Hatch / Hatching Time (Control, Solvent Control and Test Item Concentrations)






























































































































































































































































































































































NominalRep.PHD -3PHD -2PHD -1PHD 0PHD 1PHD 2
test item conc.Study day 2Study day 3Study day 4Study day 5Study day 6Study day 7
[µg/L]Cumulative hatching rate [%] 
Control10575909090
202060100100100
356590100100100
403590909090
Mean13179959595
Solvent control101065100100100
2055100100100100
304585909090
401055959595
Mean03076969696
Feb 5010359090100100
202095100100100
3055100100100100
403580909090
Mean0369195 (ns)98 (ns)98 (ns)
5.0010156095100100
20555100100100
30570100100100
401075959595
Mean096598 (ns)99 (ns)99 (ns)
10.0102090100100100
20555100100100
305095100100100
4055595100100
Mean0207499 (ns)100 (ns)100 (ns)
20.01055585100100
201030859595
301075959595
404090959595
Mean0166390 (ns)96 (ns)96 (ns)
40.0105458595100
20025859090
301035959595
405095100100100
Mean0165091 (ns)95 (ns)96 (ns)

(ns) = No 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 2.50 to 10.0 µg/L

























































































































































































































































































































NominalRep.PHD -1PHD 0PHD 1PHD 2PHD 3
test item conc.Study day 4Study day 5Study day 6Study day 7Study day 8
[µg/L]Cumulative swim up [%]
Control1094100100100
2090100100100
30100100100100
4083100100100
Mean092100100100
Solvent control1095100100100
2080100100100
307894100100
4089100100100
Mean08699100100
Feb 5010100100100100
2095100100100
30100100100100
4094100100100
Mean097100100100
5.001089100100100
2090100100100
3085100100100
40100100100100
Mean091100100100
10.010100100100100
2090100100100
30100100100100
40959595100
Mean0969999100
20.0107695100100
207195100100
3079100100100
40100100100100
Mean08298100100
40.0108210010050
20829410080
30741009357
408510010092
Mean081999870

Post-Hatch Survival on Study Day 35 (PHD 30) of the Control, Solvent Control and the Nominal Test Item Concentrations 2.50 and 5.00 µg/L







































































































































































NominalRep.Eggs introduced on study day 0Cumulative number of hatched larvaeVital Larvae on study day 35Post-Hatch survival
test item conc.(PHD 30)on study day 35
[µg/L] [%]
Control120181689
220201995
320201680
420181689
Mean20191788
Solvent control120201890
220201785
320181689
420191895
Mean20191790
Feb 50120201995
220201995
3202020100
420181372
Mean20201891 (ns)
5.00120201680
220201995
320201680
420191684
Mean20201785 (ns)

(ns) = No statistically significant difference from pooled control groups


 


Post-Hatch Survival on Study Day 35 (PHD 30) of the Nominal Test Item Concentrations 10.0 to 40.0 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] [%]
10.0120201890
220201890
320201890
420201575
Mean2020.017.086 (ns)
20.012020420
22019316
32019632
42019526
Mean2019.3524 (s)
40.01202000
2201800
3201900
4202000
Mean2019.300 (s)

(ns) / (s) = No statistically / statistically significant difference from pooled control groups


 


Overall Survival and Overall Mortality on Study Day 35 (PHD 30) of the Control, Solvent
Control and the Nominal Test Item Concentrations 2.50 and 5.00 µg/L


























































































































































NominalRep.Vital larvae onOverall survivalOverall mortality
test item conc.study day 35  
[µg/L](PHD 30)  
  [%][%]
Control1168020
219955
3168020
4168020
Mean178416
Solvent control1189010
2178515
3168020
4189010
Mean178614
2.50119955
219955
3201000
4136535
Mean188911 (ns)
5.001168020
219955
3168020
4168020
Mean178416 (ns)

(ns) = No statistically significant difference from pooled control groups


 


Overall Survival and Overall Mortality on Study Day 35 (PHD 30) of the Nominal Test
Item Concentrations 10.0 to 40.0 µg/L



























































































































NominalRep.Vital larvae onOverall survivalOverall mortality
test item conc.study day 35  
[µg/L](PHD 30)  
  [%][%]
10.01189010
2189010
3189010
4157525
Mean178614 (ns)
20.0142080
231585
363070
452575
Mean52377 (s)
40.0100100
200100
300100
400100
Mean00100 (s)

(ns) / (s) = No statistically / statistically significant difference from pooled control groups


 


Behavioral Effects observed in the Nominal Test Concentrations 2.50 and 5.00 µg/L (Study Day 11 – 32)






























































































































































Study Day2.50 µg/L5.00 µg/L
12341234
11----1 T-1 Q-
1 G
12-1 G------
1 Q
13 - 19--------
20------1 T-
21------1 SP-
22------1 SP-
23------1 SP-
24------1 SP-
25 - 29--------
30---1 A----
31---1 A----
32---1 A----

G = Arresting on the ground Q = Quiescence
T = Tumbling A = Swollen Abdomen
SP = Side position


 


Behavioral Effects observed in the Nominal Test Concentrations 20.0 µg/L (Study Day 11 – 19)








































































































Study Day20.0 µg/L
1234
11-1 T--
121 G--1 G
131 T1 T-1 S
2 Q1 G
2 G2 Q
144 S4 G--
2 G4 Q
6 Q 
154 G5 G-2 T
4 Q5 Q
161 G1 T1 G-
3 G
3 Q
17-2 G3 G2 G
18-1 G--
19--2 G1 G

 


Behavioral Effects observed in the Nominal Test Concentration 40.0 µg/L
(Study Day 7 – 12)










































































































































Study Day40.0 µg/L
1234
7--1 Q-
1 G
1 T
82 T1 T1 T1 G
4 SE2 G2 G1 SE
4 S2 Q2 SE12 Q
6 G 4 Q 
6 ME   
6 F   
91 T1 T100%2 T
3 G2 GMortality1 S
3 ME2 ME 1 SE
3 F2 F 2 G
1 S1 S 2 ME
1 SE1 SE 2 F
101 S2 F 1 S
1 SE2 S  1 SE
 2 SE  
11100%1 ME 100%
Mortality1 F Mortality
12100 % Mortality

G = Arresting on the ground Q = Quiescence
T = Tumbling S = Arresting under the surface
SE = Slowed escape reflex ME = Missing escape reflex
F = No food uptake


 


Overview of Fry Growth: Length and Wet Weight on Study Day 35 (PHD 30) of the Control, Solvent Control and Nominal Test Item Concentrations 2.50 and 5.00 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]
Control116.039.1
214.630.7
316.041.5
416.147.0
Mean15.739.6
± SD0.6225.87
CV [%]3.9614.8
Solvent control115.939.0
216.034.7
316.038.7
415.836.4
Mean15.937.2
± SD0.0831.76
CV [%]0.524.73
Feb 50115.136.3
215.734.7
315.734.9
417.048.2
Mean15.9 (ns)38.5 (ns)
± SD0.6945.62
CV [%]4.3614.6
5.00114.931.3
214.732.5
315.233.7
415.132.4
Mean15.0 (s)32.5 (s)
± SD0.1920.850
CV [%]1.282.62

(ns) / (s) = No statistically / statistically significant difference from pooled control groups


 


Overview of Fry Growth: Length and Wet Weight on Study Day 35 (PHD 30) of the Nominal Test Item Concentrations 10.0 and 20.0 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]
10.0112.924.1
214.629.6
313.627.5
414.834.6
Mean14.0 (s)29.0 (s)
± SD0.7693.81
CV [%]5.4913.1
20.0114.531.6
215.337.6
314.531.2
414.733.2
Mean14.8 (s)33.4 (s)
± SD0.3282.54
CV [%]2.227.60

(s) = Statistically significant difference from pooled controls


 


Individual Length on Study Day 35 (PHD 30) of the Control and the Solvent Control




















































































































































































































































































Fish No.ControlSolvent control
Total length of individual fish in [mm]
12341234
117.011.015.513.013.015.015.520.0
219.012.513.020.018.516.017.015.0
316.013.017.017.014.014.514.014.0
415.016.017.014.019.518.011.011.0
514.011.019.018.017.518.513.016.0
611.015.015.518.020.014.521.514.0
714.513.519.018.018.014.022.015.0
815.021.019.018.516.016.514.017.0
918.019.018.020.020.016.016.020.0
1018.518.014.018.018.016.020.017.0
1114.012.517.017.016.019.518.020.5
1220.012.016.015.015.015.516.017.0
1315.017.018.012.011.018.019.017.0
1415.014.016.014.517.012.016.016.5
1518.518.012.015.018.016.010.016.0
1615.015.010.010.08.516.013.014.0
17-13.0--16.016.0-11.5
18-13.0--10.0--12.5
19-12.0------
20--------
Mean16.014.616.016.115.916.016.015.8
± SD2.272.802.542.803.311.763.392.66
CV %14.219.215.917.420.811.021.216.8

- = Fish died before end of the study


 


Individual Length on Study Day 35 (PHD 30) of the Nominal Test Item Concentrations 2.50 and 5.00 µg/L























































































































































































































































































Fish No.Nominal test item conc. [µg/L]
Feb 505.00
Total length of individual fish in [mm]
12341234
117.014.017.517.017.020.015.012.5
219.513.516.013.518.513.014.515.0
320.015.013.018.516.018.020.018.0
414.016.019.516.018.014.512.021.0
515.013.019.017.011.011.520.517.0
613.017.013.517.010.014.013.016.0
710.016.013.017.09.015.019.011.0
814.511.016.020.020.018.516.014.0
916.017.015.019.016.018.013.018.0
1012.017.515.017.017.516.018.517.0
1115.517.515.021.016.013.516.013.0
1211.013.514.512.516.011.513.016.0
1314.017.517.015.514.013.013.011.0
1413.019.018.0-14.015.014.018.0
1513.018.017.0-12.014.013.510.0
1617.515.015.5-13.018.512.513.5
1720.512.511.0--10.5--
1816.515.018.0--14.5--
1915.520.017.0--10.5--
20--14.0-----
Mean15.115.715.717.014.914.715.215.1
± SD2.842.312.142.273.092.762.732.99
CV %18.814.713.613.420.718.818.019.8

- = Fish died before end of the study


 


Individual Length on Study Day 35 (PHD 30) of the Nominal Test Item Concentrations 10.0 and 20.0 µg/L























































































































































































































































































Fish No.Nominal test item conc. [µg/L]
10.020.0
Total length of individual fish in [mm]
12341234
19.019.017.511.516.017.511.013.0
29.015.018.014.018.010.014.011.5
317.512.014.011.512.518.515.517.5
47.517.014.515.511.5-17.019.5
517.020.011.014.0--12.012.0
618.017.013.516.0--17.5-
719.016.514.515.0----
811.011.012.517.0----
910.015.510.023.0----
1016.012.011.014.5----
1117.013.09.516.0----
1213.018.520.512.5----
1310.013.017.016.0----
149.015.010.016.0----
1513.510.510.010.0----
1614.014.012.0-----
1712.012.516.0-----
1810.010.513.0-----
19--------
20--------
Mean12.914.613.614.814.515.314.514.7
± SD3.602.893.122.942.623.792.423.20
CV %27.919.822.919.918.124.816.721.8

- = Fish died before end of the study


 


Pooled Wet Weights on Study Day 35 (PHD 30) of the Control, Solvent Control and the Nominal Test Item Concentrations 2.50 to 20.0 µg/L












































































































































































































NominalReplicateNumber of fish alivePooledMeanMean± SDCV %
test item conc.on study day 35wet weightwet weight 
[µg/L] per replicateper fish per replicate 
  [mg][mg][mg]
Control116625.839.139.65.8714.8
219584.030.7
316663.341.5
416751.547.0
Solvent control118701.239.037.21.764.73
217590.434.7
316619.538.7
418654.636.4
Feb 50119689.836.338.55.6214.6
219659.134.7
320697.434.9
413626.448.2
5.00116500.131.332.50.8502.62
219617.532.5
316538.633.7
416518.432.4
10.0118433.724.129.03.8113.1
218531.929.6
318495.827.5
415518.934.6
20.014126.231.633.42.547.60
23112.737.6
36186.931.2
45166.133.2

 


Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 0 -16)






























































































































































































































































































































































Study dayReplicateDissolved oxygen [%]
Nominal test item concentrations [µg/L]
ControlSolvent control2.505.0010.020.040.0
01100100100100100100100
299100100100100100100
3100100100100100100100
4100100100100100100100
21100100100100100100100
2100100100100100100100
3100100100100100100100
4100100100100100100100
51100100100100100100100
2100100100100100100100
3100100100100100100100
4100100100100100100100
7110010098989910099
299999999999898
31009898100999899
4991001001001009999
9197979695979796
297979797969694
397979697969697
497979696969696
12197969494959595
295969596959597
396969596959595
496969696959696
141979692959594-
2959593949594-
3969592959594-
4969796949595-
161989794969796-
2979796979696-
3979796979796-
4969896949596-

- = No measurement, due to 100 % mortality


 


Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 19 - 34)





























































































































































































































































































































































Study dayReplicateDissolved oxygen [%]
Nominal test item concentrations [µg/L]
ControlSolvent control2.505.0010.020.040.0
191969492939494-
2949492959392-
3949492929292-
4949593919094-
211989794959593-
2969795959592-
3969695939393-
4969793919094-
231959592939394-
2959592939492-
3959492919293-
4959587929192-
261969594929495-
2969494929294-
3949494929092-
4959491929093-
281929090869091-
2919190878889-
3898988888791-
4918591888889-
301939088869090-
2918989878887-
3898887868890-
4908988868590-
341858680828688-
2858283778286-
3797880818288-
4858380788085-
Mean95959494949598
± SD 4.404.955.205.735.114.032.04
Min.79788077808594
Max.100100100100100100100

- = 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 measurements2022-05-04 to 2022-06-08
Minimum temperature [°C]25.7
Maximum temperature [°C]26.8
Mean temperature [°C]26.4

 


Water Temperature in the Test Media











































































































































































































































































Study dayRep.Water temperature [°C]
Nominal test item concentrations [µg/L]
ControlSolvent control2.505.0010.020.040.0
0126.426.426.526.626.626.426.5
226.426.426.626.526.826.526.4
326.426.526.726.726.626.526.5
426.426.526.726.526.526.526.7
7126.426.426.526.626.626.426.5
226.426.526.626.526.826.626.5
326.426.526.726.726.626.526.5
426.326.426.626.526.426.426.7
14126.726.726.827.026.926.8-
226.726.826.827.027.026.8-
326.726.827.127.026.926.8-
426.726.726.926.826.826.7-
21126.226.326.326.726.526.4-
226.226.326.526.426.626.5-
326.326.226.526.526.526.4-
426.226.226.626.326.326.4-
28126.126.326.426.626.526.4-
226.226.326.526.426.526.4-
326.226.326.626.526.426.3-
426.226.226.526.326.326.3-
Mean26.426.426.626.626.626.526.5
± SD 0.1920.1900.1820.2140.2010.1590.106
Min.26.126.226.326.326.326.326.4
Max.26.726.827.127.027.026.826.7

- = 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 control2.505.0010.020.040.0
017.607.727.707.657.717.687.68
27.627.737.717.687.717.697.67
37.717.757.697.687.687.727.73
47.687.757.697.697.707.777.70
717.647.987.977.917.967.968.01
27.977.927.957.987.957.927.96
38.077.967.948.037.977.968.03
48.008.087.978.057.977.948.00
1417.657.777.717.657.627.63-
27.707.777.687.627.657.62-
37.777.777.647.657.647.59-
47.767.747.717.687.637.62-
2117.727.867.887.817.837.77-
27.837.927.807.867.837.80-
37.917.827.847.767.777.84-
47.857.867.807.797.727.76-
2817.587.677.627.607.617.72-
27.657.687.627.637.607.64-
37.667.597.627.657.617.67-
47.687.627.657.637.587.68-
Mean7.757.807.767.757.747.757.85
± SD 0.1410.1260.1240.1430.1350.1200.165
Min.7.587.597.627.607.587.597.67
Max.8.078.087.978.057.977.968.03

- = 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]
Control20.040.0
0170 69
8267 68
1536465 
2146466 
2916868 
Mean676669
± SD2.611.530.71
Min.646568
Max.706869

= 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]
01.15
81.25
151.25
211.29
291.34
Mean1.26
SD0.070
Min.1.15
Max.1.34

Min./Max. = Minimum/Maximum measured TOC


 


Flow Rates








































































Flow rateNominal test item concentrations [µg/L]
ControlSolvent control2.505.0010.020.040.0
Measured flow rates of the membrane piston pumps1)
[L/h]
Minimum3.003.003.003.00343663.003.00
Maximum3.363.243.243.243.243.243.24
Mean3.133.113.123.113.123.133.11
± Standard deviation ± 0.055± 0.046± 0.051± 0.053± 0.046± 0.051± 0.060
Overall mean3.12
± Standard deviation± 0.052

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



























































































Sampling dayDay -1
ReplicateRepl. 2
Nominal2,2’-(Octadec-9-enylimino)bisethanol
concentration of theC18:1 Amine + 2 EO
 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]
40.030.220.76910.033
20.015.18.19544.0427
10.07.544.40582.3231
5.003.771.50400.58716
2.501.891.07570.42122
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 (0.4 µg/L of the test item, corresponding to 0.302 µg a.i./L)


 


Measured Concentrations and Percent of Nominal Concentration in the Stock Solutions















































































 Freshly prepared stock solutions7 Days aged stock solutions
Nominal2,2’-(Octadec-9-enylimino)bisethanol
concentration of theC18:1 Amine + 2 EO
Test itema.i.Meas.%Meas.% 
[mg/L][mg a.i./L]conc. conc. 
  [mg a.i./L][mg a.i./L]
1600120713491121347112
800603639106672111
400302322107340113
200151160106173115
10075.481.110885.4113

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 (0.4 µg/L of the test item, corresponding to 0.302 µg a.i./L)


 


Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day 0 to Day 34
















































































































































































































Sampling dayDay 0Day 7Day 14
ReplicateRepl. 3Repl. 4Repl. 1
Nominal2,2’-(Octadec-9-enylimino)bisethanol
concentration of theC18:1 Amine + 2 EO
Test itema.i.Meas.%Meas.%Meas.%
[µg/L][µg a.i./L]conc. conc. conc. 
  [µg a.i./L][µg a.i./L][µg a.i./L]
40.030.221.17018.76221.270
20.015.112.2819.386210.066
10.07.545.33714.08544.6562
5.003.772.05541.66441.5341
2.501.890.963511.03551.0556
Solvent Control< LOQ< LOQ< LOQ
Control< LOQ< LOQ< LOQ
Sampling dayDay 21Day 34 
ReplicateRepl. 2Repl. 4 
Nominal2,2’-(Octadec-9-enylimino)bisethanol 
concentration of theC18:1 Amine + 2 EO
Test itema.i.Meas.%Meas.% 
[µg/L][µg a.i./L]conc. conc. 
  [µg a.i./L][µg a.i./L]
40.030.2-1)-1) 
20.015.17.75519.4763 
10.07.544.59613.9953 
5.003.771.19311.8248 
2.501.890.807431.0053 
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 (0.4 µg/L of the test item, corresponding to 0.302 µg a.i./L)
- = No determination of this replicate
1) = No samples due to 100% mortality


 


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 34)



































































 2,2’-(Octadec-9-enylimino)bisethanol
C18:1 Amine + 2 EO
NominalOverall Arithmetic Mean Measured Concentrations of the
concentration of the
Test itema.i.Test itema.i.%
[µg/L][µg a.i./L][µg/L][µg a.i./L]
40.030.227.020.367
20.015.112.99.7665
10.07.546.004.5360
5.003.772.191.6544
2.501.891.290.97051

a.i. = Active ingredient
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (0.4 µg/L of the test item, corresponding to 0.302 µg a.i./L)


 


Gradient Table








































Time [min]A [%]B [%]
0.007030
0.307030
1.30595
2.00595
2.107030
2.507030

 


Dilution steps (Test Vessel)







































































NominalDilutionSampleFinal
test itemFactor*volumevolume
concentration [mL][mL]
[µg/L]   
40.0100.21.0
20.0100.21.0
10.021.01.0
5.021.01.0
2.5021.01.0
Solvent Control21.01.0
Control21.01.0

*including factor 2


 


Dilution steps (Stock Solution)

















































































































NominalDilutionSampleFinal
test itemFactorvolumevolume
concentration [mL][mL]
[mg/L]   
1600250 0000.041)4.01)
0.042)4.02)
0.043)1.03)
800125 0000.041)4.01)
0.042)4.02)
0.083)1.03)
40062 5000.041)4.01)
0.042)4.02)
0.163)1.03)
20031 2500.041)4.01)
0.042)4.02)
0.323)1.03)
10015 6250.041)4.01)
0.042)4.02)
0.643)1.03)
Control15 6250.041)4.01)
0.042)4.02)
0.643)1.03)

1) first dilution step
2) second dilution step
3) third dilution step


 


Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day 0 to Day 34 (centrifuged)
















































































































































































































Sampling dayDay 0Day 7Day 14
ReplicateRepl. 3Repl. 4Repl. 1
Nominal2,2’-(Octadec-9-enylimino)bisethanol
concentration of theC18:1 Amine + 2 EO
Test itema.i.Meas.%Meas.%Meas.%
[µg/L][µg a.i./L]conc. conc. conc. 
  [µg a.i./L][µg a.i./L][µg a.i./L]
40.030.27.17245.36187.6926
20.015.13.81253.37224.7131
10.07.541.84241.34181.6121
5.003.770.568150.565150.43011
2.501.890.325170.320170.30216
Solvent Control< LOQ< LOQ< LOQ
Control< LOQ< LOQ< LOQ
Sampling dayDay 21Day 34 
ReplicateRepl. 2Repl. 4 
Nominal2,2’-(Octadec-9-enylimino)bisethanol 
concentration of theC18:1 Amine + 2 EO
Test itema.i.Meas.%Meas.% 
[µg/L][µg a.i./L]conc. conc. 
  [µg a.i./L][µg a.i./L]
40.030.2-1)-1) 
20.015.15.71383.2922 
10.07.543.03401.4920 
5.003.770.648170.67118 
2.501.890.459240.29115 
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 (0.4 µg/L of the test item, corresponding to 0.302 µg a.i./L)
- = No determination of this replicate
1) = No samples due to 100% mortality


 


Vital Eggs and Larvae from Study Days 0 to 35 (Control, Solvent Control and
2.50 µg/L)



























































































































































































































































































































































































































































































































































































Study dayNominal test item concentration [µg/L]
ControlSolvent control2.50
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
118/020/020/019/020/020/019/019/020/020/020/018/0
218/020/019/119/020/020/018/019/020/020/020/018/0
317/116/47/1312/718/29/119/917/213/716/49/1111/7
43/158/122/181/187/130/201/178/112/181/190/202/16
50/180/200/200/180/200/200/180/192/180/200/200/18
60/180/200/190/180/200/190/170/190/200/200/200/17
70/180/200/190/180/200/190/170/190/200/200/200/17
80/180/200/190/180/200/190/170/190/200/200/200/17
90/180/200/190/180/200/190/160/180/200/200/200/17
100/180/200/190/180/200/190/160/180/200/200/200/17
110/180/200/190/180/200/190/160/180/190/200/200/16
120/180/200/190/180/200/190/160/180/190/200/200/16
130/180/200/190/160/190/190/160/180/190/190/200/16
140/170/200/190/160/190/180/160/180/190/190/200/16
150/170/200/190/160/190/180/160/180/190/190/200/16
160/170/200/180/160/190/180/160/180/190/190/200/16
170/170/200/180/160/190/180/160/180/190/190/200/16
180/170/200/180/160/180/180/160/180/190/190/200/15
190/170/200/170/160/180/180/160/180/190/190/200/15
200/170/200/170/160/180/180/160/180/190/190/200/15
210/170/200/170/160/180/180/160/180/190/190/200/15
220/170/200/170/160/180/180/160/180/190/190/200/15
230/170/200/170/160/180/180/160/180/190/190/200/15
240/170/200/170/160/180/180/160/180/190/190/200/15
250/170/200/170/160/180/180/160/180/190/190/200/15
260/160/190/160/160/180/170/160/180/190/190/200/14
270/160/190/160/160/180/170/160/180/190/190/200/14
280/160/190/160/160/180/170/160/180/190/190/200/14
290/160/190/160/160/180/170/160/180/190/190/200/14
300/160/190/160/160/180/170/160/180/190/190/200/14
310/160/190/160/160/180/170/160/180/190/190/200/14
320/160/190/160/160/180/170/160/180/190/190/200/14
330/160/190/160/160/180/170/160/180/190/190/200/13
340/160/190/160/160/180/170/160/180/190/190/200/13
350/160/190/160/160/180/170/160/180/190/190/200/13

 


Vital Eggs and Larvae from Study Days 0 to 35 (5.00 to 20.0 µg/L)



























































































































































































































































































































































































































































































































































































Study dayNominal test item concentration [µg/L]
5.0010.020.0
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
120/020/020/020/020/020/020/020/020/019/019/020/0
220/020/020/019/020/020/020/020/020/019/019/019/0
317/319/119/117/216/419/110/1019/119/117/217/211/8
48/129/116/144/152/189/111/199/119/1113/64/151/18
51/190/200/200/190/200/200/201/193/172/170/190/19
60/200/200/200/190/200/200/200/200/200/190/190/19
70/200/200/200/190/200/190/200/200/200/190/190/19
80/200/200/200/190/200/190/200/200/200/190/190/19
90/200/200/200/190/200/190/200/200/200/190/190/19
100/200/200/200/190/200/190/200/200/200/180/190/19
110/200/200/200/190/200/190/200/200/200/180/180/18
120/180/200/190/190/190/190/200/200/200/140/180/17
130/170/200/190/190/190/190/200/190/190/140/180/17
140/170/200/190/190/190/190/200/190/150/130/160/14
150/170/200/190/190/190/190/200/190/110/120/160/14
160/170/200/190/190/190/190/200/190/80/100/160/12
170/170/200/190/190/190/190/200/190/60/80/130/12
180/170/200/190/190/190/190/200/180/60/70/100/9
190/170/200/190/190/190/190/200/170/60/60/90/9
200/170/200/180/190/180/190/200/170/60/50/90/7
210/170/200/180/180/180/190/200/160/50/40/80/7
220/160/200/180/180/180/190/200/160/50/30/70/6
230/160/190/180/180/180/190/200/160/40/30/60/6
240/160/190/180/180/180/190/200/160/40/30/60/5
250/160/190/170/180/180/190/200/160/40/30/60/5
260/160/190/160/160/180/180/180/150/40/30/60/5
270/160/190/160/160/180/180/180/150/40/30/60/5
280/160/190/160/160/180/180/180/150/40/30/60/5
290/160/190/160/160/180/180/180/150/40/30/60/5
300/160/190/160/160/180/180/180/150/40/30/60/5
310/160/190/160/160/180/180/180/150/40/30/60/5
320/160/190/160/160/180/180/180/150/40/30/60/5
330/160/190/160/160/180/180/180/150/40/30/60/5
340/160/190/160/160/180/180/180/150/40/30/60/5
350/160/190/160/160/180/180/180/150/40/30/60/5

 


Vital Eggs and Larvae from Study Days 0 to 12 (40.0 µg/L)
















































































































Study dayNominal test item concentration [µg/L]
40.0
Number of vital eggs and larvae (listed eggs/larvae) by study day
1234
020/020/020/020/0
120/018/020/020/0
220/018/020/020/0
319/118/018/210/10
411/913/513/71/19
53/171/171/190/20
61/190/180/180/20
70/200/170/150/20
80/120/150/70/13
90/50/40/00/5
100/10/2-0/1
110/00/1-0/0
12-0/0--

- = 100% mortality


 


Method Validation (non-GLP)


The analytical method was validated according to SANTE/2020/12830, Rev. 1 (2021), Guidance Document on Pesticide Analytical Methods for Risk Assessment and Post-approval Control and Monitoring Purposes using the criteria in the table below.


Parameter, Acceptance Criteria and Results of the Method Validation








































































































ParameterAcceptance criteriaResult
Calibration≥ 5 standard concentrations,0.05 to 10 µg test item/Lü
r ≥ 0.99 (R2 ≥ 0.9801 for 2nd order regression)(n = 9),
Calibration range from ≤ 30% LOQ to 20% above highest concentration level after sample preparationr ≥ 0.99
Matrix effectsShould be ≤ 20%Matrix effects: 22%ü
Matrix matched standards have been used
Lowest calibration level (LCL)S/N ≥ 9 for quantifier ion traceS/N for 0.05 µg test item/L-
S/N ≥ 3 for qualifier ion trace 583 (quantifier)
 1326 (qualifier)
Limit of Quantification (LOQ)Should be at or below lowest test concentration0.400 µg test item/L (1 x LOQ)ü
50.0 µg test item/L (125 x LOQ)
Accuracy (Fortified samples)1)Mean recovery rate of 70-120%1 x LOQ: 71% (n = 5)ü
per fortification level (2 levels)125 x LOQ: 102% (n = 5)
Precision1)Relative standard deviation ≤ 20% per fortification level1 x LOQ: 20%ü
125 x LOQ: 5.1%
StabilitySamples: Stable if 70 – 120% of nominal concentrationSee section 18.1ü
Calibration standards: Stable if ±10% of freshly prepared standard
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). quantifier [m/z]: 356.31 > 88.05ü
qualifier [m/z]: 356.31 > 55.04
Blank values < 30% of the LOQBlank values < 30% of LOQü
Procedural recoveryProcedural recoveries with experimental samples. The recovery has to be 70-120% of the nominal value.See section 18.2ü

 


Preparation of Fortified Samples of the Test Item
















































































































LOQ LevelControl1125
Stock solution-1000 mg test item/L in Methanol
Spiking solution-40.05000
[µg test item/L](Methanol)(Methanol)
(Medium)  
Replicates255
Concentration of the LOQ-0.40050.0
[µg test item/L]
Medium for preparationFish dilution water
Volume of spiking solution [mL]-0.050.05
Volume of medium [mL]5.04.954.95
Dilution factor2210
Dilution mediumMethanol containing 0.2% formic acid1)
Dilution medium2)
Sample volume [mL]5.01)5.01)5.01)
0.22)
Finale volume [mL]101)101)101)
1.02)
Final concentration of the test item for analysis -0.2005.00
[µg/L]
Final concentration of the a.i. for analysis -0.1513.77
[µg a.i./L]

1) First dilution step
2) Second dilution step
Dilution medium = Methanol : fish dilution water (50 : 50 v/v) containing 0.1% formic acid


 


Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of 2,2’-(Octadec-9-enylimino)bisethanol
Fortified concentrations*: 0.409 µg test item/L (1 x LOQ) and 51.1 µg test item/L (125 x LOQ), corresponding to 0.308 µg a.i./L (1 x LOQ) and 38.5 µg a.i./L (125 x LOQ).






















































































Replicate2,2’-(Octadec-9-enylimino)bisethanol
C18:1 Amine + 2 EO
1 x LOQ125 x LOQ
Meas.%Meas.%
conc.conc.
[µg a.i./L][µg a.i./L]
10.2648642.8111
20.1916238.399
30.1765738.4100
40.2738938.399
50.1966438.399
Mean0.227139102
SD ±0.04 2 
CV [%]20 5.1 

Meas. conc. = measured concentration C18:1 Amine + 2 EO in the test item, dilution factor taken into account
a.i. = active substance
% = percent concentration of the fortified sample
SD = standard deviation
CV = coefficient of variation


 


Measured Concentrations and Percent of Nominal Concentration of the Quality Control during the Definitive Test



















































Quality Control
Sampling dateMeas. conc.%
[µg a.i./L]
Day -11)0.317103
Day 01)0.358116
Day 71)0.343111
Day 142)0.335110
Day 212)0.335111
Day 282)0.28895
Day 342)0.325107

Meas. conc. = measured concentration of the a.i., dilution factors taken into account
% = percent of the nominal concentration
a.i. = active ingredient (C18:1 Amine + 2 EO)
1) = 0.409 µg test item/L, corresponding to 0.308 µg a.i./L (weighing factor taken into account)
2) = 0.402 µg test item/L, corresponding to 0.303 µg a.i./L (weighing factor taken into account)


 


Mortality [%] and Hatch [%] in the Range Finding Test
(n = 40 per treatment group with 2 replicates and 20 eggs each under flow-through conditions)













































































Nominal test item concentrationCumulativePost hatchOverallMortality [%]
[µg/L]hatch [%]survival [%]survival [%]on study day 14
 (study day 6)on study day 14on study day 14 
 ReplicateReplicateReplicateReplicate
 12121212
50.010090000*0*100*100*
5.009095941008595155
Solvent control9590100949585515

* 100 % mortality of hatched larvae was observed on study day 9


 


Measured Concentrations of the compound C18:1 Amine + 2EO of the Test Item
2,2’-(Octadec-9-enylimino)bisethanol on Study Days -1, 0, 7 and 14 of the Range Finding Test
Determination of the test item via LC-MS/MS, with a preliminary non-validated analytical method































































































































































Sampling dateDay -1
Nominal concentration of theC18:1 Amine + 2EO
test item [µg/L]active substance Meas. conc.%
[µg a.s./L][µg a.s./L]
50.037.131.483
(Replicate 1)
50.025.568
(Replicate 2)
5.003.771.9151
(Replicate 1)
5.001.7647
(Replicate 2)
Solvent control< LCL
(Replicate 1)
Solvent control< LCL
(Replicate 2)
Sampling dateDay 0Day 7 Day 14
Nominal concentration of theC18:1 Amine + 2EO
test item [µg/L]active substance Meas. conc.%Meas. conc.%Meas. conc.%
[µg a.s./L][µg a.s./L][µg a.s./L][µg a.s./L]
50.037.125.768--
(Replicate 1)
50.0-22.860-
(Replicate 2)
5.003.772.5868-1.9251
(Replicate 1)
5.00-1.07281.9151
(Replicate 2)
Solvent control< LCL< LCL< LCL
(Replicate 1)
Solvent control< LCL< LCL< LCL
(Replicate 2)

Meas. Conc. = Measured concentration of the test item, dilution factor taken into account
% = Percent of the nominal test item concentration
LCL = Lowest Calibration Level, 0.1 µg test item/L
a.s. = Active substance


 


Measured Concentrations of the Compound C18:1 Amine + 2EO of the Test Item
2,2’-(Octadec-9-enylimino)bisethanol on Study Days -1, 0, 7 and 14 of the Range Finding Test (Truly Dissolved Test Item)
Determination of the test item via LC-MS/MS, with a preliminary non-validated analytical method





























































































Sampling dateDay 0Day 7 Day 14
Nominal concentration of theC18:1 Amine + 2EO
test item [µg/L]active substance Meas. conc.%Meas. conc.%Meas. conc.%
[µg a.s./L][µg a.s./L][µg a.s./L][µg a.s./L]
50.037.111.831--
(Replicate 1)
50.0-9.4025-
(Replicate 2)
5.003.770.38510-0.63917
(Replicate 1)
5.00-0.860230.70219
(Replicate 2)
Solvent control< LCL< LCL< LCL
(Replicate 1)
Solvent control< LCL< LCL< LCL
(Replicate 2)

Meas. Conc. = Measured concentration of the test item, dilution factor taken into account
% = Percent of the nominal test item concentration
LCL = Lowest Calibration Level, 0.1 µg test item/L
a.s. = Active substance


 


 


 


 


 


 


 


 


 


 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
2,2’-(Octadec-9-enylimino)bisethanol caused significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal concentrations of 2.50, 5.00, 10.0, 20.0 and 40.0 µg/L, corresponding to overall arithmetic mean measured test item concentrations of 1.29 – 2.19 – 6.00 – 12.9 - 27.0 µg/L.

For hatchability on study days 5 and 6, the NOEC and the LOEC (based on nominal test item concentrations) for this endpoint were determined to be ≥ 40.0 and > 40.0 µg/L, respectively. The corresponding NOEC and LOEC (based on the overall arithmetic mean measured test item concentrations) were ≥ 27.0 and > 27.0 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 10.0 µg/L. Therefore, the respective nominal LOECs were determined to be 20.0 µg/L. The corresponding NOEC and LOEC values based on the overall arithmetic mean measured test item concentrations were NOEC 6.00 µg/L and LOEC 12.9 µg/L, respectively.

For the parameter fry growth (expressed as length and fresh weight) the nominal NOECs were 2.50 µg/L (for both parameters). Therefore, the nominal LOECs for length and weight were determined to be 5.00 µg/L, respectively. The corresponding NOEC and LOEC values based on the overall arithmetic mean measured test item concentrations were NOEC 1.29 µg/L and LOEC 2.19 µg/L, respectively.


All effect values are given based on the nominal and the overall arithmetic mean measured concentrations of the test item 2,2’-(Octadec-9-enylimino)bisethanol.
Executive summary:

The effects of the test item 2,2’-(Octadec-9-enylimino)bisethanol (Batch-No. 1950627) on the early-life stage of fish (Danio rerio / Zebrafish) were determined at the test facility according to OECD Guideline 210 from 2022-05-04 to 2022-06-08.


The test item is a yellow liquid UVCB substance which is dispersible/insoluble in water. Methanol was used as solvent with a concentration of 0.025 mL/L dilution water. Stock solutions in methanol with nominal concentrations of 100, 200, 400, 800 and 1600 mg/L were prepared in appropriate intervals of 7 days and continuously dosed to the dilution water in a flow-through system (for detailed descriptions, please refer to section 4.2). Based on the results of a range finding test the test was conducted as a dose-response test with the nominal test item concentrations 2.50, 5.00, 10.0, 20.0 and 40.0 µg/L, corresponding to the overall arithmetic mean measured test item concentrations of 1.29 – 2.19 – 6.00 – 12.9 - 27.0 µg/L. The term overall arithmetic mean means that all measured samples (stabilized samples, from study days 0, 7, 14, 21 and 34) were taken into account for calculation of the arithmetic mean measured concentrations.


The test was started by placing fertilized eggs into the test vessels and it lasted 35 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 5, 95% of the control and 96% the solvent control larvae had hatched. Therefore, study day 5 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 2,2’-(Octadec-9-enylimino)bisethanol in the test media and the controls was carried out via LC-MS/MS.
The concentrations of the test item 2,2’-(Octadec-9-enylimino)bisethanol, the solvent control and the control were determined during exposure on days 0, 7, 14, 21 and 34. The constituent C18:1 Amine + 2EO was 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 stock solutions prepared in methanol (see section 4.2 for details) were sampled and analyzed from the freshly prepared and corresponding 7 days aged stock solutions. Measured concentrations of the freshly prepared stock solution were 106 to 112% of the nominal values. Measured concentrations of the 7 days aged stock solution were in the range of 111 to 115% of the nominal values.


All effect values are given based on the nominal and the overall arithmetic mean measured concentrations of the test item 2,2’-(Octadec-9-enylimino)bisethanol.


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.


NOEC, LOEC, ECx Values of Hatching Success and Fry Growth
Based on nominal and overall arithmetric mean measured test item concentrations [µg/L]
with 95% Confidence intervals in brackets


















































































ParameterBased on nominal test item concentrations [µg/L]Based on overall arithmetric mean measured test item concentrations [µg/L]
Hatching success afterFry GrowthHatching success afterFry Growth
7 days1)expressed as:6 days1)expressed as:
 LengthWeight LengthWeight
NOEC≥ 40.02.502.50≥ 27.01.291.29
LOEC> 40.05.005.00> 27.02.192.19
EC10n.d.> 20.03.54n.d.> 12.91.61
(< 2.50 to > 20.0)(< 1.29 to > 12.9)
EC20 n.d.n.d.> 20.0n.d.n.d.13.6
(n.d.)
EC50n.d.n.d.n.d.n.d.n.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 arithmetric mean measured test item concentrations [µg/L]
Post-hatch survivalOverall survivalPost-hatch survivalOverall survival
NOEC10.010.06.006.00
LOEC20.020.012.912.9
LC10n.d.n.d.n.d.n.d.
LC2012.715.67.8310.0
(9.56 – 17.2)(9.63 – 20.0)(5.71 – 11.0)(5.76 – 20.2)
LC5016.818.310.711.7
(12.6 – 18.7)(10.9 – 30.8)(7.76 – 12.0)(6.60 – 21.0)

n.d. = not determinable