2,2'-(octadec-9-enylimino)bisethanol

Long-term toxicity to fish

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

Test material information

Constituent 1

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 concentrations

Effect concentrations 1

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

Effect concentrations 2

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

Effect concentrations 3

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

Effect concentrations 4

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

Effect concentrations 5

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

Effect concentrations 6

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

Effect concentrations 7

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

Effect concentrations 8

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

Effect concentrations 9

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

Effect concentrations 10

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

Effect concentrations 11

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

Effect concentrations 12

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

Effect concentrations 13

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

Effect concentrations 14

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

Effect concentrations 15

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

Effect concentrations 16

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

Effect concentrations 17

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

Effect concentrations 18

Duration:

35 d

Dose descriptor:

other: EC20

Effect conc.:

> 20 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Effect concentrations 19

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

Effect concentrations 20

Duration:

35 d

Dose descriptor:

EC10

Effect conc.:

3.54 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Effect concentrations 21

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

Effect concentrations 22

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Effect concentrations 23

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

Effect concentrations 24

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

2.5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Effect concentrations 25

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

Effect concentrations 26

Duration:

35 d

Dose descriptor:

EC10

Effect conc.:

> 20 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Effect concentrations 27

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

Effect concentrations 28

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Effect concentrations 29

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

Effect concentrations 30

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

2.5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Effect concentrations 31

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

Effect concentrations 32

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

Effect concentrations 33

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

Effect concentrations 34

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)

Nominal	Rep.	PHD -3	PHD -2	PHD -1	PHD 0	PHD 1	PHD 2
test item conc.		Study day 2	Study day 3	Study day 4	Study day 5	Study day 6	Study day 7
[µg/L]		Cumulative hatching rate [%]
Control	1	0	5	75	90	90	90
	2	0	20	60	100	100	100
	3	5	65	90	100	100	100
	4	0	35	90	90	90	90
	Mean	1	31	79	95	95	95
Solvent control	1	0	10	65	100	100	100
	2	0	55	100	100	100	100
	3	0	45	85	90	90	90
	4	0	10	55	95	95	95
	Mean	0	30	76	96	96	96
Feb 50	1	0	35	90	90	100	100
	2	0	20	95	100	100	100
	3	0	55	100	100	100	100
	4	0	35	80	90	90	90
	Mean	0	36	91	95 (ns)	98 (ns)	98 (ns)
5.00	1	0	15	60	95	100	100
	2	0	5	55	100	100	100
	3	0	5	70	100	100	100
	4	0	10	75	95	95	95
	Mean	0	9	65	98 (ns)	99 (ns)	99 (ns)
10.0	1	0	20	90	100	100	100
	2	0	5	55	100	100	100
	3	0	50	95	100	100	100
	4	0	5	55	95	100	100
	Mean	0	20	74	99 (ns)	100 (ns)	100 (ns)
20.0	1	0	5	55	85	100	100
	2	0	10	30	85	95	95
	3	0	10	75	95	95	95
	4	0	40	90	95	95	95
	Mean	0	16	63	90 (ns)	96 (ns)	96 (ns)
40.0	1	0	5	45	85	95	100
	2	0	0	25	85	90	90
	3	0	10	35	95	95	95
	4	0	50	95	100	100	100
	Mean	0	16	50	91 (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

Nominal	Rep.	PHD -1	PHD 0	PHD 1	PHD 2	PHD 3
test item conc.		Study day 4	Study day 5	Study day 6	Study day 7	Study day 8
[µg/L]		Cumulative swim up [%]
Control	1	0	94	100	100	100
	2	0	90	100	100	100
	3	0	100	100	100	100
	4	0	83	100	100	100
	Mean	0	92	100	100	100
Solvent control	1	0	95	100	100	100
	2	0	80	100	100	100
	3	0	78	94	100	100
	4	0	89	100	100	100
	Mean	0	86	99	100	100
Feb 50	1	0	100	100	100	100
	2	0	95	100	100	100
	3	0	100	100	100	100
	4	0	94	100	100	100
	Mean	0	97	100	100	100
5.00	1	0	89	100	100	100
	2	0	90	100	100	100
	3	0	85	100	100	100
	4	0	100	100	100	100
	Mean	0	91	100	100	100
10.0	1	0	100	100	100	100
	2	0	90	100	100	100
	3	0	100	100	100	100
	4	0	95	95	95	100
	Mean	0	96	99	99	100
20.0	1	0	76	95	100	100
	2	0	71	95	100	100
	3	0	79	100	100	100
	4	0	100	100	100	100
	Mean	0	82	98	100	100
40.0	1	0	82	100	100	50
	2	0	82	94	100	80
	3	0	74	100	93	57
	4	0	85	100	100	92
	Mean	0	81	99	98	70

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

Nominal	Rep.	Eggs introduced on study day 0	Cumulative number of hatched larvae	Vital Larvae on study day 35	Post-Hatch survival
test item conc.				(PHD 30)	on study day 35
[µg/L]					[%]
Control	1	20	18	16	89
	2	20	20	19	95
	3	20	20	16	80
	4	20	18	16	89
	Mean	20	19	17	88
Solvent control	1	20	20	18	90
	2	20	20	17	85
	3	20	18	16	89
	4	20	19	18	95
	Mean	20	19	17	90
Feb 50	1	20	20	19	95
	2	20	20	19	95
	3	20	20	20	100
	4	20	18	13	72
	Mean	20	20	18	91 (ns)
5.00	1	20	20	16	80
	2	20	20	19	95
	3	20	20	16	80
	4	20	19	16	84
	Mean	20	20	17	85 (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

Nominal	Rep.	Eggs introduced on study day 0	Cumulative number of hatched larvae	Vital Larvae on study day 34	Post-Hatch survival
test item conc.				(PHD 30)	on study day 34
[µg/L]					[%]
10.0	1	20	20	18	90
	2	20	20	18	90
	3	20	20	18	90
	4	20	20	15	75
	Mean	20	20.0	17.0	86 (ns)
20.0	1	20	20	4	20
	2	20	19	3	16
	3	20	19	6	32
	4	20	19	5	26
	Mean	20	19.3	5	24 (s)
40.0	1	20	20	0	0
	2	20	18	0	0
	3	20	19	0	0
	4	20	20	0	0
	Mean	20	19.3	0	0 (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

Nominal	Rep.	Vital larvae on	Overall survival	Overall mortality
test item conc.		study day 35
[µg/L]		(PHD 30)
			[%]	[%]
Control	1	16	80	20
	2	19	95	5
	3	16	80	20
	4	16	80	20
	Mean	17	84	16
Solvent control	1	18	90	10
	2	17	85	15
	3	16	80	20
	4	18	90	10
	Mean	17	86	14
2.50	1	19	95	5
	2	19	95	5
	3	20	100	0
	4	13	65	35
	Mean	18	89	11 (ns)
5.00	1	16	80	20
	2	19	95	5
	3	16	80	20
	4	16	80	20
	Mean	17	84	16 (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

Nominal	Rep.	Vital larvae on	Overall survival	Overall mortality
test item conc.		study day 35
[µg/L]		(PHD 30)
			[%]	[%]
10.0	1	18	90	10
	2	18	90	10
	3	18	90	10
	4	15	75	25
	Mean	17	86	14 (ns)
20.0	1	4	20	80
	2	3	15	85
	3	6	30	70
	4	5	25	75
	Mean	5	23	77 (s)
40.0	1	0	0	100
	2	0	0	100
	3	0	0	100
	4	0	0	100
	Mean	0	0	100 (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 Day	2.50 µg/L				5.00 µg/L
	1	2	3	4	1	2	3	4
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 Day	20.0 µg/L
	1	2	3	4
11	-	1 T	-	-
12	1 G	-	-	1 G
13	1 T	1 T	-	1 S
		2 Q		1 G
		2 G		2 Q
14	4 S	4 G	-	-
	2 G	4 Q
	6 Q
15	4 G	5 G	-	2 T
	4 Q	5 Q
16	1 G	1 T	1 G	-
		3 G
		3 Q
17	-	2 G	3 G	2 G
18	-	1 G	-	-
19	-	-	2 G	1 G

 

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

Study Day	40.0 µg/L
	1	2	3	4
7	-	-	1 Q	-
			1 G
			1 T
8	2 T	1 T	1 T	1 G
	4 SE	2 G	2 G	1 SE
	4 S	2 Q	2 SE	12 Q
	6 G		4 Q
	6 ME
	6 F
9	1 T	1 T	100%	2 T
	3 G	2 G	Mortality	1 S
	3 ME	2 ME		1 SE
	3 F	2 F		2 G
	1 S	1 S		2 ME
	1 SE	1 SE		2 F
10	1 S	2 F		1 S
	1 SE	2 S		1 SE
		2 SE
11	100%	1 ME		100%
	Mortality	1 F		Mortality
12	100 % 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

Nominal	Rep.	PHD 30 (End of exposure)
test item conc.[µg/L]		Mean total length per fish [mm]	Mean wet weight per fish [mg]
Control	1	16.0	39.1
	2	14.6	30.7
	3	16.0	41.5
	4	16.1	47.0
	Mean	15.7	39.6
	± SD	0.622	5.87
	CV [%]	3.96	14.8
Solvent control	1	15.9	39.0
	2	16.0	34.7
	3	16.0	38.7
	4	15.8	36.4
	Mean	15.9	37.2
	± SD	0.083	1.76
	CV [%]	0.52	4.73
Feb 50	1	15.1	36.3
	2	15.7	34.7
	3	15.7	34.9
	4	17.0	48.2
	Mean	15.9 (ns)	38.5 (ns)
	± SD	0.694	5.62
	CV [%]	4.36	14.6
5.00	1	14.9	31.3
	2	14.7	32.5
	3	15.2	33.7
	4	15.1	32.4
	Mean	15.0 (s)	32.5 (s)
	± SD	0.192	0.850
	CV [%]	1.28	2.62

(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

Nominal	Rep.	PHD 30 (End of exposure)
test item conc.[µg/L]		Mean total length per fish [mm]	Mean wet weight per fish [mg]
10.0	1	12.9	24.1
	2	14.6	29.6
	3	13.6	27.5
	4	14.8	34.6
	Mean	14.0 (s)	29.0 (s)
	± SD	0.769	3.81
	CV [%]	5.49	13.1
20.0	1	14.5	31.6
	2	15.3	37.6
	3	14.5	31.2
	4	14.7	33.2
	Mean	14.8 (s)	33.4 (s)
	± SD	0.328	2.54
	CV [%]	2.22	7.60

(s) = Statistically significant difference from pooled controls

 

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

Fish No.	Control				Solvent control
	Total length of individual fish in [mm]
	1	2	3	4	1	2	3	4
1	17.0	11.0	15.5	13.0	13.0	15.0	15.5	20.0
2	19.0	12.5	13.0	20.0	18.5	16.0	17.0	15.0
3	16.0	13.0	17.0	17.0	14.0	14.5	14.0	14.0
4	15.0	16.0	17.0	14.0	19.5	18.0	11.0	11.0
5	14.0	11.0	19.0	18.0	17.5	18.5	13.0	16.0
6	11.0	15.0	15.5	18.0	20.0	14.5	21.5	14.0
7	14.5	13.5	19.0	18.0	18.0	14.0	22.0	15.0
8	15.0	21.0	19.0	18.5	16.0	16.5	14.0	17.0
9	18.0	19.0	18.0	20.0	20.0	16.0	16.0	20.0
10	18.5	18.0	14.0	18.0	18.0	16.0	20.0	17.0
11	14.0	12.5	17.0	17.0	16.0	19.5	18.0	20.5
12	20.0	12.0	16.0	15.0	15.0	15.5	16.0	17.0
13	15.0	17.0	18.0	12.0	11.0	18.0	19.0	17.0
14	15.0	14.0	16.0	14.5	17.0	12.0	16.0	16.5
15	18.5	18.0	12.0	15.0	18.0	16.0	10.0	16.0
16	15.0	15.0	10.0	10.0	8.5	16.0	13.0	14.0
17	-	13.0	-	-	16.0	16.0	-	11.5
18	-	13.0	-	-	10.0	-	-	12.5
19	-	12.0	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-
Mean	16.0	14.6	16.0	16.1	15.9	16.0	16.0	15.8
± SD	2.27	2.80	2.54	2.80	3.31	1.76	3.39	2.66
CV %	14.2	19.2	15.9	17.4	20.8	11.0	21.2	16.8

- = 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 50				5.00
	Total length of individual fish in [mm]
	1	2	3	4	1	2	3	4
1	17.0	14.0	17.5	17.0	17.0	20.0	15.0	12.5
2	19.5	13.5	16.0	13.5	18.5	13.0	14.5	15.0
3	20.0	15.0	13.0	18.5	16.0	18.0	20.0	18.0
4	14.0	16.0	19.5	16.0	18.0	14.5	12.0	21.0
5	15.0	13.0	19.0	17.0	11.0	11.5	20.5	17.0
6	13.0	17.0	13.5	17.0	10.0	14.0	13.0	16.0
7	10.0	16.0	13.0	17.0	9.0	15.0	19.0	11.0
8	14.5	11.0	16.0	20.0	20.0	18.5	16.0	14.0
9	16.0	17.0	15.0	19.0	16.0	18.0	13.0	18.0
10	12.0	17.5	15.0	17.0	17.5	16.0	18.5	17.0
11	15.5	17.5	15.0	21.0	16.0	13.5	16.0	13.0
12	11.0	13.5	14.5	12.5	16.0	11.5	13.0	16.0
13	14.0	17.5	17.0	15.5	14.0	13.0	13.0	11.0
14	13.0	19.0	18.0	-	14.0	15.0	14.0	18.0
15	13.0	18.0	17.0	-	12.0	14.0	13.5	10.0
16	17.5	15.0	15.5	-	13.0	18.5	12.5	13.5
17	20.5	12.5	11.0	-	-	10.5	-	-
18	16.5	15.0	18.0	-	-	14.5	-	-
19	15.5	20.0	17.0	-	-	10.5	-	-
20	-	-	14.0	-	-	-	-	-
Mean	15.1	15.7	15.7	17.0	14.9	14.7	15.2	15.1
± SD	2.84	2.31	2.14	2.27	3.09	2.76	2.73	2.99
CV %	18.8	14.7	13.6	13.4	20.7	18.8	18.0	19.8

- = 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]
	10.0				20.0
	Total length of individual fish in [mm]
	1	2	3	4	1	2	3	4
1	9.0	19.0	17.5	11.5	16.0	17.5	11.0	13.0
2	9.0	15.0	18.0	14.0	18.0	10.0	14.0	11.5
3	17.5	12.0	14.0	11.5	12.5	18.5	15.5	17.5
4	7.5	17.0	14.5	15.5	11.5	-	17.0	19.5
5	17.0	20.0	11.0	14.0	-	-	12.0	12.0
6	18.0	17.0	13.5	16.0	-	-	17.5	-
7	19.0	16.5	14.5	15.0	-	-	-	-
8	11.0	11.0	12.5	17.0	-	-	-	-
9	10.0	15.5	10.0	23.0	-	-	-	-
10	16.0	12.0	11.0	14.5	-	-	-	-
11	17.0	13.0	9.5	16.0	-	-	-	-
12	13.0	18.5	20.5	12.5	-	-	-	-
13	10.0	13.0	17.0	16.0	-	-	-	-
14	9.0	15.0	10.0	16.0	-	-	-	-
15	13.5	10.5	10.0	10.0	-	-	-	-
16	14.0	14.0	12.0	-	-	-	-	-
17	12.0	12.5	16.0	-	-	-	-	-
18	10.0	10.5	13.0	-	-	-	-	-
19	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-
Mean	12.9	14.6	13.6	14.8	14.5	15.3	14.5	14.7
± SD	3.60	2.89	3.12	2.94	2.62	3.79	2.42	3.20
CV %	27.9	19.8	22.9	19.9	18.1	24.8	16.7	21.8

- = 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

Nominal	Replicate	Number of fish alive	Pooled	Mean	Mean	± SD	CV %
test item conc.		on study day 35	wet weight	wet weight
[µg/L]			per replicate	per fish per replicate
			[mg]	[mg]	[mg]
Control	1	16	625.8	39.1	39.6	5.87	14.8
	2	19	584.0	30.7
	3	16	663.3	41.5
	4	16	751.5	47.0
Solvent control	1	18	701.2	39.0	37.2	1.76	4.73
	2	17	590.4	34.7
	3	16	619.5	38.7
	4	18	654.6	36.4
Feb 50	1	19	689.8	36.3	38.5	5.62	14.6
	2	19	659.1	34.7
	3	20	697.4	34.9
	4	13	626.4	48.2
5.00	1	16	500.1	31.3	32.5	0.850	2.62
	2	19	617.5	32.5
	3	16	538.6	33.7
	4	16	518.4	32.4
10.0	1	18	433.7	24.1	29.0	3.81	13.1
	2	18	531.9	29.6
	3	18	495.8	27.5
	4	15	518.9	34.6
20.0	1	4	126.2	31.6	33.4	2.54	7.60
	2	3	112.7	37.6
	3	6	186.9	31.2
	4	5	166.1	33.2

 

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

Study day	Replicate	Dissolved oxygen [%]
		Nominal test item concentrations [µg/L]
		Control	Solvent control	2.50	5.00	10.0	20.0	40.0
0	1	100	100	100	100	100	100	100
	2	99	100	100	100	100	100	100
	3	100	100	100	100	100	100	100
	4	100	100	100	100	100	100	100
2	1	100	100	100	100	100	100	100
	2	100	100	100	100	100	100	100
	3	100	100	100	100	100	100	100
	4	100	100	100	100	100	100	100
5	1	100	100	100	100	100	100	100
	2	100	100	100	100	100	100	100
	3	100	100	100	100	100	100	100
	4	100	100	100	100	100	100	100
7	1	100	100	98	98	99	100	99
	2	99	99	99	99	99	98	98
	3	100	98	98	100	99	98	99
	4	99	100	100	100	100	99	99
9	1	97	97	96	95	97	97	96
	2	97	97	97	97	96	96	94
	3	97	97	96	97	96	96	97
	4	97	97	96	96	96	96	96
12	1	97	96	94	94	95	95	95
	2	95	96	95	96	95	95	97
	3	96	96	95	96	95	95	95
	4	96	96	96	96	95	96	96
14	1	97	96	92	95	95	94	-
	2	95	95	93	94	95	94	-
	3	96	95	92	95	95	94	-
	4	96	97	96	94	95	95	-
16	1	98	97	94	96	97	96	-
	2	97	97	96	97	96	96	-
	3	97	97	96	97	97	96	-
	4	96	98	96	94	95	96	-

- = No measurement, due to 100 % mortality

 

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

Study day	Replicate	Dissolved oxygen [%]
		Nominal test item concentrations [µg/L]
		Control	Solvent control	2.50	5.00	10.0	20.0	40.0
19	1	96	94	92	93	94	94	-
	2	94	94	92	95	93	92	-
	3	94	94	92	92	92	92	-
	4	94	95	93	91	90	94	-
21	1	98	97	94	95	95	93	-
	2	96	97	95	95	95	92	-
	3	96	96	95	93	93	93	-
	4	96	97	93	91	90	94	-
23	1	95	95	92	93	93	94	-
	2	95	95	92	93	94	92	-
	3	95	94	92	91	92	93	-
	4	95	95	87	92	91	92	-
26	1	96	95	94	92	94	95	-
	2	96	94	94	92	92	94	-
	3	94	94	94	92	90	92	-
	4	95	94	91	92	90	93	-
28	1	92	90	90	86	90	91	-
	2	91	91	90	87	88	89	-
	3	89	89	88	88	87	91	-
	4	91	85	91	88	88	89	-
30	1	93	90	88	86	90	90	-
	2	91	89	89	87	88	87	-
	3	89	88	87	86	88	90	-
	4	90	89	88	86	85	90	-
34	1	85	86	80	82	86	88	-
	2	85	82	83	77	82	86	-
	3	79	78	80	81	82	88	-
	4	85	83	80	78	80	85	-
Mean		95	95	94	94	94	95	98
± SD		4.40	4.95	5.20	5.73	5.11	4.03	2.04
Min.		79	78	80	77	80	85	94
Max.		100	100	100	100	100	100	100

- = No measurement, due to 100 % mortality
Min./Max. = Minimum/Maximum measured dissolved oxygen concentration

 

Water Temperature (Continuous Measuring) in Replicate 2 of the Control Group

Period of measurements	2022-05-04 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 day	Rep.	Water temperature [°C]
		Nominal test item concentrations [µg/L]
		Control	Solvent control	2.50	5.00	10.0	20.0	40.0
0	1	26.4	26.4	26.5	26.6	26.6	26.4	26.5
	2	26.4	26.4	26.6	26.5	26.8	26.5	26.4
	3	26.4	26.5	26.7	26.7	26.6	26.5	26.5
	4	26.4	26.5	26.7	26.5	26.5	26.5	26.7
7	1	26.4	26.4	26.5	26.6	26.6	26.4	26.5
	2	26.4	26.5	26.6	26.5	26.8	26.6	26.5
	3	26.4	26.5	26.7	26.7	26.6	26.5	26.5
	4	26.3	26.4	26.6	26.5	26.4	26.4	26.7
14	1	26.7	26.7	26.8	27.0	26.9	26.8	-
	2	26.7	26.8	26.8	27.0	27.0	26.8	-
	3	26.7	26.8	27.1	27.0	26.9	26.8	-
	4	26.7	26.7	26.9	26.8	26.8	26.7	-
21	1	26.2	26.3	26.3	26.7	26.5	26.4	-
	2	26.2	26.3	26.5	26.4	26.6	26.5	-
	3	26.3	26.2	26.5	26.5	26.5	26.4	-
	4	26.2	26.2	26.6	26.3	26.3	26.4	-
28	1	26.1	26.3	26.4	26.6	26.5	26.4	-
	2	26.2	26.3	26.5	26.4	26.5	26.4	-
	3	26.2	26.3	26.6	26.5	26.4	26.3	-
	4	26.2	26.2	26.5	26.3	26.3	26.3	-
Mean		26.4	26.4	26.6	26.6	26.6	26.5	26.5
± SD		0.192	0.190	0.182	0.214	0.201	0.159	0.106
Min.		26.1	26.2	26.3	26.3	26.3	26.3	26.4
Max.		26.7	26.8	27.1	27.0	27.0	26.8	26.7

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

 

pH-Values in the Test Media

Study day	Rep.	pH-value
		Nominal test item concentrations [µg/L]
		Control	Solvent control	2.50	5.00	10.0	20.0	40.0
0	1	7.60	7.72	7.70	7.65	7.71	7.68	7.68
	2	7.62	7.73	7.71	7.68	7.71	7.69	7.67
	3	7.71	7.75	7.69	7.68	7.68	7.72	7.73
	4	7.68	7.75	7.69	7.69	7.70	7.77	7.70
7	1	7.64	7.98	7.97	7.91	7.96	7.96	8.01
	2	7.97	7.92	7.95	7.98	7.95	7.92	7.96
	3	8.07	7.96	7.94	8.03	7.97	7.96	8.03
	4	8.00	8.08	7.97	8.05	7.97	7.94	8.00
14	1	7.65	7.77	7.71	7.65	7.62	7.63	-
	2	7.70	7.77	7.68	7.62	7.65	7.62	-
	3	7.77	7.77	7.64	7.65	7.64	7.59	-
	4	7.76	7.74	7.71	7.68	7.63	7.62	-
21	1	7.72	7.86	7.88	7.81	7.83	7.77	-
	2	7.83	7.92	7.80	7.86	7.83	7.80	-
	3	7.91	7.82	7.84	7.76	7.77	7.84	-
	4	7.85	7.86	7.80	7.79	7.72	7.76	-
28	1	7.58	7.67	7.62	7.60	7.61	7.72	-
	2	7.65	7.68	7.62	7.63	7.60	7.64	-
	3	7.66	7.59	7.62	7.65	7.61	7.67	-
	4	7.68	7.62	7.65	7.63	7.58	7.68	-
Mean		7.75	7.80	7.76	7.75	7.74	7.75	7.85
± SD		0.141	0.126	0.124	0.143	0.135	0.120	0.165
Min.		7.58	7.59	7.62	7.60	7.58	7.59	7.67
Max.		8.07	8.08	7.97	8.05	7.97	7.96	8.03

- = 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 day	Replicate	Total hardness [mg CaCO3/L]
		Nominal test item conc. [µg/L]
		Control	20.0	40.0
0	1	70		69
8	2	67		68
15	3	64	65
21	4	64	66
29	1	68	68
Mean		67	66	69
± SD		2.61	1.53	0.71
Min.		64	65	68
Max.		70	68	69

= No measurement, due to 100 % mortality
n.a. = Not applicable
Min./Max. = Minimum/Maximum measured hardness

 

Total Organic Carbon (TOC) of the Dilution Water

Study day	TOC [mg/L]
0	1.15
8	1.25
15	1.25
21	1.29
29	1.34
Mean	1.26
SD	0.070
Min.	1.15
Max.	1.34

Min./Max. = Minimum/Maximum measured TOC

 

Flow Rates

Flow rate	Nominal test item concentrations [µg/L]
	Control	Solvent control	2.50	5.00	10.0	20.0	40.0
	Measured flow rates of the membrane piston pumps^1)
	[L/h]
Minimum	3.00	3.00	3.00	3.00	34366	3.00	3.00
Maximum	3.36	3.24	3.24	3.24	3.24	3.24	3.24
Mean	3.13	3.11	3.12	3.11	3.12	3.13	3.11
± Standard deviation	± 0.055	± 0.046	± 0.051	± 0.053	± 0.046	± 0.051	± 0.060
Overall mean	3.12
± 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 day		Day -1
Replicate		Repl. 2
Nominal		2,2’-(Octadec-9-enylimino)bisethanol
concentration of the		C18:1 Amine + 2 EO
		Test Vessel		Test vessel (centrifuged)
Test item	a.i.	Meas. conc.	%	Meas. conc.	%
[µg/L]	[µg a.i./L]	[µg a.i./L]		[µg a.i./L]
40.0	30.2	20.7	69	10.0	33
20.0	15.1	8.19	54	4.04	27
10.0	7.54	4.40	58	2.32	31
5.00	3.77	1.50	40	0.587	16
2.50	1.89	1.07	57	0.421	22
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 solutions		7 Days aged stock solutions
Nominal		2,2’-(Octadec-9-enylimino)bisethanol
concentration of the		C18:1 Amine + 2 EO
Test item	a.i.	Meas.	%	Meas.	%
[mg/L]	[mg a.i./L]	conc.		conc.
		[mg a.i./L]		[mg a.i./L]
1600	1207	1349	112	1347	112
800	603	639	106	672	111
400	302	322	107	340	113
200	151	160	106	173	115
100	75.4	81.1	108	85.4	113

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 day		Day 0		Day 7		Day 14
Replicate		Repl. 3		Repl. 4		Repl. 1
Nominal		2,2’-(Octadec-9-enylimino)bisethanol
concentration of the		C18:1 Amine + 2 EO
Test item	a.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]
40.0	30.2	21.1	70	18.7	62	21.2	70
20.0	15.1	12.2	81	9.38	62	10.0	66
10.0	7.54	5.33	71	4.08	54	4.65	62
5.00	3.77	2.05	54	1.66	44	1.53	41
2.50	1.89	0.963	51	1.03	55	1.05	56
Solvent Control		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ
Sampling day		Day 21		Day 34
Replicate		Repl. 2		Repl. 4
Nominal		2,2’-(Octadec-9-enylimino)bisethanol
concentration of the		C18:1 Amine + 2 EO
Test item	a.i.	Meas.	%	Meas.	%
[µg/L]	[µg a.i./L]	conc.		conc.
		[µg a.i./L]		[µg a.i./L]
40.0	30.2	-^1)		-^1)
20.0	15.1	7.75	51	9.47	63
10.0	7.54	4.59	61	3.99	53
5.00	3.77	1.19	31	1.82	48
2.50	1.89	0.807	43	1.00	53
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
Nominal		Overall Arithmetic Mean Measured Concentrations of the
concentration of the
Test item	a.i.	Test item	a.i.	%
[µg/L]	[µg a.i./L]	[µg/L]	[µg a.i./L]
40.0	30.2	27.0	20.3	67
20.0	15.1	12.9	9.76	65
10.0	7.54	6.00	4.53	60
5.00	3.77	2.19	1.65	44
2.50	1.89	1.29	0.970	51

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 [%]
0.00	70	30
0.30	70	30
1.30	5	95
2.00	5	95
2.10	70	30
2.50	70	30

 

Dilution steps (Test Vessel)

Nominal	Dilution	Sample	Final
test item	Factor*	volume	volume
concentration		[mL]	[mL]
[µg/L]
40.0	10	0.2	1.0
20.0	10	0.2	1.0
10.0	2	1.0	1.0
5.0	2	1.0	1.0
2.50	2	1.0	1.0
Solvent Control	2	1.0	1.0
Control	2	1.0	1.0

*including factor 2

 

Dilution steps (Stock Solution)

Nominal	Dilution	Sample	Final
test item	Factor	volume	volume
concentration		[mL]	[mL]
[mg/L]
1600	250 000	0.04^1)	4.0^1)
		0.04^2)	4.0^2)
		0.04^3)	1.0^3)
800	125 000	0.04^1)	4.0^1)
		0.04^2)	4.0^2)
		0.08^3)	1.0^3)
400	62 500	0.04^1)	4.0^1)
		0.04^2)	4.0^2)
		0.16^3)	1.0^3)
200	31 250	0.04^1)	4.0^1)
		0.04^2)	4.0^2)
		0.32^3)	1.0^3)
100	15 625	0.04^1)	4.0^1)
		0.04^2)	4.0^2)
		0.64^3)	1.0^3)
Control	15 625	0.04^1)	4.0^1)
		0.04^2)	4.0^2)
		0.64^3)	1.0^3)

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 day		Day 0		Day 7		Day 14
Replicate		Repl. 3		Repl. 4		Repl. 1
Nominal		2,2’-(Octadec-9-enylimino)bisethanol
concentration of the		C18:1 Amine + 2 EO
Test item	a.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]
40.0	30.2	7.17	24	5.36	18	7.69	26
20.0	15.1	3.81	25	3.37	22	4.71	31
10.0	7.54	1.84	24	1.34	18	1.61	21
5.00	3.77	0.568	15	0.565	15	0.430	11
2.50	1.89	0.325	17	0.320	17	0.302	16
Solvent Control		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ
Sampling day		Day 21		Day 34
Replicate		Repl. 2		Repl. 4
Nominal		2,2’-(Octadec-9-enylimino)bisethanol
concentration of the		C18:1 Amine + 2 EO
Test item	a.i.	Meas.	%	Meas.	%
[µg/L]	[µg a.i./L]	conc.		conc.
		[µg a.i./L]		[µg a.i./L]
40.0	30.2	-^1)		-^1)
20.0	15.1	5.71	38	3.29	22
10.0	7.54	3.03	40	1.49	20
5.00	3.77	0.648	17	0.671	18
2.50	1.89	0.459	24	0.291	15
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 day

Nominal test item concentration [µg/L]

Control

Solvent control

2.50

Number of vital eggs and larvae (listed eggs/larvae) by study day

1

2

3

4

1

2

3

4

1

2

3

4

0

20/0

20/0

20/0

20/0

20/0

20/0

20/0

20/0

20/0

20/0

20/0

20/0

1

18/0

20/0

20/0

19/0

20/0

20/0

19/0

19/0

20/0

20/0

20/0

18/0

2

18/0

20/0

19/1

19/0

20/0

20/0

18/0

19/0

20/0

20/0

20/0

18/0

3

17/1

16/4

7/13

12/7

18/2

9/11

9/9

17/2

13/7

16/4

9/11

11/7

4

3/15

8/12

2/18

1/18

7/13

0/20

1/17

8/11

2/18

1/19

0/20

2/16

5

0/18

0/20

0/20

0/18

0/20

0/20

0/18

0/19

2/18

0/20

0/20

0/18

6

0/18

0/20

0/19

0/18

0/20

0/19

0/17

0/19

0/20

0/20

0/20

0/17

7

0/18

0/20

0/19

0/18

0/20

0/19

0/17

0/19

0/20

0/20

0/20

0/17

8

0/18

0/20

0/19

0/18

0/20

0/19

0/17

0/19

0/20

0/20

0/20

0/17

9

0/18

0/20

0/19

0/18

0/20

0/19

0/16

0/18

0/20

0/20

0/20

0/17

10

0/18

0/20

0/19

0/18

0/20

0/19

0/16

0/18

0/20

0/20

0/20

0/17

11

0/18

0/20

0/19

0/18

0/20

0/19

0/16

0/18

0/19

0/20

0/20

0/16

12

0/18

0/20

0/19

0/18

0/20

0/19

0/16

0/18

0/19

0/20

0/20

0/16

13

0/18

0/20

0/19

0/16

0/19

0/19

0/16

0/18

0/19

0/19

0/20

0/16

14

0/17

0/20

0/19

0/16

0/19

0/18

0/16

0/18

0/19

0/19

0/20

0/16

15

0/17

0/20

0/19

0/16

0/19

0/18

0/16

0/18

0/19

0/19

0/20

0/16

16

0/17

0/20

0/18

0/16

0/19

0/18

0/16

0/18

0/19

0/19

0/20

0/16

17

0/17

0/20

0/18

0/16

0/19

0/18

0/16

0/18

0/19

0/19

0/20

0/16

18

0/17

0/20

0/18

0/16

0/18

0/18

0/16

0/18

0/19

0/19

0/20

0/15

19

0/17

0/20

0/17

0/16

0/18

0/18

0/16

0/18

0/19

0/19

0/20

0/15

20

0/17

0/20

0/17

0/16

0/18

0/18

0/16

0/18

0/19

0/19

0/20

0/15

21

0/17

0/20

0/17

0/16

0/18

0/18

0/16

0/18

0/19

0/19

0/20

0/15

22

0/17

0/20

0/17

0/16

0/18

0/18

0/16

0/18

0/19

0/19

0/20

0/15

23

0/17

0/20

0/17

0/16

0/18

0/18

0/16

0/18

0/19

0/19

0/20

0/15

24

0/17

0/20

0/17

0/16

0/18

0/18

0/16

0/18

0/19

0/19

0/20

0/15

25

0/17

0/20

0/17

0/16

0/18

0/18

0/16

0/18

0/19

0/19

0/20

0/15

26

0/16

0/19

0/16

0/16

0/18

0/17

0/16

0/18

0/19

0/19

0/20

0/14

27

0/16

0/19

0/16

0/16

0/18

0/17

0/16

0/18

0/19

0/19

0/20

0/14

28

0/16

0/19

0/16

0/16

0/18

0/17

0/16

0/18

0/19

0/19

0/20

0/14

29

0/16

0/19

0/16

0/16

0/18

0/17

0/16

0/18

0/19

0/19

0/20

0/14

30

0/16

0/19

0/16

0/16

0/18

0/17

0/16

0/18

0/19

0/19

0/20

0/14

31

0/16

0/19

0/16

0/16

0/18

0/17

0/16

0/18

0/19

0/19

0/20

0/14

32

0/16

0/19

0/16

0/16

0/18

0/17

0/16

0/18

0/19

0/19

0/20

0/14

33

0/16

0/19

0/16

0/16

0/18

0/17

0/16

0/18

0/19

0/19

0/20

0/13

34

0/16

0/19

0/16

0/16

0/18

0/17

0/16

0/18

0/19

0/19

0/20

0/13

35

0/16

0/19

0/16

0/16

0/18

0/17

0/16

0/18

0/19

0/19

0/20

0/13

 

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

Study day

Nominal test item concentration [µg/L]

5.00

10.0

20.0

Number of vital eggs and larvae (listed eggs/larvae) by study day

1

2

3

4

1

2

3

4

1

2

3

4

0

20/0

20/0

20/0

20/0

20/0

20/0

20/0

20/0

20/0

20/0

20/0

20/0

1

20/0

20/0

20/0

20/0

20/0

20/0

20/0

20/0

20/0

19/0

19/0

20/0

2

20/0

20/0

20/0

19/0

20/0

20/0

20/0

20/0

20/0

19/0

19/0

19/0

3

17/3

19/1

19/1

17/2

16/4

19/1

10/10

19/1

19/1

17/2

17/2

11/8

4

8/12

9/11

6/14

4/15

2/18

9/11

1/19

9/11

9/11

13/6

4/15

1/18

5

1/19

0/20

0/20

0/19

0/20

0/20

0/20

1/19

3/17

2/17

0/19

0/19

6

0/20

0/20

0/20

0/19

0/20

0/20

0/20

0/20

0/20

0/19

0/19

0/19

7

0/20

0/20

0/20

0/19

0/20

0/19

0/20

0/20

0/20

0/19

0/19

0/19

8

0/20

0/20

0/20

0/19

0/20

0/19

0/20

0/20

0/20

0/19

0/19

0/19

9

0/20

0/20

0/20

0/19

0/20

0/19

0/20

0/20

0/20

0/19

0/19

0/19

10

0/20

0/20

0/20

0/19

0/20

0/19

0/20

0/20

0/20

0/18

0/19

0/19

11

0/20

0/20

0/20

0/19

0/20

0/19

0/20

0/20

0/20

0/18

0/18

0/18

12

0/18

0/20

0/19

0/19

0/19

0/19

0/20

0/20

0/20

0/14

0/18

0/17

13

0/17

0/20

0/19

0/19

0/19

0/19

0/20

0/19

0/19

0/14

0/18

0/17

14

0/17

0/20

0/19

0/19

0/19

0/19

0/20

0/19

0/15

0/13

0/16

0/14

15

0/17

0/20

0/19

0/19

0/19

0/19

0/20

0/19

0/11

0/12

0/16

0/14

16

0/17

0/20

0/19

0/19

0/19

0/19

0/20

0/19

0/8

0/10

0/16

0/12

17

0/17

0/20

0/19

0/19

0/19

0/19

0/20

0/19

0/6

0/8

0/13

0/12

18

0/17

0/20

0/19

0/19

0/19

0/19

0/20

0/18

0/6

0/7

0/10

0/9

19

0/17

0/20

0/19

0/19

0/19

0/19

0/20

0/17

0/6

0/6

0/9

0/9

20

0/17

0/20

0/18

0/19

0/18

0/19

0/20

0/17

0/6

0/5

0/9

0/7

21

0/17

0/20

0/18

0/18

0/18

0/19

0/20

0/16

0/5

0/4

0/8

0/7

22

0/16

0/20

0/18

0/18

0/18

0/19

0/20

0/16

0/5

0/3

0/7

0/6

23

0/16

0/19

0/18

0/18

0/18

0/19

0/20

0/16

0/4

0/3

0/6

0/6

24

0/16

0/19

0/18

0/18

0/18

0/19

0/20

0/16

0/4

0/3

0/6

0/5

25

0/16

0/19

0/17

0/18

0/18

0/19

0/20

0/16

0/4

0/3

0/6

0/5

26

0/16

0/19

0/16

0/16

0/18

0/18

0/18

0/15

0/4

0/3

0/6

0/5

27

0/16

0/19

0/16

0/16

0/18

0/18

0/18

0/15

0/4

0/3

0/6

0/5

28

0/16

0/19

0/16

0/16

0/18

0/18

0/18

0/15

0/4

0/3

0/6

0/5

29

0/16

0/19

0/16

0/16

0/18

0/18

0/18

0/15

0/4

0/3

0/6

0/5

30

0/16

0/19

0/16

0/16

0/18

0/18

0/18

0/15

0/4

0/3

0/6

0/5

31

0/16

0/19

0/16

0/16

0/18

0/18

0/18

0/15

0/4

0/3

0/6

0/5

32

0/16

0/19

0/16

0/16

0/18

0/18

0/18

0/15

0/4

0/3

0/6

0/5

33

0/16

0/19

0/16

0/16

0/18

0/18

0/18

0/15

0/4

0/3

0/6

0/5

34

0/16

0/19

0/16

0/16

0/18

0/18

0/18

0/15

0/4

0/3

0/6

0/5

35

0/16

0/19

0/16

0/16

0/18

0/18

0/18

0/15

0/4

0/3

0/6

0/5

 

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

Study day	Nominal test item concentration [µg/L]
	40.0
	Number of vital eggs and larvae (listed eggs/larvae) by study day
	1	2	3	4
0	20/0	20/0	20/0	20/0
1	20/0	18/0	20/0	20/0
2	20/0	18/0	20/0	20/0
3	19/1	18/0	18/2	10/10
4	11/9	13/5	13/7	1/19
5	3/17	1/17	1/19	0/20
6	1/19	0/18	0/18	0/20
7	0/20	0/17	0/15	0/20
8	0/12	0/15	0/7	0/13
9	0/5	0/4	0/0	0/5
10	0/1	0/2	-	0/1
11	0/0	0/1	-	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

Parameter	Acceptance criteria	Result
Calibration	≥ 5 standard concentrations,	0.05 to 10 µg test item/L	ü
	r ≥ 0.99 (R^2≥ 0.9801 for 2^nd order regression)	(n = 9),
	Calibration range from ≤ 30% LOQ to 20% above highest concentration level after sample preparation	r ≥ 0.99
Matrix effects	Should be ≤ 20%	Matrix effects: 22%	ü
		Matrix matched standards have been used
Lowest calibration level (LCL)	S/N ≥ 9 for quantifier ion trace	S/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 concentration	0.400 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)
Precision^1)	Relative standard deviation ≤ 20% per fortification level	1 x LOQ: 20%	ü
		125 x LOQ: 5.1%
Stability	Samples: Stable if 70 – 120% of nominal concentration	See section 18.1	ü
	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 > 88.05	ü
		qualifier [m/z]: 356.31 > 55.04
	Blank values < 30% of the 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 Fortified Samples of the Test Item

LOQ Level	Control	1	125
Stock solution	-	1000 mg test item/L in Methanol
Spiking solution	-	40.0	5000
[µg test item/L]		(Methanol)	(Methanol)
(Medium)
Replicates	2	5	5
Concentration of the LOQ	-	0.400	50.0
[µg test item/L]
Medium for preparation	Fish dilution water
Volume of spiking solution [mL]	-	0.05	0.05
Volume of medium [mL]	5.0	4.95	4.95
Dilution factor	2	2	10
Dilution medium	Methanol containing 0.2% formic acid^1)
	Dilution medium^2)
Sample volume [mL]	5.0^1)	5.0^1)	5.0^1)
			0.2^2)
Finale volume [mL]	10^1)	10^1)	10^1)
			1.0^2)
Final concentration of the test item for analysis	-	0.200	5.00
[µg/L]
Final concentration of the a.i. for analysis	-	0.151	3.77
[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).

Replicate	2,2’-(Octadec-9-enylimino)bisethanol
	C18:1 Amine + 2 EO
	1 x LOQ		125 x LOQ
	Meas.	%	Meas.	%
	conc.		conc.
	[µg a.i./L]		[µg a.i./L]
1	0.264	86	42.8	111
2	0.191	62	38.3	99
3	0.176	57	38.4	100
4	0.273	89	38.3	99
5	0.196	64	38.3	99
Mean	0.22	71	39	102
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 date	Meas. conc.	%
	[µg a.i./L]
Day -1^1)	0.317	103
Day 0^1)	0.358	116
Day 7^1)	0.343	111
Day 14^2)	0.335	110
Day 21^2)	0.335	111
Day 28^2)	0.288	95
Day 34^2)	0.325	107

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 concentration	Cumulative		Post hatch		Overall		Mortality [%]
[µg/L]	hatch [%]		survival [%]		survival [%]		on study day 14
	(study day 6)		on study day 14		on study day 14
	Replicate		Replicate		Replicate		Replicate
	1	2	1	2	1	2	1	2
50.0	100	90	0	0	0*	0*	100*	100*
5.00	90	95	94	100	85	95	15	5
Solvent control	95	90	100	94	95	85	5	15

* 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 date		Day -1
Nominal concentration of the		C18:1 Amine + 2EO
test item [µg/L]	active substance	Meas. conc.					%
	[µg a.s./L]	[µg a.s./L]
50.0	37.1	31.4					83
(Replicate 1)
50.0		25.5					68
(Replicate 2)
5.00	3.77	1.91					51
(Replicate 1)
5.00		1.76					47
(Replicate 2)
Solvent control		< LCL
(Replicate 1)
Solvent control		< LCL
(Replicate 2)
Sampling date		Day 0		Day 7		Day 14
Nominal concentration of the		C18:1 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]
50.0	37.1	25.7	68	-		-
(Replicate 1)
50.0		-		22.8	60	-
(Replicate 2)
5.00	3.77	2.58	68	-		1.92		51
(Replicate 1)
5.00		-		1.07	28	1.91		51
(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 date		Day 0		Day 7		Day 14
Nominal concentration of the		C18:1 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]
50.0	37.1	11.8	31	-		-
(Replicate 1)
50.0		-		9.40	25	-
(Replicate 2)
5.00	3.77	0.385	10	-		0.639	17
(Replicate 1)
5.00		-		0.860	23	0.702	19
(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

Parameter	Based on nominal test item concentrations [µg/L]			Based on overall arithmetric mean measured test item concentrations [µg/L]
	Hatching success after	Fry Growth		Hatching success after	Fry Growth
	7 days^1)	expressed as:		6 days^1)	expressed as:
		Length	Weight		Length	Weight
NOEC	≥ 40.0	2.50	2.50	≥ 27.0	1.29	1.29
LOEC	> 40.0	5.00	5.00	> 27.0	2.19	2.19
EC10	n.d.	> 20.0	3.54	n.d.	> 12.9	1.61
			(< 2.50 to > 20.0)			(< 1.29 to > 12.9)
EC20	n.d.	n.d.	> 20.0	n.d.	n.d.	13.6
						(n.d.)
EC50	n.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

Parameter	Based on nominal test item concentrations [µg/L]		Based on overall arithmetric mean measured test item concentrations [µg/L]
	Post-hatch survival	Overall survival	Post-hatch survival	Overall survival
NOEC	10.0	10.0	6.00	6.00
LOEC	20.0	20.0	12.9	12.9
LC10	n.d.	n.d.	n.d.	n.d.
LC20	12.7	15.6	7.83	10.0
	(9.56 – 17.2)	(9.63 – 20.0)	(5.71 – 11.0)	(5.76 – 20.2)
LC50	16.8	18.3	10.7	11.7
	(12.6 – 18.7)	(10.9 – 30.8)	(7.76 – 12.0)	(6.60 – 21.0)

n.d. = not determinable