Amines, N-(3-aminopropyl)-N'-(C16-18 and C18-unsatd. alkyl)trimethylenedi-

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2022-08-31 to 2022-10-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

(2013)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item
N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl-propane 1,3-diamine (CAS No.: 1219458-14-6)

Chemical name
N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine

Common name Tallow dipropylenetriamine

Batch number 1929319

CAS No. 1219458-14-6

Purity (certified) 99.6 FID-area-%.
The substance is a 100% UVCB (Purity = 100-water content).

Water content (certified) 0.41%

Appearance White solid

Water Solubility Slightly soluble

Stability under test conditions Not specified

Expiry date 2023-01-29

Recommended storage
Store container tightly closed in a dry, well-ventilated place. Avoid elevated temperatures.

Analytical monitoring:

yes

Remarks:

with an LC-MS/MS method

Details on sampling:

Sampling schedule
Samples of test media including control group and solvent control group were taken from alternating test replicates (i.e. the outlet of the test vessel) of these replicates on days -1, 0 and twice per week thereafter until end of exposure. The exact number of samples and sampling points was documented in the raw data and given in the report.
Stock solutions were sampled and analyzed from freshly prepared and 7 days aged solutions of one application interval.

 
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 to minimize any adsorption effects.

On each sampling date an organic solvent was added in order to capture all analytes despite of being truly dissolved or dispersed and to stabilize the samples and limit the sorption to the sample vials.

The samples taken from the test vessels were completely clear and did not contain any particles or turbidity. On each sampling point the following constituents of the test item C16:0 propylenediamine, C18:0 propylenediamine, C18:1 propylenediamine, C16:0 dipropylenetriamine, C18:0 dipropylenetriamine and C18:1 dipropylenetriamine were analytically determined to distinguish how much test item is truly dissolved.

From 2 sampling dates (study day 2 and 30) the supernatant of centrifuged sample of test medium was measured. This amount of test item was considered to represent a conservative measure of the most bioavailable fraction, which may include some colloidal material not removed by centrifugation in addition to the truly dissolved fraction. Headspace vials were used for sampling. Vials were closed with a crimp cap with septum. The samples were centrifuged in these vials ‘upside-down’. After centrifugation the centrifugate was taken from the inverted vials by extraction with a capillary through the septum. Since the density of the test item is < 1, undissolved material was supposed to be on the surface. The upside-down centrifugation prevents possible uptake of substance by conventional centrifuged samples.
Additional samples for determination of the losses due to adsorption to the centrifuge tubes were taken and analysed.

Vehicle:

yes

Remarks:

With regard to the limited solubility of the test item in water, methanol was used as a solvent. The solvent concentration was the same in all concentration levels and the solvent control (0.025 mL/L).

Details on test solutions:

Test concentrations
0.893 - 2.05 - 4.73 - 10.9 - 25.0 µg test item/L (factor: 2.3) corresponding to overall arithmetic mean measured test item concentrations of 0.395 – 1.08 – 3.03 – 5.82 – 14.9 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 maximum 15 days.
The test concentrations were prepared by adding the dissolved test item to dilution water as described above.

Solvent
With regard to the limited solubility of the test item in water, methanol (VWR, 100% HPLC gradient grade, batch 22G184026 and 22G254008) was used as a solvent. The solvent concentration was the same in all concentration levels and the solvent control (0.025 mL/L).

Stock solution
A stock solution of 1000 mg/L was prepared in methanol. An appropriate amount of the test item was weighed out and transferred with an appropriate amount of the solvent into a glass flask. The solution was agitated until it is 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 solution was prepared in intervals of 7 days. The stability of the test item in the solvent was confirmed during the range finding test.
Syringes will be filled with the freshly prepared stock solutions or pure methanol for the solvent control in corresponding intervals.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Test organism
Danio rerio (zebrafish)
Vertebrata, Gnathostomata, Pisces, Osteichthyes, Teleostei, Cypriniformes, Cyprinidae

Reason for the selection of the test system
According to the guideline Danio rerio is recommended for this type of study.

Origin
All fish used in the test were reared at Noack Laboratorien GmbH from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin, Germany)

 
Maintenance of brood fish
A breeding stock of unexposed, mature zebrafish with an age of approx. 7 - 11 months was used for the egg production. Fish were free of macroscopically discernable symptoms of infection and disease. Spawners were maintained in aquaria with a loading capacity of a minimum of 1 L water per fish.
- Temperature: 25 ± 2 °C
- Dissolved oxygen concentration > 60% of air saturation value
- pH value: 6 – 8.5
- Photoperiod: 16 h light / 8 h dark cycle
(2 transition periods, 30 minutes each)
- Diffuse light (7 – 750 lux on water surface)
- Food: Artemia salina nauplii, 48 hours old, ad libitum;
Daphnia magna, juvenile and adult daphnids, ad libitum;
dry food sera vipan SERA, ad libitum.
- No disease treatments were administered.

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:

On study day 5, 95% of the control and 93% the solvent control larvae had hatched. Therefore, study day 5 was defined as post hatch day 0 (= PHD 0).

Hardness:

see section "Any other information on results incl. tables" below

Test temperature:

see section "Any other information on results incl. tables" below

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:

Based on the results of a range finding test the test was conducted as a dose-response test with the nominal test item concentrations 0.893 - 2.05 - 4.73 - 10.9 - 25.0 µg/L, corresponding to the arithmetic mean measured test item concentrations of 0.395 – 1.08 – 3.03 – 5.82 – 14.9 µg/L.

Details on test conditions:

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 1100 eggs were taken and washed in dilution water. Eggs originated from 3 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 97%.

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.


Experimental Procedure

Test item
N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl-propane 1,3-diamine (CAS No.: 1219458-14-6)

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.
Precision syringe pumps were used for the introduction of stock solutions. The stock solutions and the dilution water were mixed in mixing chambers (approx. volume 0.7 L) 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.
One mixing chamber per test replicate was used.
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 21 days was carried out prior to start of the exposure. The measured concentrations were analysed nine times during this period until no trend of increasing or decreasing was shown.

Equilibration period
Test solutions flowed through the test vessels for 21 days prior to the start of the exposure. The measured concentrations were analysed nine times during this period until no trend of increasing or decreasing was shown.

Control Dilution water (without test item and without solvent)

Solvent control
Additionally, a solvent control with the same concentration of solvent but without test item was prepared and tested under the same conditions as the test groups.

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

Test duration
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) 639 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 7.

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 2 days after the beginning of hatch on study day 5 (post-hatch day 0, where all non-affected larvae swum up). Larvae were fed with a starter food (ST-1 (AQUA SCHWARZ GMBH, 37081 Göttingen, Germany) (2 – 3 times daily), 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 (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 48 hours at approx. 22°C). 48 h old brine shrimp nauplii were harvested, washed in a stainless-steel mesh and resuspended in tap water.
Feeding ad libitum was carried out.

Water temperature (target) 26 ± 1.5 °C

Dissolved oxygen
Not less than 60% of air saturation value
concentration (target)

Light intensity (target) 300 ± 150 Lux

Photoperiod
A daily 16 / 8 h photoperiod (light / dark) was maintained throughout exposure.
 
Type and Frequency of Measurements
Biological Parameters

All biological parameters were observed daily. Dead larvae/fish and coagulated or dead eggs were removed daily, if observed as described below.

Hatching
The number of hatched larvae was determined daily until study day 6. All embryos hatched were counted as hatched, even if they had died directly afterwards. Eggs were only removed when mortality of eggs/embryos was observed as specified below.
On study day 5, 95% of the control and 93% of the solvent control larvae had hatched. Therefore, study day 5 was defined as post-hatch day 0 (= PHD 0). For evaluation of hatch, all hatched larvae (even dead ones) were counted.
The cumulative number of hatched larvae up to study day 6 was used for evaluation.

Mortality Criteria for mortality vary according to life stage:

For eggs/embryos: If fungus growth on eggs was observed, these eggs were removed and counted. Mortality as discerned by a distinct change in coloration or a marked loss of translucency and change in coloration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance and change in coloration was checked daily. Mortality caused by absence of heartbeat was checked, if applicable. Dead eggs/embryos were discarded.

For larvae and juvenile fish: Immobility and/or lack of reaction to mechanical stimulus. Dead larvae or juvenile fish were discarded.

Further effects
Abnormal appearance and behavior were also recorded daily.
Abnormalities, e.g. quiescence, hyperventilation, uncoordinated swimming, swim-up behavior, atypical quiescence and atypical feeding behavior were recorded by visually inspecting each replicate.

Measurement of fish size
At the end of exposure (post-hatch day 30) the fish were euthanized in a Benzocaine solution and the individual total length of all survivors was measured to the nearest 0.5 mm with millimeter paper. The total length (from the tip of the snout to the tip of the longer lobe of the caudal fin) was measured.

 
Measurement of fish wet weight
At the end of exposure (post-hatch day 30) all surviving fish were weighed on replicate basis to the nearest 0.1 mg. Fish were blotted on paper towels to remove excess moisture prior to weighing. The mean wet weight per animal was calculated from the number of surviving fish.

Physical Properties

Water quality measurements were carried out during exposure in the following intervals:

Once per hour Temperature in the dilution water, measured in one control vessel


At least 3 times per week
Determination of
- Dissolved oxygen in all replicates of each test group
- Check of flow rates of the test media (variation < 10% throughout exposure)

Weekly
- pH-value and temperature in all replicates of each test group
- TOC and Chlorine from dilution water
- Total hardness in one replicate of control, solvent control and the remaining highest test item concentration

The light intensity on the surface of the test aquaria was measured at the start of the exposure.

Reference substance (positive control):

no

Remarks:

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

Key result

Duration:

35 d

Dose descriptor:

LC50

Effect conc.:

5.67 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.:

10.4 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Overall survival

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

5.82 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.:

10.9 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.:

3.03 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.:

4.73 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Overall survival

Duration:

35 d

Dose descriptor:

LC50

Effect conc.:

5.7 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.:

10.6 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.:

4.99 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.:

8.94 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Post-hatch survival

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

5.82 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.:

10.9 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.:

3.03 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.:

4.73 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Post-hatch survival

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

14.9 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

25 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

5.82 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

10.9 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

14.9 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

25 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

5.82 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

10.9 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Duration:

6 d

Dose descriptor:

LOEC

Effect conc.:

> 14.9 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

time to hatch

Remarks:

hatching success

Duration:

6 d

Dose descriptor:

LOEC

Effect conc.:

> 25 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

time to hatch

Remarks:

hatching success

Duration:

6 d

Dose descriptor:

NOEC

Effect conc.:

>= 14.9 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

time to hatch

Remarks:

hatching success

Duration:

6 d

Dose descriptor:

NOEC

Effect conc.:

>= 25 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

time to hatch

Remarks:

hatching success

Details on results:

Biological Data

Egg Fertilization Rate
The mean egg fertilization rate determined on study day 0 (start of the exposure) was 97%.
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 3 in all test groups. The hatch of larvae was completed until study day 6. Study day 5 was determined to be post hatch day 0 (PHD 0) with a hatching rate of 95% in the control and 93% in the solvent control.

Statistical procedures were applied for the total number of test organisms that have hatched on study days 5 and 6.
The Dunnett`s Multiple t-test procedure for hatch data after 5 and 6 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 25.0 µg/L.
For hatching success on study days 5 and 6, the NOEC and the LOEC (based on nominal test item concentrations) for this endpoint were determined to be ≥ 25.0 and > 25.0 µg/L, respectively. The corresponding NOEC and LOEC (based on the overall arithmetic mean measured dissolved test item concentrations) were ≥ 14.9 and > 14.9 µg/L, respectively.

Swim-up
The swim-up period of the test groups was observed from study day 4 to 5. First swim-up of larvae was observed on study day 4 in the solvent control and the nominal test concentrations of 4.73 and 25.0 µg/L. On study day 5 all live larvae had swum up. 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 82% in the control and 83% 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 10.9 and 25.0 µg/L.
The NOEC and the LOEC for this endpoint were 4.73 µg/L and 10.9 µg/L (nominal concentrations), respectively. The corresponding NOEC and LOEC (based on the overall arithmetic mean measured dissolved test item concentrations) were 3.03 µg/L and 5.82 µg/L, respectively.
The LC50-value (based on nominal concentrations) for post-hatch survival on study day 35 (PHD30) was 10.6 (5.00 to 22.9) µg/L, corresponding to 5.70 (3.16 to 10.5) µg/L (based on the overall arithmetic mean measured dissolved test item concentrations).

Overall Survival
The cumulative mortality at the end of the exposure, related to the number of eggs introduced on day 0, was 22% per control and 24% 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 10.9 and 25.0 µg/L.
The NOEC and the LOEC for this endpoint were 4.73 µg/L and 10.9 µg/L (nominal concentrations), respectively. The corresponding NOEC and LOEC (based on the overall arithmetic mean measured dissolved test item concentrations) were 3.03 µg/L and 5.82 µg/L, respectively.
The LC50-value (based on nominal concentrations) for overall survival on study day 35 (PHD 30) was 10.4 (5.06 to 21.9) µg/L, corresponding to 5.67 (3.14 to 10.5) µg/L (based on the overall arithmetic mean measured dissolved test item concentrations).

Morphological and Behavioral Effects
Observation of abnormal appearance and behavior of hatched larvae were carried out daily until the end of the exposure. Several few isolated events of morphological and behavioural effects occurred during exposure in most of the test groups. No morphological nor behavioral effects were observed in the nominal test concentrations of 2.05 and 25.0 µg/L

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 concentration of 25.0 µg/L for the growth parameters fresh weight and mean total length.
Therefore, the NOEC and the LOEC (nominal test item concentrations) for the growth parameters fresh weight and total length were determined to be 10.9 and 25.0 µg/L, respectively. The corresponding NOEC and LOEC (based on overall arithmetic mean measured dissolved test item concentrations) were 5.82 and 14.9 µg/L, respectively.
No ECx values were calculated, due to too few data points in the highest test concentration (only 1 fish survived until test end).

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 (see Table 18).

The maximum biomass at the end of the exposure was determined in replicate 4 of the control group: 727.2 mg total fish weight. The maximum biomass loading based on the 7.5 liter volume of a single growth chamber was 97.0 mg/L.

Maximum loading rate: biomass/(volume of test solution) = (727.2 mg)/(7.5 L) = 97.0 mg/L

The biomass loading rate based upon a flow of 75 liters per day through each single test aquaria was 9.70 mg per liter and day.

Maximum loading rate per day: biomass/(volume of test solution per day) = (727.2 mg)/(75 L) = 9.70 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 91 – 95% and ranged from 73 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 26.2 °C . The minimum temperature was 25.2 °C and the maximum temperature was 27.0 °C.
The mean water temperature measured once per week from all replicates during the exposure period was 26.1 °C for the control. The minimum and maximum measured temperature for the control were 25.8 and 26.6 °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 7.81 and ranged from 7.23 to 8.07 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 25.0 µg/L (day 1 to 28)). The mean total hardness was 75 mg CaCO3/L and ranged from 70 to 78 mg CaCO3/L in the control group.

Residual Chlorine

Residual chlorine, measured from the dilution water supply tank on study days 1, 7, 14, 21 and 28 was < 0.01 mg/L.

Total Organic Carbon (TOC) of the Dilution Water

The total organic carbon (TOC) sampled from the dilution water supply tank was determined on study days 1, 7, 15, 21 and 28. The mean measured TOC was 1.11 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 3.13 ± 0.076 L/h and the individual values ranged from 3.00 to 3.24 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 241 to 293 lux (mean: 271 lux).

 
Measured Test Concentrations during Equilibration Phase

The concentrations of the test item N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl-propane 1,3-diamine, the solvent control and the control were determined in the equilibration phase on day -1 from the test vessels of the test replicates 3 of all tested concentration levels via LC MS/MS.

Measured Concentrations in the Stock Solutions

Measured concentrations of the freshly prepared stock solution were 101 to 134% of the nominal values. Measured concentrations of the 7 day aged stock solutions were 99 to 134% of the nominal value.

Measured Concentrations in the Test Vessels during Exposure
The concentrations of the test item, the solvent control and the control were determined during exposure on days 0, 2, 7, 9, 14, 16, 21, 23, 28 and 30 from the test vessels of a defined test replicate of all tested concentration levels via LC MS/MS.

From two sampling dates (day 2 and 30) the supernatant of centrifuged samples of test medium and the adsorption to glass vials used during the centrifugation step were measured to evaluate how much test item is lost due to the centrifugation step. The concentration of samples taken from the test vessels of days 2 and 30 was quantified with and without centrifugation step, before the samples were subjected to the analytical procedure.

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 dissolved concentrations. The lower recoveries of the centrifuged samples are explained by losses due to adsorption to the centrifuge tubes and are therefore not further used. This effect was also observed with similar substances in aqueous matrix.
Fingerprint of the Test Item (Non-GLP)

The peak distribution of the highest test item concentration (25 µg test item/L) and the control was analyzed. A solution of the analytical standard of the test item was prepared and diluted to 35 µg/L with methanol. The sample and the control were diluted factor 2 with acetonitrile containing 2% formic acid to avoid an inhomogeneous sample. The solutions were analytically verified via high resolution MS and evaluated by the software. The detected signals of the analytical standards and the sample solution were compared. In general, however, the concentrations and solubility of the test item is too low for significant MS spectra.

Reported statistics and error estimates:

All calculations are based on the nominal test item concentrations of 0.893 – 2.06 – 4.73 – 10.9 – 25.0 µg/L, corresponding to the overall arithmetic mean measured test item concentrations of 0.395 – 1.08 – 3.03 – 5.82 – 14.9 µg/L.
Replicate means were used for statistical analysis since each test vessel is the experimental unit based on the design of the test. For each parameter analyzed (hatching success, post hatch survival, overall survival and fry growth (expressed as mean total length and fresh weight measured on PHD 30), the statistical tests given below were conducted.
Since a solvent was used, both control groups were included for statistical analysis. The two controls were compared by Fisher’s Exact Binomial Test (significance level 0.05, for the parameters hatching success, post hatch survival and overall survival) and Student-t Test (significance level 0.05, for the parameter fry growth (expressed as length and fresh weight)).
No statistically significant difference was detected between the dilution water control and the solvent control for any parameter. Therefore, the controls were pooled for statistical analysis.
The hatching success is defined as the total number of test organisms that have hatched in a replicate divided by the number of eggs introduced into the replicate at start of exposure.
The number of hatched larvae was determined daily until study day 6, when the last hatching occurred. However, not 100% of the larvae hatched successfully in all replicates.
Post-hatch survival is defined as the number of surviving fish in a replicate at the end of exposure divided by the number of test organisms in the replicate that have hatched.

NOEC/LOEC

The data of the parameters hatching success, post hatch survival and overall survival were arcsine transformed prior to statistical analysis.
Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01.
Levene’s test on variance was done with a significance level of 0.01.
Monotonicity was done by trend analysis by contrasts (significance level 0.05).
The Dunnett’s multiple t-test-procedure was done with a significance level of 0.05 for the parameter hatching success on study day 5 and 6.
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 arithmetic mean measured concentrations of the test item. 
ECx/LCx-calculation The ECx/LCX-values and the corresponding confidence intervals were calculated by standard procedures with GraphPad Prism (GRAPHPAD SOFTWARE, INC.).

Arithmetic mean measured concentrations

The arithmetic mean measured concentrations were determined by the sumproduct function of excel, referring to the recoveries and purities of six constituents of the test item samples. This procedure takes into account the mean measured concentrations of main constituents as well as their content in the product.

Software

The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- Microsoft Excel, MICROSOFT CORPORATION
- ToxRat, TOXRAT SOLUTIONS GMBH

 

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

Nominal test item concentration [µg/L]	Rep.	PHD -3	PHD -2	PHD -1	PHD 0	PHD 1
		Study day 2	Study day 3	Study day 4	Study day 5	Study day 6
		Cumulative hatching rate [%]
Control	1	0	40	90	100	100
	2	0	40	95	95	95
	3	0	25	85	90	90
	4	0	35	95	95	95
	Mean	0	35	91	95	95
Solvent control	1	0	40	80	90	90
	2	0	45	85	85	85
	3	0	30	95	95	95
	4	0	65	100	100	100
	Mean	0	45	90	93	93
0.893	1	0	75	95	95	95
	2	0	10	80	95	95
	3	0	30	90	90	90
	4	0	25	75	95	95
	Mean	0	35	85	94 (ns)	94 (ns)
2.05	1	0	10	100	100	100
	2	0	20	100	100	100
	3	0	50	95	95	95
	4	0	50	85	85	85
	Mean	0	34	95	95 (ns)	95 (ns)
4.73	1	0	10	70	100	100
	2	0	25	90	100	100
	3	0	55	100	100	100
	4	0	15	50	95	95
	Mean	0	26	78	99 (ns)	99 (ns)
10.9	1	0	35	85	100	100
	2	0	20	55	80	90
	3	0	10	75	95	95
	4	0	10	70	95	95
	Mean	0	19	71	93 (ns)	95 (ns)
25.0	1	0	40	100	100	100
	2	0	10	90	90	90
	3	0	25	65	90	90
	4	0	10	95	95	95
	Mean	0	21	88	94 (ns)	94 (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 0.893 to 25.0 µg/L

Nominal test item conc. [µg/L]	Rep.	PHD -2	PHD -1	PHD 0
		Study day 3	Study day 4	Study day 5
		Cumulative swim up [%]
Control	1	0	0	100
	2	0	0	100
	3	0	0	100
	4	0	0	100
	Mean	0	0	100
Solvent control	1	0	0	100
	2	0	0	100
	3	0	11	100
	4	0	0	100
	Mean	0	3	100
0.893	1	0	0	100
	2	0	0	100
	3	0	0	100
	4	0	0	100
	Mean	0	0	100
2.05	1	0	0	100
	2	0	0	100
	3	0	0	100
	4	0	0	100
	Mean	0	0	100
4.73	1	0	7	100
	2	0	0	100
	3	0	5	100
	4	0	0	100
	Mean	0	3	100
10.9	1	0	0	100
	2	0	0	100
	3	0	0	100
	4	0	0	100
	Mean	0	0	100
25.0	1	0	0	100
	2	0	0	100
	3	0	0	100
	4	0	5	100
	Mean	0	1	100

 

Post-Hatch Survival on Study Day 35 (PHD 30) of the Control, Solvent Control and the Nominal Test Item Concentrations 0.893 µg/L

Nominal  test item conc. [µg/L]	Rep.	Eggs introduced on study day 0	Cumulative number of hatched larvae	Vital Larvae on study day 35 (PHD 30)	Post-Hatch survival on study day 35 [%]
Control	1	20	20	14	70
	2	20	19	15	79
	3	20	18	15	83
	4	20	19	18	95
	Mean	20	19.0	15.5	82
Solvent control	1	20	18	15	83
	2	20	17	15	88
	3	20	19	16	84
	4	20	20	15	75
	Mean	20	18.5	15.3	83
0.893	1	20	19	15	79
	2	20	19	17	89
	3	20	18	16	89
	4	20	19	13	68
	Mean	20	18.8	15.3	81 (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 2.05 to 25.0 µg/L

 

Nominal  test item conc. [µg/L]	Rep.	Eggs introduced on study day 0	Cumulative number of hatched larvae	Vital Larvae on study day 35 (PHD 30)	Post-Hatch survival on study day 35 [%]
2.05	1	20	20	16	80
	2	20	20	13	65
	3	20	19	14	74
	4	20	17	13	76
	Mean	20	19.0	14.0	74 (ns)
4.73	1	20	20	17	85
	2	20	20	19	95
	3	20	20	19	95
	4	20	19	15	79
	Mean	20	19.8	17.5	89 (ns)
10.9	1	20	20	9	45
	2	20	18	7	39
	3	20	19	7	37
	4	20	19	6	32
	Mean	20	19.0	7.3	38 (s)
25.0	1	20	20	0	0
	2	20	18	0	0
	3	20	18	1	6
	4	20	19	0	0
	Mean	20	18.8	0.3	2 (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 Concentration 0.893 µg/L

 

Nominal test item conc. [µg/L]	Rep.	Eggs introduced on study day 0	Vital larvae on  study day 35 (PHD 30)	Overall survival [%]	Overall mortality [%]
Control	1	20	14	70	30
	2	20	15	75	25
	3	20	15	75	25
	4	20	18	90	10
	Mean	20	15.5	78	22
Solvent control	1	20	15	75	25
	2	20	15	75	25
	3	20	16	80	20
	4	20	15	75	25
	Mean	20	15.3	76	24
0.893	1	20	15	75	25
	2	20	17	85	15
	3	20	16	80	20
	4	20	13	65	35
	Mean	20	15.3	76 (ns)	24

(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 2.05 to 25.0 µg/L

 

Nominal test item conc. [µg/L]	Rep.	Eggs introduced on study day 0	Vital larvae on  study day 35 (PHD 30)	Overall survival [%]	Overall mortality [%]
2.05	1	20	16	80	20
	2	20	13	65	35
	3	20	14	70	30
	4	20	13	65	35
	Mean	20	14.0	70 (ns)	30
4.73	1	20	17	85	15
	2	20	19	95	5
	3	20	19	95	5
	4	20	15	75	25
	Mean	20	17.5	88 (ns)	12
10.9	1	20	9	45	55
	2	20	7	35	65
	3	20	7	35	65
	4	20	6	30	70
	Mean	20	7.3	36 (s)	64
25.0	1	20	0	0	100
	2	20	0	0	100
	3	20	1	5	95
	4	20	0	0	100
	Mean	20	0.3	1 (s)	99

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

 

Behavioral Effects observed in the Control and Solvent Control (Study Day 17 – 22)

 

Study Day	Control				Solvent Control
	1	2	3	4	1	2	3	4
17	-	-	1 T	-	-	-	-	-
18 – 19	-	-	1 T	-	1 T	-	-	-
20 – 22	-	-	1 T	-	-	-	-	-

 

Behavioral Effects observed in the Nominal Test Concentrations 0.893 and 4.73 µg/L (Study Day 10 – 22)

 

Study Day	0.893 µg/L				4.73 µg/L
	1	2	3	4	1	2	3	4
10 – 12	-	-	-	-	-	-	-	1 S
15 – 16	-	-	-	1 T	-	-	-	-
17	-	-	-	-	-	-	-	-
18 – 20	-	-	-	-	-	-	1 A 1 Sw 1 P	-
21 – 22	-	-	-	-	-	-	1 Sw 1 P	-

 

Behavioral Effects observed in the Nominal Test Concentrations 10.9 µg/L 
(Study Day 18 – 22)

 

Study Day	10.9 µg/L
	1	2	3	4
18	2 G 2 SE 1 T	-	-	-
19 – 21	-	-	-	-
22	-	-	-	1 NSw

 

T    = Tumbling                                  A    = Swollen Abdomen

S    = Side position                           Sw = Abnormal Swimming Behavior

P    = Pale Appearance                     G   = Arresting on the ground       

NSw = No swim up                           SE        = Slowed escape reflex

Overview of Fry Growth: Length and Wet Weight on Study Day 35 (PHD 30) of the Control, Solvent Control and Nominal Test Item Concentrations 0.893 and 2.05 µg/L

Nominal test item conc.[µg/L]	Rep.	PHD 30 (End of exposure)
		Mean total length per fish [mm]	Mean wet weight per fish [mg]
Control	1	16.9	44.2
	2	16.1	40.2
	3	14.7	33.6
	4	15.4	40.4
	Mean	15.8	39.6
	± SD	0.817	3.81
	CV [%]	5.17	9.63
Solvent control	1	14.7	36.6
	2	14.6	35.5
	3	15.2	36.3
	4	16.0	42.7
	Mean	15.1	37.8
	± SD	0.554	2.87
	CV [%]	3.67	7.60
0.893	1	16.0	42.7
	2	14.9	35.9
	3	16.1	42.0
	4	16.0	41.7
	Mean	15.8 (ns)	40.6 (ns)
	± SD	0.492	2.72
	CV [%]	3.11	6.71
2.05	1	15.5	38.9
	2	16.2	41.6
	3	16.8	46.9
	4	15.8	37.8
	Mean	16.1 (ns)	41.3 (ns)
	± SD	0.487	3.52
	CV [%]	3.02	8.51

(ns) = No 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 4.73 to 25.0 µg/L

Nominal test item conc.[µg/L]	Rep.	PHD 30 (End of exposure)
		Mean total length per fish [mm]	Mean wet weight per fish [mg]
4.73	1	14.7	33.0
	2	14.4	32.8
	3	15.6	37.2
	4	15.4	42.2
	Mean	15.0 (ns)	36.3 (ns)
	± SD	0.492	3.833
	CV [%]	3.28	10.6
10.9	1	15.9	45.0
	2	15.6	45.0
	3	16.2	53.6
	4	12.7	28.0
	Mean	15.1 (ns)	42.9 (ns)
	± SD	1.40	9.29
	CV [%]	9.28	21.7
25.0	1	-	-
	2	-	-
	3	6.0 (s)	0.8 (s)
	4	-	-
	Mean	n.d.	n.d.
	± SD	n.d.	n.d.
	CV [%]	n.d.	n.d.

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

- = 100 % mortality

n.d. = Not determinable

 

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	18.0	20.0	12.0	22.0	10.5	20.0	20.0	20.0
2	16.0	19.5	19.0	20.0	11.5	12.0	17.0	16.0
3	18.5	17.5	21.0	15.0	19.0	20.0	17.0	16.0
4	21.0	17.5	18.0	13.0	11.0	13.0	14.0	20.5
5	11.0	14.0	13.0	23.0	11.0	9.0	19.0	20.0
6	14.5	16.0	10.0	15.0	20.0	13.0	20.0	18.0
7	16.0	13.0	15.0	16.0	24.0	17.0	17.5	12.0
8	15.0	12.5	14.5	18.0	16.5	21.0	12.5	9.0
9	19.0	12.5	16.0	9.0	18.5	21.0	10.5	17.0
10	11.0	15.0	12.5	15.0	11.0	10.0	12.0	18.0
11	22.5	16.0	13.0	9.0	10.5	10.5	14.0	13.0
12	12.0	18.0	15.0	10.0	17.0	21.0	18.0	19.5
13	19.0	21.5	17.0	15.5	13.5	11.0	16.0	17.5
14	22.5	18.0	12.0	16.0	15.0	12.0	15.0	14.0
15	-	10.5	12.5	14.0	11.0	9.0	10.5	9.5
16	-	-	-	18.5	-	-	10.5	-
17	-	-	-	13.0	-	-	-	-
18	-	-	-	16.0	-	-	-	-
19	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-
Mean	16.9	16.1	14.7	15.4	14.7	14.6	15.2	16.0
± SD	3.75	3.05	2.93	3.84	4.15	4.61	3.24	3.62
CV %	22.2	19.0	19.9	24.9	28.2	31.6	21.3	22.6

                                                       -   = Fish died before end of the study

 

 

Individual Length on Study Day 35 (PHD 30) of the Nominal Test Item Concentrations 0.893 and 2.05 µg/L

 

Fish No.	Nominal test item conc. [µg/L]
	0.893				2.05
	Total length of individual fish in [mm]
	1	2	3	4	1	2	3	4
1	21.0	20.5	20.0	17.0	18.0	21.0	18.0	15.5
2	18.5	20.0	15.5	11.0	17.5	19.5	19.0	19.0
3	18.0	16.0	13.0	18.0	15.0	12.0	15.0	15.5
4	18.5	15.0	17.0	19.0	18.5	14.0	15.0	13.0
5	16.0	14.0	16.0	18.5	14.0	18.0	20.0	15.5
6	11.0	17.0	20.0	14.0	8.0	18.0	15.0	20.0
7	17.0	12.0	22.0	18.5	19.0	12.0	21.0	16.5
8	17.5	14.5	18.0	8.0	14.5	21.5	19.5	18.5
9	16.0	15.0	15.0	20.5	20.0	18.5	15.0	15.0
10	11.5	12.5	17.0	19.5	22.5	18.0	13.5	11.0
11	15.0	13.0	14.0	16.0	18.0	8.0	16.0	10.5
12	18.0	15.0	17.0	18.0	15.0	12.0	12.5	20.0
13	12.0	15.5	12.5	10.0	14.0	18.0	22.0	16.0
14	20.0	17.0	11.0	-	9.0	-	13.0	-
15	10.0	12.5	15.0	-	14.0	-	-	-
16	-	13.0	14.0	-	11.0	-	-	-
17	-	11.5	-	-	-	-	-	-
18	-	-	-	-	-	-	-	-
19	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-
Mean	16.0	14.9	16.1	16.0	15.5	16.2	16.8	15.8
± SD	3.30	2.51	2.87	3.84	3.82	3.97	3.00	2.95
CV %	20.6	16.9	17.8	24.0	24.7	24.5	17.9	18.7

                                                       -   = Fish died before end of the study

 

Individual Length on Study Day 35 (PHD 30) of the Nominal Test Item Concentrations 4.73 and 10.9 µg/L

 

Fish No.	Nominal test item conc. [µg/L]
	4.73				10.9
	Total length of individual fish in [mm]
	1	2	3	4	1	2	3	4
1	20.5	13.0	14.0	14.0	18.0	10.5	18.0	16.0
2	16.0	10.0	17.0	14.0	16.0	21.0	19.5	13.0
3	13.0	13.0	14.0	16.0	20.0	18.0	12.5	11.0
4	11.0	10.0	20.5	15.0	18.0	15.0	19.5	14.5
5	12.0	13.0	18.0	10.0	13.0	17.0	22.0	9.0
6	14.0	20.5	18.0	14.0	14.0	15.0	13.0	12.5
7	14.5	18.5	13.0	19.5	16.0	12.5	9.0	-
8	14.5	10.5	14.0	10.0	12.0	-	-	-
9	12.0	12.0	16.0	13.5	16.0	-	-	-
10	20.0	14.5	8.5	20.5	-	-	-	-
11	19.5	18.0	14.0	15.0	-	-	-	-
12	17.0	14.0	15.0	18.5	-	-	-	-
13	14.0	16.0	21.0	17.0	-	-	-	-
14	12.0	11.0	20.0	17.0	-	-	-	-
15	16.0	18.0	19.0	17.0	-	-	-	-
16	12.0	14.5	15.0	-	-	-	-	-
17	12.0	19.0	18.0	-	-	-	-	-
18	-	14.0	9.0	-	-	-	-	-
19	-	15.0	12.5	-	-	-	-	-
20	-	-	-	-	-	-	-	-
Mean	14.7	14.4	15.6	15.4	15.9	15.6	16.2	12.7
± SD	2.95	3.09	3.44	2.93	2.42	3.23	4.38	2.27
CV %	20.0	21.5	22.1	19.0	15.3	20.7	27.1	17.9

                                                       -   = Fish died before end of the study

 

 

In the highest test concentration of nominal 25.0 µg/L only one single fish survived in replicate 3 until study day 35. The measured length and weight of this fish were 6 mm and 0.8 mg, respectively.

 

 

Pooled Wet Weights on Study Day 35 (PHD 30) of the Control, Solvent Control and the Nominal Test Item Concentrations 0.893 to 25.0 µg/L

 

Nominal test item conc. [µg/L]	Replicate	Number of fish alive  on study day 35	Pooled wet weight  per replicate [mg]	Mean  wet weight  per fish per replicate [mg]	Mean [mg]	± SD	CV %
Control	1	14	618.4	44.2	39.6	3.81	9.63
	2	15	603.1	40.2
	3	15	504.7	33.6
	4	18	727.2	40.4
Solvent control	1	15	549.2	36.6	37.8	2.87	7.60
	2	15	532.4	35.5
	3	16	581.2	36.3
	4	15	640.1	42.7
0.893	1	15	639.8	42.7	40.6	2.72	6.71
	2	17	610.4	35.9
	3	16	672.0	42.0
	4	13	541.8	41.7
2.05	1	16	622.7	38.9	41.3	3.52	8.51
	2	13	540.6	41.6
	3	14	657.2	46.9
	4	13	491.4	37.8
4.73	1	17	561.7	33.0	36.3	3.83	10.6
	2	19	622.5	32.8
	3	19	707.7	37.2
	4	15	632.3	42.2
10.9	1	9	405.4	45.0	42.9	9.29	21.7
	2	7	315.0	45.0
	3	7	375.3	53.6
	4	6	167.7	28.0
25.0	1	0	-	-	n.d.	n.d.	n.d.
	2	0	-	-
	3	1	0.8	0.8
	4	0	-	-

- = 100 % mortality

n.d. = Not determinable

 

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	0.893	2.05	4.73	10.9	25.0
0	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
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	99	100	100
	2	100	100	100	99	99	99	99
	3	99	99	100	100	100	99	100
	4	100	100	100	100	100	100	100
7	1	95	93	94	91	91	90	92
	2	93	92	92	92	93	95	92
	3	96	94	93	94	93	92	92
	4	96	93	91	96	90	92	92
9	1	95	94	95	95	93	91	91
	2	96	94	93	97	93	90	91
	3	96	95	95	95	93	91	91
	4	97	94	94	96	93	82	91
12	1	96	97	94	96	96	92	90
	2	96	97	95	97	95	91	91
	3	97	95	91	97	97	90	89
	4	98	95	96	97	97	93	90
14	1	95	95	95	94	94	88	89
	2	94	94	93	95	93	89	87
	3	95	95	94	91	93	90	89
	4	97	93	93	95	93	89	89
16	1	95	94	73	94	95	89	88
	2	93	93	92	93	94	79	87
	3	95	86	94	94	95	90	88
	4	96	93	94	93	94	89	87

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	0.893	2.05	4.73	10.9	25.0
19	1	92	91	92	92	93	87	-
	2	90	91	90	92	90	89	-
	3	92	91	90	90	93	88	89
	4	93	90	88	90	91	88	-
21	1	87	89	90	92	90	90	-
	2	88	91	85	90	89	85	-
	3	92	91	90	91	92	91	91
	4	93	89	90	89	92	86	-
23	1	90	88	91	95	90	92	-
	2	92	94	90	92	88	87	-
	3	94	92	95	93	88	94	91
	4	95	88	90	90	92	85	-
26	1	93	88	88	94	89	93	-
	2	92	92	90	93	90	92	-
	3	94	92	93	92	89	89	90
	4	94	91	93	90	93	91	-
28	1	90	83	85	92	81	88	-
	2	88	91	86	90	84	87	-
	3	91	87	89	90	88	90	89
	4	88	84	86	85	88	88	-
30	1	95	88	83	93	86	91	-
	2	92	92	90	90	89	89	-
	3	92	91	85	87	90	90	92
	4	92	92	86	90	91	87	-
33	1	91	84	82	93	87	90	-
	2	89	90	91	91	87	85	-
	3	92	89	87	85	90	91	90
	4	91	92	87	88	88	91	-
Mean		95	93	92	94	93	91	93
± SD		3.64	4.48	5.50	4.03	4.57	4.99	4.86
Min.		87	83	73	85	81	79	87
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 one replicate of the Control Group

 

Period of measurements	2022-08-31 to 2022-10-05
Minimum temperature [°C]	25.2
Maximum temperature [°C]	27.0
Mean temperature [°C]	26.2

 

Water Temperature in the Test Media

 

Study day	Rep.	Water temperature [°C]
		Nominal test item concentrations [µg/L]
		Control	Solvent control	0.893	2.05	4.73	10.9	25.0
0	1	26.1	26.1	26.2	26.1	26.1	26.2	26.2
	2	25.9	26.1	26.1	26.2	25.9	26.0	26.1
	3	25.8	25.8	26.2	26.3	26.0	26.2	26.1
	4	26.0	26.0	26.0	26.0	26.1	26.0	26.2
7	1	26.6	26.7	26.7	26.7	26.8	26.9	26.9
	2	26.4	26.6	26.7	26.8	26.5	26.7	26.8
	3	26.4	26.5	26.9	26.8	26.7	26.8	26.8
	4	26.6	26.6	26.6	26.8	26.7	26.7	26.9
14	1	26.3	26.4	26.4	26.3	26.4	26.4	26.4
	2	26.0	26.3	26.3	26.4	26.2	26.2	26.4
	3	26.0	26.1	26.5	26.6	26.3	26.4	26.3
	4	26.2	26.3	26.2	26.3	26.3	26.3	26.5
21	1	26.2	26.3	26.3	26.3	26.4	26.4	-
	2	25.9	26.2	26.2	26.4	26.1	26.2	-
	3	26.0	26.0	26.4	26.5	26.2	26.4	26.2
	4	26.2	26.1	26.2	26.2	26.3	26.3	-
28	1	26.4	26.2	26.3	26.2	26.3	26.3	-
	2	25.9	26.2	26.2	26.3	26.1	26.1	-
	3	26.0	26.0	26.4	26.5	26.2	26.3	26.2
	4	26.0	26.1	26.0	26.1	26.1	26.2	-
30	1	26.2	26.3	26.3	26.3	26.3	26.3	-
	2	26.0	26.2	26.3	26.3	26.1	26.1	-
	3	26.0	26.1	26.2	26.3	26.2	26.3	26.2
	4	26.2	26.0	26.3	26.2	26.2	26.2	-
Mean		26.1	26.2	26.3	26.4	26.3	26.3	26.4
± SD		0.220	0.220	0.220	0.227	0.224	0.235	0.295
Min.		25.8	25.8	26.0	26.0	25.9	26.0	26.1
Max.		26.6	26.7	26.9	26.8	26.8	26.9	26.9

              -        = 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	0.893	2.05	4.73	10.9	25.0
0	1	7.72	7.86	7.79	7.85	7.85	7.86	7.83
	2	7.81	7.83	7.78	7.91	7.81	7.85	7.84
	3	7.88	7.86	7.89	7.87	7.81	7.86	7.84
	4	7.91	7.82	7.84	7.91	7.83	7.86	7.80
7	1	7.91	7.93	7.93	7.89	7.86	7.94	7.89
	2	8.02	7.97	7.91	7.93	7.83	7.92	7.89
	3	7.96	8.02	7.90	7.82	7.83	7.90	7.87
	4	8.03	7.97	7.90	7.92	7.85	7.87	7.89
14	1	8.02	7.93	7.85	7.88	7.87	7.79	7.80
	2	8.02	7.92	7.89	7.91	7.90	7.85	7.76
	3	8.07	7.94	7.86	7.91	7.84	7.81	7.79
	4	8.02	7.87	7.86	7.92	7.84	7.87	7.78
21	1	7.23	7.63	7.60	7.66	7.64	7.62	-
	2	7.60	7.66	7.58	7.62	7.65	7.63	-
	3	7.71	7.69	7.60	7.63	7.64	7.62	7.67
	4	7.81	7.80	7.63	7.66	7.62	7.60	-
28	1	7.52	7.61	7.55	7.63	7.51	7.55	-
	2	7.60	7.61	7.56	7.64	7.53	7.54	-
	3	7.66	7.64	7.59	7.63	7.52	7.57	7.61
	4	7.67	7.56	7.62	7.60	7.57	7.61	-
30	1	7.55	7.80	7.73	7.83	7.78	7.83	-
	2	7.72	7.76	7.77	7.83	7.78	7.79	-
	3	7.79	7.78	7.75	7.66	7.76	7.82	7.85
	4	7.85	7.81	7.58	7.72	7.76	7.80	-
Mean		7.80	7.80	7.75	7.78	7.75	7.77	7.81
± SD		0.207	0.133	0.136	0.126	0.124	0.131	0.080
Min.		7.23	7.56	7.55	7.60	7.51	7.54	7.61
Max.		8.07	8.02	7.93	7.93	7.90	7.94	7.89

              -        = 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	25.0
1	1	75	54
7	2	70	68
14	3	76	61
21	4 (control)  3 (25 µg/L)	78	80
28	1 (control) 3 (25 µg/L)	78	72
Mean		75	67
± SD		3.29	10.0
Min.		70	54
Max.		78	80

 

Min./Max.                = Minimum/Maximum measured hardness

 

Total Organic Carbon (TOC) of the Dilution Water

 Values given in () were < LOQ (2 mgC/L)

 

 

Study day	TOC [mg/L]
1	(1.06)
7	(1.14)
15	(1.10)
21	(1.01)
28	(1.24)
Mean	(1.11)
SD	0.087
Min.	(1.01)
Max.	(1.24)

 

Min./Max.         = Minimum/Maximum measured TOC

 

 

Flow Rates

 

Flow rate	Nominal test item concentrations [µg/L]
	Control	Solvent control	0.893	2.05	4.73	10.9	25.0
	Measured flow rates of the membrane piston pumps^1)[L/h]
Minimum	3.00	3.00	3.00	3.00	3.00	3.00	3.00
Maximum	3.24	3.24	3.24	3.24	3.24	3.24	3.24
Mean  ± Standard deviation	3.13 ± 0.075	3.13 ± 0.074	3.13  ± 0.082	3.14  ± 0.074	3.13  ± 0.081	3.12 ± 0.076	3.13  ± 0.074
Overall mean ± Standard deviation	3.13 ± 0.076

1)   Nominal flow rates of dilution water (for preparation of the test solutions): 3.125 (2.81 – 3.44 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.

 

 Nominal Concentrations of the components and limit of quantification of the test item

	Nominal concentration of the test item [µg/L]
Component	25.0	10.9	4.73	2.05	0.893	LOQ [µg a.s./L]
	Corresponding to [µg a.s./L]
C16:0 propylenediamine	1.43	0.621	0.270	0.117	0.0509	0.0200
C18:0 propylenediamine	1.68	0.730	0.317	0.137	0.0598	0.0235
C18:1 propylenediamine	2.10	0.916	0.397	0.172	0.0750	0.0294
C16:0 dipropylenetriamine	5.33	2.32	1.01	0.437	0.190	0.0746
C18:0 dipropylenetriamine	6.08	2.65	1.15	0.498	0.217	0.0851
C18:1 dipropylenetriamine	5.05	2.20	0.955	0.414	0.180	0.0707

LOQ              = limit of quantification (0.350 µg test item/L)

a.s.                = active substance

 

Measured Concentrations and Percent of Nominal Concentration of the Test Item compounds C16:0 propylenediamine, C18:0 propylenediamine and C18:1 propylenediamine in the Test Vessel on Study Day -1

Sampling day	Day -1
Replicate	3
Nominal conc. of the test item [µg/L]	N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl-propane 1,3-diamine
	C16:0 propylenediamine			C18:0 propylenediamine			C18:1 propylenediamine
	Nominalconc. [µg a.s./L]	Meas. conc. [µg a.s./L]	%	Nominal conc. [µg a.s./L]	Meas. conc. [µg a.s./L]	%	Nominal conc. [µg a.s./L]	Meas. conc. [µg a.s./L]	%
25.0	1.43	0.738	52	1.68	0.899	54	2.10	0.945	45
10.9	0.621	0.292	47	0.730	0.313	43	0.916	0.321	35
4.73	0.270	0.141	52	0.317	0.196	62	0.397	0.182	46
2.05	0.117	0.0561	48	0.137	0.0768	56	0.172	0.0658	38
0.893	0.0509	0.0310	61	0.0598	0.0401	67	0.0750	0.0347	46
Solvent Control	< LOQ			< LOQ			< LOQ
Control	< LOQ			< LOQ			< LOQ

Meas. conc. = measured concentration of the different compounds of the test item, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L)

 

Measured Concentrations and Percent of Nominal Concentration of the Test Item compounds C16:0 dipropylenetriamine, C18:0 dipropylenetriamine and C18:1 dipropylenetriamine in the Test Vessel on Study Day -1

Sampling day	Day -1
Replicate	3
Nominal conc. of the test item [µg/L]	N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl-propane 1,3-diamine
	C16:0 dipropylenetriamine			C18:0 dipropylenetriamine			C18:1 dipropylenetriamine
	Nominal conc. [µg a.s./L]	Meas. conc. [µg a.s./L]	%	Nominal conc. [µg a.s./L]	Meas. conc. [µg a.s./L]	%	Nominal conc. [µg a.s./L]	Meas. conc. [µg a.s./L]	%
25.0	5.33	2.38	45	6.08	2.94	48	5.05	2.10	41
10.9	2.32	1.03	44	2.65	1.08	41	2.20	0.818	37
4.73	1.01	0.512	51	1.15	0.573	50	0.955	0.483	51
2.05	0.437	0.213	49	0.498	0.227	46	0.414	0.184	45
0.893	0.190	0.124	65	0.217	0.130	60	0.180	0.108	60
Solvent Control	< LOQ			< LOQ			< LOQ
Control	< LOQ			< LOQ			< LOQ

Meas. conc. = measured concentration of the different compounds of the test item, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance in the test item

LOQ              = limit of quantification (0.350 µg test item/L)

 

Nominal Concentrations of the components of the stock solutions

	Nominal concentration of the stock solution [mg/L]
Component	1000	435	189	82.2	35.7
	Corresponding to [mg a.s./L]
C16:0 propylenediamine	57.0	24.8	10.8	4.69	2.03
C18:0 propylenediamine	67.0	29.1	12.7	5.51	2.39
C18:1 propylenediamine	84.0	36.5	15.9	6.90	3.00
C16:0 dipropylenetriamine	213	92.7	40.3	17.5	7.60
C18:0 dipropylenetriamine	243	106	45.9	20.0	8.68
C18:1 dipropylenetriamine	202	87.9	38.2	16.6	7.21

a.s.          = active substance

 

Measured Concentrations and Percent of Nominal Concentration in the Stock Solutions

                

	N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl-propane 1,3-diamine
Nominal conc. of the test item [mg/L]	Freshly prepared stock solutions
	C16:0 propylenediamine			C18:0 propylenediamine			C18:1 propylenediamine
	Nominal conc. [mg a.s./L]	Meas. conc. [mg a.s./L]	%	Nominal conc. [mg a.s./L]	Meas. conc. [mg a.s./L]	%	Nominal conc. [mg a.s./L]	Meas. conc. [mg a.s./L]	%
1000	57.0	60.5	106	67.0	74.5	111	84.0	91.3	109
435	24.8	25.8	104	29.1	32.7	112	36.5	39.0	107
189	10.8	11.1	103	12.7	13.9	110	15.9	16.7	105
82.2	4.69	5.08	108	5.51	6.17	112	6.90	7.54	109
35.7	2.03	2.14	105	2.39	2.66	111	3.00	3.16	106
Nominal conc. of the test item [mg/L]	7 Days aged stock solutions
	C16:0 propylenediamine			C18:0 propylenediamine			C18:1 propylenediamine
	Nominal conc. [mg a.s./L]	Meas. conc. [mg a.s./L]	%	Nominal conc. [mg a.s./L]	Meas. conc. [mg a.s./L]	%	Nominal conc. [mg a.s./L]	Meas. conc. [mg a.s./L]	%
1000	57.0	59.2	104	67.0	74.1	111	84.0	57	106
435	24.8	26.0	105	29.1	33.0	113	36.5	38.7	106
189	10.8	11.2	104	12.7	14.0	111	15.9	16.7	105
82.2	4.69	4.81	103	5.51	5.96	108	6.90	7.22	105
35.7	2.03	2.02	99	2.39	2.50	104	3.00	3.06	102

Meas. Conc.           =              Measured concentration of the different compounds of the test item (dilution

=              factor taken into account)

%                             =              Percent of nominal concentration of the active substance in the test item

 

Measured Concentrations and Percent of Nominal Concentration in the Stock Solutions

	N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl-propane 1,3-diamine
Nominal conc. of the test item [mg/L]	Freshly prepared stock solutions
	C16:0 dipropylenetriamine			C18:0 dipropylenetriamine			C18:1 dipropylenetriamine
	Nominal conc. [mg a.s./L]	Meas. conc. [mg a.s./L]	%	Nominal conc. [mg a.s./L]	Meas. conc. [mg a.s./L]	%	Nominal conc. [mg a.s./L]		Meas. conc. [mg a.s./L]	%
1000	213	246	115	243	306	126	202		224	111
435	92.7	97.5	105	106	125	118	87.9		94.6	108
189	40.3	40.7	101	45.9	51.9	113	38.2		39.0	102
82.2	17.5	19.6	112	20.0	25.3	127	16.6		22.2	134
35.7	7.60	7.72	102	8.68	10.6	122	7.21		8.48	118
Nominal conc. of the test item [mg/L]	7 Days aged stock solutions
	C16:0 dipropylenetriamine			C18:0 dipropylenetriamine				C18:1 dipropylenetriamine
	Nominal conc. [mg a.s./L]	Meas. conc. [mg a.s./L]	%	Nominal conc. [mg a.s./L]	Meas. conc. [mg a.s./L]	%		Nominal conc. [mg a.s./L]	Meas. conc. [mg a.s./L]	%
1000	213	261	123	243	311	128		202	264	131
435	92.7	120	129	106	114	107		87.9	118	134
189	40.3	47.5	118	45.9	54.9	119		38.2	46.1	121
82.2	17.5	17.7	101	20.0	22.2	111		16.6	19.5	117
35.7	7.60	8.95	118	8.68	10.7	123		7.21	9.67	134

Meas. Conc.           =              Measured concentration of the different compounds of the test item (dilution

=              factor taken into account)

%                             =              Percent of nominal concentration of the active substance in the test item

 

 Measured Concentrations of C16:0 propylenediamine of the Test Item during the Definitive Test

Sampling		Day 0		Day 2		Day 7		Day 9
Replicate		4		1		2		3
Nominal concentration of		C16:0 propylenediamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
25.0	1.43	0.861	60	0.428	30	0.812	57	0.828	58
10.9	0.621	0.382	61	0.195	31	0.271	44	0.290	47
4.73	0.270	0.159	59	0.0701	26	0.158	59	0.202	75
2.05	0.117	0.0673	58	0.0394	34	0.0731	63	0.0689	59
0.893	0.0509	< LOQ		< LOQ		< LOQ		0.0317	62
Solvent Control		< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ
Sampling		Day 14		Day 16		Day 21		Day 23
Replicate		4		1		2		3
Nominal concentration of		C16:0 propylenediamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
25.0	1.43	0.628	44	0.880	62	0.373	26	0.735	52
10.9	0.621	0.117	19	0.253	41	0.147	24	0.322	52
4.73	0.270	0.165	61	0.156	58	0.0722	27	0.178	66
2.05	0.117	0.0390	33	0.0526	45	< LOQ		0.0550	47
0.893	0.0509	< LOQ		0.0180	35	< LOQ		0.0237	47
Solvent Control		< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ^1)		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the compound C16:0 propylenediamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0200 a.s./L)

¹⁾                              = due to a sampling mistake, the first analysis was > LOQ; a new sample was taken of the same vessel and analyzed on day 16, which then was < LOQ

 

 

 

Table continued

Sampling		Day 28		Day 30
Replicate		4		1
Nominal concentration of		C16:0 propylenediamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
25.0	1.43	0.618	43	1.28	90
10.9	0.621	0.298	48	0.559	90
4.73	0.270	0.132	49	0.171	63
2.05	0.117	0.0279	24	0.0675	58
0.893	0.0509	< LOQ		0.0202	40
Solvent Control		< LOQ		< LOQ
Control		< LOQ		< LOQ

Meas. conc. = measured concentration of the compound C16:0 propylenediamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0200 a.s./L)

 

Measured Concentrations of C16:0 propylenediamine of the Test Item during the Definitive Test - Truly dissolved and Glass Adsorption of the centrifugation tube

Sampling		Day 2		Day 30		Day 2		Day 30
		Truly dissolved (supernatant)				Glass adsorption (centrifuge tube)
Replicate		1		1		1		1
Nominal concentration of		C16:0 propylenediamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
25.0	1.43	0.154	11	0.524	37	0.0843	6	0.499	35
10.9	0.621	0.0955	15	0.231	37	0.0407	7	0.172	28
4.73	0.270	0.0358	13	0.102	38	< LOQ		0.0301	11
2.05	0.117	< LOQ		0.0418	36	< LOQ		< LOQ
0.893	0.0509	< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the compound C16:0 propylenediamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0200 a.s./L)

 

Measured Concentrations of C18:0 propylenediamine of the Test Item during the Definitive Test

Sampling		Day 0			Day 2		Day 7			Day 9
Replicate		4			1		2			3
Nominal concentration of		C18:0 propylenediamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%
25.0	1.68	1.03		61	0.569	34	1.11		66	1.23	74
10.9	0.730	0.437		60	0.275	38	0.423		58	0.429	59
4.73	0.317	0.205		65	0.106	34	0.216		68	0.284	89
2.05	0.137	0.0927		67	0.0615	45	0.104		76	0.111	81
0.893	0.0598	0.0254	42		< LOQ		0.0282	47		0.0468	78
Solvent Control		< LOQ			< LOQ		< LOQ			< LOQ
Control		< LOQ			< LOQ		< LOQ			< LOQ
Sampling		Day 14			Day 16		Day 21			Day 23
Replicate		4			1		2			3
Nominal concentration of		C18:0 propylenediamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%
25.0	1.68	0.697		42	1.07	64	0.439		26	0.978	58
10.9	0.730	0.157		21	0.423	58	0.160		22	0.425	58
4.73	0.317	0.200		63	0.187	59	0.0796		25	0.240	76
2.05	0.137	0.0488		36	0.0633	46	< LOQ			0.0900	65
0.893	0.0598	< LOQ			0.0323	54	< LOQ			0.0348	58
Solvent Control		< LOQ			< LOQ		< LOQ			< LOQ
Control		< LOQ^1)			< LOQ		< LOQ			< LOQ

Meas. conc. = measured concentration of the compound C18:0 propylenediamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0235 a.s./L)

¹⁾                              = due to a sampling mistake, the first analysis was > LOQ; a new sample was taken of the same vessel and analyzed on day 16, which then was < LOQ

 

 

Table continued

Sampling		Day 28			Day 30
Replicate		4			1
Nominal concentration of		C18:0 propylenediamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%
25.0	1.68	1.01		60	1.42	84
10.9	0.730	0.250		34	0.596	82
4.73	0.317	0.161		51	0.215	68
2.05	0.137	0.0445		32	0.0991	72
0.893	0.0598	0.0316	53		0.0272	45
Solvent Control		< LOQ			< LOQ
Control		< LOQ			< LOQ

Meas. conc. = measured concentration of the compound C18:0 propylenediamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0235 a.s./L)

 

Measured Concentrations of C18:0 propylenediamine of the Test Item during the Definitive Test - Truly dissolved and Glass Adsorption of the centrifugation tube

Sampling		Day 2			Day 30		Day 2		Day 30
		Truly dissolved (supernatant)					Glass adsorption (centrifuge tube)
Replicate		1			1		1		1
Nominal concentration of		C18:0 propylenediamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
25.0	1.68	0.304		18	0.682	41	0.0922	6	0.324	19
10.9	0.730	0.188		26	0.431	59	0.0325	4	0.0872	12
4.73	0.317	0.0654		21	0.174	55	< LOQ		< LOQ
2.05	0.137	0.0277	20		0.0826	60	< LOQ		< LOQ
0.893	0.0598	< LOQ			< LOQ		< LOQ		< LOQ
Control		< LOQ			< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the compound C18:0 propylenediamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0235 a.s./L)

 

Measured Concentrations of C18:1 propylenediamine of the Test Item during the Definitive Test

Sampling		Day 0		Day 2		Day 7		Day 9
Replicate		4		1		2		3
Nominal concentration of		C18:1 propylenediamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
25.0	2.10	0.928	44	0.528	25	0.990	47	1.13	54
10.9	0.916	0.406	44	0.260	28	0.322	35	0.378	41
4.73	0.397	0.170	43	0.0897	23	0.210	53	0.277	70
2.05	0.172	0.0669	39	0.0486	28	0.0891	52	0.0909	53
0.893	0.0750	< LOQ		< LOQ		< LOQ		0.0443	59
Solvent Control		< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ
Sampling		Day 14		Day 16		Day 21		Day 23
Replicate		4		1		2		3
Nominal concentration of		C18:1 propylenediamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
25.0	2.10	0.815	39	1.14	54	0.442	21	0.979	47
10.9	0.916	0.148	16	0.356	39	0.174	19	0.404	44
4.73	0.397	0.221	56	0.194	49	0.0847	21	0.255	64
2.05	0.172	0.0449	26	0.0657	38	< LOQ		0.0799	46
0.893	0.0750	< LOQ		< LOQ		< LOQ		0.0326	43
Solvent Control		< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ^1)		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the compound C18:1 propylenediamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0294 a.s./L)

¹⁾                              = due to a sampling mistake, the first analysis was > LOQ; a new sample was taken of the same vessel and analyzed on day 16, which then was < LOQ

 

 

 

Table continued

Sampling		Day 28		Day 30
Replicate		4		1
Nominal concentration of		C18:1 propylenediamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
25.0	2.10	0.822	39	1.57	75
10.9	0.916	0.296	32	0.697	76
4.73	0.397	0.155	39	0.218	55
2.05	0.172	0.0339	20	0.0863	50
0.893	0.0750	< LOQ		< LOQ
Solvent Control		< LOQ		< LOQ
Control		< LOQ		< LOQ

Meas. conc. = measured concentration of the compound C18:1 propylenediamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0294 a.s./L)

 

Measured Concentrations of C18:1 propylenediamine of the Test Item during the Definitive Test - Truly dissolved and Glass Adsorption of the centrifugation tube

Sampling		Day 2		Day 30		Day 2		Day 30
		Truly dissolved (supernatant)				Glass adsorption (centrifuge tube)
Replicate		1		1		1		1
Nominal concentration of		C18:1 propylenediamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]		%
25.0	2.10	0.207	10	0.722	34	0.114	5	0.414		20
10.9	0.916	0.140	15	0.335	37	0.0496	5	0.195		21
4.73	0.397	0.0491	12	0.146	37	< LOQ		0.0330	8
2.05	0.172	< LOQ		0.0648	38	< LOQ		< LOQ
0.893	0.0750	< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the compound C18:1 propylenediamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0294 a.s./L)

 

Measured Concentrations of C16:0 dipropylenetriamine of the Test Item during the Definitive Test

Sampling		Day 0			Day 2		Day 7			Day 9
Replicate		4			1		2			3
Nominal concentration of		C16:0 dipropylenetriamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%
25.0	5.33	2.26		43	1.47	28	3.29		62	3.48	65
10.9	2.32	1.41		61	0.646	28	1.08		46	1.27	55
4.73	1.01	0.502		50	0.239	24	0.657		65	0.852	85
2.05	0.437	0.294		67	0.137	31	0.282		65	0.278	64
0.893	0.190	< LOQ			< LOQ		< LOQ			0.133	70
Solvent Control		< LOQ			< LOQ		< LOQ			< LOQ
Control		< LOQ			< LOQ		< LOQ			< LOQ
Sampling		Day 14			Day 16		Day 21			Day 23
Replicate		4			1		2			3
Nominal concentration of		C16:0 dipropylenetriamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%
25.0	5.33	2.52		47	3.85	72	2.60		49	3.55	67
10.9	2.32	0.623		27	1.31	56	0.987		43	1.90	82
4.73	1.01	0.794		79	0.720	71	0.491		49	0.908	90
2.05	0.437	0.175		40	0.254	58	0.111	25		0.287	66
0.893	0.190	0.0844	44		0.0797	42	0.0827	43		0.117	61
Solvent Control		< LOQ			< LOQ		< LOQ			< LOQ
Control		< LOQ^1)			< LOQ		< LOQ			< LOQ

Meas. conc. = measured concentration of the compound C16:0 dipropylenetriamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0746 a.s./L)

¹⁾                              = due to a sampling mistake, the first analysis was > LOQ; a new sample was taken of the same vessel and analyzed on day 16, which then was < LOQ

 

 

Table continued

Sampling		Day 28			Day 30
Replicate		4			1
Nominal concentration of		C16:0 dipropylenetriamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%
25.0	5.33	2.82		53	4.57	86
10.9	2.32	1.21		52	1.98	85
4.73	1.01	0.587		58	0.789	78
2.05	0.437	0.108		25	0.285	65
0.893	0.190	0.0751	39		0.107	56
Solvent Control		< LOQ			< LOQ
Control		< LOQ			< LOQ

Meas. conc. = measured concentration of the compound C16:0 dipropylenetriamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0746 a.s./L)

 

Measured Concentrations of C16:0 dipropylenetriamine of the Test Item during the Definitive Test - Truly dissolved and Glass Adsorption of the centrifugation tube

Sampling		Day 2			Day 30		Day 2		Day 30
		Truly dissolved (supernatant)					Glass adsorption (centrifuge tube)
Replicate		1			1		1		1
Nominal concentration of		C16:0 dipropylenetriamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
25.0	5.33	0.385		7	2.11	40	0.376	7	0.831	16
10.9	2.32	0.271		12	1.34	58	0.126	5	0.478	21
4.73	1.01	0.110		11	0.655	65	< LOQ		< LOQ
2.05	0.437	0.0380	9		0.260	60	< LOQ		< LOQ
0.893	0.190	< LOQ			0.0864	45	< LOQ		< LOQ
Control		< LOQ			< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the compound C16:0 dipropylenetriamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0746 a.s./L)

 

 Measured Concentrations of C18:0 dipropylenetriamine of the Test Item during the Definitive Test

Sampling		Day 0			Day 2		Day 7			Day 9
Replicate		4			1		2			3
Nominal concentration of		C18:0 dipropylenetriamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%
25.0	6.08	3.59		59	2.23	37	4.41		73	4.54	75
10.9	2.65	1.84		70	1.07	40	1.67		63	1.61	61
4.73	1.15	0.771		67	0.408	36	0.798		69	0.949	83
2.05	0.498	0.375		75	0.212	43	0.384		77	0.388	78
0.893	0.217	0.100	46		0.0875	40	0.104	48		0.186	86
Solvent Control		< LOQ			< LOQ		< LOQ			< LOQ
Control		< LOQ			< LOQ		< LOQ			< LOQ
Sampling		Day 14			Day 16		Day 21			Day 23
Replicate		4			1		2			3
Nominal concentration of		C18:0 dipropylenetriamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%
25.0	6.08	3.13		51	4.64	76	3.11		51	4.68	77
10.9	2.65	0.755		29	2.08	78	1.17		44	2.29	86
4.73	1.15	0.749		65	0.809	70	0.549		48	1.11	97
2.05	0.498	0.215		43	0.257	52	0.149	30		0.43	86
0.893	0.217	0.105	49		0.141	65	0.100	46		0.139	64
Solvent Control		< LOQ			< LOQ		< LOQ			< LOQ
Control		< LOQ^1)			< LOQ		< LOQ			< LOQ

Meas. conc. = measured concentration of the compound C18:0 dipropylenetriamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0815 a.s./L)

¹⁾                              = due to a sampling mistake, the first analysis was > LOQ; a new sample was taken of the same vessel and analyzed on day 16, which then was < LOQ

 

 

Table continued

Sampling		Day 28			Day 30
Replicate		4			1
Nominal concentration of		C18:0 dipropylenetriamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%
25.0	6.08	4.52		74	5.79	95
10.9	2.65	1.02		39	2.32	88
4.73	1.15	0.763		66	1.03	90
2.05	0.498	0.176		35	0.422	85
0.893	0.217	0.123	57		0.120	55
Solvent Control		< LOQ			< LOQ
Control		< LOQ			< LOQ

Meas. conc. = measured concentration of the compound C18:0 dipropylenetriamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0851 a.s./L)

 

Measured Concentrations of C18:0 dipropylenetriamine of the Test Item during the Definitive Test - Truly dissolved and Glass Adsorption of the centrifugation tube

Sampling		Day 2			Day 30		Day 2		Day 30
		Truly dissolved (supernatant)					Glass adsorption (centrifuge tube)
Replicate		1			1		1		1
Nominal concentration of		C18:0 dipropylenetriamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]	%
25.0	6.08	1.22		20	2.44	40	0.379	6	1.19	20
10.9	2.65	0.733		28	1.96	74	< LOQ		0.239	9
4.73	1.15	0.261		23	0.963	84	< LOQ		< LOQ
2.05	0.498	0.109	22		0.416	83	< LOQ		< LOQ
0.893	0.217	0.0517	24		0.106	49	< LOQ		< LOQ
Control		< LOQ			< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the compound C18:0 dipropylenetriamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0851 a.s./L)

 

Measured Concentrations of C18:1 dipropylenetriamine of the Test Item during the Definitive Test

Sampling		Day 0			Day 2		Day 7			Day 9
Replicate		4			1		2			3
Nominal concentration of		C18:1 dipropylenetriamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%
25.0	5.05	2.45		49	1.42	28	2.88		57	3.30	65
10.9	2.20	1.26		57	0.650	30	0.950		43	1.12	51
4.73	0.955	0.488		51	0.238	25	0.591		62	0.804	84
2.05	0.414	0.258		62	0.134	32	0.238		57	0.280	68
0.893	0.180	< LOQ			< LOQ		< LOQ			0.129	71
Solvent Control		< LOQ			< LOQ		< LOQ			< LOQ
Control		< LOQ			< LOQ		< LOQ			< LOQ
Sampling		Day 14			Day 16		Day 21			Day 23
Replicate		4			1		2			3
Nominal concentration of		C18:1 dipropylenetriamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%
25.0	5.05	2.85		56	3.68	73	2.40		47	4.18	83
10.9	2.20	0.618		28	1.39	63	0.911		41	2.00	91
4.73	0.955	0.759		79	0.677	71	0.432		45	1.04	109
2.05	0.414	0.180		44	0.229	55	0.105	25		0.359	87
0.893	0.180	0.0805	45		0.0877	49	0.0725	40		0.130	72
Solvent Control		< LOQ			< LOQ		< LOQ			< LOQ
Control		< LOQ^1)			< LOQ		< LOQ			< LOQ

Meas. conc. = measured concentration of the compound C18:1 dipropylenetriamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0707 a.s./L)

¹⁾                              = due to a sampling mistake, the first analysis was > LOQ; a new sample was taken of the same vessel and analyzed on day 16, which then was < LOQ

 

 

 

Table continued

Sampling		Day 28			Day 30
Replicate		4			1
Nominal concentration of		C18:1 dipropylenetriamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%
25.0	5.05	3.08		61	5.08	101
10.9	2.20	1.11		50	2.31	105
4.73	0.955	0.612		64	0.900	94
2.05	0.414	0.133		32	0.357	86
0.893	0.180	0.0821	46		0.124	68
Solvent Control		< LOQ			< LOQ
Control		< LOQ			< LOQ

Meas. conc. = measured concentration of the compound C18:1 dipropylenetriamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0707 a.s./L)

 

Measured Concentrations of C18:1 dipropylenetriamine of the Test Item during the Definitive Test - Truly dissolved and Glass Adsorption of the centrifugation tube

Sampling		Day 2			Day 30		Day 2			Day 30
		Truly dissolved (supernatant)					Glass adsorption (centrifuge tube)
Replicate		1			1		1			1
Nominal concentration of		C18:1 dipropylenetriamine
the test item [µg/L]	a.s. [µg a.s./L]	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%	Meas. conc. [µg a.s./L]		%	Meas. conc. [µg a.s./L]	%
25.0	5.05	0.431		9	2.06	41	0.502		10	0.706	14
10.9	2.20	0.321		15	1.63	74	0.128	6		0.494	22
4.73	0.955	0.139		15	0.786	82	< LOQ			< LOQ
2.05	0.414	0.0422	10		0.316	76	< LOQ			< LOQ
0.893	0.180	< LOQ			0.0872	48	< LOQ			< LOQ
Control		< LOQ			< LOQ		< LOQ			< LOQ

Meas. conc. = measured concentration of the compound C18:1 dipropylenetriamine, dilution factors taken into account

a.s.                = active substance

%                   = percentage of the nominal concentration of the active substance

LOQ              = limit of quantification (0.350 µg test item/L, corresponding to 0.0707 a.s./L)

 

 

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 30)

Sampling day	Day 0	Day 2		Day 7		Day 9		Day 14		Day 16		Day 21	Day 23	Day 28	Day 30
Nominal Conc. of the test item [µg/L]	N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl -propane 1,3-diamine															Overall arithmetic mean measured concentration of the test item [µg/L]
	Calculated mean measured concentration per study day [µg/L]
0.893	0.259		0.229		0.267		0.659		0.356		0.432	0.336	0.549	0.390	0.474	0.395
2.05	1.33		0.730		1.35		1.41		0.813		1.06	0.462	1.50	0.603	1.52	1.08
4.73	2.65		1.34		3.03		3.90		3.33		3.16	1.98	4.31	2.77	3.84	3.03
10.9	6.63		3.58		5.43		5.91		2.81		6.68	4.10	8.47	4.83	9.78	5.82
25.0	12.9		7.71		15.6		16.7		12.2		17.6	10.8	17.5	14.8	22.8	14.9

 

 

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

The test item is a multi-component substance (UVCB) which is slightly soluble/dispersible in water. On each sampling point the constituents C16:0 propylenediamine, C18:0 propylenediamine, C18:1 propylenediamine, C16:0 dipropylenetriamine, C18:0 dipropylenetriamine and C18:1 dipropylenetriamine were analytically determined.
The samples taken from the test vessels were completely clear and did not contain any particles or turbidity. These samples were subjected to the analytical procedure and the results are used for the calculation of the effect values.
Therefore, all effect values were based on the nominal and the overall arithmetic mean measured dissolved concentrations of the test item N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl-propane 1,3-diamine.

N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl-propane 1,3-diamine caused significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with the nominal concentrations of 0.893 - 2.05 - 4.73 - 10.9 - 25.0 µg/L, corresponding to the overall arithmetic mean measured dissolved test item concentrations of 0.395 – 1.08 – 3.03 – 5.82 – 14.9 µg/L.

For hatching success on study day 5 and 6, the NOEC and the LOEC (based on nominal test item concentrations) for this endpoint were determined to be ≥ 25.0 and > 25.0 µg/L, respectively. The corresponding NOEC and LOEC (based on overall arithmetic mean measured dissolved test item concentrations) were ≥ 14.9 and > 14.9 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 4.73 µg/L (for both parameters). Therefore, the respective nominal LOECs were determined to be 10.9 µg/L. The corresponding NOEC and LOEC values based on overall arithmetic mean measured dissolved test item concentrations were NOEC 3.03 µg/L and LOEC 5.82 µg/L, respectively. The EC10 values were not determinable.

For the parameter fry growth (expressed as length and fresh weight) the nominal NOECs were 10.9 µg/L (for both parameters). Therefore, the nominal LOECs for length and weight were determined to be 25.0 µg/L, respectively. The corresponding NOEC and LOEC values based on overall arithmetic mean measured dissolved test item concentrations were NOEC 5.82 µg/L and LOEC 14.9 µg/L, respectively. The ECx values were not determinable.

Executive summary:

The effects of the test item N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl-propane 1,3-diamine (CAS No.: 1219458-14-6) (Batch-No. 1929319) on the early-life stage of fish (Danio rerio / Zebrafish) were determined at the test facility according to OECD Guideline 210.

 

The test item is a white solid which is slightly soluble 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 35.7 – 82.2 – 189 – 435 – 1000 mg/L were prepared in appropriate intervals of 7 days and continuously dosed to the dilution water in a flow-through system. Based on the results of a range finding test the test was conducted as a dose-response test with the nominal test item concentrations 0.893 - 2.05 - 4.73 - 10.9 - 25.0 µg/L, corresponding to the arithmetic mean measured test item concentrations of 0.395 – 1.08 – 3.03 – 5.82 – 14.9 µg/L.

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 93% 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 N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl-propane 1,3-diamine (CAS No.: 1219458-14-6) in the test media and the controls was carried out via LC-MS/MS.

The concentrations of the test item, the solvent control and the control were determined during exposure on day 0, day 2, day 7, day 9, day 14, day 16, day 21, day 23, day 28 and day 30 from the test vessels of a defined test replicate of all tested concentration levels via LC‑MS/MS.

Six constituents of the test item (C16:0 propylenediamine, C18:0 propylenediamine, C18:1 propylenediamine, C16:0 dipropylenetriamine, C18:0 dipropylenetriamine and C18:1 dipropylenetriamine) were determined.

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 were sampled and analyzed from the freshly prepared and corresponding 7 days aged stock solutions. A stability of the stock solutions over 7 days was confirmed.

All effect values are given based on the nominal and the overall arithmetic mean measured dissolved concentrations of the test item N-(3-aminopropyl)-N'-C16 - 18 (evennumbered), C18 unsaturated alkyl-propane 1,3-diamine (CAS No.: 1219458-14-6).

 

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, EC_x, LC_x values were determined based on the statistical results.

The results are presented in the table below:

 NOEC, LOEC, EC_x Values of Hatching Success and Fry Growth

Based on nominal and overall arithmetic mean measured dissolved test item concentrations [µg/L] with 95% Confidence intervals in brackets

 

Parameter	Based on nominal  test item concentrations [µg/L]			Based on overall arithmetic mean measured  test item concentrations [µg/L]
	Hatching success after  6 days^1)	Fry Growth  expressed as:		Hatching success after  6 days^1)	Fry Growth  expressed as:
		Length	Weight		Length	Weight
NOEC	≥ 25.0	10.9	10.9	≥ 14.9	5.82	5.82
LOEC	> 25.0	25.0	25.0	> 14.9	14.9	14.9
EC10	n.d.	n.d	n.d	n.d.	n.d	n.d
EC20	n.d.	n.d	n.d	n.d.	n.d	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, LC_x values of Post Hatch Survival and Overall Survival

 Based on nominal and 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 arithmetic mean measured test item concentrations [µg/L]
	Post-hatch survival	Overall survival	Post-hatch survival	Overall survival
NOEC	4.73	4.73	3.03	3.03
LOEC	10.9	10.9	5.82	5.82
LC10	n.d.^1)	n.d.^1)	n.d.^1)	n.d.^1)
LC20	8.94 (< 0.893 – 22.4)	n.d.	4.99 (< 0.395 – 10.2)	n.d.
LC50	10.6 (5.00 – 22.9)	10.4 (5.06 – 21.9)	5.70 (3.16 – 10.5)	5.67 (3.14 – 10.5)

n.d.   =        not determinable

      1)                          =              The mortality of the control group was in the valid range but exceeded 10%.

                                   Therefore, no reliable LC₁₀ can be given.

Description of key information

One valid long-term fish study is available performed with the substance. The study was performed according to OECD TG 210 using a flow-through test design. The study with N-(3-aminopropyl)-N'-C16-18 (evennumbered), C18 unsaturated alkyl-propane-1,3-diamine (Triamine T, CAS 1219458-14-6) resulted in a NOEC for overall survival and post-hatch survival of 3.03 µg/L based on mean measured test item concentrations and 4.73 µg/L based on nominal test item concentrations. 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

3.03 µg/L