Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 233-520-3 | CAS number: 10213-78-2
Long-term toxicity to fish
Administrative data
Link to relevant study record(s)
- Endpoint:
- fish early-life stage toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- from 2021-06-08 to 2021-07-30, with the definitive exposure phase from 2021-06-23 to 2021-07-27
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Klimish 1 study performed with Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (PFAEO-C, CAS no. 71786-60-2) an analogue substance of 2,2'-(octadec-9-enylimino)bisethanol (PFAEO-O, CAS no.: 25307-17-9)
Read-across is justifified in the read across document included in chapter 13. - Reason / purpose for cross-reference:
- read-across source
- 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 Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
Batch number 20201158
CAS No. 71786-60-2
Purity (certified) 100% UVCB
Chemical name Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs
Density 910 kg/m3 at 20 °C
Appearance Light yellow clear liquid
Water Solubility Dispersible / insoluble
Stability under test Not specified
conditions
Expiry date 2023-02-10
Recommended storage Store container tightly closed in a dry, well-ventilated place. Avoid elevated temperatures. - Analytical monitoring:
- yes
- Remarks:
- via LC-MS/MS
- Details on sampling:
- Determination of the test item
The samples were analyzed under GLP with an LC-MS/MS method which was implemented under non-GLP and documented finally in the GLP raw data. The method was validated according to Annex I.
The peak distribution (fingerprint) of the highest stock solution was analyzed, once during the test. The signal distribution was compared with an analytical standard prepared in solvent.
Sampling schedule
Samples of test media including solvent control group and control group were taken from alternating test replicates and the mixing chamber supply of these replicates on study days -1, 0, and weekly thereafter until end of exposure. The changing intervals of the stock solution were taken into account.
The stock solution was sampled and analyzed from freshly prepared and corresponding 7 days aged stock solution of one application interval.
Sampling and pre-treatment
At each sampling day 2 samples were taken per (alternating) test replicate. For each sample 50 mL of each test item concentration, the solvent control and the control were sampled. For analysis of fresh and aged stock solutions, approx. 4 mL of each stock solution were sampled. - Vehicle:
- yes
- Remarks:
- With regard to the limited solubility of the test item in water, methanol (VWR, ≥ 99.9% ultra LC-MS grade, batch 200827C002 and VWR, 100% HPLC gradient grade, batch 21C044012) was used as a solvent.
- Details on test solutions:
- Test design
A randomized block design with each treatment being present in each block was established.
A flow-through exposure design was carried out. Membrane piston pumps provided the water flow-through via silicone tubing.
Precision syringe pumps were used for the introduction of stock solution. The stock solution and the dilution water were mixed in a mixing chamber (approx. volume 0.7 L, one mixing chamber per test replicate) by magnetic stirring before passing the test aquaria (approx. volume 7.5 L; four replicates per test concentration, control and solvent control) where the eggs/fish were exposed.
The accuracy of the water flow-through was checked prior to start of the exposure and three times per week thereafter.
Water exchange in the test aquaria was about 10 times per day (3.125 L/h).
An equilibration period of at least 22 days was carried out prior to start of the exposure. The measured concentrations showed no trend of increasing or decreasing.
Test concentrations
5.00 - 10.0 - 20.0 - 40.0 - 80.0 µg test item/L (factor: 2), corresponding to overall mean measured test item concentrations of 4.61 - 10.4 - 21.0 - 39.0 - 80.7 µg test item/L.
The test concentrations were based on the results of a preliminary range finding test (non-GLP) conducted under flow-through conditions over 15 days.
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, ≥ 99.9% ultra LC-MS grade, batch 200827C002 and VWR, 100% HPLC gradient grade, batch 21C044012) was used as a solvent. The solvent concentration was the same in all concentration levels and the solvent control (0.025 mL/L).
Stock solution
A stock solution of 3200 mg/L was prepared in methanol. An appropriate amount of the test item was weighed out and transferred with the solvent into a glass flask. The solution was agitated until it was visually clear dissolved.
Further stock solutions were prepared by dilution with methanol.
The stock solutions were prepared in appropriate intervals of 7 days.
Syringes were filled with the freshly prepared stock solutions or pure methanol for the solvent control in corresponding intervals.
Equilibration period
Test solutions flowed through the test vessels for 6 days prior to the start of the exposure. The measured concentrations were in the range of ± 20% of the nominal concentrations, with no trend of increasing or decreasing.
Control Dilution water (without test item and without solvent)
Solvent control
Additionally, a solvent control with the same concentration of solvent but without test item was prepared and tested under the same conditions as the test groups. - Test organisms (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. 10 months was used for the egg production. Fish were free of macroscopically discernable symptoms of infection and disease. Spawners were maintained in aquaria with a loading capacity of a minimum of 1 L water per fish.
- Temperature: 25 ± 2 °C
- Dissolved oxygen concentration > 60% of air saturation value
- pH value: 6 – 8.5
- Photoperiod: 16 h light / 8 h dark cycle
(2 transition periods, 30 minutes each)
- Diffuse light (7 – 750 lux on water surface)
- Food: Artemia salina nauplii, 48 hours old, ad libitum;
Daphnia magna, juvenile and adult daphnids, ad libitum;
dry food sera vipan SERA, ad libitum.
- No disease treatments were administered.
Water
Tap water of local origin was used for holding. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
Nominal water parameters of an acceptable dilution water for holding and testing:
Total hardness: 10 – 250 mg CaCO3/L
pH-value: 6.0 – 8.5
Alkalinity: 0.8 mmol/L (recent measurement: 2021-04-26)
Acidity: 0.8 mmol/L (recent measurement: 2021-04-26)
Conductivity: 174 µS/cm (recent measurement: 2021-04-26)
Spawning
15 – 35 adult zebrafish were kept in at least 3 separate aquaria. The fish were healthy with a mortality rate < 5% during the last 7 days and thus not medically treated for at least 7 days. About 15 minutes before start of artificial dawning rectangular dishes covered with a stainless-steel mesh and provided with artificial plants (plastic), were introduced into the aquaria. After 1.5 hours the glass dishes were gently removed. Eggs were checked carefully for abnormalities like fungus infections. These eggs as well as coagulated and not fertilized eggs were discarded (less than 30%). About 900 eggs were taken and washed in dilution water. Eggs originated from 3 different spawnings. - Test type:
- flow-through
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 34 d
- Remarks on exposure duration:
- 34 days (30 days post-hatch)
- Hardness:
- See "Any other information on results incl. tables" below.
- Test temperature:
- See "Any other information on results incl. tables" below.
- pH:
- See "Any other information on results incl. tables" below.
- Dissolved oxygen:
- See "Any other information on results incl. tables" below.
- Nominal and measured concentrations:
- 5.00 - 10.0 - 20.0 - 40.0 - 80.0 µg test item/L (factor: 2), corresponding to overall mean measured test item concentrations of 4.61 - 10.4 - 21.0 - 39.0 - 80.7 µg test item/L.
- Details on test conditions:
- Test design
A randomized block design with each treatment being present in each block was established. A flow-through exposure design was carried out. Membrane piston pumps provided the water flow-through via silicone tubing.
Precision syringe pumps were used for the introduction of stock solution. The stock solution and the dilution water were mixed in a mixing chamber (approx. volume 0.7 L, one mixing chamber per test replicate) by magnetic stirring before passing the test aquaria (approx. volume 7.5 L; four replicates per test concentration, control and solvent control) where the eggs/fish were exposed.
The accuracy of the water flow-through was checked prior to start of the exposure and three times per week thereafter.
Water exchange in the test aquaria was about 10 times per day (3.125 L/h).
An equilibration period of at least 22 days was carried out prior to start of the exposure. The measured concentrations showed no trend of increasing or decreasing.
Equilibration period
Test solutions flowed through the test vessels for 6 days prior to the start of the exposure. The measured concentrations were in the range of ± 20% of the nominal concentrations, with no trend of increasing or decreasing.
Control Dilution water (without test item and without solvent)
Solvent control
Additionally, a solvent control with the same concentration of solvent but without test item was prepared and tested under the same conditions as the test groups.
Reference item
No reference item is recommended for this test according to the guideline.
Test duration
34 days (30 days post hatch), depending on post-hatch day 0 (study day 4).
Replicates, number of eggs
Four replicates per test concentration and control, with 20 eggs each (80 eggs per test concentration, solvent control and control) were tested.
For the whole study (including the range finding test and definitive tests) 1182 healthy eggs/fish were used.
Loading
A loading rate not exceeding 0.5 g/L wet weight fish per 24 hours and not exceeding 5 g/L of solution at any time was maintained.
Test vessels
Glass aquaria of 8.7 L provided with mesh coated fittings allowing flow-through of test media (dimensions: 22/22/18 cm) were used. Test vessels were covered by glass lids. The volume of the test media was approximately 7.5 L.
Cleaning
The test vessels were siphoned as needed to remove excess fecal material and uneaten food, also to minimize microbial growth and biodegradation of the test item. Furthermore, the mesh coated fittings were cleaned once per day. Cleaning started on study day 5.
Aeration
The dilution water supply tank was aerated.
No additional aeration of the test vessels was provided.
Dilution water
Tap water was used for testing. The water was filtered on activated charcoal to remove residual chlorine.
Feeding of test fish
The feeding regime was ad libitum during the whole feeding period (study day 5 to 33). Feeding started 3 days after the beginning of hatch on study day 5 (post-hatch day 1, where almost all non-affected larvae swum up). Larvae were fed with a starter food (ST-1 (AQUA SCHWARZ GMBH, 37081 Göttingen, Germany), as well as a suspension of the starter food ST-1 and fine milled brine shrimp nauplii (2 – 6 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 24 - 48 hours at approx. 22 °C). 24 - 48 h old brine shrimp nauplii were harvested, washed in a stainless-steel mesh and resuspended in tap water.
Feeding ad libitum was carried out.
Water temperature (target) 26 ± 1.5 °C
Dissolved oxygen concentration (target) Not less than 60% of air saturation value
Light intensity (target) 300 ± 150 Lux
One measuring point was slight out of range (measured value 466 lux). However mean measured light intensity was 313 lux and within the given range.
Photoperiod
A daily 16 / 8 h photoperiod (light / dark) was maintained throughout exposure.
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 4, 91% of the control and 96% of the solvent control larvae had hatched. Therefore, study day 4 was defined as post-hatch day 0 (= PHD 0). For evaluation of hatch, all hatched larvae (even dead ones) were counted.
The cumulative number of hatched larvae up to study day 6 was used for evaluation.
Mortality
Criteria for mortality vary according to life stage:
For eggs/embryos: If fungus growth on eggs was observed, these eggs were removed and counted. Mortality as discerned by a distinct change in coloration or a marked loss of translucency and change in coloration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance and change in coloration was checked daily. Mortality caused by absence of heartbeat was checked, if applicable. Dead eggs/embryos were discarded.
For larvae and juvenile fish: Immobility and/or lack of reaction to mechanical stimulus. Dead larvae or juvenile fish were discarded.
Further effects
Abnormal appearance and behavior were also recorded daily.
The number of larvae or fish showing abnormality of body form was recorded. Abnormal animals were only removed from the test vessels on death. 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 graph 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
- Duration:
- 34 d
- Dose descriptor:
- LC50
- Effect conc.:
- 34.2 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- other: Overall survival
- Duration:
- 34 d
- Dose descriptor:
- LC50
- Effect conc.:
- 35.2 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- other: Post-hatch survival
- Duration:
- 34 d
- Dose descriptor:
- LC50
- Effect conc.:
- 34.6 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- other: Overall survival
- Duration:
- 34 d
- Dose descriptor:
- LC50
- Effect conc.:
- 35.6 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- other: Post-hatch survival
- Duration:
- 34 d
- Dose descriptor:
- other: LC20
- Effect conc.:
- 27.5 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- other: Overall survival
- Duration:
- 34 d
- Dose descriptor:
- other: LC20
- Effect conc.:
- 39.8 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- other: Post-hatch survival
- Duration:
- 34 d
- Dose descriptor:
- other: LC20
- Effect conc.:
- 27.1 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- other: Overall survival
- Duration:
- 34 d
- Dose descriptor:
- other: LC20
- Effect conc.:
- 29.7 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- other: Post-hatch survival
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LC10
- Effect conc.:
- 23.2 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- other: Overall survival
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LC10
- Effect conc.:
- 27.1 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- other: Post-hatch survival
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LC10
- Effect conc.:
- 22.4 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- other: Overall survival
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LC10
- Effect conc.:
- 26.6 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- other: Post-hatch survival
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 39 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- other: Post-hatch and Overall survival
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 40 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- other: Post-hatch and Overall survival
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 21 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- other: Post-hatch and Overall survival
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 20 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- other: Post-hatch and Overall survival
- Duration:
- 34 d
- Dose descriptor:
- EC50
- Effect conc.:
- 66.8 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- weight
- Duration:
- 34 d
- Dose descriptor:
- EC50
- Effect conc.:
- > 80.7 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- length
- Duration:
- 34 d
- Dose descriptor:
- EC50
- Effect conc.:
- 70.6 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- weight
- Duration:
- 34 d
- Dose descriptor:
- EC50
- Effect conc.:
- > 80 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- length
- Duration:
- 34 d
- Dose descriptor:
- other: EC20
- Effect conc.:
- 24.3 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- weight
- Duration:
- 34 d
- Dose descriptor:
- other: EC20
- Effect conc.:
- 43.8 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- length
- Duration:
- 34 d
- Dose descriptor:
- other: EC20
- Effect conc.:
- 23.8 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- weight
- Duration:
- 34 d
- Dose descriptor:
- other: EC20
- Effect conc.:
- 45.2 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- length
- Key result
- Duration:
- 34 d
- Dose descriptor:
- EC10
- Effect conc.:
- 14.3 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- weight
- Key result
- Duration:
- 34 d
- Dose descriptor:
- EC10
- Effect conc.:
- 13.4 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- weight
- Key result
- Duration:
- 34 d
- Dose descriptor:
- EC10
- Effect conc.:
- 27.8 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- length
- Key result
- Duration:
- 34 d
- Dose descriptor:
- EC10
- Effect conc.:
- 27.6 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Purity = 100% (UVCB)
- Basis for effect:
- length
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 21 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Fry growth expressed as length and weight
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 20 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Fry growth expressed as length and weight
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 10.4 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Fry growth expressed as length and weight
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 10 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Fry growth expressed as length and weight
- Duration:
- 6 d
- Dose descriptor:
- LOEC
- Effect conc.:
- > 80.7 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- time to hatch
- Remarks:
- hatching success after 6 days
- Duration:
- 6 d
- Dose descriptor:
- LOEC
- Effect conc.:
- > 80 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- time to hatch
- Remarks:
- hatching success after 6 days
- Duration:
- 6 d
- Dose descriptor:
- NOEC
- Effect conc.:
- >= 80.7 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- time to hatch
- Remarks:
- hatching success after 6 days
- Duration:
- 6 d
- Dose descriptor:
- NOEC
- Effect conc.:
- >= 80 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- time to hatch
- Remarks:
- hatching success after 6 days
- Details on results:
- Biological Data
Egg Fertilization Rate
The mean egg fertilization rate determined on study day 0 (start of the exposure) was 91%. Eggs were fully covered with the respective test solutions during fertilization check.
Hatch and Definition of Post Hatch Day 0
Hatch began on study day 2 in the test concentration of 40.0 µg/L. The hatch of larvae in other test concentrations and control groups started on study day 3. The hatch of larvae was completed until study day 6. Study day 4 was determined to be post hatch day 0 (PHD 0) with a hatching rate of 91% in the control and 96% in the solvent control.
Statistical procedures were applied for the total number of test organisms that have hatched on study days 4 and 6.
The Dunnett’s multiple t-test for hatch data after 4 and 6 days was done with a significance level of 0.05. No statistically significant differences were found between the pooled controls and the nominal test concentrations of 5.00 to 80.0 µg/L.
The NOEC and the LOEC (based on nominal test item concentrations) for this endpoint were determined to be ≥ 80.0 and > 80.0 µg/L, respectively. The corresponding NOEC and LOEC (based on overall mean measured test item concentrations) were ≥ 80.7 and > 80.7 µg/L, respectively.
Swim-up
The swim-up period of the control groups and the nominal test concentrations of 5.00 to 40.0 µg/L was observed from study day 5 to 7. First swim-up of larvae was observed on study day 5 in all test groups. However completion of swim-up was not observed for the highest nominal test concentration of 80.0 µg/L, since behavioural and non-lethal effects interfered with the swim-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 91% in the control and 88% in the solvent control, thus fully meeting the validity criteria of the guideline. A concentration-related decrease of the post-hatch survival was detected with increasing test concentrations.
The Williams Multiple sequential t-test procedure (alpha = 0.05) was performed for statistical analysis of post-hatch survival data on study day 34 (PHD 30). Statistically significant differences were found between the pooled control groups and the nominal test item concentrations of 40.0 and 80.0 µg/L.
The NOEC and the LOEC for this endpoint were 20.0 µg/L and 40.0 µg/L (nominal concentrations), respectively. The corresponding NOEC and LOEC (based on overall mean measured test item concentrations) were 21.0 µg/L and 39.0 µg/L, respectively.
The LC50-value (based on nominal concentrations) for post-hatch survival on study day 34 (PHD30) was 35.6 (27.8 to 38.1) µg/L, corresponding to 35.2 (28.2 to 37.4) µg/L (based on overall mean measured test item concentrations).
Overall Survival
The cumulative mortality at the end of the exposure, related to the number of eggs introduced on day 0, was 14% per control and solvent control. A concentration-related decrease of the overall survival (increase of overall mortality) was detected in the test item concentrations.
The William’s multiple sequential t-test procedure (alpha = 0.05) was performed for statistical analysis of overall survival data on study day 34 (PHD 30). Statistically significant differences were found between the pooled control groups and the nominal test item concentrations of 40.0 and 80.0 µg/L.
The NOEC and the LOEC for this endpoint were 20.0 µg/L and 40.0 µg/L (nominal concentrations), respectively. The corresponding NOEC and LOEC (based on overall mean measured test item concentrations) were 21.0 µg/L and 39.0 µg/L, respectively.
The LC50-value for overall survival on study day 34 (PHD 30) was 34.6 (29.0 to 37.1) µg/L, corresponding to 34.2 (29.3 to 36.5) µg/L (based on overall mean measured test item concentrations).
Morphological and Behavioral Effects
Observation of abnormal appearance and behavior of hatched larvae were carried out daily until the end of the exposure. No morphological and behavioral effects were observed in the control, solvent control and in the nominal test concentrations of 5.00 to 20.0 µg/L. The nominal test concentration of 40.0 µg/L showed an increase of observed non-lethal effects with ongoing exposure from study day 12 up to study day 27. The highest nominal test concentration of 80.0 µg/L showed non-lethal effects for a short period after hatch from study day 7 to 10 resulting in 100 % mortality.
Fry Growth
Fry growth, expressed as length and wet weight measurements, was measured on study day 34 (PHD 30) from all survivors.
The Williams Multiple sequential t-test procedure (alpha = 0.05) showed statistically significant differences for the surviving fish of the nominal test concentrations of 20.0 and 40.0 µg/L for the growth parameters fresh weight and mean total length.
Therefore, the NOEC and the LOEC (nominal test item concentrations) for the growth parameters fresh weight and total length were determined to be 10.0 and 20.0 µg/L, respectively. The corresponding NOEC and LOEC (based on overall mean measured test item concentrations) were 10.4 and 21.0 µg/L, respectively.
Biomass Loading
The biomass-loading factor for the study was determined from the fresh weights of the control and solvent control fish at the end of the exposure.
The maximum biomass at the end of the exposure was determined in replicate 2 of the solvent control group: 685.5 mg total fish weight. The maximum biomass loading based on the 7.5 liter volume of a single growth chamber was 91.4 mg/L.
Maximum loading rate:
biomass/(volume of test solution) = (685.5 mg)/(7.5 L) =91.4 mg/L
The biomass loading rate based upon a flow of 75 liters per day through each single test aquaria was 9.14 mg per liter and day.
Maximum loading rate per day:
biomass/(volume of test solution per day) = (685.5 mg)/(75 L) = 9.14 mg/L per day
These loadings were well within the requirements to ensure adequate dissolved oxygen levels and to avoid crowding of the fish.
Physico-chemical Data
Dissolved Oxygen
The dissolved oxygen concentrations in the control, solvent control and the test item groups, expressed in percent saturation, were in the mean 94 – 99% and ranged from 74 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 25.7 °C and the maximum temperature was 26.5 °C.
The mean water temperature measured once per week from all replicates during the exposure period was 26.4 °C for the control. The minimum and maximum measured temperature for the control were 26.0 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 7.73 and 7.86 and ranged from 7.56 to 8.12 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 80.0 µg/L (day 1 and 8), 40.0 µg/L (day 16, 22 and 30). The mean total hardness was 68 mg CaCO3/L and ranged from 66 to 69 mg CaCO3/L in the control group.
Residual Chlorine
Residual chlorine, measured from the dilution water supply tank on study days 1, 8, 16, 22 and 30 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, 8, 16, 22 and 30. The mean measured TOC was 0.65 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 and control groups was 3.12 ± 0.083 L/h and the individual values ranged from 2.94 to 3.36 L/h.
A precision syringe pump was used for introduction of the stock solution to the mixing chambers. At any renewal of the syringes the proper function of the pump and the applied volume was checked by the syringe volume indicator.
Light intensity
Light intensity was measured at the start of exposure on the surface of all test vessels and ranged from 237 to 466 lux (mean: 313 lux). One measuring point was slight out of range (measured value 466 lux). However mean measured light intensity was 313 lux and within the given range.
Measured Test Concentrations during Equilibration Phase
The concentrations of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2), the solvent control and the control were determined in the equilibration phase on day -1 from the test vessels of the test replicates 1 of all tested concentration levels and for the four compounds C10 Amine + 2EO, C12 Amine + 2EO, C14 Amine + 2EO and C16 Amine + 2EO via LC MS/MS.
Measured Concentrations in the Stock Solutions
Measured concentrations of the freshly prepared stock solution were 101 to 105% of the nominal values. Measured concentrations of the 7 day aged stock solutions were in the range of 100 to 109% of the nominal values.
Measured Concentrations in the Test Vessels during Exposure
The concentrations of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2), the solvent control and the control were determined during exposure on day 0, day 7, day 14, day 21 and day 28 from the test vessels of a defined test replicate of all tested concentration levels and for the four compounds C10 Amine + 2EO, C12 Amine + 2EO, C14 Amine + 2EO and C16 Amine + 2EO via LC MS/MS.
Fingerprint of the Test Item (non-GLP)
The peak distribution of the test concentration was analyzed in freshly prepared medium in the nominal test item concentration 80 µg/L. A solution of the analytical standard of the test item was prepared in acetonitrile and diluted to 40 µg/L with acetonitrile. A sample of the nominal test item concentration 80 µg/L and a 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 standard and the test item solution were compared. In test item and standard solutions 6 masses were observed and could be assigned to the test item. One additional signal could be observed in the test item solution which was observed to be related to the matrix used for this study. Compared to the standard in acetonitrile the test item in fresh media showed a relative increase in the amounts of the compounds with 274 Da, 302 Da and 358 Da, whereas the relative amounts of compounds < 274 Da are decreased. In general, however, the concentrations and solubility of the test item is too low for significant MS spectra.
Validity Criteria
The study was performed according to OECD 210 and met the validity criteria:
• Dissolved oxygen saturation was between 74 and 100% of the air saturation value (required: > 60%).
• Water temperature was in the recommended range for the test species (i.e. 26 °C ± 1.5 °C): 25.7 – 26.5 °C. It did not differ by more than 1.5 °C between test vessels or between successive days at any time during the test.
• The cumulative hatching success was 95% in the control group and 98% in the solvent control group (required: ≥ 70%).
• Post-hatch survival was 91% in the control group and 88% in solvent control group (required: ≥ 75%).
• Concentration analysis: the concentrations of the test item in the test solutions should be maintained within 80 – 120% of the nominal values. The test item is a multi-component substance with compounds of different carbon chain length and different solubility. Although the application system worked correctly throughout the test, the measured values were partly distinctly out of the nominal range. This effect was also observed in the preliminary test.
Preliminary Range Finding Test (non-GLP)
A non-GLP preliminary range finding test was conducted at the test facility as a shortened early-life stage test under flow-through conditions over a period of 15 days. An equilibration period over 12 days was carried out prior to the start of the exposure. Exposure was started by placing fertilized eggs into the test replicates. A solvent control and two test concentrations of the test item of 10.0 and 100 µg/L were tested. Two replicates per solvent control and test concentration were tested. 20 eggs per replicate were exposed to each concentration level and the solvent control. The biological results are presented in Table 45.
The test item concentrations were analytically verified via LC-MS/MS on days 0, 5, 7 and 12. The following constituents of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) were quantified: C10 Component + 2EO; C12 Component + 2EO; C14 Component + 2 EO; C16 Component + 2EO.
Fingerprint – MS spectrum
The peak distribution of the test concentration was analyzed in freshly prepared medium in the nominal test item concentration 80 µg/L. A solution of the analytical standard of the test item was prepared in acetonitrile and diluted to 40 µg/L with acetonitrile. A sample of the nominal test item concentration 80 µg/L and a 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 standard and the test item solution were compared. In test item and standard solutions 6 masses were observed and could be assigned to the test item. One additional signal could be observed in the test item solution which was observed to be related to the matrix used for this study. Compared to the standard in acetonitrile the test item in fresh media showed a relative increase in the amounts of the compounds with 274 Da, 302 Da and 358 Da, whereas the relative amounts of compounds < 274 Da are decreased. In general, however, the concentrations and solubility of the test item is too low for significant MS spectra. - Validity criteria fulfilled:
- yes
- Conclusions:
- Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) caused significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal concentrations of 5.00, 10.0, 20.0, 40.0 and 80.0 µg/L, corresponding to overall mean measured test item concentrations of 4.61, 10.4, 21.0, 39.0 and 80.7 µg/L.
For the parameter hatching success, the nominal NOEC was ≥ 80.0 µg/L. Therefore, the respective nominal LOEC was determined to be > 80.0 µg/L. The corresponding NOEC and LOEC values based on overall mean measured test item concentrations were NOEC ≥ 80.7 µg/L and LOEC > 80.7 µg/L, respectively. Therefore no EC10 value could be calculated for this parameter.
For the parameters post hatch survival and overall survival, the nominal NOECs were 20.0 µg/L, respectively. Therefore, the respective nominal LOECs were determined to be 40.0 µg/L. The corresponding NOEC and LOEC values based on overall mean measured test item concentrations were NOEC 21.0 µg/L and LOEC 39.0 µg/L, respectively.
The LC10 value for the parameter post hatch survival was calculated to be 26.6 (20.4 – 34.4) µg/L (based on nominal test item concentrations), corresponding to 27.1 (21.4 – 34.1) µg/L (based on overall mean measured test item concentrations), respectively.
The LC10 value for the parameter overall survival was calculated to be 22.4 (17.6 – 30.7) µg/L (based on nominal test item concentrations), corresponding to 23.2 (18.7 – 30.8) µg/L (based on overall mean measured test item concentrations), respectively.
For the parameter fry growth (expressed as length and fresh weight) the nominal NOECs were 10.0 µg/L (for both parameters). Therefore, the nominal LOECs for length and weight were determined to be 20.0 µg/L, respectively. The corresponding NOEC and LOEC values based on overall mean measured test item concentrations were NOEC 10.4 µg/L and LOEC 21.0 µg/L, respectively.
The EC10 value for the growth parameter length was calculated to be 27.6 (21.7 – 35.0) µg/L (based on nominal test item concentrations), corresponding to 27.8 (22.5 – 34.4) µg/L (based on overall mean measured test item concentrations), respectively.
The EC10 value for the growth parameter fresh weight was calculated to be 13.4 (6.86 – 26.3) µg/L (based on nominal test item concentrations), corresponding to 14.3 (7.59 – 27.1) µg/L (based on overall mean measured test item concentrations), respectively.
All effect values are given based on the nominal and the overall mean measured concentrations of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2). - Executive summary:
The effects of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) (Batch-No. 20201158) on the early-life stage of fish (Danio rerio / Zebrafish) were determined at the test facility according to OECD Guideline 210 from 2021-06-08 to 2021-07-30, with the definitive exposure phase from 2021-06-23 to 2021-07-27.
The test item is a light yellow clear liquid. Methanol was used as solvent with a concentration of 0.025 mL/L dilution water. Stock solutions in methanol with nominal concentrations of 200, 400, 800, 1600 and 3200 mg/L were prepared in appropriate intervals of 7 days and continuously dosed to the dilution water in a flow-through system. Based on the results of a range finding test the test was conducted as a dose-response test with the nominal test item concentrations 5.00, 10.0, 20.0, 40.0 and 80.0 µg/L, corresponding to the overall mean measured test item concentrations of 4.61, 10.4, 21.0, 39.0 and 80.7 µg/L.
The test was started by placing fertilized eggs into the test vessels and it lasted 34 days (30 days post-hatch). 80 eggs of Danio rerio / zebrafish were exposed to each test concentration, the solvent control and the control (4 replicates with 20 eggs each).
The water quality parameters pH-value, oxygen concentration, temperature and total hardness were within the acceptable limits.
On study day 4, 91% of the control and 96% the solvent control larvae had hatched. Therefore, study day 4 was defined as post hatch day 0 (= PHD 0).
Different toxicological endpoints were determined: hatching success, fry growth (assessed via length and fresh weight measurements on PHD 30), morphological and behavioral effects, post-hatch survival and overall survival.
Specific analysis of various concentrations of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) in the test media and the controls was carried out via LC-MS/MS.
The test media were sampled and analyzed from alternating test vessels prior to exposure on study day -1 and during the exposure on study days 0, 7, 14, 21 and 28.
The analytical results of the range finding tests showed that for single compounds (especially with higher carbon chain length) the measured concentrations were partly out of range of the nominal concentrations in some of the replicates. The reason is not known but inhomogenous distribution of the compounds in the test replicates was suspected.The methanol stock solutions (see section 4.2 for details) were sampled and analyzed from freshly prepared and corresponding 7 days aged stock solutions. Measured concentrations of the freshly prepared stock solutions were 101 to 105% of the nominal values. Measured concentrations of the 7 days aged stock solutions in the range of 100 to 109% of the nominal values.
All effect values are given based on the nominal and the overall mean measured concentrations of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
Findings and Observations
The results of the parameters hatching success, fry growth (expressed as weight and length measurement at PHD 30), post-hatch survival and overall survival were checked for statistically significant differences.
No statistically significant difference was detected between the dilution water control and the solvent control for all parameters (hatching success, fry growth expressed as length or weight on PHD 30, post-hatch survival and overall survival). Therefore, both controls were pooled for statistical analysis.
The effect values NOEC, LOEC, ECx, LCx values were determined based on the statistical results. The results are presented in the tables below:NOEC, LOEC, ECx Values of Hatching Success and Fry Growth
Based on nominal and overall mean measured test item concentrations [µg/L]
with 95% Confidence intervals in bracketsParameter Based on nominal test item concentrations [µg/L] Based on overall mean measured test item concentrations [µg/L] Hatching success after Fry Growth Hatching success after Fry Growth 6 days1) expressed as: 6 days1) expressed as: Length Weight Length Weight NOEC ≥ 80.0 10.0 10.0 ≥ 80.7 10.4 10.4 LOEC > 80.0 20.0 20.0 > 80.7 21.0 21.0 EC10 n.d. 27.6 13.4 n.d. 27.8 14.3 (21.7 - 35.0) (6.86 - 26.3) (22.5 - 34.4) (7.59 - 27.1) EC20 n.d. 45.2 23.8 n.d. 43.8 24.3 (32.8 - 61.1) (11.8 - 49.0) (32.7 - 57.5) (12.5 - 48.4) EC50 n.d. > 80.0 70.6 n.d. > 80.7 66.8 (22.5 - (22.2 - > 80.0) > 80.7) 1) end of hatching period
n.d. not determinable as no dose-response was foundNOEC, LOEC, LCx values of Post Hatch Survival and Overall Survival
Based on nominal and overall mean measured test item concentrations [µg/L]
with 95% Confidence intervals in bracketsParameter Based on nominal test item concentrations [µg/L] Based on overall mean measured test item concentrations [µg/L] Post-hatch survival Overall survival Post-hatch survival Overall survival NOEC 20.0 20.0 21.0 21.0 LOEC 40.0 40.0 39.0 39.0 LC10 26.6 22.4 27.1 23.2 (20.4 – 34.4) (17.6 – 30.7) (21.4 – 34.1) (18.7 – 30.8) LC20 29.7 27.1 29.8 27.5 (23.0 – 35.3) (21.8 – 32.5) (23.8 – 34.9) (22.7 – 32.4) LC50 35.6 34.6 35.2 34.2 (27.8 – 38.1) (29.0 – 37.1) (28.2 – 37.4) (29.3 – 36.5) - Endpoint:
- fish early-life stage toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- from 2022-05-04 to 2022-06-08
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See chapter 13 for read-across justification - Reason / purpose for cross-reference:
- read-across source
- 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 2,2’-(Octadec-9-enylimino)bisethanol
Batch number 1950627
CAS No. 25307-17-9
Purity (certified) 100% UVCB
Density 905 kg/m3 at 20 °C
Appearance Yellow liquid
Water Solubility Dispersible / insoluble
Stability under test Not specified
conditions
Expiry date 2023-03-18
Recommended storage Keep container in a dry and well-ventilated place.
Avoid elevated temperatures. - Analytical monitoring:
- yes
- Remarks:
- LC-MS/MS
- Details on sampling:
- Determination of the test item
The samples were analyzed under GLP with an LC-MS/MS method which was implemented under non-GLP and documented finally in the GLP raw data. The method was validated.
The peak distribution (fingerprint) of the highest test concentration was analyzed, once during the test (under non-GLP). The signal distribution was compared with an analytical standard prepared in solvent.
Sampling procedure and pre-treatment
On each time of sampling one alternating replicate of the test vessels (i.e. the outlet of the test vessel) was sampled. The water samples for analysis were obtained by siphoning through inert tubing. These samples did not contain any surface particulates or material dislodged from the bottom or sides of the test vessels. On each sampling point the compound C18:1 Amine + 2EO was analytically determined to distinguish how much test item is truly dissolved.
Organic solvent was added to the samples to stabilize the samples and limit the sorption to the sample vials.
Sampling schedule
Samples of test media including control group and solvent control group were taken from alternating test replicates (the outlet of the test vessel of these replicates) on days -1, 0 and weekly thereafter until end of exposure.
Stock solutions were sampled and analysed from freshly prepared and aged solutions of one application interval on study day 9 and 16. - Vehicle:
- yes
- Remarks:
- With regard to the limited solubility of the test item in water, methanol (VWR, 100% HPLC gradient grade) was used as a solvent. The solvent concentration was the same in all concentration levels and the solvent control (0.025 mL/L)
- Details on test solutions:
- A stock solution of 1600 mg/L was prepared in methanol. An appropriate volume of the test item was pipetted into a glass flask and filled up with the solvent. Density of the test item was taken into account. The solution was agitated until it was visually clear dissolved. Typically, this covers a time period of 20 to 120 seconds. Further stock solutions were prepared by dilution with methanol.
The stock solutions were prepared in appropriate intervals of 7 days.
Syringes were filled with the freshly prepared stock solutions or pure methanol for the solvent control in corresponding intervals.
Equilibration period
Test solutions flowed through the test vessels for 12 days prior to the start of the exposure. The measured concentrations showed no trend of increasing or decreasing.
Control
Dilution water (without test item and without solvent)
Solvent control
Additionally, a solvent control with the same concentration of solvent but without test item was prepared and tested under the same conditions as the test groups. - Test organisms (species):
- Danio rerio (previous name: Brachydanio rerio)
- Details on test organisms:
- Danio rerio (zebrafish)
Vertebrata, Gnathostomata, Pisces, Osteichthyes, Teleostei, Cypriniformes, Cyprinidae
Reason for the selection of the test system
According to the guideline Danio rerio is recommended for this type of study.
Origin
All fish used in the test were reared at Noack Laboratorien GmbH from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin, Germany)
Maintenance of brood fish
A breeding stock of unexposed, mature zebrafish with an age of approx. 10-13 months was used for the egg production. Fish were free of macroscopically discernable symptoms of infection and disease. Spawners were maintained in aquaria with a loading capacity of a minimum of 1 L water per fish.
- Temperature: 25 ± 2 °C
- Dissolved oxygen concentration > 60% of air saturation value
- pH value: 6 – 8.5
- Photoperiod: 16 h light / 8 h dark cycle
(2 transition periods, 30 minutes each)
- Diffuse light (7 – 750 lux on water surface)
- Food: Artemia salina nauplii, 48 hours old, ad libitum;
Daphnia magna, juvenile and adult daphnids, ad libitum;
dry food sera vipan SERA, ad libitum.
- No disease treatments were administered.
Water
Tap water of local origin was used for holding. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
Nominal water parameters of an acceptable dilution water for holding and testing:
Total hardness: 10 – 250 mg CaCO3/L
pH-value: 6.0 – 8.5
Alkalinity: 0.6 mmol/L (recent measurement: 2022-05-30)
Acidity: 0.1 mmol/L (recent measurement: 2022-05-30)
Conductivity: 153 µS/cm (recent measurement: 2022-05-30)
Spawning
15 – 35 adult zebrafish were kept in at least 3 separate aquaria. The fish were healthy with a mortality rate < 5% during the last 7 days and thus not medically treated for at least 7 days. About 15 minutes before start of artificial dawning rectangular dishes covered with a stainless-steel mesh and provided with artificial plants (plastic), were introduced into the aquaria. After 1.5 hours the glass dishes were gently removed. Eggs were checked carefully for abnormalities like fungus infections. These eggs as well as coagulated and not fertilized eggs were discarded (less than 30%). About 900 eggs were taken and washed in dilution water. Eggs originated from 4 different spawnings.
Start of exposure
The eggs that were used to start the exposure were pooled and attributed randomly (eggs were placed in alternating groups into each of the test groups) to the test groups in crystallization dishes containing test solutions (two dishes per test group, each dish loaded with at least 60 eggs, resulting in a total of about 120 eggs per test concentration).
Fertilization check
Immediately after exposing the eggs to the test solutions (start of exposure), the eggs were checked for fertilization. Eggs were fully covered with the respective test solutions. Every embryo was checked under a stereo microscope for its stage. Cleavages which form 4, 8, 16 and 32 cell blastomers can be clearly identified by the development of the blastula and were regarded to be fertilized. Eggs with only a 2 cell stage were regarded as not fertilized and discarded.
Fertilization rate The mean fertilization rate was 89%.
Introduction of eggs
The eggs were placed in the middle of the water phase of the test vessels directly after the fertilization check at a stage before cleavage of the blastodisc commences or as close as possible to this stage. The eggs were transferred randomly into test vessels containing the respective exposure solutions. The distribution of eggs to the concentration groups was carried out indiscriminately by adding 5 eggs to the first test group, the 2nd 5 eggs to the next test group and so on, until all test groups contained the necessary number of eggs. Distribution started with the control group, followed by the solvent control group and from the lowest to the highest test concentration. - Test type:
- flow-through
- Water media type:
- freshwater
- Remarks:
- Tap water was used for testing. The water was filtered on activated charcoal to remove residual chlorine.
- Limit test:
- no
- Total exposure duration:
- 35 d
- Remarks on exposure duration:
- 35 days (30 days post-hatch)
- Hardness:
- See section "Any other information on results incl. tables" below.
- Test temperature:
- See section "Any other information on results incl. tables" below.
During the exposure the water temperature was recorded continuously (once per hour) with a data logger.
The mean temperature was 26.4 °C. The minimum temperature was 25.7 °C and the maximum temperature was 26.8 °C.
The mean water temperature measured once per week from all replicates during the exposure period was 26.4 °C for the control. The minimum and maximum measured temperature for the control were 26.1 and 26.7 °C, respectively - pH:
- See section "Any other information on results incl. tables" below.
- Dissolved oxygen:
- See section "Any other information on results incl. tables" below.
- Nominal and measured concentrations:
- 2.50 - 5.00 - 10.0 - 20.0 - 40.0 µg test item/L (factor: 2), corresponding to the overall arithmetic mean measured test item concentrations of 1.29 – 2.19 – 6.00 – 12.9 - 27.0 µg test item/L.
The test concentrations were based on the results of a preliminary range finding test (non-GLP) conducted under flow-through conditions over 14 days.
The test concentrations were prepared by adding the dissolved test item to dilution water as described above. - Details on test conditions:
- Test design
A randomized block design with each treatment being present in each block was established.
A flow-through exposure design was carried out. Membrane piston pumps provided the water flow-through via silicone tubing.
Precision syringe pumps were used for the introduction of stock solution. The stock solution and the dilution water were mixed in a mixing chamber (approx. volume 0.7 L, one mixing chamber per test replicate) by magnetic stirring at a sufficient speed to ensure a small vortex depth of about 10 % of the dispersion height in the mixing chamber before passing the test aquaria (approx. volume 7.5 L; four replicates per test concentration, control and solvent control) where the eggs/fish were exposed.
The accuracy of the water flow-through was checked prior to start of the exposure and three times per week thereafter.
Water exchange in the test aquaria was about 10 times per day (3.125 L/h).
An equilibration period of 12 days was carried out prior to start of the exposure. The measured concentrations were analysed five times during this period and showed no trend of increasing or decreasing.
Reference item
No reference item is recommended for this test according to the guideline.
Test duration
35 days (30 days post hatch), depending on post-hatch day 0 (study day 5).
Replicates, number of eggs
Four replicates per test concentration and control, with 20 eggs each (80 eggs per test concentration, solvent control and control) were tested.
For the whole study (including the range finding test and definitive tests) 653 healthy eggs/fish were used.
Loading
A loading rate not exceeding 0.5 g/L wet weight fish per 24 hours and not exceeding 5 g/L of solution at any time was maintained.
Test vessels
Glass aquaria of 8.7 L provided with mesh coated fittings allowing flow-through of test media (dimensions: 22/22/18 cm) were used. Test vessels were covered by glass lids. The volume of the test media was approximately 7.5 L.
Cleaning
The test vessels were siphoned as needed to remove excess fecal material and uneaten food, also to minimize microbial growth and biodegradation of the test item. Furthermore, the mesh coated fittings were cleaned once per day during exposure. Cleaning started on study day 2.
Aeration
The dilution water supply tank was aerated.
No additional aeration of the test vessels was provided.
Dilution water
Tap water was used for testing. The water was filtered on activated charcoal to remove residual chlorine.
Feeding of test fish
The feeding regime was ad libitum during the whole feeding period (study day 5 to 34).
Feeding started 3 days after the beginning of hatch on study day 5 (post-hatch day 1, where almost all non-affected larvae swum up). Larvae were fed with a starter food (ST-1 (AQUA SCHWARZ GMBH, 37081 Göttingen, Germany), as well as a suspension of the starter food ST-1 and fine milled brine shrimp nauplii (2 – 7 times daily). 1 day after start of feeding brine shrimp nauplii (24 -48 h old) were fed until the end of the test (2 – 7 times daily).
Brine shrimp nauplii origin, breeding conditions:
Artemia salina (Brine shrimp eggs) were purchased from Kessler Zoologiegroßhandel GmbH & Co. KG, D 67122 Altrip, Germany. Fresh cultures were prepared with salt water (NaCl 20 g/L, ca. 2 g eggs to 1 L salt water, gentle aeration for 24 - 48 hours at approx. 22 °C). 24 - 48 h old brine shrimp nauplii were harvested, washed in a stainless-steel mesh and resuspended in tap water.
Feeding ad libitum was carried out.
Water temperature (target) 26 ± 1.5 °C
Dissolved oxygen
Not less than 60% of air saturation value concentration (target)
Light intensity (target) 300 ± 150 Lux
Photoperiod
A daily 16 / 8 h photoperiod (light / dark) was maintained throughout exposure. - Reference substance (positive control):
- no
- Remarks:
- No reference item is recommended for this test according to the guideline.
- Key result
- Duration:
- 35 d
- Dose descriptor:
- LC50
- Effect conc.:
- 11.7 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Overall survival
- Duration:
- 35 d
- Dose descriptor:
- LC50
- Effect conc.:
- 18.3 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Overall survival
- Duration:
- 35 d
- Dose descriptor:
- other: LC20
- Effect conc.:
- 10 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Overall survival
- Duration:
- 35 d
- Dose descriptor:
- other: LC20
- Effect conc.:
- 15.6 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Overall survival
- Key result
- Duration:
- 35 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 12.9 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Overall survival
- Duration:
- 35 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 20 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Overall survival
- Key result
- Duration:
- 35 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 6 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Overall survival
- Duration:
- 35 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 10 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Overall survival
- Key result
- Duration:
- 35 d
- Dose descriptor:
- LC50
- Effect conc.:
- 10.7 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Post-hatch survival
- Duration:
- 35 d
- Dose descriptor:
- LC50
- Effect conc.:
- 16.8 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Post-hatch survival
- Duration:
- 35 d
- Dose descriptor:
- other: LC20
- Effect conc.:
- 7.83 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Post-hatch survival
- Duration:
- 35 d
- Dose descriptor:
- other: LC20
- Effect conc.:
- 12.7 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Post-hatch survival
- Key result
- Duration:
- 35 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 12.9 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Post-hatch survival
- Duration:
- 35 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 20 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Post-hatch survival
- Key result
- Duration:
- 35 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 6 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Post-hatch survival
- Duration:
- 35 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 10 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Post-hatch survival
- Duration:
- 35 d
- Dose descriptor:
- other: EC20
- Effect conc.:
- 13.6 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- weight
- Duration:
- 35 d
- Dose descriptor:
- other: EC20
- Effect conc.:
- > 20 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- weight
- Key result
- Duration:
- 35 d
- Dose descriptor:
- EC10
- Effect conc.:
- 1.61 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- weight
- Duration:
- 35 d
- Dose descriptor:
- EC10
- Effect conc.:
- 3.54 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- weight
- Key result
- Duration:
- 35 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 2.19 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- weight
- Duration:
- 35 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 5 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- weight
- Key result
- Duration:
- 35 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 1.29 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- weight
- Duration:
- 35 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 2.5 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- weight
- Key result
- Duration:
- 35 d
- Dose descriptor:
- EC10
- Effect conc.:
- > 12.9 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- length
- Duration:
- 35 d
- Dose descriptor:
- EC10
- Effect conc.:
- > 20 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- length
- Key result
- Duration:
- 35 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 2.19 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- length
- Duration:
- 35 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 5 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- length
- Key result
- Duration:
- 35 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 1.29 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- length
- Duration:
- 35 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 2.5 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- length
- Key result
- Duration:
- 7 d
- Dose descriptor:
- LOEC
- Effect conc.:
- > 27 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- time to hatch
- Remarks:
- hatching success
- Key result
- Duration:
- 7 d
- Dose descriptor:
- NOEC
- Effect conc.:
- >= 27 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- time to hatch
- Remarks:
- hatching success
- Duration:
- 7 d
- Dose descriptor:
- LOEC
- Effect conc.:
- > 40 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- time to hatch
- Remarks:
- hatching success
- Duration:
- 7 d
- Dose descriptor:
- NOEC
- Effect conc.:
- >= 40 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- time to hatch
- Remarks:
- hatching success
- Details on results:
- Results
Biological Data
Egg Fertilization Rate
The mean egg fertilization rate determined on study day 0 (start of the exposure) was 89%. Eggs were fully covered with the respective test solutions during fertilization check.
Hatch and Definition of Post Hatch Day 0
Hatch began on study day 2 in the control goup. The hatch of larvae in other test concentrations and solvent control group started on study day 3. The hatch of larvae was completed until study day 7. Study day 5 was determined to be post hatch day 0 (PHD 0) with a hatching rate of 95% in the control and 96% in the solvent control.
Statistical procedures were applied for the total number of test organisms that have hatched on study days 5, 6 and 7.
The Dunnett`s Multiple t-test procedure for hatch data after 5 days was done with a significance level of 0.05. No statistically significant differences were found between the pooled controls and all tested concentrations up to 40.0 µg/L.
The Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm for data of hatch after 6 and 7 days was done with a significance level of 0.05. No statistically significant differences were found between the pooled controls and all tested nominal concentrations up to 40.0 µg/L.
For hatchability on study day 5 and 6, the NOEC and the LOEC (based on nominal test item concentrations) for this endpoint were determined to be ≥ 40.0 and > 40.0 µg/L, respectively. The corresponding NOEC and LOEC (based on the overall arithmetic mean measured test item concentrations) were ≥ 27.0 and > 27.0 µg/L, respectively.
Swim-up
The swim-up period of the control groups and the nominal test concentrations of 5.00 to 40.0 µg/L was observed from study day 5 to 7. First swim-up of larvae was observed on study day 5 in all test groups. From study day 7 on, swim-up was reduced in the highest test concentration of 40 µg/L due to observed non-lethal effects or mortality. No statistical analysis of swim-up data was carried out.
Fry Survival (Post-Hatch Survival)
The post-hatch survival in the control replicates met the validity criteria of the guideline (required: ≥ 75%). The fry survival (post-hatch survival) at the end of the study was 88% in the control and 90% in the solvent control. A concentration-related decrease of the post-hatch survival was detected with increasing test concentrations.
The Williams Multiple sequential t-test procedure (alpha = 0.05) was performed for statistical analysis of post-hatch survival data on study day 35 (PHD 30). Statistically significant differences were found between the pooled control groups and the nominal test item concentrations of 20.0 and 40.0 µg/L.
The NOEC and the LOEC for this endpoint were 10.0 µg/L and 20.0 µg/L (nominal concentrations), respectively. The corresponding NOEC and LOEC (based on the overall arithmetic mean measured test item concentrations) were 6.00 µg/L and 12.9 µg/L, respectively.
The LC50-value (based on nominal concentrations) for post-hatch survival on study day 35 (PHD30) was 16.8 (12.6 to 18.7) µg/L, corresponding to 10.7 (7.76 to 12.0) µg/L (based on the overall arithmetic mean measured test item concentrations).
Overall Survival
The cumulative mortality at the end of the exposure, related to the number of eggs introduced on day 0, was 16% per control and 14% per solvent control. A concentration-related decrease of the overall survival (increase of overall mortality) was detected in the test item concentrations.
The William’s multiple sequential t-test procedure (alpha = 0.05) was performed for statistical analysis of overall survival data on study day 35 (PHD 30). Statistically significant differences were found between the pooled control groups and the nominal test item concentrations of 20.0 and 40.0 µg/L.
The NOEC and the LOEC for this endpoint were 10.0 µg/L and 20.0 µg/L (nominal concentrations), respectively. The corresponding NOEC and LOEC (based on the overall arithmetic mean measured test item concentrations) were 6.00 µg/L and 12.9 µg/L, respectively.
The LC50-value (based on nominal concentrations) for overall survival on study day 35 (PHD 30) was 18.3 (10.9 to 30.8) µg/L, corresponding to 11.7 (6.60 to 21.0) µg/L (based on the overall arithmetic mean measured test item concentrations).
Morphological and Behavioral Effects
Observation of abnormal appearance and behavior of hatched larvae were carried out daily until the end of the exposure. As an isolated event quiescence and an unusual long arresting on the ground of one larvae of the control group was observed on study days 11 and 12. In the solvent control group one larvae showed tumbling from study day 10 to 13. Quiecscence and unusual long arresting on the ground was observed from two larvae on study day 13 and from one larvae on study day 14. No further morphological nor behavioral effects were observed in the control and solvent groups throughout exposure.
In the nominal test concentrations of 2.50 and 5.00 µg/L occasional non-lethal effects were observed. The nominal test concentration of 10.0 µg/L showed no morphological nor behavioural effects throughout exposure. The test concentration of 20.0 µg/L showed non-lethal effects with ongoing exposure from study day 11 up to study day 19. The highest nominal test concentration of 40.0 µg/L showed non-lethal effects for a short period after hatch from study day 7 to 12. This resulted in 100 % mortality.
Fry Growth
Fry growth, expressed as length and wet weight, was measured on study day 35 (PHD 30) from all survivors.
The Williams Multiple sequential t-test procedure (alpha = 0.05) showed statistically significant differences for the surviving fish of the nominal test concentrations of 5.00 to 20.0 µg/L for the growth parameters fresh weight and mean total length.
Therefore, the NOEC and the LOEC (nominal test item concentrations) for the growth parameters fresh weight and total length were determined to be 2.50 and 5.00 µg/L, respectively. The corresponding NOEC and LOEC (based on overall arithmetic mean measured test item concentrations) were 1.29 and 2.19 µg/L, respectively.
Biomass Loading
The biomass-loading factor for the study was determined from the fresh weights of the control and solvent control fish at the end of the exposure.
The maximum biomass at the end of the exposure was determined in replicate 4 of the control group: 751.5 mg total fish weight. The maximum biomass loading based on the 7.5 liter volume of a single growth chamber was 100.2 mg/L.
Maximum loading rate:
biomass/(volume of test solution) = (751.5 mg)/(7.5 L) = 100.2 mg/L
The biomass loading rate based upon a flow of 75 liters per day through each single test aquaria was 10.0 mg per liter and day.
Maximum loading rate per day:
biomass/(volume of test solution per day) = (751.5 mg)/(75 L) = 10.0 mg/L per day
These loadings were well within the requirements to ensure adequate dissolved oxygen levels and to avoid crowding of the fish.
Physico-chemical Data
Dissolved Oxygen
The dissolved oxygen concentrations in the control, solvent control and the test item groups, expressed in percent saturation, were in the mean 94 – 98% and ranged from 77 to 100% during the exposure period.
Water Temperature
During the exposure the water temperature was recorded continuously (once per hour) with a data logger. The mean temperature was 26.4 °C. The minimum temperature was 25.7 °C and the maximum temperature was 26.8 °C.
The mean water temperature measured once per week from all replicates during the exposure period was 26.4 °C for the control. The minimum and maximum measured temperature for the control were 26.1 and 26.7 °C, respectively.
The validity criterion for the parameter temperature was fulfilled.
pH-Values
The mean pH-values in the control, solvent control and test item groups were between 7.75 and 7.80 and ranged from 7.58 to 8.08 during the exposure period.
Total Hardness
Total hardness of the test media was measured once per week from alternating replicates of the control and the remaining highest test concentration of 40.0 µg/L (day 0 and 8) and 20.0 µg/L (day 15, 21 and 29). The mean total hardness was 67 mg CaCO3/L and ranged from 64 to 68 mg CaCO3/L in the control group.
Residual Chlorine
Residual chlorine, measured from the dilution water supply tank on study days 0, 8, 15, 21 and 29 was < 0.01 mg/L.
Total Organic Carbon (TOC) of the Dilution Water
The total organic carbon (TOC) sampled from the dilution water supply tank was determined on study days 0, 8, 15, 21 and 29. The mean measured TOC was 1.26 mgC/L throughout exposure. All measured concentrations were below the LOQ of 2 mgC/L.
Flow Rates
The mean flow rate through the mixing chambers of all test item, control and solvent groups was 3.12 ± 0.052 L/h and the individual values ranged from 2.94 to 3.36 L/h.
A precision syringe pump was used for introduction of the stock solution to the mixing chambers. At any renewal of the syringes the proper function of the pump and the applied volume was checked by the syringe volume indicator.
Light intensity
Light intensity was measured at the start of exposure on the surface of all test vessels and ranged from 188 to 406 lux (mean: 275 lux).
Measured Test Concentrations during Equilibration Phase
The concentrations of the test item 2,2’-(Octadec-9-enylimino)bisethanol, the solvent control and the control were determined in the equilibration phase on day -1 from the test vessels of the test replicates 2 of all tested concentration levels via LC MS/MS.
To evaluate how much test item is lost due to the centrifiguation step, the concentration in samples taken from the test vessels of day -1 was quantified with and without centrifugation step, before the samples were subjected to the analytical procedure as described.
The samples analysed without centrifugation showed a homogeneous distribution and no turbidity. Although the recoveries were below the nominal concentration, no precipitate could be observed. Therefore, these samples were used for the calculation of the overall arithmetic mean measured concentrations. The lower recoveries of the centrifuged samples are explained by losses due to adsorption to the centrifuge tubes and are therefore not further used. This effect was observed with similar substances in aquaeous matrix.
Measured Concentrations in the Stock Solutions
Measured concentrations of the freshly prepared stock solution were 106 to 112% of the nominal values. Measured concentrations of the 7 days aged stock solution were in the range of 111 to 115% of the nominal values.
Measured Concentrations in the Test Vessels during Exposure
The concentrations of the test item 2,2’-(Octadec-9-enylimino)bisethanol, the solvent control and the control were determined during exposure on days 0, 7, 14, 21 and 34 from the test vessels of a defined test replicate of all tested concentration levels via LC MS/MS.
Fingerprint of the Test Item (Non-GLP)
The peak distribution of the highest test item concentration (40.0 µg test item/L) and the control was analyzed. A solution of the analytical standard of the test item was prepared and diluted to 1.00 mg/L and 20 µg/L with methanol : fish dilution water (50 : 50) containing 0.1% formic acid. The sample and the control were diluted by factor 2 with methanol containing 0.2% formic acid to avoid an inhomogeneous sample. The detected signals of the analytical standards and the sample solution were compared. In sample and standard solution the mass 356 m/z was observed and could be assigned to the test item. In general, the MS spectra of the sample solution and the analytical standard were quite similar. However, in the highest test item concentration the intensity of the signal corresponding to the test item was slightly lower compared to the 20 µg/L standard solution indicating a lower solubility of the test item in the aquaeous test medium.
Validity Criteria
The study was performed according to OECD 210 and met the validity criteria:
• Dissolved oxygen saturation was between 77 and 100% of the air saturation value
(required: > 60%).
• Water temperature was in the recommended range for the test species (i.e. 26 °C ± 1.5 °C): 26.1 – 27.1 °C. It did not differ by more than 1.5 °C between test vessels or between successive days at any time during the test.
• The cumulative hatching success was 95% in the control group and 96% in the solvent control group (required: ≥ 70%).
• Post-hatch survival was 88% in the control group and 90% in solvent control group (required: ≥ 75%).
• Concentration analysis: the concentrations of the test item in the test solutions should be maintained within 80 – 120% of the nominal values. The test item is a multi-component substance (UVCB) which is insoluble/dispersible in water. On each sampling point the compound C18:1 Amine + 2EO was analytically determined to distinguish how much of the test item is truly dissolved and dispersed in the test media. The measured concentrations of the test item (truly dissolved and dispersed fraction) were constantly below the nominal range. This effect was also observed in the preliminary test. Additionally all effect values were based on the overall arithmetic mean measured concentrations of the test item 2,2’-(Octadec-9-enylimino)bisethanol.
Stability
The stability of the standard stock solution (1000 mg test item/L in methanol) was checked after 23 days and was 92% of the nominal concentration.
The responses of a freshly prepared calibration standard (6 µg test item/L) was compared with a 19 days old calibration standard with 5 injections each. The deviation was < 10% and the calibration standards were found to be stable over this period.
The stability of the fortified samples at the 1 x LOQ was checked after 7 days. The recovery was 101% of the nominal concentration.
Procedural Recovery
A procedural recovery (Quality Control) on 1 x LOQ Level was freshly prepared on each day of analysis. For preparation of the quality contro. It was treated in parallel to the test samples and measured in each sample set.
Preliminary Range Finding Test (non-GLP)
A non-GLP preliminary range finding test was conducted at the test facility as a shortened early-life stage test over a period of 14 days under flow-through conditions as described. An equilibration period over 12 days was carried out prior to the start of the exposure. Exposure was started by placing fertilized eggs into the test replicates. A solvent control and two test concentrations of the test item of 5.00 and 50.0 µg/L were included in the test. Two replicates per solvent control and test concentration were tested. 20 eggs per replicate were exposed to each concentration level and the solvent control.
The test item concentrations were analytically verified via LC-MS/MS on days -1, 0, 7 and 14. The sampling was done from the test vessels and from the outlet of the test vessels. Additionally, the samples for analysis of the truly dissolved test item in the test vessels were centrifuged and analyzed. - Reported statistics and error estimates:
- The data of the parameters hatching success, post hatch survival and overall survival were arcsine transformed prior to statistical analysis.
Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01.
Levene’s test on variance was done with a significance level of 0.01.
Monotonicity was done by trend analysis by contrasts (significance level 0.05).
The Dunnett’s multiple t-test-procedure was done with a significance level of 0.05 for the parameter hatching success on study day 5.
The multiple sequentially-rejective Welsh-t-test after Bonferroni-Holm was done with a significance level of 0.05 for the parameter hatching success on study days 6 and 7.
The Williams multiple sequential t-test procedure was done with a significance level of 0.05 for the parameters post hatch survival, overall survival, fresh weight and mean total length.
All calculations were done with ToxRat Professional and based on the nominal and overall arithmetic mean measured concentrations of the test item. - Validity criteria fulfilled:
- yes
- Conclusions:
- 2,2’-(Octadec-9-enylimino)bisethanol caused significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal concentrations of 2.50, 5.00, 10.0, 20.0 and 40.0 µg/L, corresponding to overall arithmetic mean measured test item concentrations of 1.29 – 2.19 – 6.00 – 12.9 - 27.0 µg/L.
For hatchability on study days 5 and 6, the NOEC and the LOEC (based on nominal test item concentrations) for this endpoint were determined to be ≥ 40.0 and > 40.0 µg/L, respectively. The corresponding NOEC and LOEC (based on the overall arithmetic mean measured test item concentrations) were ≥ 27.0 and > 27.0 µg/L, respectively. Therefore, no EC10 value could be calculated for this parameter.
For both parameters post hatch survival and overall survival, the nominal NOECs were 10.0 µg/L. Therefore, the respective nominal LOECs were determined to be 20.0 µg/L. The corresponding NOEC and LOEC values based on the overall arithmetic mean measured test item concentrations were NOEC 6.00 µg/L and LOEC 12.9 µg/L, respectively.
For the parameter fry growth (expressed as length and fresh weight) the nominal NOECs were 2.50 µg/L (for both parameters). Therefore, the nominal LOECs for length and weight were determined to be 5.00 µg/L, respectively. The corresponding NOEC and LOEC values based on the overall arithmetic mean measured test item concentrations were NOEC 1.29 µg/L and LOEC 2.19 µg/L, respectively.
All effect values are given based on the nominal and the overall arithmetic mean measured concentrations of the test item 2,2’-(Octadec-9-enylimino)bisethanol. - Executive summary:
The effects of the test item 2,2’-(Octadec-9-enylimino)bisethanol (Batch-No. 1950627) on the early-life stage of fish (Danio rerio / Zebrafish) were determined at the test facility according to OECD Guideline 210 from 2022-05-04 to 2022-06-08.
The test item is a yellow liquid UVCB substance which is dispersible/insoluble in water. Methanol was used as solvent with a concentration of 0.025 mL/L dilution water. Stock solutions in methanol with nominal concentrations of 100, 200, 400, 800 and 1600 mg/L were prepared in appropriate intervals of 7 days and continuously dosed to the dilution water in a flow-through system (for detailed descriptions, please refer to section 4.2). Based on the results of a range finding test the test was conducted as a dose-response test with the nominal test item concentrations 2.50, 5.00, 10.0, 20.0 and 40.0 µg/L, corresponding to the overall arithmetic mean measured test item concentrations of 1.29 – 2.19 – 6.00 – 12.9 - 27.0 µg/L. The term overall arithmetic mean means that all measured samples (stabilized samples, from study days 0, 7, 14, 21 and 34) were taken into account for calculation of the arithmetic mean measured concentrations.
The test was started by placing fertilized eggs into the test vessels and it lasted 35 days (30 days post-hatch). 80 eggs of Danio rerio / zebrafish were exposed to each test concentration, the solvent control and the control (4 replicates with 20 eggs each).
The water quality parameters pH-value, oxygen concentration, temperature and total hardness were within the acceptable limits.
On study day 5, 95% of the control and 96% the solvent control larvae had hatched. Therefore, study day 5 was defined as post hatch day 0 (= PHD 0).
Different toxicological endpoints were determined: hatching success, fry growth (assessed via length and fresh weight measurements on PHD 30), morphological and behavioral effects, post-hatch survival and overall survival.
Specific analysis of various concentrations of 2,2’-(Octadec-9-enylimino)bisethanol in the test media and the controls was carried out via LC-MS/MS.
The concentrations of the test item 2,2’-(Octadec-9-enylimino)bisethanol, the solvent control and the control were determined during exposure on days 0, 7, 14, 21 and 34. The constituent C18:1 Amine + 2EO was determined via LC MS/MS.
The samples taken from the test vessels were completely clear and did not contain any particles or turbidity. These samples were subjected to the analytical procedure and the results are used for the calculation of the dose response.
The stock solutions prepared in methanol (see section 4.2 for details) were sampled and analyzed from the freshly prepared and corresponding 7 days aged stock solutions. Measured concentrations of the freshly prepared stock solution were 106 to 112% of the nominal values. Measured concentrations of the 7 days aged stock solution were in the range of 111 to 115% of the nominal values.All effect values are given based on the nominal and the overall arithmetic mean measured concentrations of the test item 2,2’-(Octadec-9-enylimino)bisethanol.
Findings and Observations
The results of the parameters hatching success, fry growth (expressed as weight and length measurement at PHD 30), post-hatch survival and overall survival were checked for statistically significant differences.
No statistically significant difference was detected between the dilution water control and the solvent control for all parameters (hatching success, fry growth expressed as length or weight on PHD 30, post-hatch survival and overall survival). Therefore, both controls were pooled for statistical analysis.
The effect values NOEC, LOEC, ECx, LCx values were determined based on the statistical results.NOEC, LOEC, ECx Values of Hatching Success and Fry Growth
Based on nominal and overall arithmetric mean measured test item concentrations [µg/L]
with 95% Confidence intervals in bracketsParameter Based on nominal test item concentrations [µg/L] Based on overall arithmetric mean measured test item concentrations [µg/L] Hatching success after Fry Growth Hatching success after Fry Growth 7 days1) expressed as: 6 days1) expressed as: Length Weight Length Weight NOEC ≥ 40.0 2.50 2.50 ≥ 27.0 1.29 1.29 LOEC > 40.0 5.00 5.00 > 27.0 2.19 2.19 EC10 n.d. > 20.0 3.54 n.d. > 12.9 1.61 (< 2.50 to > 20.0) (< 1.29 to > 12.9) EC20 n.d. n.d. > 20.0 n.d. n.d. 13.6 (n.d.) EC50 n.d. n.d. n.d. n.d. n.d. n.d. 1) = end of hatching period
n.d. = not determinableNOEC, LOEC, LCx values of Post Hatch Survival and Overall Survival
Based on nominal and overall arithmetic mean measured test item concentrations [µg/L]
with 95% Confidence intervals in bracketsParameter Based on nominal test item concentrations [µg/L] Based on overall arithmetric mean measured test item concentrations [µg/L] Post-hatch survival Overall survival Post-hatch survival Overall survival NOEC 10.0 10.0 6.00 6.00 LOEC 20.0 20.0 12.9 12.9 LC10 n.d. n.d. n.d. n.d. LC20 12.7 15.6 7.83 10.0 (9.56 – 17.2) (9.63 – 20.0) (5.71 – 11.0) (5.76 – 20.2) LC50 16.8 18.3 10.7 11.7 (12.6 – 18.7) (10.9 – 30.8) (7.76 – 12.0) (6.60 – 21.0) n.d. = not determinable
Referenceopen allclose all
Hatch / Hatching Time (Control, Solvent Control and Test Item Concentrations)
| Nominal | Rep. | PHD -2 | PHD -1 | PHD 0 | PHD 1 | PHD 2 |
| test item conc. | Study day 2 | Study day 3 | Study day 4 | Study day 5 | Study day 6 | |
| [µg/L] | Cumulative hatching rate [%] | |||||
| Control | 1 | 0 | 25 | 85 | 95 | 95 |
| 2 | 0 | 35 | 95 | 95 | 95 | |
| 3 | 0 | 35 | 100 | 100 | 100 | |
| 4 | 0 | 70 | 85 | 90 | 90 | |
| Mean | 0 | 41 | 91 | 95 | 95 | |
| Solvent control | 1 | 0 | 50 | 95 | 95 | 95 |
| 2 | 0 | 55 | 95 | 100 | 100 | |
| 3 | 0 | 70 | 95 | 95 | 95 | |
| 4 | 0 | 70 | 100 | 100 | 100 | |
| Mean | 0 | 61 | 96 | 98 | 98 | |
| 5.00 | 1 | 0 | 80 | 100 | 100 | 100 |
| 2 | 0 | 90 | 90 | 90 | 90 | |
| 3 | 0 | 70 | 95 | 95 | 95 | |
| 4 | 0 | 40 | 95 | 95 | 95 | |
| Mean | 0 | 70 | 95 (ns) | 95 | 95 (ns) | |
| 10.0 | 1 | 0 | 85 | 100 | 100 | 100 |
| 2 | 0 | 65 | 95 | 95 | 95 | |
| 3 | 0 | 90 | 100 | 100 | 100 | |
| 4 | 0 | 40 | 95 | 95 | 95 | |
| Mean | 0 | 70 | 98 (ns) | 98 | 98 (ns) | |
| 20.0 | 1 | 0 | 35 | 95 | 95 | 95 |
| 2 | 0 | 70 | 85 | 85 | 85 | |
| 3 | 0 | 75 | 100 | 100 | 100 | |
| 4 | 0 | 70 | 95 | 95 | 95 | |
| Mean | 0 | 63 | 94 (ns) | 94 | 94 (ns) | |
| 40.0 | 1 | 0 | 45 | 90 | 90 | 95 |
| 2 | 0 | 40 | 90 | 90 | 100 | |
| 3 | 0 | 75 | 95 | 95 | 95 | |
| 4 | 5 | 80 | 100 | 100 | 100 | |
| Mean | 1 | 60 | 94 (ns) | 94 | 98 (ns) | |
| 80.0 | 1 | 0 | 55 | 90 | 95 | 100 |
| 2 | 0 | 65 | 95 | 95 | 95 | |
| 3 | 0 | 55 | 80 | 90 | 90 | |
| 4 | 0 | 25 | 90 | 100 | 100 | |
| Mean | 0 | 50 | 89 (ns) | 95 | 96 (ns) | |
(ns) = No statistically significant difference from pooled control groups
Percent Swim-up of Hatched Live Fry of the Control, Solvent Control and the Nominal Test Item Concentrations 5.00 to 20.0 µg/L
| Nominal | Rep. | PHD 0 | PHD 1 | PHD 2 | PHD 3 | PHD 4 | PHD 5 |
| test item conc. | Study day 4 | Study day 5 | Study day 6 | Study day 7 | Study day 8 | Study day 9 | |
| [µg/L] | Cumulative swim up [%] | ||||||
| Control | 1 | 0 | 100 | 100 | 100 | 100 | 100 |
| 2 | 0 | 100 | 100 | 100 | 100 | 100 | |
| 3 | 0 | 100 | 100 | 100 | 100 | 100 | |
| 4 | 0 | 100 | 100 | 100 | 100 | 100 | |
| Mean | 0 | 100 | 100 | 100 | 100 | 100 | |
| Solvent control | 1 | 0 | 100 | 100 | 100 | 100 | 100 |
| 2 | 0 | 95 | 95 | 100 | 100 | 100 | |
| 3 | 0 | 95 | 95 | 100 | 100 | 100 | |
| 4 | 0 | 100 | 100 | 100 | 100 | 100 | |
| Mean | 0 | 98 | 98 | 100 | 100 | 100 | |
| 5.00 | 1 | 0 | 100 | 100 | 100 | 100 | 100 |
| 2 | 0 | 100 | 100 | 100 | 100 | 100 | |
| 3 | 0 | 100 | 100 | 100 | 100 | 100 | |
| 4 | 0 | 100 | 100 | 100 | 100 | 100 | |
| Mean | 0 | 100 | 100 | 100 | 100 | 100 | |
| 10.0 | 1 | 0 | 100 | 100 | 100 | 100 | 100 |
| 2 | 0 | 100 | 100 | 100 | 100 | 100 | |
| 3 | 0 | 95 | 100 | 100 | 100 | 100 | |
| 4 | 0 | 100 | 100 | 100 | 100 | 100 | |
| Mean | 0 | 99 | 100 | 100 | 100 | 100 | |
| 20.0 | 1 | 0 | 100 | 100 | 100 | 100 | 100 |
| 2 | 0 | 100 | 100 | 100 | 100 | 100 | |
| 3 | 0 | 95 | 100 | 100 | 100 | 100 | |
| 4 | 0 | 79 | 100 | 100 | 100 | 100 | |
| Mean | 0 | 94 | 100 | 100 | 100 | 100 | |
Percent Swim-up of Hatched Live Fry of the Nominal Test Item Concentrations 40.0 and 80.0 µg/L
| Nominal | Rep. | PHD 0 | PHD 1 | PHD 2 | PHD 3 | PHD 4 | PHD 5 |
| test item conc. | Study day 4 | Study day 5 | Study day 6 | Study day 7 | Study day 8 | Study day 9 | |
| [µg/L] | Cumulative swim up [%] | ||||||
| 40.0 | 1 | 0 | 100 | 100 | 100 | 100 | 100 |
| 2 | 0 | 100 | 100 | 100 | 100 | 100 | |
| 3 | 0 | 100 | 100 | 100 | 100 | 100 | |
| 4 | 0 | 95 | 100 | 100 | 100 | 100 | |
| Mean | 0 | 99 | 100 | 100 | 100 | 100 | |
| 80.0 | 1 | 0 | 95 | 90 | 90 | 63 | 50 |
| 2 | 0 | 95 | 100 | 100 | 29 | 0 | |
| 3 | 0 | 100 | 100 | 100 | 53 | 0 | |
| 4 | 0 | 95 | 85 | 74 | 44 | 0 | |
| Mean | 0 | 96 | 94 | 91 | 47 | 13 | |
Post-Hatch Survival on Study Day 34 (PHD 30) of the Control, Solvent Control and the Nominal Test Item Concentrations 5.00 and 10.0 µg/L
| Nominal | Rep. | Eggs introduced on study day 0 | Cumulative number of hatched larvae | Vital Larvae on study day 34 | Post-Hatch survival |
| test item conc. | (PHD 30) | on study day 34 | |||
| [µg/L] | [%] | ||||
| Control | 1 | 20 | 19 | 16 | 84 |
| 2 | 20 | 19 | 18 | 95 | |
| 3 | 20 | 20 | 19 | 95 | |
| 4 | 20 | 18 | 16 | 89 | |
| Mean | 20 | 19 | 17 | 91 | |
| Solvent control | 1 | 20 | 19 | 16 | 84 |
| 2 | 20 | 20 | 18 | 90 | |
| 3 | 20 | 19 | 16 | 84 | |
| 4 | 20 | 20 | 19 | 95 | |
| Mean | 20 | 20 | 17 | 88 | |
| 5.00 | 1 | 20 | 20 | 19 | 95 |
| 2 | 20 | 18 | 17 | 94 | |
| 3 | 20 | 19 | 17 | 89 | |
| 4 | 20 | 19 | 19 | 100 | |
| Mean | 20 | 19 | 18 | 95 (ns) | |
| 10.0 | 1 | 20 | 20 | 18 | 90 |
| 2 | 20 | 19 | 19 | 100 | |
| 3 | 20 | 20 | 20 | 100 | |
| 4 | 20 | 19 | 17 | 89 | |
| Mean | 20 | 20 | 19 | 95 (ns) |
(ns) = No statistically significant difference from pooled control groups
Post-Hatch Survival on Study Day 34 (PHD 30) of the Nominal Test Item Concentrations 20.0 to 80.0 µg/L
| Nominal | Rep. | Eggs introduced on study day 0 | Cumulative number of hatched larvae | Vital Larvae on study day 34 | Post-Hatch survival |
| test item conc. | (PHD 30) | on study day 34 | |||
| [µg/L] | [%] | ||||
| 20.0 | 1 | 20 | 19 | 19 | 100 |
| 2 | 20 | 17 | 17 | 100 | |
| 3 | 20 | 20 | 19 | 95 | |
| 4 | 20 | 19 | 19 | 100 | |
| Mean | 20 | 19 | 19 | 99 (ns) | |
| 40.0 | 1 | 20 | 19 | 5 | 26 |
| 2 | 20 | 20 | 6 | 30 | |
| 3 | 20 | 19 | 9 | 47 | |
| 4 | 20 | 20 | 3 | 15 | |
| Mean | 20 | 20 | 6 | 30 (s) | |
| 80.0 | 1 | 20 | 20 | 0 | 0 |
| 2 | 20 | 19 | 0 | 0 | |
| 3 | 20 | 18 | 0 | 0 | |
| 4 | 20 | 20 | 0 | 0 | |
| Mean | 20 | 19 | 0 | 0 (s) |
(ns) / (s) = No statistically / statistically significant difference from pooled control groups
Overall Survival and Overall Mortality on Study Day 34 (PHD 30) of the Control, Solvent
Control and the Nominal Test Item Concentrations 5.00 and 10. µg/L
| Nominal | Rep. | Vital larvae on | Overall survival | Overall mortality |
| test item conc. | study day 34 | |||
| [µg/L] | (PHD 30) | |||
| [%] | [%] | |||
| Control | 1 | 16 | 80 | 20 |
| 2 | 18 | 90 | 10 | |
| 3 | 19 | 95 | 5 | |
| 4 | 16 | 80 | 20 | |
| Mean | 17 | 86 | 14 | |
| Solvent control | 1 | 16 | 80 | 20 |
| 2 | 18 | 90 | 10 | |
| 3 | 16 | 80 | 20 | |
| 4 | 19 | 95 | 5 | |
| Mean | 17 | 86 | 14 | |
| 5.00 | 1 | 19 | 95 | 5 |
| 2 | 17 | 85 | 15 | |
| 3 | 17 | 85 | 15 | |
| 4 | 19 | 95 | 5 | |
| Mean | 18 | 90 (ns) | 10 | |
| 10.0 | 1 | 18 | 90 | 10 |
| 2 | 19 | 95 | 5 | |
| 3 | 20 | 100 | 0 | |
| 4 | 17 | 85 | 15 | |
| Mean | 19 | 93 (ns) | 7 |
(ns) = No statistically significant difference from pooled control groups
Overall Survival and Overall Mortality on Study Day 34 (PHD 30) of the Nominal Test
Item Concentrations 20.0 to 80.0 µg/L
| Nominal | Rep. | Vital larvae on | Overall survival | Overall mortality |
| test item conc. | study day 34 | |||
| [µg/L] | (PHD 30) | |||
| [%] | [%] | |||
| 20.0 | 1 | 19 | 95 | 5 |
| 2 | 17 | 85 | 15 | |
| 3 | 19 | 95 | 5 | |
| 4 | 19 | 95 | 5 | |
| Mean | 19 | 93 (ns) | 7 | |
| 40.0 | 1 | 5 | 25 | 75 |
| 2 | 6 | 30 | 70 | |
| 3 | 9 | 45 | 55 | |
| 4 | 3 | 15 | 85 | |
| Mean | 6 | 29 (s) | 71 | |
| 80.0 | 1 | 0 | 0 | 100 |
| 2 | 0 | 0 | 100 | |
| 3 | 0 | 0 | 100 | |
| 4 | 0 | 0 | 100 | |
| Mean | 0 | 0 (s) | 100 |
(ns) / (s) = No statistically / statistically significant difference from pooled control groups
Behavioral Effects observed in the Nominal Test Concentrations 40.0 and 80.0 µg/L (Study Day 7 – 27)
| Study Day | 40.0 µg/L | 80.0 µg/L | ||||||
| 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
| 7 | - | - | - | - | - | 4 Q | 2 Q | 12 Q |
| 5 G | ||||||||
| 8 | - | - | - | - | 16 Q | 17 Q | 15 Q | 16 Q |
| 6 G | 12 G | 7 G | 9 G | |||||
| 9 | - | - | - | - | 4 Q | 1 Q | 4 Q | 2 Q |
| 2 G | 1 G | 4 G | 2 G | |||||
| 10 | - | - | - | - | 3 M | 2 M | 2 M | 1 M |
| 3 T | 2 T | 2 T | 1 T | |||||
| 3 V | 2 V | 2 V | 1 V | |||||
| 11 | - | - | - | - | 1 M | 100 % Mortality | ||
| 1 T | ||||||||
| 1 V | ||||||||
| 12 | 2 Q | 4 Q | 3 Q | 4 Q | 100 % Mortality | |||
| 1 T | 4 G | 3 G | 4 G | |||||
| 1 P | ||||||||
| 13 | 4 Q | 5 Q | 1 Q | 2 Q | ||||
| 4 G | 4 G | 1 G | 2 G | |||||
| 1 T | 1 T | |||||||
| 1 S | 1 S | |||||||
| 14 | 4 Q | 2 Q | - | 1 Q | ||||
| 1 G | 2 G | 1 G | ||||||
| 3 T | 2 F | 1 F | ||||||
| 3 S | ||||||||
| 15 | 3 M | 4 M | 1 M | 1 M | ||||
| 3 G | 4 G | 1 G | 1 G | |||||
| 3 F | 4 F | 1 F | 1 F | |||||
| 16 | - | 2 M | 2 Q | 1 M | ||||
| 2 G | 2 S | 1 G | ||||||
| 2 F | 1 F | |||||||
| 17 | - | - | 1 Q | - | ||||
| 1 G | ||||||||
| 18 | - | - | 3 Q | - | ||||
| 3 G | ||||||||
| 19 | - | - | 1 Q | - | ||||
| 1 G | ||||||||
| 20 to 26 | - | - | - | - | ||||
| 27 | - | - | 1 Q | |||||
| 1 G | ||||||||
| 1 F | ||||||||
Q = Quiescence T = Tumbling P = Side position
G = Arresting on the ground S = Arresting at the surface
F = No food uptake M = Missing escape reflex
Overview of Fry Growth: Length and Wet Weight on Study Day 34 (PHD 30) of the Control, Solvent Control and Nominal Test Item Concentrations 5.00 and 10.0 µg/L
Mean total length per fish [mm]Mean wet weight per fish [mg]
| Nominal | Rep. | PHD 30 (End of exposure) | ||||||
| test item conc.[µg/L] | ||||||||
| Control | 1 | 16.6 | 39.4 | 2 | 15.3 | 35.8 | ||
| 3 | 15.7 | 31.9 | ||||||
| 4 | 16.5 | 37.7 | ||||||
| Mean | 16.0 | 36.2 | ||||||
| ± SD | 0.545 | 2.79 | ||||||
| CV [%] | 3.41 | 7.71 | ||||||
| Solvent control | 1 | 16.5 | 34.3 | |||||
| 2 | 16.4 | 38.1 | ||||||
| 3 | 16.5 | 39.0 | ||||||
| 4 | 15.9 | 33.4 | Mean | 16.3 | 36.2 | |||
| ± SD | 0.249 | 2.39 | ||||||
| CV [%] | 1.53 | 6.61 | ||||||
| 5.00 | 1 | 16.3 | 33.0 | |||||
| 2 | 16.4 | 36.5 | ||||||
| 3 | 16.3 | 36.5 | 4 | 16.1 | 35.4 | Mean | 16.3 (ns) | 35.4 (ns) |
| ± SD | 0.109 | 1.43 | ||||||
| CV [%] | 0.670 | 4.04 | 10.0 | 1 | 16.1 | 35.4 | ||
| 2 | 16.3 | 34.3 | ||||||
| 3 | 15.4 | 28.7 | ||||||
| 4 | 16.9 | 39.0 | Mean | 16.2 (ns) | 34.4 (ns) | |||
| ± SD | 0.536 | 3.70 | ||||||
| CV [%] | 3.31 | 10.7 | ||||||
(ns) = No statistically significant difference from pooled controls
Overview of Fry Growth: Length and Wet Weight on Study Day 34 (PHD 30) of the Nominal Test Item Concentrations 20.0 and 40.0 µg/L
Mean total length per fish [mm]Mean wet weight per fish [mg]
| Nominal | Rep. | PHD 30 (End of exposure) | ||||
| test item conc.[µg/L] | ||||||
| 20.0 | 1 | 15.1 | 29.4 | 2 | 15.6 | 31.2 |
| 3 | 14.9 | 29.1 | ||||
| 4 | 15.1 | 29.6 | ||||
| Mean | 15.2 (s) | 29.8 (s) | ||||
| ± SD | 0.259 | 0.814 | ||||
| CV [%] | 1.70 | 2.73 | ||||
| 40.0 | 1 | 13.1 | 23.7 | |||
| 2 | 13.3 | 21.6 | ||||
| 3 | 12.8 | 20.9 | ||||
| 4 | 14.8 | 31.4 | Mean | 13.5 (s) | 24.4 (s) | |
| ± SD | 0.771 | 4.17 | ||||
| CV [%] | 5.71 | 17.1 | ||||
(s) = Statistically significant difference from pooled controls
Individual Length on Study Day 34 (PHD 30) of the Control and the Solvent Control
| Fish No. | Control | Solvent control | ||||||
| Total length of individual fish in [mm] | ||||||||
| 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
| 1 | 16.5 | 13.5 | 15.5 | 15.5 | 14.0 | 17.0 | 20.0 | 16.0 |
| 2 | 14.0 | 15.5 | 19.5 | 20.0 | 16.0 | 18.0 | 14.5 | 14.5 |
| 3 | 18.0 | 12.0 | 13.0 | 16.0 | 20.0 | 15.5 | 18.5 | 17.5 |
| 4 | 13.0 | 17.0 | 13.5 | 22.0 | 13.5 | 14.0 | 15.5 | 18.0 |
| 5 | 20.0 | 14.5 | 16.0 | 17.0 | 12.0 | 14.5 | 17.0 | 15.0 |
| 6 | 19.0 | 14.5 | 15.5 | 12.0 | 15.5 | 15.5 | 16.5 | 16.0 |
| 7 | 17.5 | 15.0 | 15.5 | 18.0 | 18.0 | 16.0 | 15.0 | 16.0 |
| 8 | 17.0 | 18.0 | 14.5 | 16.5 | 16.0 | 16.5 | 15.5 | 18.0 |
| 9 | 17.0 | 18.5 | 13.0 | 15.5 | 18.5 | 15.0 | 16.5 | 19.5 |
| 10 | 16.5 | 14.0 | 17.0 | 18.0 | 15.5 | 15.5 | 18.0 | 16.0 |
| 11 | 16.5 | 16.0 | 17.0 | 15.5 | 17.0 | 16.0 | 19.0 | 16.0 |
| 12 | 15.0 | 19.0 | 18.0 | 18.0 | 16.0 | 17.0 | 14.5 | 16.0 |
| 13 | 17.0 | 17.0 | 13.0 | 15.0 | 19.0 | 19.0 | 16.0 | 16.0 |
| 14 | 15.5 | 15.0 | 14.5 | 16.0 | 15.0 | 16.0 | 12.0 | 16.0 |
| 15 | 16.5 | 13.5 | 15.5 | 12.5 | 18.0 | 18.0 | 16.0 | 15.5 |
| 16 | 16.0 | 17.0 | 15.0 | 17.0 | 20.0 | 20.0 | 19.5 | 18.0 |
| 17 | - | 15.5 | 17.0 | - | - | 18.5 | - | 11.0 |
| 18 | - | 10.0 | 20.5 | - | - | 14.0 | - | 13.0 |
| 19 | - | - | 15.0 | - | - | - | - | 14.5 |
| 20 | - | - | - | - | - | - | - | - |
| Mean | 16.6 | 15.3 | 15.7 | 16.5 | 16.5 | 16.4 | 16.5 | 15.9 |
| ± SD | 1.67 | 2.22 | 2.04 | 2.39 | 2.24 | 1.67 | 2.05 | 1.87 |
| CV % | 10.0 | 14.5 | 13.0 | 14.5 | 13.6 | 10.2 | 12.4 | 11.7 |
- = Fish died before end of the study
Individual Length on Study Day 34 (PHD 30) of the Nominal Test Item Concentrations 5.00 and 10.0 µg/L
| Fish No. | Nominal test item conc. [µg/L] | |||||||
| 5.00 | 10.0 | |||||||
| Total length of individual fish in [mm] | ||||||||
| 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
| 1 | 18.0 | 16.0 | 15.0 | 15.0 | 20.0 | 17.0 | 12.0 | 14.5 |
| 2 | 17.5 | 17.0 | 13.0 | 12.0 | 12.0 | 16.5 | 19.0 | 19.0 |
| 3 | 15.0 | 19.5 | 19.0 | 15.5 | 18.0 | 14.5 | 15.0 | 19.0 |
| 4 | 14.0 | 15.0 | 19.0 | 19.0 | 17.5 | 18.0 | 15.0 | 18.5 |
| 5 | 16.0 | 15.5 | 16.5 | 20.5 | 17.0 | 18.5 | 13.5 | 18.0 |
| 6 | 17.5 | 19.0 | 18.5 | 17.0 | 18.5 | 17.5 | 15.5 | 18.5 |
| 7 | 16.5 | 20.5 | 18.0 | 15.0 | 17.0 | 15.0 | 15.0 | 18.0 |
| 8 | 18.0 | 17.5 | 17.0 | 16.0 | 12.0 | 14.5 | 15.0 | 15.5 |
| 9 | 16.0 | 17.0 | 14.0 | 15.5 | 17.5 | 20.5 | 17.0 | 18.0 |
| 10 | 14.5 | 15.5 | 19.5 | 18.0 | 10.5 | 14.0 | 14.5 | 16.0 |
| 11 | 19.0 | 15.0 | 13.5 | 15.0 | 16.0 | 19.0 | 15.5 | 16.5 |
| 12 | 16.0 | 13.0 | 19.5 | 16.0 | 21.0 | 17.0 | 12.0 | 21.0 |
| 13 | 19.0 | 15.0 | 17.0 | 17.5 | 16.0 | 17.0 | 18.0 | 16.0 |
| 14 | 17.5 | 14.5 | 13.0 | 16.0 | 16.0 | 16.0 | 15.5 | 13.0 |
| 15 | 14.0 | 15.0 | 14.5 | 17.5 | 13.5 | 15.0 | 17.5 | 15.5 |
| 16 | 16.0 | 15.0 | 16.0 | 17.0 | 14.0 | 15.5 | 13.0 | 16.0 |
| 17 | 15.5 | 18.0 | 14.0 | 12.0 | 17.0 | 13.5 | 15.0 | 14.0 |
| 18 | 18.0 | - | - | 17.5 | 16.0 | 15.0 | 15.0 | - |
| 19 | 12.5 | - | - | 14.0 | - | 15.5 | 17.5 | - |
| 20 | - | - | - | - | - | - | 18.0 | - |
| Mean | 16.3 | 16.4 | 16.3 | 16.1 | 16.1 | 16.3 | 15.4 | 16.9 |
| ± SD | 1.76 | 1.95 | 2.29 | 2.07 | 2.709 | 1.80 | 1.90 | 2.03 |
| CV % | 10.8 | 11.9 | 14.0 | 12.8 | 16.8 | 11.1 | 12.3 | 12.0 |
- = Fish died before end of the study
Individual Length on Study Day 34 (PHD 30) of the Nominal Test Item Concentrations 20.0 and 40.0 µg/L
| Fish No. | Nominal test item conc. [µg/L] | |||||||
| 20.0 | 40.0 | |||||||
| Total length of individual fish in [mm] | ||||||||
| 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
| 1 | 13.5 | 17.5 | 13.0 | 16.0 | 13.5 | 14.0 | 15.0 | 15.0 |
| 2 | 12.0 | 13.5 | 14.0 | 16.0 | 11.5 | 15.5 | 11.0 | 14.0 |
| 3 | 11.0 | 18.0 | 16.0 | 14.5 | 12.0 | 13.0 | 14.0 | 15.5 |
| 4 | 15.5 | 16.5 | 12.5 | 18.0 | 16.0 | 13.5 | 11.0 | - |
| 5 | 11.0 | 17.0 | 16.5 | 17.0 | 12.5 | 12.5 | 11.0 | - |
| 6 | 17.0 | 16.5 | 14.0 | 16.5 | - | 11.0 | 17.0 | - |
| 7 | 17.0 | 18.0 | 16.0 | 15.5 | - | - | 10.0 | - |
| 8 | 18.0 | 14.5 | 13.5 | 15.0 | - | - | 16.5 | - |
| 9 | 18.0 | 16.0 | 17.0 | 14.0 | - | - | 9.5 | - |
| 10 | 12.5 | 18.0 | 13.0 | 13.0 | - | - | - | - |
| 11 | 11.0 | 14.0 | 17.5 | 15.5 | - | - | - | - |
| 12 | 18.5 | 16.0 | 13.5 | 15.0 | - | - | - | - |
| 13 | 16.0 | 16.5 | 10.5 | 17.0 | - | - | - | - |
| 14 | 17.5 | 11.0 | 13.0 | 13.0 | - | - | - | - |
| 15 | 12.5 | 15.0 | 17.0 | 15.5 | - | - | - | - |
| 16 | 14.5 | 12.5 | 20.0 | 16.0 | - | - | - | - |
| 17 | 16.0 | 15.5 | 16.0 | 14.0 | - | - | - | - |
| 18 | 18.0 | - | 15.0 | 13.5 | - | - | - | - |
| 19 | 16.5 | - | 16.0 | 12.0 | - | - | - | - |
| 20 | - | - | - | - | - | - | - | - |
| Mean | 15.1 | 15.6 | 14.9 | 15.1 | 13.1 | 13.3 | 12.8 | 14.8 |
| ± SD | 2.62 | 1.95 | 2.18 | 1.53 | 1.59 | 1.38 | 2.71 | 0.62 |
| CV % | 17.3 | 12.5 | 14.6 | 10.1 | 12.2 | 10.4 | 21.2 | 4.22 |
- = Fish died before end of the study
Pooled Wet Weights on Study Day 34 (PHD 30) of the Control, Solvent Control and the Nominal Test Item Concentrations 5.00 to 40.0 µg/L
[mg][mg]
| Nominal | Replicate | Number of fish alive | Pooled | Mean | Mean | ± SD | CV % | ||||||
| test item conc. | on study day 34 | wet weight | wet weight | ||||||||||
| [µg/L] | per replicate | per fish | |||||||||||
| [mg] | |||||||||||||
| Control | 1 | 16 | 630.2 | 39.4 | 36.2 | 2.79 | 7.71 | ||||||
| 2 | 18 | 645.2 | 35.8 | ||||||||||
| 3 | 19 | 605.8 | 31.9 | ||||||||||
| 4 | 16 | 603.1 | 37.7 | ||||||||||
| Solvent control | 1 | 16 | 548.4 | 34.3 | 36.2 | 2.39 | 6.61 | ||||||
| 2 | 18 | 685.5 | 38.1 | 3 | 16 | 623.9 | 39.0 | ||||||
| 4 | 19 | 634.1 | 33.4 | ||||||||||
| 5.00 | 1 | 19 | 627.2 | 33.0 | 35.4 | 1.43 | 4.04 | ||||||
| 2 | 17 | 620.2 | 36.5 | ||||||||||
| 3 | 17 | 619.9 | 36.5 | ||||||||||
| 4 | 19 | 672.3 | 35.4 | ||||||||||
| 10.0 | 1 | 18 | 636.6 | 35.4 | 34.4 | 3.70 | 10.7 | ||||||
| 2 | 19 | 651.2 | 34.3 | ||||||||||
| 3 | 20 | 573.2 | 28.7 | ||||||||||
| 4 | 17 | 662.2 | 39.0 | ||||||||||
| 20.0 | 1 | 19 | 558.9 | 29.4 | 29.8 | 0.814 | 2.73 | ||||||
| 2 | 17 | 529.7 | 31.2 | ||||||||||
| 3 | 19 | 553.8 | 29.1 | ||||||||||
| 4 | 19 | 563.0 | 29.6 | ||||||||||
| 40.0 | 1 | 5 | 118.4 | 23.7 | 24.4 | 4.17 | 17.1 | ||||||
| 2 | 6 | 129.8 | 21.6 | ||||||||||
| 3 | 9 | 188.0 | 20.9 | ||||||||||
| 4 | 3 | 94.1 | 31.4 |
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 | 5.00 | 10.0 | 20.0 | 40.0 | 80.0 | ||
| 0 | 1 | 100 | 99 | 99 | 99 | 100 | 100 | 100 |
| 2 | 100 | 100 | 99 | 100 | 99 | 99 | 99 | |
| 3 | 99 | 100 | 99 | 99 | 99 | 100 | 100 | |
| 4 | 100 | 100 | 100 | 100 | 99 | 99 | 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 | 99 | 100 | 100 | 100 | 100 | 100 |
| 2 | 100 | 100 | 100 | 100 | 99 | 99 | 99 | |
| 3 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
| 4 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
| 7 | 1 | 100 | 98 | 98 | 99 | 99 | 99 | 99 |
| 2 | 99 | 99 | 99 | 99 | 98 | 99 | 98 | |
| 3 | 100 | 99 | 99 | 99 | 99 | 100 | 98 | |
| 4 | 100 | 98 | 99 | 99 | 99 | 100 | 99 | |
| 9 | 1 | 100 | 97 | 97 | 97 | 98 | 98 | 98 |
| 2 | 98 | 97 | 98 | 98 | 98 | 97 | 96 | |
| 3 | 99 | 97 | 98 | 98 | 98 | 99 | 96 | |
| 4 | 98 | 97 | 98 | 97 | 99 | 99 | 98 | |
| 12 | 1 | 100 | 96 | 93 | 94 | 97 | 96 | - |
| 2 | 98 | 96 | 94 | 96 | 94 | 94 | - | |
| 3 | 98 | 92 | 96 | 96 | 97 | 97 | - | |
| 4 | 98 | 97 | 97 | 97 | 95 | 97 | - | |
| 14 | 1 | 100 | 96 | 96 | 95 | 98 | 98 | - |
| 2 | 97 | 98 | 96 | 97 | 96 | 94 | - | |
| 3 | 98 | 98 | 97 | 97 | 96 | 97 | - | |
| 4 | 98 | 98 | 98 | 96 | 96 | 97 | - | |
| 16 | 1 | 100 | 96 | 95 | 95 | 97 | 98 | - |
| 2 | 98 | 98 | 97 | 97 | 96 | 95 | - | |
| 3 | 98 | 97 | 98 | 97 | 95 | 97 | - | |
| 4 | 97 | 98 | 98 | 95 | 97 | 97 | - | |
- = No measurement, due to 100 % mortality
Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 19 - 33)
| Study day | Replicate | Dissolved oxygen [%] | ||||||
| Nominal test item concentrations [µg/L] | ||||||||
| Control | Solvent control | 5.00 | 10.0 | 20.0 | 40.0 | 80.0 | ||
| 19 | 1 | 99 | 94 | 94 | 95 | 96 | 97 | - |
| 2 | 96 | 96 | 94 | 95 | 94 | 95 | - | |
| 3 | 96 | 95 | 96 | 94 | 91 | 96 | - | |
| 4 | 96 | 96 | 96 | 93 | 95 | 96 | - | |
| 20 | 1 | 97 | 93 | 93 | 95 | 95 | 96 | - |
| 2 | 94 | 95 | 94 | 95 | 93 | 95 | - | |
| 3 | 94 | 94 | 96 | 94 | 94 | 95 | - | |
| 4 | 95 | 95 | 96 | 92 | 94 | 96 | - | |
| 23 | 1 | 96 | 91 | 94 | 91 | 93 | 94 | - |
| 2 | 93 | 94 | 91 | 93 | 94 | 94 | - | |
| 3 | 92 | 90 | 92 | 93 | 95 | 94 | - | |
| 4 | 90 | 90 | 93 | 90 | 93 | 93 | - | |
| 26 | 1 | 96 | 90 | 92 | 89 | 92 | 94 | - |
| 2 | 94 | 93 | 89 | 92 | 91 | 94 | - | |
| 3 | 93 | 90 | 91 | 92 | 93 | 94 | - | |
| 4 | 94 | 89 | 88 | 92 | 91 | 94 | - | |
| 28 | 1 | 93 | 89 | 91 | 89 | 90 | 93 | - |
| 2 | 92 | 89 | 89 | 90 | 92 | 93 | - | |
| 3 | 92 | 91 | 89 | 90 | 93 | 95 | - | |
| 4 | 91 | 89 | 85 | 86 | 90 | 93 | - | |
| 30 | 1 | 93 | 87 | 89 | 87 | 88 | 92 | - |
| 2 | 90 | 89 | 86 | 88 | 89 | 90 | - | |
| 3 | 89 | 86 | 88 | 88 | 88 | 94 | - | |
| 4 | 89 | 84 | 83 | 82 | 87 | 92 | - | |
| 33 | 1 | 90 | 81 | 86 | 82 | 82 | 87 | - |
| 2 | 90 | 83 | 78 | 83 | 86 | 86 | - | |
| 3 | 89 | 78 | 84 | 80 | 86 | 89 | - | |
| 4 | 88 | 77 | 74 | 77 | 82 | 90 | - | |
| Mean | 96 | 94 | 94 | 94 | 95 | 96 | 99 | |
| ± SD | 3.77 | 5.69 | 5.74 | 5.57 | 4.56 | 3.38 | 1.30 | |
| Min. | 88 | 77 | 74 | 77 | 82 | 86 | 96 | |
| Max. | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
- = No measurement, due to 100 % mortality
Min./Max. = Minimum/Maximum measured dissolved oxygen concentration
Water Temperature (Continuous Measuring) in Replicate 2 of the Control Group
| Period of measurements | 2021-06-23 to 2021-07-27 |
| Minimum temperature [°C] | 25.0 |
| Maximum temperature [°C] | 26.7 |
| 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 | 5.00 | 10.0 | 20.0 | 40.0 | 80.0 | ||
| 0 | 1 | 26.0 | 26.4 | 26.6 | 26.6 | 26.5 | 26.5 | 26.5 |
| 2 | 26.4 | 26.6 | 26.5 | 26.5 | 26.5 | 26.5 | 26.4 | |
| 3 | 26.4 | 26.7 | 26.5 | 26.4 | 26.4 | 26.4 | 26.4 | |
| 4 | 26.2 | 26.40 | 26.40 | 26.60 | 26.30 | 26.30 | 26.5 | |
| 7 | 1 | 26.5 | 26.7 | 26.8 | 26.9 | 26.8 | 26.7 | 26.7 |
| 2 | 26.4 | 26.7 | 26.8 | 26.7 | 26.7 | 26.7 | 26.7 | |
| 3 | 26.5 | 26.9 | 26.7 | 26.7 | 26.7 | 26.7 | 26.7 | |
| 4 | 26.4 | 26.6 | 26.6 | 26.7 | 26.5 | 26.5 | 26.7 | |
| 14 | 1 | 26.4 | 26.7 | 26.8 | 26.9 | 26.8 | 26.8 | - |
| 2 | 26.5 | 26.8 | 26.8 | 26.7 | 26.7 | 26.7 | - | |
| 3 | 26.6 | 27.0 | 26.7 | 26.7 | 26.7 | 26.7 | - | |
| 4 | 26.4 | 26.6 | 26.6 | 26.7 | 26.6 | 26.6 | - | |
| 20 | 1 | 26.5 | 26.6 | 26.7 | 26.8 | 26.7 | 26.4 | - |
| 2 | 26.4 | 26.7 | 26.7 | 26.6 | 26.6 | 26.5 | - | |
| 3 | 26.5 | 26.9 | 26.6 | 26.6 | 26.6 | 26.6 | - | |
| 4 | 26.4 | 26.5 | 26.5 | 26.6 | 26.4 | 26.6 | - | |
| 28 | 1 | 26.2 | 26.5 | 26.5 | 26.7 | 26.5 | 26.5 | - |
| 2 | 26.2 | 26.5 | 26.5 | 26.5 | 26.4 | 26.4 | - | |
| 3 | 26.3 | 26.7 | 26.5 | 26.5 | 26.4 | 26.5 | - | |
| 4 | 26.1 | 26.3 | 26.4 | 26.4 | 26.4 | 26.3 | - | |
| Mean | 26.4 | 26.6 | 26.6 | 26.6 | 26.6 | 26.6 | 26.6 | |
| ± SD | 0.15 | 0.18 | 0.13 | 0.14 | 0.15 | 0.14 | 0.14 | |
| Min. | 26.0 | 26.3 | 26.4 | 26.4 | 26.3 | 26.3 | 26.4 | |
| Max. | 26.6 | 27.0 | 26.8 | 26.9 | 26.8 | 26.8 | 26.7 | |
- = No measurement, due to 100 % mortality
Min./Max. = Minimum/Maximum measured water temperature
pH-Values in the Test Media
| Study day | Rep. | pH-value | ||||||
| Nominal test item concentrations [µg/L] | ||||||||
| Control | Solvent control | 5.00 | 10.0 | 20.0 | 40.0 | 80.0 | ||
| 0 | 1 | 7.88 | 7.84 | 7.82 | 7.79 | 7.81 | 7.84 | 7.80 |
| 2 | 7.87 | 7.84 | 7.79 | 7.80 | 7.78 | 7.83 | 7.80 | |
| 3 | 7.83 | 7.78 | 7.79 | 7.80 | 7.79 | 7.81 | 7.77 | |
| 4 | 7.83 | 7.82 | 7.81 | 7.80 | 7.88 | 7.81 | 7.79 | |
| 7 | 1 | 8.03 | 7.97 | 7.92 | 7.91 | 7.92 | 7.92 | 7.95 |
| 2 | 8.05 | 8.00 | 7.93 | 7.93 | 7.96 | 7.93 | 7.95 | |
| 3 | 8.12 | 8.02 | 7.98 | 7.93 | 7.94 | 7.95 | 7.89 | |
| 4 | 8.03 | 7.98 | 7.98 | 7.92 | 8.05 | 8.02 | 7.91 | |
| 14 | 1 | 7.56 | 7.64 | 7.60 | 7.66 | 7.64 | 7.65 | - |
| 2 | 7.67 | 7.68 | 7.63 | 7.66 | 7.65 | 7.61 | - | |
| 3 | 7.76 | 7.66 | 7.69 | 7.67 | 7.62 | 7.64 | - | |
| 4 | 7.74 | 7.67 | 7.66 | 7.65 | 7.63 | 7.63 | - | |
| 20 | 1 | 7.58 | 7.61 | 7.59 | 7.61 | 7.62 | 7.63 | - |
| 2 | 7.62 | 7.64 | 7.58 | 7.62 | 7.61 | 7.63 | - | |
| 3 | 7.65 | 7.63 | 7.62 | 7.62 | 7.58 | 7.61 | - | |
| 4 | 7.65 | 7.64 | 7.62 | 7.57 | 7.63 | 7.65 | - | |
| 28 | 1 | 7.56 | 7.66 | 7.67 | 7.71 | 7.67 | 7.69 | - |
| 2 | 7.71 | 7.70 | 7.66 | 7.66 | 7.68 | 7.76 | - | |
| 3 | 7.77 | 7.69 | 7.70 | 7.65 | 7.69 | 7.74 | - | |
| 4 | 7.71 | 7.66 | 7.62 | 7.58 | 7.69 | 7.71 | - | |
| Mean | 7.78 | 7.76 | 7.73 | 7.73 | 7.74 | 7.75 | 7.86 | |
| ± SD | 0.171 | 0.140 | 0.135 | 0.122 | 0.140 | 0.129 | 0.075 | |
| Min. | 7.56 | 7.61 | 7.58 | 7.57 | 7.58 | 7.61 | 7.77 | |
| Max. | 8.12 | 8.02 | 7.98 | 7.93 | 8.05 | 8.02 | 7.95 | |
- = No measurement, due to 100 % mortality
Min./Max. = Minimum/Maximum measured pH-value
Total Hardness in the Control and the Remaining Highest Test Concentration
| Study day | Replicate | Total hardness [mg CaCO3/L] | ||
| Nominal test item conc. [µg/L] | ||||
| Control | 40.0 | 80.0 | ||
| 1 | 1 | 66 | 66 | |
| 8 | 2 | 69 | 67 | |
| 16 | 3 | 69 | 68 | |
| 22 | 4 | 68 | 69 | |
| 30 | 1 | 69 | 69 | |
| Mean | 68 | 69 | 67 | |
| ± SD | 1.30 | 0.58 | 0.71 | |
| Min. | 66 | 68 | 66 | |
| Max. | 69 | 69 | 67 | |
= No measurement, due to 100 % mortality
n.a. = Not applicable
Min./Max. = Minimum/Maximum measured hardness
Total Organic Carbon (TOC) of the Dilution Water
| Study day | TOC [mg/L] |
| 1 | 0.78 |
| 8 | 0.42 |
| 16 | 0.52 |
| 22 | 0.39 |
| 30 | 1.12 |
| Mean | 0.65 |
| SD | 0.31 |
| Min. | 0.39 |
| Max. | 1.12 |
Min./Max. = Minimum/Maximum measured TOC
Flow Rates
| Flow rate | Nominal test item concentrations [µg/L] | ||||||
| Control | Solvent control | 5.00 | 10.0 | 20.0 | 40.0 | 80.0 | |
| Measured flow rates of the membrane piston pumps1) | |||||||
| [L/h] | |||||||
| Minimum | 2.94 | 3.00 | 3.00 | 3.00 | 3.00 | 3.00 | 3.00 |
| Maximum | 3.36 | 3.24 | 3.36 | 3.36 | 3.36 | 3.36 | 3.36 |
| Mean | 3.13 | 3.08 | 3.13 | 3.12 | 3.13 | 3.13 | 3.14 |
| ± Standard deviation | ± 0.091 | ± 0.063 | ± 0.067 | ± 0.084 | ± 0.102 | ± 0.071 | ± 0.098 |
| Overall mean | 3.12 | ||||||
| ± Standard deviation | ± 0.083 | ||||||
1) Nominal flow rates of dilution water (for preparation of the test solutions): 3.125 (2.81 – 3.44 L/h)
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessel on Study Day -1
| Sampling day / Replicate | Day -1 | ||
| Repl. 1 | |||
| Nominal | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | ||
| concentration of the | C10 Amine + 2 EO | ||
| Test item | a.i. | Meas. conc. | % |
| [µg/L] | [µg a.i./L] | [µg a.i./L] | |
| 5.00 | 0.364 | 0.390 | 107 |
| 10.0 | 0.727 | 0.724 | 100 |
| 20.0 | 1.45 | 1.49 | 103 |
| 40.0 | 2.91 | 3.25 | 112 |
| 80.0 | 5.82 | 6.07 | 104 |
| Solvent Control | < LOQ | ||
| Control | < LOQ | ||
| Sampling day / Replicate | Day -1 | ||
| Repl. 1 | |||
| Nominal | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | ||
| concentration of the | C12 Amine + 2 EO | ||
| Test item | a.i. | Meas. conc. | % |
| [µg/L] | [µg a.i./L] | [µg a.i./L] | |
| 5.00 | 2.63 | 2.83 | 108 |
| 10.0 | 5.26 | 5.45 | 104 |
| 20.0 | 10.5 | 10.6 | 101 |
| 40.0 | 21.1 | 23.9 | 114 |
| 80.0 | 42.1 | 44.2 | 105 |
| Solvent Control | < LOQ | ||
| Control | < LOQ | ||
Meas. Conc. = Measured concentration (dilution factor taken into account)
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (2.0 µg/L of the test item)
Continuation Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessel on Study Day -1
| Sampling day / Replicate | Day -1 | ||
| Repl. 1 | |||
| Nominal | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | ||
| concentration of the | C14 Amine + 2 EO | ||
| Test item | a.i. | Meas. conc. | % |
| [µg/L] | [µg a.i./L] | [µg a.i./L] | |
| 5.00 | 0.932 | 0.904 | 97 |
| 10.0 | 1.86 | 1.79 | 96 |
| 20.0 | 3.73 | 3.68 | 99 |
| 40.0 | 7.46 | 8.46 | 114 |
| 80.0 | 14.9 | 15.7 | 105 |
| Solvent Control | < LOQ | ||
| Control | < LOQ | ||
| Sampling day / Replicate | Day -1 | ||
| Repl. 1 | |||
| Nominal | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | ||
| concentration of the | C16 Amine + 2 EO | ||
| Test item | a.i. | Meas. conc. | % |
| [µg/L] | [µg a.i./L] | [µg a.i./L] | |
| 5.00 | 0.423 | 0.354 | 84 |
| 10.0 | 0.846 | 0.773 | 91 |
| 20.0 | 1.69 | 1.60 | 95 |
| 40.0 | 3.38 | 5.46 | 161 |
| 80.0 | 6.77 | 9.19 | 136 |
| Solvent Control | < LOQ | ||
| Control | < LOQ | ||
Measured Concentrations and Percent of Nominal Concentration in the Stock Solutions
| Freshly prepared stock solutions | 7 Days aged stock solutions | |||||
| Nominal | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | |||||
| concentration of the | C10 Amine + 2 EO | |||||
| Test item | a.i. | Meas. | % | Meas. | % | |
| [mg/L] | [mg a.i./L] | conc. | conc. | |||
| [mg a.i./L] | [mg a.i./L] | |||||
| 3200 | 233 | 240 | 103 | 242 | 104 | |
| 1600 | 116 | 120 | 103 | 116 | 100 | |
| 800 | 58.2 | 60.8 | 105 | 60.5 | 104 | |
| 400 | 29.1 | 30.2 | 104 | 30.8 | 106 | |
| 200 | 14.5 | 14.8 | 102 | 15.1 | 104 | |
| Solvent Control | < LOQ | < LOQ | ||||
| Freshly prepared stock solutions | 7 Days aged stock solutions | |||||
| Nominal | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | |||||
| concentration of the | C12 Amine + 2 EO | |||||
| Test item | a.i. | Meas. | % | Meas. | % | |
| [mg/L] | [mg a.i./L] | conc. | conc. | |||
| [mg a.i./L] | [mg a.i./L] | |||||
| 3200 | 1684 | 1767 | 105 | 1804 | 107 | |
| 1600 | 842 | 874 | 104 | 871 | 103 | |
| 800 | 421 | 439 | 104 | 449 | 107 | |
| 400 | 210 | 220 | 105 | 227 | 108 | |
| 200 | 105 | 109 | 104 | 111 | 105 | |
| Solvent Control | < LOQ | < LOQ | ||||
Meas. Conc. = Measured concentration (dilution factor taken into account)
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (2.0 µg/L of the test item)
Continuation: Measured Concentrations and Percent of Nominal Concentration in the Stock Solutions
| Freshly prepared stock solutions | 7 Days aged stock solutions | |||||
| Nominal | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | |||||
| concentration of the | C10 Amine + 2 EO | |||||
| Test item | a.i. | Meas. | % | Meas. | % | |
| [mg/L] | [mg a.i./L] | conc. | conc. | |||
| [mg a.i./L] | [mg a.i./L] | |||||
| 3200 | 596 | 608 | 102 | 642 | 108 | |
| 1600 | 298 | 302 | 101 | 309 | 104 | |
| 800 | 149 | 153 | 103 | 158 | 106 | |
| 400 | 74.6 | 76.8 | 103 | 81.3 | 109 | |
| 200 | 37.3 | 37.5 | 101 | 39.1 | 105 | |
| Solvent Control | < LOQ | < LOQ | ||||
| Freshly prepared stock solutions | 7 Days aged stock solutions | |||||
| Nominal | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | |||||
| concentration of the | C12 Amine + 2 EO | |||||
| Test item | a.i. | Meas. | % | Meas. | % | |
| [mg/L] | [mg a.i./L] | conc. | conc. | |||
| [mg a.i./L] | [mg a.i./L] | |||||
| 3200 | 271 | 277 | 102 | 279 | 103 | |
| 1600 | 135 | 137 | 102 | 136 | 100 | |
| 800 | 67.7 | 69.6 | 103 | 70.3 | 104 | |
| 400 | 33.8 | 35.0 | 103 | 35.4 | 105 | |
| 200 | 16.9 | 17.3 | 102 | 17.4 | 103 | |
| Solvent Control | < LOQ | < LOQ | ||||
Meas. Conc. = Measured concentration (dilution factor taken into account)
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (2.0 µg/L of the test item)
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day 0 to Day 28 (C10 Amine + 2 EO)
| Sampling day / Replicate | Day 0 | Day 7 | Day 14 | |||||
| Repl. 2 | Repl. 4 | Repl. 4 | ||||||
| Nominal | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | |||||||
| concentration of the | C10 Amine + 2 EO | |||||||
| Test item | a.i. | Meas. | % | Meas. | % | Meas. | % | |
| [µg/L] | [µg a.i./L] | conc. | conc. | conc. | ||||
| [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | ||||||
| 80.0 | 5.82 | 5.62 | 97 | 6.12 | 105 | - 1) | ||
| 40.0 | 2.91 | 2.40 | 82 | 2.91 | 100 | 3.15 | 108 | |
| 20.0 | 1.45 | 1.52 | 105 | 1.99 | 137 | 1.39 | 95 | |
| 10.0 | 0.727 | 0.745 | 102 | 0.654 | 90 | 0.676 | 93 | |
| 5.00 | 0.364 | 0.441 | 121 | 0.285 | 79 | 0.358 | 98 | |
| Solvent Control | < LOQ | < LOQ | < LOQ | |||||
| Control | < LOQ | < LOQ | < LOQ | |||||
| Sampling day / Replicate | Day 21 | Day 28 | ||||||
| Repl. 1 | Repl. 2 | |||||||
| Nominal | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | |||||||
| concentration of the | C10 Amine + 2 EO | |||||||
| Test item | a.i. | Meas. | % | Meas. | % | |||
| [µg/L] | [µg a.i./L] | conc. | conc. | |||||
| [µg a.i./L] | [µg a.i./L] | |||||||
| 80.0 | 5.82 | - 1) | - 1) | |||||
| 40.0 | 2.91 | 2.71 | 93 | 2.48 | 85 | |||
| 20.0 | 1.45 | 1.23 | 85 | 1.66 | 114 | |||
| 10.0 | 0.727 | 0.726 | 100 | 0.807 | 111 | |||
| 5.00 | 0.364 | 0.321 | 88 | 0.316 | 87 | |||
| Solvent Control | < LOQ | < LOQ | ||||||
| Control | < LOQ | < LOQ | ||||||
Meas. Conc. = Measured concentration (dilution factor taken into account)
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (2.0 µg/L of the test item, corresponding to 0.147 µg a.i./L)
1) = no samples due to 100% mortality
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day 0 to Day 28 (C12 Amine + 2 EO)
| Sampling day / Replicate | Day 0 | Day 7 | Day 14 | |||||
| Repl. 2 | Repl. 3 | Repl. 4 | ||||||
| Nominal | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | |||||||
| concentration of the | C12 Amine + 2 EO | |||||||
| Test item | a.i. | Meas. | % | Meas. | % | Meas. | % | |
| [µg/L] | [µg a.i./L] | conc. | conc. | conc. | ||||
| [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | ||||||
| 80.0 | 42.1 | 40.3 | 96 | 43.7 | 104 | - 1) | ||
| 40.0 | 21.0 | 17.9 | 85 | 21.8 | 104 | 23.1 | 110 | |
| 20.0 | 10.5 | 10.7 | 102 | 13.5 | 129 | 9.82 | 93 | |
| 10.0 | 5.26 | 5.47 | 104 | 4.84 | 92 | 4.81 | 91 | |
| 5.00 | 2.63 | 3.26 | 124 | 2.15 | 82 | 2.44 | 93 | |
| Solvent Control | < LOQ | < LOQ | < LOQ | |||||
| Control | < LOQ | < LOQ | < LOQ | |||||
| Sampling day / Replicate | Day 21 | Day 28 | ||||||
| Repl. 1 | Repl. 2 | |||||||
| Nominal | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | |||||||
| concentration of the | C12 Amine + 2 EO | |||||||
| Test item | a.i. | Meas. | % | Meas. | % | |||
| [µg/L] | [µg a.i./L] | conc. | conc. | |||||
| [µg a.i./L] | [µg a.i./L] | |||||||
| 80.0 | 42.1 | - 1) | - 1) | |||||
| 40.0 | 21.0 | 20.3 | 97 | 16.7 | 79 | |||
| 20.0 | 10.5 | 8.57 | 81 | 11.4 | 108 | |||
| 10.0 | 5.26 | 5.12 | 97 | 5.57 | 106 | |||
| 5.00 | 2.63 | 2.25 | 85 | 2.14 | 81 | |||
| Solvent Control | < LOQ | < LOQ | ||||||
| Control | < LOQ | < LOQ | ||||||
Meas. Conc. = Measured concentration (dilution factor taken into account)
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (2.0 µg/L of the test item, corresponding to 1.06 µg a.i./L)
1) = no samples due to 100% mortality
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day 0 to Day 28 (C14 Amine + 2 EO)
| Sampling day / Replicate | Day 0 | Day 7 | Day 14 | |||||
| Repl. 2 | Repl. 3 | Repl. 4 | ||||||
| Nominal | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | |||||||
| concentration of the | C14 Amine + 2 EO | |||||||
| Test item | a.i. | Meas. | % | Meas. | % | Meas. | % | |
| [µg/L] | [µg a.i./L] | conc. | conc. | conc. | ||||
| [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | ||||||
| 80.0 | 14.9 | 14.6 | 98 | 14.4 | 97 | - 1) | ||
| 40.0 | 7.46 | 6.68 | 90 | 7.81 | 105 | 7.92 | 106 | |
| 20.0 | 3.73 | 3.80 | 102 | 4.79 | 129 | 3.49 | 94 | |
| 10.0 | 1.86 | 2.02 | 108 | 1.96 | 105 | 1.61 | 86 | |
| 5.00 | 0.932 | 1.05 | 113 | 0.795 | 85 | 0.887 | 95 | |
| Solvent Control | < LOQ | < LOQ | < LOQ | |||||
| Control | < LOQ | < LOQ | < LOQ | |||||
| Sampling day / Replicate | Day 21 | Day 28 | ||||||
| Repl. 1 | Repl. 2 | |||||||
| Nominal | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | |||||||
| concentration of the | C14 Amine + 2 EO | |||||||
| Test item | a.i. | Meas. | % | Meas. | % | |||
| [µg/L] | [µg a.i./L] | conc. | conc. | |||||
| [µg a.i./L] | [µg a.i./L] | |||||||
| 80.0 | 14.9 | - 1) | - 1) | |||||
| 40.0 | 7.46 | 7.05 | 95 | 5.46 | 73 | |||
| 20.0 | 3.73 | 3.00 | 81 | 3.72 | 100 | |||
| 10.0 | 1.86 | 1.73 | 93 | 2.08 | 111 | |||
| 5.00 | 0.932 | 0.761 | 82 | 0.679 | 73 | |||
| Solvent Control | < LOQ | < LOQ | ||||||
| Control | < LOQ | < LOQ | ||||||
Meas. Conc. = Measured concentration (dilution factor taken into account)
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (2.0 µg/L of the test item, corresponding to 0.376 µg a.i./L)
1) = no samples due to 100% mortality
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day 0 to Day 28 (C16 Amine + 2 EO)
| Sampling day / Replicate | Day 0 | Day 7 | Day 14 | |||||
| Repl. 2 | Repl. 3 | Repl. 4 | ||||||
| Nominal | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | |||||||
| concentration of the | C16 Amine + 2 EO | |||||||
| Test item | a.i. | Meas. | % | Meas. | % | Meas. | % | |
| [µg/L] | [µg a.i./L] | conc. | conc. | conc. | ||||
| [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | ||||||
| 80.0 | 6.77 | 9.36 | 138 | 6.18 | 91 | - 1) | ||
| 40.0 | 3.38 | 3.81 | 113 | 4.67 | 138 | 4.82 | 142 | |
| 20.0 | 1.69 | 2.30 | 136 | 2.99 | 177 | 1.72 | 102 | |
| 10.0 | 0.846 | 1.07 | 127 | 1.80 | 213 | 0.743 | 88 | |
| 5.00 | 0.423 | 0.453 | 107 | 0.428 | 101 | 0.420 | 99 | |
| Solvent Control | < LOQ | < LOQ | < LOQ | |||||
| Control | < LOQ | < LOQ | < LOQ | |||||
| Sampling day / Replicate | Day 21 | Day 28 | ||||||
| Repl. 1 | Repl. 2 | |||||||
| Nominal | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | |||||||
| concentration of the | C16 Amine + 2 EO | |||||||
| Test item | a.i. | Meas. | % | Meas. | % | |||
| [µg/L] | [µg a.i./L] | conc. | conc. | |||||
| [µg a.i./L] | [µg a.i./L] | |||||||
| 80.0 | 6.77 | - 1) | - 1) | |||||
| 40.0 | 3.38 | 3.31 | 98 | 4.86 | 144 | |||
| 20.0 | 1.69 | 1.42 | 84 | 2.39 | 141 | |||
| 10.0 | 0.846 | 0.797 | 94 | 2.15 | 254 | |||
| 5.00 | 0.423 | 0.340 | 80 | 0.284 | 67 | |||
| Solvent Control | < LOQ | < LOQ | ||||||
| Control | < LOQ | < LOQ | ||||||
Meas. Conc. = Measured concentration (dilution factor taken into account)
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (2.0 µg/L of the test item, corresponding to 0.171 µg a.i./L)
1) = no samples due to 100% mortality
Overview of Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels and Calculated Overall Mean Measured Test Item Concentrations (Study Day 0 to 29)
| Sampling day / Replicate | Day 0 | Day 7 | Day 14 | Day 21 | Day 28 | |
| Repl. 2 | Repl. 3 | Repl. 4 | Repl. 1 | Repl. 2 | ||
| Nominal | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | Overall mean measured concentration of the test item [µg/L] | ||||
| concentration of the test item | ||||||
| [µg/L] | Calculated mean measured concentration per study day [µg/L] | |||||
| 80.0 | 80.3 | 81.1 | 80.7 | |||
| 40.0 | 35.4 | 42.8 | 44.8 | 38.5 | 33.7 | 39.0 |
| 20.0 | 21.1 | 26.8 | 18.8 | 16.3 | 22.0 | 21.0 |
| 10.0 | 10.7 | 10.6 | 8.99 | 9.61 | 12.1 | 10.4 |
| 5.00 | 6.00 | 4.21 | 4.72 | 4.21 | 3.93 | 4.61 |
Fingerprint of the Test Item (non-GLP)
The peak distribution of the test concentration was analyzed in freshly prepared medium in the nominal test item concentration 80 µg/L. A solution of the analytical standard of the test item was prepared in acetonitrile and diluted to 40 µg/L with acetonitrile. A sample of the nominal test item concentration 80 µg/L and a 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 standard and the test item solution were compared. In test item and standard solutions 6 masses were observed and could be assigned to the test item. One additional signal could be observed in the test item solution which was observed to be related to the matrix used for this study. Compared to the standard in acetonitrile the test item in fresh media showed a relative increase in the amounts of the compounds with 274 Da, 302 Da and 358 Da, whereas the relative amounts of compounds < 274 Da are decreased. In general, however, the concentrations and solubility of the test item is too low for significant MS spectra.
Percentage of the Relevant Amine
| [%] | |
| C10 Amine + 2EO | 7.27 |
| C12 Amine + 2EO | 52.62 |
| C14 Amine + 2EO | 18.64 |
| C16 Amine + 2EO | 8.46 |
Gradient Table
| Time [min] | A [%] | B [%] |
| 0.00 | 75 | 25 |
| 0.30 | 75 | 25 |
| 1.30 | 10 | 90 |
| 2.00 | 10 | 90 |
| 2.10 | 75 | 25 |
| 2.50 | 75 | 25 |
Dilution steps
| Nominal | Dilution | Sample | Final |
| test item | Factor* | volume | volume |
| concentration | [mL] | [mL] | |
| [µg/L] | |||
| 80.0 | 10 | 0.2 | 1.0 |
| 40.0 | 10 | 0.2 | 1.0 |
| 20.0 | 2 | 1.0 | 1.0 |
| 10.0 | 2 | 1.0 | 1.0 |
| 5.00 | 2 | 1.0 | 1.0 |
| Solvent Control | 2 | 1.0 | 1.0 |
| Control | 2 | 1.0 | 1.0 |
*including factor 2
Dilution steps
| Nominal | Dilution | Sample | Final |
| test item | Factor* | volume | volume |
| concentration | [mL] | [mL] | |
| [mg/L] | |||
| 3200 | 320 000 | 0.051) | 101) |
| 0.125 | 1.0 | ||
| 1600 | 160 000 | 0.051) | 101) |
| 0.25 | 1.0 | ||
| 800 | 80 000 | 0.051) | 101) |
| 0.52) | 1.02) | ||
| 400 | 40 000 | 0.05 | 10 |
| 200 | 20 000 | 0.1 | 10 |
| Solvent Control | 203) | 0.05 | 1.0 |
*including factor 200
1) first dilution step
2) second dilution step
3) excluding dilution factor 200
Vital Eggs and Larvae from Study Days 0 to 34 (Control, Solvent Control and 5.00 µg/L)
| Study day | Nominal test item concentration [µg/L] | |||||||||||
| Control | Solvent control | 5.00 | ||||||||||
| Number of vital eggs and larvae (listed eggs/larvae) by study day | ||||||||||||
| 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
| 0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 |
| 1 | 19/0 | 20/0 | 20/0 | 18/0 | 19/0 | 20/0 | 20/0 | 20/0 | 20/0 | 19/0 | 19/0 | 19/0 |
| 2 | 19/0 | 20/0 | 20/0 | 18/0 | 19/0 | 20/0 | 20/0 | 20/0 | 20/0 | 19/0 | 19/0 | 19/0 |
| 3 | 14/5 | 13/7 | 13/7 | 4/14 | 9/10 | 9/11 | 6/14 | 6/14 | 4/16 | 1/18 | 5/14 | 11/8 |
| 4 | 2/17 | 0/19 | 0/20 | 1/17 | 0/19 | 1/19 | 1/19 | 0/20 | 0/20 | 0/18 | 0/19 | 0/19 |
| 5 | 0/19 | 0/19 | 0/20 | 0/17 | 0/19 | 0/20 | 0/19 | 0/20 | 0/20 | 0/18 | 0/19 | 0/19 |
| 6 | 0/19 | 0/19 | 0/20 | 0/17 | 0/19 | 0/20 | 0/19 | 0/20 | 0/20 | 0/18 | 0/19 | 0/19 |
| 7 | 0/19 | 0/19 | 0/20 | 0/17 | 0/19 | 0/19 | 0/18 | 0/20 | 0/20 | 0/18 | 0/19 | 0/19 |
| 8 | 0/19 | 0/19 | 0/20 | 0/17 | 0/19 | 0/19 | 0/18 | 0/20 | 0/20 | 0/18 | 0/19 | 0/19 |
| 9 | 0/19 | 0/19 | 0/20 | 0/17 | 0/19 | 0/19 | 0/18 | 0/20 | 0/20 | 0/18 | 0/19 | 0/19 |
| 10 | 0/19 | 0/19 | 0/20 | 0/17 | 0/19 | 0/19 | 0/18 | 0/20 | 0/20 | 0/18 | 0/19 | 0/19 |
| 11 | 0/18 | 0/19 | 0/20 | 0/17 | 0/17 | 0/19 | 0/17 | 0/20 | 0/20 | 0/18 | 0/19 | 0/19 |
| 12 | 0/17 | 0/19 | 0/20 | 0/16 | 0/16 | 0/19 | 0/17 | 0/20 | 0/20 | 0/18 | 0/19 | 0/19 |
| 13 | 0/16 | 0/19 | 0/20 | 0/16 | 0/16 | 0/19 | 0/17 | 0/20 | 0/20 | 0/18 | 0/18 | 0/19 |
| 14 | 0/16 | 0/19 | 0/20 | 0/16 | 0/16 | 0/19 | 0/17 | 0/20 | 0/19 | 0/18 | 0/18 | 0/19 |
| 15 | 0/16 | 0/19 | 0/20 | 0/16 | 0/16 | 0/18 | 0/16 | 0/20 | 0/19 | 0/18 | 0/18 | 0/19 |
| 16 | 0/16 | 0/19 | 0/20 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/18 | 0/18 | 0/19 |
| 17 | 0/16 | 0/19 | 0/20 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/18 | 0/18 | 0/19 |
| 18 | 0/16 | 0/18 | 0/20 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/18 | 0/18 | 0/19 |
| 19 | 0/16 | 0/18 | 0/20 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/18 | 0/18 | 0/19 |
| 20 | 0/16 | 0/18 | 0/20 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/18 | 0/18 | 0/19 |
| 21 | 0/16 | 0/18 | 0/20 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/18 | 0/18 | 0/19 |
| 22 | 0/16 | 0/18 | 0/20 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/18 | 0/18 | 0/19 |
| 23 | 0/16 | 0/18 | 0/20 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/18 | 0/18 | 0/19 |
| 24 | 0/16 | 0/18 | 0/20 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/17 | 0/18 | 0/19 |
| 25 | 0/16 | 0/18 | 0/20 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/17 | 0/18 | 0/19 |
| 26 | 0/16 | 0/18 | 0/20 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/17 | 0/18 | 0/19 |
| 27 | 0/16 | 0/18 | 0/20 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/17 | 0/18 | 0/19 |
| 28 | 0/16 | 0/18 | 0/19 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/17 | 0/17 | 0/19 |
| 29 | 0/16 | 0/18 | 0/19 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/17 | 0/17 | 0/19 |
| 30 | 0/16 | 0/18 | 0/19 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/17 | 0/17 | 0/19 |
| 31 | 0/16 | 0/18 | 0/19 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/17 | 0/17 | 0/19 |
| 32 | 0/16 | 0/18 | 0/19 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/17 | 0/17 | 0/19 |
| 33 | 0/16 | 0/18 | 0/19 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/17 | 0/17 | 0/19 |
| 34 | 0/16 | 0/18 | 0/19 | 0/16 | 0/16 | 0/18 | 0/16 | 0/19 | 0/19 | 0/17 | 0/17 | 0/19 |
Vital Eggs and Larvae from Study Days 0 to 34 (10.0 to 40.0 µg/L)
| Study day | Nominal test item concentration [µg/L] | |||||||||||
| 10.0 | 20.0 | 40.0 | ||||||||||
| Number of vital eggs and larvae (listed eggs/larvae) by study day | ||||||||||||
| 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
| 0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 |
| 1 | 20/0 | 19/0 | 20/0 | 19/0 | 19/0 | 18/0 | 20/0 | 19/0 | 19/0 | 20/0 | 19/0 | 20/0 |
| 2 | 20/0 | 19/0 | 20/0 | 19/0 | 19/0 | 18/0 | 20/0 | 19/0 | 19/0 | 20/0 | 19/0 | 19/1 |
| 3 | 3/17 | 6/13 | 2/18 | 11/8 | 12/7 | 4/14 | 5/15 | 5/14 | 10/9 | 12/8 | 4/15 | 4/16 |
| 4 | 0/20 | 0/19 | 0/20 | 0/18 | 0/19 | 0/17 | 0/20 | 0/19 | 1/18 | 2/18 | 0/19 | 0/20 |
| 5 | 0/20 | 0/19 | 0/20 | 0/18 | 0/19 | 0/17 | 0/20 | 0/19 | 1/18 | 2/18 | 0/19 | 0/20 |
| 6 | 0/20 | 0/19 | 0/20 | 0/18 | 0/19 | 0/17 | 0/20 | 0/19 | 0/19 | 0/20 | 0/19 | 0/20 |
| 7 | 0/20 | 0/19 | 0/20 | 0/18 | 0/19 | 0/17 | 0/20 | 0/19 | 0/19 | 0/20 | 0/19 | 0/20 |
| 8 | 0/20 | 0/19 | 0/20 | 0/18 | 0/19 | 0/17 | 0/20 | 0/19 | 0/19 | 0/20 | 0/19 | 0/20 |
| 9 | 0/20 | 0/19 | 0/20 | 0/18 | 0/19 | 0/17 | 0/20 | 0/19 | 0/19 | 0/20 | 0/19 | 0/20 |
| 10 | 0/20 | 0/19 | 0/20 | 0/18 | 0/19 | 0/17 | 0/20 | 0/19 | 0/19 | 0/20 | 0/19 | 0/18 |
| 11 | 0/20 | 0/19 | 0/20 | 0/18 | 0/19 | 0/17 | 0/20 | 0/19 | 0/19 | 0/20 | 0/19 | 0/18 |
| 12 | 0/19 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/20 | 0/19 | 0/17 | 0/18 | 0/19 | 0/15 |
| 13 | 0/19 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/14 | 0/16 | 0/17 | 0/10 |
| 14 | 0/19 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/13 | 0/12 | 0/16 | 0/7 |
| 15 | 0/19 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/8 | 0/11 | 0/16 | 0/6 |
| 16 | 0/19 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/8 | 0/13 | 0/5 |
| 17 | 0/19 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/12 | 0/3 |
| 18 | 0/19 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/12 | 0/3 |
| 19 | 0/19 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/11 | 0/3 |
| 20 | 0/19 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/10 | 0/3 |
| 21 | 0/19 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/10 | 0/3 |
| 22 | 0/18 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/10 | 0/3 |
| 23 | 0/18 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/10 | 0/3 |
| 24 | 0/18 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/10 | 0/3 |
| 25 | 0/18 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/10 | 0/3 |
| 26 | 0/18 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/10 | 0/3 |
| 27 | 0/18 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/10 | 0/3 |
| 28 | 0/18 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/9 | 0/3 |
| 29 | 0/18 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/9 | 0/3 |
| 30 | 0/18 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/9 | 0/3 |
| 31 | 0/18 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/9 | 0/3 |
| 32 | 0/18 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/9 | 0/3 |
| 33 | 0/18 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/9 | 0/3 |
| 34 | 0/18 | 0/19 | 0/20 | 0/17 | 0/19 | 0/17 | 0/19 | 0/19 | 0/5 | 0/6 | 0/9 | 0/3 |
Vital Eggs and Larvae from Study Days 0 to 8 (80.0 µg/L)
| Study day | Nominal test item concentration [µg/L] | |||
| 80.0 | ||||
| Number of vital eggs and larvae (listed eggs/larvae) by study day | ||||
| 1 | 2 | 3 | 4 | |
| 0 | 20/0 | 20/0 | 20/0 | 20/0 |
| 1 | 20/0 | 19/0 | 18/0 | 20/0 |
| 2 | 20/0 | 19/0 | 18/0 | 20/0 |
| 3 | 9/11 | 6/13 | 7/11 | 15/5 |
| 4 | 2/18 | 0/19 | 2/16 | 2/18 |
| 5 | 1/19 | 0/19 | 0/18 | 0/20 |
| 6 | 0/20 | 0/19 | 0/18 | 0/20 |
| 7 | 0/20 | 0/19 | 0/18 | 0/19 |
| 8 | 0/16 | 0/17 | 0/15 | 0/16 |
| 9 | 0/3 | 0/2 | 0/2 | 0/1 |
| 10 | 0/1 | 0/0 | 0/0 | 0/0 |
| 11 | 0/0 | - | - | - |
Parameter, Acceptance Criteria and Results of the Method Validation
| Parameter | Acceptance criteria | Result | |
| Linearity | ≥ 5 standard concentrations, | 0.2 to 20 µg test item/L (n = 8), | ü |
| r ≥ 0.99 (R2 ≥ 0.9801 for 2nd order regression) | r ≥ 0.99 | ||
| Calibration range from ≤ 30% LOQ to 20% above highest concentration level after sample preparation | |||
| Lowest calibration level (LCL) | S/N ≥ 9 for quantifier ion trace | S/N for 0.2 µg test item/L | ü |
| S/N ≥ 3 for qualifier ion trace | C10 Amine + 2 EO | ||
| 187 (Quantifier), 110 (Qualifier) | |||
| C12 Amine + 2 EO | |||
| 937 (Quantifier), 460 (Qualifier) | |||
| C14 Amine + 2 EO | |||
| 493 (Quantifier), 354 (Qualifier) | |||
| C16 Amine + 2 EO | |||
| 185 (Quantifier), 176 (Qualifier) | |||
| Matrix effects | Should be ≤ 20% | Matrix effects: < 20% | ü |
| Limit of Quantification (LOQ) | Should be at or below lowest test concentration | 2.0 µg test item/L (1 x LOQ) | ü |
| 100 µg test item/L (50 x LOQ) | |||
| Accuracy1) | Mean recovery rate of 70-120% | C10 Amine + 2 EO | ü |
| (Fortified samples) | per fortification level (2 levels) | 1 x LOQ: 88% (n = 5) | |
| 50 x LOQ: 92% (n = 5) | |||
| C12 Amine + 2 EO | |||
| 1 x LOQ: 92% (n = 5) | |||
| 50 x LOQ: 99% (n = 5) | |||
| C14 Amine + 2 EO | |||
| 1 x LOQ: 91% (n = 5) | |||
| 50 x LOQ: 95% (n = 5) | |||
| C16 Amine + 2 EO | |||
| 1 x LOQ: 89% (n = 5) | |||
| 50 x LOQ: 92% (n = 5) | |||
| Precision1) | Relative standard deviation ≤ 20% per fortification level | C10 Amine + 2 EO | ü |
| 1 x LOQ: 7.9% | |||
| 50 x LOQ: 3.8% | |||
| C12 Amine + 2 EO | |||
| 1 x LOQ: 7.8% | |||
| 50 x LOQ: 3.3% | |||
| C14 Amine + 2 EO | |||
| 1 x LOQ: 8.2% | |||
| 50 x LOQ: 3.2% | |||
| C16 Amine + 2 EO | |||
| 1 x LOQ: 14% | |||
| 50 x LOQ: 4.0% | |||
continuation: Parameter, Acceptance Criteria and Results of the Method Validation
| Specificity: | Measurement of two transitions of the same precursor ion - one quantifier (used for evaluation) and one qualifier (for confirmation of the analyte identity). | C10 Amine + 2 EO | ü |
| LC-MS/(MS) | (Compounds: | quantifier [m/z]: 246.20 > 88.13 | |
| C10 Component + 2EO; | qualifier [m/z]: 246.20 > 70.17 | ||
| C12 Component + 2EO; C14 Component + 2EO; | C12 Amine + 2 EO | ||
| C16 Component + 2EO) | quantifier [m/z]: 274.30 > 88.13 | ||
| qualifier [m/z]: 274.30 > 70.23 | |||
| C14 Amine + 2 EO | |||
| quantifier [m/z]: 302.36 > 88.12 | |||
| qualifier [m/z]: 302.36 > 70.29 | |||
| C16 Amine + 2 EO | |||
| quantifier [m/z]: 330.36 > 88.12 | |||
| qualifier [m/z]: 330.36 > 70.29 | |||
| Blank values < 30% of LOQ | Blank values < 30% of LOQ | ü | |
| Procedural recovery | Procedural recoveries with experimental samples. The recovery has to be 70-120% of the nominal value. | See section 18.1 | ü |
| Stability | Samples: Stable if 70 – 120% of freshly prepared sample | Samples: stable for at least 3 days | ü |
| Standards: Stable if ±10% of freshly prepared | Standards: stability < 3 days2) |
= criterion fulfilled
- = not determined
1) = For details, see tables below
2) = calibration standards were freshly prepared at each day of analysis
Preparation of the The method was validated at the 1 x LOQ (2.0 µg test item/L)
fortified samples and at the 50 x LOQ ( 100 µg test item/L).
Preparation of Fortified Samples
| LOQ Level | Control | 1 | 50 |
| Stock solution | - | 100 mg test item/L in Acetonitrile | |
| Spiking solution | - | 0.2 | 10 |
| [mg test item/L] | (Dilution medium) | (Dilution medium) | |
| (Medium) | |||
| Replicates | 2 | 5 | 5 |
| Concentration of the LOQ | - | 2.0 | 100 |
| [µg test item/L] | |||
| Medium for preparation | Fish dilution medium | ||
| Volume of spiking solution [mL] | - | 0.05 | 0.05 |
| Volume of medium [mL] | 5.0 | 4.95 | 4.95 |
| Dilution factor | - | 2 | 20 |
| Dilution medium | Acetonitrile containing 2% formic acid 1) | ||
| Dilution medium 2) | |||
| Sample volume [mL] | 5.01) | 5.01) | 5.01) |
| 0.12) | |||
| Finale volume [mL] | 101) | 101) | 101) |
| 1.02) | |||
| Test item concentration in the final solution [µg/L] | - | 1.0 | 5.0 |
Dilution medium Acetonitrile : ultra-pure water (50 : 50 v/v) containing 1 % formic acid
1) First dilution step
2) Second dilution step
Nominal Concentrations of the Fortified Samples of the active ingredients of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
Fortified concentrations*: 2.02 µg test item/L (1 x LOQ) and 101 µg test item/L (50 x LOQ).
| Active ingredient | 1 x LOQ | 50 x LOQ |
| [µg a.s./L] | [µg a.s./L] | |
| C10 Amine + 2 EO | 0.147 | 7.34 |
| C12 Amine + 2 EO | 1.06 | 53.1 |
| C14 Amine + 2 EO | 0.376 | 18.8 |
| C16 Amine + 2 EO | 0.171 | 8.54 |
* = weighing factor taken into account
a.s. = active substance
Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
Active ingredient: C10 Amine + 2 EO and C12 Amine + 2 EO
| Replicate | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | |||
| C10 Amine + 2 EO | ||||
| 1 x LOQ | 50 x LOQ | |||
| Meas.conc. | % | Meas.conc. | % | |
| [µg a.i./L] | [µg a.i./L] | |||
| 1 | 0.147 | 101 | 6.56 | 89 |
| 2 | 0.125 | 85 | 7.00 | 95 |
| 3 | 0.123 | 84 | 6.72 | 92 |
| 4 | 0.127 | 87 | 7.00 | 95 |
| 5 | 0.125 | 85 | 6.45 | 88 |
| Mean | 0.13 | 88 | 6.7 | 92 |
| SD ± | 0.01 | 0.3 | ||
| CV [%] | 7.9 | 3.8 | ||
| Replicate | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | |||
| C12 Amine + 2 EO | ||||
| 1 x LOQ | 50 x LOQ | |||
| Meas. conc. | % | Meas. conc. | % | |
| [mg a.i./L] | [mg a.i./L] | |||
| 1 | 1.11 | 104 | 50.1 | 94 |
| 2 | 0.956 | 90 | 54.5 | 103 |
| 3 | 0.941 | 89 | 52.1 | 98 |
| 4 | 0.922 | 87 | 51.8 | 98 |
| 5 | 0.948 | 89 | 53.6 | 101 |
| Mean | 0.98 | 92 | 52 | 99 |
| SD ± | 0.08 | 2 | ||
| CV [%] | 7.8 | 3.3 | ||
Meas. conc. = measured concentration of each component of the test item, dilution factor taken into account
% = percent concentration of the fortified sample
* = weighing factor taken into account
SD = standard deviation
CV = coefficient of variation
Continuation: Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
Active ingredient: C14 Amine + 2 EO and C16 Amine + 2 EO
| Replicate | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | |||
| C14 Amine + 2 EO | ||||
| 1 x LOQ | 50 x LOQ | |||
| Meas. conc. | % | Meas. conc. | % | |
| [µg a.i./L] | [µg a.i./L] | |||
| 1 | 0.389 | 103 | 17.6 | 94 |
| 2 | 0.346 | 92 | 18.8 | 100 |
| 3 | 0.318 | 85 | 17.5 | 93 |
| 4 | 0.327 | 87 | 17.5 | 93 |
| 5 | 0.329 | 87 | 17.7 | 94 |
| Mean | 0.34 | 91 | 17.8 | 95 |
| SD ± | 0.03 | 0.6 | ||
| CV [%] | 8.2 | 3.2 | ||
| Replicate | Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) | |||
| C16 Amine + 2 EO | ||||
| 1 x LOQ | 50 x LOQ | |||
| Meas. conc. | % | Meas. conc. | % | |
| [µg a.i./L] | [µg a.i./L] | |||
| 1 | 0.187 | 109 | 7.62 | 89 |
| 2 | 0.149 | 87 | 8.35 | 98 |
| 3 | 0.135 | 79 | 7.73 | 91 |
| 4 | 0.138 | 81 | 7.55 | 88 |
| 5 | 0.151 | 89 | 7.87 | 92 |
| Mean | 0.15 | 89 | 7.8 | 92 |
| SD ± | 0.02 | 0.3 | ||
| CV [%] | 14 | 4.0 | ||
Meas. conc. = measured concentration of each component of the test item, dilution factor taken into account
% = percent concentration of the fortified sample
* = weighing factor taken into account
SD = standard deviation
CV = coefficient of variation
The stability of the fortified samples at the 1 x LOQ was checked after 3 days and was for C10 Amine + 2 EO, C12 Amine + 2 EO, C14 Amine + 2 EO and C16 Amine + 2 EO between 105 and 109% of the nominal concentration for each component.
The stability of the standard stock solution (1000 mg test item/L in acetonitrile) was checked after 7 days and was 103 to 106% of the nominal concentration.
Procedural Recovery
A procedural recovery (Quality Control) on 1x LOQ Level was freshly prepared on each day of analysis. It was treated in parallel to the test samples.
Measured Concentrations and Percent of Nominal Concentration of the Quality Control during the Definitive Test
| Sampling | Day -1 | Day 0 | Day 7 | Day 14 | ||||
| date | ||||||||
| Component | Meas. | % | Meas. | % | Meas. | % | Meas. | % |
| conc. | conc. | conc. | conc. | |||||
| [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | |||||
| C10 Amine + 2 EO | 0.156 | 108 | 0.152 | 105 | 0.109 | 75 | 0.126 | 87 |
| C12 Amine + 2 EO | 1.17 | 111 | 1.10 | 104 | 1.04 | 99 | 0.911 | 87 |
| C14 Amine + 2 EO | 0.399 | 107 | 0.351 | 94 | 0.342 | 92 | 0.283 | 76 |
| C16 Amine + 2 EO | 0.182 | 107 | 0.134 | 79 | 0.118 | 70 | 0.102 | 60 |
| Sampling | Day 21 | Day 28 | ||||||
| date | ||||||||
| Component | Meas. | % | Meas. | % | ||||
| conc. | conc. | |||||||
| [µg a.i./L]] | [µg a.i./L] | |||||||
| C10 Amine + 2 EO | 0.119 | 82 | 0.134 | 92 | ||||
| C12 Amine + 2 EO | 0.911 | 87 | 0.909 | 86 | ||||
| C14 Amine + 2 EO | 0.306 | 82 | 0.297 | 80 | ||||
| C16 Amine + 2 EO | 0.114 | 68 | 0.114 | 68 | ||||
Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration
Quality Control = 2.00 µg test item/L, weighing factor taken into account
Preliminary Range Finding Test (non-GLP)
A non-GLP preliminary range finding test was conducted at the test facility as a shortened early-life stage test under flow-through conditions over a period of 15 days. An equilibration period over 12 days was carried out prior to the start of the exposure. Exposure was started by placing fertilized eggs into the test replicates. A solvent control and two test concentrations of the test item of 10.0 and 100 µg/L were tested. Two replicates per solvent control and test concentration were tested. 20 eggs per replicate were exposed to each concentration level and the solvent control.
The test item concentrations were analytically verified via LC-MS/MS on days 0, 5, 7 and 12. The following constituents of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) were quantified: C10 Component + 2EO; C12 Component + 2EO; C14 Component + 2 EO; C16 Component + 2EO.
Mortality [%] and Hatch [%] in the Range Finding Test
(n = 40 per treatment group with 2 replicates and 20 eggs each under flow-through conditions)
| Nominal test item concentration | Hatch [%] | Post hatch survival [%] on study day 15 | Overall survival [%] | Mortality [%] |
| [µg/L] | on study day 6 | on study day 15 | on study day 15 | |
| 100 | 93 | 0* | 0* | 100* |
| 10 | 98 | 100 | 98 | 2 |
| Solvent control | 93 | 95 | 88 | 12 |
* 100 % mortality of hatched larvae was already observed on study day 10
Measured Exposure Concentrations on Study Day 0 to Day 12 of the Range Finding Test (C10-Component + 2EO and C12-Component + 2EO)
Determination of the test item via LC-MS/MS, with a preliminary non-validated analytical method
| Sampling date | Day 0 | Day 5 | Day 7 | Day 12 | |||||
| Nominal concentration | Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chloride | ||||||||
| C10-Component + 2EO | |||||||||
| of the test item | of the active substance | Meas. conc. | % | Meas. conc. | % | Meas. conc. | % | Meas. conc. | % |
| [µg/L] | [µg a.s./L] | [µg a.s./L] | [µg a.s./L] | [µg a.s./L] | [µg a.s./L] | ||||
| 100 | 7.27 | 8.75 | 120 | 6.63 | 91 | 6.38 | 88 | not determined | |
| 10.0 | 0.727 | 0.677 | 93 | 0.725 | 100 | 0.667 | 92 | 0.0864 | 119 |
| Solvent Control | < LCL | < LCL | < LCL | < LCL | |||||
| Sampling date | Day 0 | Day 5 | Day 7 | Day 12 | |||||
| Nominal concentration | Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chloride | ||||||||
| C12-Component + 2EO | |||||||||
| of the test item | of the active substance | Meas. conc. | % | Meas. conc. | % | Meas. conc. | % | Meas. conc. | % |
| [µg/L] | [µg a.s./L] | [µg a.s./L] | [µg a.s./L] | [µg a.s./L] | [µg a.s./L] | ||||
| 100 | 52.6 | 55.3 | 105 | 48.2 | 92 | 47.4 | 90 | not determined | |
| 10.0 | 5.26 | 4.62 | 88 | 4.78 | 91 | 4.57 | 87 | 5.99 | 114 |
| Solvent Control | < LCL | < LCL | < LCL | < LCL | |||||
Meas. Conc. = Measured concentration of the active substance, dilution factor taken into account
% = Percent of the nominal test item concentration
LCL = Lowest Calibration Level, 0.5 µg test item/L, corresponding to
0.0364 µg C10-Component + 2EO/L and 0.263 µg C12-Component + 2 EO/L
a.s. = active substance
Measured Exposure Concentrations on Study Day 0 to Day 12 of the Range Finding Test (C14-Component + 2EO and C16-Component + 2EO)
Determination of the test item via LC-MS/MS, with a preliminary non-validated analytical method
| Sampling date | Day 0 | Day 5 | Day 7 | Day 12 | |||||
| Nominal concentration | Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chloride | ||||||||
| C14-Component + 2EO | |||||||||
| of the test item | of the active substance | Meas. conc. | % | Meas. conc. | % | Meas. conc. | % | Meas. conc. | % |
| [µg/L] | [µg a.s./L] | [µg a.s./L] | [µg a.s./L] | [µg a.s./L] | [µg a.s./L] | ||||
| 100 | 18.6 | 17.4 | 93 | 15.9 | 85 | 14.6 | 78 | not determined | |
| 10.0 | 1.86 | 1.70 | 91 | 1.58 | 85 | 1.29 | 69 | 1.91 | 102 |
| Solvent Control | < LCL | < LCL | < LCL | < LCL | |||||
| Sampling date | Day 0 | Day 5 | Day 7 | Day 12 | |||||
| Nominal concentration | Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chloride | ||||||||
| C16-Component + 2EO | |||||||||
| of the test item | of the active substance | Meas. conc. | % | Meas. conc. | % | Meas. conc. | % | Meas. conc. | % |
| [µg/L] | [µg a.s./L] | [µg a.s./L] | [µg a.s./L] | [µg a.s./L] | [µg a.s./L] | ||||
| 100 | 8.46 | 9.32 | 110 | 6.62 | 78 | 5.61 | 66 | not determined | |
| 10.0 | 0.846 | 0.876 | 104 | 0.890 | 105 | 0.456 | 54 | 0.787 | 93 |
| Solvent Control | < LCL | < LCL | < LCL | < LCL | |||||
Meas. Conc. = Measured concentration of the active substance, dilution factor taken into account
% = Percent of the nominal test item concentration
LCL = Lowest Calibration Level, 0.5 µg test item/L, corresponding to
0.0930 µg C14-Component + 2EO/L and 0.0423 µg C16-Component + 2 EO/L
a.s. = active substance
Fingerprint – MS spectrum
The peak distribution of the test concentration was analyzed in freshly prepared medium in the nominal test item concentration 80 µg/L. A solution of the analytical standard of the test item was prepared in acetonitrile and diluted to 40 µg/L with acetonitrile. A sample of the nominal test item concentration 80 µg/L and a 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 standard and the test item solution were compared. In test item and standard solutions 6 masses were observed and could be assigned to the test item. One additional signal could be observed in the test item solution which was observed to be related to the matrix used for this study. Compared to the standard in acetonitrile the test item in fresh media showed a relative increase in the amounts of the compounds with 274 Da, 302 Da and 358 Da, whereas the relative amounts of compounds < 274 Da are decreased. In general, however, the concentrations and solubility of the test item is too low for significant MS spectra.
Hatch / Hatching Time (Control, Solvent Control and Test Item Concentrations)
| Nominal | Rep. | PHD -3 | PHD -2 | PHD -1 | PHD 0 | PHD 1 | PHD 2 |
| test item conc. | Study day 2 | Study day 3 | Study day 4 | Study day 5 | Study day 6 | Study day 7 | |
| [µg/L] | Cumulative hatching rate [%] | ||||||
| Control | 1 | 0 | 5 | 75 | 90 | 90 | 90 |
| 2 | 0 | 20 | 60 | 100 | 100 | 100 | |
| 3 | 5 | 65 | 90 | 100 | 100 | 100 | |
| 4 | 0 | 35 | 90 | 90 | 90 | 90 | |
| Mean | 1 | 31 | 79 | 95 | 95 | 95 | |
| Solvent control | 1 | 0 | 10 | 65 | 100 | 100 | 100 |
| 2 | 0 | 55 | 100 | 100 | 100 | 100 | |
| 3 | 0 | 45 | 85 | 90 | 90 | 90 | |
| 4 | 0 | 10 | 55 | 95 | 95 | 95 | |
| Mean | 0 | 30 | 76 | 96 | 96 | 96 | |
| Feb 50 | 1 | 0 | 35 | 90 | 90 | 100 | 100 |
| 2 | 0 | 20 | 95 | 100 | 100 | 100 | |
| 3 | 0 | 55 | 100 | 100 | 100 | 100 | |
| 4 | 0 | 35 | 80 | 90 | 90 | 90 | |
| Mean | 0 | 36 | 91 | 95 (ns) | 98 (ns) | 98 (ns) | |
| 5.00 | 1 | 0 | 15 | 60 | 95 | 100 | 100 |
| 2 | 0 | 5 | 55 | 100 | 100 | 100 | |
| 3 | 0 | 5 | 70 | 100 | 100 | 100 | |
| 4 | 0 | 10 | 75 | 95 | 95 | 95 | |
| Mean | 0 | 9 | 65 | 98 (ns) | 99 (ns) | 99 (ns) | |
| 10.0 | 1 | 0 | 20 | 90 | 100 | 100 | 100 |
| 2 | 0 | 5 | 55 | 100 | 100 | 100 | |
| 3 | 0 | 50 | 95 | 100 | 100 | 100 | |
| 4 | 0 | 5 | 55 | 95 | 100 | 100 | |
| Mean | 0 | 20 | 74 | 99 (ns) | 100 (ns) | 100 (ns) | |
| 20.0 | 1 | 0 | 5 | 55 | 85 | 100 | 100 |
| 2 | 0 | 10 | 30 | 85 | 95 | 95 | |
| 3 | 0 | 10 | 75 | 95 | 95 | 95 | |
| 4 | 0 | 40 | 90 | 95 | 95 | 95 | |
| Mean | 0 | 16 | 63 | 90 (ns) | 96 (ns) | 96 (ns) | |
| 40.0 | 1 | 0 | 5 | 45 | 85 | 95 | 100 |
| 2 | 0 | 0 | 25 | 85 | 90 | 90 | |
| 3 | 0 | 10 | 35 | 95 | 95 | 95 | |
| 4 | 0 | 50 | 95 | 100 | 100 | 100 | |
| Mean | 0 | 16 | 50 | 91 (ns) | 95 (ns) | 96 (ns) | |
(ns) = No statistically significant difference from pooled control groups
Percent Swim-up of Hatched Live Fry of the Control, Solvent Control and the Nominal Test Item Concentrations 2.50 to 10.0 µg/L
| Nominal | Rep. | PHD -1 | PHD 0 | PHD 1 | PHD 2 | PHD 3 |
| test item conc. | Study day 4 | Study day 5 | Study day 6 | Study day 7 | Study day 8 | |
| [µg/L] | Cumulative swim up [%] | |||||
| Control | 1 | 0 | 94 | 100 | 100 | 100 |
| 2 | 0 | 90 | 100 | 100 | 100 | |
| 3 | 0 | 100 | 100 | 100 | 100 | |
| 4 | 0 | 83 | 100 | 100 | 100 | |
| Mean | 0 | 92 | 100 | 100 | 100 | |
| Solvent control | 1 | 0 | 95 | 100 | 100 | 100 |
| 2 | 0 | 80 | 100 | 100 | 100 | |
| 3 | 0 | 78 | 94 | 100 | 100 | |
| 4 | 0 | 89 | 100 | 100 | 100 | |
| Mean | 0 | 86 | 99 | 100 | 100 | |
| Feb 50 | 1 | 0 | 100 | 100 | 100 | 100 |
| 2 | 0 | 95 | 100 | 100 | 100 | |
| 3 | 0 | 100 | 100 | 100 | 100 | |
| 4 | 0 | 94 | 100 | 100 | 100 | |
| Mean | 0 | 97 | 100 | 100 | 100 | |
| 5.00 | 1 | 0 | 89 | 100 | 100 | 100 |
| 2 | 0 | 90 | 100 | 100 | 100 | |
| 3 | 0 | 85 | 100 | 100 | 100 | |
| 4 | 0 | 100 | 100 | 100 | 100 | |
| Mean | 0 | 91 | 100 | 100 | 100 | |
| 10.0 | 1 | 0 | 100 | 100 | 100 | 100 |
| 2 | 0 | 90 | 100 | 100 | 100 | |
| 3 | 0 | 100 | 100 | 100 | 100 | |
| 4 | 0 | 95 | 95 | 95 | 100 | |
| Mean | 0 | 96 | 99 | 99 | 100 | |
| 20.0 | 1 | 0 | 76 | 95 | 100 | 100 |
| 2 | 0 | 71 | 95 | 100 | 100 | |
| 3 | 0 | 79 | 100 | 100 | 100 | |
| 4 | 0 | 100 | 100 | 100 | 100 | |
| Mean | 0 | 82 | 98 | 100 | 100 | |
| 40.0 | 1 | 0 | 82 | 100 | 100 | 50 |
| 2 | 0 | 82 | 94 | 100 | 80 | |
| 3 | 0 | 74 | 100 | 93 | 57 | |
| 4 | 0 | 85 | 100 | 100 | 92 | |
| Mean | 0 | 81 | 99 | 98 | 70 | |
Post-Hatch Survival on Study Day 35 (PHD 30) of the Control, Solvent Control and the Nominal Test Item Concentrations 2.50 and 5.00 µg/L
| Nominal | Rep. | Eggs introduced on study day 0 | Cumulative number of hatched larvae | Vital Larvae on study day 35 | Post-Hatch survival |
| test item conc. | (PHD 30) | on study day 35 | |||
| [µg/L] | [%] | ||||
| Control | 1 | 20 | 18 | 16 | 89 |
| 2 | 20 | 20 | 19 | 95 | |
| 3 | 20 | 20 | 16 | 80 | |
| 4 | 20 | 18 | 16 | 89 | |
| Mean | 20 | 19 | 17 | 88 | |
| Solvent control | 1 | 20 | 20 | 18 | 90 |
| 2 | 20 | 20 | 17 | 85 | |
| 3 | 20 | 18 | 16 | 89 | |
| 4 | 20 | 19 | 18 | 95 | |
| Mean | 20 | 19 | 17 | 90 | |
| Feb 50 | 1 | 20 | 20 | 19 | 95 |
| 2 | 20 | 20 | 19 | 95 | |
| 3 | 20 | 20 | 20 | 100 | |
| 4 | 20 | 18 | 13 | 72 | |
| Mean | 20 | 20 | 18 | 91 (ns) | |
| 5.00 | 1 | 20 | 20 | 16 | 80 |
| 2 | 20 | 20 | 19 | 95 | |
| 3 | 20 | 20 | 16 | 80 | |
| 4 | 20 | 19 | 16 | 84 | |
| Mean | 20 | 20 | 17 | 85 (ns) |
(ns) = No statistically significant difference from pooled control groups
Post-Hatch Survival on Study Day 35 (PHD 30) of the Nominal Test Item Concentrations 10.0 to 40.0 µg/L
| Nominal | Rep. | Eggs introduced on study day 0 | Cumulative number of hatched larvae | Vital Larvae on study day 34 | Post-Hatch survival |
| test item conc. | (PHD 30) | on study day 34 | |||
| [µg/L] | [%] | ||||
| 10.0 | 1 | 20 | 20 | 18 | 90 |
| 2 | 20 | 20 | 18 | 90 | |
| 3 | 20 | 20 | 18 | 90 | |
| 4 | 20 | 20 | 15 | 75 | |
| Mean | 20 | 20.0 | 17.0 | 86 (ns) | |
| 20.0 | 1 | 20 | 20 | 4 | 20 |
| 2 | 20 | 19 | 3 | 16 | |
| 3 | 20 | 19 | 6 | 32 | |
| 4 | 20 | 19 | 5 | 26 | |
| Mean | 20 | 19.3 | 5 | 24 (s) | |
| 40.0 | 1 | 20 | 20 | 0 | 0 |
| 2 | 20 | 18 | 0 | 0 | |
| 3 | 20 | 19 | 0 | 0 | |
| 4 | 20 | 20 | 0 | 0 | |
| Mean | 20 | 19.3 | 0 | 0 (s) |
(ns) / (s) = No statistically / statistically significant difference from pooled control groups
Overall Survival and Overall Mortality on Study Day 35 (PHD 30) of the Control, Solvent
Control and the Nominal Test Item Concentrations 2.50 and 5.00 µg/L
| Nominal | Rep. | Vital larvae on | Overall survival | Overall mortality |
| test item conc. | study day 35 | |||
| [µg/L] | (PHD 30) | |||
| [%] | [%] | |||
| Control | 1 | 16 | 80 | 20 |
| 2 | 19 | 95 | 5 | |
| 3 | 16 | 80 | 20 | |
| 4 | 16 | 80 | 20 | |
| Mean | 17 | 84 | 16 | |
| Solvent control | 1 | 18 | 90 | 10 |
| 2 | 17 | 85 | 15 | |
| 3 | 16 | 80 | 20 | |
| 4 | 18 | 90 | 10 | |
| Mean | 17 | 86 | 14 | |
| 2.50 | 1 | 19 | 95 | 5 |
| 2 | 19 | 95 | 5 | |
| 3 | 20 | 100 | 0 | |
| 4 | 13 | 65 | 35 | |
| Mean | 18 | 89 | 11 (ns) | |
| 5.00 | 1 | 16 | 80 | 20 |
| 2 | 19 | 95 | 5 | |
| 3 | 16 | 80 | 20 | |
| 4 | 16 | 80 | 20 | |
| Mean | 17 | 84 | 16 (ns) |
(ns) = No statistically significant difference from pooled control groups
Overall Survival and Overall Mortality on Study Day 35 (PHD 30) of the Nominal Test
Item Concentrations 10.0 to 40.0 µg/L
| Nominal | Rep. | Vital larvae on | Overall survival | Overall mortality |
| test item conc. | study day 35 | |||
| [µg/L] | (PHD 30) | |||
| [%] | [%] | |||
| 10.0 | 1 | 18 | 90 | 10 |
| 2 | 18 | 90 | 10 | |
| 3 | 18 | 90 | 10 | |
| 4 | 15 | 75 | 25 | |
| Mean | 17 | 86 | 14 (ns) | |
| 20.0 | 1 | 4 | 20 | 80 |
| 2 | 3 | 15 | 85 | |
| 3 | 6 | 30 | 70 | |
| 4 | 5 | 25 | 75 | |
| Mean | 5 | 23 | 77 (s) | |
| 40.0 | 1 | 0 | 0 | 100 |
| 2 | 0 | 0 | 100 | |
| 3 | 0 | 0 | 100 | |
| 4 | 0 | 0 | 100 | |
| Mean | 0 | 0 | 100 (s) |
(ns) / (s) = No statistically / statistically significant difference from pooled control groups
Behavioral Effects observed in the Nominal Test Concentrations 2.50 and 5.00 µg/L (Study Day 11 – 32)
| Study Day | 2.50 µg/L | 5.00 µg/L | ||||||
| 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
| 11 | - | - | - | - | 1 T | - | 1 Q | - |
| 1 G | ||||||||
| 12 | - | 1 G | - | - | - | - | - | - |
| 1 Q | ||||||||
| 13 - 19 | - | - | - | - | - | - | - | - |
| 20 | - | - | - | - | - | - | 1 T | - |
| 21 | - | - | - | - | - | - | 1 SP | - |
| 22 | - | - | - | - | - | - | 1 SP | - |
| 23 | - | - | - | - | - | - | 1 SP | - |
| 24 | - | - | - | - | - | - | 1 SP | - |
| 25 - 29 | - | - | - | - | - | - | - | - |
| 30 | - | - | - | 1 A | - | - | - | - |
| 31 | - | - | - | 1 A | - | - | - | - |
| 32 | - | - | - | 1 A | - | - | - | - |
G = Arresting on the ground Q = Quiescence
T = Tumbling A = Swollen Abdomen
SP = Side position
Behavioral Effects observed in the Nominal Test Concentrations 20.0 µg/L (Study Day 11 – 19)
| Study Day | 20.0 µg/L | |||
| 1 | 2 | 3 | 4 | |
| 11 | - | 1 T | - | - |
| 12 | 1 G | - | - | 1 G |
| 13 | 1 T | 1 T | - | 1 S |
| 2 Q | 1 G | |||
| 2 G | 2 Q | |||
| 14 | 4 S | 4 G | - | - |
| 2 G | 4 Q | |||
| 6 Q | ||||
| 15 | 4 G | 5 G | - | 2 T |
| 4 Q | 5 Q | |||
| 16 | 1 G | 1 T | 1 G | - |
| 3 G | ||||
| 3 Q | ||||
| 17 | - | 2 G | 3 G | 2 G |
| 18 | - | 1 G | - | - |
| 19 | - | - | 2 G | 1 G |
Behavioral Effects observed in the Nominal Test Concentration 40.0 µg/L
(Study Day 7 – 12)
| Study Day | 40.0 µg/L | |||
| 1 | 2 | 3 | 4 | |
| 7 | - | - | 1 Q | - |
| 1 G | ||||
| 1 T | ||||
| 8 | 2 T | 1 T | 1 T | 1 G |
| 4 SE | 2 G | 2 G | 1 SE | |
| 4 S | 2 Q | 2 SE | 12 Q | |
| 6 G | 4 Q | |||
| 6 ME | ||||
| 6 F | ||||
| 9 | 1 T | 1 T | 100% | 2 T |
| 3 G | 2 G | Mortality | 1 S | |
| 3 ME | 2 ME | 1 SE | ||
| 3 F | 2 F | 2 G | ||
| 1 S | 1 S | 2 ME | ||
| 1 SE | 1 SE | 2 F | ||
| 10 | 1 S | 2 F | 1 S | |
| 1 SE | 2 S | 1 SE | ||
| 2 SE | ||||
| 11 | 100% | 1 ME | 100% | |
| Mortality | 1 F | Mortality | ||
| 12 | 100 % Mortality | |||
G = Arresting on the ground Q = Quiescence
T = Tumbling S = Arresting under the surface
SE = Slowed escape reflex ME = Missing escape reflex
F = No food uptake
Overview of Fry Growth: Length and Wet Weight on Study Day 35 (PHD 30) of the Control, Solvent Control and Nominal Test Item Concentrations 2.50 and 5.00 µg/L
| Nominal | Rep. | PHD 30 (End of exposure) | |
| test item conc.[µg/L] | Mean total length per fish [mm] | Mean wet weight per fish [mg] | |
| Control | 1 | 16.0 | 39.1 |
| 2 | 14.6 | 30.7 | |
| 3 | 16.0 | 41.5 | |
| 4 | 16.1 | 47.0 | |
| Mean | 15.7 | 39.6 | |
| ± SD | 0.622 | 5.87 | |
| CV [%] | 3.96 | 14.8 | |
| Solvent control | 1 | 15.9 | 39.0 |
| 2 | 16.0 | 34.7 | |
| 3 | 16.0 | 38.7 | |
| 4 | 15.8 | 36.4 | |
| Mean | 15.9 | 37.2 | |
| ± SD | 0.083 | 1.76 | |
| CV [%] | 0.52 | 4.73 | |
| Feb 50 | 1 | 15.1 | 36.3 |
| 2 | 15.7 | 34.7 | |
| 3 | 15.7 | 34.9 | |
| 4 | 17.0 | 48.2 | |
| Mean | 15.9 (ns) | 38.5 (ns) | |
| ± SD | 0.694 | 5.62 | |
| CV [%] | 4.36 | 14.6 | |
| 5.00 | 1 | 14.9 | 31.3 |
| 2 | 14.7 | 32.5 | |
| 3 | 15.2 | 33.7 | |
| 4 | 15.1 | 32.4 | |
| Mean | 15.0 (s) | 32.5 (s) | |
| ± SD | 0.192 | 0.850 | |
| CV [%] | 1.28 | 2.62 | |
(ns) / (s) = No statistically / statistically significant difference from pooled control groups
Overview of Fry Growth: Length and Wet Weight on Study Day 35 (PHD 30) of the Nominal Test Item Concentrations 10.0 and 20.0 µg/L
| Nominal | Rep. | PHD 30 (End of exposure) | |
| test item conc.[µg/L] | Mean total length per fish [mm] | Mean wet weight per fish [mg] | |
| 10.0 | 1 | 12.9 | 24.1 |
| 2 | 14.6 | 29.6 | |
| 3 | 13.6 | 27.5 | |
| 4 | 14.8 | 34.6 | |
| Mean | 14.0 (s) | 29.0 (s) | |
| ± SD | 0.769 | 3.81 | |
| CV [%] | 5.49 | 13.1 | |
| 20.0 | 1 | 14.5 | 31.6 |
| 2 | 15.3 | 37.6 | |
| 3 | 14.5 | 31.2 | |
| 4 | 14.7 | 33.2 | |
| Mean | 14.8 (s) | 33.4 (s) | |
| ± SD | 0.328 | 2.54 | |
| CV [%] | 2.22 | 7.60 | |
(s) = Statistically significant difference from pooled controls
Individual Length on Study Day 35 (PHD 30) of the Control and the Solvent Control
| Fish No. | Control | Solvent control | ||||||
| Total length of individual fish in [mm] | ||||||||
| 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
| 1 | 17.0 | 11.0 | 15.5 | 13.0 | 13.0 | 15.0 | 15.5 | 20.0 |
| 2 | 19.0 | 12.5 | 13.0 | 20.0 | 18.5 | 16.0 | 17.0 | 15.0 |
| 3 | 16.0 | 13.0 | 17.0 | 17.0 | 14.0 | 14.5 | 14.0 | 14.0 |
| 4 | 15.0 | 16.0 | 17.0 | 14.0 | 19.5 | 18.0 | 11.0 | 11.0 |
| 5 | 14.0 | 11.0 | 19.0 | 18.0 | 17.5 | 18.5 | 13.0 | 16.0 |
| 6 | 11.0 | 15.0 | 15.5 | 18.0 | 20.0 | 14.5 | 21.5 | 14.0 |
| 7 | 14.5 | 13.5 | 19.0 | 18.0 | 18.0 | 14.0 | 22.0 | 15.0 |
| 8 | 15.0 | 21.0 | 19.0 | 18.5 | 16.0 | 16.5 | 14.0 | 17.0 |
| 9 | 18.0 | 19.0 | 18.0 | 20.0 | 20.0 | 16.0 | 16.0 | 20.0 |
| 10 | 18.5 | 18.0 | 14.0 | 18.0 | 18.0 | 16.0 | 20.0 | 17.0 |
| 11 | 14.0 | 12.5 | 17.0 | 17.0 | 16.0 | 19.5 | 18.0 | 20.5 |
| 12 | 20.0 | 12.0 | 16.0 | 15.0 | 15.0 | 15.5 | 16.0 | 17.0 |
| 13 | 15.0 | 17.0 | 18.0 | 12.0 | 11.0 | 18.0 | 19.0 | 17.0 |
| 14 | 15.0 | 14.0 | 16.0 | 14.5 | 17.0 | 12.0 | 16.0 | 16.5 |
| 15 | 18.5 | 18.0 | 12.0 | 15.0 | 18.0 | 16.0 | 10.0 | 16.0 |
| 16 | 15.0 | 15.0 | 10.0 | 10.0 | 8.5 | 16.0 | 13.0 | 14.0 |
| 17 | - | 13.0 | - | - | 16.0 | 16.0 | - | 11.5 |
| 18 | - | 13.0 | - | - | 10.0 | - | - | 12.5 |
| 19 | - | 12.0 | - | - | - | - | - | - |
| 20 | - | - | - | - | - | - | - | - |
| Mean | 16.0 | 14.6 | 16.0 | 16.1 | 15.9 | 16.0 | 16.0 | 15.8 |
| ± SD | 2.27 | 2.80 | 2.54 | 2.80 | 3.31 | 1.76 | 3.39 | 2.66 |
| CV % | 14.2 | 19.2 | 15.9 | 17.4 | 20.8 | 11.0 | 21.2 | 16.8 |
- = Fish died before end of the study
Individual Length on Study Day 35 (PHD 30) of the Nominal Test Item Concentrations 2.50 and 5.00 µg/L
| Fish No. | Nominal test item conc. [µg/L] | |||||||
| Feb 50 | 5.00 | |||||||
| Total length of individual fish in [mm] | ||||||||
| 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
| 1 | 17.0 | 14.0 | 17.5 | 17.0 | 17.0 | 20.0 | 15.0 | 12.5 |
| 2 | 19.5 | 13.5 | 16.0 | 13.5 | 18.5 | 13.0 | 14.5 | 15.0 |
| 3 | 20.0 | 15.0 | 13.0 | 18.5 | 16.0 | 18.0 | 20.0 | 18.0 |
| 4 | 14.0 | 16.0 | 19.5 | 16.0 | 18.0 | 14.5 | 12.0 | 21.0 |
| 5 | 15.0 | 13.0 | 19.0 | 17.0 | 11.0 | 11.5 | 20.5 | 17.0 |
| 6 | 13.0 | 17.0 | 13.5 | 17.0 | 10.0 | 14.0 | 13.0 | 16.0 |
| 7 | 10.0 | 16.0 | 13.0 | 17.0 | 9.0 | 15.0 | 19.0 | 11.0 |
| 8 | 14.5 | 11.0 | 16.0 | 20.0 | 20.0 | 18.5 | 16.0 | 14.0 |
| 9 | 16.0 | 17.0 | 15.0 | 19.0 | 16.0 | 18.0 | 13.0 | 18.0 |
| 10 | 12.0 | 17.5 | 15.0 | 17.0 | 17.5 | 16.0 | 18.5 | 17.0 |
| 11 | 15.5 | 17.5 | 15.0 | 21.0 | 16.0 | 13.5 | 16.0 | 13.0 |
| 12 | 11.0 | 13.5 | 14.5 | 12.5 | 16.0 | 11.5 | 13.0 | 16.0 |
| 13 | 14.0 | 17.5 | 17.0 | 15.5 | 14.0 | 13.0 | 13.0 | 11.0 |
| 14 | 13.0 | 19.0 | 18.0 | - | 14.0 | 15.0 | 14.0 | 18.0 |
| 15 | 13.0 | 18.0 | 17.0 | - | 12.0 | 14.0 | 13.5 | 10.0 |
| 16 | 17.5 | 15.0 | 15.5 | - | 13.0 | 18.5 | 12.5 | 13.5 |
| 17 | 20.5 | 12.5 | 11.0 | - | - | 10.5 | - | - |
| 18 | 16.5 | 15.0 | 18.0 | - | - | 14.5 | - | - |
| 19 | 15.5 | 20.0 | 17.0 | - | - | 10.5 | - | - |
| 20 | - | - | 14.0 | - | - | - | - | - |
| Mean | 15.1 | 15.7 | 15.7 | 17.0 | 14.9 | 14.7 | 15.2 | 15.1 |
| ± SD | 2.84 | 2.31 | 2.14 | 2.27 | 3.09 | 2.76 | 2.73 | 2.99 |
| CV % | 18.8 | 14.7 | 13.6 | 13.4 | 20.7 | 18.8 | 18.0 | 19.8 |
- = Fish died before end of the study
Individual Length on Study Day 35 (PHD 30) of the Nominal Test Item Concentrations 10.0 and 20.0 µg/L
| Fish No. | Nominal test item conc. [µg/L] | |||||||
| 10.0 | 20.0 | |||||||
| Total length of individual fish in [mm] | ||||||||
| 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
| 1 | 9.0 | 19.0 | 17.5 | 11.5 | 16.0 | 17.5 | 11.0 | 13.0 |
| 2 | 9.0 | 15.0 | 18.0 | 14.0 | 18.0 | 10.0 | 14.0 | 11.5 |
| 3 | 17.5 | 12.0 | 14.0 | 11.5 | 12.5 | 18.5 | 15.5 | 17.5 |
| 4 | 7.5 | 17.0 | 14.5 | 15.5 | 11.5 | - | 17.0 | 19.5 |
| 5 | 17.0 | 20.0 | 11.0 | 14.0 | - | - | 12.0 | 12.0 |
| 6 | 18.0 | 17.0 | 13.5 | 16.0 | - | - | 17.5 | - |
| 7 | 19.0 | 16.5 | 14.5 | 15.0 | - | - | - | - |
| 8 | 11.0 | 11.0 | 12.5 | 17.0 | - | - | - | - |
| 9 | 10.0 | 15.5 | 10.0 | 23.0 | - | - | - | - |
| 10 | 16.0 | 12.0 | 11.0 | 14.5 | - | - | - | - |
| 11 | 17.0 | 13.0 | 9.5 | 16.0 | - | - | - | - |
| 12 | 13.0 | 18.5 | 20.5 | 12.5 | - | - | - | - |
| 13 | 10.0 | 13.0 | 17.0 | 16.0 | - | - | - | - |
| 14 | 9.0 | 15.0 | 10.0 | 16.0 | - | - | - | - |
| 15 | 13.5 | 10.5 | 10.0 | 10.0 | - | - | - | - |
| 16 | 14.0 | 14.0 | 12.0 | - | - | - | - | - |
| 17 | 12.0 | 12.5 | 16.0 | - | - | - | - | - |
| 18 | 10.0 | 10.5 | 13.0 | - | - | - | - | - |
| 19 | - | - | - | - | - | - | - | - |
| 20 | - | - | - | - | - | - | - | - |
| Mean | 12.9 | 14.6 | 13.6 | 14.8 | 14.5 | 15.3 | 14.5 | 14.7 |
| ± SD | 3.60 | 2.89 | 3.12 | 2.94 | 2.62 | 3.79 | 2.42 | 3.20 |
| CV % | 27.9 | 19.8 | 22.9 | 19.9 | 18.1 | 24.8 | 16.7 | 21.8 |
- = Fish died before end of the study
Pooled Wet Weights on Study Day 35 (PHD 30) of the Control, Solvent Control and the Nominal Test Item Concentrations 2.50 to 20.0 µg/L
| Nominal | Replicate | Number of fish alive | Pooled | Mean | Mean | ± SD | CV % |
| test item conc. | on study day 35 | wet weight | wet weight | ||||
| [µg/L] | per replicate | per fish per replicate | |||||
| [mg] | [mg] | [mg] | |||||
| Control | 1 | 16 | 625.8 | 39.1 | 39.6 | 5.87 | 14.8 |
| 2 | 19 | 584.0 | 30.7 | ||||
| 3 | 16 | 663.3 | 41.5 | ||||
| 4 | 16 | 751.5 | 47.0 | ||||
| Solvent control | 1 | 18 | 701.2 | 39.0 | 37.2 | 1.76 | 4.73 |
| 2 | 17 | 590.4 | 34.7 | ||||
| 3 | 16 | 619.5 | 38.7 | ||||
| 4 | 18 | 654.6 | 36.4 | ||||
| Feb 50 | 1 | 19 | 689.8 | 36.3 | 38.5 | 5.62 | 14.6 |
| 2 | 19 | 659.1 | 34.7 | ||||
| 3 | 20 | 697.4 | 34.9 | ||||
| 4 | 13 | 626.4 | 48.2 | ||||
| 5.00 | 1 | 16 | 500.1 | 31.3 | 32.5 | 0.850 | 2.62 |
| 2 | 19 | 617.5 | 32.5 | ||||
| 3 | 16 | 538.6 | 33.7 | ||||
| 4 | 16 | 518.4 | 32.4 | ||||
| 10.0 | 1 | 18 | 433.7 | 24.1 | 29.0 | 3.81 | 13.1 |
| 2 | 18 | 531.9 | 29.6 | ||||
| 3 | 18 | 495.8 | 27.5 | ||||
| 4 | 15 | 518.9 | 34.6 | ||||
| 20.0 | 1 | 4 | 126.2 | 31.6 | 33.4 | 2.54 | 7.60 |
| 2 | 3 | 112.7 | 37.6 | ||||
| 3 | 6 | 186.9 | 31.2 | ||||
| 4 | 5 | 166.1 | 33.2 |
Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 0 -16)
| Study day | Replicate | Dissolved oxygen [%] | ||||||
| Nominal test item concentrations [µg/L] | ||||||||
| Control | Solvent control | 2.50 | 5.00 | 10.0 | 20.0 | 40.0 | ||
| 0 | 1 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| 2 | 99 | 100 | 100 | 100 | 100 | 100 | 100 | |
| 3 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
| 4 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
| 2 | 1 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| 2 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
| 3 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
| 4 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
| 5 | 1 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| 2 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
| 3 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
| 4 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
| 7 | 1 | 100 | 100 | 98 | 98 | 99 | 100 | 99 |
| 2 | 99 | 99 | 99 | 99 | 99 | 98 | 98 | |
| 3 | 100 | 98 | 98 | 100 | 99 | 98 | 99 | |
| 4 | 99 | 100 | 100 | 100 | 100 | 99 | 99 | |
| 9 | 1 | 97 | 97 | 96 | 95 | 97 | 97 | 96 |
| 2 | 97 | 97 | 97 | 97 | 96 | 96 | 94 | |
| 3 | 97 | 97 | 96 | 97 | 96 | 96 | 97 | |
| 4 | 97 | 97 | 96 | 96 | 96 | 96 | 96 | |
| 12 | 1 | 97 | 96 | 94 | 94 | 95 | 95 | 95 |
| 2 | 95 | 96 | 95 | 96 | 95 | 95 | 97 | |
| 3 | 96 | 96 | 95 | 96 | 95 | 95 | 95 | |
| 4 | 96 | 96 | 96 | 96 | 95 | 96 | 96 | |
| 14 | 1 | 97 | 96 | 92 | 95 | 95 | 94 | - |
| 2 | 95 | 95 | 93 | 94 | 95 | 94 | - | |
| 3 | 96 | 95 | 92 | 95 | 95 | 94 | - | |
| 4 | 96 | 97 | 96 | 94 | 95 | 95 | - | |
| 16 | 1 | 98 | 97 | 94 | 96 | 97 | 96 | - |
| 2 | 97 | 97 | 96 | 97 | 96 | 96 | - | |
| 3 | 97 | 97 | 96 | 97 | 97 | 96 | - | |
| 4 | 96 | 98 | 96 | 94 | 95 | 96 | - | |
- = No measurement, due to 100 % mortality
Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 19 - 34)
| Study day | Replicate | Dissolved oxygen [%] | ||||||
| Nominal test item concentrations [µg/L] | ||||||||
| Control | Solvent control | 2.50 | 5.00 | 10.0 | 20.0 | 40.0 | ||
| 19 | 1 | 96 | 94 | 92 | 93 | 94 | 94 | - |
| 2 | 94 | 94 | 92 | 95 | 93 | 92 | - | |
| 3 | 94 | 94 | 92 | 92 | 92 | 92 | - | |
| 4 | 94 | 95 | 93 | 91 | 90 | 94 | - | |
| 21 | 1 | 98 | 97 | 94 | 95 | 95 | 93 | - |
| 2 | 96 | 97 | 95 | 95 | 95 | 92 | - | |
| 3 | 96 | 96 | 95 | 93 | 93 | 93 | - | |
| 4 | 96 | 97 | 93 | 91 | 90 | 94 | - | |
| 23 | 1 | 95 | 95 | 92 | 93 | 93 | 94 | - |
| 2 | 95 | 95 | 92 | 93 | 94 | 92 | - | |
| 3 | 95 | 94 | 92 | 91 | 92 | 93 | - | |
| 4 | 95 | 95 | 87 | 92 | 91 | 92 | - | |
| 26 | 1 | 96 | 95 | 94 | 92 | 94 | 95 | - |
| 2 | 96 | 94 | 94 | 92 | 92 | 94 | - | |
| 3 | 94 | 94 | 94 | 92 | 90 | 92 | - | |
| 4 | 95 | 94 | 91 | 92 | 90 | 93 | - | |
| 28 | 1 | 92 | 90 | 90 | 86 | 90 | 91 | - |
| 2 | 91 | 91 | 90 | 87 | 88 | 89 | - | |
| 3 | 89 | 89 | 88 | 88 | 87 | 91 | - | |
| 4 | 91 | 85 | 91 | 88 | 88 | 89 | - | |
| 30 | 1 | 93 | 90 | 88 | 86 | 90 | 90 | - |
| 2 | 91 | 89 | 89 | 87 | 88 | 87 | - | |
| 3 | 89 | 88 | 87 | 86 | 88 | 90 | - | |
| 4 | 90 | 89 | 88 | 86 | 85 | 90 | - | |
| 34 | 1 | 85 | 86 | 80 | 82 | 86 | 88 | - |
| 2 | 85 | 82 | 83 | 77 | 82 | 86 | - | |
| 3 | 79 | 78 | 80 | 81 | 82 | 88 | - | |
| 4 | 85 | 83 | 80 | 78 | 80 | 85 | - | |
| Mean | 95 | 95 | 94 | 94 | 94 | 95 | 98 | |
| ± SD | 4.40 | 4.95 | 5.20 | 5.73 | 5.11 | 4.03 | 2.04 | |
| Min. | 79 | 78 | 80 | 77 | 80 | 85 | 94 | |
| Max. | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
- = No measurement, due to 100 % mortality
Min./Max. = Minimum/Maximum measured dissolved oxygen concentration
Water Temperature (Continuous Measuring) in Replicate 2 of the Control Group
| Period of measurements | 2022-05-04 to 2022-06-08 |
| Minimum temperature [°C] | 25.7 |
| Maximum temperature [°C] | 26.8 |
| Mean temperature [°C] | 26.4 |
Water Temperature in the Test Media
| Study day | Rep. | Water temperature [°C] | ||||||
| Nominal test item concentrations [µg/L] | ||||||||
| Control | Solvent control | 2.50 | 5.00 | 10.0 | 20.0 | 40.0 | ||
| 0 | 1 | 26.4 | 26.4 | 26.5 | 26.6 | 26.6 | 26.4 | 26.5 |
| 2 | 26.4 | 26.4 | 26.6 | 26.5 | 26.8 | 26.5 | 26.4 | |
| 3 | 26.4 | 26.5 | 26.7 | 26.7 | 26.6 | 26.5 | 26.5 | |
| 4 | 26.4 | 26.5 | 26.7 | 26.5 | 26.5 | 26.5 | 26.7 | |
| 7 | 1 | 26.4 | 26.4 | 26.5 | 26.6 | 26.6 | 26.4 | 26.5 |
| 2 | 26.4 | 26.5 | 26.6 | 26.5 | 26.8 | 26.6 | 26.5 | |
| 3 | 26.4 | 26.5 | 26.7 | 26.7 | 26.6 | 26.5 | 26.5 | |
| 4 | 26.3 | 26.4 | 26.6 | 26.5 | 26.4 | 26.4 | 26.7 | |
| 14 | 1 | 26.7 | 26.7 | 26.8 | 27.0 | 26.9 | 26.8 | - |
| 2 | 26.7 | 26.8 | 26.8 | 27.0 | 27.0 | 26.8 | - | |
| 3 | 26.7 | 26.8 | 27.1 | 27.0 | 26.9 | 26.8 | - | |
| 4 | 26.7 | 26.7 | 26.9 | 26.8 | 26.8 | 26.7 | - | |
| 21 | 1 | 26.2 | 26.3 | 26.3 | 26.7 | 26.5 | 26.4 | - |
| 2 | 26.2 | 26.3 | 26.5 | 26.4 | 26.6 | 26.5 | - | |
| 3 | 26.3 | 26.2 | 26.5 | 26.5 | 26.5 | 26.4 | - | |
| 4 | 26.2 | 26.2 | 26.6 | 26.3 | 26.3 | 26.4 | - | |
| 28 | 1 | 26.1 | 26.3 | 26.4 | 26.6 | 26.5 | 26.4 | - |
| 2 | 26.2 | 26.3 | 26.5 | 26.4 | 26.5 | 26.4 | - | |
| 3 | 26.2 | 26.3 | 26.6 | 26.5 | 26.4 | 26.3 | - | |
| 4 | 26.2 | 26.2 | 26.5 | 26.3 | 26.3 | 26.3 | - | |
| Mean | 26.4 | 26.4 | 26.6 | 26.6 | 26.6 | 26.5 | 26.5 | |
| ± SD | 0.192 | 0.190 | 0.182 | 0.214 | 0.201 | 0.159 | 0.106 | |
| Min. | 26.1 | 26.2 | 26.3 | 26.3 | 26.3 | 26.3 | 26.4 | |
| Max. | 26.7 | 26.8 | 27.1 | 27.0 | 27.0 | 26.8 | 26.7 | |
- = No measurement, due to 100 % mortality
Min./Max. = Minimum/Maximum measured water temperature
pH-Values in the Test Media
| Study day | Rep. | pH-value | ||||||
| Nominal test item concentrations [µg/L] | ||||||||
| Control | Solvent control | 2.50 | 5.00 | 10.0 | 20.0 | 40.0 | ||
| 0 | 1 | 7.60 | 7.72 | 7.70 | 7.65 | 7.71 | 7.68 | 7.68 |
| 2 | 7.62 | 7.73 | 7.71 | 7.68 | 7.71 | 7.69 | 7.67 | |
| 3 | 7.71 | 7.75 | 7.69 | 7.68 | 7.68 | 7.72 | 7.73 | |
| 4 | 7.68 | 7.75 | 7.69 | 7.69 | 7.70 | 7.77 | 7.70 | |
| 7 | 1 | 7.64 | 7.98 | 7.97 | 7.91 | 7.96 | 7.96 | 8.01 |
| 2 | 7.97 | 7.92 | 7.95 | 7.98 | 7.95 | 7.92 | 7.96 | |
| 3 | 8.07 | 7.96 | 7.94 | 8.03 | 7.97 | 7.96 | 8.03 | |
| 4 | 8.00 | 8.08 | 7.97 | 8.05 | 7.97 | 7.94 | 8.00 | |
| 14 | 1 | 7.65 | 7.77 | 7.71 | 7.65 | 7.62 | 7.63 | - |
| 2 | 7.70 | 7.77 | 7.68 | 7.62 | 7.65 | 7.62 | - | |
| 3 | 7.77 | 7.77 | 7.64 | 7.65 | 7.64 | 7.59 | - | |
| 4 | 7.76 | 7.74 | 7.71 | 7.68 | 7.63 | 7.62 | - | |
| 21 | 1 | 7.72 | 7.86 | 7.88 | 7.81 | 7.83 | 7.77 | - |
| 2 | 7.83 | 7.92 | 7.80 | 7.86 | 7.83 | 7.80 | - | |
| 3 | 7.91 | 7.82 | 7.84 | 7.76 | 7.77 | 7.84 | - | |
| 4 | 7.85 | 7.86 | 7.80 | 7.79 | 7.72 | 7.76 | - | |
| 28 | 1 | 7.58 | 7.67 | 7.62 | 7.60 | 7.61 | 7.72 | - |
| 2 | 7.65 | 7.68 | 7.62 | 7.63 | 7.60 | 7.64 | - | |
| 3 | 7.66 | 7.59 | 7.62 | 7.65 | 7.61 | 7.67 | - | |
| 4 | 7.68 | 7.62 | 7.65 | 7.63 | 7.58 | 7.68 | - | |
| Mean | 7.75 | 7.80 | 7.76 | 7.75 | 7.74 | 7.75 | 7.85 | |
| ± SD | 0.141 | 0.126 | 0.124 | 0.143 | 0.135 | 0.120 | 0.165 | |
| Min. | 7.58 | 7.59 | 7.62 | 7.60 | 7.58 | 7.59 | 7.67 | |
| Max. | 8.07 | 8.08 | 7.97 | 8.05 | 7.97 | 7.96 | 8.03 | |
- = No measurement, due to 100 % mortality
Min./Max. = Minimum/Maximum measured pH-value
Total Hardness in the Control and the Remaining Highest Test Concentration
| Study day | Replicate | Total hardness [mg CaCO3/L] | ||
| Nominal test item conc. [µg/L] | ||||
| Control | 20.0 | 40.0 | ||
| 0 | 1 | 70 | 69 | |
| 8 | 2 | 67 | 68 | |
| 15 | 3 | 64 | 65 | |
| 21 | 4 | 64 | 66 | |
| 29 | 1 | 68 | 68 | |
| Mean | 67 | 66 | 69 | |
| ± SD | 2.61 | 1.53 | 0.71 | |
| Min. | 64 | 65 | 68 | |
| Max. | 70 | 68 | 69 | |
= No measurement, due to 100 % mortality
n.a. = Not applicable
Min./Max. = Minimum/Maximum measured hardness
Total Organic Carbon (TOC) of the Dilution Water
| Study day | TOC [mg/L] |
| 0 | 1.15 |
| 8 | 1.25 |
| 15 | 1.25 |
| 21 | 1.29 |
| 29 | 1.34 |
| Mean | 1.26 |
| SD | 0.070 |
| Min. | 1.15 |
| Max. | 1.34 |
Min./Max. = Minimum/Maximum measured TOC
Flow Rates
| Flow rate | Nominal test item concentrations [µg/L] | ||||||
| Control | Solvent control | 2.50 | 5.00 | 10.0 | 20.0 | 40.0 | |
| Measured flow rates of the membrane piston pumps1) | |||||||
| [L/h] | |||||||
| Minimum | 3.00 | 3.00 | 3.00 | 3.00 | 34366 | 3.00 | 3.00 |
| Maximum | 3.36 | 3.24 | 3.24 | 3.24 | 3.24 | 3.24 | 3.24 |
| Mean | 3.13 | 3.11 | 3.12 | 3.11 | 3.12 | 3.13 | 3.11 |
| ± Standard deviation | ± 0.055 | ± 0.046 | ± 0.051 | ± 0.053 | ± 0.046 | ± 0.051 | ± 0.060 |
| Overall mean | 3.12 | ||||||
| ± Standard deviation | ± 0.052 | ||||||
1) Nominal flow rates of dilution water (for preparation of the test solutions): 3.125 (2.81 – 3.44 L/h)
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessel on Study Day -1
| Sampling day | Day -1 | |||||
| Replicate | Repl. 2 | |||||
| Nominal | 2,2’-(Octadec-9-enylimino)bisethanol | |||||
| concentration of the | C18:1 Amine + 2 EO | |||||
| Test Vessel | Test vessel (centrifuged) | |||||
| Test item | a.i. | Meas. conc. | % | Meas. conc. | % | |
| [µg/L] | [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | |||
| 40.0 | 30.2 | 20.7 | 69 | 10.0 | 33 | |
| 20.0 | 15.1 | 8.19 | 54 | 4.04 | 27 | |
| 10.0 | 7.54 | 4.40 | 58 | 2.32 | 31 | |
| 5.00 | 3.77 | 1.50 | 40 | 0.587 | 16 | |
| 2.50 | 1.89 | 1.07 | 57 | 0.421 | 22 | |
| Solvent Control | < LOQ | < LOQ | ||||
| Control | < LOQ | < LOQ | ||||
Meas. Conc. = Measured concentration (dilution factor taken into account)
a.i. = Active ingredient
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (0.4 µg/L of the test item, corresponding to 0.302 µg a.i./L)
Measured Concentrations and Percent of Nominal Concentration in the Stock Solutions
| Freshly prepared stock solutions | 7 Days aged stock solutions | |||||
| Nominal | 2,2’-(Octadec-9-enylimino)bisethanol | |||||
| concentration of the | C18:1 Amine + 2 EO | |||||
| Test item | a.i. | Meas. | % | Meas. | % | |
| [mg/L] | [mg a.i./L] | conc. | conc. | |||
| [mg a.i./L] | [mg a.i./L] | |||||
| 1600 | 1207 | 1349 | 112 | 1347 | 112 | |
| 800 | 603 | 639 | 106 | 672 | 111 | |
| 400 | 302 | 322 | 107 | 340 | 113 | |
| 200 | 151 | 160 | 106 | 173 | 115 | |
| 100 | 75.4 | 81.1 | 108 | 85.4 | 113 | |
Meas. Conc. = Measured concentration (dilution factor taken into account)
a.i. = Active ingredient
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (0.4 µg/L of the test item, corresponding to 0.302 µg a.i./L)
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day 0 to Day 34
| Sampling day | Day 0 | Day 7 | Day 14 | ||||
| Replicate | Repl. 3 | Repl. 4 | Repl. 1 | ||||
| Nominal | 2,2’-(Octadec-9-enylimino)bisethanol | ||||||
| concentration of the | C18:1 Amine + 2 EO | ||||||
| Test item | a.i. | Meas. | % | Meas. | % | Meas. | % |
| [µg/L] | [µg a.i./L] | conc. | conc. | conc. | |||
| [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | |||||
| 40.0 | 30.2 | 21.1 | 70 | 18.7 | 62 | 21.2 | 70 |
| 20.0 | 15.1 | 12.2 | 81 | 9.38 | 62 | 10.0 | 66 |
| 10.0 | 7.54 | 5.33 | 71 | 4.08 | 54 | 4.65 | 62 |
| 5.00 | 3.77 | 2.05 | 54 | 1.66 | 44 | 1.53 | 41 |
| 2.50 | 1.89 | 0.963 | 51 | 1.03 | 55 | 1.05 | 56 |
| Solvent Control | < LOQ | < LOQ | < LOQ | ||||
| Control | < LOQ | < LOQ | < LOQ | ||||
| Sampling day | Day 21 | Day 34 | |||||
| Replicate | Repl. 2 | Repl. 4 | |||||
| Nominal | 2,2’-(Octadec-9-enylimino)bisethanol | ||||||
| concentration of the | C18:1 Amine + 2 EO | ||||||
| Test item | a.i. | Meas. | % | Meas. | % | ||
| [µg/L] | [µg a.i./L] | conc. | conc. | ||||
| [µg a.i./L] | [µg a.i./L] | ||||||
| 40.0 | 30.2 | -1) | -1) | ||||
| 20.0 | 15.1 | 7.75 | 51 | 9.47 | 63 | ||
| 10.0 | 7.54 | 4.59 | 61 | 3.99 | 53 | ||
| 5.00 | 3.77 | 1.19 | 31 | 1.82 | 48 | ||
| 2.50 | 1.89 | 0.807 | 43 | 1.00 | 53 | ||
| Solvent Control | < LOQ | < LOQ | |||||
| Control | < LOQ | < LOQ | |||||
Meas. Conc. = Measured concentration (dilution factor taken into account)
a.i. = Active ingredient
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (0.4 µg/L of the test item, corresponding to 0.302 µg a.i./L)
- = No determination of this replicate
1) = No samples due to 100% mortality
Overview of Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels and Calculated Overall Arithmetic Mean Measured Test Item Concentrations (Study Day 0 to 34)
| 2,2’-(Octadec-9-enylimino)bisethanol | ||||
| C18:1 Amine + 2 EO | ||||
| Nominal | Overall Arithmetic Mean Measured Concentrations of the | |||
| concentration of the | ||||
| Test item | a.i. | Test item | a.i. | % |
| [µg/L] | [µg a.i./L] | [µg/L] | [µg a.i./L] | |
| 40.0 | 30.2 | 27.0 | 20.3 | 67 |
| 20.0 | 15.1 | 12.9 | 9.76 | 65 |
| 10.0 | 7.54 | 6.00 | 4.53 | 60 |
| 5.00 | 3.77 | 2.19 | 1.65 | 44 |
| 2.50 | 1.89 | 1.29 | 0.970 | 51 |
a.i. = Active ingredient
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (0.4 µg/L of the test item, corresponding to 0.302 µg a.i./L)
Gradient Table
| Time [min] | A [%] | B [%] |
| 0.00 | 70 | 30 |
| 0.30 | 70 | 30 |
| 1.30 | 5 | 95 |
| 2.00 | 5 | 95 |
| 2.10 | 70 | 30 |
| 2.50 | 70 | 30 |
Dilution steps (Test Vessel)
| Nominal | Dilution | Sample | Final |
| test item | Factor* | volume | volume |
| concentration | [mL] | [mL] | |
| [µg/L] | |||
| 40.0 | 10 | 0.2 | 1.0 |
| 20.0 | 10 | 0.2 | 1.0 |
| 10.0 | 2 | 1.0 | 1.0 |
| 5.0 | 2 | 1.0 | 1.0 |
| 2.50 | 2 | 1.0 | 1.0 |
| Solvent Control | 2 | 1.0 | 1.0 |
| Control | 2 | 1.0 | 1.0 |
*including factor 2
Dilution steps (Stock Solution)
| Nominal | Dilution | Sample | Final |
| test item | Factor | volume | volume |
| concentration | [mL] | [mL] | |
| [mg/L] | |||
| 1600 | 250 000 | 0.041) | 4.01) |
| 0.042) | 4.02) | ||
| 0.043) | 1.03) | ||
| 800 | 125 000 | 0.041) | 4.01) |
| 0.042) | 4.02) | ||
| 0.083) | 1.03) | ||
| 400 | 62 500 | 0.041) | 4.01) |
| 0.042) | 4.02) | ||
| 0.163) | 1.03) | ||
| 200 | 31 250 | 0.041) | 4.01) |
| 0.042) | 4.02) | ||
| 0.323) | 1.03) | ||
| 100 | 15 625 | 0.041) | 4.01) |
| 0.042) | 4.02) | ||
| 0.643) | 1.03) | ||
| Control | 15 625 | 0.041) | 4.01) |
| 0.042) | 4.02) | ||
| 0.643) | 1.03) |
1) first dilution step
2) second dilution step
3) third dilution step
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day 0 to Day 34 (centrifuged)
| Sampling day | Day 0 | Day 7 | Day 14 | ||||
| Replicate | Repl. 3 | Repl. 4 | Repl. 1 | ||||
| Nominal | 2,2’-(Octadec-9-enylimino)bisethanol | ||||||
| concentration of the | C18:1 Amine + 2 EO | ||||||
| Test item | a.i. | Meas. | % | Meas. | % | Meas. | % |
| [µg/L] | [µg a.i./L] | conc. | conc. | conc. | |||
| [µg a.i./L] | [µg a.i./L] | [µg a.i./L] | |||||
| 40.0 | 30.2 | 7.17 | 24 | 5.36 | 18 | 7.69 | 26 |
| 20.0 | 15.1 | 3.81 | 25 | 3.37 | 22 | 4.71 | 31 |
| 10.0 | 7.54 | 1.84 | 24 | 1.34 | 18 | 1.61 | 21 |
| 5.00 | 3.77 | 0.568 | 15 | 0.565 | 15 | 0.430 | 11 |
| 2.50 | 1.89 | 0.325 | 17 | 0.320 | 17 | 0.302 | 16 |
| Solvent Control | < LOQ | < LOQ | < LOQ | ||||
| Control | < LOQ | < LOQ | < LOQ | ||||
| Sampling day | Day 21 | Day 34 | |||||
| Replicate | Repl. 2 | Repl. 4 | |||||
| Nominal | 2,2’-(Octadec-9-enylimino)bisethanol | ||||||
| concentration of the | C18:1 Amine + 2 EO | ||||||
| Test item | a.i. | Meas. | % | Meas. | % | ||
| [µg/L] | [µg a.i./L] | conc. | conc. | ||||
| [µg a.i./L] | [µg a.i./L] | ||||||
| 40.0 | 30.2 | -1) | -1) | ||||
| 20.0 | 15.1 | 5.71 | 38 | 3.29 | 22 | ||
| 10.0 | 7.54 | 3.03 | 40 | 1.49 | 20 | ||
| 5.00 | 3.77 | 0.648 | 17 | 0.671 | 18 | ||
| 2.50 | 1.89 | 0.459 | 24 | 0.291 | 15 | ||
| Solvent Control | < LOQ | < LOQ | |||||
| Control | < LOQ | < LOQ | |||||
Meas. Conc. = Measured concentration (dilution factor taken into account)
a.i. = Active ingredient
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (0.4 µg/L of the test item, corresponding to 0.302 µg a.i./L)
- = No determination of this replicate
1) = No samples due to 100% mortality
Vital Eggs and Larvae from Study Days 0 to 35 (Control, Solvent Control and
2.50 µg/L)
| Study day | Nominal test item concentration [µg/L] | |||||||||||
| Control | Solvent control | 2.50 | ||||||||||
| Number of vital eggs and larvae (listed eggs/larvae) by study day | ||||||||||||
| 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
| 0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 |
| 1 | 18/0 | 20/0 | 20/0 | 19/0 | 20/0 | 20/0 | 19/0 | 19/0 | 20/0 | 20/0 | 20/0 | 18/0 |
| 2 | 18/0 | 20/0 | 19/1 | 19/0 | 20/0 | 20/0 | 18/0 | 19/0 | 20/0 | 20/0 | 20/0 | 18/0 |
| 3 | 17/1 | 16/4 | 7/13 | 12/7 | 18/2 | 9/11 | 9/9 | 17/2 | 13/7 | 16/4 | 9/11 | 11/7 |
| 4 | 3/15 | 8/12 | 2/18 | 1/18 | 7/13 | 0/20 | 1/17 | 8/11 | 2/18 | 1/19 | 0/20 | 2/16 |
| 5 | 0/18 | 0/20 | 0/20 | 0/18 | 0/20 | 0/20 | 0/18 | 0/19 | 2/18 | 0/20 | 0/20 | 0/18 |
| 6 | 0/18 | 0/20 | 0/19 | 0/18 | 0/20 | 0/19 | 0/17 | 0/19 | 0/20 | 0/20 | 0/20 | 0/17 |
| 7 | 0/18 | 0/20 | 0/19 | 0/18 | 0/20 | 0/19 | 0/17 | 0/19 | 0/20 | 0/20 | 0/20 | 0/17 |
| 8 | 0/18 | 0/20 | 0/19 | 0/18 | 0/20 | 0/19 | 0/17 | 0/19 | 0/20 | 0/20 | 0/20 | 0/17 |
| 9 | 0/18 | 0/20 | 0/19 | 0/18 | 0/20 | 0/19 | 0/16 | 0/18 | 0/20 | 0/20 | 0/20 | 0/17 |
| 10 | 0/18 | 0/20 | 0/19 | 0/18 | 0/20 | 0/19 | 0/16 | 0/18 | 0/20 | 0/20 | 0/20 | 0/17 |
| 11 | 0/18 | 0/20 | 0/19 | 0/18 | 0/20 | 0/19 | 0/16 | 0/18 | 0/19 | 0/20 | 0/20 | 0/16 |
| 12 | 0/18 | 0/20 | 0/19 | 0/18 | 0/20 | 0/19 | 0/16 | 0/18 | 0/19 | 0/20 | 0/20 | 0/16 |
| 13 | 0/18 | 0/20 | 0/19 | 0/16 | 0/19 | 0/19 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/16 |
| 14 | 0/17 | 0/20 | 0/19 | 0/16 | 0/19 | 0/18 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/16 |
| 15 | 0/17 | 0/20 | 0/19 | 0/16 | 0/19 | 0/18 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/16 |
| 16 | 0/17 | 0/20 | 0/18 | 0/16 | 0/19 | 0/18 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/16 |
| 17 | 0/17 | 0/20 | 0/18 | 0/16 | 0/19 | 0/18 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/16 |
| 18 | 0/17 | 0/20 | 0/18 | 0/16 | 0/18 | 0/18 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/15 |
| 19 | 0/17 | 0/20 | 0/17 | 0/16 | 0/18 | 0/18 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/15 |
| 20 | 0/17 | 0/20 | 0/17 | 0/16 | 0/18 | 0/18 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/15 |
| 21 | 0/17 | 0/20 | 0/17 | 0/16 | 0/18 | 0/18 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/15 |
| 22 | 0/17 | 0/20 | 0/17 | 0/16 | 0/18 | 0/18 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/15 |
| 23 | 0/17 | 0/20 | 0/17 | 0/16 | 0/18 | 0/18 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/15 |
| 24 | 0/17 | 0/20 | 0/17 | 0/16 | 0/18 | 0/18 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/15 |
| 25 | 0/17 | 0/20 | 0/17 | 0/16 | 0/18 | 0/18 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/15 |
| 26 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/17 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/14 |
| 27 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/17 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/14 |
| 28 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/17 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/14 |
| 29 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/17 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/14 |
| 30 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/17 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/14 |
| 31 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/17 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/14 |
| 32 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/17 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/14 |
| 33 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/17 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/13 |
| 34 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/17 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/13 |
| 35 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/17 | 0/16 | 0/18 | 0/19 | 0/19 | 0/20 | 0/13 |
Vital Eggs and Larvae from Study Days 0 to 35 (5.00 to 20.0 µg/L)
| Study day | Nominal test item concentration [µg/L] | |||||||||||
| 5.00 | 10.0 | 20.0 | ||||||||||
| Number of vital eggs and larvae (listed eggs/larvae) by study day | ||||||||||||
| 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | |
| 0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 |
| 1 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 19/0 | 19/0 | 20/0 |
| 2 | 20/0 | 20/0 | 20/0 | 19/0 | 20/0 | 20/0 | 20/0 | 20/0 | 20/0 | 19/0 | 19/0 | 19/0 |
| 3 | 17/3 | 19/1 | 19/1 | 17/2 | 16/4 | 19/1 | 10/10 | 19/1 | 19/1 | 17/2 | 17/2 | 11/8 |
| 4 | 8/12 | 9/11 | 6/14 | 4/15 | 2/18 | 9/11 | 1/19 | 9/11 | 9/11 | 13/6 | 4/15 | 1/18 |
| 5 | 1/19 | 0/20 | 0/20 | 0/19 | 0/20 | 0/20 | 0/20 | 1/19 | 3/17 | 2/17 | 0/19 | 0/19 |
| 6 | 0/20 | 0/20 | 0/20 | 0/19 | 0/20 | 0/20 | 0/20 | 0/20 | 0/20 | 0/19 | 0/19 | 0/19 |
| 7 | 0/20 | 0/20 | 0/20 | 0/19 | 0/20 | 0/19 | 0/20 | 0/20 | 0/20 | 0/19 | 0/19 | 0/19 |
| 8 | 0/20 | 0/20 | 0/20 | 0/19 | 0/20 | 0/19 | 0/20 | 0/20 | 0/20 | 0/19 | 0/19 | 0/19 |
| 9 | 0/20 | 0/20 | 0/20 | 0/19 | 0/20 | 0/19 | 0/20 | 0/20 | 0/20 | 0/19 | 0/19 | 0/19 |
| 10 | 0/20 | 0/20 | 0/20 | 0/19 | 0/20 | 0/19 | 0/20 | 0/20 | 0/20 | 0/18 | 0/19 | 0/19 |
| 11 | 0/20 | 0/20 | 0/20 | 0/19 | 0/20 | 0/19 | 0/20 | 0/20 | 0/20 | 0/18 | 0/18 | 0/18 |
| 12 | 0/18 | 0/20 | 0/19 | 0/19 | 0/19 | 0/19 | 0/20 | 0/20 | 0/20 | 0/14 | 0/18 | 0/17 |
| 13 | 0/17 | 0/20 | 0/19 | 0/19 | 0/19 | 0/19 | 0/20 | 0/19 | 0/19 | 0/14 | 0/18 | 0/17 |
| 14 | 0/17 | 0/20 | 0/19 | 0/19 | 0/19 | 0/19 | 0/20 | 0/19 | 0/15 | 0/13 | 0/16 | 0/14 |
| 15 | 0/17 | 0/20 | 0/19 | 0/19 | 0/19 | 0/19 | 0/20 | 0/19 | 0/11 | 0/12 | 0/16 | 0/14 |
| 16 | 0/17 | 0/20 | 0/19 | 0/19 | 0/19 | 0/19 | 0/20 | 0/19 | 0/8 | 0/10 | 0/16 | 0/12 |
| 17 | 0/17 | 0/20 | 0/19 | 0/19 | 0/19 | 0/19 | 0/20 | 0/19 | 0/6 | 0/8 | 0/13 | 0/12 |
| 18 | 0/17 | 0/20 | 0/19 | 0/19 | 0/19 | 0/19 | 0/20 | 0/18 | 0/6 | 0/7 | 0/10 | 0/9 |
| 19 | 0/17 | 0/20 | 0/19 | 0/19 | 0/19 | 0/19 | 0/20 | 0/17 | 0/6 | 0/6 | 0/9 | 0/9 |
| 20 | 0/17 | 0/20 | 0/18 | 0/19 | 0/18 | 0/19 | 0/20 | 0/17 | 0/6 | 0/5 | 0/9 | 0/7 |
| 21 | 0/17 | 0/20 | 0/18 | 0/18 | 0/18 | 0/19 | 0/20 | 0/16 | 0/5 | 0/4 | 0/8 | 0/7 |
| 22 | 0/16 | 0/20 | 0/18 | 0/18 | 0/18 | 0/19 | 0/20 | 0/16 | 0/5 | 0/3 | 0/7 | 0/6 |
| 23 | 0/16 | 0/19 | 0/18 | 0/18 | 0/18 | 0/19 | 0/20 | 0/16 | 0/4 | 0/3 | 0/6 | 0/6 |
| 24 | 0/16 | 0/19 | 0/18 | 0/18 | 0/18 | 0/19 | 0/20 | 0/16 | 0/4 | 0/3 | 0/6 | 0/5 |
| 25 | 0/16 | 0/19 | 0/17 | 0/18 | 0/18 | 0/19 | 0/20 | 0/16 | 0/4 | 0/3 | 0/6 | 0/5 |
| 26 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/18 | 0/18 | 0/15 | 0/4 | 0/3 | 0/6 | 0/5 |
| 27 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/18 | 0/18 | 0/15 | 0/4 | 0/3 | 0/6 | 0/5 |
| 28 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/18 | 0/18 | 0/15 | 0/4 | 0/3 | 0/6 | 0/5 |
| 29 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/18 | 0/18 | 0/15 | 0/4 | 0/3 | 0/6 | 0/5 |
| 30 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/18 | 0/18 | 0/15 | 0/4 | 0/3 | 0/6 | 0/5 |
| 31 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/18 | 0/18 | 0/15 | 0/4 | 0/3 | 0/6 | 0/5 |
| 32 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/18 | 0/18 | 0/15 | 0/4 | 0/3 | 0/6 | 0/5 |
| 33 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/18 | 0/18 | 0/15 | 0/4 | 0/3 | 0/6 | 0/5 |
| 34 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/18 | 0/18 | 0/15 | 0/4 | 0/3 | 0/6 | 0/5 |
| 35 | 0/16 | 0/19 | 0/16 | 0/16 | 0/18 | 0/18 | 0/18 | 0/15 | 0/4 | 0/3 | 0/6 | 0/5 |
Vital Eggs and Larvae from Study Days 0 to 12 (40.0 µg/L)
| Study day | Nominal test item concentration [µg/L] | |||
| 40.0 | ||||
| Number of vital eggs and larvae (listed eggs/larvae) by study day | ||||
| 1 | 2 | 3 | 4 | |
| 0 | 20/0 | 20/0 | 20/0 | 20/0 |
| 1 | 20/0 | 18/0 | 20/0 | 20/0 |
| 2 | 20/0 | 18/0 | 20/0 | 20/0 |
| 3 | 19/1 | 18/0 | 18/2 | 10/10 |
| 4 | 11/9 | 13/5 | 13/7 | 1/19 |
| 5 | 3/17 | 1/17 | 1/19 | 0/20 |
| 6 | 1/19 | 0/18 | 0/18 | 0/20 |
| 7 | 0/20 | 0/17 | 0/15 | 0/20 |
| 8 | 0/12 | 0/15 | 0/7 | 0/13 |
| 9 | 0/5 | 0/4 | 0/0 | 0/5 |
| 10 | 0/1 | 0/2 | - | 0/1 |
| 11 | 0/0 | 0/1 | - | 0/0 |
| 12 | - | 0/0 | - | - |
- = 100% mortality
Method Validation (non-GLP)
The analytical method was validated according to SANTE/2020/12830, Rev. 1 (2021), Guidance Document on Pesticide Analytical Methods for Risk Assessment and Post-approval Control and Monitoring Purposes using the criteria in the table below.
Parameter, Acceptance Criteria and Results of the Method Validation
| Parameter | Acceptance criteria | Result | |
| Calibration | ≥ 5 standard concentrations, | 0.05 to 10 µg test item/L | ü |
| r ≥ 0.99 (R2 ≥ 0.9801 for 2nd order regression) | (n = 9), | ||
| Calibration range from ≤ 30% LOQ to 20% above highest concentration level after sample preparation | r ≥ 0.99 | ||
| Matrix effects | Should be ≤ 20% | Matrix effects: 22% | ü |
| Matrix matched standards have been used | |||
| Lowest calibration level (LCL) | S/N ≥ 9 for quantifier ion trace | S/N for 0.05 µg test item/L | - |
| S/N ≥ 3 for qualifier ion trace | 583 (quantifier) | ||
| 1326 (qualifier) | |||
| Limit of Quantification (LOQ) | Should be at or below lowest test concentration | 0.400 µg test item/L (1 x LOQ) | ü |
| 50.0 µg test item/L (125 x LOQ) | |||
| Accuracy (Fortified samples)1) | Mean recovery rate of 70-120% | 1 x LOQ: 71% (n = 5) | ü |
| per fortification level (2 levels) | 125 x LOQ: 102% (n = 5) | ||
| Precision1) | Relative standard deviation ≤ 20% per fortification level | 1 x LOQ: 20% | ü |
| 125 x LOQ: 5.1% | |||
| Stability | Samples: Stable if 70 – 120% of nominal concentration | See section 18.1 | ü |
| Calibration standards: Stable if ±10% of freshly prepared standard | |||
| Specificity: LC-MS/(MS) | Measurement of two transitions of the same precursor ion - one quantifier (used for evaluation) and one qualifier (for confirmation of the analyte identity). | quantifier [m/z]: 356.31 > 88.05 | ü |
| qualifier [m/z]: 356.31 > 55.04 | |||
| Blank values < 30% of the LOQ | Blank values < 30% of LOQ | ü | |
| Procedural recovery | Procedural recoveries with experimental samples. The recovery has to be 70-120% of the nominal value. | See section 18.2 | ü |
Preparation of Fortified Samples of the Test Item
| LOQ Level | Control | 1 | 125 |
| Stock solution | - | 1000 mg test item/L in Methanol | |
| Spiking solution | - | 40.0 | 5000 |
| [µg test item/L] | (Methanol) | (Methanol) | |
| (Medium) | |||
| Replicates | 2 | 5 | 5 |
| Concentration of the LOQ | - | 0.400 | 50.0 |
| [µg test item/L] | |||
| Medium for preparation | Fish dilution water | ||
| Volume of spiking solution [mL] | - | 0.05 | 0.05 |
| Volume of medium [mL] | 5.0 | 4.95 | 4.95 |
| Dilution factor | 2 | 2 | 10 |
| Dilution medium | Methanol containing 0.2% formic acid1) | ||
| Dilution medium2) | |||
| Sample volume [mL] | 5.01) | 5.01) | 5.01) |
| 0.22) | |||
| Finale volume [mL] | 101) | 101) | 101) |
| 1.02) | |||
| Final concentration of the test item for analysis | - | 0.200 | 5.00 |
| [µg/L] | |||
| Final concentration of the a.i. for analysis | - | 0.151 | 3.77 |
| [µg a.i./L] | |||
1) First dilution step
2) Second dilution step
Dilution medium = Methanol : fish dilution water (50 : 50 v/v) containing 0.1% formic acid
Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of 2,2’-(Octadec-9-enylimino)bisethanol
Fortified concentrations*: 0.409 µg test item/L (1 x LOQ) and 51.1 µg test item/L (125 x LOQ), corresponding to 0.308 µg a.i./L (1 x LOQ) and 38.5 µg a.i./L (125 x LOQ).
| Replicate | 2,2’-(Octadec-9-enylimino)bisethanol | |||
| C18:1 Amine + 2 EO | ||||
| 1 x LOQ | 125 x LOQ | |||
| Meas. | % | Meas. | % | |
| conc. | conc. | |||
| [µg a.i./L] | [µg a.i./L] | |||
| 1 | 0.264 | 86 | 42.8 | 111 |
| 2 | 0.191 | 62 | 38.3 | 99 |
| 3 | 0.176 | 57 | 38.4 | 100 |
| 4 | 0.273 | 89 | 38.3 | 99 |
| 5 | 0.196 | 64 | 38.3 | 99 |
| Mean | 0.22 | 71 | 39 | 102 |
| SD ± | 0.04 | 2 | ||
| CV [%] | 20 | 5.1 | ||
Meas. conc. = measured concentration C18:1 Amine + 2 EO in the test item, dilution factor taken into account
a.i. = active substance
% = percent concentration of the fortified sample
SD = standard deviation
CV = coefficient of variation
Measured Concentrations and Percent of Nominal Concentration of the Quality Control during the Definitive Test
| Quality Control | ||
| Sampling date | Meas. conc. | % |
| [µg a.i./L] | ||
| Day -11) | 0.317 | 103 |
| Day 01) | 0.358 | 116 |
| Day 71) | 0.343 | 111 |
| Day 142) | 0.335 | 110 |
| Day 212) | 0.335 | 111 |
| Day 282) | 0.288 | 95 |
| Day 342) | 0.325 | 107 |
Meas. conc. = measured concentration of the a.i., dilution factors taken into account
% = percent of the nominal concentration
a.i. = active ingredient (C18:1 Amine + 2 EO)
1) = 0.409 µg test item/L, corresponding to 0.308 µg a.i./L (weighing factor taken into account)
2) = 0.402 µg test item/L, corresponding to 0.303 µg a.i./L (weighing factor taken into account)
Mortality [%] and Hatch [%] in the Range Finding Test
(n = 40 per treatment group with 2 replicates and 20 eggs each under flow-through conditions)
| Nominal test item concentration | Cumulative | Post hatch | Overall | Mortality [%] | ||||
| [µg/L] | hatch [%] | survival [%] | survival [%] | on study day 14 | ||||
| (study day 6) | on study day 14 | on study day 14 | ||||||
| Replicate | Replicate | Replicate | Replicate | |||||
| 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | |
| 50.0 | 100 | 90 | 0 | 0 | 0* | 0* | 100* | 100* |
| 5.00 | 90 | 95 | 94 | 100 | 85 | 95 | 15 | 5 |
| Solvent control | 95 | 90 | 100 | 94 | 95 | 85 | 5 | 15 |
* 100 % mortality of hatched larvae was observed on study day 9
Measured Concentrations of the compound C18:1 Amine + 2EO of the Test Item
2,2’-(Octadec-9-enylimino)bisethanol on Study Days -1, 0, 7 and 14 of the Range Finding Test
Determination of the test item via LC-MS/MS, with a preliminary non-validated analytical method
| Sampling date | Day -1 | |||||||
| Nominal concentration of the | C18:1 Amine + 2EO | |||||||
| test item [µg/L] | active substance | Meas. conc. | % | |||||
| [µg a.s./L] | [µg a.s./L] | |||||||
| 50.0 | 37.1 | 31.4 | 83 | |||||
| (Replicate 1) | ||||||||
| 50.0 | 25.5 | 68 | ||||||
| (Replicate 2) | ||||||||
| 5.00 | 3.77 | 1.91 | 51 | |||||
| (Replicate 1) | ||||||||
| 5.00 | 1.76 | 47 | ||||||
| (Replicate 2) | ||||||||
| Solvent control | < LCL | |||||||
| (Replicate 1) | ||||||||
| Solvent control | < LCL | |||||||
| (Replicate 2) | ||||||||
| Sampling date | Day 0 | Day 7 | Day 14 | |||||
| Nominal concentration of the | C18:1 Amine + 2EO | |||||||
| test item [µg/L] | active substance | Meas. conc. | % | Meas. conc. | % | Meas. conc. | % | |
| [µg a.s./L] | [µg a.s./L] | [µg a.s./L] | [µg a.s./L] | |||||
| 50.0 | 37.1 | 25.7 | 68 | - | - | |||
| (Replicate 1) | ||||||||
| 50.0 | - | 22.8 | 60 | - | ||||
| (Replicate 2) | ||||||||
| 5.00 | 3.77 | 2.58 | 68 | - | 1.92 | 51 | ||
| (Replicate 1) | ||||||||
| 5.00 | - | 1.07 | 28 | 1.91 | 51 | |||
| (Replicate 2) | ||||||||
| Solvent control | < LCL | < LCL | < LCL | |||||
| (Replicate 1) | ||||||||
| Solvent control | < LCL | < LCL | < LCL | |||||
| (Replicate 2) | ||||||||
Meas. Conc. = Measured concentration of the test item, dilution factor taken into account
% = Percent of the nominal test item concentration
LCL = Lowest Calibration Level, 0.1 µg test item/L
a.s. = Active substance
Measured Concentrations of the Compound C18:1 Amine + 2EO of the Test Item
2,2’-(Octadec-9-enylimino)bisethanol on Study Days -1, 0, 7 and 14 of the Range Finding Test (Truly Dissolved Test Item)
Determination of the test item via LC-MS/MS, with a preliminary non-validated analytical method
| Sampling date | Day 0 | Day 7 | Day 14 | ||||
| Nominal concentration of the | C18:1 Amine + 2EO | ||||||
| test item [µg/L] | active substance | Meas. conc. | % | Meas. conc. | % | Meas. conc. | % |
| [µg a.s./L] | [µg a.s./L] | [µg a.s./L] | [µg a.s./L] | ||||
| 50.0 | 37.1 | 11.8 | 31 | - | - | ||
| (Replicate 1) | |||||||
| 50.0 | - | 9.40 | 25 | - | |||
| (Replicate 2) | |||||||
| 5.00 | 3.77 | 0.385 | 10 | - | 0.639 | 17 | |
| (Replicate 1) | |||||||
| 5.00 | - | 0.860 | 23 | 0.702 | 19 | ||
| (Replicate 2) | |||||||
| Solvent control | < LCL | < LCL | < LCL | ||||
| (Replicate 1) | |||||||
| Solvent control | < LCL | < LCL | < LCL | ||||
| (Replicate 2) | |||||||
Meas. Conc. = Measured concentration of the test item, dilution factor taken into account
% = Percent of the nominal test item concentration
LCL = Lowest Calibration Level, 0.1 µg test item/L
a.s. = Active substance
Description of key information
Long-term toxicity to fish
For the primary fatty amine ethoxylates (2EO) there are two long-term toxicity test with fish available. Both studies were performed according to OECD 210 using a flow through test setup.
The first study with Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (PFAEO-C; CAS no.: 71786-60-2) resulted in an EC10 for fry growth (weight) of 14.3 µg/L based on the mean measured test item concentrations and 13.4 µg/L based on nominal test item concentrations.
The second study with item 2,2’-(Octadec-9-enylimino)bisethanol (PFAEO-O; CAS no.: 25307-17-9)
resulted in an EC10 based on fry growth (weight) is 1.61 µg/L based on the mean measured test item concentrations and 3.54 µg/L based on nominal test item concentrations.
A detailed summary of both studies is presented below.
Summary Study 1:
The effects of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (PFAEO-C; CAS: 71786-60-2) on the early-life stage of fish (Danio rerio / Zebrafish) were determined according to OECD Guideline 210 using a flow-through test setup under GLP conditions.
The test item is a light-yellow clear liquid. Methanol was used as solvent with a concentration of 0.025 mL/L dilution water. Stock solutions in methanol with nominal concentrations of 200, 400, 800, 1600 and 3200 mg/L were prepared in appropriate intervals of 7 days and continuously dosed to the dilution water in a flow-through system. Based on the results of a range finding test the test was conducted as a dose-response test with the nominal test item concentrations 5.00, 10.0, 20.0, 40.0 and 80.0 µg/L, corresponding to the overall mean measured test item concentrations of 4.61, 10.4, 21.0, 39.0 and 80.7 µg/L.
The test was started by placing fertilized eggs into the test vessels and it lasted 34 days (30 days post-hatch). 80 eggs of Danio rerio / zebrafish were exposed to each test concentration, the solvent control and the control (4 replicates with 20 eggs each).
The water quality parameters pH-value, oxygen concentration, temperature and total hardness were within the acceptable limits.
On study day 4, 91% of the control and 96% the solvent control larvae had hatched. Therefore, study day 4 was defined as post hatch day 0 (= PHD 0).
Different toxicological endpoints were determined: hatching success, fry growth (assessed via length and fresh weight measurements on PHD 30), morphological and behavioral effects, post-hatch survival and overall survival.
Specific analysis of various concentrations of PFAEO-C in the test media and the controls was carried out via LC-MS/MS.
The test media were sampled and analyzed from alternating test vessels prior to exposure on study day -1 and during the exposure on study days 0, 7, 14, 21 and 28.
The analytical results of the range finding tests showed that for single compounds (especially with higher carbon chain length) the measured concentrations were partly out of range of the nominal concentrations in some of the replicates. The reason is not known but inhomogeneous distribution of the compounds in the test replicates was suspected.
The methanol stock solutions were sampled and analyzed from freshly prepared and corresponding 7 days aged stock solutions. Measured concentrations of the freshly prepared stock solutions were 101 to 105% of the nominal values. Measured concentrations of the 7 days aged stock solutions in the range of 100 to 109% of the nominal values.
All effect values are given based on the nominal and the overall mean measured concentrations of the test item PFAEO-C (see Table 1 and Table 2).
Findings and Observations
The results of the parameters hatching success, fry growth (expressed as weight and length measurement at PHD 30), post-hatch survival and overall survival were checked for statistically significant differences.
No statistically significant difference was detected between the dilution water control and the solvent control for all parameters (hatching success, fry growth expressed as length or weight on PHD 30, post-hatch survival and overall survival). Therefore, both controls were pooled for statistical analysis.
The effect values NOEC, LOEC, ECx, LCx values were determined based on the statistical results. The results are presented in the tables below:
Table 1: NOEC, LOEC, ECx Values of Hatching Success and Fry Growth
Based on nominal and overall 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 mean measured test item concentrations [µg/L] | ||||
Hatching success after | Fry Growth | Hatching success after | Fry Growth | |||
Length | Weight | Length | Weight | |||
NOEC | ≥ 80.0 | 10.0 | 10.0 | ≥ 80.7 | 10.4 | 10.4 |
LOEC | > 80.0 | 20.0 | 20.0 | > 80.7 | 21.0 | 21.0 |
EC10 | n.d. | 27.6 | 13.4 | n.d. | 27.8 | 14.3 |
EC20 | n.d. | 45.2 | 23.8 | n.d. | 43.8 | 24.3 |
EC50 | n.d. | > 80.0 | 70.6 | n.d. | > 80.7 | 66.8 |
n.d. not determinable as no dose-response was found
Table 2: NOEC, LOEC, LCx values of Post Hatch Survival and Overall Survival
Based on nominal and overall 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 mean measured test item concentrations [µg/L] | ||
Post-hatch survival | Overall survival | Post-hatch survival | Overall survival | |
NOEC | 20.0 | 20.0 | 21.0 | 21.0 |
LOEC | 40.0 | 40.0 | 39.0 | 39.0 |
LC10 | 26.6 | 22.4 | 27.1 | 23.2 |
LC20 | 29.7 | 27.1 | 29.8 | 27.5 |
LC50 | 35.6 | 34.6 | 35.2 | 34.2 |
Summary Study 2:
The effects of the test item 2,2’-(Octadec-9-enylimino)bisethanol (PFAEO-O, UVCB, purity 99.97%) on the early-life stage of fish (Danio rerio / Zebrafish) were determined according to OECD Guideline 210 using a flow-through test setup under GLP conditions.
The test item is a yellow liquid UVCB substance is poorly soluble in water (CMC = 5.9 mg/L, pH 7 and 23 °C). Methanol was used as solvent with a concentration of 0.025 mL/L dilution water. Stock solutions in methanol with nominal concentrations of 100, 200, 400, 800 and 1600 mg/L were prepared in appropriate intervals of 7 days and continuously dosed to the dilution water in a flow-through system. Based on the results of a range finding test the test was conducted as a dose-response test with the nominal test item concentrations 2.50, 5.00, 10.0, 20.0 and 40.0 µg/L, corresponding to the overall arithmetic mean measured test item concentrations of 1.29 – 2.19 – 6.00 – 12.9 - 27.0 µg/L. The term overall arithmetic mean means that all measured samples (stabilized samples, from study days 0, 7, 14, 21 and 34) were taken into account for calculation of the arithmetic mean measured concentrations.
The test was started by placing fertilized eggs into the test vessels and it lasted 35 days (30 days post-hatch). 80 eggs of Danio rerio / zebrafish were exposed to each test concentration, the solvent control and the control (4 replicates with 20 eggs each).
The water quality parameters pH-value, oxygen concentration, temperature and total hardness were within the acceptable limits.
On study day 5, 95% of the control and 96% the solvent control larvae had hatched. Therefore, study day 5 was defined as post hatch day 0 (= PHD 0).
Different toxicological endpoints were determined: hatching success, fry growth (assessed via length and fresh weight measurements on PHD 30), morphological and behavioral effects, post-hatch survival and overall survival.
Specific analysis of various concentrations of PFAEO-O in the test media and the controls was carried out via LC-MS/MS.
The concentrations of the test item PFAEO-O, the solvent control and the control were determined during exposure on days 0, 7, 14, 21 and 34. The constituent C18:1 Amine + 2EO was determined via LC-MS/MS.
The samples taken from the test vessels were completely clear and did not contain any particles or turbidity. These samples were subjected to the analytical procedure and the results are used for the calculation of the dose response.
The stock solutions prepared in methanol were sampled and analyzed from the freshly prepared and corresponding 7 days aged stock solutions. Measured concentrations of the freshly prepared stock solution were 106 to 112% of the nominal values. Measured concentrations of the 7 days aged stock solution were in the range of 111 to 115% of the nominal values.
All effect values are given based on the nominal and the overall arithmetic mean measured concentrations of the test item PFAEO-O.
Findings and Observations
The results of the parameters hatching success, fry growth (expressed as weight and length measurement at PHD 30), post-hatch survival and overall survival were checked for statistically significant differences. No statistically significant difference was detected between the dilution water control and the solvent control for all parameters (hatching success, fry growth expressed as length or weight on PHD 30, post-hatch survival and overall survival). Therefore, both controls were pooled for statistical analysis.
The effect values NOEC, LOEC, ECx, LCx values were determined based on the statistical results.
The EC10 based on fry growth (weight) is 1.61 µg/L based on arithmetic mean measured test item concentrations and 3.54 µg/L based on nominal test item concentrations.
The LC50 based on overall survival is 11.7 µg/L based on arithmetic mean measured test item concentrations and 18.3 µg/L based on nominal test item concentrations.
NOEC, LOEC, ECx Values of Hatching Success and Fry Growth
Based on nominal and overall arithmetric mean measured test item concentrations [µg/L]
with 95% Confidence intervals in brackets
Parameter | Based on nominal test item concentrations [µg/L] | Based on overall arithmetric mean measured test item concentrations [µg/L] | ||||
Hatching success after | Fry Growth | Hatching success after | Fry Growth | |||
Length | Weight | Length | Weight | |||
NOEC | ≥ 40.0 | 2.50 | 2.50 | ≥ 27.0 | 1.29 | 1.29 |
LOEC | > 40.0 | 5.00 | 5.00 | > 27.0 | 2.19 | 2.19 |
EC10 | n.d. | > 20.0 | 3.54 | n.d. | > 12.9 | 1.61 |
EC20 | n.d. | n.d. | > 20.0 | n.d. | n.d. | 13.6 |
EC50 | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. |
1) = end of hatching period
n.d. = not determinable
NOEC, LOEC, LCx values of Post Hatch Survival and Overall Survival
Based on nominal and overall arithmetic mean measured test item concentrations [µg/L]
with 95% Confidence intervals in brackets
Parameter | Based on nominal test item concentrations [µg/L] | Based on overall arithmetric mean measured test item concentrations [µg/L] | ||
Post-hatch survival | Overall survival | Post-hatch survival | Overall survival | |
NOEC | 10.0 | 10.0 | 6.00 | 6.00 |
LOEC | 20.0 | 20.0 | 12.9 | 12.9 |
LC10 | n.d. | n.d. | n.d. | n.d. |
LC20 | 12.7 | 15.6 | 7.83 | 10.0 |
LC50 | 16.8 | 18.3 | 10.7 | 11.7 |
n.d. = not determinable
Key value for chemical safety assessment
Fresh water fish
Fresh water fish
- Dose descriptor:
- EC10
- Effect concentration:
- 1.61 µg/L
Additional information
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
