Registration Dossier
Registration Dossier
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EC number: 269-924-1 | CAS number: 68391-05-9 This substance is identified by SDA Substance Name: C12-C18 dialkyl dimethyl ammonium chloride and SDA Reporting Number: 16-047-00.
Long-term toxicity to fish
Administrative data
Link to relevant study record(s)
- Endpoint:
- fish early-life stage toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- from 2020-11-10 to 2020-12-14
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- Test item Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
Batch number SPA19005
CAS No. 61789-77-3; 68391-05-9
Purity (certified) 95.6% m/m
Density 880 kg/m3 at 20°C
Appearance Yellow, viscous liquid, paste
Water Solubility Dispersible
Stability under test
conditions Not specified
Expiry date 2021-03-12
Recommended storage Keep in a well ventilated place. - Analytical monitoring:
- yes
- Details on sampling:
- Chemical Analysis of Test Item Concentrations
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.
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 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 and mixing chamber supply. For each sample 40 mL of each test item concentration, the solvent control and the control were sampled.
For analysis of fresh and aged stock solutions, approx. 10 mL of each stock solution were sampled. - Vehicle:
- yes
- Remarks:
- Methanol
- 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).
Test concentrations
0.0100 - 0.0220 - 0.0484 - 0.106 - 0.233 mg test item/L (nominal, factor: 2.2), corresponding to overall mean measured test item concentrations of 0.0117, 0.0309, 0.0606, 0.109 and 0.225 mg 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 16 days.
The test concentrations were prepared by adding the dissolved test item to dilution water as described above.
Solvent
With regard to the limited solubility of the test item in water, methanol (VWR, 100% gradient grade, batch 20G064020) was used as a solvent. The solvent concentration was the same in all concentration levels and the solvent control (0.05 mL/L).
Stock solution
A stock solution of 4.67 g/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.
Dilution Table of the Stock Solutions
Nominal
test item Dilution water
concentration flow rate Stock solution
Flow rate Concentration
[mg/L] [L/h] [mL/h] [g/L]
0.233 3.125 0.1563 4.67
0.106 3.125 0.1563 2.12
0.0484 3.125 0.1563 0.968
0.0220 3.125 0.1563 0.440
0.0100 3.125 0.1563 0.200
Solvent control 3.125 0.1563 ---
Control 3.125 --- ---
Equilibration period
Test solutions flowed through the test vessels for 19 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:
Total hardness: 10 – 250 mg CaCO3/L
pH-value: 6.0 – 8.5
Alkalinity: 0.6 mmol/L (recent measurement: 2020-10-28)
Acidity: 0.1 mmol/L (recent measurement: 2020-10-28)
Conductivity: 161 µS/cm (recent measurement: 2020-10-28)
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.
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 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 95%.
- Test type:
- flow-through
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 34 d
- Remarks on exposure duration:
- 30 days post hatch
- 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 0.0220 mg/L (day 16, 23 and 30), 0.106 mg/L (day 8) and 0.233 mg/L (day 1). The mean total hardness was 69 mg CaCO3/L and ranged from 64 to 73 mg CaCO3/L in the control.
- Test temperature:
- mean temperature was 26.2 °C
- pH:
- pH-Values
The mean pH-values in the control, solvent control and test item groups were between 7.87 and 8.16 and ranged from 7.44 to 8.19 during the exposure period. - Dissolved oxygen:
- Dissolved Oxygen
The dissolved oxygen concentrations in the control, solvent control and the test item groups, expressed in percent saturation, were in the mean 95 – 100% and ranged from 87 to 100% during the exposure period. - Nominal and measured concentrations:
- Test concentrations
0.0100 - 0.0220 - 0.0484 - 0.106 - 0.233 mg test item/L (nominal, factor: 2.2), corresponding to overall mean measured test item concentrations of 0.0117, 0.0309, 0.0606, 0.109 and 0.225 mg 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 16 days.
The test concentrations were prepared by adding the dissolved test item to dilution water as described above.
Solvent With regard to the limited solubility of the test item in water, methanol (VWR, 100% gradient grade, batch 20G064020) was used as a solvent. The solvent concentration was the same in all concentration levels and the solvent control (0.05 mL/L).
Stock solution
A stock solution of 4.67 g/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.
Dilution Table of the Stock Solutions
Nominal
test item concentration Dilution water
flow rate Stock solution
Flow rate Concentration
[mg/L] [L/h] [mL/h] [g/L]
0.233 3.125 0.1563 4.67
0.106 3.125 0.1563 2.12
0.0484 3.125 0.1563 0.968
0.0220 3.125 0.1563 0.440
0.0100 3.125 0.1563 0.200
Solvent control 3.125 0.1563 ---
Control 3.125 --- --- - 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).
Equilibration period
Test solutions flowed through the test vessels for 19 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 tests) 570 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. For results, see part 7.1.8.
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 6.
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 (48 h old) were fed until the end of the test (2 – 8 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.
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.
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. - Key result
- Duration:
- 34 d
- Dose descriptor:
- EC10
- Effect conc.:
- 0.196 mg/L
- 95% CI:
- ca. 0.108 - ca. 0.233
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Hatching success after 7 days
- Remarks on result:
- other: 0.187 (CI: 0.103-0.223) mg a.i./L
- Key result
- Duration:
- 34 d
- Dose descriptor:
- EC10
- Effect conc.:
- 0.196 mg/L
- 95% CI:
- ca. 0.115 - ca. 0.225
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Hatchin success after 7 days
- Remarks on result:
- other: 0.187 (CI: 0.111-0.215) mg a.i./L
- Key result
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 0.106 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Hatching success after 7 days
- Remarks on result:
- other: 0.101 mg a.i./L
- Key result
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 0.109 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Hatching success after 7 days
- Remarks on result:
- other: 0.104 mg a.i./L
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 0.233 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Hatching success after 7 days
- Remarks on result:
- other: 0.222 mg a.i./L
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 0.225 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Hatching success after 7 days
- Remarks on result:
- other: 0.215 mg a.i./L
- Key result
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 0.01 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Fry Growth expressed as Length and Weight
- Remarks on result:
- other: 0.00956 mg a.i./L
- Key result
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 0.012 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Fry Growth expresses as: Length and Weight
- Remarks on result:
- other: 0.0112 mg a.i./L
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- > 0.01 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Fry Growth expressed as Lenght and Weight
- Remarks on result:
- other: 0.00956 mg a.i./L
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- > 0.012 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Fry Growth expressed as Length and Weight
- Remarks on result:
- other: 0.0112 mg a.i./L
- Key result
- Duration:
- 30 d
- Dose descriptor:
- LC10
- Effect conc.:
- 0.014 mg/L
- 95% CI:
- ca. 0.01 - ca. 0.018
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Post-hatch survival
- Remarks on result:
- other: 0.0131 (CI: 0.00975-0.0176) mg a.i./L
- Key result
- Duration:
- 30 d
- Dose descriptor:
- LC10
- Effect conc.:
- 0.018 mg/L
- 95% CI:
- ca. 0.012 - ca. 0.025
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Post-htach survival
- Remarks on result:
- other: 0.0168 (CI: 0.0117-0.0241) mg a.i./L
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LC10
- Effect conc.:
- 0.013 mg/L
- 95% CI:
- ca. 0.01 - 0.017
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Overall survival
- Remarks on result:
- other: 0.0122 (CI: 0.00956-0.0161) mg a.i./L
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LC10
- Effect conc.:
- 0.016 mg/L
- 95% CI:
- 0.012 - 0.022
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Overall survival
- Remarks on result:
- other: 0.0153 (CI: 0.0112 -0.0215) mg a.i./L
- Key result
- Duration:
- 30 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 0.01 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Post-hatch survival
- Remarks on result:
- other: 0.00956 mg a.i./L
- Key result
- Duration:
- 30 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 0.012 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Post-hatch survival
- Remarks on result:
- other: 0.0112 mg a.i./L
- Key result
- Duration:
- 30 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 0.022 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Post-hatch survival
- Remarks on result:
- other: 0.021 mg a.i./L
- Key result
- Duration:
- 30 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 0.031 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Post-hatch survival
- Remarks on result:
- other: 0.0295 mg a.i./L
- Key result
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 0.01 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Overall survival
- Remarks on result:
- other: 0.00956 mg a.i./L
- Key result
- Duration:
- 34 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 0.012 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Overall survival
- Remarks on result:
- other: 0.0112 mg a.i./L
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 0.022 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: Overall survival
- Remarks on result:
- other: 0.021 mg a.i./L
- Key result
- Duration:
- 34 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 0.031 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: Overall survival
- Remarks on result:
- other: 0.0295 mg a.i./L
- Details on results:
- Biological Data
Egg Fertilization Rate
The mean egg fertilization rate determined on study day 0 (start of the exposure) was 95%.
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 groups and test concentrations of 0.484 and 0.106 mg/L. The hatch of larvae in other test concentrations started on study day 3. The hatch of larvae of the control, solvent control and all test item concentrations was completed until study day 7. Study day 4 was determined to be post hatch day 0 (PHD 0) with a hatching rate of 91% in the control and 98% in the solvent control.
Statistical procedures were applied for the total number of test organisms that have hatched on study days 4 and 7.
The Step-down Rao-Scott-Cochran-Armitage Test Procedure for hatch data after 4 days was done with a significance level of 0.05. Statistically significant differences were found between the pooled controls and the test concentrations of 0.106 and 0.233 mg/L on this study day, since hatch of larvae in these test concentrations was delayed.
The Chi²-2 x 2 Test with Bonferroni Correction with hatch data after 7 days was done with a significance level of 0.05. Statistically significant differences were found between the pooled controls and the nominal test concentration of 0.233 mg/L on this study day.
The NOEC and the LOEC (based on nominal test item concentrations) for this endpoint were determined to be 0.106 mg/L and 0.233 mg/L, respectively. The corresponding NOEC and LOEC (based on overall mean measured test item concentrations) were 0.109 mg/L and 0.225 mg/L, respectively.
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 98% in the control and 97% 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 Step-down Chochran-Armitage Test (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 0.0220 up to 0.233 mg/L.
The NOEC and the LOEC for this endpoint were 0.0100 mg/L and 0.0220 mg/L (nominal concentrations), respectively. The corresponding NOEC and LOEC (based on overall mean measured test item concentrations) were 0.0117 mg/L and 0.0309 mg/L, respectively.
The LC50-value (based on nominal concentrations) for post-hatch survival on study day 34 (PHD30) was 0.0188 (0.0142 to 0.0206) mg/L, corresponding to 0.0257 (0.0180 to 0.0286) mg/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 7% in the control group and 5% in the solvent control group. A concentration-related decrease of the overall survival (increase of overall mortality) was detected in the test item concentrations.
The Step-down Chochran-Armitage Test (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 0.0220 up to 0.233 mg/L.
The NOEC and the LOEC for this endpoint were 0.0100 mg/L and 0.0220 mg/L (nominal concentrations), respectively. The corresponding NOEC and LOEC (based on overall mean measured test item concentrations) were 0.0117 mg/L and 0.0309 mg/L, respectively.
The LC50-value for overall survival on study day 34 (PHD 30) was 0.0184 (0.0149 to 0.0200) mg/L, corresponding to 0.0248 (0.0191 to 0.0276) mg/L (based on overall mean measured test item concentrations).
Morphological and Behavioral Effects
No morphological and behavioral effects were observed in the control and solvent control. In the lowest nominal test item concentration of 0.0100 mg/L only on one day (study day 6) temporarily non-lethal effects were observed. The nominal test concentration of 0.0220 showed an increase of observed non-lethal effects with ongoing exposure from study day 9 up to study day 21. The nominal test concentrations of 0.0484 and 0.106 mg/L showed non-lethal effects for a short period after hatch from study day 6 to 9 resulting in 100 % mortality a few days after hatch and swim-up of larvae. The highest nominal test concentration of 0.233 mg/l showed no morphological and behavioural effects since hatched larvae died within 1 to 2 days after hatch.
Fry Growth
Fry growth, expressed as length and wet weight measurements, was measured on study day 34 (PHD 30) from all survivors.. The growth data of the nominal test concentration of 0.0220 mg/L were excluded from stastistical analysis, since the high mortality in this test concentration affected the biomass loading and the related swarm and feeding behavior of surviving fish.
The two-sample t-test procedure (alpha = 0.05) showed no statistically significant differences for the suriving fish of the nominal test concentration of 0.0100 mg/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 0.0100 and > 0.0100 mg/L, respectively. The corresponding NOEC and LOEC (based on overall mean measured test item concentrations) were 0.0117 mg/L and > 0.0117 mg/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 3 of the control group: 862.4 mg total fish weight. The maximum biomass loading based on the 7.5 liter volume of a single growth chamber was 115 mg/L.
Maximum loading rate: biomass/(volume of test solution) = (862.4 mg)/(7.5 L) = 115 mg/L
The biomass loading rate based upon a flow of 75 liters per day through each single test aquaria was 15.7 mg per liter and day.
Maximum loading rate per day: biomass/(volume of test solution per day) = (862.4 mg)/(75 L) = 11.5 mg/L per day
These loadings were well within the requirements to ensure adequate dissolved oxygen levels and to avoid crowding of the fish. - Nominal flow rates of dilution water (for preparation of the test solutions): 3.125 (2.81 – 3.44 L/h)
- = reanalyzed and confirmed
- = reanalyzed and confirmed
- = reanalyzed and confirmed
- = reanalyzed and confirmed
- = reanalyzed and confirmed
- = reanalyzed and confirmed
- = reanalyzed and confirmed
- = reanalyzed and confirmed
- = reanalyzed and confirmed
- Validity criteria fulfilled:
- yes
- Conclusions:
- Under the conditions of the study, the test substance caused significant effects on Zebrafish in an early life stage test, 30 days post-hatch. Based on overall mean measured test substance concentrations, the EC10, NOEC and LOEC values for the parameter hatch were determined to be 0.196 mg/L (i.e., 0.187 mg a.i./L), 0.109 mg/L (i.e., 0.104 mg a.i./L) and 0.225 mg/L (i.e., 0.215 mg a.i./L), respectively. The NOEC and LOEC values for the parameters post-hatch survival and overall survival were determined to be 0.0117 mg/L (i.e., 0.0112 mg a.i./L) and 0.0309 mg/L (i.e., 0.0295 mg a.i./L), respectively. The corresponding LC10 values were 0.0176 mg/L (0.0168 mg a.i./L) and 0.0160 mg/L (i.e., 0.0153 mg a.i./L), respectively. The NOEC and LOEC values for the parameter fry growth (expressed as length and fresh weight) were determined to be 0.0117 mg/L (i.e., 0.0112 mg a.i./L) and >0.0117 mg/L (i.e., >0.0112 mg a.i./L), respectively.
- Executive summary:
A study was conducted to determine the long-term aquatic toxicity of the test substance,C12-18 DAQ (95.6% active) with Danio rerio / Zebrafish, according to the OECD guideline 210, in compliance with GLP. The test substance is a yellow viscous liquid (paste). Methanol was used as solvent with a concentration of 0.05 mL/L dilution water. Stock solutions in methanol with nominal concentrations of 4.67, 2.12, 0.968, 0.440 and 0.200 g/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 substance concentrations 0.0100, 0.0220, 0.0484, 0.106 and 0.233 mg/L, corresponding to the overall mean measured test substance concentrations of 0.0117, 0.0309, 0.0606, 0.109 and 0.225 mg/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 98% 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 behavioural effects, post-hatch survival and overall survival. Specific analysis of various concentrations of the test substance in the test media and the controls was carried out via LC-MS/MS. The test media were sampled and analysed from alternating test vessels and the corresponding mixing chamber supply prior to exposure on days -1 and during the exposure on study days 0, 7, 8, 9, 16, 23 and 30. The analytical results of the range-finding tests showed that for single compounds (especially with higher carbon chain length) the measured concentrations were above the nominal concentrations in some of the replicates. The reason is not known but the inhomogeneous distribution of the compounds in the test replicates was suspected. In contrast, the measured values from the mixing chambers nearly met the nominal concentrations. Therefore, it was decided to include samples from the mixing chambers in the analytical programme to demonstrate the correct dosage into the flow-through system. The methanol stock solutions were sampled and analysed from freshly prepared and corresponding 7 days aged stock solutions. Measured concentrations of the freshly prepared stock solutions were 96 to 130% of the nominal values. Measured concentrations of the 7 days aged stock solutions in the range of 86 to 127% of the nominal values. All effect values are given based on the nominal and the overall mean measured concentrations of the test substance (see Table 1 and Table 2). The results were also corrected for active ingredient content.
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 substance concentrations [mg/L] with 95% Confidence intervals in brackets)
Parameter
Based on nominal substance concentrations [mg/L]
Based on overall mean measured test substance concentrations [mg/L]
Hatchingsuccess after 7days1)
Frygrowth
expressedas
Hatchingsuccessafter
7days1)
Fry growth expressed as
Length
Weight
Length
Weight
NOEC
0.106
0.0100
0.0100
0.109
0.0117
0.0117
LOEC
0.233
>0.0100
>0.0100
0.225
>0.0117
>0.0117
EC10
0.196
(0.108->0.233)
n.d.
n.d.
0.196
(0.115->0.225)
n.d.
n.d.
EC50
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1)end of hatching period
n.d. not determinable 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 substance concentrations [mg/L] with 95% Confidence intervals in brackets)
Parameter
Based on nominal test substance concentrations [mg/L]
Based on overall mean measured test substance concentrations [mg/L]
Post-hatchsurvival
Overallsurvival
Post-hatchsurvival
Overallsurvival
NOEC
0.0100
0.0100
0.0117
0.0117
LOEC
0.0220
0.0220
0.0309
0.0309
LC10
0.0137
(0.0102–0.0184)
0.0128
(<0.0100–0.0168)
0.0176
(0.0122–0.0252)
0.0160
(0.0117–0.0225)
LC50
0.0188
(0.0142–0.0206)
0.0184
(0.0149–0.0200)
0.0257
(0.0180–0.0286)
0.0248
(0.0191–0.0276)
The test substance caused significant effects on Zebrafish in an early life stage test, 30 days post-hatch. For the parameter hatch, the nominal EC10, NOEC and LOEC were determined to be 0.196 mg/L (i.e., 0.187 mg a.i./L), 0.106 mg/L (i.e., 0.101 mg a.i./L) and 0.233 mg/L (i.e., 0.222 mg a.i./L) respectively. The corresponding EC10, NOEC and LOEC values based on overall mean measured test substance concentrations were 0.196 mg/L (i.e., 0.187 mg a.i./L), 0.109 mg/L (i.e., 0.104 mg a.i./L) and 0.225 mg/L (i.e., 0.215 mg a.i./L), respectively.
For the parameters post-hatch survival and overall survival, the nominal NOECs and LOECs for both parameters were determined to be 0.0100 mg/L (i.e., 0.00956 mg a.i./L) and 0.0220 mg/L (i.e., 0.021 mg a.i./L), respectively. The corresponding NOEC and LOEC values based on overall mean measured test substance concentrations were 0.0117 mg/L (i.e., 0.0112 mg a.i./L) and 0.0309 mg/L (i.e., 0.0295 mg a.i./L), respectively. The nominal LC10 values post-hatch survival and overall survival were determined to be 0.0137 mg/L (i.e., 0.0131 mg a.i./L) and 0.0128 mg/L (i.e., 0.0122 mg a.i./L), respectively. The corresponding LC10 values based on overall mean measured test substance concentrations were 0.0176 mg/L (0.0168 mg a.i./L) and 0.0160 mg/L (i.e., 0.0153 mg a.i./L), respectively.
For the parameter fry growth (expressed as length and fresh weight) the nominal NOECs and LOECs for both parameters were determined to be 0.0100 mg/L (i.e., 0.00956 mg a.i./L) and >0.0100 mg/L (i.e., >0.00956 mg a.i./L), respectively. The corresponding NOEC and LOEC values based on overall mean measured test substance concentrations were 0.0117 mg/L (i.e., 0.0112 mg a.i./L) and >0.0117 mg/L (i.e., >0.0112 mg a.i./L), respectively (Scheerbaum, 2021).
- Endpoint:
- fish early-life stage toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Study period:
- Not available
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Justification for type of information:
- Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- other: US EPA: Methods for acute toxicity tests with fish, macroinvertebrates and amphibians. Ecological Research Service EPA-66013-75-00
- Deviations:
- not specified
- GLP compliance:
- no
- Analytical monitoring:
- yes
- Remarks:
- The test concentrations were confirmed, separated and quantified using HPLC with a conductivity detector.
- Vehicle:
- yes
- Details on test solutions:
- No data
- Test organisms (species):
- Pimephales promelas
- Details on test organisms:
- TEST ORGANISM
- Common name: Fathead minnow
- Source: U.S. Fish and Wildlife Service at Columbia, Missouri - Test type:
- not specified
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 35 d
- Post exposure observation period:
- None
- Hardness:
- Well water: 315-318 ; Town river: 14-38 as mg CaCO3
- Test temperature:
- No data
- pH:
- Well water: 7.1-7.9 ; Town river: 6.4- 7.7
- Dissolved oxygen:
- No data
- Salinity:
- No data
- Nominal and measured concentrations:
- Mean measured concentration:
Well water: Control, 0.006, 0.013, 0.024, 0.053, 0.090 mg/L
Town River water: Control, 0.040, 0.061, 0.130, 0.230, 0.450 mg/L - Details on test conditions:
- - Detailed description of the test substance (purity, nature and quantification of impurities etc.); (71.4% active, 8% monoalkyl impurities)
- Test procedure used (e.g. Semi-static or flow-through); flow through system
- Test design (e.g. Number of test chambers and replicates, number of eggs per replicate, material and size of the test chamber (height, width, volume), water volume per test chamber);
o Two replicates at each test concentration and control
o In the river water study, the five test concentrations were: 0.040, 0.061, 0.130, 0.230 and 0.45 mg a.i./l (measured conc); in the well water study, the five test concentrations were: 0.006, 0.013, 0.024, 0.053 and 0.090 mg a.i./l (measured conc)
o Exposure was intiated within 48 h after fertilisation and continued through 30 d post hatch.
o The river water had the following characteristics: ph = 6.4-6.9, total hardness = 62 mg/l caco3, suspended solids = 9.4 mg/l. The well water had a hardness of 28-31 mg/l caco3, ph = 6.8-7.6
-Method of preparation of stock solutions and frequency of renewal (the solubilising agent and its concentration should be given);
o For the river water study, 0.59 mg/l methylene blue active substance (mbas) and triethyleneglycol was used as carrier solvent.
o For well water study, isopropanol was used as carrier solvent.
- Detailed description of the preparation of test sample for analytical quantification; details not reported
- The recovery efficiency of the method and the nominal test concentrations, the limit of quantification, the means of the measured values and their standard deviations in the test vessels and the method by which these were attained and evidence that the measurements refer to the concentrations of the test chemical in true solution;
o In this river water study, the measured concentrations averaged 45-67% of the nominal concentrations.
o In the well water study, the measured concentrations were equal to the nominal concentrations
- Evidence that controls survival was acceptable; see below table
- Data on mortality at each stage (embryo, larval and juvenile) and cumulative mortality; see below table
- Days to hatch, numbers of larvae hatched each day, and end of hatching; not reported
- Number of healthy fish at end of test; not reported
- Incidence, description and number of morphological abnormalities, if any; not reported
- Incidence, description and number of behavioural effects, if any; not reported
- Approach for the statistical analysis (regression analysis or analysis of the variance) and treatment of data (statistical test or model used). Not reported
o In well water, the most sensitive parameters were mean percent survival, length and weight of larvae. Effects were seen only at the highest mean test conc. Of 0.09 mg/l; the noec was 0.053 mg/l (measured conc)
o In river water, effects on hatchability and mean weight of larvae were seen only at the highest mean test conc. Of 0.45 mg/l; therefore, the noec was determined at 0.23 mg/l after 33 d test duration; see below table for details. - Reference substance (positive control):
- no
- Key result
- Duration:
- 35 d
- Dose descriptor:
- NOEC
- Effect conc.:
- ca. 0.23 mg/L
- Nominal / measured:
- meas. (not specified)
- Conc. based on:
- test mat.
- Basis for effect:
- other: hatchability, survival, mean length and mean weight
- Remarks on result:
- other: in Town River water
- Key result
- Duration:
- 35 d
- Dose descriptor:
- NOEC
- Effect conc.:
- ca. 0.053 mg/L
- Nominal / measured:
- meas. (not specified)
- Conc. based on:
- test mat.
- Basis for effect:
- other: hatchability, survival, mean length and mean weight
- Remarks on result:
- other: in Well water
- Details on results:
- - In the river water test, hatchability and mean weight of fry were reduced only at the highest mean test concentration of 0.45 mg/L test material
- For the laboratory water test, effects on fry survival and mean length and weight of fry were reduced only at the highest mean test concentration of 0.09 mg/L test material. - Results with reference substance (positive control):
- No data
- Reported statistics and error estimates:
- No data
- Validity criteria fulfilled:
- not specified
- Conclusions:
- Based on the results of the read across study, the 35 d NOEC of the test substance was determined to be 0.23 and 0.053 mg/L in Town water and Well water respectively.
- Executive summary:
A study was conducted to determine the long-term aquatic toxicity of the read across substance, C16-18 DAQ (71.4% active, 8% TMAC T) with Pimephales promelas (fathead minnow), according to the US EPA Guidelines. In a 35-day embryo-larval study eggs were exposed to 5 concentrations of the read across substance in filtered well water as well as in river water. The following concentrations were used besides the controls: 0.006; 0.013; 0.024; 0.053; 0.090 mg/L (laboratory water); 0.040; 0.061; 0.130; 0.230; 0.450 mg/L (river water). The test concentrations were confirmed, separated and quantified using HPLC with a conductivity detector. All effect concentrations were based on measured levels of the read across substance. In the study with laboratory water, survival of embryos as well as growth of larvae (length and weight) was reduced at 0.090 mg/L whereas the NOEC was determined to be at 0.053 mg/L. In the study with river water, hatching of embryos as well as growth of larvae (weight) was reduced at 0.450 mg/L whereas the NOEC was determined to be at 0.230 mg/L. Under the study conditions, the 35 d NOEC of the read across substance was determined to be 0.23 and 0.053 mg/L in Town water and Well water respectively (Lewis & Wee, 1983). Based on the results of the read across study, similar effect concentrations can be considered for the test substance.
Referenceopen allclose all
Results
Biological Data
Egg Fertilization Rate
The mean egg fertilization rate determined on study day 0 (start of the exposure) was 95%.
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 groups and test concentrations of 0.484 and 0.106 mg/L. The hatch of larvae in other test concentrations started on study day 3. The hatch of larvae of the control, solvent control and all test item concentrations was completed until study day 7. Study day 4 was determined to be post hatch day 0 (PHD 0) with a hatching rate of 91% in the control and 98% in the solvent control.
Statistical procedures were applied for the total number of test organisms that have hatched on study days 4 and 7.
The Step-down Rao-Scott-Cochran-Armitage Test Procedure for hatch data after 4 days was done with a significance level of 0.05. Statistically significant differences were found between the pooled controls and the test concentrations of 0.106 and 0.233 mg/L on this study day, since hatch of larvae in these test concentrations was delayed.
The Chi²-2 x 2 Test with Bonferroni Correction with hatch data after 7 days was done with a significance level of 0.05. Statistically significant differences were found between the pooled controls and the nominal test concentration of 0.233 mg/L on this study day.
The NOEC and the LOEC (based on nominal test item concentrations) for this endpoint were determined to be 0.106 mg/L and 0.233 mg/L, respectively. The corresponding NOEC and LOEC (based on overall mean measured test item concentrations) were 0.109 mg/L and 0.225 mg/L, respectively.
Hatch / Hatching Time (Control, Solvent Control and Test Item Concentrations)
Nominal |
Rep. |
PHD -2 |
PHD -1 |
PHD 0 |
PHD 1 |
PHD 2 |
PHD 3 |
Study day 2 |
Study day 3 |
Study day 4 |
Study day 5 |
Study day 6 |
Study day 7 |
||
Cumulative hatching rate [%] |
|||||||
Control |
1 |
5 |
70 |
95 |
95 |
95 |
95 |
2 |
0 |
35 |
95 |
100 |
100 |
100 |
|
3 |
0 |
45 |
75 |
85 |
85 |
85 |
|
4 |
0 |
45 |
100 |
100 |
100 |
100 |
|
Mean |
1 |
49 |
91 |
95 |
95 |
95 |
|
Solvent control |
1 |
0 |
95 |
95 |
95 |
95 |
95 |
2 |
0 |
55 |
95 |
95 |
95 |
95 |
|
3 |
5 |
100 |
100 |
100 |
100 |
100 |
|
4 |
0 |
75 |
100 |
100 |
100 |
100 |
|
Mean |
1 |
81 |
98 |
98 |
98 |
98 |
|
0.0100 |
1 |
0 |
45 |
100 |
100 |
100 |
100 |
2 |
0 |
60 |
100 |
100 |
100 |
100 |
|
3 |
0 |
65 |
95 |
95 |
95 |
95 |
|
4 |
0 |
45 |
100 |
100 |
100 |
100 |
|
Mean |
0 |
54 |
99 (-) |
99 |
99 |
99 (-) |
|
0.0220 |
1 |
0 |
15 |
45 |
85 |
95 |
100 |
2 |
0 |
30 |
90 |
95 |
95 |
95 |
|
3 |
0 |
20 |
100 |
100 |
100 |
100 |
|
4 |
0 |
35 |
85 |
95 |
95 |
95 |
|
Mean |
0 |
25 |
80 (-) |
94 |
96 |
98 (-) |
|
0.0484 |
1 |
0 |
35 |
100 |
100 |
100 |
100 |
2 |
0 |
35 |
75 |
85 |
100 |
100 |
|
3 |
5 |
75 |
95 |
95 |
95 |
95 |
|
4 |
0 |
40 |
100 |
100 |
100 |
100 |
|
Mean |
1 |
46 |
93 (-) |
95 |
99 |
99 (-) |
|
0.106 |
1 |
0 |
30 |
90 |
90 |
90 |
95 |
2 |
0 |
35 |
75 |
90 |
90 |
90 |
|
3 |
5 |
20 |
85 |
100 |
100 |
100 |
|
4 |
0 |
25 |
65 |
80 |
85 |
90 |
|
Mean |
1 |
28 |
79 (+) |
90 |
91 |
94 (-) |
|
0.233 |
1 |
0 |
45 |
90 |
95 |
95 |
95 |
2 |
0 |
25 |
75 |
85 |
85 |
85 |
|
3 |
0 |
25 |
60 |
70 |
85 |
85 |
|
4 |
0 |
25 |
55 |
70 |
75 |
85 |
|
Mean |
0 |
30 |
70 (+) |
80 |
85 |
88 (+) |
|
(-) / (+) = No statistically / statistically significant difference from pooled control groups
Swim-up
The swim-up period of the control groups and the two lowest nominal test concentrations of 0.0100 and 0.0220 mg/L was observed from study day 4 to 7. First swim-up of few larvae of the nominal test concentrations 0.0484 and 0.106 mg/L was observed on study day 4. However completion of swim-up was not observed for these concentrations, since behavioural and non-lethal effects interfered with the swim-up. For the highest tested concentration no swim-up of hatched larvae was obsereved. No statistical analysis of swim-up data was carried out.
Percent Swim-up of Hatched Live Fry of the Control, Solvent Control and the Nominal Test Item Concentrations 0.0100 to 0.0484 mg/L
Nominal |
Rep. |
PHD 0 |
PHD 1 |
PHD 2 |
PHD 3 |
PHD 4 |
Study day 4 |
Study day 5 |
Study day 6 |
Study day 7 |
Study day 8 |
||
Cumulative swim up [%] |
||||||
Control |
1 |
0 |
100 |
100 |
100 |
100 |
2 |
0 |
100 |
100 |
100 |
100 |
|
3 |
13 |
100 |
100 |
100 |
100 |
|
4 |
15 |
100 |
100 |
100 |
100 |
|
Mean |
7 |
100 |
100 |
100 |
100 |
|
Solvent control |
1 |
0 |
95 |
100 |
100 |
100 |
2 |
0 |
95 |
84 |
100 |
100 |
|
3 |
0 |
100 |
100 |
100 |
100 |
|
4 |
0 |
95 |
100 |
100 |
100 |
|
Mean |
0 |
96 |
96 |
100 |
100 |
|
0.0100 |
1 |
5 |
100 |
100 |
100 |
100 |
2 |
0 |
100 |
80 |
100 |
100 |
|
3 |
11 |
100 |
95 |
100 |
100 |
|
4 |
10 |
100 |
100 |
100 |
100 |
|
Mean |
7 |
100 |
94 |
100 |
100 |
|
0.0220 |
1 |
11 |
82 |
100 |
100 |
100 |
2 |
17 |
95 |
95 |
100 |
100 |
|
3 |
0 |
95 |
95 |
100 |
100 |
|
4 |
6 |
100 |
100 |
100 |
100 |
|
Mean |
9 |
93 |
98 |
100 |
100 |
|
0.0484 |
1 |
0 |
90 |
95 |
35 |
0 |
2 |
0 |
76 |
95 |
29 |
0 |
|
3 |
0 |
100 |
89 |
11 |
0 |
|
4 |
5 |
10 |
90 |
61 |
13 |
|
Mean |
1 |
69 |
92 |
34 |
3 |
|
Percent Swim-up of Hatched Live Fry of the Nominal Test Item Concentrations 0.106 and 0.233 mg/L
Nominal |
Rep. |
PHD 0 |
PHD 1 |
PHD 2 |
PHD 3 |
PHD 4 |
Study day 4 |
Study day 5 |
Study day 6 |
Study day 7 |
Study day 8 |
||
Cumulative swim up [%] |
||||||
0.106 |
1 |
6 |
22 |
7 |
0 |
n.d. |
2 |
0 |
22 |
6 |
0 |
||
3 |
0 |
30 |
16 |
0 |
||
4 |
0 |
6 |
17 |
0 |
||
Mean |
2 |
20 |
12 |
0 |
- |
|
0.233 |
1 |
0 |
0 |
n.d. |
n.d. |
n.d. |
2 |
0 |
0 |
||||
3 |
0 |
0 |
||||
4 |
0 |
0 |
0 |
|||
Mean |
0 |
0 |
- |
- |
- |
|
n.d. = not determinable, due to 100 % mortality
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 98% in the control and 97% 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 Step-down Chochran-Armitage Test (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 0.0220 up to 0.233 mg/L.
The NOEC and the LOEC for this endpoint were 0.0100 mg/L and 0.0220 mg/L (nominal concentrations), respectively. The corresponding NOEC and LOEC (based on overall mean measured test item concentrations) were 0.0117 mg/L and 0.0309 mg/L, respectively.
The LC50-value (based on nominal concentrations) for post-hatch survival on study day 34 (PHD30) was 0.0188 (0.0142 to 0.0206) mg/L, corresponding to 0.0257 (0.0180 to 0.0286) mg/L (based on overall mean measured test item concentrations).
Post-Hatch Survival on Study Day 34 (PHD 30) of the Control, Solvent Control and the Nominal Test Item Concentrations 0.0100 and 0.0220 mg/L
Nominal |
Rep. |
Eggs introduced on study day 0 |
Cumulative number of hatched larvae |
Vital Larvae on study day 34 |
Post-Hatch survival |
Control |
1 |
20 |
19 |
18 |
95 |
2 |
20 |
20 |
19 |
95 |
|
3 |
20 |
17 |
17 |
100 |
|
4 |
20 |
20 |
20 |
100 |
|
Mean |
20 |
19 |
19 |
98 |
|
Solvent control |
1 |
20 |
19 |
19 |
100 |
2 |
20 |
19 |
17 |
89 |
|
3 |
20 |
20 |
20 |
100 |
|
4 |
20 |
20 |
20 |
100 |
|
Mean |
20 |
19 |
19 |
97 |
|
0.0100 |
1 |
20 |
20 |
20 |
100 |
2 |
20 |
20 |
20 |
100 |
|
3 |
20 |
19 |
18 |
95 |
|
4 |
20 |
20 |
20 |
100 |
|
Mean |
20 |
19 |
20 |
99 (-) |
|
0.0220 |
1 |
20 |
20 |
2 |
10 |
2 |
20 |
19 |
4 |
21 |
|
3 |
20 |
20 |
10 |
50 |
|
4 |
20 |
19 |
4 |
21 |
|
Mean |
20 |
19 |
5 |
26 (+) |
(-) / (+) = No statistically / statistically significant difference from pooled control groups
Post-Hatch Survival on Study Day 34 (PHD 30) of the Nominal Test Item Concentrations 0.0484, 0.106 and 0.233 mg/L
Nominal |
Rep. |
Eggs introduced on study day 0 |
Cumulative number of hatched larvae |
Vital Larvae on study day 34 |
Post-Hatch survival |
0.0484 |
1 |
20 |
20 |
0 |
0 |
2 |
20 |
20 |
0 |
0 |
|
3 |
20 |
19 |
0 |
0 |
|
4 |
20 |
20 |
0 |
0 |
|
Mean |
20 |
19 |
0 |
0 (+) |
|
0.106 |
1 |
20 |
19 |
0 |
0 |
2 |
20 |
18 |
0 |
0 |
|
3 |
20 |
20 |
0 |
0 |
|
4 |
20 |
18 |
0 |
0 |
|
Mean |
20 |
19 |
0 |
0 (+) |
|
0.233 |
1 |
20 |
19 |
0 |
0 |
2 |
20 |
17 |
0 |
0 |
|
3 |
20 |
17 |
0 |
0 |
|
4 |
20 |
17 |
0 |
0 |
|
Mean |
20 |
18 |
0 |
0 (+) |
(+) = Statistically significant difference from pooled control groups
Overall Survival
The cumulative mortality at the end of the exposure, related to the number of eggs introduced on day 0, was 7% in the control group and 5% in the solvent control group. A concentration-related decrease of the overall survival (increase of overall mortality) was detected in the test item concentrations.
The Step-down Chochran-Armitage Test (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 0.0220 up to 0.233 mg/L.
The NOEC and the LOEC for this endpoint were 0.0100 mg/L and 0.0220 mg/L (nominal concentrations), respectively. The corresponding NOEC and LOEC (based on overall mean measured test item concentrations) were 0.0117 mg/L and 0.0309 mg/L, respectively.
LC50-value for overall survival on study day 34 (PHD 30) was 0.0184 (0.0149 to 0.0200) mg/L, corresponding to 0.0248 (0.0191 to 0.0276) mg/L (based on overall mean measured test item concentrations).
Overall Survival and Overall Mortality on Study Day 34 (PHD 30) of the Control, Solvent Control and the Nominal Test Item Concentrations 0.0100 and 0.0220 mg/L
Nominal |
Rep. |
Vital larvae on |
Overall survival |
Overall mortality |
Control |
1 |
18 |
90 |
10 |
2 |
19 |
95 |
5 |
|
3 |
17 |
85 |
15 |
|
4 |
20 |
100 |
0 |
|
Mean |
19 |
93 |
7 |
|
Solvent control |
1 |
19 |
95 |
5 |
2 |
17 |
85 |
15 |
|
3 |
20 |
100 |
0 |
|
4 |
20 |
100 |
0 |
|
Mean |
19 |
95 |
5 |
|
0.0100 |
1 |
20 |
100 |
0 |
2 |
20 |
100 |
0 |
|
3 |
18 |
90 |
10 |
|
4 |
20 |
100 |
0 |
|
Mean |
20 |
98 (-) |
2 (-) |
|
0.0220 |
1 |
2 |
10 |
90 |
2 |
4 |
20 |
80 |
|
3 |
10 |
50 |
50 |
|
4 |
4 |
20 |
80 |
|
Mean |
5 |
25 (+) |
75 (+) |
(-) / (+) = No statistically / statistically significant difference from pooled control groups
Overall Survival and Overall Mortality on Study Day 34 (PHD 30) of the Nominal Test Item Concentrations 0.0484, 0.106 amd 0.233 mg/L
Nominal |
Rep. |
Vital larvae on |
Overall survival |
Overall mortality |
0.0484 |
1 |
0 |
0 |
100 |
2 |
0 |
0 |
100 |
|
3 |
0 |
0 |
100 |
|
4 |
0 |
0 |
100 |
|
Mean |
0 |
0 (+) |
100 (+) |
|
0.106 |
1 |
0 |
0 |
100 |
2 |
0 |
0 |
100 |
|
3 |
0 |
0 |
100 |
|
4 |
0 |
0 |
100 |
|
Mean |
0 |
0 (+) |
100 (+) |
|
0.233 |
1 |
0 |
0 |
100 |
2 |
0 |
0 |
100 |
|
3 |
0 |
0 |
100 |
|
4 |
0 |
0 |
100 |
|
Mean |
0 |
0 (+) |
100 (+) |
(+) = Statistically significant difference from pooled control groups
Morphological and Behavioral Effects
No morphological and behavioral effects were observed in the control and solvent control. In the lowest nominal test item concentration of 0.0100 mg/L only on one day (study day 6) temporarily non-lethal effects were observed. The nominal test concentration of 0.0220 showed an increase of observed non-lethal effects with ongoing exposure from study day 9 up to study day 21. The nominal test concentrations of 0.0484 and 0.106 mg/L showed non-lethal effects for a short period after hatch from study day 6 to 9 resulting in 100 % mortality a few days after hatch and swim-up of larvae. The highest nominal test concentration of 0.233 mg/l showed no morphological and behavioural effects since hatched larvae died within 1 to 2 days after hatch.
Behavioral Effects observed in the Nominal Test Concentrations 0.0100 to 0.106 mg/L (Study Day 6 – 21)
Study Day |
0.0100 mg/L |
0.0220 mg/L |
0.0484 mg/L |
0.106 mg/L |
||||||||||||
1 |
2 |
3 |
4 |
1 |
2 |
3 |
4 |
1 |
2 |
3 |
4 |
1 |
2 |
3 |
4 |
|
6 |
- |
4 Q |
1 Q |
- |
- |
- |
- |
- |
- |
- |
2 Q |
- |
- |
- |
- |
- |
7 |
- |
- |
- |
- |
- |
- |
- |
- |
13 Q |
12 Q |
17 Q |
7 Q |
2 Q |
2 Q |
3 Q |
3 Q |
8 |
- |
- |
- |
- |
- |
- |
- |
- |
8 Q |
3 Q |
5 Q |
7 Q |
|
|||
9 |
- |
- |
- |
- |
1 Q |
- |
1 Q |
- |
1 Q |
1 Q |
- |
- |
||||
10 |
- |
- |
- |
- |
1 T |
- |
- |
- |
100 % mortality |
|||||||
11 |
- |
- |
- |
- |
4 Q |
- |
- |
4 Q |
||||||||
12 |
- |
- |
- |
- |
6 Q |
- |
2 Q |
2 Q |
||||||||
13 |
- |
- |
- |
- |
7 Q |
4 Q |
1 Q |
5 Q |
||||||||
14 |
- |
- |
- |
- |
5 Q |
7 Q |
1 Q |
3 Q |
||||||||
15 |
- |
- |
- |
- |
4 Q |
5 Q |
2 Q |
- |
||||||||
16 |
- |
- |
- |
- |
1 Q |
2 Q |
3 Q |
2 Q |
||||||||
17 |
- |
- |
- |
- |
1 Q |
1 Q |
3 Q |
2 Q |
||||||||
18 |
- |
- |
- |
- |
- |
1 Q |
- |
- |
||||||||
19 |
- |
- |
- |
- |
- |
- |
- |
2 Q |
||||||||
20 |
- |
- |
- |
- |
- |
- |
- |
2 Q |
||||||||
21 |
- |
- |
- |
- |
- |
- |
- |
1 Q |
||||||||
Q = Quiescence T = Tumbling
G = Arresting on the ground S = Arresting at the surface
F = No food uptake
Fry Growth
Fry growth, expressed as length and wet weight measurements, was measured on study day 34 (PHD 30) from all survivors. The growth data of the nominal test concentration of 0.0220 mg/L were excluded from stastistical analysis, since the high mortality in this test concentration affected the biomass loading and the related swarm and feeding behavior of surviving fish.
The two-sample t-test procedure (alpha = 0.05) showed no statistically significant differences for the suriving fish of the nominal test concentration of 0.0100 mg/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 0.0100 and > 0.0100 mg/L, respectively. The corresponding NOEC and LOEC (based on overall mean measured test item concentrations) were 0.0117 mg/L and > 0.0117 mg/L, respectively.
Overview of Fry Growth: Length and Wet Weight on Study Day 34 (PHD 30) of the Control, Solvent Control and Nominal Test Item Concentrations 0.0100 and 0.0220 mg/L
Nominal |
Rep. |
PHD 30 (End of exposure) |
|
Mean total length per fish [mm] |
Mean wet weight per fish [mg] |
||
Control |
1 |
17.6 |
42.5 |
2 |
17.1 |
42.6 |
|
3 |
18.4 |
50.7 |
|
4 |
16.3 |
35.0 |
|
Mean |
17.4 |
42.7 |
|
± SD |
0.763 |
5.55 |
|
CV [%] |
4.39 |
13.0 |
|
Solvent control |
1 |
15.8 |
34.7 |
2 |
17.0 |
41.3 |
|
3 |
16.2 |
37.9 |
|
4 |
16.6 |
41.2 |
|
Mean |
16.4 |
38.8 |
|
± SD |
0.447 |
2.72 |
|
CV [%] |
2.73 |
7.02 |
|
0.0100 |
1 |
15.8 |
34.7 |
2 |
16.1 |
38.2 |
|
3 |
16.2 |
40.1 |
|
4 |
16.3 |
39.7 |
|
Mean |
16.1 (-) |
38.2 (-) |
|
± SD |
0.187 |
2.13 |
|
CV [%] |
1.16 |
5.57 |
|
0.02201) |
1 |
21.3 |
94.5 |
2 |
19.3 |
78.4 |
|
3 |
18.3 |
57.8 |
|
4 |
17.5 |
63.1 |
|
Mean |
19.1 |
73.5 |
|
± SD |
1.42 |
14.3 |
|
CV [%] |
7.44 |
19.5 |
|
(-) = No statistically significant difference from pooled controls 1) Excluded from statistical analysis
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 |
18.0 |
18.5 |
21.0 |
16.0 |
16.5 |
18.0 |
18.0 |
12.0 |
2 |
16.0 |
17.0 |
21.0 |
15.0 |
17.5 |
17.0 |
18.0 |
10.0 |
3 |
17.0 |
18.0 |
17.0 |
13.5 |
17.0 |
16.0 |
20.0 |
16.0 |
4 |
17.0 |
18.0 |
20.0 |
15.0 |
16.0 |
16.5 |
15.5 |
18.0 |
5 |
16.0 |
19.0 |
18.0 |
16.0 |
15.0 |
19.5 |
15.0 |
10.0 |
6 |
18.0 |
18.0 |
17.0 |
17.5 |
15.0 |
16.0 |
17.0 |
20.0 |
7 |
18.0 |
18.0 |
19.5 |
17.0 |
19.0 |
15.0 |
13.0 |
13.0 |
8 |
18.0 |
17.5 |
17.5 |
16.0 |
19.0 |
19.0 |
16.5 |
18.0 |
9 |
16.0 |
11.0 |
17.5 |
14.5 |
18.0 |
19.5 |
17.0 |
17.0 |
10 |
21.0 |
16.0 |
17.0 |
14.0 |
8.0 |
16.0 |
16.0 |
17.0 |
11 |
18.5 |
18.0 |
20.0 |
18.0 |
13.5 |
19.0 |
14.0 |
20.0 |
12 |
17.5 |
16.0 |
19.0 |
19.0 |
19.0 |
13.0 |
13.0 |
20.0 |
13 |
15.5 |
13.5 |
19.0 |
15.0 |
20.0 |
19.5 |
16.0 |
19.0 |
14 |
18.0 |
18.0 |
15.5 |
18.0 |
18.0 |
14.5 |
20.0 |
18.0 |
15 |
18.0 |
19.0 |
17.0 |
16.0 |
17.0 |
19.0 |
14.0 |
13.0 |
16 |
17.5 |
17.0 |
17.0 |
18.5 |
11.0 |
14.0 |
16.0 |
18.0 |
17 |
18.0 |
16.5 |
20.0 |
16.0 |
12.0 |
17.5 |
17.0 |
18.0 |
18 |
19.0 |
18.0 |
- |
17.5 |
11.0 |
- |
16.0 |
21.0 |
19 |
- |
17.0 |
- |
18.0 |
18.0 |
- |
13.0 |
17.0 |
20 |
- |
- |
- |
16.0 |
- |
- |
18.0 |
17.0 |
Mean |
17.6 |
17.1 |
18.4 |
16.3 |
15.8 |
17.0 |
16.2 |
16.6 |
± SD |
1.25 |
1.89 |
1.59 |
1.52 |
3.22 |
2.04 |
2.04 |
3.20 |
CV % |
7.12 |
11.1 |
8.66 |
9.32 |
20.4 |
12.0 |
12.6 |
19.3 |
- = Fish died before end of the study
Individual Length on Study Day 34 (PHD 30) of the Nominal Test Item Concentrations 0.0100 and 0.0220 mg/L
Fish No. |
Nominal test item conc. [mg/L] |
|||||||
0.0100 |
0.0220 |
|||||||
Total length of individual fish in [mm] |
||||||||
1 |
2 |
3 |
4 |
1 |
2 |
3 |
4 |
|
1 |
16.0 |
16.0 |
16.0 |
16.0 |
22.0 |
15.0 |
19.0 |
17.0 |
2 |
17.0 |
19.0 |
17.0 |
19.0 |
20.5 |
16.0 |
18.5 |
21.0 |
3 |
15.5 |
12.0 |
16.0 |
13.0 |
- |
21.0 |
21.0 |
10.0 |
4 |
16.0 |
17.5 |
17.0 |
17.0 |
- |
25.0 |
22.0 |
22.0 |
5 |
18.0 |
18.0 |
14.0 |
15.0 |
- |
- |
19.5 |
- |
6 |
17.0 |
18.0 |
15.0 |
17.5 |
- |
- |
18.0 |
- |
7 |
17.5 |
17.0 |
17.0 |
17.0 |
- |
- |
19.0 |
- |
8 |
17.0 |
14.0 |
13.0 |
13.0 |
- |
- |
12.0 |
- |
9 |
11.0 |
14.0 |
14.0 |
15.0 |
- |
- |
16.0 |
- |
10 |
12.5 |
17.5 |
14.0 |
17.0 |
- |
- |
17.5 |
- |
11 |
9.0 |
18.0 |
18.0 |
18.0 |
- |
- |
- |
- |
12 |
16.0 |
17.0 |
18.0 |
18.0 |
- |
- |
- |
- |
13 |
17.5 |
15.0 |
19.0 |
19.0 |
- |
- |
- |
- |
14 |
17.0 |
17.0 |
20.0 |
13.0 |
- |
- |
- |
- |
15 |
16.5 |
13.0 |
20.0 |
17.0 |
- |
- |
- |
- |
16 |
16.0 |
15.0 |
16.0 |
16.0 |
- |
- |
- |
- |
17 |
16.5 |
15.0 |
12.5 |
15.0 |
- |
- |
- |
- |
18 |
21.0 |
17.0 |
15.0 |
18.0 |
- |
- |
- |
- |
19 |
14.0 |
14.0 |
- |
16.0 |
- |
- |
- |
- |
20 |
15.0 |
18.0 |
- |
16.0 |
- |
- |
- |
- |
Mean |
15.8 |
16.1 |
16.2 |
16.3 |
21.3 |
19.3 |
18.3 |
17.5 |
± SD |
2.54 |
1.91 |
2.19 |
1.80 |
0.750 |
4.02 |
2.63 |
4.72 |
CV % |
16.1 |
11.9 |
13.5 |
11.0 |
3.52 |
20.8 |
14.4 |
27.0 |
- = 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 0.0100 and 0.0220 mg/L
Nominal |
Replicate |
Number of fish alive |
Pooled |
Mean |
Mean |
± SD |
CV % |
Control |
1 |
18 |
765.0 |
42.5 |
42.7 |
5.55 |
13.0 |
2 |
19 |
808.9 |
42.6 |
||||
3 |
17 |
862.4 |
50.7 |
||||
4 |
20 |
700.0 |
35.0 |
||||
Solvent control |
1 |
19 |
658.9 |
34.7 |
38.8 |
2.72 |
7.02 |
2 |
17 |
702.4 |
41.3 |
||||
3 |
20 |
758.7 |
37.9 |
||||
4 |
20 |
824.5 |
41.2 |
||||
0.0100 |
1 |
20 |
694.7 |
34.7 |
38.2 |
2.13 |
5.57 |
2 |
20 |
764.7 |
38.2 |
||||
3 |
18 |
721.1 |
40.1 |
||||
4 |
20 |
794.6 |
39.7 |
||||
0.02201) |
1 |
2 |
189.0 |
94.5 |
73.5 |
14.3 |
19.5 |
2 |
4 |
313.6 |
78.4 |
||||
3 |
10 |
577.8 |
57.8 |
||||
4 |
4 |
252.2 |
63.1 |
1) Excluded from statistical analysis, for details see part 7.1.7
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 3 of the control group: 862.4 mg total fish weight. The maximum biomass loading based on the 7.5 liter volume of a single growth chamber was 115 mg/L.
Maximum loading rate: = = 115 mg/L
The biomass loading rate based upon a flow of 75 liters per day through each single test aquaria was 15.7 mg per liter and day.
Maximum loading rate per day: = = 11.5 mg/L per day
These loadings were well within the requirements to ensure adequate dissolved oxygen levels and to avoid crowding of the fish.
Physico-chemical Data
Dissolved Oxygen
The dissolved oxygen concentrations in the control, solvent control and the test item groups, expressed in percent saturation, were in the mean 95 – 100% and ranged from 87 to 100% during the exposure period.
Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 0 -15)
Study day |
Replicate |
Dissolved oxygen [%] |
||||||
Nominal test item concentrations [mg/L] |
||||||||
Control |
Solvent control |
0.0100 |
0.0220 |
0.0484 |
0.106 |
0.233 |
||
0 |
1 |
100 |
100 |
100 |
100 |
100 |
100 |
100 |
2 |
100 |
100 |
100 |
100 |
100 |
100 |
100 |
|
3 |
100 |
100 |
100 |
100 |
100 |
100 |
100 |
|
4 |
100 |
100 |
100 |
100 |
100 |
100 |
100 |
|
1 |
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 |
|
3 |
1 |
100 |
100 |
100 |
100 |
99 |
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 |
|
6 |
1 |
100 |
99 |
99 |
99 |
98 |
99 |
99 |
2 |
99 |
99 |
100 |
99 |
99 |
97 |
100 |
|
3 |
100 |
100 |
98 |
99 |
98 |
98 |
98 |
|
4 |
100 |
99 |
99 |
97 |
98 |
99 |
100 |
|
8 |
1 |
100 |
99 |
99 |
99 |
98 |
100 |
- |
2 |
100 |
99 |
100 |
98 |
100 |
99 |
- |
|
3 |
99 |
99 |
98 |
98 |
98 |
100 |
- |
|
4 |
99 |
99 |
98 |
99 |
99 |
96 |
- |
|
10 |
1 |
97 |
96 |
96 |
96 |
97 |
- |
- |
2 |
97 |
97 |
96 |
96 |
97 |
- |
- |
|
3 |
98 |
97 |
96 |
97 |
- |
- |
- |
|
4 |
98 |
97 |
96 |
97 |
- |
- |
- |
|
13 |
1 |
96 |
96 |
96 |
96 |
- |
- |
- |
2 |
97 |
96 |
97 |
96 |
- |
- |
- |
|
3 |
97 |
95 |
96 |
96 |
- |
- |
- |
|
4 |
97 |
95 |
96 |
95 |
- |
- |
- |
|
15 |
1 |
96 |
96 |
95 |
96 |
- |
- |
- |
2 |
97 |
96 |
96 |
96 |
- |
- |
- |
|
3 |
97 |
95 |
94 |
96 |
- |
- |
- |
|
4 |
96 |
95 |
96 |
96 |
- |
- |
- |
|
- = No measurement, due to 100 % mortality
Dissolved Oxygen in Percent Air Saturation Value in the Test Media (Study Day 17 - 31)
Study day |
Replicate |
Dissolved oxygen [%] |
||||||
Nominal test item concentrations [mg/L] |
||||||||
Control |
Solvent control |
0.0100 |
0.0220 |
0.0484 |
0.106 |
0.233 |
||
17 |
1 |
98 |
96 |
95 |
96 |
- |
- |
- |
2 |
98 |
96 |
97 |
96 |
- |
- |
- |
|
3 |
97 |
95 |
94 |
96 |
- |
- |
- |
|
4 |
97 |
95 |
95 |
96 |
- |
- |
- |
|
20 |
1 |
97 |
96 |
96 |
97 |
- |
- |
- |
2 |
97 |
96 |
96 |
96 |
- |
- |
- |
|
3 |
97 |
95 |
94 |
95 |
- |
- |
- |
|
4 |
97 |
95 |
96 |
96 |
- |
- |
- |
|
22 |
1 |
96 |
95 |
96 |
97 |
- |
- |
- |
2 |
96 |
94 |
94 |
95 |
- |
- |
- |
|
3 |
97 |
94 |
93 |
94 |
- |
- |
- |
|
4 |
96 |
94 |
95 |
95 |
- |
- |
- |
|
24 |
1 |
95 |
94 |
94 |
95 |
- |
- |
- |
2 |
95 |
93 |
93 |
94 |
- |
- |
- |
|
3 |
95 |
93 |
93 |
93 |
- |
- |
- |
|
4 |
94 |
93 |
95 |
94 |
- |
- |
- |
|
27 |
1 |
96 |
89 |
93 |
95 |
- |
- |
- |
2 |
95 |
90 |
89 |
93 |
- |
- |
- |
|
3 |
96 |
88 |
89 |
91 |
- |
- |
- |
|
4 |
95 |
90 |
91 |
93 |
- |
- |
- |
|
29 |
1 |
93 |
89 |
93 |
93 |
- |
- |
- |
2 |
94 |
89 |
89 |
93 |
- |
- |
- |
|
3 |
94 |
87 |
90 |
91 |
- |
- |
- |
|
4 |
94 |
90 |
91 |
92 |
- |
- |
- |
|
31 |
1 |
94 |
89 |
92 |
94 |
- |
- |
- |
2 |
93 |
90 |
91 |
94 |
- |
- |
- |
|
3 |
94 |
89 |
91 |
91 |
- |
- |
- |
|
4 |
93 |
89 |
92 |
92 |
- |
- |
- |
|
Mean |
97 |
95 |
96 |
96 |
99 |
99 |
100 |
|
± SD |
2.24 |
3.89 |
3.31 |
2.68 |
1.08 |
1.14 |
0.54 |
|
Min. |
93 |
87 |
89 |
91 |
97 |
96 |
98 |
|
Max. |
100 |
100 |
100 |
100 |
100 |
100 |
100 |
|
- = No measurement, due to 100 % mortality
Min./Max. = Minimum/Maximum measured dissolved oxygen concentration
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 (Table 18). The minimum temperature was 24.5 °C and the maximum temperature was 27.1 °C.
The mean water temperature measured once per week from all replicates during the exposure period was 26.5 °C for the control. The minimum and maximum measured temperature for the control were 25.9 and 26.8 °C, respectively.
The recommended water temperature between the test vessels or between successive days at any time during the test differed more than ± 1.5 °C (2.2 °C) once during the test on study day 12. This deviation is considered to have no impact on quality and integrity of the study.
The validity criterion for the parameter temperature was fulfilled with one restriction.
Water Temperature (Continuous Measuring) in Replicate 2 of the Control Group
Period of measurements |
2020-11-10 to 2020-12-14 |
Minimum temperature [°C] |
24.5 |
Maximum temperature [°C] |
27.1 |
Mean temperature [°C] |
26.2 |
Water Temperature in the Test Media
Study day |
Rep. |
Water temperature [°C] |
||||||
Nominal test item concentrations [mg/L] |
||||||||
Control |
Solvent control |
0.0100 |
0.0220 |
0.0484 |
0.106 |
0.233 |
||
1 |
1 |
26.8 |
26.5 |
26.6 |
26.5 |
26.5 |
26.7 |
26,,4 |
2 |
26.6 |
26.7 |
26.5 |
26.5 |
26.4 |
26.5 |
26.6 |
|
3 |
26.6 |
26.5 |
26.7 |
26.6 |
26.6 |
26.7 |
26.7 |
|
4 |
26.6 |
26.6 |
26.5 |
26.6 |
26.7 |
26.6 |
26.7 |
|
8 |
1 |
26.3 |
26.2 |
26.4 |
26.3 |
26.2 |
26.4 |
- |
2 |
25.9 |
26.6 |
26.3 |
26.7 |
26.6 |
26.4 |
- |
|
3 |
26.4 |
26.5 |
26.2 |
26.4 |
26.7 |
26.5 |
- |
|
4 |
26.1 |
26.4 |
26.3 |
26.6 |
26.3 |
26.6 |
- |
|
15 |
1 |
26.6 |
26.6 |
26.8 |
26.7 |
- |
- |
- |
2 |
26.4 |
26.8 |
26.7 |
27.0 |
- |
- |
- |
|
3 |
26.6 |
27.0 |
26.7 |
26.8 |
- |
- |
- |
|
4 |
26.4 |
26.7 |
26.6 |
26.8 |
- |
- |
- |
|
22 |
1 |
26.7 |
26.6 |
26.8 |
26.5 |
- |
- |
- |
2 |
26.3 |
26.8 |
26.7 |
26.7 |
- |
- |
- |
|
3 |
26.7 |
27.0 |
26.3 |
26.9 |
- |
- |
- |
|
4 |
26.5 |
26.7 |
26.4 |
26.9 |
- |
- |
- |
|
29 |
1 |
26.6 |
26.7 |
26.8 |
26.7 |
- |
- |
- |
2 |
26.4 |
26.8 |
26.6 |
26.9 |
- |
- |
- |
|
3 |
26.7 |
27.0 |
26.6 |
26.8 |
- |
- |
- |
|
4 |
26.5 |
26,,8 |
26.7 |
26.7 |
- |
- |
- |
|
Mean |
26.5 |
26.7 |
26.6 |
26.7 |
26.5 |
26.6 |
26.7 |
|
± SD |
0.218 |
0.208 |
0.188 |
0.182 |
0.185 |
0.120 |
0.058 |
|
Min. |
25.9 |
26.2 |
26.2 |
26.3 |
26.2 |
26.4 |
26.6 |
|
Max. |
26.8 |
27.0 |
26.8 |
27.0 |
26.7 |
26.7 |
26.7 |
|
- = No measurement, due to 100 % mortality
Min./Max. = Minimum/Maximum measured water temperature
pH-Values
The mean pH-values in the control, solvent control and test item groups were between 7.87 and 8.16 and ranged from 7.44 to 8.19 during the exposure period .
pH-Values in the Test Media
Study day |
Rep. |
pH-value |
||||||
Nominal test item concentrations [mg/L] |
||||||||
Control |
Solvent control |
0.0100 |
0.0220 |
0.0484 |
0.106 |
0.233 |
||
1 |
1 |
8.14 |
8.16 |
8.15 |
8.19 |
8.16 |
8.11 |
8.12 |
2 |
8.17 |
8.17 |
8.14 |
8.12 |
8.15 |
8.15 |
8.16 |
|
3 |
8.15 |
8.16 |
8.16 |
8.14 |
8.16 |
8.17 |
8.17 |
|
4 |
8.11 |
8.14 |
8.17 |
8.15 |
8.16 |
8.17 |
8.17 |
|
8 |
1 |
7.90 |
7.92 |
7.93 |
7.93 |
7.95 |
8.04 |
- |
2 |
7.96 |
7.96 |
7.94 |
7.94 |
8.05 |
7.96 |
- |
|
3 |
7.96 |
7.89 |
7.93 |
7.93 |
7.98 |
7.97 |
- |
|
4 |
7.96 |
7.95 |
7.93 |
7.99 |
7.96 |
7.89 |
- |
|
15 |
1 |
7.76 |
7.83 |
7.82 |
7.83 |
- |
- |
- |
2 |
7.87 |
7.84 |
7.84 |
7.82 |
- |
- |
- |
|
3 |
7.86 |
7.81 |
7.81 |
7.84 |
- |
- |
- |
|
4 |
7.85 |
7.81 |
7.82 |
7.84 |
- |
- |
- |
|
22 |
1 |
7.44 |
7.71 |
7.72 |
7.73 |
- |
- |
- |
2 |
7.67 |
7.70 |
7.71 |
7.78 |
- |
- |
- |
|
3 |
7.72 |
7.73 |
7.72 |
7.72 |
- |
- |
- |
|
4 |
7.73 |
7.72 |
7.71 |
7.73 |
- |
- |
- |
|
29 |
1 |
7.74 |
7.76 |
7.76 |
7.81 |
- |
- |
- |
2 |
7.80 |
7.75 |
7.72 |
7.81 |
- |
- |
- |
|
3 |
7.83 |
7.72 |
7.72 |
7.78 |
- |
- |
- |
|
4 |
7.82 |
7.74 |
7.72 |
7.78 |
- |
- |
- |
|
Mean |
7.87 |
7.87 |
7.87 |
7.89 |
8.07 |
8.06 |
8.16 |
|
± SD |
0.182 |
0.166 |
0.166 |
0.151 |
0.097 |
0.108 |
0.024 |
|
Min. |
7.44 |
7.70 |
7.71 |
7.72 |
7.95 |
7.89 |
8.12 |
|
Max. |
8.17 |
8.17 |
8.17 |
8.19 |
8.16 |
8.17 |
8.17 |
|
- = No measurement, due to 100 % mortality
Min./Max. = Minimum/Maximum measured pH-value
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 0.0220 mg/L (day 16, 23 and 30), 0.106 mg/L (day 8) and 0.233 mg/L (day 1). The mean total hardness was 69 mg CaCO3/L and ranged from 64 to 73 mg CaCO3/L in the control.
Total Hardness in the Control and the Remaining Highest Test Concentration
Study day |
Replicate |
Total hardness [mg CaCO3/L] |
|||
Nominal test item conc. [mg/L] |
|||||
Control |
0.0220 |
0.106 |
0.233 |
||
1 |
1 |
73 |
|
|
66 |
8 |
2 |
72 |
|
70 |
- |
16 |
3 |
64 |
77 |
- |
- |
23 |
4 |
66 |
73 |
- |
- |
30 |
1 |
69 |
70 |
- |
- |
Mean |
69 |
73 |
70 |
66 |
|
± SD |
3.83 |
3.51 |
n.a. |
n.a. |
|
Min. |
64 |
70 |
n.a. |
n.a. |
|
Max. |
73 |
77 |
n.a. |
n.a. |
|
- = No measurement, due to 100 % mortality
n.a. = Not applicable
Min./Max. = Minimum/Maximum measured hardness
Residual Chlorine
Residual chlorine, measured from the dilution water supply tank on study days 1, 8, 16, 23 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, 23 and 30. The mean measured TOC was 0.627 mgC/L throughout exposure (see Table 22). Measured concentrations were below the LOQ of 2 mgC/L.
Total Organic Carbon (TOC) of the Dilution Water
Study day |
TOC [mg/L] |
1 |
0.794 |
8 |
0.530 |
16 |
0.601 |
23 |
0.598 |
30 |
0.613 |
Mean |
0.627 |
SD |
0.099 |
Min. |
0.530 |
Max. |
0.794 |
Min./Max. = Minimum/Maximum measured TOC
Flow Rates
The mean flow rate through the mixing chambers of all test item and control groups was 3.09 ± 0.131 L/h and the individual values ranged from 3.00 to 3.16 L/h.
Flow Rates
Flow rate |
Nominal test item concentrations [mg/L] |
||||||
Control |
Solvent control |
0.0100 |
0.0220 |
0.0484 |
0.106 |
0.233 |
|
Measured flow rates of the membrane piston pumps1) |
|||||||
Minimum |
2.88 |
2.82 |
2.82 |
2.88 |
2.88 |
2.88 |
3.00 |
Maximum |
3.36 |
3.36 |
3.36 |
3.24 |
3.36 |
3.24 |
3.24 |
Mean |
3.13 |
3.09 |
3.16 |
3.00 |
3.08 |
3.08 |
3.08 |
Overall mean |
3.10 |
||||||
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 183 to 326 lux (mean: 221 lux).
Measured Test Concentrations during Equilibration Phase
The test item concentration in the equilibration phase was measured on day -1 from the test vessels and mixing chambers of the test replicates 1 and 4.
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day -1
Sampling day / Replicate |
Day -1 |
||||
Repl. 1 |
Repl. 4 |
||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides |
||||
C24H52N |
|||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
0.233 |
0.0161 |
0.0188 |
117 |
0.0170 |
106 |
0.106 |
0.00731 |
0.00682 |
93 |
0.00800 |
110 |
0.0484 |
0.00334 |
0.00397 |
119 |
0.00371 |
111 |
0.0220 |
0.00152 |
0.001881) |
1241) |
0.00170 |
1121) |
0.0100 |
0.000690 |
0.000676 |
98 |
0.000752 |
109 |
Solvent Control |
< LOQ |
< LOQ |
|||
Control |
< LOQ |
< LOQ |
|||
Sampling day / Replicate |
Day -1 |
||||
Repl. 1 |
Repl. 4 |
||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides |
||||
C26H56N |
|||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
0.233 |
0.0596 |
0.0666 |
112 |
0.0625 |
105 |
0.106 |
0.0271 |
0.0266 |
98 |
0.0323 |
118 |
0.0484 |
0.0124 |
0.0146 |
118 |
0.0170 |
137 |
0.0220 |
0.00563 |
0.008601) |
1531) |
0.009041) |
1611) |
0.0100 |
0.00256 |
0.00251 |
98 |
0.00294 |
115 |
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 (0.00200 mg/L of the test item)
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day -1
Sampling day / Replicate |
Day -1 |
||||
Repl. 1 |
Repl. 4 |
||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides |
||||
C28H60N |
|||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
0.233 |
0.0417 |
0.0469 |
112 |
0.0435 |
104 |
0.106 |
0.0190 |
0.0193 |
102 |
0.0242 |
127 |
0.0484 |
0.00866 |
0.0105 |
121 |
0.0163 |
188 |
0.0220 |
0.00394 |
0.007591) |
1931) |
0.01031) |
2621) |
0.0100 |
0.00179 |
0.00181 |
101 |
0.00206 |
115 |
Solvent Control |
< LOQ |
< LOQ |
|||
Control |
< LOQ |
< LOQ |
|||
Sampling day / Replicate |
Day -1 |
||||
Repl. 1 |
Repl. 4 |
||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides |
||||
C30H64N |
|||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
0.233 |
0.0308 |
0.0355 |
115 |
0.0315 |
103 |
0.106 |
0.0140 |
0.0141 |
101 |
0.0164 |
117 |
0.0484 |
0.00639 |
0.00771 |
121 |
0.00954 |
149 |
0.0220 |
0.00290 |
0.004511) |
1551) |
0.006021) |
2071) |
0.0100 |
0.00132 |
0.00137 |
104 |
0.00147 |
111 |
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 (0.00200 mg/L of the test item)
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day -1
Sampling day / Replicate |
Day -1 |
||||
|
Repl. 1 |
Repl. 4 |
|||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides |
||||
C32H68N |
|||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
0.233 |
0.0242 |
0.0271 |
112 |
0.0239 |
99 |
0.106 |
0.0110 |
0.0108 |
99 |
0.0118 |
107 |
0.0484 |
0.00503 |
0.00584 |
116 |
0.00638 |
127 |
0.0220 |
0.00229 |
0.003161) |
1381) |
0.004131) |
1801) |
0.0100 |
0.00104 |
0.00106 |
102 |
0.00116 |
112 |
Solvent Control |
< LOQ |
< LOQ |
|||
Control |
< LOQ |
< LOQ |
|||
Sampling day / Replicate |
Day -1 |
||||
Repl. 1 |
Repl. 4 |
||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides |
||||
C34H72N |
|||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
0.233 |
0.00792 |
0.00891 |
113 |
0.0792 |
100 |
0.106 |
0.00360 |
0.00340 |
94 |
0.00402 |
112 |
0.0484 |
0.00165 |
0.00189 |
115 |
0.02098 |
128 |
0.0220 |
0.000748 |
0.001071) |
1441) |
0.001331) |
1781) |
0.0100 |
0.000340 |
0.000335 |
98 |
0.000374 |
110 |
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 (0.00200 mg/L of the test item)
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Mixing chambers on Study Day -1
Sampling day / Replicate |
Day -1 |
||||
Repl. 1 |
Repl. 4 |
||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides |
||||
C24H52N |
|||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
0.233 |
0.0161 |
0.0171 |
107 |
0.0162 |
101 |
0.106 |
0.00731 |
0.00800 |
110 |
0.00752 |
103 |
0.0484 |
0.00334 |
0.00337 |
101 |
0.00349 |
104 |
0.0220 |
0.00152 |
0.00193 |
127 |
0.00166 |
109 |
0.0100 |
0.000690 |
0.00644 |
93 |
0.000731 |
106 |
Solvent Control |
< LOQ |
< LOQ |
|||
Control |
< LOQ |
< LOQ |
|||
Sampling day / Replicate |
Day -1 |
||||
Repl. 1 |
Repl. 4 |
||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides |
||||
C26H56N |
|||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
0.233 |
0.0596 |
0.0530 |
89 |
0.0515 |
86 |
0.106 |
0.0271 |
0.0266 |
98 |
0.0217 |
80 |
0.0484 |
0.0124 |
0.00983 |
79 |
0.0124 |
100 |
0.0220 |
0.00563 |
0.00527 |
94 |
0.00461 |
82 |
0.0100 |
0.00256 |
0.00192 |
75 |
0.00217 |
85 |
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 (0.00200 mg/L of the test item)
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Mixing Chambers on Study Day -1
Sampling day / Replicate |
Day -1 |
||||
Repl. 1 |
Repl. 4 |
||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides |
||||
C28H60N |
|||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
0.233 |
0.0417 |
0.0406 |
98 |
0.410 |
98 |
0.106 |
0.0190 |
0.0200 |
106 |
0.0179 |
95 |
0.0484 |
0.00866 |
0.00807 |
93 |
0.00967 |
112 |
0.0220 |
0.00394 |
0.00388 |
99 |
0.00345 |
88 |
0.0100 |
0.00179 |
0.00158 |
88 |
0.00170 |
95 |
Solvent Control |
< LOQ |
< LOQ |
|||
Control |
< LOQ |
< LOQ |
|||
Sampling day / Replicate |
Day -1 |
||||
Repl. 1 |
Repl. 4 |
||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides |
||||
C30H64N |
|||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
0.233 |
0.0308 |
0.0312 |
101 |
0.0318 |
103 |
0.106 |
0.0140 |
0.0156 |
111 |
0.0149 |
107 |
0.0484 |
0.00639 |
0.00681 |
107 |
0.00730 |
114 |
0.0220 |
0.00290 |
0.00341 |
117 |
0.00296 |
102 |
0.0100 |
0.00132 |
0.00145 |
110 |
0.00140 |
106 |
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 (0.00200 mg/L of the test item)
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Mixing Chambers on Study Day -1
Sampling day / Replicate |
Day -1 |
||||
|
Repl. 1 |
Repl. 4 |
|||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides |
||||
C32H68N |
|||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
0.233 |
0.0242 |
0.0261 |
108 |
0.0255 |
105 |
0.106 |
0.0110 |
0.0127 |
115 |
0.0120 |
109 |
0.0484 |
0.00503 |
0.00539 |
107 |
0.00545 |
108 |
0.0220 |
0.00229 |
0.00280 |
122 |
0.00245 |
107 |
0.0100 |
0.00104 |
0.00113 |
109 |
0.00116 |
112 |
Solvent Control |
< LOQ |
< LOQ |
|||
Control |
< LOQ |
< LOQ |
|||
Sampling day / Replicate |
Day -1 |
||||
Repl. 1 |
Repl. 4 |
||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides |
||||
C34H72N |
|||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
0.233 |
0.00792 |
0.00843 |
107 |
0.00853 |
108 |
0.106 |
0.00360 |
0.00391 |
109 |
0.00384 |
107 |
0.0484 |
0.00165 |
0.00187 |
114 |
0.00186 |
113 |
0.0220 |
0.000748 |
0.000976 |
131 |
0.000867 |
116 |
0.0100 |
0.000340 |
0.000401 |
118 |
0.000388 |
114 |
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 (0.00200 mg/L of the test item)
Measured Concentrations in the Stock Solutions
Measured concentrations of the freshly prepared stock solution were 96 to 130% of the nominal values. Measured concentrations of the 7 day aged stock solutions in the range of 86 to 127% of the nominal values.
Measured Concentrations and Percent of Nominal Concentration in the Stock Solutions
|
Freshly prepared stock solutions |
7 Days aged stock solutions |
|||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C24H52N |
||||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
||||
4.67 |
0.322 |
0.342 |
106 |
0.308 |
96 |
||||
2.12 |
0.146 |
0.174 |
119 |
0.126 |
86 |
||||
0.968 |
0.0668 |
0.0718 |
108 |
0.0624 |
94 |
||||
0.440 |
0.0304 |
0.0322 |
106 |
0.0358 |
118 |
||||
0.200 |
0.0138 |
0.0150 |
109 |
0.0140 |
101 |
||||
Solvent Control |
< LOQ |
< LOQ |
|||||||
|
Freshly prepared stock solutions |
7 Days aged stock solutions |
|||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C26H56N |
||||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
||||
4.67 |
1.20 |
1.24 |
104 |
1.17 |
98 |
||||
2.12 |
0.543 |
0.540 |
100 |
0.504 |
93 |
||||
0.968 |
0.248 |
0.271 |
109 |
0.241 |
97 |
||||
0.440 |
0.113 |
0.123 |
109 |
0.141 |
125 |
||||
0.200 |
0.0512 |
0.0556 |
109 |
0.0535 |
104 |
||||
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 (0.00200 mg/L of the test item)
Measured Concentrations and Percent of Nominal Concentration in the Stock Solutions
|
Freshly prepared stock solutions |
7 Days aged stock solutions |
|||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C28H60N |
||||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
||||
4.67 |
0.836 |
0.927 |
111 |
0.845 |
101 |
||||
2.12 |
0.379 |
0.371 |
98 |
0.347 |
91 |
||||
0.968 |
0.173 |
0.199 |
115 |
0.171 |
99 |
||||
0.440 |
0.0788 |
0.0893 |
113 |
0.100 |
127 |
||||
0.200 |
0.0358 |
0.0403 |
112 |
0.0363 |
102 |
||||
Solvent Control |
< LOQ |
< LOQ |
|||||||
|
Freshly prepared stock solutions |
7 Days aged stock solutions |
|||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C30H64N |
||||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
||||
4.67 |
0.616 |
0.694 |
113 |
0.609 |
99 |
||||
2.12 |
0.280 |
0.269 |
96 |
0.253 |
90 |
||||
0.968 |
0.128 |
0.152 |
119 |
0.118 |
93 |
||||
0.440 |
0.0581 |
0.0684 |
118 |
0.0702 |
121 |
||||
0.200 |
0.0264 |
0.0308 |
117 |
0.0263 |
100 |
||||
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 (0.00200 mg/L of the test item)
Measured Concentrations and Percent of Nominal Concentration in the Stock Solutions
|
Freshly prepared stock solutions |
7 Days aged stock solutions |
|
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C32H68N |
|
|||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
|||
4.67 |
0.486 |
0.545 |
112 |
0.465 |
96 |
|
|||
2.12 |
0.220 |
0.213 |
97 |
0.196 |
89 |
|
|||
0.968 |
0.101 |
0.115 |
115 |
0.0924 |
92 |
|
|||
0.440 |
0.0458 |
0.0534 |
116 |
0.0519 |
113 |
|
|||
0.200 |
0.0208 |
0.0242 |
116 |
0.0206 |
99 |
|
|||
Solvent Control |
< LOQ |
< LOQ |
|
||||||
|
Freshly prepared stock solutions |
7 Days aged stock solutions |
|
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C34H72N |
|
|||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
||||
4.67 |
0.159 |
0.199 |
125 |
0.156 |
99 |
|
|||
2.12 |
0.0721 |
0.0694 |
96 |
0.0653 |
90 |
|
|||
0.968 |
0.0329 |
0.0388 |
118 |
0.0311 |
95 |
|
|||
0.440 |
0.0150 |
0.0181 |
121 |
0.0168 |
112 |
|
|||
0.200 |
0.00680 |
0.00884 |
130 |
0.00673 |
99 |
|
|||
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 (0.00200 mg/L of the test item)
Measured Concentrations in the Test Vessels and Mixing Chambers during Exposure
The analytical results of the range finding tests showed that for single compounds (especially with higher carbon chain length) the measured concentrations were above 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. In contrast, the measured values from the mixing chambers nearly met the nominal concentrations. Therefore, it was decided to include samples from the mixing chambers in the analytical programme to demonstrate the correct dosage into the flow through system.
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day 0 to Day 30
Sampling day / Replicate |
Day 0 |
Day 9 |
Day 16 |
|||||
Repl. 2 |
Repl. 4 |
Repl. 4 |
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C24H52N |
|||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
0.233 |
0.0161 |
0.0162 |
101 |
0.01652) |
102 |
|
||
0.106 |
0.00731 |
0.00794 |
109 |
0.007253) |
99 |
|||
0.0484 |
0.00334 |
0.00359 |
107 |
0.00395 |
118 |
|||
0.0220 |
0.00152 |
0.00181 |
119 |
0.00168 |
110 |
0.00154 |
106 |
|
0.0100 |
0.000690 |
0.000718 |
104 |
0.000671 |
97 |
0.000644 |
93 |
|
Solvent Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Sampling day / Replicate |
Day 23 |
Day 30 |
|
|||||
Repl. 1 |
Repl. 2 |
|
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C24H52N |
|
||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
||
0.0220 |
0.00152 |
0.00168 |
111 |
0.00162 |
107 |
|
||
0.0100 |
0.000690 |
0.000670 |
97 |
0.000640 |
93 |
|
||
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 (0.00200 mg/L of the test item, corresponding to 0.000138 mg a.i./L)
2) = samples were analyzed on day 7 due 100% mortality
3) = samples were analyzed on day 8 due 100% mortality
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Mixing Chambers on Study Day 0 to Day 30
Sampling day / Replicate |
Day 0 |
Day 9 |
Day 16 |
|||||
Repl. 2 |
Repl. 4 |
Repl. 4 |
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C24H52N |
|||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
0.233 |
0.0161 |
0.0166 |
103 |
0.01562) |
97 |
|
||
0.106 |
0.00731 |
0.007181) |
981) |
0.007313) |
100 |
|||
0.0484 |
0.00334 |
0.00353 |
106 |
0.00321 |
96 |
|||
0.0220 |
0.00152 |
0.0008631) |
571) |
0.00165 |
108 |
0.00130 |
86 |
|
0.0100 |
0.000690 |
0.0005981) |
871) |
0.0004391) |
641) |
0.000517 |
75 |
|
Solvent Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Sampling day / Replicate |
Day 23 |
Day 30 |
|
|||||
Repl. 1 |
Repl. 2 |
|
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C24H52N |
|
||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
||
0.0220 |
0.00152 |
0.00120 |
79 |
0.001881) |
1231) |
|
||
0.0100 |
0.000690 |
0.000541 |
78 |
0.0005971) |
861) |
|
||
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 (0.00200 mg/L of the test item, corresponding to 0.000138 mg a.i./L)
2) = samples were analyzed on day 7 due 100% mortality
3) = samples were analyzed on day 8 due 100% mortality
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day 0 to Day 30
Sampling day / Replicate |
Day 0 |
Day 9 |
Day 16 |
|||||
Repl. 2 |
Repl. 4 |
Repl. 4 |
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C26H56N |
|||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
0.233 |
0.0596 |
0.0589 |
99 |
0.05732) |
96 |
|
||
0.106 |
0.0271 |
0.0320 |
118 |
0.02533) |
94 |
|||
0.0484 |
0.0124 |
0.0151 |
122 |
0.00134 |
108 |
|||
0.0220 |
0.00563 |
0.00904 |
160 |
0.00607 |
108 |
0.00660 |
117 |
|
0.0100 |
0.00256 |
0.00445 |
174 |
0.00202 |
791) |
0.00315 |
123 |
|
Solvent Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Sampling day / Replicate |
Day 23 |
Day 30 |
|
|||||
Repl. 1 |
Repl. 2 |
|
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C26H56N |
|
||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
||
0.0220 |
0.00563 |
0.00909 |
161 |
0.00855 |
152 |
|
||
0.0100 |
0.00256 |
0.00254 |
99 |
0.00315 |
123 |
|
||
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 (0.00200 mg/L of the test item, corresponding to 0.000512 mg a.i./L)
2) = samples were analyzed on day 7 due 100% mortality
3) = samples were analyzed on day 8 due 100% mortality
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Mixing Chambers on Study Day 0 to Day 30
Sampling day / Replicate |
Day 0 |
Day 9 |
Day 16 |
|||||
Repl. 2 |
Repl. 4 |
Repl. 4 |
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C26H56N |
|||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
0.233 |
0.0596 |
0.0571 |
96 |
0.09272) |
155 |
|
||
0.106 |
0.0271 |
0.02111) |
781) |
0.02323) |
85 |
|||
0.0484 |
0.0124 |
0.0103 |
83 |
0.00952 |
77 |
|||
0.0220 |
0.00563 |
0.003841) |
681) |
0.00522 |
93 |
0.00422 |
75 |
|
0.0100 |
0.00256 |
0.001911) |
741) |
0.001631) |
641) |
0.00173 |
67 |
|
Solvent Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Sampling day / Replicate |
Day 23 |
Day 30 |
|
|||||
Repl. 1 |
Repl. 2 |
|
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C26H56N |
|
||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
||
0.0220 |
0.00563 |
0.00399 |
71 |
0.01361) |
2421) |
|
||
0.0100 |
0.00256 |
0.00180 |
70 |
0.002921) |
1141) |
|
||
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 (0.00200 mg/L of the test item, corresponding to 0.000512 mg a.i./L)
2) = samples were analyzed on day 7 due 100% mortality
3) = samples were analyzed on day 8 due 100% mortality
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day 0 to Day 30
Sampling day / Replicate |
Day 0 |
Day 9 |
Day 16 |
|||||
Repl. 2 |
Repl. 4 |
Repl. 4 |
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C28H60N |
|||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
0.233 |
0.0417 |
0.0413 |
99 |
0.03922) |
94 |
|
||
0.106 |
0.0190 |
0.0236 |
124 |
0.01743) |
92 |
|||
0.0484 |
0.00866 |
0.0165 |
190 |
0.00900 |
104 |
|||
0.0220 |
0.00394 |
0.00614 |
156 |
0.00439 |
111 |
0.00734 |
186 |
|
0.0100 |
0.00179 |
0.00349 |
195 |
0.00211) |
1171) |
0.00217 |
121 |
|
Solvent Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Sampling day / Replicate |
Day 23 |
Day 30 |
|
|||||
Repl. 1 |
Repl. 2 |
|
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C28H60N |
|
||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
||
0.0220 |
0.00394 |
0.00788 |
200 |
0.00730 |
186 |
|
||
0.0100 |
0.00179 |
0.00178 |
100 |
0.00208 |
116 |
|
||
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 (0.00200 mg/L of the test item, corresponding to 0.000358 mg a.i./L)
2) = samples were analyzed on day 7 due 100% mortality
3) = samples were analyzed on day 8 due 100% mortality
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Mixing Chambers on Study Day 0 to Day 30
Sampling day / Replicate |
Day 0 |
Day 9 |
Day 16 |
|||||
Repl. 2 |
Repl. 4 |
Repl. 4 |
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C28H60N |
|||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
0.233 |
0.0417 |
0.0424 |
102 |
0.06232) |
149 |
|
||
0.106 |
0.0190 |
0.0160 |
84 |
0.01783) |
94 |
|||
0.0484 |
0.00866 |
0.00725 |
84 |
0.00748 |
86 |
|||
0.0220 |
0.00394 |
0.00317 |
80 |
0.00406 |
103 |
0.00358 |
91 |
|
0.0100 |
0.00179 |
0.00144 |
81 |
0.001401) |
781) |
0.00158 |
88 |
|
Solvent Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Sampling day / Replicate |
Day 23 |
Day 30 |
|
|||||
Repl. 1 |
Repl. 2 |
|
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C28H60N |
|
||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
||
0.0220 |
0.00394 |
0.00274 |
70 |
0.00943 |
240 |
|
||
0.0100 |
0.00179 |
0.00151 |
84 |
0.00245 |
137 |
|
||
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 (0.00200 mg/L of the test item, corresponding to 0.000358 mg a.i./L)
2) = samples were analyzed on day 7 due 100% mortality
3) = samples were analyzed on day 8 due 100% mortality
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day 0 to Day 30
Sampling day / Replicate |
Day 0 |
Day 9 |
Day 16 |
|||||
Repl. 2 |
Repl. 4 |
Repl. 4 |
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C30H64N |
|||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
0.233 |
0.0308 |
0.0306 |
99 |
0.02682) |
87 |
|
||
0.106 |
0.0140 |
0.0158 |
113 |
0.01143) |
81 |
|||
0.0484 |
0.00639 |
0.00985 |
154 |
0.00620 |
97 |
|||
0.0220 |
0.00290 |
0.00378 |
130 |
0.00286 |
99 |
0.00466 |
160 |
|
0.0100 |
0.00132 |
0.00210 |
159 |
0.00108 |
82 |
0.00145 |
110 |
|
Solvent Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Sampling day / Replicate |
Day 23 |
Day 30 |
|
|||||
Repl. 1 |
Repl. 2 |
|
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C30H64N |
|
||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
||
0.0220 |
0.00290 |
0.00421 |
145 |
0.00432 |
148 |
|
||
0.0100 |
0.00132 |
0.00130 |
98 |
0.00136 |
103 |
|
||
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 (0.00200 mg/L of the test item, corresponding to 0.000264 mg a.i./L)
2) = samples were analyzed on day 7 due 100% mortality
3) = samples were analyzed on day 8 due 100% mortality
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Mixing Chambers on Study Day 0 to Day 30
Sampling day / Replicate |
Day 0 |
Day 9 |
Day 16 |
|||||
Repl. 2 |
Repl. 4 |
Repl. 4 |
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C30H64N |
|||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
0.233 |
0.0308 |
0.0318 |
103 |
0.03462) |
113 |
|
||
0.106 |
0.0140 |
0.0140 |
100 |
0.01323) |
95 |
|||
0.0484 |
0.00639 |
0.00615 |
96 |
0.00590 |
92 |
|||
0.0220 |
0.00290 |
0.00297 |
102 |
0.00315 |
109 |
0.00306 |
106 |
|
0.0100 |
0.00132 |
0.00124 |
94 |
0.001191) |
901) |
0.00133 |
101 |
|
Solvent Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Sampling day / Replicate |
Day 23 |
Day 30 |
|
|||||
Repl. 1 |
Repl. 2 |
|
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C30H64N |
|
||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
||
0.0220 |
0.00290 |
0.00248 |
86 |
0.00483 |
166 |
|
||
0.0100 |
0.00132 |
0.00123 |
93 |
0.00198 |
150 |
|
||
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 (0.00200 mg/L of the test item, corresponding to 0.000264 mg a.i./L)
2) = samples were analyzed on day 7 due 100% mortality
3) = samples were analyzed on day 8 due 100% mortality
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day 0 to Day 30
Sampling day / Replicate |
Day 0 |
Day 9 |
Day 16 |
|||||
Repl. 2 |
Repl. 4 |
Repl. 4 |
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C32H68N |
|||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
0.233 |
0.0242 |
0.0236 |
97 |
0.0226 |
93 |
|
||
0.106 |
0.0110 |
0.0123 |
111 |
0.0093 |
84 |
|||
0.0484 |
0.00503 |
0.00724 |
144 |
0.00506 |
101 |
|||
0.0220 |
0.00229 |
0.00261 |
114 |
0.00212 |
93 |
0.00368 |
161 |
|
0.0100 |
0.00104 |
0.00147 |
141 |
0.000925 |
89 |
0.00116 |
111 |
|
Solvent Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Sampling day / Replicate |
Day 23 |
Day 30 |
|
|||||
Repl. 1 |
Repl. 2 |
|
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C32H64N |
|
||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
||
0.0220 |
0.00229 |
0.00276 |
120 |
0.003041) |
1331) |
|
||
0.0100 |
0.00104 |
0.00104 |
100 |
0.001061) |
1021) |
|
||
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 (0.00200 mg/L of the test item, corresponding to 0.000208 mg a.i./L)
1) = reanalyzed and confirmed
2) = samples were analyzed on day 7 due 100% mortality
3) = samples were analyzed on day 8 due 100% mortality
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Mixing Chambers on Study Day 0 to Day 30
Sampling day / Replicate |
Day 0 |
Day 9 |
Day 16 |
|||||
Repl. 2 |
Repl. 4 |
Repl. 4 |
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C32H68N |
|||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
0.233 |
0.0242 |
0.0249 |
102 |
0.02792) |
115 |
|
||
0.106 |
0.0110 |
0.0111 |
101 |
0.01143) |
103 |
|||
0.0484 |
0.00503 |
0.00491 |
98 |
0.00493 |
98 |
|||
0.0220 |
0.00229 |
0.00244 |
107 |
0.00252 |
110 |
0.00235 |
103 |
|
0.0100 |
0.00104 |
0.000960 |
92 |
0.0009681) |
931) |
0.00111 |
107 |
|
Solvent Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Sampling day / Replicate |
Day 23 |
Day 30 |
|
|||||
Repl. 1 |
Repl. 2 |
|
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C32H64N |
|
||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
||
0.0220 |
0.00229 |
0.00230 |
101 |
0.00327 |
143 |
|
||
0.0100 |
0.00104 |
0.00103 |
99 |
0.00123 |
118 |
|
||
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 (0.00200 mg/L of the test item, corresponding to 0.000208 mg a.i./L)
1) = reanalyzed and confirmed
2) = samples were analyzed on day 7 due 100% mortality
3) = samples were analyzed on day 8 due 100% mortality
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Test Vessels on Study Day 0 to Day 30
Sampling day / Replicate |
Day 0 |
Day 9 |
Day 16 |
|||||
Repl. 2 |
Repl. 4 |
Repl. 4 |
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C34H72N |
|||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
0.233 |
0.00792 |
0.00809 |
102 |
0.00711 |
90 |
|
||
0.106 |
0.00360 |
0.00398 |
111 |
0.00283 |
79 |
|||
0.0484 |
0.00165 |
0.00233 |
142 |
0.00169 |
103 |
|||
0.0220 |
0.000748 |
0.000942 |
126 |
0.000636 |
85 |
0.00116 |
155 |
|
0.0100 |
0.000340 |
0.000490 |
144 |
0.000377 |
111 |
0.000350 |
103 |
|
Solvent Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Sampling day / Replicate |
Day 23 |
Day 30 |
|
|||||
Repl. 1 |
Repl. 2 |
|
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C34H72N |
|
||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
||
0.0220 |
0.000748 |
0.000881 |
118 |
0.001031) |
1371) |
|
||
0.0100 |
0.000340 |
0.000338 |
99 |
0.000332 |
98 |
|
||
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 (0.00200 mg/L of the test item, corresponding to 0.0000680 mg a.i./L)
1) = reanalyzed and confirmed
2) = samples were analyzed on day 7 due 100% mortality
3) = samples were analyzed on day 8 due 100% mortality
Measured Concentrations and Percent of Nominal Concentration of the Test item in the Mixing Chambers on Study Day 0 to Day 30
Sampling day / Replicate |
Day 0 |
Day 9 |
Day 16 |
|||||
Repl. 2 |
Repl. 4 |
Repl. 4 |
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C34H72N |
|||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
0.233 |
0.00792 |
0.00823 |
104 |
0.009552) |
121 |
|
||
0.106 |
0.00360 |
0.00377 |
105 |
0.003773) |
105 |
|||
0.0484 |
0.00165 |
0.00165 |
100 |
0.00161 |
98 |
|||
0.0220 |
0.000748 |
0.000843 |
113 |
0.000700 |
94 |
0.000847 |
113 |
|
0.0100 |
0.000340 |
0.000340 |
100 |
0.0003211) |
951) |
0.000347 |
102 |
|
Solvent Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Control |
< LOQ |
< LOQ |
< LOQ |
|||||
Sampling day / Replicate |
Day 23 |
Day 30 |
|
|||||
Repl. 1 |
Repl. 2 |
|
||||||
Nominal concentration of the |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides C34H72N |
|
||||||
Test item [mg/L] |
a.i. [mg a.i./L] |
Meas. conc. [mg a.i./L] |
% |
Meas. conc. [mg a.i./L] |
% |
|
||
0.0220 |
0.000748 |
0.000646 |
86 |
0.00109 |
146 |
|
||
0.0100 |
0.000340 |
0.000339 |
100 |
0.000428 |
126 |
|
||
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 (0.00200 mg/L of the test item, corresponding to 0.0000680 mg a.i./L)
1) = reanalyzed and confirmed
2) = samples were analyzed on day 7 due 100% mortality
3) = samples were analyzed on day 8 due 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 30)
Sampling day / Replicate |
Day 0 |
Day 9 |
Day 16 |
Day 23 |
Day 30 |
|
Repl. 2 |
Repl. 3 |
Repl. 4 |
Repl. 1 |
Repl. 2 |
|
|
Nominal concentration of the test item [mg/L] |
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides |
Overall mean measured concentration of the test item [mg/L] |
||||
Calculated mean measured concentration per study day [mg/L] |
||||||
0.233 |
0.231 |
0.219 |
|
|
|
0.225 |
0.106 |
0.123 |
0.0951 |
|
|
|
0.109 |
0.0484 |
0.0705 |
0.0508 |
|
|
|
0.0606 |
0.0220 |
0.0314 |
0.0229 |
0.0323 |
0.0342 |
0.0334 |
0.0309 |
0.0100 |
0.0164 |
0.00927 |
0.0115 |
0.00990 |
0.0111 |
0.0117 |
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 0.0220 mg/L. A solution of the analytical standard of the test item was prepared in methanol and diluted to 11 µg/L with acetonitrile : ultra-pure water (50 : 50) containing 1% formic acid. A sample of the nominal test item concentration 0.0220 mg/L was diluted factor 2 with acetonitrile containing 2% formic acid to avoid an inhomogeneous sample. Both solutions were analytically verified via high resolution MS (Q-ToF) and evaluated by the software. The detected signals of the analytical standard and the test item solution were compared. In both solutions 6 masses were observed and could be assigned to the test item. No additional signals except those analyzed during the study and related to the test item could be observed in the analytical standard or test item solution.
None
Description of key information
There are two long term fish test results available. The Key study (Scheerbaum, 2021) was performed with C12-18 DAQ according to OECD 210 under flow through conditions. In this study, the lowest 34d LC10 of 15.3 µg a.i./L (95% CI: 11.2 - 21..5 µg a.i./L) was observed for overall survival based on overall mean measured test concentration.
The second study was performed with C16-18 DAQ and is read across to C12-18 DAQ as supporting evidence. In this study, the 35 d NOEC value for long-term toxicity in fish were determined to be 0.23 and 0.053 mg/L (nominal) in Town water and Well water respectively.
Therefore, based on the key study, 34 d the LC10 = 15.3 µg a.i./L (mean measured concentration) for effects on overall survival has been considered for the classification and labelling (C&L) as well as risk assessment.
Key value for chemical safety assessment
Fresh water fish
Fresh water fish
- Dose descriptor:
- LC10
- Remarks:
- based on mean measured concentrations for the endpoint: Overall survival
- Effect concentration:
- 15.3 µg/L
Additional information
Study 1: A study was conducted to determine the long-term aquatic toxicity of the test substance, C12-18 DAQ (95.6% active) with Danio rerio / Zebrafish, according to the OECD guideline 210, in compliance with GLP. The test substance is a yellow viscous liquid (paste). Methanol was used as solvent with a concentration of 0.05 mL/L dilution water. Stock solutions in methanol with nominal concentrations of 4.67, 2.12, 0.968, 0.440 and 0.200 g/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 substance concentrations 0.0100, 0.0220, 0.0484, 0.106 and 0.233 mg/L, corresponding to the overall mean measured test substance concentrations of 0.0117, 0.0309, 0.0606, 0.109 and 0.225 mg/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 98% of 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 behavioural effects, post-hatch survival and overall survival. Specific analysis of various concentrations of the test substance in the test media and the controls was carried out via LC-MS/MS. The test media were sampled and analysed from alternating test vessels and the corresponding mixing chamber supply prior to exposure on days -1 and during the exposure on study days 0, 7, 8, 9, 16, 23 and 30. The analytical results of the range-finding tests showed that for single compounds (especially with higher carbon chain length) the measured concentrations were above the nominal concentrations in some of the replicates. The reason is not known but the inhomogeneous distribution of the compounds in the test replicates was suspected. In contrast, the measured values from the mixing chambers nearly met the nominal concentrations. Therefore, it was decided to include samples from the mixing chambers in the analytical programme to demonstrate the correct dosage into the flow-through system. The methanol stock solutions were sampled and analysed from freshly prepared and corresponding 7 days aged stock solutions. Measured concentrations of the freshly prepared stock solutions were 96 to 130% of the nominal values. Measured concentrations of the 7 days aged stock solutions in the range of 86 to 127% of the nominal values. All effect values are given based on the nominal and the overall mean measured concentrations of the test substance (see Table 1 and Table 2). The results were also corrected for active ingredient content.
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 substance concentrations [mg/L] with 95% Confidence intervals in brackets)
Parameter |
Based on nominal substance concentrations [mg/L] |
Based on overall mean measured test substance concentrations [mg/L] |
||||
Hatchingsuccess after 7days1) |
Frygrowth expressedas |
Hatchingsuccessafter 7days1) |
Fry growth expressed as |
|||
Length |
Weight |
Length |
Weight |
|||
NOEC |
0.106 |
0.0100 |
0.0100 |
0.109 |
0.0117 |
0.0117 |
LOEC |
0.233 |
>0.0100 |
>0.0100 |
0.225 |
>0.0117 |
>0.0117 |
EC10 |
0.196 (0.108->0.233) |
n.d. |
n.d. |
0.196 (0.115->0.225) |
n.d. |
n.d. |
EC50 |
n.d. |
n.d. |
n.d. |
n.d. |
n.d. |
n.d. |
1)end of hatching period
n.d. not determinable 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 substance concentrations [mg/L] with 95% Confidence intervals in brackets)
Parameter |
Based on nominal test substance concentrations [mg/L] |
Based on overall mean measured test substance concentrations [mg/L] |
||
Post-hatchsurvival |
Overallsurvival |
Post-hatchsurvival |
Overallsurvival |
|
NOEC |
0.0100 |
0.0100 |
0.0117 |
0.0117 |
LOEC |
0.0220 |
0.0220 |
0.0309 |
0.0309 |
LC10 |
0.0137 (0.0102–0.0184) |
0.0128 (<0.0100–0.0168) |
0.0176 (0.0122–0.0252) |
0.0160 (0.0117–0.0225) |
LC50 |
0.0188 (0.0142–0.0206) |
0.0184 (0.0149–0.0200) |
0.0257 (0.0180–0.0286) |
0.0248 (0.0191–0.0276) |
The test substance caused significant effects on Zebrafish in an early life stage test, 30 days post-hatch. For the parameter hatch, the nominal EC10, NOEC and LOEC were determined to be 0.196 mg/L (i.e., 0.187 mg a.i./L), 0.106 mg/L (i.e., 0.101 mg a.i./L) and 0.233 mg/L (i.e., 0.222 mg a.i./L) respectively. The corresponding EC10, NOEC and LOEC values based on overall mean measured test substance concentrations were 0.196 mg/L (i.e., 0.187 mg a.i./L), 0.109 mg/L (i.e., 0.104 mg a.i./L) and 0.225 mg/L (i.e., 0.215 mg a.i./L), respectively.
For the parameters post-hatch survival and overall survival, the nominal NOECs and LOECs for both parameters were determined to be 0.0100 mg/L (i.e., 0.00956 mg a.i./L) and 0.0220 mg/L (i.e., 0.021 mg a.i./L), respectively. The corresponding NOEC and LOEC values based on overall mean measured test substance concentrations were 0.0117 mg/L (i.e., 0.0112 mg a.i./L) and 0.0309 mg/L (i.e., 0.0295 mg a.i./L), respectively. The nominal LC10 values post-hatch survival and overall survival were determined to be 0.0137 mg/L (i.e., 0.0131 mg a.i./L) and 0.0128 mg/L (i.e., 0.0122 mg a.i./L), respectively. The corresponding LC10 values based on overall mean measured test substance concentrations were 0.0176 mg/L (0.0168 mg a.i./L) and 0.0160 mg/L (i.e., 0.0153 mg a.i./L), respectively.
For the parameter fry growth (expressed as length and fresh weight) the nominal NOECs and LOECs for both parameters were determined to be 0.0100 mg/L (i.e., 0.00956 mg a.i./L) and >0.0100 mg/L (i.e., >0.00956 mg a.i./L), respectively. The corresponding NOEC and LOEC values based on overall mean measured test substance concentrations were 0.0117 mg/L (i.e., 0.0112 mg a.i./L) and >0.0117 mg/L (i.e., >0.0112 mg a.i./L), respectively (Scheerbaum, 2021).
Study 2: A study was conducted to determine the long-term aquatic toxicity of the read across substance, C16-18 DAQ (71.4% active, 8% C16-18 and C18-unsatd. TMAC) with Pimephales promelas (fathead minnow), according to the US EPA Guidelines. In a 35-day embryo-larval study eggs were exposed to 5 concentrations of the read across substance in filtered well water as well as in river water. The following concentrations were used besides the controls: 0.006; 0.013; 0.024; 0.053; 0.090 mg/L (laboratory water); 0.040; 0.061; 0.130; 0.230; 0.450 mg/L (river water). The test concentrations were confirmed, separated and quantified using HPLC with a conductivity detector. All effect concentrations were based on measured levels of the read across substance. In the study with laboratory water, survival of embryos as well as growth of larvae (length and weight) was reduced at 0.090 mg/L whereas the NOEC was determined to be at 0.053 mg/L. In the study with river water, hatching of embryos as well as growth of larvae (weight) was reduced at 0.450 mg/L whereas the NOEC was determined to be at 0.230 mg/L. Under the study conditions, the 35 d NOEC of the read across substance was determined to be 0.23 and 0.053 mg/L in Town water and Well water respectively (Lewis & Wee, 1983). Based on the results of the read across study, similar effect concentrations can be considered for the test substance.
Therefore, based on the key study, 34 d the LC10 = 15.3 µg a.i./L (mean measured concentration) for effects on overall survival has been considered for the classification and labelling (C&L) as well as risk assessment.
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