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Long-term toxicity to fish

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Endpoint:
fish early-life stage toxicity
Remarks:
embryolarval toxicity tests
Type of information:
experimental study
Adequacy of study:
key 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
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

None

Validity criteria fulfilled:
not specified
Conclusions:
Under the study conditions, 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 test substance, C16-18 DAQ (71.4% active, 8% C16-18 and C18-unsatd. TMAC) with Pimephales promelas (fathead minnow), according to US EPA guidelines. In a 35-day embryo-larval study eggs were exposed to 5 concentrations of the test 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 test 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 test substance was determined to be 0.23 and 0.053 mg/L in Town water and Well water respectively (Lewis & Wee, 1983).

Endpoint:
fish early-life stage toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
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

None

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% 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.

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.


Duration:
34 d
Dose descriptor:
EC10
Effect conc.:
0.196 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Hatching success after 7 days
Remarks on result:
other: Confidence intervals (0.108 - > 0.233) mg/L
Key result
Duration:
34 d
Dose descriptor:
EC10
Effect conc.:
0.196 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Hatchin success after 7 days
Remarks on result:
other: Confidence intervals (0.115 - > 0.225) mg/L
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
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
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
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
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
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
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 succes after 7 days
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 succes after 7 days
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.

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
test item conc.
[mg/L]

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
test item conc.
[mg/L]

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
test item conc.
[mg/L]

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
test item conc.
[mg/L]

Rep.



Eggs introduced on study day 0

Cumulative number of hatched larvae

Vital Larvae on study day 34
(PHD 30)

Post-Hatch survival
on study day 34
[%]

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
test item conc.
[mg/L]

Rep.



Eggs introduced on study day 0

Cumulative number of hatched larvae

Vital Larvae on study day 34
(PHD 30)

Post-Hatch survival
on study day 34
[%]

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
test item conc.
[mg/L]

Rep.



Vital larvae on
study day 34
(PHD 30)

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
test item conc.
[mg/L]

Rep.



Vital larvae on
study day 34
(PHD 30)

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
4 G

1 Q
1 G

-

-

-

-

-

-

-

2 Q
2 G

-

-

-

-

-

7

-

-

-

-

-

-

-

-

13 Q
13 G

12 Q
12 G
  1 T

17 Q
17 G

7 Q
7 G
1 T

2 Q
2 G

2 Q
2 G

3 Q
3 G

3 Q
3 G

8

-

-

-

-

-

-

-

-

8 Q
8 G

3 Q
3 G

5 Q
5 G

7 Q
7 G






100 % mortality

9

-

-

-

-

1 Q
1 G
1 T

-

1 Q
1 G

-

1 Q
1 G 1 F

1 Q
1 G 1 F

-

-

10

-

-

-

-

1 T

-

-

-

100 % mortality

11

-

-

-

-

4 Q
4 G

-

-

4 Q
4 G

12

-

-

-

-

6 Q
6 G

-

2 Q
2 G

2 Q
2 G

13

-

-

-

-

7 Q
3 S
4 G

4 Q
4 G

1 Q
1 G

5 Q
5 G

14

-

-

-

-

5 Q
5 G

7 Q
7 G

1 Q
1 G

3 Q
3 G

15

-

-

-

-

4 Q
4 G

5 Q
5 G

2 Q
2 G

-

16

-

-

-

-

1 Q
1 G

2 Q
2 G

3 Q
3 G

2 Q
2 G

17

-

-

-

-

1 Q
1 G

1 Q
1 G

3 Q
3 G

2 Q
2 G

18

-

-

-

-

-

1 Q
1 G

-

-

19

-

-

-

-

-

-

-

2 Q
2 G

20

-

-

-

-

-

-

-

2 Q
1 S
1 G

21

-

-

-

-

-

-

-

1 Q
1 G

 

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
test item conc.[mg/L]

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
test itemconc.
[mg/L]

Replicate


Number of fish alive
on study day 34

Pooled
wet weight
per replicate
[mg]

Mean
wet weight
per fish
[mg]

Mean


[mg]

± 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)
[L/h]

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
± Standard deviation

3.13
± 0.130

3.09
± 0.140

3.16
± 0.121

3.00
± 0.081

3.08
± 0.139

3.08
± 0.127

3.08
± 0.075

Overall mean
± Standard deviation

3.10
± 0.169

  • Nominal flow rates of dilution water (for preparation of the test solutions): 3.125 (2.81 – 3.44 L/h)

 

A precision syringe pump was used for introduction of the stock solution to the mixing chambers. At any renewal of the syringes the proper function of the pump and the applied volume was checked by the syringe volume indicator.

 

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)

  • = reanalyzed and confirmed

 

 

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)

  • = reanalyzed and confirmed

 

 

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)

  • = reanalyzed and confirmed

 

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)

  • = 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

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)

  • = 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

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)

  • = 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

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)

  • = 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

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)

  • = 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

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)

  • = 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

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.

 

Validity criteria fulfilled:
yes
Conclusions:
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides caused significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal concentrations of 0.0100, 0.0220, 0.0484, 0.106 and 0.233 mg/L, corresponding to overall mean measured test item concentrations of 0.0117, 0.0309, 0.0606, 0.109 and 0.225 mg/L.

For the parameter hatch, the nominal NOEC was 0.106 mg/L. Therefore, the respective nominal LOEC was determined to be 0.233 mg/L. The corresponding NOEC and LOEC values based on overall mean measured test item concentrations were NOEC 0.109 mg/L and LOEC 0.225 mg/L, respectively.

For the parameters post hatch survival and overall survival, the nominal NOECs were 0.0100 mg/L, respectively. Therefore, the respective nominal LOECs were determined to be 0.0220 mg/L. The corresponding NOEC and LOEC values based on overall mean measured test item concentrations were NOEC 0.0117 mg/L and LOEC 0.0309 mg/L, respectively.

For the parameter fry growth (expressed as length and fresh weight) the nominal NOECs were 0.0100 mg/L (for both parameters). Therefore, the nominal LOECs for length and weight were determined to be > 0.0100 mg/L, respectively. The corresponding NOEC and LOEC values based on overall mean measured test item concentrations were NOEC 0.0117 mg/L and LOEC > 0.0117 mg/L, respectively.

All effect values are given based on the nominal and the overall mean measured concentrations of the test item Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides.
Executive summary:

The effects of the test item Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides (Batch-No. SPA19005) on the early-life stage of fish (Danio rerio / Zebrafish) were determined at the test facility according to OECD Guideline 210 from 2020-11-10 to 2020-12-14.

 

The test item 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 item concentrations 0.0100, 0.0220, 0.0484, 0.106 and 0.233 mg/L, corresponding to the overall mean measured test item 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 behavioral effects, post-hatch survival and overall survival.

 

Specific analysis of various concentrations of Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides in the test media and the controls was carried out via LC-MS/MS. 

The test media were sampled and analyzed from alternating test vessels 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 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.

The methanol stock solutions were sampled and analyzed from freshly prepared and corresponding 7 days aged stock solutions. Measured concentrations of the freshly prepared stock solutions were 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 item Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides (see Table 1 and Table 2).

 

Findings and Observations

 

The results of the parameters hatching success, fry growth (expressed as weight and length measurement at PHD 30), post-hatch survival and overall survival were checked for statistically significant differences.

No statistically significant difference was detected between the dilution water control and the solvent control for all parameters (hatching success, fry growth expressed as length or weight on PHD 30, post-hatch survival and overall survival). Therefore, both controls were pooled for statistical analysis.

The effect values NOEC, LOEC, ECx, LCx values were determined based on the statistical results. The results are presented in the tables below.

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

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

 

Parameter

Based on nominal test item concentrations [mg/L]

Based on overall mean measured test item concentrations [mg/L]

Hatching success after
7 days1)

Fry Growth
expressed as:

Hatching success after
7 days1)

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.

EC20

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

EC50

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

 

  • end of hatching period

n.d. not determinable as no dose-response was found

NOEC, LOEC, LCx values of Post Hatch Survival and Overall Survival

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

 

Parameter

Based on nominal test item concentrations [mg/L]

Based on overall mean measured test item concentrations [mg/L]

Post-hatch survival

Overall survival

Post-hatch survival

Overall survival

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)

LC20

0.0154
(0.0116 – 0.0189)

0.0146
(0.0115 – 0.0176)

0.0202
(0.0142 – 0.0260)

0.0189
(0.0140 – 0.0238)

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)

 

Description of key information

Based on the results of read across study with C16-18 DAQ, the 35 d NOEC value of the test substance for long-term toxicity in fish is considered to be 0.23 and 0.053 mg/L (nominal) in Town water and Well water respectively.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
NOEC
Remarks:
based on mean measured concentrations for the endpoint: Fry Growth
Effect concentration:
0.012 mg/L

Additional information

Study 1:

The effects of Quaternary ammonium compounds, di-C12 -18 -alkyldimethyl, chlorides on the early-life stage of fish (Danio rerio/ Zebrafish) were determined according to OECD Guideline 210 from 2020-11-10 to 2020-12-14.

 

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. Based on the results of a range finding test the test was conducted as a dose-response test with the nominal test item concentrations 0.0100, 0.0220, 0.0484, 0.106 and 0.233 mg/L, corresponding to the overall mean measured test item 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 behavioral effects, post-hatch survival and overall survival.

 

Specific analysis of various concentrations of Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides in the test media and the controls was carried out via LC-MS/MS. 

The test media were sampled and analyzed from alternating test vessels 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 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.

The methanol stock solutions were sampled and analyzed from freshly prepared and corresponding 7 days aged stock solutions. Measured concentrations of the freshly prepared stock solutions were 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 item Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides

 

Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides caused significant effects on Zebrafish in an early life stage test, 30 days post hatch.

For the parameter hatch, the nominal NOEC was 0.106 mg/L. Therefore, the respective nominal LOEC was determined to be 0.233 mg/L. The corresponding NOEC and LOEC values based on overall mean measured test item concentrations were NOEC 0.109 mg/L and LOEC 0.225 mg/L, respectively.

For the parameters post hatch survival and overall survival, the nominal NOECs were 0.0100 mg/L, respectively. Therefore, the respective nominal LOECs were determined to be 0.0220 mg/L. The corresponding NOEC and LOEC values based on overall mean measured test item concentrations were NOEC 0.0117 mg/L and LOEC 0.0309 mg/L, respectively.

For the parameter fry growth (expressed as length and fresh weight) the nominal NOECs were 0.0100 mg/L (for both parameters). Therefore, the nominal LOECs for length and weight were determined to be > 0.0100 mg/L, respectively. The corresponding NOEC and LOEC values based on overall mean measured test item concentrations were NOEC 0.0117 mg/L and LOEC > 0.0117 mg/L, respectively.

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.