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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiation date: 30 August 2018 and Study completion date: 29 November 2018 and Definitive exposure dates: 17 to 19 October 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Alcohols, C12-14 (even numbered), propoxylated, aminated, ethoxylated
EC Number:
Molecular formula:
C12H27N(C3H6O)n(C2H4O)m, C14H31N(C3H6O)n(C2H4O)m
Alcohols, C12-14 (even numbered), propoxylated, aminated, ethoxylated
Test material form:
Details on test material:
Name: XTJ-785, experimental
Lot No.: 9570-2-6738
CAS No.: Not listed
Purity: >92% C1214 alcohol, propoxylated, aminated, ethyoxylated
Expiry Date: No date established

Sampling and analysis

Analytical monitoring:
Details on sampling:
Sampling method:
- Sampling Intervals and Solution Composition: 0 and 24 Hour (new solutions) / 24 and 48 hour (aged solutions)
- Exposure solutions in intermediate mixing vessels prior to division into replicate vessels
- Composite of all replicates within each concentration and control
- Number of Samples Taken from Each Solution: One sample at each interval. In addition, subsamples of the test solutions were also collected at each sampling interval and stored frozen as archive samples.
- Sampling Location: Approximate midpoint from the surface, bottom, and sides of the vessel
- Archive Samples Collected: Yes
- Sampling Device: Pipet
- Quality Control (QC) Samples: Three samples at each sampling interval, prepared in dilution water at nominal concentrations which approximated the test concentration range

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

Primary Stock Solution Preparation:
- Primary Stock Concentration: 100 mg/L
- Stock Vessel: Glass beaker
- Stock Volume: 2.5 L
- Amount of Test Substance Added (for example): 0.2510 g, tested as 100%
- Diluent: Fortified laboratory well water

The primary stock solution was prepared as a water-accomodated fraction (WAF) by adding the aliquot of test substance directly to the total volume of dilution water in a glass beaker. The solution was observed to be clear and colorless with oily particulates of undissolved test substance on the surface of the solution. The solution was covered with plastic wrap and allowed to mix slowly overnight using a magnetic stir plate and Teflon-coated stir bar. Following overnight mixing and an approximately 30-minute settling period, the solution was observed to be clear and colorless with oily, particulates on the surface of the solution and on the sides of the glass beaker. The solution was then siphoned, avoiding the sides, surface, and bottom of the vessel, and was observed to be clear and colorless with no visible undissolved test substance. Exposure solutions were prepared from the WAF solution as follows.

Exposure Solution Preparation
Stock Concentration Volume of stock used Total volume with dilution water Nominal exposure Concentration
(% of 100 mg/L WAF) (mL) (L) (% of 100 mg/L WAF)
100 1000 1.0 100
100 500 1.0 50
100 250 1.0 25
100 130 1.0 13
100 63 1.0 6.3
NA NA 1.0 Control
NA = Not applicable

The resulting exposure solutions were observed to be clear and colorless with no visible undissolved test substance following mixing with a glass rod for one minute. Control vessels were maintained under the same conditions as the treatment level solutions.

Exposure solutions were renewed at the 24-hour interval following the procedures outlined above.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Species: Daphnia magna
- Source: Smithers Viscient culture
- Location: Wareham, Massachusetts
- Age: <24 hours old; immature daphnids were removed from culture 24 hours prior to exposure initiation, thus isolating sexually mature adults. Young produced by the parent organisms were used for the exposure.
- Culture Vessel: 1.0-L glass beaker containing 0.80 L of water
- Food Type: Unicellular green algae, Ankistrodesmus falcatus (4 × 107 cells/mL) and YCT suspension (yeast, cereal leaves, and flaked fish food)
Representative samples of the food source were analyzed periodically for the presence of pesticides, PCBs, and toxic metals by Eurofins Lancaster Laboratories Environmental, Pennsylvania (U.S. EPA, 1997). None of these compounds have been detected at concentrations that are considered toxic in the food samples analyzed, in agreement with ASTM (2007) standard practice. Based on the analyses, food sources were considered to be of acceptable quality since all analyte concentrations were below levels of concern.
- Feeding Frequency: Once daily during culture

- Acclimation period: 2 weeks

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Remarks on exposure duration:
Post exposure observation period:
not applicable

Test conditions

Total hardness as CaCO3 (mg/L): 170 to 180
Test temperature:
Temperature (°C): 20 to 22
pH: 7.7 to 7.9
Dissolved oxygen:
Dissolved Oxygen (mg/L): 7.8 to 8.8
Not applicable as freshwater was used
Conductivity (µS/cm): 830 to 840
Nominal and measured concentrations:
- Nominal concentrations: 6.3 / 13 / 25 / 50 and 100% of a 100 mg/L water accommodated fraction (WAF).
- Mean measured concentrations: 0.16 / 0.36 / 0.69 /1.4 and 2.7 mg/L.
Measured concentrations were generally consistent across the treatment levels at each sampling interval.

Nominal Concentration Measured Concentration (mg/L)
(% of a
100 mg/L WAF) 0-Hour 24-Hour(Aged) 24-Hour(New) 48-Hour Mean Measured Concentration
Control <0.0050b <0.0050 <0.0050 <0.0050 NAc
6.3 0.19 0.15 0.18 0.14 0.16
13 0.42 0.33 0.36 0.31 0.36
25 0.72 0.68 0.71 0.63 0.69
50 1.5 1.4 1.4 1.3 1.4
100 3.0 2.6 2.8 2.5 2.7

QCd #1 0.098 0.095 0.97
0.10 (98) (95) (97)

QC #2 0.96 0.96 0.95
1.0 (96) (96) (95)

QC #3 11 10 11
10 (110) (100) (110)

a :Mean measured concentration values were calculated using the actual analytical results (unrounded values) and not the rounded values presented in this table.
b :Concentrations expressed as less than values were below the method detection limit (MDL). The MDL is dependent upon the lowest concentration calibration standard and the dilution factor of the controls..
c : NA = Not Applicable
d : QC = Quality Control. Concentrations are presented as mg/L. Percent recovery is presented in parentheses.
Details on test conditions:
- Vessel Size/Material: 250-mL glass beakers
- Vessel Solution Volume: 200 mL
- Vessel Cleaning: Cleaned prior to initiation
- Biomass Loading Rate: 25 daphnids/L (5 daphnids per 200 mL)- Exposure Solution Renewal Frequency: At the 24-hour interval
- Food Type: None
- Feeding Frequency: Not applicable
- Replication: Four replicates per treatment level and the control
- Control of Bias: Impartial addition of daphnids to intermediate vessel prior to transfer to exposure vessels
- Aeration: None

- Water Type: Fortified laboratory well water
- pH: 7.7 to 7.9
- Dissolved Oxygen (mg/L): 7.8 to 8.8
- Conductivity (µS/cm): 830 to 840
- Total Hardness as CaCO3 (mg/L): 170 to 180
- Total Alkalinity as CaCO3 (mg/L): 84 to 86
- TOC: 0.27 mg/L

- Adjustment of pH: no
- Photoperiod: 16 hours light: 8 hours dark
- Transition Period: 15- to 30-minute transition period controlled with an automatic timer
- Light Intensity: 66 to 99 footcandles (710 to 1100 lux) measured around the test area once during the exposure with a Fisher Scientific traceable light meter
- Bulb Type: Fluorescent bulbs


The pH, dissolved oxygen, and temperature measurements made during the definitive toxicity exposure were measured.
Throughout the exposure period, all water quality parameters measured were unaffected by the concentrations of test substance tested and remained within acceptable ranges for the survival of daphnids.

Biological observations, number of immobilized daphnids, and observations of the physical characteristics of the exposure and control solutions (e.g., precipitate, film on solution’s surface) were recorded at exposure initiation, at the 24-hour interval, and at exposure termination (48 hours), if applicable. Immobilization was defined as the inability to swim within 15 seconds after gentle agitation of the exposure vessel. Immobilized organisms were removed and discarded at each observation interval.

Prior to initiating the definitive study, a preliminary range-finding exposure was conducted during which daphnids were exposed under static-renewal conditions.
Two exposure vessels were established for each concentration and a control.
Exposure solutions were prepared in the same manner as in the definitive exposure.
- Test concentrations: 0.01 / 0.10 / 1.0 / 10 and 100 (% of 100 mg/L WAF)
% immobilization after 24 and 48 hours exposure time: 0% in the comtrol, 0.010 / 0.10 / 1.0/ and 10 % of 100 mg/L WAF. (48h exposure at 10 % of 100 mg/L WAF: one surviving daphnid was observed to be lethargic).
% immobilization after 24 and 48 hours exposure time: 100% at 100 % of 100 mg/L WAF.
- Results used to determine the conditions for the definitive study: yes
Based on these results and consultation with the Study Sponsor, nominal concentrations of 6.3, 13, 25, 50, and 100% of a 100 mg/L WAF, and a control were selected for the definitive exposure.
Reference substance (positive control):
Potassium Dichromate

Results and discussion

Effect concentrations
Key result
48 h
Dose descriptor:
Effect conc.:
0.95 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% confidence Intervals (0.89 - 1.0 mg/L)
Determined by Spearman-K!arber.
Details on results:
- Highest concentration producing 0% immobilization: 0.36 mg/l
- Lowest concentration producing 100% immobilization: 1.4 mg/L
- NOEC through 48 hours: 0.36 mg/L
- 24-h EC50: 1.9 mg/L (95% CL: 1.4-2.7 mg/L) (EC50 value and 95% confidence interval estimated by binomial probability)
Results with reference substance (positive control):
The reference test was performed outside of the scope of this study and was used to evaluate the sensitivity of the daphnid population at Smithers Viscient. This result was within the historical range for daphnids and potassium dichromate (0.68 to 3.5 mg/L) ; therefore, the culture is considered of appropriate health and sensitivity for use in toxicity testing.

Testing perfoemd on 27 to 28 September 2018.
Duration: 24 hours
Age of Daphnids: < 24 hours old

EC50: 2.5 mg/L, with a 95% confidence interval of 2.0 to 3.1 mg/L

Reported statistics and error estimates:
The 48-h EC50 value and 95% confidence interval were determined by Spearman-Kärber Estimates.

Applicant's summary and conclusion

Validity criteria fulfilled:
The acute toxicity (EC50) of alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated to daphnids (Daphnia magna) was tested under static renewal test conditions following the OECD 202 guideline. The results (based on mean measured concentrations) were as follows:
- 48-hour EC50 value: Determined by Spearman-Kärber Estimates to be 0.95 mg/L, with a 95% confidence interval of 0.89 to 1.0 mg/L.
- No-Observed-Effect Concentration (NOEC): 0.36 mg/L
- Highest concentration producing 0% immobilization: 0.36 mg/L
- Lowest concentration producing 100% immobilization: 1.4 mg/L