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Ecotoxicological information

Short-term toxicity to fish

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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-04-07 to 2015-12-09
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to GLP requirements. No deviations was issued.
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
Test water samples were collected from each test chamber of each treatment and control group 7 and 4 days prior to the start of exposure to confirm concentrations after conditioning the diluter system for 1 and 4 days, respectively. Test water samples also were collected from each replicate test chamber in each treatment and control group at the beginning of the test and at 24, 48, 72, and 96 hours (± 1 hour) to measure concentrations of the test substance. The samples were collected from mid-depth, placed in glass vials containing equal volumes of methanol, and processed immediately for analysis.
Details on test solutions:
Individual stock solutions were prepared for each of the five concentrations tested, and were prepared once during pretest period and once during the test. Test solution concentrations are based on the test substance as received, with no adjustments made for purity as the test substance is a UVCB (chemical substance of unknown or variable composition, complex reaction products and biological materials). A 150-mL primary stock solution was prepared by mixing a calculated amount of test substance into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 157 mg/mL. The primary stock solution was sonicated for approximately 15 minutes, stirred on a stir plate for approximately 15 minutes, and then inverted. The primary stock solution appeared clear and colorless with no visible precipitates. Four secondary stock solutions (60 mL each) were prepared in DMF at nominal concentrations of 98, 61.1, 38.1, and 24.0 mg/mL by proportional dilution of the primary stock. The secondary stock solutions were mixed by stirring for approximately 15 minutes on a stir plate followed by inversion, and were all clear and colorless with no evidence of precipitates. Solvent was used in this study as the solubility of the test substance in water, the amount of mixing required to prepare stock solutions in water, and the volume of water that would be required to make such stocks would be unfeasible in the facilities in which the study was conducted.
The stock solutions were held under ambient conditions in the syringes by the diluter system. Fresh aliquots of each stock were placed on the delivery system pumps once during the pre-test equilibration period and once on the day before exposure of the test organisms began. During the exposure period, the stock solutions were pumped into the diluter mixing chambers assigned to the treatment groups at a target rate of 4.00 µL/minute and were mixed with dilution water in the mixing chambers, delivered at a target rate of 200 mL/minute to achieve the desired nominal test concentrations. The negative control received dilution water only. The solvent control was prepared by delivering HPLC grade DMF to the mixing chamber for the solvent control at the same rate as the test substance stock solutions. The concentration of DMF in the solvent control and all treatment groups was 20 µL/L.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
The zebrafish (Danio rerio) was selected as the test species for this study. This species is representative of an important group of aquatic vertebrates and was selected for use in the test based upon past history of use in the laboratory. Zebrafish used in the test were received as juveniles from Aquatic Research Organisms of Hampton, New Hampshire. Identification of the species was verified by the supplier. All fish used in the test were juveniles from the same source and year class, and the length of the longest fish measured was no more than twice the length of the shortest. The average total length of 10 negative control fish measured at the end of the test was 2.7 cm, with a range of 2.2 to 3.0 cm. The average wet weight (blotted dry) of 10 negative control fish measured at the end of the test was 0.16 grams, with a range of 0.11 to 0.24 grams. Loading was defined as the total wet weight of fish per liter of test solution, and was 0.011 g fish/L that passed through the test chamber in 24 hours. Approximately 10 volume additions of test solution went through each test chamber per day. Instantaneous loading was 0.11 g fish/L of test water present in the test chambers at any given time.
The fish were held for at least 14 days prior to the test in water from the same source and at approximately the same temperature as used during the test. During the 2 week period immediately preceding the test, water temperatures in the cultures ranged from 21.8 to 23.6°C, the pH of the water ranged from 8.4 to 8.5, and the dissolved oxygen concentrations were >=8.1 mg/L (>=93% of saturation). During this 2-week period, the fish in the lot used for the test showed no signs of disease or stress and there were no mortalities.
Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
144 mg/L as CaCO3
Test temperature:
Dissolved oxygen:
5.2 mg/L (dissolved oxygen) and 60% of Air Saturation Value at 22°C
Nominal and measured concentrations:
Nominal: 0.48, 0.76, 1.22, 1.96 and 3.1 mg/L
Measured: 0.42, 0.63, 1.15, 1.6 and 2.12 mg/L
Details on test conditions:
The test systems were illuminated using fluorescent tubes that emit wavelengths similar to natural sunlight. The lights were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30 minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in light intensity. Light intensity was measured at the water surface of one representative test chamber at the beginning of the test using a SPER Scientific Model 840006 light meter.

The test was conducted at a target water temperature of 22 ± 1°C. Temperature was measured in each test chamber at the beginning and end of the test using a digital thermometer. Water temperature also was monitored continuously in one negative control test chamber using a validated environmental monitoring system (AmegaView Central Monitoring System). The system measurements were verified prior to exposure initiation with a digital thermometer.

Dissolved oxygen and pH were measured in one replicate test chamber of each treatment and control group at the beginning of the test, at approximately 24 hour intervals during the test, and at the end of the test, with measurements typically alternating between replicates in each group at each measurement interval. Dissolved oxygen was measured using a Thermo Orion Star A213 dissolved oxygen meter, and measurements of pH were made using a Thermo Orion Dual Star pH/ISE meter.

Hardness, alkalinity and specific conductance in the dilution water were measured at the beginning of the test. Hardness and alkalinity measurements were made by titration based on methods in Standard Methods for the Examination of Water and Wastewater (4). Specific conductance was measured using an Acorn Series Model CON6 conductivity-temperature meter.

Reference substance (positive control):
96 h
Dose descriptor:
Effect conc.:
1.65 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95% CI: 1.51-1.79
96 h
Dose descriptor:
Effect conc.:
0.63 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Results with reference substance (positive control):
24-Hour LC50: 316 mg/L
95% Confidence Interval: 244 - 411 mg/L
*based on a non-GLP 24-hour screening test using nominal concentrations
Sublethal observations / clinical signs:

Measurement of Test Concentrations

Nominal concentrations selected for use in this study were 0.48, 0.76, 1.22, 1.96, and 3.1 mg/L. During the course of the test, the appearance of the test solutions at these nominal concentrations was observed in the test chambers, as well as in the diluter mixing chambers where test substance stocks anddilution water were combined prior to delivery to the test chambers. At test initiation, the test solutions in the mixing chambers and test chambers appeared clear and colorless, with no evidence of precipitation observed in any control or treatment solution. At test termination, test solutions in all the control and treatment test chambers as well as in diluter mixing chambers that delivered test solution for the nominal 0.48, 0.76, and 1.22 mg/L test concentrations also appeared clear and colorless with no precipitates observed; the solutions in the diluter mixing chambers that delivered test solutions for the 1.96 and 3.1 mg/L test concentrations also appeared clear and colorless, but a light oil sheen was observed on the solutions’ surfaces. 


Measured concentrations of the samples ranged from approximately 54.7 to 115% of nominal. Measured concentrations of the samples ranged from approximately 60.3 to 99% of nominal. When measured concentrations of the samples collected during the test were averaged, the mean measured test concentrations for this study were 0.42, 0.63, 1.15, 1.60, and 2.12 mg/L, representing 88, 83, 94, 82, and 68% of nominal concentrations, respectively. The results of the study were based on the arithmetic mean, measured concentrations.


Observations and Measurements

Water temperatures were within the 22 ± 1ºC range established for the test. Measurements of pH ranged from 8.0 to 8.1 during the test. Dissolved oxygen concentrations remained =7.1 mg/L (=82% of saturation) throughout the test. The measurements of hardness, alkalinity and specific conductance in the dilution water at the beginning of the test were typical of Wildlife International well water. Light intensity at the beginning of the test was 330 lux at the surface of the water of one representative test chamber.


All zebrafish in the negative and solvent control groups appeared normal throughout the test. All fish in the 0.42 mg/L treatment group also appeared normal throughout the test, with no mortalities orovert signs of toxicity observed. No mortality was also noticed in the 0.63 mg/L treatment group throughout the test. Percent mortality in the 1.15, 1.60, and 2.12 mg/L treatment groups at test termination was 5, 35, and 95%, respectively. Signs of toxicity observed among the surviving fish in the 0.63, 1.15, 1.60, and 2.12 mg/L treatment groups at test termination included lethargy, surfacing, discoloration, and loss of equilibrium. Fish were also observed to be lying on the bottom of the test chambers in the 1.60 and 2.12 mg/L treatment groups at 72 hours after initiation. Therefore in this study, the NOEC was 0.63 mg/L based on the mortality data. LC50 values at 24, 48, 72 and 96 hours were determined from the mortality data. 
Validity criteria fulfilled:
See above.
The 96h-LC50 obtained after exposure of Danio rerio to the test substance according to OECD 203 testing guideline is 1.65 mg/L.
Executive summary:

Zebrafish (Danio rerio) were exposed for 96 hours under flow-through conditions to five mean measured concentrations of tricyclodecane dimethanol, esters with acrylic acid ranging from 0.42 to 2.12 mg/L acoording to OECD 203 testing guideline. Based on the mean measured concentrations, the 96-hour LC50value was 1.65 mg/L, with a 95% confidence interval of 1.51 to 1.79 mg/L. The slope of the concentration-response curve was 12.6. The NOEC was 0.63 mg/L based solely on the mortality data. 

Description of key information

Zebrafish (Danio rerio) were exposed for 96 hours under flow-through conditions to five concentrations of the test substance in a study according to the OECD 203 testing guideline. Based on the mean measured concentrations, the 96-hour LC50 value was 1.65 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
Effect concentration:
1.65 mg/L

Additional information