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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14th November 2017 to 6th December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Isomeric mixture of alkenyl succinate with 99.06%. All test solutions and results were corrected for this purity.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Range finding Test

A range-finding test with X-19924 was initiated on November 14, 2017. Nominal concentrationsof 0.1, 1, 10, 100 and 1000 mg/L were prepared by adding 0.035, 0.350, 3.5, 35, and 350 mL of a nominal 1000 mg/L stock solution, respectively into a graduated cylinder and making this up to be a volume of 350 mL with dilution water. The nominal 1000 mg/L solution was prepared by adding 1.1381 g of the test item into a 1 L glass aspirator bottle and made up to a 1 L volume with dilution water. The individual test solutions were made up in a graduated cylinder hand mixed by inverting the cylinder several times, prior to dispensing the test solution into each of two 100 mL test vessels (75 mL per test vessel). A total of 10 daphnids (i.e., 2 replicates with 5 daphnids each) were exposed to each test level
under static test conditions. All test organisms were transferred to fresh test and the negative control solutions using a pipette and bulb.

Definitive test
The definitive test with X-19924 was initiated on December 4, 2017. Results of the rangefinding test indicated that the 48-hour EC50 for daphnid immobilization was between 10 and 100 mg/L concentration of the test. For the definitive test, a nominal 1000 mg/L stock solution was prepared by adding 1.1380 g of X-19924 into a 1 L glass aspirator bottle and made up to a 1 L volume in dilution water. Appropriate amounts of the 1000 mg/L stock solution were mixed with control/dilution water to achieve the desired nominal test concentrations (i.e., 9.38, 18.8, 37.5, 75.0, 150 and 300 mg/L). The test solutions were hand mixed by inverting the cylinder several times, prior to dispensing the test solution into each of four 100 mL test vessels (with 75 mL per vessel). A total of 20 daphnids (i.e., 4 replicates with 5 daphnids each) were exposed to the test and control solution under static test conditions. All test organisms were transferred to the control and test solutions using a pipette and bulb. Test results were based on immobility over a 48-hour exposure period.

Test solutions

Vehicle:
no
Details on test solutions:
Individual test solutions of the test item for both range-finding and definitive tests were prepared from stock solutions (1000 mg/L nominal concentration) prepared in dilution water and without the use of any solubilizing agent. All stock and test solutions were corrected for purity of the test item (i.e., 87.9% test item, 12.1%KOH) Stock solutions were prepared in glass aspirator bottles and stirred for approximately 24 h using a stir bar and stir plate, and then settled for approximately 24 and 48 h for the range-finding and definitive tests, respectively. The first ~ 100 mL of stock solution removed from each glass aspirator bottle was discarded. The individual test solutions were prepared by adding the appropriate amounts of stock solution and dilution water to achieve the desired test concentrations. The individual test solutions were hand mixed by inverting mixing vessel several times prior to being divided equally into the replicate test vessels. All tests (rangefinding and definitive) included a negative control consisting of dilution water with no test item added.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnia magna STRAUS were cultured continuously from a population that has been actively reproducing in AquaTox’s laboratory since 1988. The organisms were originally obtained from the Ministry of the Environment (Toronto, Ontario, Canada). Certification of the taxonomic identity of the D. magna is provided in Appendix D. Culturing and holding of the test species prior to testing was conducted according to AquaTox SOP # 230 (AquaTox, 2016).

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
200-220 mg/L as CaCO3
Test temperature:
20 ± 2 °C,
pH:
8.2 to 8.5 of dilution water
Dissolved oxygen:
The concentration of dissolved oxygen in the water was maintained at >80% of the air saturation value
Nominal and measured concentrations:
Range finding test
Nominal Concentration (mg/L) 1000, 100, 10, 1, 0.1, 0

Definitive test

Nominal Concentration (mg/L) 300, 150, 75, 37.5, 18.75, 9.375, 0
Details on test conditions:
Parameter Condition
Test type Static
Test duration 48 hour
Temperature 20 ± 2 °C, as recorded daily with a maximum/minimum thermometer (constant within ± 1 °C)
Light quality Ambient laboratory illumination
Light intensity 400 to 800 lux
Photoperiod 16-hour light, 8-hour dark (with 30 minute transition period)
Feeding prior to test Once/day: 1) yeast, Cerophyll broth, Trout Chow (YCT) and 2) Pseudokirchneriella subcapitata algae
Feeding regime None during test
Test vessel Glass beaker, 150 mL (range-finding test), 100 mL (definitive test)
Loading rate 15 mL/daphnid
Test volume 75 mL (min)
Source of test organisms In-house culture
Age of test organisms First instar, neonate (<24-hours old)
Number of concentrations Range-finding test: 5 + a negative control; Definitive multiple concentration test: 6 + a negative control;
Number of organisms per replicate 2 (Range-finding test) 4 (Definitive test)

Total number of organisms 10 (Range-finding test) 20 (Definitive multiple concentration test)
per test level

Aeration None
Dilution water Reconstituted water (hardness 210 - 220 mg/L as CaCO3)

Monitoring parameters and Temperature daily; dissolved oxygen, pH, and conductivity on all solutions at start and end of test; dilution water
observations made during the test hardness at 0-hours; and visual observations of immobilisation of D. magna at 24 and 48 hours.

Measured endpoints Immobility, stressed behaviour
Calculated endpoints 24 and 48-hour EC50s; NOEC and LOEC
Reference substance (positive control):
yes

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
43.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
254 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
18.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
37.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Immobility of the controls did not exceed 10% and dissolved oxygen concentration at the end of the test were ≥ 3 mg/L in the control and test solutions.
Conclusions:
Results of the range-finding test indicated that the 48-hour EC50 for immobility of D. magna was between 10 and 100 mg/L. In terms of the definitive test, the 24 and 48-hour EC50s for D. magna were reported to be 254 and 43.6 mg/L, respectively. The 48-hour NOEC and LOEC for immobility of daphnids were estimated to be 18.8 mg/L and 37.5 mg/L, respectively
Executive summary:

The toxicity of the test substance to the Daphnia Magna was investigated under static test conditions, according to OECD Guideline 202,“ Daphnia sp., Acute Immobilisation Test” (OECD, 2004). This study were conducted in accordance with the requirements of OECD Principles of Good Laboratory Practice (GLP).

 

Both the range-finding and definitive tests met all the validity criteria and the test results were based on measured concentrations at the beginning and end of the test, or more specifically, the Time-Weighted Mean (TWM) concentrations. Results of the range-finding test indicated that the 48-hour EC50 for immobility of D. magna

was between 10 and 100 mg/L.

Results of the definitive test showed that the test item caused immobility of daphnids at nominal concentrations ≥ 37.5 mg/L, whereas immobility of daphnids was not observed at concentrations ≤ 18.8 mg/l.

The 24- and 48-hour EC50s for immobility of D. magna determined from the definitive test were estimated to be 254 and 43.6 mg/L, respectively. The NOEC and LOEC for immobility of daphnids were estimated to be 18.8 and 37.5 mg/L, respectively.

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