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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
Analytical monitoring:
Details on sampling:
Standards and samples of Mn-hexyl acrylate were analyzed by headspace gas chromatography with flame ionization detection (HS GC-FID). Analysis was performed on a Perkin Elmer TurboMatrix 40 Trap Headspace Sampler and Perkin Elmer AutoSystem XL gas chromatograph equipped with a 15 m × 0.53 mm id, 1.5 µm film Rtx-1 (Restek) analytical column connected to a 1 m × 0.2 mm id capillary restrictor. The headspace transfer line was connected directly to the analytical column. Standards and samples were equilibrated for 45 minutes at 95°C. The Perkin Elmer TurboMatrix 40 was equipped with a Carbopack C analytical trap. The pressurization time was 3 minutes at 40 psi, and was dry purged for 5 minutes. The trap was then thermally desorbed at 280°C for 0.5 minutes. Needle and transfer line temperatures were both 180 °C. The FID temperature was 300°C and the column pressure was 28 psi. The oven temperature was held at 45 °C for 2.0 minutes and then ramped to 300 °C at 20 °C/minute. The oven was held at a final temperature of 300 °C for 3 minutes. The signal attenuation setting was -2.

Water samples were collected in ca. 20 mL volatile organic analysis (VOA) vials with no headspace. On Day 0, “New” samples were collected directly from the respective control or test substance mixing vessels. At 48 hours, the termination of the study, “Old” samples were collected from a composite of replicate exposure chambers except for the two highest concentrations (4.7 and 15 mg/L). There was complete immobilization observed in these two concentrations following 24 hours of exposure and therefore analytical samples were collected and analyzed on day 1. Samples were prepared for analysis by pipetting 0.050 to 10 mL portions to ca. 20-mL headspace vials containing five grams sodium sulfate and sufficient moderately hard diluent water so that the sum of sample plus diluent totaled 10 mL. Five microliters of an approximately 81.6 µg/mL 2-ethylhexyl acrylate internal standard in acetone was also added. The headspace vials were immediately capped and shaken for 30 minutes on a mechanical wrist-action shaker before being placed on the automated headspace sampler. Analytical standards were similarly prepared by spiking microliter aliquots of n-hexyl acrylate solutions in acetone, along with internal standard solution, directly into headspace vials containing 10 mL of moderately hard water and sodium sulfate as described above. MRD-18-435 standards in water were prepared and analyzed at concentrations 4.08, 12.2, 40.8 and 122 ng/mL; each with a constant 40.8 ng/mL concentration of 2-ethylhexyl acrylate internal standard.

Data were acquired and processed using Perkin Elmer TotalChrom Workstation software (version 6.3.1) and a Dell OptiPlex 745 PC. Standard analyses resulted in a linear response over the standard concentration range. MRD-18-435 eluted as single peak with an approximate retention time 5.83 minutes under the analytical conditions utilized. The 2-ethylhexyl acrylate internal standard eluted at approximately 6.97 minutes. The practical quantitation limit (PQL) was approximately 0.0041 µg/mL; corresponding to the lowest analyzed standard. All reported concentrations were derived from the use of the standard curve and the internal standard and reported as n-hexyl acrylate in milligrams per liter of water (mg/L), which is also equivalent to µg/mL.

Test organisms (species):
Ceriodaphnia dubia
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
102 mg/L CaCO3
Nominal and measured concentrations:
Measured Concentration (mg/L) of n-hexyl acrylate
Nominal 4-Mar-18 5-March-18 6-Mar-18 Geomean(mg/L)
Day 0 Day 1 Day 2
0 (control) nd nd
0.06 0.0553 0.00732
0.0558 0.00754
0.0556 0.00743 0.02
0.19 0.222 0.0911
0.225 0.0904
0.224 0.0908 0.14
0.61 0.843 0.493
0.864 0.478
0.854 0.486 0.64
2.0 2.08 1.22
1.89 1.18
1.99 1.20 1.5
6.3 5.58
5.14* 4.32
5.05* 4.23
mean 5.26 4.28 4.7
20 16.3* 14.5
16.1* 14.2
mean 16.2 14.4 15
nd = not detected
practical quantitation limit ~0.0041 mg/L
*Reported results from retain; original analysis had low internal standard recovery.
Details on test conditions:
There are no known contaminants in the feed used for the study, in culturing the organisms or the dilution water, believed to be at levels high enough to interfere with this study. The algae and yeast-cereal leaves-trout chow mixture (YCT) were not analyzed. The reconstituted water is prepared from salts and UV-sterilized deionized well water that is treated and distributed throughout the testing facility via PVC and stainless-steel pipes. The deionized water is monitored annually for priority pollutants, unionized ammonia, total suspended solids, and for bacteria by Accutest® Laboratories, Inc., Dayton, NJ 08810 USA. Contaminant analysis results are maintained at the testing facility. The laboratory is accredited by the National Environmental Laboratory Accreditation Conference (NELAC) and has been audited by ExxonMobil Biomedical Sciences using the Quality Practices and Guidelines (QP & G v. 5.3). The analyses are performed using standard US EPA methods (e.g., EPA 200.7, 200.8, 245.1, 335.4, 608, 624, 625; SW846 3510C, SW846 8151).

Individual treatment solutions were prepared by adding the appropriate mass of test substance to dilution water in glass aspirator bottles sealed with Teflon® screw caps and stirred on a magnetic stir plates for 23 hours and 30 minutes. A control solution was prepared in the same manner without the addition of test substance. Vortex was set at > 10% to facilitate mixing as all treatment loadings were below water solubility. All treatment solutions and the control were allowed to settle for 2 hours and 30 minutes. At the end of the settling period, separate samples of the control and treatment solutions were removed from the mixing vessel through the outlet at the bottom of the vessel and added to each corresponding replicate test chamber. The geometric mean (geomean) measured concentrations were, 0 (Control), 0.14, 0.64, 1.5, 4.7 and 15 mg/L based on head space gas chromatograph flame ionization detection (HS GC-FID) analysis.
Reference substance (positive control):
Key result
48 h
Dose descriptor:
Effect conc.:
1.82 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
Details on results:
Percent immobilization observed at 48 hours was 0, 0, 0, 5, 35, 100 and 100% for the control, 0.02 0.14, 0.64, 1.5, 4.7 and 15 mg/L treatment groups, respectively. The 48 hour EC50 value for MRD-18-435 (N-Hexyl Acrylate) was calculated as 1.82 mg/L with 95% confidence intervals of 1.77 to 1.87 mg/L based on geomean measured concentrations. Geomean measured concentrations were used as there was greater than 20% loss in test substance detection between day 0 samples and 48 hour samples as indicated in OECD 201 (OECD, 2011).
Validity criteria fulfilled:
This study met the acceptability criteria for mortality (not to exceed 10%) in the control group at the end of the test and the dissolved oxygen was above 4 mg/L.

Headspace gas chromatography coupled with flame ionization detection (HS GC-FID) was used to analyze treatment concentrations on day 0, day 1 and day 2 old samples.

Water quality measurements are presented in Appendix A. The pH values ranged from 8.26-8.40. The dissolved oxygen remained above 7.4 mg/L and the temperature ranged from 24.8-25.5°C.

No observation of test substance insolubility (surface slicks, precipitates, and/or adherence to the test chamber) was noted during the time of organism observations. No abnormal observation of behavior or appearance occurred in the control treatment. The control survival was 100% throughout the 48 hour exposure. Percent immobilization is presented below. The 48 hour EC50 value for N-Hexyl Acrylate was calculated as 1.82 mg/L with 95% confidence intervals of 1.77 to 1.87 mg/L based on Geomean measured concentrations. Geomean measured concentrations were used as there was greater than 20% loss in test substance detection between day 0 samples and 48 hour samples as indicated in OECD 201 (OECD, 2011).

Description of key information

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
1.82 mg/L

Additional information