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Ecotoxicological information

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
Study period:
17 Feb - 04 Mar 2014
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study under GLP, analytical monitoring. Due to rapid hydrolysis, the ultimate degradation product, not the parent substance was monitored in test solutions.
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
- Concentrations: all solutions at initiation and conclusion of test.
- Sampling method: Samples (ca. 50 mL) were collected from excess solution at test initiation. At termination, samples were collected from each chamber and pooled.
- Sample storage conditions before analysis: Samples were stored at 1 - 6 °C until analysis.
-Other: In a preliminary test, the test substance was determined to be hydrolytically unstable. The sole detectable degradant was trifluoroacetic acid (TFA). TFA was the analyte monitored, and the parent substance concentration determined by stoichiometry
Details on test solutions:
- Method: The test substance (CAS# 756-12-7) is very volatile. To minimize losses, ca. 250 μL of CAS# 756-12-7 removed from its stainless steel cylinder through a septum, using a 250 μL airtight glass syringe. 0.2041 g (by analytical balance) was quickly dispensed into a ca. 20 mL glass vial containing ca. 10 mL of dilution water. During this process, the tip of the syringe was held just above the surface of the dilution water. The vial was closed immediately and tightly with a screw cap and the contents of the vial were vigorously mixed for approximately 45 seconds. The vial was then shaken for ca. 21 minutes at ca. 75 RPM on a horizontal shaker, after which time the contents of the vial were transferred to a 1 L glass volumetric flask containing ca. 500 mL of dilution water. The vial was rinsed three times with ca. 20 mL dilution water. Dilution water was used to fill the volumetric flask to the mark. The solution was then mixed well and transferred to a larger glass jar. An additional 1 L of dilution water was measured using the same glass volumetric flask and added to the stock solution. The diluted stock was sealed with Parafilm and mixed ca. 20x by inversion
- Controls: Dilution water only
- Evidence of undissolved material: None. All solutions were clear and colorless throughout the test.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Water flea
- Source: In-house culture reared from stock obtained ca. 8 months before study
- Age at study initiation: < 24h
- Method of breeding: The Daphnia cultures were fed approximately 2.5 mL each of Pseudokirchneriella subcapitata and yeast‐cereal‐grass fermented‐trout‐chow (YCT) three times per week. The neonates used in the test were fed approximately 2.0 mL of P. subcapitata prior to test initiation. Daphnia were reared and tested in moderately hard reconstituted water
- Feeding during test: No
- Health/mortality: 15.4% in 7 days prior to test
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
104 mg/L as CaCO3
Test temperature:
18.7 - 20.0 °C
7.5 - 8.1
Dissolved oxygen:
8.8 - 9.2 mg/L
Nominal and measured concentrations:
Nominal: 0 (negative control), 6.4 mg/L, 12.8 mg/L, 25.5 mg/L, 51 mg/L, 102 mg/L
Calculated: < 2.33 mg/L, 3.62 mg/L, 8.61 mg/L, 16.8 mg/L, 30.3 mg/L, 56.5 mg/L (see Table 1)
Calculated concentrations are based on measured concentrations of TFA (surrogate) and stoichiometric ratio.
Details on test conditions:
- Test vessel: 125 mL glass jar sealed with parafilm and covered with plexiglass.
- Type: closed
- Headspace, fill volume: Indicated 125-mL lines were verified with a graduated cylinder, fill volume 125 mL. Headspace minimal.
- Aeration: Medium aerated before use, no aeration during test.
- No. of organisms per vessel: Five
- No. of vessels per concentration: Four
- No. of vessels per control: Four

- Source/preparation of dilution water: Deionized water containing
MgSO4 (anh) 1.1444 g / 20 L
CaSO4 1.3198 g / 20 L
NaHCO3 2.1120 g / 20 L
KCl 0.0878 g / 20 L
4 mg/L vitamin B12 solution, 10 mL / 20 L
1 mg/L Na2SeO4 solution, 40 mL / 20 L

- Conductivity: 335 - 336 µS/cm
- Metals: See Fig. 1
- Alkalinity: 63.5 mg/L as CaCO3
- Ca/mg ratio: 1.1
- Culture medium different from test medium: No
- Intervals of water quality measurement: At test termination

- Adjustment of pH: No
- Photoperiod: 16:8 light:dark with 30 minute transition periods
- Light intensity: 590 ‐ 618 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: Blank, 3.13 mg/L, 6.25 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L, and 100 mg/L (nominal concentration)
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
Zinc sulfate test was started one day prior to the toxicity study
48 h
Dose descriptor:
Effect conc.:
39 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% CI, 36.0 - 42.2 mg/L. Test substance concentrations calculated using measured concentrations of degradant and reaction stoicheometry
Details on results:
- Effects: All affected organisms were confirmed dead by microscopy. Mortality was 10% at 51 mg/L (nominal) and 100% at 102 mg/L (nominal). No other indications of toxicity, including immobilization, were observed.
- Behavioral abnormalities: No
- Mortality of control: None
- Other adverse effects control: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No, all solutions remained clear and colorless throughout the test
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50/LC50: 0.77 mg/L
- Other: Historical norm 0.598 mg/L
Reported statistics and error estimates:
Statistical analysis is based on equivalent concentrations as parent compound. The 48‐h EC50 for immobilization was calculated using the statistical program CETIS™ (Version 1.7.0), Comprehensive Environmental Toxicity Information System, recommended by Environment Canada. EC50 and 95% CI were calculated using the trimmed Spearman-Kärber method.
The 48-h EC50 (OECD 202) of CAS# 756-12-7 to D. magna was 39.0 mg/L (95% CI, 36.0 - 42.2 mg/L) based on measured concentration of degradation product and stoichiometry.
Executive summary:

The toxicity of CAS# 756-12-7 was assessed in a study done according to OECD guideline 202. CAS# 756-12-7 is volatile and hydrolytically active. In a preliminary study, the parent compound could not be detected at any time point, and the sole degradation product detected was trifluoroacetic acid (TFA). Analytical confirmation was therefore of TFA concentrations, with equivalent concentration of CAS# 756-12-7 calculated by stoichiometry and molecular weight ratios. Equivalent concentrations were 55 -67% of nominal concentrations, and so the equivalent concentrations were used to calculate EC50. 10% immobilization was seen at 30.6 mg/L and 100% immobilization was seen at 56.0 mg/L. All immobilized organisms were confirmed dead by microscopy. The EC50 was 39.0 mg/L (95% CI, 36.0 - 42.2 mg/L).

The test was conducted according to an internationally accepted guideline under GLP criteria, with analytical confirmation of test substance concentration (by surrogate). However, the parent compound was itself not analyzed due to rapid hydrolysis. Therefore, the test is considered reliable with restrictions. It is suitable for Risk Assessment, Classification & Labelling, and PBT Analysis.

Results summary:

48-h EC50 (immobilization): 39.0 mg/L

95% CI: 36.0 - 42.2 mg/L

Description of key information

The 48-hour EC50 (immobilization) of CAS# 756-12-7 to Daphnia magna is 39 mg/L (95% CI, 36 - 42 mg/L) (OECD 202).  All immobilized organisms were confirmed dead at end of study.

Key value for chemical safety assessment

Additional information

The toxicity of CAS# 756-12-7 to Daphnia magna was examined in two studies done according to OECD 202 guidelines. Both studies featured NMR spectroscopic analysis that demonstrated that the parent compound was not stable in daphnid test media. The parent compound, initial hydrolysis product, and one stable degradant were demonstrably absent during the test. Only the water soluble degradant trifluoroacetic acid (TFA) was present during the test. However, a published risk assessment for TFA (1) conclusively shows that TFA has insignificant toxicity to D. magna (EC50 >1000 mg/L as the sodium salt). Accordingly, the analytical result for TFA was expressed as the stoicheometric equivalent concentration of parent. The key study had an EC50 of 39 mg/L (95% CI, 36-42 mg/L) as parent. The supporting study had 48-hour EC50 of 17 mg/L (95% C.I. 14 to 21 mg/L) as parent. Both studies are considered reliable with restrictions, however the key study is considered to have more reliable chemical analysis. The supporting study featured NMR quantitation of all known degradation products in test chambers at initiation and termination of the test, with analysis by a non-GLP lab. Only TFA was detected. Analytical determination of concentrations was by LC/MS in the key study and was conducted by a GLP-accredited lab. Stability of CAS# 756-12-7 in culture medium by NMR was done as a separate phase in this study, during which only TFA was detected in the culture medium. Further, the key study was done later than the supporting study, and test conditions were monitored more closely due to the unexpected toxicity (observed toxicity higher than expected from the measured TFA concentration).


1)   Boutonnet (Ed.), 1999. Environmental Risk Assessment of Trifluoroacetic Acid. Human and Ecological Risk Assessment: Vol. 5, No. 1, pp. 59-124.