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Aquatic toxicity was evaluated by short-term toxicity tests on fathead minnow (Pimephales promelas) (Minderhout et al. 2012b (K1)), waterflea (Daphnia magna) (Minderhout et al. 2012a (K1)) and a toxicity test on freshwater algae (Pseudokirchneriella subcapitata) (Arnie et al. 2013 (K1)).

The tests have been performed with materials designated as 3 G FF and 3.5 G FF, both different substances but both fall within the substance definition as defined in Chapter 1.2.


In the short-term toxicity study with fathead minnows (Minderhout et al. 2012b), the fish were exposed for 96 h under static conditions to five mean measured concentrations of 3 G FF ranging from 3.6 to 76 μg/l, based on measured concentrations of C4. Nominal concentrations ranged from 7.5 to 120 μg/l. The 96 -h LC50 value was > 76 μg/l, the highest measured concentration tested. Fish exposed to 3 G FF at concentrations 76μg/l, appeared normal with no mortality or overt signs of toxicity. The NOEC was concluded to be 76 μg/l, the highest concentration tested.


In the short-term toxicity with Daphnia magna (Minderhout et al. 2012a), the cladocerans were exposed for 48 h under static conditions to five mean concentrations of 3 G FF ranging from 10 to 73 μg/l, based on measured C4 or 16 to 127 μg/l, based on measured TPP. The 48 -h EC50 was 73 μg/l or 127 μg/l, based on measured C4 or TPP, respectively. The no-immobility concentration and NOEC were both 73 μg/l or 127 μg/l 3 G FF, respectively based on C4 or TPP.


In the 96 h toxicity test with Pseudokirchneriella subcapitata (Arnie et al. 2013), the freshwater algae were exposed to five treatment levels of 3 G FF ranging from 4.56 to 220 μg/l, based on day 0 measured concentrations of C4. On day 0, concentrations ranged from 89 to 110% of the nominal concentrations. After four days measured concentrations were declined to 5 to 40% of the nominal concentrations.

Toxicity was assessed based on effects on area under the growth curve, yield and growth rate relative to the negative control group. None of the treatment groups mean responses for area under the growth curve, yield and growth rate were significantly reduced relative to the mean control responses.

Consequently, the 72 and 96 h NOEC values were determined to be 220 μg/l for both intervals and the 72 and 96 h EC50 values were higher than 220 μg/l, the highest concentration tested.


For all three tests, it can be concluded that 3 G FF was tested in the aquatic acute toxicity tests at concentrations up to water solubility or slightly higher. The water solubility of 3 G FF, based on the primary component (C4), in water at 20 °C, was determined to be 116 μg/l. In the three evaluated aquatic toxicity tests, concentrations of 3 G FF were determined based on measured C4, the primary component (representing approx. 76% of the test substance), in the test solutions during the study. Recovery based on C4 was lower to slightly lower at the beginning of the conducted tests. The measured concentrations based on C4 in the test solution samples declined during the study in all three studies.

In the three aquatic toxicity studies the less hydrophobic TPP was measured as well and concentrations of TPP in the test solutions were consistent during the tests. The fact that TPP concentration remain stable throughout the different tests is of importance as TPP is the constituent for which the highest level of aquatic toxicity is expected to occur based on the fact that TPP is classified as an aquatic toxicant.

The lower to slightly lower recoveries seen at the beginning of the test and the declines during the test of the 3 G FF concentration based on measured C4 were likely due to adsorption and/or hydrophobicity of the C4 component.

However, despite the lower to slightly lower recovery of 3 G FF based on measured C4, the starting measured concentrations were up to or close to the water solubility limit. No toxicity was observed at the highest concentration in any of the aquatic toxicity tests.