Ammonium iron(3+) hexakis(cyano-C)ferrate(4-)

Long-term toxicity to aquatic invertebrates

Description of key information

NOEC (21 d): 0.142 mg/L (meas. (TWA)) based on reproduction of Daphnia magna (OECD 211)

Additional information

Two experimental studies are available investigating the effects of Milori blue (CAS 25869-00-5) to aquatic invertebrates. The first study (Kuhl, 2013) was considered not to be reliable for the assessment as explained below. A second study with a modified test design was performed and the hazard assessment was based on the result of this study (Niino, 2014).

The first study was performed according to OECD 211 (GLP). Daphnia magna was exposed to a nominal concentration of 100 mg/L and dilutions of 1:2, 1:4, 1:8 and 1:16 (Water Accommodated Fractions). In the main test, ahighmortality of the adult daphnids was observed (except in the lowest concentration, dilution 1:16). Since the effect is not expected to be related to the soluble fraction of the test substance the study was stopped and will be repeated with a modified test design.

According to our study monitor, Dr. Knoell Consult GmbH, the study has been conducted using the correct OECD guidanceincl. the recommendations given in the OECD Monograph No. 23 for difficult substances.A pre-test with the filtrate (filter size: 0.45 µm) of the water accommodated fraction showed no effects, means no mortality. In the main test, apretendeddose-dependent mortality of the adult daphnids was observed (except in the lowest concentration, dilution 1:16). At the same time, there was no offspring at all, again except in the lowest concentration. But also in this concentration there was a strong inhibition in offspring number (> 50%) and no offspring has been produced after day 15. Thus, the test was stoppedby the lab at day 15as it wasobvious that it wouldnotbepossible to derive reliable (no-) effect levels out of this study based on the preliminary results.

To the understanding of our study monitor, Dr. Knoell Consult GmbH, the strong inhibiting effect on Daphnia mortality and reproductionis unlikely to be related to an intrinsictoxicity of the test item. In fact, the effect isconsidered to bean artefact as result of a physical effect. An explanation could be that the particle size of the substance is so low that considerable amounts of particles passed theusedfilter. It is known that Daphnia magna reacts very sensitive to the occurrence of particles in the test solution, as these particles can injure the antennae of the daphnids, and, if filtrated by the animal, can to some extent block the gut of the daphnids. This can lead to a substantially loweredmobility,food intakeandingestion rate which could explain the mortality (merely due to starving) as well as the reduced reproduction of the daphnids.This would also explain the dose-dependent effect, as the number of particles was directly dependent on the dilution factor.Therefore, this study was not considered to be reliable for the evaluation of chronic effects to aquatic invertebrates and the study was repeated with a modified test design.

The second study was repeated following OECD 211 (GLP) with the freshwater invertebrate Daphnia magna (Niino, 2014). A stock solution of the test substance was prepared by stirring and filtration (100 mg/L) to prepare the loading rates of 0.82, 1.8, 4.0 9.0 and 20 mg/L. No additional solvent was used for preparation of test solutions. ICP-MS analysis was used for the analytical measurement of actual test solutions. After 21 d effects on reproduction of D. magna were recorded with a corresponding NOEC of 0.142 mg/L (meas. (TWA)) and an EC10 of 0.168 mg/L (meas. (TWA)). 100% mortality of parent animals was observed in the highest test concentration (1.29 mg/L (meas. TWA)). This might have resulted from physical effects since precipitation was observed in the highest test concentration. Moreover, the test solution was completely blue. The test concentrations relevant for calculation/derivation of effect concentrations were completely clear. Thus, physical effects on reproduction can be excluded and the effect values are reliable and can be used for hazard assessment.