An internationally benchmarked approach towards the standardised aquatic ecotoxicity testing of nanomaterials

As part of the EU FP7 project NANoREG, we addressed the need for development or modification of exposure and (eco)toxicity methods to generate more reliable data for regulatory risk assessment of manufactured nanomaterials (MNMs). The key challenges identified were: (i) generation of reproducible MNM dispersions, (ii) measurement of dynamic MNM properties during standard aquatic ecotoxicity tests, and (iii) a lack of specificity in existing standard tests for MNMs. Using synthetic amorphous silica (NM-200) from the OECD Working Party on Manufactured Nanomaterials’ sponsorship programme as a reference, we present a benchmarked standard operating procedure (SOP) for the calibration of probe sonicators that allows generation of comparable and reproducible dispersions. We also present a MNM dispersion SOP that is performance tested using 14 different reference materials representing metal, metal oxide and carbon nanotube (CNT) MNMs. A minimum of three data sets for each MNM were generated by different laboratories using in-house calibrated probe sonicators. Measurement of the resulting MNM dispersions is by simple dynamic light scattering (DLS). The results show that reproducible MNM dispersions can be generated, both with the same laboratory and across different laboratories, for all MNMs that are suitable to DLS (i.e. granular MNMs). There are limitations with the DLS analysis in the case of high aspect ratio MNMs such as CNTs and silver rods, which may also be linked to the stability of the dispersions. A technical guidance document (TGD) on procedures for the quantification of MNM exposure and fate in dispersions for aquatic ecotoxicological studies has also been developed. The TGD has the goal of identifying the minimum level of characterisation that allows meaningful interpretation of ecotoxicity data generated. The TGD uses a decision tree approach for identifying how and when to determine changes in MNM particle size and morphology over time, nominal and total measured MNM concentrations over time, MNM dissolution and changes in dissolution over time, and the amount of MNM lost from the exposure due to deposition. The probe sonication and MNM dispersion SOPs, together with the quantification TGD, have been applied to the testing of one silver MNM (NM-300K), three TiO₂ MNMs (NM-100, NM-101 and NM-103), and three CNT MNMs (NM-400, NM-401 and NM-411) in optimised freshwater microalgae and Daphnia magna acute ecotoxicity studies.