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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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long-term toxicity to aquatic invertebrates
Data waiving:
study technically not feasible
Justification for data waiving:
Justification for type of information:
Acute testing to the aquatic environment has been waived in accordance with column 2 of Annexes VII and VIII on the basis of low solubility. Normally, in such cases, long term aquatic toxicity testing is then considered to cover the possibility that low aqueous solubility means increased lipophilic nature and the possibility of bioaccumulation. However, this is not the case for inorganic substances such as these fibers. They have low fat solubility, are not lipophilic and are not considered likely to accumulate in aquatic species. Additionally, as outlined below, they have low possibility to cross biological membranes, further reducing their potential bioavailability.

Within the environment, microorganisms found in soils, water columns and waste water treatment processes (activated sludge) may encounter a range of exogenous compounds. These can include antimicrobial compounds such as biocides, pollutants, drugs, pharmaceutical ingredients and pesticides [12]. Where such agents are able to cross biological membranes, they may result in toxicity leading to possible adverse impacts on the local environment. However, microorganisms such as bacteria are well adapted to the constantly changing chemical and physical agencies of the environment. Their outer cell wall or membrane acts as an efficient permeability barrier, preventing internal exposure and subsequent exposure related toxicity. Despite this, there are various compounds that through properties such as relevant size and surface charge may penetrate this outer line of defence. As such, consideration of the ability of substances to cross such biological membranes is prudent as part of assessment of environmental safety.

Size-restricted, Selectively Permeable Barrier

Micro-organisms basically consist of a cell wall and one or two lipid membranes [132] while archaea and eubacteria, which are commonly found in soils, are also surrounded by a crystalline surface layer (S-layer) [143]. For eubacteria, there are two major groups based on differences in their cell envelope and these are gram-positive and gram-negative bacteria. Gram positive bacteria have a single cytoplasmic membrane encased in a thick rigid cell wall while gram-negative bacteria have an outer membrane in addition to the cytoplasmic membrane. Between the inner cytoplasmic membrane and outer membrane/ cell wall is a thin peptidoglycan layer. The composition, properties and role each of these layers plays in maintaining a selectively permeable barrier for a variety of micro-organisms provided in Table 1.

The first barrier to penetration is, if present, the S-layer of glycoproteins bound to the outermost layer of the organism. The S-layer possess a pore size of 2 – 8 nm meaning that small molecules and, theoretically, very small (<8 nm) nanoparticles may pass through this layer. The geometric mean diameter of PAS fibers as assessed by the Fraunhofer Institute is several orders of magnitude greater than this physical size cut-off at 0.56 µm (± 1.78).

The second barrier in the case of Gram-negative is the outer membrane which has membrane spanning porins which serve as a molecular sieve, preventing penetration of molecules with a molecular mass greater than 600 to 1000 Daltons [4]. Such a size cut-off for membrane permeability has been demonstrated using libraries of cyclic peptides of different sizes in the 800 – 1200 Da size range. It was found that there is a steep drop-off in membrane permeability at molecular weights above 1000 Da and it appears likely that this cutoff constitutes an upper size limit also for more drug-like compounds [5]. Indeed, in relation to the ecotoxicological hazards of polymers, those with small molecular weights (<1000 Dalton) are considered to be more likely to cross biological membranes and therefore raise environmental concerns. From a hazard screening perspective, the US EPA ‘Safer Choice Program’ promotes closer review of potential hazards if the average MW is greater than 10,000 Daltons, or if the low MW portion (percent MW that is <1,000) is more than 5% [6].

Whilst porins may allow low molecular weight compounds to penetrate this outer layer, at <1nm in diameter, the porins are too small to allow passage of nanomaterials [7] let alone micron-scale PAS fibres. It is also relevant to note that the inner surface of porin channels often exhibit charged amino acids [8] that form a selective barrier to metal ions released by particle dissolution [9]. As such, given the low solubility of PAS fibres and selective nature of the porin channel, K+ ions would likely be excluded from passive diffusion across the outer membrane.

The murein layer either follows the outer layer in the case of gram-negative bacteria or completely replaces it in the case of gram-positive bacteria. It is formed of a variety of sugars and amino acids that form a rigid exoskeleton and is hydrophilic in nature forming a selective barrier to hydrophobic moieties. Following this layer is the cytoplasmic membrane of the microorganism which is again, size restrictive with the pore size of membrane spanning proteins being <1 nm.

Table 1: The prokaryote envelope as a barrier to particulates. Reproduced and adapted from Handy, van den Brink [9]

Structure Archaea Gram-positive bacteria Gram-negative bacteria Particle Issue
Cytoplasmic membrane Lipid bilayer of mainly glycerol-ether lipids. Contains membrane spanning proteins Lipid bilayer of mainly glycerol-ester lipids. Contains membrane spanning proteins Lipid bilayer of mainly glycerol-ester lipids. Contains membrane spanning proteins Hydrophobic layers, pore sizes in proteins <1 nm. Only lipid dispersible, or lipid coated nanomaterials may associate with latter
Murein layer Absent Relatively thick layer, 10–50 nm wide. Peptidoglycan, teichoic
acids and polysaccharides.
Contains fixed polyanions and hydrophilic Relatively thin layer, 2–3 nm wide. Mostly peptidoglycan. Contains fixed polyanions and hydrophilic Hydrophobic particulates are less likely to be able to penetrate this layer
Outer membrane Absent Absent A thin peptidoglycan layer, 7–8 nm thick. Contains
lipopolysaccharides. Membrane spanning porins. Contains fixed polyanions and hydrophilic Hydrophilic nanoparticles likely to associate with the outer membrane. Porins too small (<1 nm pore) for nanoparticles.
S-layer Glycoprotein coat forming the outer-most cell envelope layer Glycoprotein layer covalently linked to the murein layer. Lattice structure with a pore size 2–8 nm Glycoprotein layer covalently
linked to the outer membrane.
Lattice structure with a pore size of 2-8nm Nanoparticles <8 nm may
theoretically penetrate the
(large pore size) lattice

The structure of micro-organisms creates a size-restricted barrier to the penetration of substances that operates effectively to maintain structure and health. Only small molecules, typically with a molecular weight <1000 Da can penetrate effectively. In terms of inorganic particles, it is theoretically possible for very small (<8 nm) nanoparticles to penetrate the outer layer but would be unlikely to cross the cytoplasmic membrane where protein pore sizes are <1 nm. As such, it is clear that concern of possible penetration of bio-membranes is largely confined to compounds of low molecular weight (e.g. drugs, certain polymers) and theoretically, nanomaterials. A large molecular size both limits and precludes uptake by biota should environmental release occur [10].

Due to the very large, macromolecular structure of PAS fibers it is clear that they are not a low molecular weight compound and are do not meet the size definition for a nanomaterial (one, two, or three dimensions are within the range from 1 to 100 nm [11]). The geometric mean diameter of PAS fibers at 0.56 µm (± 1.78) is orders of magnitude above the size cut-off for bio-membrane penetration and is in fact, larger than the size of many prokaryotes [12]. As such, it is highly unlikely that PAS fibres will penetrate bio-membranes and as such, are of low concern for subsequent ecotoxicity and/or bioaccumulation. The only form in which constituents of PAS fibres are likely to permeate biological membranes is in the ionic form and in relation to PAS fibres, this is likely to be K+. The permeability coefficient of K+ measured using an artificial membrane is 4.7 x 10-14 cm/s [24]. However, it should be considered that the charge amino acids of the porin channels restricts the passive penetration of metal ions meaning that controlled active, transporter-mediated entry is required. Potassium (as well as SiO2 and Al2O3) is an integral constituent of soils and intracellular concentrations of K+ are controlled via ion pumps whereby the sodium/ potassium pump transports three Na+ ions extracellularly and two K+ ions intracellularly for every molecule of ATP hydrolyzed [25].
1. Russell, A.D., Bacterial outer membrane and cell wall penetration and cell destruction by polluting chemical agents and physical conditions. Sci Prog, 2003. 86(Pt 4): p. 283-311.
2. Beveridge, T.J. and L.L. Graham, Surface layers of bacteria. Microbiol Rev, 1991. 55(4): p. 684-705.
3. Sára, M. and U. Sleytr, Crystalline bacterial cell surface layers (S-layers): from cell structure to biomimetics. Progress in biophysics and molecular biology, 1996. 65(1-2): p. 83-111.
4. Sikkema, J., J.A. de Bont, and B. Poolman, Mechanisms of membrane toxicity of hydrocarbons. Microbiological reviews, 1995. 59(2): p. 201-222.
5. Matsson, P. and J. Kihlberg, How Big Is Too Big for Cell Permeability? Journal of Medicinal Chemistry, 2017. 60(5): p. 1662-1664.
6. EPA. Safer Choice Criteria for Colorants, Polymers, Preservatives, and Related Chemicals. Safer Choice Program 2019 [cited 2019 5/11/2019]; Available from:
7. Fan, L., et al., Synthesis of magnetic beta-cyclodextrin-chitosan/graphene oxide as nanoadsorbent and its application in dye adsorption and removal. Colloids Surf B Biointerfaces, 2013. 103: p. 601-7.
8. Neidhardt, F.C., J.L. Ingraham, and M. Schaechter, Physiology of the bacterial cell: a molecular approach. Vol. 20. 1990: Sinauer Associates Sunderland, MA.
9. Handy, R.D., et al., Practical considerations for conducting ecotoxicity test methods with manufactured nanomaterials: what have we learnt so far? Ecotoxicology, 2012. 21(4): p. 933-972.
10. Venkatesan, A.K. and R.U. Halden, Effective Strategies for Monitoring and Regulating Chemical Mixtures and Contaminants Sharing Pathways of Toxicity. International journal of environmental research and public health, 2015. 12(9): p. 10549-10557.
11. ISO, ISO/TR 18401:2017. Nanotechnologies — Plain language explanation of selected terms from the ISO/IEC 80004 series. 2017, International Organization for Standardization (ISO): Geneva.
12. Koch, A.L., What size should a bacterium be? A question of scale. Annu Rev Microbiol, 1996. 50: p. 317-48.

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