Registration Dossier

Diss Factsheets

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The REACH registration of silver (powder and massive forms of zero-valent, elemental, silver) is underpinned, in common with other metals, by a read-across (or analogue) approach from the properties of the free ion. This principle of read-across from the free ion has been extended to also include nanosilver.
The scientific validity of read-across from the hazard properties of ionic silver (source) to nanosilver (target) under REACH is underpinned by both theoretical and empirical considerations.
The theoretical basis for the use of ionic silver data as the foundation of the risk assessment of nanosilver is based on the premise that the free metal ion (Me+) is the most toxic metal form/species (Starodub et al. 1987). This consideration was implicit in the development of the Free Ion Activity Model [FIAM] (Morel 1983, Paquin et al. 2002, Campbell 1985, Brown and Markich 2000) and, more recently, the Biotic Ligand Model [BLM] (Paquin et al 2002, Niyogi and Wood 2004) that has underpinned the risk assessment of several metals (e.g. Cu, Ni, Zn) under the Existing Substance Regulations and REACH; and most recently the development of the Environmental Quality Standard (EQS) for nickel and nickel substances under the Water Framework Directive (WFD). When considered on an equal mass basis ionic silver would therefore be expected to have greater toxicity than nanosilver simply on the basis that silver ions are released over time from the surface of particles (via oxidative dissolution). As the properties of nanosilver are read-across directly from ionic silver (not just to the fraction of silver ions released from nanosilver), this read-across is also expected to introduce considerable precaution into the hazard component of the risk assessment of nanosilver as all nanosilver, irrespective of coating, morphology or particle size distribution is assumed to behave similarly to the free ion.
This theoretical consideration has been tested by conducting a comprehensive review of the available scientific literature for nanosilver, with particular emphasis on the comparative effects on REACH relevant biotic systems (REACH information requirement) of ionic silver and nanosilver. This review is described for each endpoint in subsequent sections of the report ‘Nanosilver: read-across justification for environmental information requirements’ (attached in IULCID Section 13) and is summarised below. Furthermore, this theoretical consideration was confirmed by a specific ecotoxicity testing programme undertaken by the EPMF following the silver substance evaluation and designed to compare the effects of the smallest nanosilver form registered under REACH (‘Nano 8.1’ or ‘Silberpulver Typ 300-30’) and silver nitrate to algae, Daphnia (long-term) and soil microorganisms. This testing programme is summarised below under the applicable endpoints in 4.2.1 and 4.2.3.

In terms of ecotoxicity, nanosilver on an equal mass basis has been found to be significantly less toxic than ionic silver for the majority of REACH endpoints and of equivalent toxicity for some others. None of the empirical information available suggests that nanosilver is consistently more hazardous than ionic silver on an equivalent mass basis, or that “nano-specific” effects that would prejudice the validity of read across from ionic silver to nanosilver occur. In addition, with very limited exceptions which are described further in the report ‘Nanosilver: read-across justification for environmental information requirements’, none of the available data suggested consistent relationships between particle morphology, size, particle size distribution (raw or agglomerated) or coating (surface treatment) and effects.
Notter et al. (2014) presents a meta-analysis of published EC50 values for ionic silver and nanosilver. The authors demonstrate that almost 94% of acute toxicity values assessed for freshwater, seawater and terrestrial systems using algae, annelid, arthropoda, bacteria, crustacea, fish, nematoda, plant, protozoa and rotatoria show that the nanoform of silver is less toxic than the dissolved metal (when normalised for total metal concentration).
In addition, a specific ecotoxicity testing programme designed to compare the effects of the smallest nanosilver form registered under REACH (‘Nano 8.1’ or ‘Silberpulver Typ 300-30’) and silver nitrate to algae, Daphnia (long-term) and soil microorganisms was undertaken following the silver substance evaluation. This demonstrated that the nanoform of silver is less toxic than ionic silver (based on EC10 and EC50 values for total, ‘conventional’ dissolved (<0.45μm) and ‘truly’ dissolved (<3kDa) silver). Therefore, taking the full body of evidence into account, the read-across use of toxicity values from ionic to nanosilver as a ‘worst case’ approach is justified and scientifically defensible for environmental endpoints. The Substance Evaluation Conclusion document for silver agreed with this conclusion for the nanosilver forms covered in the REACH dossier (RIVM 2018).

Short-term toxicity to aquatic invertebrates
Published data from several short-term toxicity to aquatic invertebrates studies using various sizes of nanoparticles and coating types are included in the REACH dossier as Endpoint Study Records. All of the nanosilver LC50 values from these studies are higher than the 48h LC50 of 0.22 μg/L for Daphnia magna (Bianchini et al. 2002) for ionic silver. Together with the theoretical basis for read-across based on the free-ion, this supports the use of ionic silver as the ‘worst case’ basis to read across properties to nanosilver, irrespective of any data available describing morphology, size, size distribution or coating. A summary of these supporting studies is available under Section 4.1.2 of the report ‘Nanosilver: read-across justification for environmental information requirements’ (attached in IUCLID Section 13).
Reason / purpose for cross-reference:
read-across source
48 h
Dose descriptor:
Effect conc.:
0.22 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
Basis for effect:
Remarks on result:
other: 95% CL 0.19–0.25 µg Ag/L. Filtered (0.45 mm), silver concentration.
Details on results:
Results also for 24 hour LC50 and with added sulphide. Reactive sulfide protects D. magna against acute silver toxicity.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The 48 hour LC50 values and the respective 95% confidence intervals were estimated on the basis of the cumulative mortality data using probit analysis. These values were estimated on the basis of both nominal and mean measured total and filtered silver concentrations over the respective periods of the test and the results were compared.
Validity criteria fulfilled:
not applicable
The freshwater 48 hour LC50 values for Daphnia magna when exposed to silver nitrate are 0.18 to 0.26 µg Ag/L based on measured total silver concentration and 0.22 µg Ag/L based on measured filtered silver concentration.
Executive summary:

The study is a non-guideline study, published in peer reviewed literature and considered suitable for use as a key study for this endpoint. The freshwater 48 hour LC50 values for Daphnia magna when exposed to silver nitrate are 0.18 to 0.26 µg Ag/L based on measured total silver concentration and 0.22 µg Ag/L based on measured filtered silver concentration.

Description of key information

Key value for chemical safety assessment

Additional information

Summary of available data for uncoated and coated nanosilver

Reliable and relevant data on the short-term toxicity of uncoated and coated nanosilver to invertebrates are available from 12 studies (Griffitt et al. 2008, Gao et al. 2009, Kennedy et al. 2010, Li et al. 2010, Gaiser et al 2011, Gaiser et al. 2012, Hoheisel et al. 2012, McLaughlin and Bonzongo 2012, Poynton 2012, Wang et al. 2012, Zhao and Wang 2012, Blinova et al. 2013).

A total of 55 LC50 values are available from studies investigating effects on five species (Daphnia magna, Daphnia pulex, Ceriodaphnia dubia, Thamnocephalus platyurus, Chydorus sphaericus). However, data predominantly relate to the crustacean species conventionally used in laboratory ecotoxicity testing i.e. Daphnia magna (seven studies – 35 LC50 values) and Ceriodaphina dubia (three studies – seven LC50 values).

The available reliable data includes various sizes of nanoparticles and, in addition to uncoated nanosilver materials, a range of coating/capping materials, including: PVP, citrate, EDTA, lactate and sodium dodecylbenzene sulfonate.

Studies predominantly report effects of spherical nanoparticles but there are also data for mixtures of spherical particles and nano rods (Kennedy et al. 2012), mixtures of spherical, prismatic and rod-shaped colloids (Li et al. 2012) and face-centred cubic crystal structures (McLaughlin and Bonzongo 2012). All studies were conducted in freshwater media.

The particle size of raw nanomaterials across the studies ranged from 8.4 to 123.9 nm (Particles >100 nm would not usually fall under the proposed definition of a nanomaterial, but as this threshold is not based on any scientific criteria toxicity data from studies with particle sizes >100 nm are included in this assessment for completeness). However, the majority of studies report effects associated with primary particle sizes between 8.4 and 51.86 nm (10th to 90th percentile). Aggregation/agglomeration behaviour of primary particles within experimental media or stock solutions varied between studies but in almost all instances authors report that some degree of aggregation/agglomeration in the test systems occurs, increasing the size of particles in environmental media. The characterisation results for nanosilver in test systems report particle sizes after aggregation/agglomeration of between 50.7 and 1,584 nm, with the majority of studies reporting particle sizes between 50.7 and 360.0 nm (10th to 90th percentile).

LC50 values range from 0.43 µg/L to 221 µg/L, after 48 hour exposure. Interestingly, both of these LC50 values are from the study by McLaughlin and Bonzongo (2012) which investigated the toxicity of uncoated nanosilver particles (25.4 nm) to Ceriodaphnia dubia in both synthetic and natural waters. The greatest toxicity (LC50 value of 0.4 µg/L) was recorded in river water, whilst the lowest toxicity was recorded in water from a wetland. A similar observation was recorded in Pseudokirchnerialla subcapitata exposed to nanosilver in the same water. Differences in observed toxicity may be the result of variable bioavailability of silver in these waters. The 10th to 90th percentile range of LC50 values for all forms of nanosilver assessed for acute invertebrate toxicity range from 1.31 to 187.8 µg/L.

All of the nanosilver LC50 values are less sensitive than the 48 hour LC50 of 0.22 µg/L for Daphnia magna (Bianchini et al. 2002) used in the REACH CSR for silver.

In addition, where studies undertook a comparative assessment of the relative toxicity of nanosilver and ionic silver within their own study designs (Griffitt et al. 2008, Kennedy et al. 2010, Zhao and Wang 2012, Poynton 2012, Hoheisel et al. 2012, Wang et al. 2012, Blinova et al. 2013) nanosilver was only observed to be more toxic than ionic silver on a single occasion (Griffitt et al. 2008 – 48 hour exposure of C. dubia neonates). The relative toxicity of bulk silver (0.6 - 1.6 µm particles) in comparison to nanosilver was tested on a single occasion (Gaiser et al. 2011). In this test, bulk silver was approximately an order of magnitude less toxic than nanosilver.

Overall, there is no statistically significant correlation between smaller particle sizes and increased toxicity (Kendall test, p>0.05). However, Hoheisel et al. 2012 report a clear reduction in toxicity (greater LC50) with increasing particle size. This relationship was effectively normalised when results were expressed on the basis of surface area rather than mass concentration, which suggests that the toxicity observed was related to the dissolution rate of silver ions from the surface of the particles. Smaller particles have much greater surface area than larger particles.

When the LC50 values from materials with different coatings were compared using a non-parametric ANOVA procedure (Kruskal-Wallis, p<0.05) a statistically significant difference between coating material and LC50 value was identified. Protein coated nanosilver materials appear to have lower toxicity than other coating materials and uncoated materials. However, this cannot be confirmed using a statistical post-hoc test as the variance and normality of the different treatment groups was homogenous.