Silver

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil microorganisms

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE READ-ACROSS APPROACH (ENDPOINT LEVEL)
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.

2. READ-ACROSS APPROACH JUSTIFICATION (ENDPOINT LEVEL)
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).

Toxicity to soil micro-organisms
Published data from one study investigating the effects of nanosilver on soil microorganisms is included in the REACH dossier as Endpoint Study Record. The study reports a NOEC for both nanosilver and silver ions in loam soil of >3 mg/L. A summary of this supporting study is available under Section 4.3.3 of the report ‘Nanosilver: read-across justification for environmental information requirements’ (attached in IUCLID Section 13). In addition, following the silver substance evaluation, the effects of the smallest nanosilver form registered under REACH (‘Nano 8.1’ or ‘Silberpulver Typ 300-30’) and silver nitrate to soil microorganisms were compared by Smolders and Willaert (2017), using a soil nitrogen (N) transformation test (OECD guideline 216). The toxicity of nanosilver and silver nitrate were evaluated in three natural soils (Rots, Poelkapelle and Lufa 2.2), based on two test endpoints: potential nitrification rate (PNR) and substrate induced nitrification (SIN). The EC50 and EC10 values for the SIN endpoint were larger than those for the PNR values, for all three soils and both silver forms, showing that the sensitivity of nitrification to silver decreases with increasing time interval. As predicted based on their soil properties, the Lufa 2.2 soil was the most sensitive to silver and the Poelkapelle soil (with highest organic matter content) the least sensitive. Based on EC10 and EC50 values for both endpoints (SIN and PNR), nanosilver was significantly less toxic than silver nitrate in the Poelkapelle soil. For the Lufa 2.2 and Rots soil, no significant difference in toxicity was observed between nanosilver and silver nitrate. These studies are included in the REACH dossier as Endpoint Study Records and a summary is available in the report ‘Summary of Comparative Ecotoxicity Testing Programme for Nanosilver and Silver Nitrate (2017)’ (attached in IUCLID Section 13).
Together with the theoretical basis for read-across based on the free-ion, above studies support the use of ionic silver as the ‘worst case’ basis to read across properties to nanosilver.


For further information and data matrix see 'CSR Annex 9 - Read Across Justification Nanosilver ENV_SUMMARY_200706' attached in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

0.65 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

other: total NO3 production

Remarks on result:

other: Bordeaux soil, unleached, 95% CL = na-1.0

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

30 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

other: total NO3 production

Remarks on result:

other: Inman Valley soil, unleached, 95% CL = 14-44

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

26 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

other: total NO3 production

Remarks on result:

other: Charleston soil, unleached, 95% CL = 23-28

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

371 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

other: total NO3 production

Remarks on result:

other: Millicent soil, unleached, 95% CL = 322-609

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

61 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

other: total NO3 production

Remarks on result:

other: Bakalava soil, unleached, 95% CL = 52-72

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

85 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

other: total NO3 production

Remarks on result:

other: Port Kenny soil, unleached, 95% CL = 78-92

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

1.2 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

other: potential nitrification rate

Remarks on result:

other: Bordeaux soil, unleached, 95% CL = na-1.5

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

26 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

other: potential nitrification rate

Remarks on result:

other: Inman Valley soil, unleached, 95% CL = 23-29

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

14 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

other: potential nitrification rate

Remarks on result:

other: Charleston soil, unleached, 95% CL = 10-19

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

488 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

other: potential nitrification rate

Remarks on result:

other: Millicent soil, unleached, 95% CL = 369-613

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

36 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

other: potential nitrification rate

Remarks on result:

other: Bakalava soil, unleached, 95% CL = 32-41

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

38 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

other: potential nitrification rate

Remarks on result:

other: Port Kenny soil, unleached, 95% CL = 37-40

Details on results:

The control samples for all the soils showed sufficient production of NO3 over the 28 days of the test with the exception of the Kingaroy soil. The Kingaroy soil produced only approximately 1 to 2 mg NO3/kg over the 28 days of the test. Due to this low NO3 production in the controls and low and highly variable results in the remaining Ag rates, no further data analysis could be completed in this soil and no ECx values could be determined.

Results with reference substance (positive control):

The Ag concentration in the digested CRM was in good agreement with the certified Ag concentration and averaged 1.24 mg/kg with a relative standard deviation (RSD) of 7%.

Reported statistics and error estimates:

The data from the test endpoints were fitted to dose response models to determine the concentration that produced a 10% and 50% reduction relative to the controls (EC10 and EC50 respectively) using GraphPab Prism®. In cases where there was no significant increase (p > 0.05) in the measured response at low Ag concentrations, a standard dose log-logistic model was used to fit the data (Equation 1) and derive ECx values. For dose response curves that showed a significant increase (p ≤ 0.05) in the response at low Ag concentrations, a non-linear model that accounted for hormesis was fitted to the data (Equation 2) (Brain and Cousens, 1989).
y=c+ (d-c)/(1+(x/e)^b ) (1)
y=c+(d-c +fx)/(1+(x/e)^b ) (2)

The EC10 and EC50 values were then determined in each case through interpolation from the fitted curve at a 10% and 50% reduction from the fitted d values (i.e. fitted response in the control).

The EC10 and EC50 values were used to determine if significant relationships could be developed with soil properties (pH(CaCl2), OC, CEC and clay). This was conducted using stepwise multiple linear regression (MLR) analysis in GenStat® (15th Edition). Prior to analysis, the EC10 and EC50 values were transformed (square root or log10) to normalise the distribution. The transformed data were tested for normality using the Shapiro-Wilk test. The final relationship was deemed significant if the regression showed a p-value ≤ 0.05 and each of the properties was significant in the model (p ≤ 0.05).

Table1: Results from analyses of the selected study soils for pH, organic carbon, cation exchange capacity, particle size distribution and total silver concentration.

Soil	pH (CaCl2)	pH (water)	Organic carbon (%)	Cation exchange capacity (cmol^+/kg)	Particle size distribution (%)			Total Silver (mg/kg)
					clay	silt	Sand
Houthalen	3.6	4.9	1.5	5.3	1.4	1.7	93	< 0.04
Bordeaux	4.6	5.6	1.9	6.4	2.5	1.6	93	< 0.04
Inman Valley	5.0	6.0	5.3	25	42	22	26	< 0.04
Charleston	5.1	6.6	6.9	12	14	12	63	< 0.04
Kingaroy	5.5	6.1	0.9	13	60	17	19	< 0.04
Millicent	6.6	6.9	12	42	19	5.2	48	< 0.04
Balaklava	7.1	8.5	1.9	27	30	20	47	< 0.04
Port Kenny*	8.0	8.8	1.8	13	12	4.2	21	< 0.04

* note that particle size distribution for Port Kenny is equal to 37.2% due to the high concentration of CaCO₃that is present in this soil (60%)

 

Conclusions:

The EC10 values for 28-day NO3 production ranged from 0.65 to 371 mg Ag/kg and for PNR were between 1.2 and 488 mg Ag/kg in the unleached treatmen. The toxicity of Ag to the soil nitrification process was primarily controlled by soil pH and OC.

Executive summary:

The toxicity of silver to soil microorganisms was investigated using a soil nitrogen (N) transformation test (OECD guideline 216, 2000) for six study soils. Two test endpoints were calculated from the results: total NO3 production and potential nitrification rate (PNR). The 28-day EC10 values for NO3 production ranged from 0.65 (Bordeaux) to 371 mg Ag/kg (Millicent) and for PNR were between 1.2 (Bordeaux) and 488 mg Ag/kg (Millicent) in unleached soils. The toxicity of silver to the soil nitrification process was primarily controlled by soil pH and OC.

Reference 2

Endpoint:

toxicity to soil microorganisms

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE READ-ACROSS APPROACH (ENDPOINT LEVEL)
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.

2. READ-ACROSS APPROACH JUSTIFICATION (ENDPOINT LEVEL)
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).

Toxicity to soil micro-organisms
Published data from one study investigating the effects of nanosilver on soil microorganisms is included in the REACH dossier as Endpoint Study Record. The study reports a NOEC for both nanosilver and silver ions in loam soil of >3 mg/L. A summary of this supporting study is available under Section 4.3.3 of the report ‘Nanosilver: read-across justification for environmental information requirements’ (attached in IUCLID Section 13). In addition, following the silver substance evaluation, the effects of the smallest nanosilver form registered under REACH (‘Nano 8.1’ or ‘Silberpulver Typ 300-30’) and silver nitrate to soil microorganisms were compared by Smolders and Willaert (2017), using a soil nitrogen (N) transformation test (OECD guideline 216). The toxicity of nanosilver and silver nitrate were evaluated in three natural soils (Rots, Poelkapelle and Lufa 2.2), based on two test endpoints: potential nitrification rate (PNR) and substrate induced nitrification (SIN). The EC50 and EC10 values for the SIN endpoint were larger than those for the PNR values, for all three soils and both silver forms, showing that the sensitivity of nitrification to silver decreases with increasing time interval. As predicted based on their soil properties, the Lufa 2.2 soil was the most sensitive to silver and the Poelkapelle soil (with highest organic matter content) the least sensitive. Based on EC10 and EC50 values for both endpoints (SIN and PNR), nanosilver was significantly less toxic than silver nitrate in the Poelkapelle soil. For the Lufa 2.2 and Rots soil, no significant difference in toxicity was observed between nanosilver and silver nitrate. These studies are included in the REACH dossier as Endpoint Study Records and a summary is available in the report ‘Summary of Comparative Ecotoxicity Testing Programme for Nanosilver and Silver Nitrate (2017)’ (attached in IUCLID Section 13).
Together with the theoretical basis for read-across based on the free-ion, above studies support the use of ionic silver as the ‘worst case’ basis to read across properties to nanosilver.

For further information and data matrix see 'CSR Annex 9 - Read Across Justification Nanosilver ENV_SUMMARY_200706' attached in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.13 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

0.3 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

nitrate formation rate

Remarks on result:

other: 95% confidence 0.16-0.41 mg/kg dw

Details on results:

Nitrate concentrations increased with test material dose, as silver nitrate was used. To determine the difference in nitrate-N concentration between day 28 and 0, average soil nitrate-N concentration measured on day 0 was subtracted from the individual soil nitrate-N concentration measured on day 28.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Hypothesis testing of the NOEC was determined using Toxstat version 3.5.

Validity criteria fulfilled:

yes

Conclusions:

The 28 day NOEC was 0.13 mg Ag/kg dw and the EC10 was 0.30 mg Ag/kg dw.

Executive summary:

The chronic toxicity of silver nitrate to indiginous soil microorganisms was tested in an OECD 216 test. The test was conducted as a static exposure with a single soil type. Six test concentrations and a control were included, and the results are expressed based on the mean measured total silver concentrations at the start and end of the test. Alfalfa was added to half the replicates as an organic substrate. The rate of nitrogen transformation was studied for 28 days. The 28 day NOEC was 0.13 mg Ag/kg dw and the EC10 was 0.30 mg Ag/kg dw.

Reference 3

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

GLP compliance:

no

Specific details on test material used for the study:

- Purity: 37% Ag

Analytical monitoring:

no

Details on sampling:

- Sampling method: Pore-waters of the soils were sampled in duplicate at day 1, 4, 7, 14 (=day 7 of the toxicity test) and 35 (=day 28 of the toxicity test) after spiking. This was done by centrifugation in a double chamber system for 30 minutes at 2000 g. To distinguish between total Ag and truly dissolved Ag in the pore-water, a fraction of the collected pore-water was filtered over a 1 kDA filter (microsep, Pall corporation). The filtration was done three times using the same filter at 3800g for 15 minutes to saturate the filter with Ag to prevent loss due to sorption on the filter membrane which would result in an underestimation of the truly dissolved Ag concentration. The Ag concentration in the 1 kDa filtrates after the third filtration was considered to be truly dissolved.

The total Ag concentrations in the soils were assessed using an HNO3 extraction and ICP-MS. Soil samples were sampled at day 7 and 28 after start of the toxicity test (14 and 35 days after spiking).

Vehicle:

no

Details on preparation and application of test substrate:

SOIL:
Soil samples from three different arable soils (Rots, Poelkapelle and Lufa 2.2) were collected from the plough layer (0-20 cm). The Rots soil was sampled in 2007, Poelkapelle in 2015 and Lufa 2.2 in 2016.


APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The soils were then spiked with AgNP and thoroughly mixed.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20°C

Moisture:

60 %

Details on test conditions:

TEST SYSTEM
- Testing facility:
- Amount of soil: 50 g
- No. of replicates per concentration: 3
- No. of replicates per control: 3


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Country of origin
Rots France
Lufa 2.2 Germany
Poelkapelle Belgium

- Depth of sampling: 0-20 cm
- Soil texture:
- % sand:
- % silt:
- % clay:
- Soil taxonomic classification:
- Soil classification system:
- pH (in water):
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight):
- Maximum water holding capacity (in % dry weigth):
- Cation exchange capacity (mmol/kg): between 9.7 and 33.9 cmolc/kg
- Pretreatment of soil: The soils had been dried in a thin layer at 25°C in a plant growth cabinet with continuous illumination. After partial drying, all soils were sieved through 4 mm.
- Storage (condition, duration): The sieved and dried soils were stored in the dark until experiments.
- Initial microbial biomass as % of total organic C: between 1.1 and 6.8%

DETAILS OF PREINCUBATION OF SOIL (if any): The soil samples were pre-incubated for 1 week at 20°C at a moisture content of 60% of the water holding capacity.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- PNR (the potential nitrification rate between 0 and 14 days, was determined based on the slope of the linear regression of soil nitrate concentration versus time for 0, 7 and 14 days for each replicate).
- SIN (the substrate induced nitrification rate between 0 and 28 days, based on the difference in soil nitrate concentration between 28 and 0 days for each replicate.)


VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: Rots soil: 49 and 196 mg Ag/kg soil, Lufa soil: 7 and 28 mg Ag/kg soil, Poelkapelle soil: 78 and 310 mg Ag/kg soil
- Results used to determine the conditions for the definitive study: yes

Nominal and measured concentrations:

Rots soil: 0.0, 7, 24, 75, 242, 773, 2474 mg added Ag/kg soil
Lufa soil: 0.0, 0.7, 2, 6, 20, 65, 209, 669 mg added Ag/kg soil
Poelkapelle: 0.0, 14, 44, 139, 446, 1427, 4563 mg added Ag/kg soil

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

68 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

other: Potential Nitrification Rate

Remarks on result:

other: soil: Rots, based on measured total concentration of Ag in soil, CI: 47 - 97 mg Ag/kg soil

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

38 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

other: Potential Nitrification Rate

Remarks on result:

other: soil: Lufa 2.2, based on measured total concentration of Ag in soil, CI: 23 - 62 mg Ag/kg soil

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

242 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

other: Potential Nitrification Rate

Remarks on result:

other: soil: Poelkapelle, based on measured total concentration of Ag in soil, CI: 165 - 356 mg Ag/kg soil

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

141 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

other: Substrate Induced Nitrification

Remarks on result:

other: soil: Rots, based on measured total concentration of Ag in soil, CI: 112 - 177 mg Ag/kg soil

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

107 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

other: Substrate Induced Nitrification

Remarks on result:

other: soil: Lufa 2.2, based on measured total concentration of Ag in soil, CI: 93 - 114 mg Ag/kg soil

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

397 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

other: Substrate Induced Nitrification

Remarks on result:

other: soil: Poelkapelle, based on measured total concentration of Ag in soil, CI: 269 - 568 mg Ag/kg soil

Duration:

14 d

Dose descriptor:

EC10

Effect conc.:

9 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

other: Potential Nitrification Rate

Remarks on result:

other: soil: Rots, based on measured total concentration of Ag in soil, CI: 4.1 - 17 mg Ag/kg soil

Duration:

14 d

Dose descriptor:

EC10

Effect conc.:

3.8 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

other: Potential Nitrification Rate

Remarks on result:

other: soil: Lufa 2.2, based on measured total concentration of Ag in soil, CI: 0.9 - 14 mg Ag/kg soil

Duration:

14 d

Dose descriptor:

EC10

Effect conc.:

29 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

other: Potential Nitrification Rate

Remarks on result:

other: soil: Poelkapelle, based on measured total concentration of Ag in soil, CI: 13 - 59 mg Ag/kg soil

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

35 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

other: Substrate Induced Nitrification

Remarks on result:

other: soil: Rots, based on measured total concentration of Ag in soil, CI: 22 - 54 mg Ag/kg soil

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

37 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

other: Substrate Induced Nitrification

Remarks on result:

other: soil: Lufa 2.2, based on measured total concentration of Ag in soil, CI: 33 - 42 mg Ag/kg soil

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

132 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

other: Substrate Induced Nitrification

Remarks on result:

other: soil: Poelkapelle, based on measured total concentration of Ag in soil, CI: 90 - 189 mg Ag/kg soil

Details on results:

- Other:
The total concentration of Ag in the pore water increased during the first days after spiking, indicating dissolution of the nanoparticles, followed by a decrease due to the ageing reactions.

In the Ag-NP spiked soils, the recovered Ag concentration showed larger variation (65-130%), likely more variable because the spiking of the particles was more heterogeneous. The average measured doses were used in all data analysis.

Reported statistics and error estimates:

The data from the nitrification assay, i.e. the PNR or SIN, were first converted to their value relative to that of the average of the control value (Y, in %). These relative responses Y were then statistically analysed using a log-logistic dose-response model fitted with the JMP Pro 12 software package to derive the 10% inhibition concentration EC10 or for the 50% inhibition concentration EC50.

Table1: The mean value and standard deviation of the PNR and the SIN (all inmg N/kg soil/day) of the control treatments for the different soils.

	PNR 0-14	SIN
soil	(mg N/kg soil/day)	(mg N/kg soil/day)
Rots	6.0 (0.3)	3.1 (0.2)
Lufa 2.2	2.9 (0.4)	2.5 (0.8)
Poelkapelle	5.7 (0.2)	3.0 (0.1)

Validity criteria fulfilled:

not specified

Conclusions:

Based on measured total concentration of Ag in soil the EC10 values for PNR were 9.0, 3.8 and 29 mg Ag/kg soil for Rots, Lufa and Poelkapelle soils, respectively. Based on measured total concentration of Ag in soil the EC10 values for SIN were 35, 37 and 132 mg Ag/kg soil for Rots, Lufa and Poelkapelle soils, respectively. Lufa soil was the most sensitive to Ag and the Poelkapelle soil, the least sensitive.

Executive summary:

The toxicity of Silberpulver typ 300-30 was measured with the nitrification assay (soil microbial assay) conducted according to OECD 216 guideline. Three uncontaminated agricultural soils were spiked with Ag NP, and the potential nitrate production rate of the soils was determined as an indicator for toxicity. Two different endpoints were determined, the Potential Nitrification Rate (PNR) and Substrate Induced Nitrification (SIN). The PNR was determined over a time period of 14 days after spiking while the SIN was determined over a time period of 28 days after spiking.

The study is a guideline, non-GLP study, with adequate description of methods and conditions and is considered suitable for use as a key study for this endpoint.

Additional information

Summary of available data for uncoated and coated nanosilver

Two reliable studies investigating the effects of nanosilver on soil microorganisms are available (Calder et al. 2012, and Smolders and Willaert, 2017)).

Calder et al. (2012) evaluated the toxicity of 10 nm spherical silver nanoparticles (1 and 3 mg/L) to Pseudomonas chlororaphis O6 (a beneficial soil bacterium) in sand and soil matrices (loam soil). In sand, both concentrations of nanosilver resulted in a loss of bacterial viability whereas in loam soil, no cell death was observed. The addition of clays (30% v/v kaolinite or bentonite) to sand did not protect the bacterium when challenged with Ag NPs. However, viability of the bacterium was maintained when sand was mixed with soil pore water or, to a lesser extent, humic acid. Despite being considered a reliable study the experimental design employed should be considered to represent rather artificial conditions that may not reflect the behaviour of nanosilver materials in conventional soils or the response of natural microbial assemblages. Specifically, this relates to the fact that the effects of nanosilver in soil matrices were tested in aqueous suspension and that only a single microbial species was present. The study reports a NOEC for nanosilver and silver ions in loam soil of >3 mg/L (note that conventionally the results of terrestrial ecotoxicity tests are expressed as units of soil mass e. g. mg/kg (dry weight).

 

The effects of nanosilver and silver nitrate to soil microorganisms were compared by Smolders and Willaert (2017), using a soil nitrogen (N) transformation test (OECD guideline 216, 2000). The toxicity of nanosilver and silver nitrate were evaluated in three natural soils (Rots, Poelkapelle and Lufa 2.2), based on two test endpoints: potential nitrification rate (PNR) and substrate induced nitrification (SIN).Soil samples from three different arable soils (Rots, Poelkapelle and Lufa 2.2) were collected from the plough layer (0-20 cm). The soils were selected to have a pH between 4.3 and 7.3, %OC between 1.1 and 6.8% and CEC between 9.7 and 33.9 cmol_c/kg.The nanosilver material used was a powder in aqueous suspension with the following particle size distribution: D25 = 7 nm, D50 = 8 nm, D75 = 9 nm (see section 4.5 of IUCLID), and contained 37% silver. Based on 0-14 day PNR, the EC10 values for nanosilver (as measured total silver) ranged from 3.8 to 29mg Ag/kg. Based on 28 day SIN,the EC10 values for nanosilver (measured total silver) ranged from 35 to 132mg Ag/kg. Corresponding 0-14 day PNR EC10 values for silver nitrate (measured total silver) ranged from 3.8 to 9.1 mg Ag/kg, and 28 day SIN EC10 values ranged from 30 to 45 mg Ag/kg. These results inicate that silver toxicity to soil microorganisms is similar for both ionic silver and nanosilver.