Silver chloride

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, available as unpublished report. Some limitations in method and/or reporting but otherwise adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

- Sampling method: At the completion of the toxicity test, all of the remaining soil was dried at 40°C, homogenised and sieved to 2 mm for chemical analysis.

Vehicle:

no

Details on preparation and application of test substrate:

APPLICATION OF TEST SUBSTANCE TO SOIL
The soils were spiked with Ag by adding a concentrated AgNO3 solution to the soil samples at a rate of 50 mL of solution per kg dry soil. This addition was done using a fine spray onto the soils, followed by thorough mixing of the sample, to ensure that there was a homogeneous distribution of the solution throughout the soil. All of the soil spiking was conducted in duplicate to allow for a leached and unleached treatment.

LEACHING PROCEDURE:
The soils were left to equilibrate overnight prior to the commencement of soil leaching. The soils were leached with an artificial rain water (ARW) solution. Some modifications were made to the solution to remove Cl- salts to prevent precipitation of AgCl. On the initial day of leaching the spiked soils were transferred to containers with holes in the base, lined with mesh or filter paper. The containers were then submerged in a larger container that contained the ARW solution (ratio 800 mL of ARW : 1 kg soil), and left overnight for the soils to reach saturation. The following day, the soils were removed from the ARW solution and additional ARW was added to the surface of the soils at a rate equivalent to 600 mL ARW per 1 kg soil and allowed to drain through the base of the container overnight. The following day all soils (leached and unleached) were transferred to trays to allow them to air dry. The unleached soils were retained at room temperature while leaching of the other samples was being conducted. Approximately 10 days was allowed for sample drying. The dried samples were then homogenised and sieved to < 2 mm for the commencement of the toxicity test.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia fetida
- Source: not reported
- Age at test initiation (mean and range, SD): adult worms
- Weight at test initiation (mean and range, SD): not reported

ACCLIMATION
- Acclimation period: no data
- Acclimation conditions (same as test or not): no data
- Health during acclimation (any mortality observed): no data

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

70 d

Remarks:

Survival and growth were determined after 28 days, the total number of juveniles was counted after 42 days from the start of the second phase of the test (started when ≥ 10 cocoons per test container were present in the controls soils).

Post exposure observation period:

None.

Test temperature:

22°C (± 2°C)

pH:

pH values for the soils used (in CaCl2):
Houthalen 3.6
Bordeaux 4.6
Inman Valley 5.0
Charleston 5.1
Kingaroy 5.5
Millicent 6.6
Balaklava 7.1
Port Kenny* 8.0
South East 7.7

Moisture:

70% of FC

Details on test conditions:

TEST SYSTEM
- Amount of soil or substrate: 500 g
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 1
- No. of replicates per control: 2

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: eight study soils used, two of the soils were collected from Europe, Houthalen (Belgium) and Bordeaux (France) and the remaining six soils (Inman Valley, Charleston, Kingaroy, South East, Balaklava and Port Kenny) were collected from locations around Australia.
- Collection procedures: not reported
- Soil texture (if natural soil): Full details provided in Table 1.
- % sand: 19 - 93%
- % silt: 1.6 - 22%
- % clay: 1.4 - 60%
- Organic carbon: 0.9 - 12%
- Maximum water holding capacity (in % dry weigth): no data
- CEC: 5.3 - 42 cmol+/kg

OTHER TEST CONDITIONS
- Photoperiod: 12 hours of light and 12 hours of dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Survival, growth were observed after 28 days and the total number of juveniles was counted after 42 days from commencing the second phase of the test (i.e. reproduction).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study: no
- Test concentrations:
The nominal Ag concentrations used were as follows (all concentrations are shown as mg Ag/kg):
Houthalen 0, 0.25, 0.5, 1, 2, 4, 8
Bordeaux 0, 0.5, 1, 2, 4, 8, 16
Inman Valley 0, 5, 10, 20, 40, 80, 160, 320
Charleston 0, 5, 10, 20, 40, 80, 160, 320
Kingaroy 0, 2, 4, 8, 16, 32, 64, 128
Balaklava 0, 5, 10, 20, 40, 80, 160, 320
Port Kenny 0, 5, 10, 20, 40, 80, 160, 320
South East 0, 5, 10, 20, 40, 80, 160, 320

Nominal and measured concentrations:

The nominal Ag concentrations used for the testing were as follows (all concentrations are shown as mg Ag/kg):
- Houthalen 0, 0.25, 0.5, 1, 2, 4, 8
- Bordeaux 0, 0.5, 1, 2, 4, 8, 16
- Inman Valley 0, 5, 10, 20, 40, 80, 160, 320
- Charleston 0, 5, 10, 20, 40, 80, 160, 320
- Kingaroy 0, 2, 4, 8, 16, 32, 64, 128
- Balaklava 0, 5, 10, 20, 40, 80, 160, 320
- Port Kenny 0, 5, 10, 20, 40, 80, 160, 320
- South East 0, 5, 10, 20, 40, 80, 160, 320
For all of the soils, with the exception of Inman Valley and Kingaroy, the measured concentrations were in close agreement with the nominal and were within the required range of 80 to 120%, therefore the nominal concentrations were used for data analysis in these soils. In the Inman Valley and Kingaroy soils some Ag concentrations were outside of this required range, therefore, in these cases the measured concentrations were used for all data analysis.

Measured concentrations (as mg Ag/kg):
- Inman Valley 5.5, 11, 30, 43, 82, 160, 325 (leached soil); 4.6, 8.2, 25, 34, 64, 139, 334 (unleached soil)
- Kingaroy 2.4, 4.3, 8.6, 16, 42, 63, 121 (leached soil); 2.3, 3.7, 10, 26, 23, 67, 101 (unleached soil)

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

14 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: Houthalen soil, unleached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

33 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: Bordeaux soil, unleached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

63 mg/kg soil dw

95% CI:

>= 41 - <= 101

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: Inman Valley soil, unleached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

77 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: Charleston soil, unleached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

141 mg/kg soil dw

95% CI:

>= 99 - <= 196

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: Balaklava soil, unleached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

36 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: Port Kenny soil, unleached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

55 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: South East soil, unleached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

46 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: Houthalen soil, leached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

15 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: Bordeaux soil, leached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

5.3 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: Kingaroy soil, unleached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

196 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: Inman Valley soil, leached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

211 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: Millicent soil, unleached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

93 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: Charleston soil, leached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

209 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: Balaklava soil, leached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

133 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: Port Kenny soil, leached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

151 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: South East soil, leached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

34 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth

Remarks on result:

other: Houthalen soil, unleached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

21 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth

Remarks on result:

other: Bordeaux soil, unleached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

83 mg/kg soil dw

95% CI:

>= 41 - <= 133

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

growth

Remarks on result:

other: Inman Valley soil, unleached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

62 mg/kg soil dw

95% CI:

>= 39 - <= 91

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth

Remarks on result:

other: Charleston soil, unleached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

93 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth

Remarks on result:

other: Millicent soil, unleached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

74 mg/kg soil dw

95% CI:

>= 35 - <= 133

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth

Remarks on result:

other: Balaklava soil, unleached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

23 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth

Remarks on result:

other: Houthalen soil, leached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

6.8 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth

Remarks on result:

other: Bordeaux soil, leached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

127 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

growth

Remarks on result:

other: Inman Valley soil, leached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

91 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth

Remarks on result:

other: Charleston soil, leached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

221 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth

Remarks on result:

other: Millicent soil, leached

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

105 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth

Remarks on result:

other: Balaklava soil, leached

Duration:

70 d

Dose descriptor:

EC10

Effect conc.:

67 mg/kg soil dw

95% CI:

>= 41 - <= 105

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: Inman Valley soil, unleached

Duration:

70 d

Dose descriptor:

EC10

Effect conc.:

5.9 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: Kingaroy soil, unleached

Duration:

70 d

Dose descriptor:

EC10

Effect conc.:

64 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: Balaklava soil, unleached

Duration:

70 d

Dose descriptor:

EC10

Effect conc.:

5.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: South East soil, unleached

Duration:

70 d

Dose descriptor:

EC10

Effect conc.:

108 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: Inman Valley soil, leached

Duration:

70 d

Dose descriptor:

EC10

Effect conc.:

74 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: Charleston soil, leached

Duration:

70 d

Dose descriptor:

EC10

Effect conc.:

34 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: Balaklava soil, leached

Duration:

70 d

Dose descriptor:

EC10

Effect conc.:

11 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: Port Kenny soil, leached

Duration:

70 d

Dose descriptor:

EC10

Effect conc.:

14 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks on result:

other: South East soil, leached

Details on results:

- Mortality at end of exposure period: There was no significant decrease in the survival of earthworms with increasing Ag concentrations in the Houthalen and Bordeaux soils. For the remaining soils a dose response relationship was observed with increasing Ag concentration. The survival of earthworms in the control soils after 28 days varied considerably across the study soils ranging from approximately 60% to 100% survival.

- Changes in body weigth of live adults (% of initial weight) at end of exposure period: The dose response analysis of the data showed that for many soils there was no decrease in earthworm growth with increasing Ag concentration. In several cases some growth was observed in the control soils, however, in the majority of cases the earthworms decreased in weight during the initial 28 days of the test.

- No. of offspring produced: The number of juveniles per adult worm at the completion of the test was highly variable and in the majority of cases below the validation level for the test (i.e. 3 juveniles/adult worm). In addition, the variability between the replicates was high and in most cases exceeded an RSD of 30%. In the Houthalen and Bordeaux soil there were no juveniles found in the controls. This is likely due to a negative effect of the low pH on the hatching and survival of juveniles. In addition, in the Charleston unleached treatment there was less than 0.2 juveniles produced per adult worm. Based on these low results in these cases, no further data analysis was conducted on these data.

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 some cases, the toxicity data showed stimulation in the response relative to the controls at low Ag concentrations (i.e. hormesis), as a result, one of two dose response models were used to fit the data and derive ECx values depending on this response. 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).
EC10 values outside the concentration range tested, i.e., below or above the lowest or highest dose tested, respectively, were considered unreliable due to their large degree of uncertainty (and are thus not added here).

Earthworm survival was presented as the percentage of worms present after 28 days relative to the initial number of worms (i.e. 10 worms). The tests were deemed valid if there was ≥ 90% survival in the control soils. The growth of the worms was presented as a relative weight change over the initial 28 days of the test. This was done by calculating the average weight per worm in each of the test containers at day 0 and day 28 and then determining the ratio of these two values (i.e. d₂₈/d₀).

The total number of juveniles that were present in each of the test containers at the completion of the second phase of the test was normalised to the number of juveniles per adult worm. For this normalisation, the number of adult worms that had been removed after the initial phase of the test was used. The test was deemed valid if there were ≥ 3 juveniles produced per adult worm and the RSD between the control samples was ≤ 30%(OECD, 2004).

Validity criteria fulfilled:

no

Remarks:

In some soils the survival in the controls was less than 90%. The number of juveniles per adult worm was highly variable and in the majority of cases was < 3 juveniles/adult worm. the variability between the replicates was high and in most cases >30%.

Conclusions:

All toxicity results should be used with caution as the validitiy criteria were not fulfilled. The most sensitive endpoint in the study was reproduction. The EC10 values based on this endpoint ranged from 5.5 (South East soil) to 108 (Inman Valley soil) mg Ag/kg.

Executive summary:

The earthworm toxicity study was conducted according to the OECD guideline 222 (Earthworm Reproduction Test) using the earthworm Eisenia fetida as the test species in a range of soils indicative of natural variability in soil conditions across the EU (pH 4.6 – 8.0, organic carbon 0.9 – 6.9, and clay 2.5 – 60%). Reproduction (number of juveniles) was found to be the most sensitive endpoint in the study. However, there were low juvenile counts in all of the soils (in most cases < 3 juveniles per worm). The EC10 values based on this endpoint ranged from 5.5 (South East soil) to 108 (Inman Valley soil) mg Ag/kg. The EC10 values for survival were in the range of 5.3 (Kingaroy soil) – 211 (Millicent soil) mg Ag/kg. The EC10 values for earthworm growth were in the range of 6.8 (Bordeaux soil) to 221 (Millicent soil) mg Ag/kg. All EC10 values were taken forward to the derivation of the PNEC according to the statistical extrapolation approach. Given the negligible effects of leaching on Ag toxicity to soil organisms, both results for leached and unleached soil treatments were used.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, summary available as an unpublished study report. Some limitations in method and/or reporting but otherwise adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Deviations:

no

GLP compliance:

not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

not specified

Vehicle:

no

Details on preparation and application of test substrate:

- Soils: There were four soils tested – Lufa 2.2, Woburn, North Wales and Chiltern soils (Table 1). Lufa 2.2 was from a batch supplied by Spreyer, Germany. The other three soils were collected from sites in the UK. All the soils were sieved to < 2mm and air-dried before use. An amount of 500 g dry weight Lufa 2.2 soil was used for each test replicate. The dry weight which amounted to an equivalent volume of 500 g Lufa 2.2 soil was used for the was used for each of the other three soils (Chiltern = 450 g, North Wales = 400 g and Woburn = 550 g). Each replicate was held in a 183 x 120 x 70mm polypropylene container.
- Spiking procedure: Five concentrations of AgNO3 were tested in Lufa 2.2, North Wales and Woburn soils - 9, 22.5, 56.3, 140.8, 352 mg Ag/kg dry soil. The Chiltern soil was spiked with the following concentrations 22.5, 56.3, 140.8, 352, 880 mg Ag/kg. This was based on preliminary data which showed low toxicity in this soil. AgNO3 was added as a salt directly to the dry soil (enough for three replicates) and mixed thoroughly. The spiked soil was then distributed evenly between three replicate containers. MilliQ water was added to the replicates to reach 50% of the WHC of each soil and then mixed. Soils were then left for one week to allow for equilibration. In addition, six controls without Ag were included in the toxicity test.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Eisenia fetida were reared at the NERC Centre for Ecology and Hydrology, UK. These were maintained in culture soil constituting 33% loamy soil, 33% peat and 33% bark on a volume basis. Cultures were kept in a temperature controlled room at 20°C in a 12:12 hour light:dark cycle. Worm cultures were fed fresh horse manure free from contamination or medication. Worms used in the tests were maintained until they were adults and of a suitable size (300-600 mg) for testing. Ten adult, fully clitellated worms were selected per replicate.

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

28 d

Post exposure observation period:

None

Test temperature:

All containers were placed in a controlled temperature room for the duration of the test at 20 ± 1 °C.

pH:

Woburn: 6.91 ± 0.11. North Wales: 5.48 ± 0.11. Chiltern: 8.31 ± 0.02. Lufa 2.2: 5.96 ± 0.07.

Moisture:

Over the duration of the test, soils were corrected for moisture loss every two weeks and additional water added as needed to maintain a consistent soil moisture level.

Details on test conditions:

- Toxicity test: To initiate the experiment, ten worms were added to each replicate container. Worms were selected taken from the larger culture, washed to remove adhering soil and blotted dry. The 10 worms were then weighed as a batch before being put onto the surface of the soil of the relevant test container. As food, 10 g (dry weight) of horse manure was added to the soil surface in each container and the total container weight recorded. All containers were placed in a controlled temperature room for the duration under a 12:12 hour light:dark cycle.
- Survival and weight change: After 14 days, the contained were sorted and the numbers of worms alive in each counted. Retrieved worms were washed, blotted dry and weight as a batch. The worms were then returned to their respective container; an additional 10 g of food was added and the containers were then returned for a second 14 days of exposure. At 28 days the worms were again sorted from the soil and weighed. These worms were retained for subsequent chemical and histological analysis.
- Reproduction: After the removal of the adult worms after 28 days, the soils were returned to the controlled climates facility for a further 28 days to allow juveniles to hatch from laid cocoons. At the end of this hatching period, the containers were placed in a water bath at 60°C for 15 minutes. This heating forces juveniles to the soil surface where they can be picked off and counted. From this number and the counts of surviving adult worms at 14 and 28 days, reproduction was expressed as a juvenile production rate (juveniles/worm/week).

Nominal and measured concentrations:

- Lufa (A): 0, 9, 22.5, 56.3, 104.8, 352 mg Ag/kg soil dw
- Lufa (B): 0, 18, 45, 112.5, 281.3, 703.1, 1757.8 mg Ag/kg soil dw
- Woburn: 0, 9, 22.5, 56.3, 104.8, 352 mg Ag/kg soil dw
- North Wales: 0, 9, 22.5, 56.3, 104.8, 352 mg Ag/kg soil dw
- Chiltern: 22.5, 56.3, 104.8, 352, 880 mg Ag/kg soil dw

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

104.8 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Silver

Basis for effect:

mortality

Remarks on result:

other: Lufa 2.2

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

104.8 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Silver

Basis for effect:

reproduction

Remarks on result:

other: Lufa 2.2

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

190.77 mg/kg soil dw

95% CI:

>= 119 - <= 233

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Silver

Basis for effect:

mortality

Remarks on result:

other: Lufa 2.2

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

57.3 mg/kg soil dw

95% CI:

>= 0 - <= 91.7

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Silver

Basis for effect:

reproduction

Remarks on result:

other: Lufa 2.2

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

104.8 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Silver

Basis for effect:

mortality

Remarks on result:

other: Woburn

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

22.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Silver

Basis for effect:

reproduction

Remarks on result:

other: Woburn

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

86.73 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Silver

Basis for effect:

mortality

Remarks on result:

other: Woburn

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

13.22 mg/kg soil dw

95% CI:

>= 0 - <= 28.56

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Silver

Basis for effect:

reproduction

Remarks on result:

other: Woburn

Details on results:

Lufa 2.2
Results from study report
Survival LC50: 231.1 (validated nominal concentrations)
Reproduction EC50: 45.4 (validated nominal concentrations)
Survival and reproduction EC50: 161.2 (95% CI: 38.22-284.2) (nominal)

Estimates from ToxCalc
Survival:
NOEC = 104.8 mg/kg dwt – Homoscedastic t-test (Bonferroni)
LOEC = 352 mg/kg dwt – Homoscedastic t-test (Bonferroni)
EC10 = 190.77 (119.0 - 233.0) mg/kg dwt – Maximum likelihood logit
EC50 = 406.15 (357.2 – 510.36) mg/kg dwt – Maximum likelihood logit
Reproduction:
NOEC = 104.8 mg/kg dwt – Heteroscedastic t-test (Bonferroni)
LOEC = 352 mg/kg dwt – Heteroscedastic t-test (Bonferroni)
EC10 = 57.3 (0 – 91.07) mg/kg dwt – ICP
EC50 = 169.17 (84.13 – 215.23) mg/kg dwt – ICP

Lufa 2.2 - Worms exposed to 2 months aged soils (no leachings, soils were only incubated at the climate rooms)
Survival LC50: 318 (95% CI: 162-475) (nominal)
Reproduction EC50: 30 (95% CI: 16-43) (nominal)

Lufa 2.2 - Worms exposed to 7 months aged soils (no leachings, soils were only incubated at the climate rooms)
Survival LC50: 682 (nominal)
Reproduction EC50: 90 (95% CI: 29-151) (nominal)

Woburn
Results from study report
Survival LC50: 254 (S.E. = 22) (measured)
Reproduction EC50: 13.2 (S.E. = 4.2) (measured)
Reproduction EC10: 3.0 (S.E. = 0.6) (measured)
Survival and reproduction EC50: 33.1 (95% CI: 21.7-44.5) (nominal)

Estimates from ToxCalc
Survival:
NOEC = 104.8 mg/kg dwt – Homoscedastic t-test (Bonferroni)
LOEC = 352 mg/kg dwt – Homoscedastic t-test (Bonferroni)
EC10 = 86.73 mg/kg dwt – Non-linear interpolation
EC50 =258.87 mg/kg dwt – Non-linear interpolation
Reproduction:
NOEC = 22.5 mg/kg dwt – Homoscedastic t-test (Bonferroni)
LOEC = 56.3 mg/kg dwt – Homoscedastic t-test (Bonferroni)
EC10 = 13.22 (0 – 28.56) mg/kg dwt – ICP
EC50 = 37.29 (25.38 – 49.72) mg/kg dwt – ICP

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Additional estimates for chronic endpoints were determined using ToxCalc.

Lufa 2.2 soil (A)

Ag conc. (mg/kg)	No. worms surviving	Juveniles/worm/wk
0	9	1.919
0	10	1.400
0	10	0.975
0	10	0.625
0	10	1.100
0	10	2.375
9	10	1.868
9	10	1.450
9	10	1.325
22.5	10	1.250
22.5	10	0.975
22.5	10	1.725
56.3	10	1.175
56.3	10	1.425
56.3	10	1.450
104.8	10	1.025
104.8	9	1.108
104.8	10	0.842
352	10	0.000
352	3	0.000
352	5	0.037

Lufa 2.2 (b)

Ag conc. (mg/kg)	No. worms surviving	Juveniles
0.0	9	46
0.0	10	57
0.0	10	41
18.0	9	51
18.0	10	36
18.0	9	34
45.0	10	30
45.0	10	17
45.0	9	30
112.5	9	13
112.5	10	3
112.5	10	13
281.3	1	0
281.3	6	0
281.3	5	0
703.1	0	0
703.1	0	0
703.1	0	0
1757.8	0	0
1757.8	0	0
1757.8	0	0

Woburn soil

Ag conc. (mg/kg)	No. worms surviving	Juveniles/worm/wk
0	10	0.875
0	10	0.875
0	10	0.825
0	10	0.600
0	10	1.200
0	10	0.975
9	9	0.816
9	10	1.150
9	10	0.650
22.5	10	0.575
22.5	10	0.800
22.5	10	0.575
56.3	10	0.200
56.3	10	0.025
56.3	10	0.325
104.8	10	0.030
104.8	10	0.000
104.8	3	0.000
352	9	0.000
352	3	0.000
352	0	0.000

Chiltern soil

Ag conc. (mg/kg)	No. worms surviving	Juveniles/worm/wk
0	10	1.150
0	10	0.900
0	9	1.564
0	10	1.125
0	10	1.050
0	10	1.250
22.5	10	1.150
22.5	10	1.375
22.5	10	1.200
56.3	9	0.872
56.3	10	0.900
56.3	9	1.026
104.8	10	0.425
104.8	10	0.550
104.8	10	0.475
352	8	0.342
352	4	0.294
352	1	0.065
880	0	0.000
880	0	0.000
880	0	0.000

North Wales soil

Ag conc. (mg/kg)	No. worms surviving	Juveniles/worm/wk
0	10	2.675
0	10	2.450
0	10	3.375
0	10	2.350
0	10	2.700
0	10	2.100
9	10	1.625
9	10	2.625
9	10	2.500
22.5	10	1.600
22.5	10	1.925
22.5	10	1.950
56.3	10	1.950
56.3	10	2.175
56.3	10	1.725
104.8	10	1.575
104.8	10	1.700
104.8	10	2.175
352	5	0.172
352	10	0.625
352	8	0.294

Validity criteria fulfilled:

not specified

Conclusions:

The NOEC for survival and reproduction in Lufa 2.2 (A) and survival Woburn soil was 104.8 mg Ag/kg soil dw . The NOEC for reproduction in Woburn soil was 22.5 mg Ag/kg soil dw.

Executive summary:

The toxicity of silver nitrate to Eisenia fetida was determined in study following OECD guideline 222. Worms were exposed to silver nitrate in different substrates (Lufa 2.2, Woburn, North Wales and Chiltern soils) for 28 days and the effects on survival and reproduction were recorded. A summary of the study report is available as unpublished nanofate material.

All EC10 values from this study were taken forward to the derivation of the PNEC according to the statistical extrapolation approach.

Reference 3

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, acceptable with restriction

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

yes

Details on sampling:

The concentrations of Ag in the test soils was measured on a sub-sample collected at the commencement of the test and an additional sub-sample collected at the completion of the test. In addition, at both the commencement and completion of the test one of the test treatments was duplicated for Ag analysis.

Vehicle:

no

Details on preparation and application of test substrate:

Soils were prepared the day before study initiation.Enough soil for replicates and analysis at each treatment level was weighed into stainless steel bowls. For each treatment water (control) or water spiked with silver nitrate (test solution) was slowly poured onto the soil in a back and forth pattern. The soil was mixed by hand for a minimum of three minutes before being placed in 1 gallon plastic containers and tumbled in a rotary tumbler for a minimum of 10 hours. Prior to test set up the soil was again mixed by hand for three minutes and distributed to the test chambers.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Redworms obtained from The Worm Farm, Durham, CA. Worms were kept in batches of ~200 in opaque plastic tubs with lids. The soil (85% sphagnum peat moss, 15% organic sheep and peat, with calcium carbonate to raise the pH to >6) was kept at 50-60% saturation. Worms were fed with grass slurry applied to the soil surface. Adult worms with a clitellum were selected for testing.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Post exposure observation period:

None

Test temperature:

20°C mean, 19.2°C-22.4°C range

pH:

7.2 mean, 6.9-7.5 range

Moisture:

57.1% WHC mean, 52.2-61.4% WHC range at day 0.
71.6% WHC mean, 67.1-78.9% WHC range at day 56.

Details on test conditions:

Artificial soil composition: 10% sphagnum peat, 20% kaolinite clay, 70% fine silica sand, 0.3-1% calcium carbonate.
Characterisation of the soil indicated that it had a pH of 7.2, organic carbon content of 4.3% and consisted of 76% sand, 9% silt and 15% clay.
Soil prepared to be at 60% WHC. Test chambers were weighed over the exposure period to monitor hydration levels. Water was added during the weekly feedings.
Control had eight replicates. Each of the eight treatment concentrations had four replicates.
Test chambers were held in an environmental chamber under fluorescent light with a photoperiod of 16h light: 8h dark.
10 worms were placed in each test chamber at random, after the group weight was recorded. Avoidance was noted from day 1-28. Surviving adult worms were removed from the chambers on day 28 counted, examined for unusual behaviour and weighed (wet and dry). Soil was returned to the chambers for an additional 28 days after which time the soil was hand searched for cocoons and juvenile worms.

Nominal and measured concentrations:

Nominal: 0, 3.27, 5.88, 10.6, 19.1, 34.3, 61.7, 111.1, 200 mg/kg
Measured: 0.2, 3.65, 5.9, 11.15, 21.25, 39.0, 60.85, 127.75, 225.95 mg/kg
Measured values were within 15% nominal.

Reference substance (positive control):

yes

Remarks:

potassium chloride

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

226 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

38.8 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

growth

Remarks:

dry and wet weight

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

11.2 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

reproduction

Remarks:

number of juveniles produced

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

39 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

reproduction

Remarks:

mean number of cocoons

Key result

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

15.05 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

reproduction

Remarks:

number of juveniles produced

Key result

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

22.2 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

growth

Remarks:

dry weight

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

109.8 mg/kg soil ww

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

growth

Remarks:

wet weight

Key result

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

194.5 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Basis for effect:

mortality

Details on results:

Avoidance was noted in the higher silver treatments (>50 mg/kg) up to day 12.

Results with reference substance (positive control):

The 96hr LC50 fell within the 95% historical control limits. Therefore, the response of the organisms was within the historical range for this laboratory.

Reported statistics and error estimates:

Hypothesis testing of the NOEC was determined using Toxstat version 3.5.
EC10s were calculated using a logistic (sigmoidal) dose-response curve.

Validity criteria fulfilled:

yes

Remarks:

Survival and reproduction in controls was within acceptable levels

Conclusions:

The most sensitive endpoint for this test was the number of juveniles produced, with a 56 day NOEC of 11 mg Ag/kg dw and an EC10 of 15.05 mg Ag/kg dw.

Executive summary:

The chronic toxicity of silver nitrate to the earthworm Eisenia fetida was tested in an OECD 222 test. The test was conducted as a static exposure with a single soil type. Eight 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. The survival and growth of the adult worms was studied for the first 28 days, and reproduction endpoints (number of juveniles and number of cocoons) was studied after 56 days. NOECs and EC10s were determined for each biological endpoint. The most sensitive endpoint for this test was the number of juveniles produced, with a 56 day NOEC of 11.2 mg Ag/kg dw and an EC10 of 15.05 mg Ag/kg dw.

All EC10 values from this study were taken forward to the derivation of the PNEC according to the statistical extrapolation approach.

Reference 4

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Non-GLP, guideline study available as a published article. Minor limitations in design and/or reporting but otherwise adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Deviations:

no

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

yes

Details on sampling:

The silver concentrations in whole earthworms and ~0.25 g soil samples were determined after digestion in concentrated trace metal grade nitric acid using a microwaved digestion system.

Vehicle:

no

Details on preparation and application of test substrate:

Silver nitrate was dissolved in deionised water and applied to dry soil.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

The adult earthworms, with fully developed clitella, had average massess of 0.450 ± 0.101 g (range: 0.243 - 0.752 g).

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Post exposure observation period:

The worms were observed for 24 hours after removal from the exposure media.

Test temperature:

Vessels were maintained at 20°C in an environmental chamber.

pH:

No data reported

Moisture:

Water was added bi-weekly to maintain constant vessel weights.

Details on test conditions:

The exposure media consisted of artificial soil, with 69.58 % dry mass quartz sand, 0.43 % dm crushed limestone, 20 % dm kaolin clay and 10 % dm <2 mm sphagnum peat moss. Tests were conducted in triplicate with 750 g of soil per test vessel, which had been allowed to equilibriate overnight in an incubator and 10 adult earthworms.
Vessels were maintained with a photoperiod of 12 hours light per day. 24 hours after the start of exposure, 5 g of a 50/50 mix of dehydrated alfalfa and commercially available chicken feed was added to the surface of the soil, along with 5 mL of deionised water. Feeding was repeated at the end of each week of exposure. After 28 days exposure, worms were observed for mortality and removed for 24 hours to Petri dishes containing wet filter paper.
After the worms had been removed, feeding was repeated and the vessels were incubated for a further 28 days with weekly moisture replenishment. After the second incubation, the soil was searched and cocoons manually collected. The hatching sucess was determined by immediately placing the cocoons in water.
Silver nanoparticles were tested in parallel.

Nominal and measured concentrations:

Nominal concentrations of 10 and 100 mg Ag/kg soil dw were applied as dissolved silver nitrate.

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

8.38 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

other: Silver

Basis for effect:

reproduction

Remarks on result:

other: Silver nitrate. Standard deviation: ± 1.26 mg/kg

Details on results:

Silver nitrate led to a concentration-dependent decrease in the number of cocoons produced per earthworm. A significant decrease in cocoon production was observed at 94.12 ± 5.56 mg/kg silver nitrate, an effect not observed at similar concentration treatments of silver nanoparticles. When compared to the controls, the rate of cocoon hatching was not singificantly affected when worms were exposed to silver nitrate.
For growth and mortality, no-concentration dependent effects were observed at up to 94.12 mg/kg, though a non-significant decrease in growth was observed. The silver nitrate treatments did not induce significant mortality but at the highest concentration tested did show some reproductive toxicity.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The data were log transformed and Shapiro-Wilk's test was used to test normality, with Levene's test used to test homogeneity of variance. Where data were normal and homogeneous, ANOVA was used to determine significant differences between treatment and controls in growth, mortality and reproduction. Duncan's Multiple Range test was used for post-hoc mulitple comparisons for ANOVA. Wilcoxon Rank-Sum test was used for non-normal data.

Validity criteria fulfilled:

not specified

Remarks:

Insufficient data to determine whether validity criteria were met

Conclusions:

The 28 day reproduction NOEC for silver nitrate to earthworms is 8.38 mg/kg dw.

Executive summary:

The study is a guideline study, published in peer reviewed literature and considered suitable for use as a supporting study for this endpoint. The 28 day reproduction NOEC for silver nitrate to earthworms is 8.38 mg/kg dw. This value was taken forward to the derivation of the PNEC according to the statistical extrapolation approach.

Reference 5

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Non-GLP, guideline study available as a published article. Minor limitations in design and/or reporting but otherwise adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Deviations:

no

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

yes

Details on sampling:

The silver concentrations in whole earthworms and ~0.25 g soil samples were determined after digestion in concentrated trace metal grade nitric acid using a microwaved digestion system.

Vehicle:

no

Details on preparation and application of test substrate:

Silver nitrate was deionised water and then applied to the soils at approximately 50 % of the moisture holding capacity (approximately 26 % v/w for the artifical soil and 19.2 % w/v for the natural soil).

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

The adult earthworms, with fully developed clitella, had average massess of 0.450 ± 0.101 g (range: 0.243 - 0.752 g).

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Post exposure observation period:

The worms were observed for 24 hours after removal from the exposure media.

Test temperature:

Vessels were maintained at 20°C in an environmental chamber.

pH:

No data reported

Moisture:

Water was added bi-weekly to maintain constant vessel weights.

Details on test conditions:

The exposure media consisted of artificial soil, with 69.58 % dry mass quartz sand, 0.43 % dm crushed limestone, 20 % dm kaolin clay and 10 % dm <2 mm sphagnum peat moss.
Tests were also run using natural soil (Yeager sandy loam) from a type site in central Kentucky (Estill County), which was air dried, sieved to <2 nmm and buffered with 0.1 % w/w CaCO3.
Tests were conducted in triplicate with 750 g of soil per test vessel, which had been allowed to equilibriate overnight in an incubator and 10 adult earthworms.
Vessels were maintained with a photoperiod of 12 hours light per day. 24 hours after the start of exposure, 5 g of a 50/50 mix of dehydrated alfalfa and commercially available chicken feed was added to the surface of the soil, along with 5 mL of deionised water. Feeding was repeated at the end of each week of exposure with 4 g of feed mixture. After 28 days exposure, worms were observed for mortality and removed for 24 hours to Petri dishes containing wet filter paper.
After the worms had been removed, feeding was repeated and the vessels were incubated for a further 28 days with weekly moisture replenishment. After the second incubation, the soil was submersed in 60°C water and the emerging juveniles counted. The soil was then searched and cocoons manually collected and hatching sucess determined by immediately placing the cocoons in water.
Silver nanoparticles were tested in parallel.

Nominal and measured concentrations:

The nominal concentrations applied to the artifical soil were 10 and 100 mg Ag/kg dry soil. The YSL was tested at a nominal concentration of 10 mg Ag/kg dw.

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

8.38 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

other: Silver

Basis for effect:

reproduction

Remarks:

Cocoons and worms

Remarks on result:

other: Soil: Artifical. Standard error: ± 1.26 mg/kg soil dw.

Details on results:

Earthworms in artificial soil exposed to silver nitrate showed no significant concentration-dependent decrease in growth or survival but a significant decrease in the number of cocoons per worm was observed at concentrations of 94.12 g/kg. In the artifical soil, the hatching rate of cocoons showed no significiant difference at any concentration. In all the treatment groups and the control, no juveniles survived to be counted.
In YSL, earthworms exposed to 7.41 ± 1.19 mg/kg ionic silver showed a signficiant decrease in growth and exposures of 7.41 mg/kg soil dw caused a significant decreases in the hatching sucess and the number of juveniles. No significant decrease was observed on the number of cocoons produced per earthworm.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The data were log transformed and Shapiro-Wilk's test was used to test normality, with Levene's test used to test homogeneity of variance. Where data were normal and homogeneous, ANOVA was used to determine significant differences between treatment and controls in growth, mortality and reproduction. Duncan's Multiple Range test was used for post-hoc mulitple comparisons for ANOVA. Wilcoxon Rank-Sum test was used for non-normal data.

Validity criteria fulfilled:

not specified

Remarks:

Insufficient data to determine whether validity criteria were met

Conclusions:

The 28 day reproduction (cocoons and worms) NOEC for silver nitrate to earthworms in artificial soil is 8.38 mg Ag/kg dw .

Executive summary:

The study is a guideline study, published in peer reviewed literature and considered suitable for use as a supporting study for this endpoint. The 28 day reproduction (cocoons and worms) NOEC for silver nitrate to earthworms in artificial soil is 8.38 mg Ag/kg dw .

Reference 6

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Three endogeic earthworms were placed in circular plastic pots containing 250 g of soil that was either not contaminated (control) or contaminated with AgNO3 (4 concentrations). Each treatment was replicated 5 times. The test duration was 14 days and biomass measurements were performed at day 0 and day 14.

GLP compliance:

not specified

Specific details on test material used for the study:

99.9+% ultra-pure, Alfa Aesar

Analytical monitoring:

yes

Details on sampling:

3 samples from each soil treatment were taken in order to obtain a dry mass of soil of about 0.1 g per sample

Vehicle:

yes

Remarks:

sand

Details on preparation and application of test substrate:

AgNO3 was first added in given amounts to achieve the desired concentrations to 50 g of oven-dried quartz sand (≤2 mm) in a 50 ml sealed plastic centrifuge tube and agitated for 1 h using a laboratory flask shaker. The AgNO3 and sand mixture was then added to the other dry constituents. A plastic rod was used to thoroughly mix the experimental substrate and water gradually added. The final substrate was composed of 10% by mass of the AgNO3 and sand mixture, 88% dried Kettering loam and 2% dried and sieved organic matter (horse manure). The substrate was wetted to a moisture content of 25%.

Test organisms (species):

other: Allolobophora chlorotica

Animal group:

annelids

Details on test organisms:

endogeic and anecic earthworm species

Study type:

laboratory study

Substrate type:

artificial soil

Remarks:

Composed of 10% by mass AgNO3 and sand mixture, 88% dried Kettering loam and 2% dried and sieved horse manure (OM). Kettering loam composition: Clay 24%, Silt 18%, Sand 58%, Organic content 6.72%, pH 6.8, obtained from Boughton Loam Ltd

Limit test:

no

Total exposure duration:

14 d

Test temperature:

15°C

pH:

7.77 - 8.07

Moisture:

25%

Details on test conditions:

300 mL plastic pots kept in constant darkness in a temperature-controlled incubator at 15 °C
3 adult worms/pot and 5 replicates/treatment

Nominal and measured concentrations:

nominal: 0, 12.5, 25, 50 and 100 mg Ag/kg
actual at the end of the test: 0, 7.19, 18.26, 35.27 and 81.94 mg Ag/kg

Reference substance (positive control):

no

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

35.27 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Ag

Basis for effect:

mortality

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

81.94 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Ag

Basis for effect:

mortality

Duration:

14 d

Dose descriptor:

LC10

Effect conc.:

66.88 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Ag

Basis for effect:

mortality

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

7.19 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Ag

Basis for effect:

growth

Remarks:

biomass

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

18.26 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Ag

Basis for effect:

growth

Remarks:

biomass

Duration:

14 d

Dose descriptor:

EC10

Effect conc.:

2.56 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Ag

Basis for effect:

growth

Remarks:

biomass

Remarks on result:

other: EC10 below lowest test concentration and therefore not considered reliable

Details on results:

LC10 value calculated with TRAP

Reported statistics and error estimates:

Welch's One-way ANOVA followed by a Games-Howell post hoc test was used to analyse earth- worm biomass results (at day 0 and day 14) across the 5 separate Ag NP/AgNO3 concentrations. Anderson-Darling Normality test and Levene's test for equality of variances were used to validate that test assumptions had been met. A significance level of 5% (p=0.05) was applied.

Validity criteria fulfilled:

not applicable

Conclusions:

The 14 d NOEC for the effect of silver (AgNO3) to the biomass (most sensitive endpoint) of the earthworm Allolobophora chlorotica amounted to 7.2 mg Ag/kg soil (14 d LOEC: 18.3 mg/kg).

Executive summary:

The toxicity of silver nitrate to the earthworm Allolobophora chlorotica was determined in a non-guideline study.

The NOEC values of 35.3 mg Ag/kg dw for mortality and 7.2 mg Ag/kg dw for growth (biomass) were taken forward to the derivation of the PNEC according to the statistical extrapolation approach.

Reference 7

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

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:

other: ISO 16387 Soil Quality - Effects of Pollutants on Enchytraeidae (Enchytraeus sp.). Determination of Effects on Reproduction.

Version / remarks:

ISO 2003

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 220 (Enchytraeid Reproduction Test)

Version / remarks:

OECD 2004

Deviations:

no

GLP compliance:

not specified

Specific details on test material used for the study:

Silver nitrate (AgNO3 >99%, Sigma Aldrich)

Analytical monitoring:

yes

Details on sampling:

The soil for the analysis (1 g dry weight) was digested using 7N HNO3 (nitric acid) and heating up to 110°C until all brown fumes were gone

Vehicle:

no

Details on preparation and application of test substrate:

Aqueous solutions added to pre-moistened soil.

Test organisms (species):

Enchytraeus crypticus

Animal group:

annelids

Study type:

laboratory study

Substrate type:

natural soil

Remarks:

Lufa 2.2 natural standard soil (Speyer, Germany) pH (0.01 M CaCl2) of 5.5, 1.61% organic matter, 10.0 meq/100 g CEC (cation exchange capacity), 43.3% WHC (water holding capacity), grain size distribution of 7.9% clay, 16.3% silt, and 75.8% sand

Limit test:

no

Total exposure duration:

21 d

Test temperature:

20°C

pH:

5.4

Moisture:

50% of WHC

Details on test conditions:

Adults (10) with well-developed clitellum were selected from cultures and introduced in each test vessel containing 20 g of moist soil and food supply. Test run at 20°C and 16:8 h photoperiod for 21 days. Four replicates per treatment were used. Food and water were replenished weekly.

Nominal and measured concentrations:

NOMINAL: 0-36-48-60-72 mg Ag/kg soil dry weight
The soil Ag spiking measurement was confirmed, being between 95 and 109% Ag recovery with no concentration dependent pattern.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

LC10

Effect conc.:

52 mg/kg soil dw

95% CI:

38 - 67

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Ag

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

75 mg/kg soil dw

95% CI:

66 - 84

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Ag

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

38 mg/kg soil dw

95% CI:

24 - 51

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Ag

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

62 mg/kg soil dw

95% CI:

57 - 68

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Ag

Basis for effect:

reproduction

Reported statistics and error estimates:

ANOVA (Analysis of variance) followed by Dunnett’s comparison post-hoc test (p <= 0.05) was done to assess differences between control and treatments (SigmaPlot 11.0). Effect Concentrations (ECx) estimates were performed for the various endpoints modelling data, using Toxicity Relationship Analysis Program (TRAP v1.22).

Validity criteria fulfilled:

yes

Conclusions:

A 21 d EC10 of 38 mg Ag/kg soil dry weight was derived for the effect of silver to the reproduction (most sensitive endpoint) of Enchytraeus crypticus.

Executive summary:

The toxicity of silver nitrate to Enchytraeus crypticus was determined in a guideline study. The EC10 values of 52 mg Ag/kg dw for mortality and 38 mg Ag/kg dw for reproduction were taken forward to the derivation of the PNEC according to the statistical extrapolation approach.

Reference 8

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

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 220 (Enchytraeid Reproduction Test)

Version / remarks:

OECD 2004

Deviations:

yes

Remarks:

3 weeks exposure

GLP compliance:

not specified

Specific details on test material used for the study:

AgNO3 (Sigma- Aldrich, > 99%) dissolved in deionized water (Milli-Q)

Analytical monitoring:

yes

Details on sampling:

After drying at 40 °C for 24 h, about 0.130 g soil samples were digested in Teflon bombs with 2 mL of a mixture of HCl (Sigma Aldrich, 37%) and HNO3 (Sigma Aldrich, %65) (4:1). The bombs were closed tightly and heated at 140 °C for 7 h in an oven (Binder FD). After cooling down, 8 mL demineralized water was added.

Vehicle:

no

Details on preparation and application of test substrate:

The soils were spiked in glass jars with AgNO3 dissolved in demineralized water, at the same time adjusting soil moisture content to 50% of the water holding capacity (WHC). After spiking, the soils were mixed to achieve an as homogeneous distribution of the Ag as possible. Control soils received demineralized water only. Four replicates were prepared for each test concentration and control. For each replicate, 30 g moist soil was placed in a 100 mL glass jar and equilibrated overnight.

Test organisms (species):

Enchytraeus crypticus

Animal group:

annelids

Details on test organisms:

Toxicity tests started with the introduction of 10 adult E. crypticus, with clearly visible clitella and approximately 1 cm length, to the jars.

Study type:

laboratory study

Substrate type:

natural soil

Remarks:

Lufa 2.2: %OC: 1.61 pH: 5.5 CEC: 10.0 meq/100 g WHCmax: 43.3% - Lufa 2.3: %OC: 0.67 pH: 5.8 CEC: 7.3 meq/100 g WHCmax: 35.6% - Lufa5M: %OC: 0.98 pH: 7.3 CEC: 16.1 meq/100 g WHCmax: 39.8%

Limit test:

no

Total exposure duration:

21 d

Test temperature:

20°C

pH:

Lufa 2.2: pH: 5.5 - Lufa 2.3: pH: 5.8 - Lufa5M: pH: 7.3

Moisture:

50% of WHC

Details on test conditions:

A few grains of oat meal were added to the test jars for food. The jars were covered with perforated aluminum foil for air transfer and incubated at 20 ± 1 °C and 12:12 h light:dark. Water content and food availability were checked twice a week and adjusted if necessary.

Nominal and measured concentrations:

NOMINAL concentrations: 0, 10.6, 25.6, 64, 160, 400, 1000 mg Ag/kg dry soil;
MEASURED in LUFA 2.2: MEASURED in LUFA 2.3: MEASURED in LUFA 5 M:

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

18.3 mg/kg soil dw

95% CI:

10.8 - 25.8

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Ag

Basis for effect:

reproduction

Remarks on result:

other: Lufa 2.2

Duration:

21 d

Dose descriptor:

EC10

Remarks:

extrapolation

Effect conc.:

8.29 mg/kg soil dw

95% CI:

5.21 - 11.4

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Ag

Basis for effect:

reproduction

Remarks on result:

other: Lufa 2.3

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

11.7 mg/kg soil dw

95% CI:

7.64 - 15.7

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Ag

Basis for effect:

reproduction

Remarks on result:

other: Lufa 5 M

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

75.2 mg/kg soil dw

95% CI:

60.8 - 90

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Ag

Basis for effect:

reproduction

Remarks on result:

other: Lufa 2.2

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

26.9 mg/kg soil dw

95% CI:

22.3 - 31.5

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Ag

Basis for effect:

reproduction

Remarks on result:

other: Lufa 2.3

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

46 mg/kg soil dw

95% CI:

39 - 52.6

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Ag

Basis for effect:

reproduction

Remarks on result:

other: Lufa 5 M

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

111 mg/kg soil dw

95% CI:

89.2 - 139

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Ag

Basis for effect:

mortality

Remarks on result:

other: Lufa 2.2

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

112 mg/kg soil dw

95% CI:

87 - 144

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Ag

Basis for effect:

mortality

Remarks on result:

other: Lufa 2.3

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

92.1 mg/kg soil dw

95% CI:

73.3 - 116

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Ag

Basis for effect:

mortality

Remarks on result:

other: Lufa 5 M

Reported statistics and error estimates:

Lethal concentrations for 50% of the test organisms (LC50) and corresponding 95% confidence intervals were calculated with the trimmed Spearman–Karber method. Effective concentrations that caused 10% and 50% reduction in the number of juveniles (EC10 and EC50, respectively) and their 95% confidence intervals were obtained by fitting a 3-parameter logistic dose-response model. All analysis was run in SPSS 21 for Windows.

Validity criteria fulfilled:

yes

Conclusions:

21 d LC50 values for the effect of silver to mortality of Enchytraeus crypticus in Lufa 2.2, Lufa 2.3, and Lufa 5 M soil, amounted to 111, 112, and 92.1 mg Ag/kg soil dry weight, respectively.
21 d EC10 values for the effect of silver to reproduction of Enchytraeus crypticus in Lufa 2.2, Lufa 2.3, and Lufa 5 M soil, amounted to 18.3, 8.29 (extrapolated), and 11.7 mg Ag/kg soil dry weight, respectively. Since the value of 8.29 was below the lowest test concentration, only the 2 other EC10 values were taken forward to the derivation of the PNEC according to the statistical extrapolation approach.

Executive summary:

The toxicity of silver nitrate to Enchytraeus crypticus was determined in a guideline study. The EC10 values of 11.7 mg Ag/kg dw and 18.3 mg Ag/kg dw for reproduction were taken forward to the derivation of the PNEC according to the statistical extrapolation approach.

Description of key information

The most sensitive effects data is an EC10 of 5.3 mg/kg Ag for mortality in Eisenia fetida from a 28 day exposure. The physico-chemical conditions of the soil were pH 5.5, organic carbon 0.9% and clay 60% (Langdon et al. 2013). 

Additional information

In total, 52 reliable EC₁₀ or NOEC values for chronic silver toxicity to terrestrial invertebrates except arthropods were identified. The data cover Ag toxicity to reproduction of 3 different species (Allolobophora chlorotica, Eisenia fetida and Enchytraeus crypticus). NOEC and EC₁₀ values vary between 5.3 mg Ag/kg dw for mortality of Eisenia fetida (Langdon et al., 2013) and 221.0 mg Ag/kg dw for growth of Eisenia fetida (Langdon et al., 2013). All NOEC and EC₁₀ values are expressed based on total elemental Ag concentrations in soil. All reliable NOEC and EC₁₀ values were taken forward to the derivation of the PNEC according to the statistical extrapolation approach.