Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to soil microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

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:
other: Guideline study, available as unpublished report.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 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
yes
Remarks:
7 soils tested (one recommended)
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:
AMENDMENT OF SOIL
After the 7 day pre-incubation period, the test was commenced by adding dried lucerne (C:N ratio = 13.6:1) to each sample at a rate of 5 mg/g dry soil.

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 by 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 (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
22°C
Moisture:
50% of its maximum water holding capacity (MWHC)
Details on test conditions:
TEST SYSTEM
- Amount of soil: 60g
- No. of replicates per concentration: 3
- No. of replicates per control: 3

- Geographic location: seven study soils used, one of the soils were collected from Europe, Bordeaux (France) and the remaining six soils (Inman Valley, Charleston, Kingaroy, Millicent, 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
- Cation Exchange Capacity: 5.3 - 42 cmol+/kg
- pH (in water): 4.9-8.8

DETAILS OF PREINCUBATION OF SOIL: soils were divided into three replicate samples, with a leached and unleached treatment. Each replicate consisted of approximately 60 g of dry soil that was hydrated to 50% of its maximum water holding capacity (MWHC). The hydrated samples were then placed on damp paper towel in an airtight container in the dark at 22°C for a pre-incubation period of 7 days. All samples were opened for aeration on a daily basis and the water content in the samples was maintained at 50% MWHC by weight.

EFFECT PARAMETERS MEASURED: total NO3 production and potential nitrification rate (PNR). The total NO3 production was determined by subtracting the time zero NO3 concentration in the soil from the day 28 NO3 concentration. The PNR was calculated as the slope of the regression line of soil nitrate to time in the linear range of the data, therefore, representing the rate of nitrification at its most rapid phase during the test. The linear regression was determined for each replicate samples using the NO3 measurements from 0, 7 and 14 days as this portion of the data produced the most linear relationship

VEHICLE CONTROL PERFORMED: no
Nominal and measured concentrations:
The nominal concentrations for each soil were as follows (all concentrations are shown as mg Ag/kg dry soil):
Bordeaux 0, 1, 2, 4, 8, 16, 32
Inman Valley 0, 10, 20, 40, 80, 160, 320
Charleston 0, 10, 20, 40, 80, 160, 320
Kingaroy 0, 0.5, 1, 2, 4, 8, 16, 32
Millicent 0, 20, 40, 80, 160, 320, 640
Balaklava 0, 10, 20, 40, 80, 160, 320
Port Kenny 0, 10, 20, 40, 80, 160, 320

The measured concentrations of Ag in the spiked soils were between 80 and 120% of the nominal concentrations, therefore nominal concentrations were used for all data analysis.
Reference substance (positive control):
yes
Remarks:
certified reference material (CRM) (PACS-2)
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
30 mg/kg soil dw
95% CI:
>= 14 - <= 44
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: substrate induced nitrification
Remarks on result:
other: Inman Valley soil, unleached
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
26 mg/kg soil dw
95% CI:
>= 23 - <= 28
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: substrate induced nitrification
Remarks on result:
other: Charleston soil, unleached
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
371 mg/kg soil dw
95% CI:
>= 322 - <= 609
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: substrate induced nitrification
Remarks on result:
other: Millicent soil, unleached
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
61 mg/kg soil dw
95% CI:
>= 52 - <= 72
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: substrate induced nitrification
Remarks on result:
other: Balaklava soil, unleached
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
85 mg/kg soil dw
95% CI:
>= 78 - <= 92
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: substrate induced nitrification
Remarks on result:
other: Port Kenny 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:
other: substrate induced nitrification
Remarks on result:
other: Inman Valley soil, leached
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
36 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: substrate induced nitrification
Remarks on result:
other: Charleston soil, leached
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
83 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: substrate induced nitrification
Remarks on result:
other: Balaklava soil, leached
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
83 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: substrate induced nitrification
Remarks on result:
other: Port Kenny soil, leached
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
1.2 mg/kg soil dw
95% CI:
<= 1.5
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: potential nitrification rate
Remarks on result:
other: Bordeaux soil, unleached
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
26 mg/kg soil dw
95% CI:
>= 23 - <= 29
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: potential nitrification rate
Remarks on result:
other: Inman Valley soil, unleached
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
14 mg/kg soil dw
95% CI:
>= 10 - <= 19
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: potential nitrification rate
Remarks on result:
other: Charleston soil, unleached
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
488 mg/kg soil dw
95% CI:
>= 369 - <= 613
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: potential nitrification rate
Remarks on result:
other: Millicent soil, unleached
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
36 mg/kg soil dw
95% CI:
>= 32 - <= 41
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: potential nitrification rate
Remarks on result:
other: Balaklava soil, unleached
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
38 mg/kg soil dw
95% CI:
>= 37 - <= 40
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: potential nitrification rate
Remarks on result:
other: Port Kenny soil, unleached
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
20 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: potential nitrification rate
Remarks on result:
other: Charleston soil, leached
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
296 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: potential nitrification rate
Remarks on result:
other: Millicent soil, leached
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
46 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: potential nitrification rate
Remarks on result:
other: Balaklava soil, leached
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
53 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, leached
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).

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).

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 substrate induced nitrification ranged from 23 to 371 mg Ag/kg and for PNR from 1.2 to 488 mg Ag/kg. 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: substrate induced nitrification (SIN) and potential nitrification rate (PNR). The 28-day EC10 values for SIN ranged from 23 (Inman Valley soil) to 371 mg Ag/kg (Millicent soil) and for PNR were between 1.2 (Bordeaux soil) and 488 mg Ag/kg (Millicent soil). The toxicity of silver to the soil nitrification process was primarily controlled by soil pH and OC. 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.

Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, acceptable with restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
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 in samples collected at the commencement of the test and at the completion of the test.
Vehicle:
no
Details on preparation and application of test substrate:
Soils were prepared the day before study initiation. Enough soil for two test treatments 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 stored. Prior to test set up the soil was again mixed by hand for three minutes and distributed to the test chambers. Half of the replicates also had alfalfa dehydrated meal added at a rate of 5g/1000g to provide an organic substrate.
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20.1°C mean, 20.0°C-20.2°C range

Moisture:
45.2% WHC mean, 44.0-48.4% WHC range at day 0.
43.4% WHC mean, 42.5-44.5% WHC range at day 56.
Details on test conditions:
Test soil composition: 1:1:1 mix of garden soil, sand and a silt clay mix.
Garden soil = Collected from Fort Collins, CO, USA. Site was used for vegetable/flower gardening, without fertiliser or pesticides. Sampling depth <25cm. 77% sand, 18% silt, 5% clay, 4% organic carbon.
Sand = silica medium sand.
Silt clay mix = 0% sand, 61% silt, 39% clay, 0% organic carbon.
Characterisation of the soil indicated that it had a pH of 7.3, organic carbon content of 1.3% and consisted of 81% sand, 8% silt and 11% clay.

Soil prepared to be at 40% WHC. Test chambers were weighed over the exposure period to monitor hydration levels. No additional water was required to maintain adequate soil hydration.

Control and each of the six treatment concentrations had one replicate (one with and one without alfalfa added). However, multiple grabs from the same chamber were used as replicates for nitrate analysis.
Test chambers were held in an environmental chamber in the dark.

Three soil subsamples collected on day 0 and 28 from each chamber, and one soil sample collected from each chamber on day 14, were extracted for nitrate using 0.1 M potassium chloride solution.
Nominal and measured concentrations:
Nominal: 0, 0.04, 0.16, 0.63, 2.5, 10, 40 mg/kg
Measured: 0.04, 0.058, 0.129, 0.45, 2.12, 9.45, 31 mg/kg
Measured values were within 45% nominal.
Reference substance (positive control):
no
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
95% CI:
>= 0.16 - <= 0.41
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Basis for effect:
nitrate formation rate
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. The EC10 value was taken forward to the derivation of the PNEC according to the statistical extrapolation approach.

Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Basal Respiration (BR):
To evaluate the effect of AgNO3 on total biological activity of the soils, BR was measured according to the alkali base trap technique. Spiked and control soil samples (80 g) were transported to screw bottles (Schott, 250 mL), in triplicates, and incubated for 60 days at 25 °C with 10 mL of 0.2 M NaOH traps. The acid titration was performed every 4 days and the total amount of CO2 evolved from each soil sample was calculated over a period of 60 days.
Substrate Induced Respiration (SIR):
The SIR involves the measurement of the soil microbial respiration after adding a simple substrate, e.g. glucose, to stimulate microbial activity for a period of time. It can be used for estimating the microbial biomass in a soil. Before SIR measurement, triplicate Ag-treated and control soil samples were incubated for 7 days at 25 °C and 60% of WHC.
GLP compliance:
not specified
Analytical monitoring:
no
Remarks:
only background Ag concentration measured
Details on sampling:
open vessel aqua regia (1:3 HNO3:HCl) digestion at 140 °C
Vehicle:
no
Details on preparation and application of test substrate:
Appropriate volumes of stock AgNO3 solutions supplemented by adequate distilled water were slowly added to the soil subsamples to reach the predefined soil Ag concentrations and water contents (60% of WHC).
Test organisms (inoculum):
soil
Total exposure duration:
60 d
Remarks:
Basal respiration determined after 60 days of incubation; Substrate induced respiration determined after 7 d of incubation
Test temperature:
25°C
Moisture:
60% of WHC
Organic carbon content (% dry weight):
0.7
Details on test conditions:
Golshahr soil:
clay 34.0%
silt 37.4%
sand 32.0%
CEC 24.83 cmolc/kg
OC 0.66%
pH 7.54

Janatabad soil:
clay 26.0%
silt 44.6%
sand 29.0%
CEC 21.56 cmolc/kg
OC 0.65%
pH 8.09
Nominal and measured concentrations:
Nominal: 0, 0.01, 0.1, 0.5, 1, 5, 10, 20 and 50 mg Ag kg− 1 soil
Reference substance (positive control):
no
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Ag
Basis for effect:
respiration rate
Remarks:
substrate induced respiration
Remarks on result:
other: Janatabad soil
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
42 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Ag
Basis for effect:
respiration rate
Remarks:
substrate induced respiration
Remarks on result:
other: Golshahr soil
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
>= 50 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Ag
Basis for effect:
respiration rate
Remarks:
substrate induced respiration
Remarks on result:
other: Janatabad soil
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
>= 50 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Ag
Basis for effect:
respiration rate
Remarks:
substrate induced respiration
Remarks on result:
other: Golshahr soil
Duration:
60 d
Dose descriptor:
NOEC
Effect conc.:
>= 50 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Ag
Basis for effect:
respiration rate
Remarks:
basal respiration
Remarks on result:
other: Janatabad soil
Duration:
60 d
Dose descriptor:
NOEC
Effect conc.:
>= 50 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Ag
Basis for effect:
respiration rate
Remarks:
basal respiration
Remarks on result:
other: Golshahr soil
Reported statistics and error estimates:
Statistical analysis was performed using SAS software version 9, and significant differences of the means were calculated by Tukey's test at probability level of 0.01 (P < 0.01).
Three different mathematical models were used to describe responses of the soil microbial activities to AgNO3 doses. The model fitting parameters were optimized and the coefficient of determination (R2) was obtained using the GraphPad Prism v.5. In addition to R2, the Akaike information criterion (AIC) was also used for comparing the efficacy of three models.
Validity criteria fulfilled:
not applicable
Conclusions:
After 60 d (basal respiration) or 7 d (substrate induced respiration) of soil incubation with silver, no effects were observed at concentrations up to 50 mg Ag/kg soil dry weight for both Golshahr and Janatabad soil.
Executive summary:

EC10 values of 42 mg/kg dw and 6 mg/kg dw for glucose induced respiration were extracted from this study for use in the PNEC derivation.

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:
other: Non-GLP, guideline study, available as a published journal article. Minor limitations in design and/or reporting but otherwise adequate for assessment.
Principles of method if other than guideline:
For the assessment of the effect of NM-300K and silver nitrate on the nitrifiers, a combination of two test guidelines was applied. The incubation was carried out in accordance with the OECD Guideline 216. The nitrite concentration was determined with a short-term potential ammonium oxidation test performed in accordance with the ISO Guideline 15685.
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:
Digestion was carried out in accordance with standardised guidelines, using soil dried at 105°C for at least 12 hours until it reached a constant weight. Approximately 3 g of the homogenised material was mixed with 28 g of aqua regia and incubated at room temperature for 16 hours without agitation. The mixture was then heated under reflux for 2 hours with glass chips and 1-octanol added to avoid over-boiling and foaming. The mixture was cooled to room temperature, made up to 100 mL and filtered using a 0.45 µm syringe filter.
Vehicle:
no
Details on preparation and application of test substrate:
Tests were carried out using sieved and spiked soil. The experiments were carried out using the reference soil RefeSol 01A (sieved ≤2 mm) as both the test and carrier soil. RefeSol 01A is a loamy, medium-acidic and lightly humic sand. RefeSol soils were selected as reference soils by the German Federal Environment Agency (Umweltbundesamt) and are known to be suitable for testing the influence of substances on the habitat function of soils (bioavailability, effects on organisms). RefeSol 01A matches the properties stated in various OECD terrestrial ecotoxicological guidelines (e.g. tests with plants and soil microflora). The soils were sampled in the field and stored in high-grade stainless steel basins with drainage and ground contact on the grounds of Fraunhofer IME in Schmallenberg. During all experiments, red clover was sown on the stored soils and no pesticides were used. Appropriate amounts of soil were sampled 1 to 4 weeks before the test. If the soil was too wet for sieving, it was dried at room temperature to 20% to 30% of the maximum water holding capacity (WHCmax) with periodic turning to avoid surface drying. If the tests did not start immediately after sieving, the soil was stored in the dark at 4°C under aerobic conditions.
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
The vessels were incubated in the dark at 20°C ± 2°C for 24 hours, after which the mineral test medium was added and the slurries were incubated on an orbital shaker at 25°C ± 2°C and the nitrite levels in the filtrate determined.
Moisture:
The test was conducted on soil prepared to 20% to 30% of the maximum water holding capacity (WHCmax).
Details on test conditions:
Four 250 mL Erlenmeyer flasks per treatment were filled with 25 g dry matter of spiked soil along with four matching controls. The vessels were incubated in the dark at 20°C ± 2°C for 24 hours, after which the mineral test medium was added to make the volume up to 100 mL. The medium consisted of KH2PO4 (0.56 mM), K2HPO4 (1.44 mM), NaClO3 (5 mM) and (NH4)2SO4 (1.50 mM). The slurries were incubated on an orbital shaker at 25°C ± 2°C, after which 10 mL samples were removed at 2 and 6 hours, supplemented with 10 mL of 4 mol/L KCl and filtered, and the nitrite levels in the filtrate determined photometrically (UV–VIS Spectrophotometer) with a wavelength of 530 nm. The effect of NM-300K and silver nitrate on nitrifying microorganisms was examined by the transformation of ammonium after 24 hours, 7 days and 28 days. Three replicates for each control (with or without dispersant) and the different contents of NM-300K and silver nitrate in soil were tested. The transformation of ammonium is expressed as nitrite production.
Nominal and measured concentrations:
Both NM-300K and silver nitrate were tested at concentrations of 0.56, 1.67, 5.0 and 15.0 mg/kg dry soil and additionally at 0.19 mg/kg dry soil for silver nitrate. All concentrations refer to the silver Ag content in the respective test substance.
Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
1.4 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: potential ammonium oxidation
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
2.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass
Details on results:
Silver nitrate caused no inhibition of nitrite production in the 0.19 and 0.56 mg/kg dry soil treatments. Instead, a stimulatory effect occurred but was only significant after 7 days at 0.19 mg/kg dry soil (19.4%). After 28 days, the silver nitrate NOEC was 0.56 mg/kg dry soil and the EC10 1.4 mg/kg dry soil. The inhibitory effects of NM-300K and silver nitrate were comparable at concentrations of 1.67 to 15 mg/kg dry soil, as silver nitrate caused 25.5% to 90.0% inhibition in nitrite production and NM-300K 25.1% to 85.9% inhibition. The effect of NM-300K slightly increased over a period of 28 days, while the effect of silver nitrate was comparable at all three points of measurement.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The NOEC were calculated by ANOVA followed by parametric pairwise comparisons of treatments to the control using the Dunnett, Welch, Williams or Fisher exact tests. Probit analysis was performed for the estimate of the ECx and dose–response curve. Due to fewer treatments and concentrations, the significance of variance between the different treatments was conducted by Student’s t tests in the long-term tests. Statistical analysis was carried out using ToxRat® Pro v2.10 software for ecotoxicity response analysis.
Validity criteria fulfilled:
not applicable
Remarks:
In all ammonium oxidation tests, the validity criterion (coefficient of variation between replicates of control ≤15%) was fulfilled.
Conclusions:
After 28 days, the EC10 for potential ammonium oxidation was 1.4 mg/kg dw. After 7 days, the EC10 for biomass was 2.5 mg/kg dw.
Executive summary:

The study is a guideline study, published in peer reviewed literature and considered suitable for use for this endpoint. For the assessment of the effect of NM-300K and silver nitrate on the nitrifiers, a combination of two test guidelines was applied. The incubation was carried out in accordance with the OECD Guideline 216. The nitrite concentration was determined with a short-term potential ammonium oxidation test performed in accordance with the ISO Guideline 15685. In all ammonium oxidation tests, the validity criterion (coefficient of variation between replicates of control ≤15%) was fulfilled.


For silver nitrate, after 28 days, the EC10 for potential ammonium oxidation was 1.4 mg/kg dw. After 7 days, the EC10 for biomass was 2.5 mg/kg dw. Both EC10 values were taken forward to the derivation of the PNEC according to the statistical extrapolation approach.

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: 63.49% Ag, purity >99.9%

FORM AS APPLIED IN THE TEST: A stock solution of 10 µg Ag/L in MQ water was prepared.

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 undertaken 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 test item 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):
Rots France
Lufa 2.2 Germany
Poelkapelle Belgium

- Depth of sampling: 0-20 cm
- 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: 36 and 72 mg Ag/kg soil, Lufa soil: 5.2 and 10.4 mg Ag/kg soil, Poelkapelle soil: 57 and 114 mg Ag/kg soil
- Results used to determine the conditions for the definitive study: yes
Nominal and measured concentrations:
Rots soil: 0.0, 4.6, 15, 47, 150, 480 and 1536 mg added Ag/kg soil
Lufa soil: 0.0, 0.5, 1.5, 4.7, 15, 48, 154 and 492 mg added Ag/kg soil
Poelkapelle: 0.0, 10, 32, 102, 328, 1049 and 3355 mg added Ag/kg soil
Reference substance (positive control):
no
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
49 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: 30 - 83 mg Ag/kg soil
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
36 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: 8 - 143 mg Ag/kg soil
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
66 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: 34 - 128 mg Ag/kg soil
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
113 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: 91 - 146 mg Ag/kg soil
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
100 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: 76 - 133 mg Ag/kg soil
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
134 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: 98-182 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: 1.2 - 11 mg Ag/kg soil
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
30 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 - 41 mg Ag/kg soil
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
42 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: 29 - 72 mg Ag/kg soil
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
45 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: 33 - 61 mg Ag/kg soil
Details on results:
- Other:
For all soils spiked with AgNO3, the total concentration of Ag in the pore water decreases with time probably due to ageing (e.g. increased sorption of Ag on soil particles)
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.
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).

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 reliable EC10 value for PNR was 3.8 mg Ag/kg soil for Lufa soil. Based on measured total concentration of Ag in soil the reliable EC10 values for SIN were 30, 42 and 45 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 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.

The toxicity of silver nitrate was measured with the nitrification assay (soil microbial assay) conducted according to OECD 216 guideline. Three uncontaminated agricultural soils were spiked with AgNO3, 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.

Based on measured total concentration of Ag in soil the reliable EC10 value for PNR was 3.8 mg Ag/kg soil for Lufa soil. Based on measured total concentration of Ag in soil the reliable EC10 values for SIN were 30, 42 and 45 mg Ag/kg soil for Rots, Lufa and Poelkapelle soils, respectively.

All EC10 values 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 0.30 mg/kg Ag for effects on nitrogen transformation in a 28 day OECD 216 test. The physico-chemical conditions of the soil were pH 7.3, Organic Carbon 1.3 % and clay <11 % (Naddy 2011).

Key value for chemical safety assessment

Additional information

In total, 28 reliable EC10 or NOEC values for chronic silver toxicity to soil microorganisms were retrieved. All tests are performed with the native soil microbial community present in the soils. The data cover effects on nitrification, glucose induced respiration and microbial biomass. The reliable NOEC and EC10 values range between 0.3 and 488.0 mg Ag/kg, both for nitrification (Langdon et al., 2013 and Naddy, 2011). 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.