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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: guidelines included in the Rotoxkit F test (MicroBioTests 2018), which follows procedure 8420 of the American Public Health Association (2017) methods
Version / remarks:
MicroBioTests. 2018. Rotoxkit F—Chronic toxicity test for freshwater: Standard operational procedure. Mariakerke, Belgium.
American Public Health Association. 2017. Standard methods for the examination of water and wastewater, 23rd ed. Washington, DC.
Deviations:
yes
Remarks:
The strong metal complexing organic ligand ethylenediaminetetraacetic acid (EDTA) was omitted from all test media and was replaced by 1 mg/L DOC (from a natural origin: Suwannee River standard natural organic matter; ID 2R101N).
GLP compliance:
no
Remarks:
Publication, GLP compliance not specified
Specific details on test material used for the study:
63.49% Ag, purity >99.9%
Analytical monitoring:
yes
Details on sampling:
Samples for analysis of total and dissolved Ag (filtered over a 0.45-µm filter; Acrodisc; Pall Life Science) of fresh media were taken for all test concentrations at test initiation (both before and after addition of P. subcapitata to the exposure medium), after 24-h exposure, and at test termination (48 h). To have an adequate volume of exposure medium for sampling, the test was set up in quadruplicate.
Vehicle:
no
Details on test solutions:
Toxicity tests were performed in moderately hard US Environmental Protection Agency (USEPA) reconstituted water with addition of 1 mg DOC/L. The test medium was prepared two days before test initiation, and aerated for 24h in total darkness. After this, the medium was divided in different aliquots which were stored in acid-washed falcon tubes (range finding) or polypropylene beakers (definitive test) until use. Ionic silver was spiked into the medium 24h before test initiation using the appropriate volume of AgNO3-stock solution, and left to equilibrate in total darkness in a 24°C incubation cabinet until use. Storage of spiked solutions were conducted in polypropylene or polycarbonate test vessels to minimize adsorption of Ag to the walls of the test vessels
Test organisms (species):
other: Brachionus calyciflorus
Details on test organisms:
The B. calyciflorus cysts were provided in the Rotoxkit. Prior to test initiation, the cysts were hatched in 10 mL of medium hard USEPA reconstituted water at 25 °C on a light box. After hatching, rotifers were fed with RotiRich food 2 h before test initiation.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
The 48-h test period covers approximately 3 full generations of this rotifer and can therefore be considered a true chronic test.
Hardness:
10.6 ± 0.4 mg Ca /L and 5.2 ± 0.1 mg Mg/L (48 mg CaCO3/L)
Test temperature:
24.9 ± 0.2 °C
pH:
pH of fresh test media: 7.8 ± 0.1
pH throughout the test: 7.9 ± 0.1
Dissolved oxygen:
85 ± 4%
Nominal and measured concentrations:
Nominal concentrations: control and 5, 10, 20, 40, 80, 160, and 320 µg Ag/L.
Measured concentrations (time-weighted average of dissolved Ag concentrations in fresh solutions at t 0 h (after addition of algae) and in old solutions at t 24 h (when available) and t 48 h): <0.01, 0.27, 0.52, 1.4, 5.2, 11, 23 and 35 µg Ag/L.

Total Ag concentrations in the fresh medium were in the 2 lowest Ag treatments up to 26% lower compared to nominal concentrations, whereas total Ag concentrations in the other Ag treatments were 4 to 16% higher than nominal concentrations.
Dissolved Ag concentrations in fresh solutions were on average 12 ± 10% lower than total concentrations in fresh solutions. A strong decrease in dissolved Ag was observed immediately after addition of the algae food source to the fresh solution, probably because of surface sorption of Ag to the algae. Dissolved samples taken just after the addition of the algae to the fresh solution contained on average only 21 ± 4% of the Ag that was present in the dissolved fraction before addition of the algae. Dissolved Ag concentrations in the 3 lowest Ag treatments could not be accurately determined (concentrations below quantification limit of the ICP-OES analysis and an insufficient sample volume remained for ICP-MS analysis). In the other Ag treatments, dissolved Ag concentrations after 24-h exposure were on average 34 ± 12% lower than dissolved Ag concentrations in fresh solutions after the algae addition. After 48-h exposure, dissolved Ag concentrations further decreased and were on average 70 ± 29% lower than dissolved Ag concentrations in fresh solutions after the algae addition. The highest decreases in dissolved Ag concentrations were observed at the 3 lowest Ag concentrations, where dissolved concentrations decreased up to 95% compared to dissolved concentrations in fresh solutions.
Details on test conditions:
Each test concentration and the control was assayed in 16 replicates. The assays were conducted in acid-washed polycarbonate 54-multiwell plates provided in the Rotoxkit. Before dividing the test media over the exposure wells, 2 × 106 P. subcapitata (delivered in the Rotoxkit as algal beads) cells/mL were added to the exposure media as a food source for the rotifers during the toxicity test. Each exposure well received 1 mL of the appropriate algae-containing exposure medium.
At test initiation, one rotifer (<2 h old) was placed in each well, and the plates were sealed with Parafilm and placed in a dark 25 °C growth cabinet.
Key result
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0.31 µg/L
95% CI:
>= 0.13 - <= 0.73
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
other: population size
Remarks on result:
other: Effect concentrations are expressed as the geometric mean of the measured dissolved Ag concentrations in fresh and old solutions.
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
2.6 µg/L
95% CI:
>= 1.5 - <= 4.7
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
other: population growth rate
Remarks on result:
other: Effect concentrations are expressed as the geometric mean of the measured dissolved Ag concentrations in fresh and old solutions.
Details on results:
Concentration–response analysis was based on the time-weighted average of dissolved Ag concentrations measured in fresh medium after algae addition (t 0 h) and those measured in the old solutions after the 24 (when available) and 48-h exposures. These time-weighted average dissolved Ag concentrations were on average 90 ± 4% lower than nominal concentrations.
Reported statistics and error estimates:
Effect concentrations were calculated based on relative responses (expressed relative to the mean control response of the respective experiment). The EC10, EC20, and EC50 values and corresponding confidence intervals were determined based on a log-logistic concentration response model with 2 parameters using Statistica software.
NOEC and LOECs were calculated with the nonparametric Jonckheere-Terpstra test.

Overview of average* population size (at end of test) and population growth rate in the different exposure treatments of the 48-h Brachionus calyciflorus reproduction test

Dissolved Ag (µg/L)

Nominal Ag concentration (µg/L)

t 0 h before algae addition

t 0 h after algae addition

t 24 h

t 48 h

Time-weighted dissolved Ag**(µg/L)

Population size

Population growth rate (d–1)

Mortality (%)

Control

<0.01

<0.01

<0.01

<0.01

<0.01

7.4 ± 0.7

0.95 ± 0.07

6

5

2.8

0.7

<3.7

0.07

0.27

6.9 ± 0.4

0.95 ± 0.03

0

10

6.6

1.4

<3.7

0.12

0.52

6.1 ± 0.4

0.89 ± 0.04

0

20

20

4.5

<3.7

0.23

1.4

5.3 ± 0.4

0.82 ± 0.03

0

40

38

9.8

5.0

2.1

5.2

4.3 ± 0.4

0.70 ± 0.04

0

80

89

18

11

4.5

11

3.6 ± 0.2

0.62 ± 0.04

0

160

172

30

21

18

23

4.0 ± 0.4

0.65 ± 0.06

0

320

296

42

33

34

35

0.3 ± 0.2

0.02 ± 0.02

75

*Average of all replicates ± standard error is reported.

**Time-weighted average of dissolved Ag concentrations in fresh solutions at t 0 h (after addition of algae) and in old solutions at t 24 h (when available) and t 48  h.

Above table shows that a clear concentration–response behavior of population size and population growth rate was observed.

Corresponding effect concentrations are shown in the table below. The EC10 and EC50 values for population size are lower than for population growth rate, and NOEC and LOEC levels for both endpoints are similar.

Effect concentrations(expressed as the geometric mean of the measured dissolved Ag concentrations in fresh and old solutions )for the 48h-Brachionus calyciflorustest.

 

Endpoint

EC10

(μg diss. Ag/L)

EC20

(μg diss. Ag/L)

EC50

(μg diss. Ag/L)

NOEC

(μg diss. Ag/L)

LOEC

(μg diss. Ag/L)

Population size

0.31

(0.13-0.73)

1.0

(0.6-1.9)

8.2

(5.9-11.3)

0.27

6.8±7.8

0.52

17.0±8.6

Population growth rate

2.6

(1.5-4.7)

5.2

(3.5-7.7)

16.7

(13.5-20.7)

0.27

0.2±4.6

0.52

6.9±5.3

EC10, EC20 and EC50’s were calculated using a log-logistic concentration response model with 2 parameters. 95% confidential intervals are reported between parentheses.

NOEC and LOECs were calculated using the non-parametric Jonkhere-Terpstra-test. The average growth rate inhibition±standard error relative to the control (%) at the NOEC or LOEC are reported between brackets.

Validity criteria fulfilled:
yes
Remarks:
The average control population size at the end of the test was 7.4 ± 0.7 rotifers. Reproduction occurred in 94% of the control replicates, and the control population growth rate was 0.95 ± 0.07 d–1. As such, the test is considered valid.
Conclusions:
An EC10 of 0.31 μg dissolved Ag /L for the endpoint population size and an EC10 of 2.6 μg dissolved Ag /L for the endpoint population growth rate was found after 48h exposure of B. calyciflorus to silver nitrate.
Executive summary:

In a 48 hour study with the rotifer Brachionus calyciflorus exposed to silver nitrate, the EC10 for the most sensitive endpoint (population size) was 0.31 µg dissolved Ag/L.

This is a guideline study considered suitable for use as a key study for this endpoint.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Non GLP, guidelines study acceptable with restrictions
Qualifier:
according to guideline
Guideline:
other: ASTM standard guidelines for conducting D. magna life cycle test (ASTM 1997)
Deviations:
not specified
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:
yes
Details on sampling:
Exposure survival and reproduction in daphnids tested daily for 21 days.
Vehicle:
not specified
Details on test solutions:
Nominal range 0.75-23 μg Ag/L (AgNO3 stock solutions 1 - 10 mg/Ag/L) acidified with 1% HNO3. Test solutions pre-equilibrated for 3-hours prior to use. Moderately hard (MH) synthetic water was used for treatment.
Test organisms (species):
Daphnia magna
Details on test organisms:
Details on test organisms - source: ARO strain, Aquatic Research Organisms, Hampton, NH USA
Details on test organisms - holding conditions prior to test: Collected from a laboratory established culture. Maintained in moderately hard water (CaCO3 0.15 mM) similar to that of the Lake Ontario water. To ensure standardised conditions for water chemistry, synthetic water used for all tests prepared as a single batch employing 1000 L of reverse osmosis purified water in a good grad polyethylene tank. Daphnia fed algae and YCT. Water not aerated, experimental medium including food renewed daily. Temperature and photoperiod fixed at 20°C and 16:8 light:dark respectively. After acclimation, reproductive rate measured to ensure it met established criteria. Neonates <24h were collected for experiments using plastic pipettes.
Details on test organisms - life stage of test species used: <24 hr neonates
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
115 and 460 mg/L (high hardness test) CaCO3
Test temperature:
20°C
pH:
pH 8.16 ± 0.07
Dissolved oxygen:
Oxygen content: 90% saturation
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal range 0.75-23 μg Ag/L (AgNO3 stock solutions 1 - 10 mg Ag/L) acidified with 1% HNO3: 0.21, 0.69, 1.16, 1.98, 2.80, 2.95, 3.56, 3.98, 10.11, 21.41 µg/L dissolved silver.
Details on test conditions:
DOC 4.8 ± 1.3. Number of replicates per concentration: 10. Number of organisms per replicate: 1.
Reference substance (positive control):
not specified
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
2.7 µg/L
95% CI:
>= 2.2 - <= 3.4
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
2.9 µg/L
95% CI:
>= 2.8 - <= 3
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
3.6 µg/L
95% CI:
>= 3.4 - <= 3.7
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth
Remarks on result:
other: High hardness
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
3.1 µg/L
95% CI:
>= 2.3 - <= 4.3
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
reproduction
Remarks on result:
other: High hardness
Details on results:
The key EC10 for Daphnia magna is 2.7 µg/L for growth (most sensitive endpoint, low hardness).
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
EC10 values have been re-calculated using Graph Digitizer and log-logistic curve fitting (TRAP software). Dose response curves are reported.
Validity criteria fulfilled:
yes
Conclusions:
For D. magna EC10 value of 2.7 μg/L is calculated for the endpoint growth. For the endpoint reproduction, EC10 of 2.9 μg/L is calculated. All toxicity valus are expressed as dissolved Ag.
Executive summary:

This is a non-GLP, chronic toxicity study on Daphnia magna following ASTM guidelines for conducting D. magna life cycle tests. EC10 values of 2.7 μg/L for the endpoint growth and 2.9 μg/L for the endpoint reproduction are calculated. All toxicity valus are expressed as dissolved Ag.

The study is considered reliable and suitable for use for this endpoint.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, Non-guideline study. Available as a published paper. Acceptable with restrictions.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Aqueous only exposure with C. tentans. Well described. 10 day water-only (flow-through) exposure with larvae of chironomus tentans. Survival and growth endpoints relative to control treatment were recorded.
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:
yes
Details on sampling:
Days 0 and 10 of exposure (total, dissolved and free ionic silver)
Vehicle:
not specified
Details on test solutions:
Type and source of the exposure medium: Dechlorinated municipal water
Test organisms (species):
other aquatic arthropod: Chironomus tentans
Details on test organisms:
11 days old (2nd to 3rd instar). Source and holding conditions not reported.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
10 d
Post exposure observation period:
None
Hardness:
52.1 ± 4.8 mg/L
Test temperature:
23 ± 0.2 °C
pH:
7.4 ± 0.1
Dissolved oxygen:
7.7 ± 0.4 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 12.5, 25.0, 50.0, 100.0, 200.0 μg/L.
Measured concentrations (dissolved silver): <5 (control), <5, <5, 13.0, 66.0, 155.0 μg/L.
Details on test conditions:
Type and source of exposure medium: Dechlorinated municipal water that had been passed through an ion-exchange resin to remove metal ions. 300 mL high-form beakers containing a thin later of quartz sand. Renewal rate: 1.0 mL/min. Ten organisms per vessel. Two replicates per concentration. Feeding: 6.0 mg of tetrafin slurry per test vessel daily.
Reference substance (positive control):
not specified
Duration:
10 d
Dose descriptor:
EC10
Effect conc.:
12.54 µg/L
95% CI:
>= 10.4 - <= 15.1
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth
Duration:
10 d
Dose descriptor:
NOEC
Effect conc.:
13 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth
Details on results:
No data reported
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
NOEC and LOEC values determined using Anova followed by Bonferroni’s test. EC10 have been re-calculated using log-logistic curve fitting (TRAP software)
Validity criteria fulfilled:
not applicable
Remarks:
No applicable criteria but water quality appears acceptable.
Conclusions:
The 10 day EC10 (growth) of AgNO3 to Chironomus tentans was determined to be 12.54 µg dissolved Ag/L.
Executive summary:

In a non-GLP, non-guideline, test the 10 day EC10 (growth) of AgNO3 to Chironomus tentans was determined to be 12.54 µg dissolved Ag/L.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline available
Principles of method if other than guideline:
14-d growth study with Lymnaea stagnalis; study reports tests with Ag alone and tests with mixtures of Ag and other metals.
A range of concentrations of AgNO3 was tested.
Tests were terminated after 14 days, and snail wet weights were measured to the nearest 0.1 mg.
GLP compliance:
no
Remarks:
Publication, GLP compliance not followed
Specific details on test material used for the study:
Purity: ACS grade
Analytical monitoring:
yes
Details on sampling:
Samples for Ag analysis taken on days 1, 7 and 14 prior to and after the water change; samples filtered through a 0.45 µm membrane
Vehicle:
no
Remarks:
Publication, GLP compliance not specified
Details on test solutions:
Test medium: dechlorinated tap water amended with salts
Exposure solutions were prepared by spiking stock solutions of AgNO3 in 2 L of the same water used for snail culture. Water was kept in polypropylene containers and was constantly aerated
via a tubing system.
Number of test concentrations: 32 different Ag treatments with concentrations varying between 0.3 and 14.4 μg/L measured dissolved Ag and 18 controls were used. Metal addition was performed 24 h prior to the test initiation.
Test organisms (species):
Lymnaea stagnalis
Details on test organisms:
Age: juveniles, 3 weeks old (9.5 mg wet weight on average)
Source: in-house culture at McMaster University (Hamilton, ON); kept at University of British Columbia at 25 ± 1 °C, under a 16 h light/8 h dark photoperiod in Vancouver dechlorinated tap water amended with salts (to increase the water hardness of the very soft Vancouver water (from 3.3 to 120 mg/L CaCO3) to promote healthier snail culture conditions)
All organisms were acclimated to the test medium
Snails were fed ad libitum with fresh and thoroughly washed romaine lettuce
Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
14 d
Hardness:
116 mg/L CaCO3
Test temperature:
25°C
pH:
7.81 ± 0.20
Dissolved oxygen:
constantly aerated
Nominal and measured concentrations:
32 different Ag treatments with concentrations varying between 0.3 and 14.4 μg/L measured dissolved Ag and 18 controls were used. Metal addition was performed 24 h prior to the test initiation.
Details on test conditions:
Final water composition (as mean ± SD, n = 252): pH = 7.81 ± 0.20, [Ca] = 0.94 ± 0.01 mM, [Mg] = 0.22 ± 0.02 mM, [Na] = 1.7 ± 0.2 mM, [K] = 0.054 ± 0.015 mM, [Cl] = 1.0 mM (nominal), [SO4] = 0.79 mM (nominal), [dissolved organic carbon, DOC] = 0.76 ± 0.08 mg/L, and alkalinity = 0.80 ± 0.05 mEq·L−1 (measured as [dissolved inorganic carbon, DIC]).
Number of replicates: 1 replicate per treatment, 6 to 8 replicates per control
Volume of test vessels: 2 L test volume
5 snails per exposure container; exposure water renewed every day
Endpoints measured: growth rate (weight)
Key result
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
1.48 µg/L
95% CI:
>= 1.2 - <= 1.78
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth
Remarks:
growth rate (wet weight)
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
2.56 µg/L
95% CI:
>= 2.3 - <= 2.82
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth
Remarks:
growth rate (wet weight)
Details on results:
The EC10 for Ag of 13.7 nM (=1.48 µg/L) is reported in Table 1 of the study and was derived by compiling all of the different single-metal toxicity tests performed in this study (see also caption under Figure 1 of the study). Since conditions between the different single-metal toxicity tests were identical, it is justified to compile the results to derive a single EC10.
Dose response: dose response curves for growth rate (weight) based on free ion and dissolved Ag are reported
Toxicity value: the manuscript reports an EC10 of 1.48 (1.20-1.78), and EC20 of 1.81 (1.56-2.05) and an EC50 of 2.56 (2.3-2.82) μg/L as dissolved Ag for the endpoint relative growth rate (wet weight).
Reported statistics and error estimates:
EC10 values were calculated based on the reported dose-response graphs using a log-logistic function. Because the toxicity data analysis was regression-based, treatments had only one replicate to maximize their numbers (32 different Ag concentrations). This allows better coverage of the response curves and surfaces and, hence, increased reliability and power in the analysis.
Validity criteria fulfilled:
not applicable
Remarks:
Validity criteria based on control performance do not exist as this is not a standard test but initial and final growth (weight) is reported in the controls
Conclusions:
Reliable EC10 of 1.48 μg/L (growth rate) could be extracted for L. stagnalis as dissolved Ag.
Executive summary:

Reliable EC10 of 1.48 μg/L (growth rate) could be extracted for L. stagnalis as dissolved Ag.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP unknown, comparable to guideline study, acceptable with restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
other: 14 d exposure
Deviations:
not applicable
Principles of method if other than guideline:
The study presents the results of a subacute toxicity test with the mayfly (Stenonema modestum). A series of effluent toxicity tests were conducted, each consisting of a 14 day exposure using a static-renewal test mode.
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:
yes
Details on sampling:
No data reported
Vehicle:
not specified
Details on test solutions:
Type and source of the exposure medium (same as holding conditions): acclimated from stream to filtered Blacksburg, VA municipal water at a temperature of 12ºC, filtered through an activated carbon, 0.2 µm ceramic filter.
Test organisms (species):
other: Stenonema modestum
Details on test organisms:
Source: Field collected larvae (collected from small streams outside of urban or industrial influences)
Holding conditions prior to test: Acclimated from stream to filtered Blacksburg, VA municipal water and a temperature of 12ºC.
Life stage of test species used: Larvae (4 – 6 mm length excluding tail; head capsule width 1 – 1.2 mm)
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
14 d
Post exposure observation period:
None
Hardness:
48.5 mg CaCO3/L
Test temperature:
12±1ºC
pH:
7.7
Dissolved oxygen:
Slow aeration provided
Salinity:
Not applicable
Nominal and measured concentrations:
0, 0.9, 1.84, 3.4, 6.6 and 12.9 μg total Ag (measured)/L
0, 0.5, 1.0, 2.2, 3.7 and 5.8 μg dissolved Ag (measured)/L
Details on test conditions:
Aeration: 10-20 mL/min (all chambers received slow aeration throughout the test to supply the necessary water current for larval respiration)
Renewal rate: Renewal 3 times per week
Number of organisms per vessel: 10
Volume of test vessels: 500 mL test volume using 800 mL beakers
Number of replicates per concentration: 3
DOC: not reported
Reference substance (positive control):
not specified
Key result
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
1 µg/L
Nominal / measured:
estimated
Conc. based on:
element (dissolved fraction)
Basis for effect:
other: molting
Remarks on result:
other: dissolved Ag calculated based on mean recovery between unfiltered and filtered samples
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
4.1 µg/L
95% CI:
>= 2.5 - <= 6.9
Nominal / measured:
estimated
Conc. based on:
element (dissolved fraction)
Basis for effect:
mortality
Remarks on result:
other: dissolved Ag calculated based on mean recovery between unfiltered and filtered samples
Details on results:
Survival NOEC of 3.7 µg/L and survival LC10 of 4.1 µg/L (dissolved Ag).
Molting NOEC of 1 µg/L and molting EC10 of 0.31 µg/L (CI: 0.003-37.1) (dissolved Ag); EC10 for molting not selected because of the too wide confidence interval.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Larval survival was analysed at 7 and 14 days to yield NOECs using ANOVA and Dunnett’s Test. Survival data were transformed (arc-sine square root) before analysing by using a significance level of 0.05. Growth was analysed by examining 3 endpoints: A) molt or exuviae production at 7 and 14 days, B) body length after 14 days of exposure and C) head capsule width after 14 days exposure. Differences in the number of molts between treatments and controls were analysed by ANOVA and Dunnett’s test at a p value of 0.05. Treatments were compared to each other with either ANOVA and Tukey’s HSD test (if data satisfied parametric test assumptions) or Kruskal-Wallis nonparametric ANOVA and multiple means test. Differences in final body length and head capsule width between controls and treatments at 14 days were analysed with ANOVA and Bonferroni’s t test (p at 0.05) because these data met parametric test assumptions.
EC10 values were calculated based on the reported dose-response data using a logistic func-tion (TRAP software).
Validity criteria fulfilled:
not applicable
Remarks:
80% survival after 14 d in the control
Conclusions:
Reliable LC10/NOEC could be extracted, i.e. the LC10 of 4.1 μg/L for the endpoint mortality and the NOEC of 1.0 μg/L for the endpoint molting, as dissolved Ag.
Executive summary:

The study presents the results of a subacute toxicity test with the mayfly (Stenonema modestum). A series of effluent toxicity tests were conducted, each consisting of a 14 day exposure using a static-renewal test mode.

Reliable LC10/NOEC could be extracted, i.e. the LC10 of 4.1 μg/L for the endpoint mortality and the NOEC of 1.0 μg/L for the endpoint molting, as dissolved Ag.

The study is considered to be reliable and suitable for use for this endpoint.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP unknown, guideline unknown, acceptable with restrictions
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
14 day sublethal effects on representative or indigenous species of New River, Virgina using Resident Species Approach. Minimum methodology given in paper, references given.
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:
yes
Details on sampling:
At start, sample renewal and end of test from surface of exposure chambers, using composite samples from low, medium and highest concentrations. Concentrations not measured were adjusted according to calculated mean recovery rate. Portion of sample filtered through 0.45 micron glass fiber filter for determination of dissolved silver. Total silver determined from unfiltered sample.
Vehicle:
not specified
Details on test solutions:
Type and source of the exposure medium: New River water, Virginia, USA (2 out of 8 samples had background Ag concentration of 0.1 µg/L)
Test organisms (species):
other: Isonychia bicolor
Details on test organisms:
Source: Field collected from unimpacted sites in the New River of tributaries
Holding conditions prior to test: Acclimated to New River water over a 2 - 4 day period. Temperature of 20±2ºC.
Life stage of test species used: < 4 instar (2 - 5 mm)
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
14 d
Post exposure observation period:
None
Hardness:
35.2 mg CaCO3/L
Test temperature:
20±2ºC
pH:
7.7
Dissolved oxygen:
No data reported
Salinity:
Not applicable
Nominal and measured concentrations:
<0.1, 0.3, 1.6, 3.1, 9.3 and 31 μg/L.
The actual mean concentration of silver measured in specific test chambers was often lower than the theoretical value. For each treatment in the test, the authors took the mean measured concentration and divided this mean by the theoretical silver concentration to compute a mean percent recovery rate. This recovery rate was then used to calculate the mean exposure silver concentration for each treatment in a given test.
Details on test conditions:
Renewal rate: Every 48 h
Number of organisms per vessel: 10
Number of replicates per concentration: 2
Feeding: Conditioned alder and maple leaves provided for food and structure
Reference substance (positive control):
not specified
Key result
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
0.16 µg/L
Nominal / measured:
estimated
Conc. based on:
element (dissolved fraction)
Basis for effect:
other: molting
Remarks on result:
other: dissolved Ag calculated based on mean recovery between unfiltered and filtered samples
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
1.02 µg/L
95% CI:
>= 0.18 - <= 5.71
Nominal / measured:
estimated
Conc. based on:
element (dissolved fraction)
Basis for effect:
other: molting
Remarks on result:
other: dissolved Ag calculated based on mean recovery between unfiltered and filtered samples
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
1.67 µg/L
Nominal / measured:
estimated
Conc. based on:
element (dissolved fraction)
Basis for effect:
mortality
Remarks on result:
other: dissolved Ag calculated based on mean recovery between unfiltered and filtered samples
Details on results:
The 14 day NOEC for Isonychia bicolor is 0.16 µg/L.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Dissolved Ag calculated based on mean recovery between unfiltered and filtered samples.
NOEC and LOEC values determined using Anova followed by Dunnett’s test, Tukey’s test or Kruskal-Wallis test. EC10 values were calculated based on the reported dose-response graphs using a logistic function (TRAP software).
Validity criteria fulfilled:
not applicable
Conclusions:
The 14 day NOEC for Isonychia bicolor is 0.16 µg/L.
Executive summary:

Phys-chem in the tests (DOC) is not reported; Estimation of dissolved Ag is based on a mean recovery rate, therefore not considering differences among measurements between samples; Reliable NOEC of 0.16 μg/L (molting) and 1.67 μg/L (mortality) could be extracted for I. bicolor as dissolved Ag. The EC10value for molting of I. bicolor could not be retained because of the too wide (> factor of 10) confidence interval.

Although not a traditional endpoint, molting is indicative of effects on growth and valid for use as part of the overall dataset.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP unknown, guideline unknown, acceptable with restrictions
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Juvenile Corbicula were exposed in a 21 day static-renewal growth test. Minimum methodology given in paper, references given.
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:
yes
Details on sampling:
At start, sample renewal and end of test from surface of exposure chambers, using composite samples from low, medium and highest concentrations.
Vehicle:
not specified
Details on test solutions:
Type and source of the exposure medium: New River water, Virginia, USA (2 out of 8 samples had background Ag concentration of 0.1 µg/L)
Test organisms (species):
other: Corbicula fluminea
Details on test organisms:
Source: Field collected from unimpacted sites in the New River of tributaries
Holding conditions prior to test: Acclimated to New River water over a 2 - 4 day period. Temperature 20±2ºC.
Life stage of test species used: Juveniles (3 - 5 mm)
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
35.2 mg CaCO3/L
Test temperature:
20±2ºC
pH:
7.7
Dissolved oxygen:
No data reported
Salinity:
Not applicable
Nominal and measured concentrations:
Test concentrations used (mean values): <0.1, 0.3, 1.3, 2.6, 7.8, 26.1 µg/L
Details on test conditions:
Renewal rate: Every 48 h
Number of organisms per vessel: 10
Number of replicates per concentration: 2
Feeding: TetraMin flakes & algal mix daily
Observations: At test termination, the number of surviving clams, individual weight and shell diameter were measured and analysed. The number of survivors was verified by transferring closed individuals to control water for 96 hours. If these individuals remained closed through this recovery period, they were considered to be dead.
Reference substance (positive control):
not specified
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1.46 µg/L
Nominal / measured:
estimated
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth
Remarks on result:
other: dissolved Ag calculated based on mean recovery between unfiltered and filtered samples
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.84 µg/L
95% CI:
>= 0.34 - <= 2.13
Nominal / measured:
estimated
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth
Remarks:
(weight)
Remarks on result:
other: dissolved Ag calculated based on mean recovery between unfiltered and filtered samples
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
4.4 µg/L
Nominal / measured:
estimated
Conc. based on:
element (dissolved fraction)
Basis for effect:
mortality
Remarks on result:
other: dissolved Ag calculated based on mean recovery between unfiltered and filtered samples
Details on results:
21 day NOEC growth (weight and length) is 2.6 μg total silver/L and the 21 day EC10 for growth (weight) is 1.68 µg/L.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Dissolved Ag calculated based on mean recovery between unfiltered and filtered samples.
NOEC and LOEC values determined using Anova followed by Dunnett’s test, Tukey’s test or Kruskal-Wallis test. EC10 values were calculated based on the reported dose-response graphs using a logistic function (TRAP software).
Validity criteria fulfilled:
yes
Conclusions:
The 21 day EC10 for growth (weight) is 0.84 µg/L.
Executive summary:

This study is a non-GLP, non-guideline study in which juvenile Corbicula were exposed in a 21 day static-renewal growth test.

Phys-chem in the tests (DOC) is not reported; Estimation of dissolved Ag is based on a mean recovery rate, therefore not considering differences among measurements between samples; ReliableEC10/NOEC of 0.84 μg/L (growth) and 4.4 μg/L (mortality) could be extracted as dissolved Ag.

The study is considered to be reliable and suitable for use for this endpoint.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP unknown, guideline, experimental study, acceptable with restrictions
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: ASTM guideline
Principles of method if other than guideline:
Aquatic Invertebrate Bioassays STP 715 American Society for Testing and Materials; Aquatic Toxicology and Hazard Assessment STP 766; Toxicity testing with freshwater macroinvertebrates; Methods and application in environmental management
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:
yes
Details on sampling:
Observations of organism survival and behaviour made during first 6 hour of test and at 24 hour intervals. Dissolved oxygen, temperature, pH and conductivity measured initially and at 24 hour intervals.
Vehicle:
not specified
Details on test solutions:
Stock solution of AgNO3 1 g/L made from distilled deionized water. Test solutions made from stock solutions diluted with New River water.
Test organisms (species):
other: Hyalella azteca juveniles
Details on test organisms:
Obtained from Carolina Biological Supply Company. Acclimatised to New River water for 4 to 8 hours. Maintained under 16:8 light: dark photoperiod. Life stage: 2-5mm (acute test), 6-8 mm (adults in sublethal test).
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
35.2 mg/L as CaCO3
Test temperature:
20°C
pH:
Average: 7.7 ± 0.2. Range: 7.2 - 8.2.
Dissolved oxygen:
No data reported
Salinity:
Not applicable
Nominal and measured concentrations:
Acute test: <0.1, 1.3, 2.5, 6.4, 12.7, 25.5
Sublethal test: <0.1, 0.2, 0.5, 1.0, 1.9
At least 5 concentrations plus a control (two replicates of each) used in all tests
Details on test conditions:
500 mL pyrex dish.
Acute test: 4 organisms per chamber, 20 per concentration
Sublethal test: 4 organisms per chamber, 20 per concentration. Adult reproduction and survival assessed for each concentration at end of day 21.
Reference substance (positive control):
not specified
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.59 µg/L
Nominal / measured:
estimated
Conc. based on:
element (dissolved fraction)
Remarks:
silver
Basis for effect:
mortality
Remarks on result:
other: dissolved Ag calculated based on mean recovery between unfiltered and filtered samples
Details on results:
Authors state that they are unable to obtain valid sublethal reproduction value due to insufficient number of offspring in test. No results included for acute tests.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
NOEC value determined using Anova followed by Dunnett’s test, Tukey’s test or Kruskal-Wallis test.
Validity criteria fulfilled:
not applicable
Conclusions:
The 21 day survival NOEC is 0.59 µg dissolved Ag/L.
Executive summary:

Phys-chem in the tests (DOC) is not reported; Estimation of dissolved Ag is based on a mean recovery rate, therefore not considering differences among measurements between samples; Reliable NOEC of 0.59 μg/L (mortality) could be extracted for H. azteca as dissolved Ag.

The study is considered to be reliable and suitable for use for this endpoint.

Endpoint:
long-term toxicity to aquatic invertebrates
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 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
The test item solution was prepared in modified Elendt M7 medium
Qualifier:
according to guideline
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
The test item solution was prepared in modified Elendt M7 medium
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: May 2017
- Purity test date: Not reported

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep tightly closed, cool and dry, in a well-ventilated place.
- Stability under test conditions: Stable under normal storage conditions.

Analytical monitoring:
yes
Details on sampling:
-Sampling: To assess the test item concentrations and the release of silver ions from the test item ‘truly’ and ‘conventional’ dissolved silver was measured by chemical analysis at test initiation, after each medium renewal and test termination. Silver was measured at test initiation, after each medium renewal and test termination, in test media samples of each treatment taken directly from test vessels (total silver), after centrifugal filtration (for ‘truly’ dissolved silver) and after filtration through 0.45 µm membrane filters (for ‘conventional’ dissolved silver).

Additionally, the particle size and the zeta potential was measured from samples of an extra analytical vessel without daphnia and algal food to characterize the test item in test media once per week.

Differentiation between nanoparticles and ions
For differentiation of particle and ions (’truly dissolve silver) the sampling of the first 10 mL of the sample was performed in special centrifugal vials (Vivaspin 20, Sartorius, 3kDa). The samples for the further pre-conditioning and the measurement were taken separately. The samples then were centrifugated at 3000 x g for 1 hour at 20 °C stepwise. In addition a separate sample was filtered through 0.45 µm polyethersulfone membrane filter for determination of the ‘conventional’ dissolved silver after precondition of the filter with an adequate amount of the sample. The determination of ‘conventional’ and ‘truly’ dissolved silver for the control and each treatment was performed at test initiation, after each medium renewal and at test termination in test vessels and once per week also in additional vessels (no Daphnia or algal food).

- Sample storage conditions before analysis: All samples for the test item concentration measurements were acidified with HNO3 (1.25 mL concentrated HNO3) and stored in a refrigerator (about 4 °C) until further analysis. The particle size was measured in 2 mL and the zeta potential in 1 mL of an aliquot of the control and the five treatments as soon as possible after sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item solution was prepared in modified Elendt M7 medium. Initially, a stock solution was prepared by pipetting an adequate amount of the test item, and transferring it to purified water. Directly after addition of the test item the stock dispersion was sonicated for 3 minutes in a sonication bath (indirect sonication; frequence: 35 kHz) and then the dispersion was shaken carefully.
The test concentrations were prepared by adding 26.4, 52.8, 105.6, 211.2 and 422.4 µL of stock solution into modified Elendt M7 medium and the solution was sonicated for 3 minutes in a sonication bath (frequence: 35 kHz) and then shaken carefully. The freshly prepared test solutions were then left to stand for 2 hours to reach equilibrium. Following equilibration, the test solutions were filled into the test vessels after more careful shaking.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Not reported
- Source: bred in the testing facility, originally from German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene.
- Age of parental stock (mean and range, SD): at least 3 weeks old
- Feeding during test: yes
- Food type: The daphnids were fed during the test with a mixture of suspensions of unicellular alga Desmodesmus subspicatus and JBL ArtemioFluid in a ratio of 9 : 1 (vol/vol).
- Amount: The content of alga food in the test media, measured at 585 nm, was kept constant at 0.2 mg C/(Daphnia x day).

ACCLIMATION
- Acclimation period: at least three weeks
- Acclimation conditions: No, daphnids were gradually acclimated to the modified Elendt M7 medium. First batch of daphnids was prepared by moving from purified drinking water to 33 %, 66 % and 100 % of the modified Elendt M7 mediumover a three week period. Second batch was acclimated by moving the daphnids from purified drinking water to 50 % and 100 % of the modified Elendt M7 medium.The gradual acclimation was accelerated based on the findings of the first acclimation period.
- Health during acclimation (any mortality observed): The first batch animals appeared to be less vital and a higher mortality of adult and juvenile animals was observed in the culture. Therefore, a new culture was established from animals held in Cu-free dilution water (second batch). No mortalities was observed in the second batch culture.


Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
Modified Elendt M7 medium: 2.2 mmol/L (Ca-hardenss)
Overlaying water: 2.7 - 2.8 mmol/L (fresh), 2.6 - 2.9 mmol/L (old)
Test temperature:
Temperature of the overlaying water: 21.0 - 21.9 °C
pH:
7.94 – 9.37
Dissolved oxygen:
8.1 - 11.3 mg/L
Salinity:
Not applicable
Conductivity:
255.0 µS/cm
Nominal and measured concentrations:
Nominal: 1.5, 3.0, 6.0, 12.0 and 24.0 µg Ag/L
Arithmetic mean measured concentrations of total Ag: 0.95, 1.99, 4.08, 8.33 and 18.8 µg Ag/L
Arithmetic mean measured conventional dissolved Ag concentrations: 0.61, 1.27, 2.52, 5.03 and 10.3 µg Ag/L
Arithmetic mean measured concentrations of truly dissolved Ag: 0.020, 0.024, 0.057, 0.163 and 0.269 µg Ag/L
Details on test conditions:
TEST SYSTEM
- Test vessel: polypropylene beakers (50 mL nominal volume) cleaned as per standard cleaning methods to remove any deposits derived from the manufacturing process or previous use, and then were cleaned with a HNO3 solution (11.5 mL of concentrated HNO3 per L of pure water) and rinsed thoroughly with ultrapure water.
- Material, size, headspace, fill volume: The containers were filled up with about 50 mL test solution and covered with glass panes to prevent evaporation as much as possible, but also permit gaseous exchange
- Aeration: no
- Renewal rate of test solution: Test dispersion was exchanged three times a week.
- No. of organisms per vessel: 1 young female Daphnia per vial
- No. of vessels per concentration (replicates): 10 (2 additional replicates per treatment and control were prepared similar to the test vessels (with daphnia and algal food) for chemical analysis purposes only; and 2 additional replicate without daphnia and algal food for the measurement of particle size and zeta potential (and additional chemical analysis)).
- No. of vessels per control (replicates): 10 (2 additional replicates per treatment and control were prepared similar to the test vessels (with daphnia and algal food) for chemical analysis purposes only; and 2 additional replicate without daphnia and algal food for the measurement of particle size and zeta potential (and additional chemical analysis)).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic, modified Elendt M7 medium. To avoid reactions of the silver with chloride (Cl-), the trace elements and macro nutrients containing Cl- were replaced by suitable elements containing nitrate (NO3-) instead of Cl¬-. EDTA was completely excluded from the test media.
- Culture medium different from test medium: yes

OTHER TEST CONDITIONS
- Photoperiod: light/dark cycle of 16/8 hours
- Light intensity: 747 – 825 lux (corresponding to 10.0 – 11.0 µE/(m² *s))

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The numbers of immobile daphnids were visually determined and any abnormalities in appearance and behaviour were recorded daily. The newborn daphnids per beaker were counted and removed daily until all daphnids start to reproduce (at least 10 days and at a maximum of 14 days). After that newborn daphnids were counted and removed at each water renewal. Abnormalities in condition (including male sex) or presence of winter eggs were checked and recorded. At study termination, length of the adults without significant mortality was measured by digital photography and image analysis and compared with the equally measured control animals.
The following endpoints were evaluated quantitatively: Immobility of parental generation daphnids, Time to the first brood, Cumulative number of live offspring at day 21 (study end), Individual length of adults

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations range finding study: three nominal test concentrations of 0.1, 1.0 and 10 µg Ag/L
- Results used to determine the conditions for the definitive study: There was a significant effect on the reproduction of Daphnia magna after 21 days of exposure to the test item silver nitrate. The EC10 for reproduction per introduced adult Daphnia was calculated to be 0.1 µg Ag/L and the EC50 was calculated to be 19.8 µg Ag/L
- Test concentrations definitive study: nominal test concentrations of 1.5, 3.0, 6.0, 12.0 and 24.0 µg Ag/L
Reference substance (positive control):
yes
Remarks:
An acute immobilization tests over 24 h was performed with K2Cr2O7 (separate study, January 2017).
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
5.37 µg/L
95% CI:
>= 2.89 - <= 7.12
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (total fraction)
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
12.01 µg/L
95% CI:
>= 9.79 - <= 14.95
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (total fraction)
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
4.08 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (total fraction)
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
8.33 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (total fraction)
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
3.49 µg/L
95% CI:
>= 1.98 - <= 4.49
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
reproduction
Remarks on result:
other: based on conventional dissolved Ag
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
6.97 µg/L
95% CI:
>= 5.81 - <= 8.44
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
reproduction
Remarks on result:
other: based on conventional dissolved Ag
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
5.03 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
reproduction
Remarks on result:
other: based on conventional dissolved Ag
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
2.52 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
reproduction
Remarks on result:
other: based on conventional dissolved Ag
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.059 µg/L
95% CI:
>= 0.022 - <= 0.088
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Basis for effect:
reproduction
Remarks on result:
other: Based on truly dissolved Ag
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.195 µg/L
95% CI:
>= 0.151 - <= 0.266
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Basis for effect:
reproduction
Remarks on result:
other: Based on truly dissolved Ag
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.163 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Basis for effect:
reproduction
Remarks on result:
other: Based on truly dissolved Ag
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.057 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Basis for effect:
reproduction
Remarks on result:
other: Based on truly dissolved Ag
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
3.97 µg/L
95% CI:
>= 1.7 - <= 5.4
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
immobilisation
Remarks on result:
other: based on conventional dissolved Ag
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
6.4 µg/L
95% CI:
>= 4.8 - <= 9.4
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth
Remarks:
length
Remarks on result:
other: based on conventional dissolved Ag
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
5 µg/L
95% CI:
>= 1.7 - <= 14.6
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
other: intrinsic rate
Remarks on result:
other: based on conventional dissolved Ag
Details on results:
- Observations on body length and weight: Adult body length showed significant differences between control and the two highest treatments.
- Other biological observations: There was a significant difference in survival of individuals between control and the highest treatments with silver nitrate. Thus, a concentration relating mortality of the adults was observed in the treatment with silver nitrate. No other clinical signs were observed in any replicate at any concentration tested. Neither any physical nor pathological symptoms were obtained.
- Mortality of control: There was 8% mortality in control.
- Particle size and zeta potential: The measurements of size and zeta potential revealed at all test concentrations that the chosen concentrations, which represented an optimal range for the growth test, were not sufficient for the size and zeta potential analysis. Especially the count rate of the size measurements indicated that the test concentrations were too low.
Results with reference substance (positive control):
24 h EC50 value (immobilization): 1.088 mg/L (95% CL: 0.632 – 1.841 mg/L)

Reported statistics and error estimates:
A LOEC was calculated by using ANOVA followed by Student-t test, Fisher's Exact Binomial test, Dunnett’s or Williams’ test or an appropriate non-parametric test. If no effect was observed up to the highest tested concentration the NOEC values were determined using appropriate statistical methods.
Since the test results of the reproduction test show a concentration-response relationship they were statistically analysed to determine an EC50, EC20 and EC10 value together with 95 % confidence intervals using Probit-analysis assuming log-normal distribution of the values.
Statistical calculations were done with the computer program ToxRat® Pro 3.2.1.

 Effective concentrations based on total Ag

Table 1: Percent reduction of survival, length and reproduction compared to controls after21 days.

Mean measured
total Ag
[µg Ag/L]

% reduction of survival (immobility)

% reduction of reproduction per introduced parent

% minimum detectable difference of age of first production

% decrease in length

% inhibition of intrinsic rate r

Control

8.3

0

0

0

0

0.95

16.7 (-)

6.5 (-)

7.5 (-)

-2.3 (-)

-1.6 (-)

1.99

0 (-)

1.0 (-)

7.8 (+)

1.0 (-)

3.8 (-)

4.08

0 (-)

5.5 (-)

7.9 (+)

2.1 (-)

6.6 (-)

8.33

25.0 (-)

22.7 (+)

8.0 (+)

6.2 (+)

5.4 (-)

18.8

 66.7 (+)

79.0 (+)

9.7 (+)

15.9 (+)

36.3 (+)

(+) statistically significant difference between controls / (-) no significant difference between controls and treatments; Cochran-Armitage (survival), Williams Multiple Sequential t-test Procedure (reproduction, age of first reproduction and intrinsic rate), Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm (Length); significance level 0.05, one-sided smaller.

 

 Table 2: Effective concentrations based on mean measured total Ag concentrations for the exposure of Daphnia magna for 21 days.

Mean measured test item concentrations [µg/L] - total Ag

Parametera

 

EC10

EC20

EC50

LOEC

NOEC

Immobility

Value

6.483

8.421

13.891

18.8

8.33

 

95 %-cl lower

2.613

10.120

10.120

 

 

 

95 %-cl upper

9.076

23.179

23.179

 

 

Reproduction

Value

5.37

7.08

12.01

8.33

4.08

 

95 %-cl lower

2.89

4.56

9.79

 

 

 

95 %-cl upper

7.12

8.85

14.95

 

 

Age of first reproduction

Value

0.0

0.0

0.0

1.99

0.95

 

95 %-cl lower

0.0

0.0

0.0

 

 

 

95 %-cl upper

0.0

0.0

0.0

 

 

Length

Value

10.93

31.90b

247.59b

18.8

8.33a

 

95 %-cl lower

8.05

20.04b

89.65b

 

 

 

95 %-cl upper

16.40

87.10b

n.d.

 

 

Intrinsic rate r

Value

9.53

13.18

24.51b

18.8

8.33

 

95 %-cl lower

0.06

1.30

18.88b

 

 

 

95 %-cl upper

13.27

16.83

n.d.

 

 

n.d.: not determined
a: The NOEC for length was calculated to be at 40.8 µg Ag/L based on arithmetic mean measured concentrations of total Ag. However, due to the low inhibition of 6.2% at a concentration of 8.33 µg Ag/L, the NOEC was set to 8.33 µg Ag/L, since effects below 10 % compared to control are generally not considered to be ecotoxicologically relevant and it is generally recommended by OECD and EFSA to use the EC10approach in preference to the NOEC approach for the environmental risk assessment .

b: The value is above the highest test concentration of 18.8 µg Ag/L and therefore extrapolated.

Effective concentrations based on conventional dissolved Ag

 

Table 3: Percent reduction of survival, length and reproduction compared to controls after 21 days.

Mean measured
conventional dissolved Ag
[µg Ag/L]

% reduction of survival (immobility)

% reduction of reproduction per introduced parent

% minimum detectable difference of age of first production

% decrease in length

% inhibition of intrinsic rate r

Control

8.3

0

0

0

0

0.612

16.7 (-)

6.5 (-)

7.5 (-)

-2.3 (-)

-1.6 (-)

1.270

0 (-)

1.0 (-)

7.8 (+)

1.0 (-)

3.8 (-)

2.520

0 (-)

5.5 (-)

7.9 (+)

2.1 (-)

6.6 (-)

5.030

25.0 (-)

22.7 (+)

8.0 (+)

6.2 (+)

5.4 (-)

10.30

 66.7 (+)

79.0 (+)

9.7 (+)

15.9 (+)

36.3 (+)

(+) statistically significant difference between controls / (-) no significant difference between controls and treatments; Cochran-Armitage (survival), Williams Multiple Sequential t-test Procedure (reproduction, age of first reproduction and intrinsic rate), Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm (Length); significance level 0.05, one-sided smaller.

Table 4: Effective concentrations based on mean measured conventional dissolved Ag concentrations for the exposure of Daphnia magna for 21 days.

Mean measured test item concentrations [µg/L] – conventional dissolved Ag

Parametera

 

EC10

EC20

EC50

LOEC

NOEC

Immobility

Value

3.968

5.020

7.872

10.3

5.03

 

95 %-cl lower

1.651

2.776

5.900

 

 

 

95 %-cl upper

5.393

6.632

12.525

 

 

Reproduction

Value

3.49

4.43

6.97

5.03

2.52

 

95 %-cl lower

1.98

2.96

5.81

 

 

 

95 %-cl upper

4.49

5.40

8.44

 

 

Age of first reproduction

Value

0.0

0.0

0.0

1.27

0.612

 

95 %-cl lower

0.0

0.0

0.0

 

 

 

95 %-cl upper

0.0

0.0

0.0

 

 

Length

Value

6.35

17.33b

118.47b

10.3

5.03

 

95 %-cl lower

4.76

11.14b

44.99b

 

 

 

95 %-cl upper

9.37

46.36b

n.d.

 

 

Intrinsic rate r

Value

5.76

7.60

12.90b

10.3

5.03

 

95 %-cl lower

n.d.

0.18

10.29b

 

 

 

95 %-cl upper

n.d.

9.41

n.d.

 

 

n.d.: not determined
a:The NOEC for length was calculated to be at 2.52 µg Ag/L based on arithmetic mean measured concentrations of conventional dissolved Ag. However, due to the low inhibition of 6.2% at a concentration of 5.03.6 µg Ag/L, the NOEC was set to 5.03 µg Ag/L, since effects below 10 % compared to control are generally not considered to be ecotoxicologically relevant and it is generally recommended by OECD and EFSA to use the EC10 approach in preference to the NOEC approach for the environmental risk assessment .

b: The value is about the highest test concentration of 10.3 µg Ag/L and therefore extrapolated.

 

 Effective concentrations based on truly dissolved Ag

 

Table 5: Percent reduction of survival, length and reproduction compared to controls after 21 days.

Mean measured
truly dissolved Ag
[µg Ag/L]

% reduction of survival (immobility)

% reduction of reproduction per introduced parent

% minimum detectable difference of age of first production

% decrease in length

% inhibition of intrinsic rate r

Control

8.3

0

0

0

0

0.020

16.7 (-)

6.5 (-)

7.5 (-)

-2.3 (-)

-1.6 (-)

0.024

0 (-)

1.0 (-)

7.8 (+)

1.0 (-)

3.8 (-)

0.057

0 (-)

5.5 (-)

7.9 (+)

2.1 (-)

6.6 (-)

0.163

25.0 (-)

22.7 (+)

8.0 (+)

6.2 (+)

5.4 (-)

0.269

 66.7 (+)

79.0 (+)

9.7 (+)

15.9 (+)

36.3 (+)

(+) statistically significant difference between controls / (-) no significant difference between controls and treatments; Cochran-Armitage (survival), Williams Multiple Sequential t-test Procedure (reproduction, age of first reproduction and intrinsic rate), Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm (Length); significance level 0.05, one-sided smaller.

Table 6: Effective concentrations based on mean measured truly dissolved Ag concentrations for the exposure of Daphnia magna for 21 days.

Mean measured test item concentrations [µg/L] – truly dissolved Ag

Parametera

 

EC10

EC20

EC50

LOEC

NOEC

Immobility

Value

0.126

0.153

0.221

0.269

0.163

 

95 %-cl lower

0.010

0.030

0.161

 

 

 

95 %-cl upper

0.169

0.195

0.389

 

 

Reproduction

Value

0.059

0.089

0.195

0.163

0.057

 

95 %-cl lower

0.022

0.046

0.151

 

 

 

95 %-cl upper

0.088

0.120

0.266

 

 

Age of first reproduction

Value

0.0

0.0

0.0

0.024

0.020

 

95 %-cl lower

0.0

0.0

0.0

 

 

 

95 %-cl upper

0.0

0.0

0.0

 

 

Length

Value

0.188

0.626b

n.d.

0.269

0.163a

 

95 %-cl lower

0.131

0.348b

n.d.

 

 

 

95 %-cl upper

0.332

2.798b

n.d.

 

 

Intrinsic rate r

Value

0.159

0.207

0.344 b

0.269

0.163

 

95 %-cl lower

n.d.

n.d.

n.d.

 

 

 

95 %-cl upper

n.d.

n.d.

n.d.

 

 

n.d.: not determined
a:The NOEC for length was calculated to be at 0.057 µg Ag/L based on arithmetic mean measured concentrations of truly dissolved Ag. However, due to the low inhibition of 6.2% at a concentration of 0.163 µg Ag/L, the NOEC was set to 0.163 µg Ag/L, since effects below 10 % compared to control are generally not considered to be ecotoxicologically relevant and it is generally recommended by OECD and EFSA to use the EC10 approach in preference to the NOEC approach for the environmental risk assessment .

b: The value is about the highest test concentration of 0.269 µg Ag/L and therefore extrapolated.

 

Validity criteria fulfilled:
yes
Remarks:
Mortality in controls was 8%; mean number of offspring in the control was 101/ female; dissolved O2 was > 3 mg/L; pH was within the range 6 - 9, and did not vary by more than 1.5 units; CV for the mean number of control offspring was 28.2 %.
Conclusions:
Based on mean measured total Ag concentrations the EC10 was 5.37 and 6.48 µg Ag/L for reproduction and immobility, respectively. The NOECs were 4.08 and 8.33 µg Ag/L for reproduction and immobility, respectively and LOECs were 8.33 and 18.80 µg Ag/L for reproduction and immobility, respectively.
Based on mean measured conventional dissolved Ag concentrations the EC10 was 3.49 and 3.97 µg Ag/L for reproduction and immobility, respectively. The NOECs were 2.52 and 5.03 µg Ag/L and LOECs were 5.03 and 10.30 µg Ag/L for reproduction and immobility, respectively.
Based on mean measured truly dissolved Ag concentrations the EC10 was 0.059 and 0.126 µg Ag/L for reproduction and immobility, respectively. The NOECs were 0.057 and 0.163 µg Ag/L and LOECs were 0.163 and 0.269 µg Ag/L for reproduction and immobility, respectively.

Executive summary:

The 21 day toxicity of silver nitrate to the test organism Daphnia magna was determined in a study according to the OECD 211 guideline. Daphnids were exposed to nominal concentrations of 1.5, 3.0, 6.0, 12.0 and 24.0 µg Ag/L in a semi-static system with renewal of test medium three times per week. The nominal test concentrations were prepared in modified Elendt M7 medium. The concentrations of the test item in the test media were determined by chemical analysis in the aqueous phase of all treatment levels by ICP-MS. The results were based on the arithmetic mean measured concentrations of total Ag, conventional dissolved Ag and truly dissolved Ag.

For PNEC derivation, reliable EC10 for D. magna on the endpoint immobility of 3.97 μg/L, on reproduction of 3.5 μg/L, on length of 6.4 μg/L, and on intrinsic rate of 5.0 μg/L could be extracted from this study. All data are expressed as conventional dissolved Ag.

This is a guideline, GLP- study and considered suitable for use as a key study for this endpoint.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study - reliable without restriction
Qualifier:
according to guideline
Guideline:
other: US-EPA 2002 Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms (EPA 821/R-02/013)
Deviations:
not specified
GLP compliance:
no
Remarks:
Well conducted study
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:
Measurement of exposure concentrations: Yes (no further details given)
Vehicle:
not specified
Details on test solutions:
Type and source of the exposure medium: US-EPA moderately hard reconstituted water.
Test organisms (species):
Ceriodaphnia dubia
Details on test organisms:
Source of the test organisms: In-house culture according to US-EPA
Holding conditions prior to test: As per test
Life stage of the test species used: <24 hours old
Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
7 d
Post exposure observation period:
None
Hardness:
80-100 mg/L CaCO3
Test temperature:
25°C
pH:
7.4-7.8
Dissolved oxygen:
No data reported
Salinity:
No data reported
Nominal and measured concentrations:
Test concentrations used: 0.11 (control), 3.71, 5.97, 14.2, 26.9, 53.8 nM (measured dissolved)
Details on test conditions:
Nature of test system: 7 day static renewal toxicity test with 24 hours test solution renewal interval. Fed yeast-Cerophyll®141 -trout chow mixture and Pseudokirchneriella subcapitata.
DOC is not reported.
Reference substance (positive control):
not specified
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
1.14 µg/L
95% CI:
>= 0.5 - <= 2.47
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
reproduction
Details on results:
The 7 day reproduction EC10 is 1.14 µg dissolved Ag/L
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
No data reported
Validity criteria fulfilled:
yes
Conclusions:
The 7 day reproduction EC10 is 1.14 µg dissolved Ag/L
Executive summary:

This is a non-GLP, long term toxicity study on Ceriodaphnia dubia following US-EPA (2002) guideline "Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms". A reliable EC10 for reproduction of 1.14 (0.5-2.47) µg/L as dissolved Ag could be extracted.

The study is considered reliable and suitable for use for this endpoint.

Endpoint:
long-term toxicity to aquatic invertebrates
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
Qualifier:
according to guideline
Guideline:
other: 7 d USEPA, 2002
Deviations:
not specified
Principles of method if other than guideline:
The chronic toxicity of silver, as silver nitrate, was investigating two freshwater aquatic cladoceran species, Ceriodaphnia dubia and Daphnia magna. Preliminary studies with C. dubia showed variable results that were related to the equilibration time between food and silver. Follow-up testing was conducted using a 3 hour equilibration time, which stabilised dissolved silver concentrations and the toxicity of silver. The chronic toxicity of silver to C. dubia was also investigated in the presence of 1) dissolved organic carbon and 2) sulphide.
GLP compliance:
not specified
Remarks:
GLP compliance stated in published article except chemical analysis
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:
Analytical samples for dissolved (0.45 µm) and total (acidified only, no digestion) silver measurements were collected using clean metal techniques. For C. dubia studies, samples were collected daily from each treatment.
Vehicle:
not specified
Details on test solutions:
Test water, prior to the addition of any water quality factors being investigated, was similar to the culture water (target hardness and alkalinity 90 and 60 mg/L as CaCO3 respectively). Culture water consisted of water from Horsetooth (HT) Reservoir (Fort Collins, CO, USA) reconstituted to moderately hard (~90 mg/L CaCO3) characteristics (amended HT water). This natural water source served as the base water used in all studies conducted during this testing program.
Test organisms (species):
Ceriodaphnia dubia
Details on test organisms:
Source of the test organisms: In-house cultures reared according to USEPA Guidelines. Holding conditions prior to test: 25 ± 1ºC. Life stage of the test species used: < 12 hours.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Post exposure observation period:
None
Hardness:
Hardness 88 mg/L as CaCO3
Test temperature:
25 ± 1ºC
pH:
pH 7.8 – 8.4
Dissolved oxygen:
DO ≥ 5.3 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
Test concentrations used: 70% dilution series with 5 concentrations. Endpoints are expressed as total or dissolved silver concentrations (µg/L average) measured in new test solutions. See table.
Details on test conditions:
Number of replicates per concentration: 10. Number of organisms per replicate: 1. The test used a 3 hour equilibrium period between test solution preparation and use. DOC: 4.74-4.76 mg/L in standard replicates and 5.11 mg/L in additional dissolved organic carbon replicate.
Reference substance (positive control):
not specified
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
9.5 µg/L
95% CI:
>= 8.7 - <= 10.3
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
reproduction
Remarks on result:
other: HT+NOM study
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
10.1 µg/L
95% CI:
>= 7.3 - <= 13.8
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
reproduction
Remarks on result:
other: HT study
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
10.3 µg/L
95% CI:
>= 6.9 - <= 15.4
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
reproduction
Remarks on result:
other: mono study
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
11.5 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
reproduction
Remarks on result:
other: mass study
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
10.4 µg/L
95% CI:
>= 10.2 - <= 10.6
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
mortality
Remarks on result:
other: HT+NOM study
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
14.8 µg/L
95% CI:
>= 14.6 - <= 14.9
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
mortality
Remarks on result:
other: HT study
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
10.9 µg/L
95% CI:
>= 9.5 - <= 12.3
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
mortality
Remarks on result:
other: mono study
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
11.2 µg/L
95% CI:
>= 11.1 - <= 11.2
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
mortality
Remarks on result:
other: mass study
Details on results:
LC10 values of 10.4 μg/L, 14.8 μg/L, 10.9 μg/L and 11.2 μg/L (mortality) are calculated. For the endpoint reproduction, toxicity values of 9.5 μg/L, 10.1 μg/L, 10.3 μg/L and 11.5 μg/L are selected. EC10 values with too wide confidence interval (> factor of 10) were not further selected. All toxicity valus are expressed as dissolved Ag.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
All data are treated with analysis of variance (ANOVA) followed by Dunnett’s test. LC10/EC10 have been re-calculated using log-logistic distribution (TRAP software)
Validity criteria fulfilled:
yes
Conclusions:
LC10 values of 10.4 μg/L, 14.8 μg/L, 10.9 μg/L and 11.2 μg/L (mortality) are calculated. For the endpoint reproduction, toxicity values of 9.5 μg/L, 10.1 μg/L, 10.3 μg/L and 11.5 μg/L are selected. EC10 values with too wide confidence interval (> factor of 10) were not further selected. All toxicity valus are expressed as dissolved Ag.
Executive summary:

LC10 values of 10.4 μg/L, 14.8 μg/L, 10.9 μg/L and 11.2 μg/L (mortality) are calculated. For the endpoint reproduction, toxicity values of 9.5 μg/L, 10.1 μg/L, 10.3 μg/L and 11.5 μg/L are selected. EC10 values with too wide confidence interval (> factor of 10) were not further selected. All toxicity valus are expressed as dissolved Ag.

The study is considered reliable and suitable for use for this endpoint.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP unknown, guideline unknown, acceptable with restrictions
Qualifier:
no guideline followed
Guideline:
other: Methodology well described
Principles of method if other than guideline:
2 year flow through saltwater toxicity test. C. fornicata was exposed for 24 month to silver as nitrate. 10 mated pairs of C. fornicata per concentration were exposed to 1, 5 and 10 µg Ag/L (1.34, 4.97 and 10.93 µg/L actual silver concentrations). After copulation and fertilization the females produced egg sacs, which allowed observation of developing larvae. The number of larval releases for each of 10 females from each test concentration of silver and controls was recorded for the entire test period. Fifteen larvae were then measured to determine their size at release. F1 larvae from each release of a single mating pair of the parental stock at each test concentration and control were held at the same silver exposure concentrations as the parents (15-25 °C). Each culture was changed daily. The larvae were reared through metamorphosis and the percentage that set was determined. F1 juveniles were then reared to adulthood. A maximum of 10 mated pairs from each release of the single mated pair of parents was retained to observe fecundity.
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:
yes
Details on sampling:
Weekly measurements throughout 2 year test period. Background concentration < 1 μg/L.
Vehicle:
not specified
Details on test solutions:
Type and source of the exposure medium: Filtered seawater
Test organisms (species):
other: Crepidula fornicata
Details on test organisms:
Source: Commercial oyster beds, Long Island Sound, off Norwalk, Connecticut, USA
Holding conditions prior to test: Acclimated in laboratory for 2 weeks
Life stage of test species used: Mated pairs (adult female length mean: 33.6 mm, range 28.7 - 41.2 mm)
Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
24 mo
Post exposure observation period:
None
Hardness:
No data reported
Test temperature:
4.5 - 25ºC
pH:
No data reported
Dissolved oxygen:
No data reported
Salinity:
25 ± 2‰
Nominal and measured concentrations:
C. fornicata per concentration were exposed to 1, 5 and 10 µg Ag/L (1.34 ± 0.4, 4.97 ± 1.14 and 10.93 ± 2.39 µg/L actual silver concentrations).
Details on test conditions:
Number of organisms per vessel: 10 pairs
Number of replicates per concentration: 3
Nature of test system: Test animals utilized food available in seawater and supplemented by limited amount of cultured unicellular algae once a week.

10 mated pairs of C. fornicata were exposed for 24 month to silver as nitrate. After copulation and fertilization the females produced egg sacs, which allowed observation of developing larvae. The number of larval releases for each of 10 females from each test concentration of silver and controls was recorded for the entire test period. Fifteen larvae were then measured to determine their size at release.
F1 larvae from each release of a single mating pair of the parental stock at each test concentration and control were held in 3 L polypropylene containers at the same silver exposure concentrations as the parents (15-25 °C, 25±2‰ salinity). Each culture was changed daily. The larvae were reared through metamorphosis and the percentage that set was determined. F1 juveniles were then reared to adulthood. A maximum of 10 mated pairs from each release of the single mated pair of parents was retained to observe fecundity.
Reference substance (positive control):
not specified
Duration:
24 mo
Dose descriptor:
NOEC
Effect conc.:
5 µg/L
Nominal / measured:
not specified
Conc. based on:
element
Basis for effect:
reproduction
Details on results:
Larval releases in the parental stock of C. fornicata were significantly reduced in the 0.010 mg/L silver exposure group. The generation times, from egg to adulthood and first spawning of females at 0.001 and 0.005 mg/L were significantly different from controls. Females at 0.005 mg/L were significantly smaller than controls upon fist larval release. No differences were found in larval size at release for F1 C. fornicata.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
No data reported
Validity criteria fulfilled:
not specified
Conclusions:
The 2 year NOEC is 5 µg/L silver.
Executive summary:

C. fornicata was exposed for 24 month to silver as nitrate. 10 mated pairs of C. fornicata per concentration were exposed to 1, 5 and 10 µg Ag/L (1.34, 4.97 and 10.93 µg/L actual silver concentrations). After copulation and fertilization the females produced egg sacs, which allowed observation of developing larvae. The number of larval releases for each of 10 females from each test concentration of silver and controls was recorded for the entire test period. Fifteen larvae were then measured to determine their size at release. F1 larvae from each release of a single mating pair of the parental stock at each test concentration and control were held at the same silver exposure concentrations as the parents (15-25 °C). Each culture was changed daily. The larvae were reared through metamorphosis and the percentage that set was determined. F1 juveniles were maintained at ambient seawater conditions (4.5 - 25 °C) and at the same silver concentrations in which they were hatched. F1 juveniles were then reared to adulthood. A maximum of 10 mated pairs from each release of the single mated pair of parents was retained to observe fecundity. Results: Larval releases in the parental stock of C. fornicata were significantly reduced in the 0.010 mg/L silver exposure group. The generation times, from egg to adulthood and first spawning of females at 0.001 and 0.005 mg/L were significantly different from controls. Females at 0.005 mg/L were significantly smaller than controls upon fist larval release. No differences were found in larval size at release for F1 C. fornicata.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP status unknown, some experimental detail missing. Although results are expressed as total measured Ag no detail on how often exposure media was sampled and analysed
Qualifier:
no guideline followed
Guideline:
other: 96 h acute and 10 d
Principles of method if other than guideline:
10 d reproduction test (mean number of offspring per female)
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
Water samples filtered (0.45 μm, Gelman) and acidified with 15 N redistilled HNO3 (Aldrich Chemical Co., Milwaukee, WI) to a pH of 1 - 2 before analysis for dissolved silver.
Details on test solutions:
Type and source of the exposure medium: U of Mississippi Biological Field Station water. A stock solution was prepared using Milli-Q water and AgNO3. Appropriate volumes of the stock solution were added to filtered pond water to achieve desired concentrations.
Test organisms (species):
other: Ceriodaphnia dubia Richard
Details on test organisms:
Source: In-house culture maintained at University of Mississippi
Life stage of test species used: 24 h
Test type:
static
Water media type:
freshwater
Total exposure duration:
10 d
Hardness:
68 - 70 mg CaC03/L
Test temperature:
20 ± 1ºC
pH:
7.5 - 8.2
Dissolved oxygen:
≥ 7.5 mg/L
Nominal and measured concentrations:
5 concentrations + control
Details on test conditions:
Type and source of exposure medium: filtered Univerisity of Mississippi Biological Field Station pond water, adjusted for hardness and alkalinity with addition of 0.1 g l-1 NaHCO3 and CaCl2
Test vessel: 50 ml borosilicate glass beaker
Aeration: none, ≥ 40% saturation DO maintained throughout
Number of organisms per vessel: 1
Number of replicates per concentration: 10
Photoperiod: LD 16:8
Feeding: 0.05 ml Selenastrum capricornutum algae daily
Key result
Duration:
10 d
Dose descriptor:
NOEC
Effect conc.:
0.53 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
other: reproduction/mortality
Details on results:
Results with AgCl and Ag(SO2O3)nabove water solubility: 10d reproduction/survival NOEC 0.53 μg total Ag/L, 10d reproduction LOEC 1.14 μg total Ag/L and 96h and 10d LC50 0.92 (0.69 - 1.23) μg total silver/L.
Reported statistics and error estimates:
All data are treated with analysis of variance (ANOVA) followed by Dunnett’s test
Validity criteria fulfilled:
not specified
Conclusions:
10d reproduction/mortality NOEC is 0.53 μg dissolved silver/L.
Executive summary:

A 10 d reproduction test with Ceriodaphnia dubia resulted in a reliable NOEC for reproduction/mortality of 0.53 μg dissolved silver/L.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP status unknown, some experimental detail missing. Although results are expressed as total measured Ag no detail on how often exposure media was sampled and analysed
Qualifier:
no guideline followed
Guideline:
other: 96 h acute and 10 d
Principles of method if other than guideline:
10 d reproduction test (mean number of offspring per female)
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
Water samples filtered (0.45 μm, Gelman) and acidified with 15 N redistilled HNO3 (Aldrich Chemical Co., Milwaukee, WI) to a pH of 1 - 2 before analysis for dissolved silver.
Details on test solutions:
Type and source of the exposure medium: U of Mississippi Biological Field Station water. A stock solution was prepared using Milli-Q water and AgNO3. Appropriate volumes of the stock solution were added to filtered pond water to achieve desired concentrations.
Test organisms (species):
Daphnia magna
Details on test organisms:
Source: In-house culture maintained at University of Mississippi
Life stage of test species used: 24 h
Test type:
static
Water media type:
freshwater
Total exposure duration:
10 d
Hardness:
68 - 70 mg CaC03/L
Test temperature:
20 ± 1ºC
pH:
7.5 - 8.2
Dissolved oxygen:
≥ 7.5 mg/L
Nominal and measured concentrations:
5 concentrations + control
Details on test conditions:
Type and source of exposure medium: filtered Univerisity of Mississippi Biological Field Station pond water, adjusted for hardness and alkalinity with addition of 0.1 g l-1 NaHCO3 and CaCl2
Test vessel: 200 mL exposure solution in 250 mL beakers
Aeration: none, ≥ 40% saturation DO maintained throughout
Number of organisms per vessel: 1
Number of replicates per concentration: 10
Photoperiod: LD 16:8
Feeding: 0.05 ml Selenastrum capricornutum algae daily
Key result
Duration:
10 d
Dose descriptor:
NOEC
Effect conc.:
0.8 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
reproduction
Details on results:
10d reproduction NOEC 0.8 μg dissolved Ag/L
Reported statistics and error estimates:
All data are treated with analysis of variance (ANOVA) followed by Dunnett’s test
Validity criteria fulfilled:
not specified
Conclusions:
10d reproduction NOEC is 0.8 μg dissolved silver/L.
Executive summary:

A 10 d reproduction test with Daphnia magna resulted in a reliable NOEC for reproduction of 0.8 μg dissolved silver/L.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP status unknown, some experimental detail missing. Although results are expressed as total measured Ag no detail on how often exposure media was sampled and analysed
Qualifier:
no guideline followed
Guideline:
other: 96 h acute and 10 d
Principles of method if other than guideline:
10 d reproduction test (mean number of offspring per female)
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
Water samples filtered (0.45 μm, Gelman) and acidified with 15 N redistilled HNO3 (Aldrich Chemical Co., Milwaukee, WI) to a pH of 1 - 2 before analysis for dissolved silver.
Details on test solutions:
Type and source of the exposure medium: U of Mississippi Biological Field Station water. A stock solution was prepared using Milli-Q water and AgNO3. Appropriate volumes of the stock solution were added to filtered pond water to achieve desired concentrations.
Test organisms (species):
other: Hyalella azteca
Details on test organisms:
Source: In-house culture maintained at University of Mississippi
Life stage of test species used: 2-3 weeks old
Test type:
static
Water media type:
freshwater
Total exposure duration:
10 d
Hardness:
10-15 mg/L CaCO3
Test temperature:
20 ± 1ºC
pH:
6.9-7.5
Dissolved oxygen:
≥ 7.5 mg/L
Nominal and measured concentrations:
5 concentrations + control
Details on test conditions:
Type and source of exposure medium: filtered Univerisity of Mississippi Biological Field Station pond water, adjusted for hardness and alkalinity with addition of 0.1 g l-1 NaHCO3 and CaCl2
Test vessel: 200 mL exposure solution in 250 mL beakers
Aeration: none, ≥ 40% saturation DO maintained throughout
Number of organisms per vessel: 1
Number of replicates per concentration: 10
Photoperiod: LD 16:8
Feeding: 0.05 ml Selenastrum capricornutum algae daily
Key result
Duration:
10 d
Dose descriptor:
NOEC
Effect conc.:
4 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
mortality
Details on results:
10d mortality NOEC 4 μg dissolved Ag/L
Reported statistics and error estimates:
All data are treated with analysis of variance (ANOVA) followed by Dunnett’s test
Validity criteria fulfilled:
not specified
Conclusions:
10d mortality NOEC is 4 μg dissolved silver/L.
Executive summary:

A 10 d reproduction test with Hyalella azteca resulted in a reliable NOEC for mortality of 4 μg dissolved silver/L.

Endpoint:
long-term toxicity to aquatic invertebrates
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, acceptable without restrictions
Qualifier:
according to guideline
Guideline:
other: ASTM E1191-97 (2003) and ASTM E1241-98 (2003).
Deviations:
not specified
GLP compliance:
not specified
Remarks:
GLP compliance stated in published article
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:
Dissolved silver measured at start, end and weekly.
Vehicle:
not specified
Details on test solutions:
Filtered seawater from Atlantic Ocean at TR Wilbury Laboratories, Marblehead, Massachusetts. Salinity adjusted by addition of deionised water. Stock solutions prepared in deionized water acidified with 0.2% nitric acid to pH < 2 and stored at room temperature in amber glass bottles.
Test organisms (species):
Americamysis bahia (previous name: Mysidopsis bahia)
Details on test organisms:
Source: In-house culture initiated in 1998
Holding conditions prior to test: Acclimated to test exposure medium
Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
28 d
Post exposure observation period:
None
Hardness:
No data reported
Test temperature:
27 ± 1ºC
pH:
7.6 - 8.2
Dissolved oxygen:
6.2 - 9.2 mg/L
Salinity:
10, 20 and 30‰ experimental variable. Salinity test variable all within 1‰ of target.
Nominal and measured concentrations:
Nominal 10% salinity: 0, 1.9, 3.4, 6.0, 13, 28 µg/L
Nominal 20% salinity: 0, 34, 60, 120, 250, 410 µg/L
Nominal 30% salinity: 0, 19, 37, 72, 160, 310 µg/L
Details on test conditions:
Number of organisms per vessel: 15
Number of replicates per concentration: 4
Feeding: daily A. salina
Reference substance (positive control):
not specified
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
19 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
mortality
Remarks on result:
other: salinity 30‰
Details on results:
The effect values from the toxicity test demonstrate that increasing salinity has a mitigating effect on silver toxicity if toxicity is determined using measured concentrations of dissolved silver.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Values for NOEC and LOEC were calculated using ANOVA and a parmetric Dunnettt's test or nonparametric William's test. The LC20 calculated by weighted least-squares nonlinear regression, and the EC50 calculated by binomial/nonlinear interpolation. (p<0.05).

Salinity

NOEC

LOEC

EC20

ACR

µg/L dissolved Ag

 

Mysids

 

 

 

 

10

6.0

13

3.9

>3.2

20

34

60

60

5.8

30

19

37

35

9.3

Validity criteria fulfilled:
yes
Conclusions:
28 day NOEC mortality is 19 μg dissolved Ag/L (salinity 30‰)
Executive summary:

This is a GLP, guideline study and is considered reliable and fully acceptable for use for this endpoint

Endpoint:
long-term toxicity to aquatic invertebrates
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, acceptable without restrictions
Qualifier:
according to guideline
Guideline:
other: ASTM E1563-98 (2003) and US EPA -600-4-91-003 (1994) guidelines
Deviations:
not specified
GLP compliance:
not specified
Remarks:
GLP compliance stated in published article
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:
Samples were collected at beginning of fertilization test; start and end of embryo test and weekly during 30 day adult test. Measured dissolved Ag ranged from 93 – 104% of nominal. Ag+ measured and calculated.
Vehicle:
not specified
Details on test solutions:
Type and source of the exposure medium: Filtered seawater from Atlantic Ocean at TR Wilbury Laboratories, Marblehead, Massachusetts. Salinity reduced to 30‰ by addition of deionised water.
Test organisms (species):
other: Arbacia punctulata
Details on test organisms:
Source: Adults obtained from Aquatic Research Organism, Hampton, NH, USA
Holding conditions prior to test: Acclimated to test exposure medium.
Life stage of test species used: Fertilization, embryo (within 4 h of fertilization) and adult
Test type:
flow-through
Water media type:
saltwater
Total exposure duration:
30 d
Post exposure observation period:
None
Hardness:
No data reported
Test temperature:
Temperature: 19.4 – 21.9ºC (fertilization/embryo), 13.9 – 16.5ºC (adult)
pH:
6.9 - 8.1
Dissolved oxygen:
7.0 - 8.5 mg/L
Salinity:
28 - 30‰
Nominal and measured concentrations:
Nominal concentrations: 0, 5, 10, 20, 40, 80 µg/L
Details on test conditions:
Number of organisms per vessel: 5 adult, embryo suspension to yield 15-30 viable embryos/mL
Number of replicates per concentration: 3 embryo test, 4 adult test
Nature of test system: Static – fertilization and embryo. Flow-through - adult, fed daily with freshly collected kelp.
Reference substance (positive control):
not specified
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
8.6 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
other: development NOEC effects on spines
Details on results:
Adult NOEC 8.6 µg/L; adult LOEC 19 µg/L. 96 h LC50 40 µg/L. Fertilization EC50 14 µg/L (95% cf 12-16 µg/L)
Results with reference substance (positive control):
Not applicable
Validity criteria fulfilled:
yes
Conclusions:
30 day development NOEC effect on spines is 8.6 μg dissolved silver/L
Endpoint:
long-term toxicity to aquatic invertebrates
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: US EPA, Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates Environmental Protection, 192 (2000) (March)https://doi.org/EPA 600/R-94/024
GLP compliance:
not specified
Analytical monitoring:
yes
Remarks:
Total and dissolved Ag in the test solutions were measured from three subsamples of stock solutions before dosing.
Details on sampling:
- Concentrations: Measured dissolved Ag values ranged from 89–100% for AgNO3 relative to their corresponding measured total Ag.

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The appropriate volume of AgNO3 solution was added to reformulated moderately hard reconstituted water to obtain 0, 1, 2, 4, 8, and 16 μg Ag/L for 28-d tests.
Test organisms (species):
other aquatic crustacea: Hyalella azteca
Details on test organisms:
Adults and juvenile H. azteca were originally obtained from the United States Geological Survey (USGS), Columbia, Missouri, and maintained in culture in a static system. The amphipods were cultured according to the USEPA standard culturing procedures (USEPA, 2000) in reformulated moderately hard reconstituted water (R-MHRW) made in the laboratory. The bottom of the culture chambers was covered with plastic mesh to serve as a substrate. Amphipods were cultured under 16 h light and 8 h dark each day at temperatures ranging from 21 to 22 °C. Dissolved oxygen, pH, hardness, alkalinity, conductivity, and ammonia were measured every week to ensure acceptable water quality. Amphipods were fed three times per week with fish flakes (Aquatic Biosystems Inc., Fort Collins, Colorado, USA) and Thalassiosira weissflogii diatoms (Reed Mariculture, Campbell, CA, USA). Culture water was renewed with R-MHRW once per week. Juveniles were separated from adults using a #25 sieve (710-mm opening) during every water renewal. To obtain juveniles for toxicity testing, less than 24 h old newborns were isolated from adults using the #25 sieve. The newborns were held in a 1 L jar for 7–8 days under the same culturing conditions as the adults before using them for toxicity testing. The narrow range in age was chosen to reduce variability in growth at the end of the test.

Test type:
other: modular water-renewable system: water-only static renewal system with clean sand as a substrate
Water media type:
freshwater
Total exposure duration:
28 d
Hardness:
86-100 mg/L CaCO3
Test temperature:
21-22 °C
pH:
7.5-7.7
Dissolved oxygen:
7.6-8.4 mg/L
Conductivity:
259-274 µS/cm
Nominal and measured concentrations:
Nominal Ag: 0, 1, 2, 4, 8, 16 µg/L
Measured dissolved Ag: 0.0, 0.9, 1.7, 3.4, 6.8, 13.6 µg/L
Details on test conditions:
An inexpensive modular water-renewable system for bulk sediment toxicity testing designed by Leppanen & Maier (1998) was used for toxicity testing. The system has a test chamber that holds 49 300-mL beakers and two of the chambers were used. Five clean 300-mL replicate beakers were assigned to each exposure concentration including the controls (culture water without a toxicant). About
135 g (equivalent to 100 mL water) natural play sand purchased from Home Depot Store was added to each replicate beaker and filled to the brim with the test solution. The beakers were left undisturbed for 24 h for the test solution to equilibrate. The natural play sand was used as a substrate for toxicity testing because our initial culturing method showed that it supports amphipod growth, reduces variability in results, and makes it easy to find live amphipods after the test. The sand was sterilized by washing it with Milli-Q water and drying overnight in an oven at 70 °C before use.
Ten juveniles (7- to 8-day-old) were randomly placed in each beaker. The test beakers were then placed in plastic chambers with a small hole at the bottom to allow the passage of excess water from the beakers and a plastic container was placed underneath each chamber to collect excess water. Amphipods in each beaker were fed daily with 150 μL T. weissflogii diatoms and 75 μL fish flake slurry. The overlying water was completely renewed every 24 h with a newly prepared test solution to reduce feed and nanoparticle settling. At the end of the study, live amphipods in each test beaker were counted, washed with culture water, and frozen in the freezer compartment of a refrigerator at the end of every test. Frozen amphipods were thawed, dried in an oven at 60 °C for 24 h, and weighed using a Mettler Toledo AT201 analytical balance (0.0001 g) to measure dry weight for growth and biomass determination.
Key result
Duration:
28 d
Dose descriptor:
LC10
Effect conc.:
1.57 µg/L
95% CI:
> 1.4 - < 1.8
Nominal / measured:
meas. (initial)
Conc. based on:
element (dissolved fraction)
Basis for effect:
mortality
Remarks on result:
other: The LC10 values have been re-calculated using the log-logistic curve fitting from the TRAP-software (Toxicity Response Analysis Program (TRAP) v1.22 software developed by Russell J. Erickson (U.S. EPA))
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
1.7 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element (dissolved fraction)
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
3.4 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
1.7 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element (dissolved fraction)
Basis for effect:
other: biomass
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
2.68 µg/L
95% CI:
> 0 - < 500 000 000 000
Nominal / measured:
meas. (initial)
Conc. based on:
element (dissolved fraction)
Basis for effect:
other: biomass
Remarks on result:
other: The EC10 values have been re-calculated using the log-logistic curve fitting from the TRAP-software (Toxicity Response Analysis Program (TRAP) v1.22 software developed by Russell J. Erickson (U.S. EPA))
Remarks:
EC10 for biomass not reliable due to large confidence interval
Validity criteria fulfilled:
yes
Conclusions:
The LC10 for Hyalella azteca is 1.57 microgram/L based on mortality.
The lowest NOEC for Hyalella azteca is 1.7 microgram/L based on mortality and biomass.
Executive summary:

The LC10 for Hyalella azteca is 1.57 microgram/L based on mortality.
The lowest NOEC for Hyalella azteca is 1.7 microgram/L based on mortality and biomass. The NOEC for Hyalella azteca based on growth equals 3.4 microgram/L.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP, guideline study, acceptable with restrictions
Qualifier:
according to guideline
Guideline:
other: 21 d ASTM 2001
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
on days 0, 2, 4, 6, 10, 13 and 18
Test organisms (species):
Daphnia magna
Details on test organisms:
Details on test organisms - source: In-house cultures reared according to USEPA guidelines
Details on test organisms - holding conditions prior to test: 20 ± 1ºC.
Details on test organisms - life stage of test species used: < 24h
Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
21 d
Hardness:
160 mg CaCO3 l-1
Test temperature:
20 - 22ºC
pH:
7.9 - 8.6
Dissolved oxygen:
≥ 6 mg l-1
Nominal and measured concentrations:
60% dilution series (5 concs)
Details on test conditions:
type and source of exposure medium: as culture
no of organisms per vessel: 5
no of replicates per concentration: 4
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
2.37 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
other: mortality, growth and reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
3.5 µg/L
95% CI:
>= 1.8 - <= 6.5
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
reproduction
Details on results:
Reliable NOEC for D. magna as dissolved Ag could be extracted, i.e. 2.37 μg/L (endpoint mortality and growth) and 3.5 μg/L (endpoint reproduction).
Reported statistics and error estimates:
all data are treated with analysis of variance (ANOVA) followed by Dunnett’s test. LC10/EC10 have been re-calculated using log-logistic distribution (TRAP software)
Validity criteria fulfilled:
no
Remarks:
2 control replicates, yes for survival no for reproduction. Data presented in paper used comparison with subsequent control study. Rationale given.
Conclusions:
Reliable NOEC for D. magna as dissolved Ag could be extracted, i.e. 2.37 μg/L (endpoint mortality and growth) and 3.5 μg/L (endpoint reproduction).
Executive summary:

Reliable 21 d NOEC for D. magna as dissolved Ag could be extracted, i.e. 2.37 μg/L (endpoint mortality and growth) and 3.5 μg/L (endpoint reproduction).

Description of key information

Nine chronic NOECs/EC10s for ionic silver (based on dissolved silver) were taken forward for use in the derivation of the freshwater PNEC by statistical extrapolation. These were a value of 4.36 µg/L Ag for Ceriodaphnia dubia (geometric mean of six NOEC/EC10 values reported by Kolts et al. (2009), Naddy et al. (2007) and Rodgers et al. (1997)), an EC10 of 0.8 µg/L Ag for Daphnia magna (Rodgers et al. 1997), a NOEC of 1.54 µg/L Ag for Hyalella azteca (geometric mean of 2 NOEC values from Diamond et al. (1990) and Rodgers et al. (1997)), a NOEC of 0.16 µg/L Ag for Isonychia bicolor (Diamond et al. 1990), an EC10 of 1.0 µg/L Ag for Stenonema modestum (Diamond et al. 1992), an EC10 of 12.54 µg/L Ag for Chironomus tentans (Call et al. 1999), an EC10 of 0.31 µg/L Ag for Brachionus calyciflorus (Arijs et al. 2021), an EC10 of 0.84 µg/L Ag for Corbicula fluminea (Diamond et al. 1990) and an EC10 of 1.48 µg/L Ag for Lymnaea stagnalis (Cremazy et al. 2018).

 

A NOEC of 8.6 µg/L Ag for Arbacia punctulata (Ward et al. 2006) is taken forward for the derivation of the marine PNEC.

Key value for chemical safety assessment

Additional information

Data on the toxicity of silver were available for several different species of freshwater invertebrates (crustaceans, insects, rotifers and molluscs).

 

Key long-term data from three species of crustacean were available: Ceriodaphnia dubia, Daphnia magna and Hyalella azteca. 

C. dubia data were selected from three available reliable studies: Kolts et al. (2009), Naddy et al. (2007) and Rodgers et al. (1997). These studies were conducted under a range of water conditions and measured concentrations of dissolved silver in the test media. As the water conditions did not appear to influence the results of the tests, the geometric mean of six available NOEC/ EC10 values for the most sensitive endpoint inhibition of reproduction (1.14, 9.5, 10.1, 10.3, 11.5 and 0.53 μg/L measured dissolved silver) was used in the SSD (4.36 μg/L dissolved silver).

Reliable long-term Daphnia magna data were available from four studies: Rodgers et al. (1997), Naddy et al. (2007), Bianchini and Wood (2008) and Mertens et al. (2019). From these studies, key data were selected from Rodgers et al. (1997) as, of the reliable studies, this study reported the lowest effect value for the most sensitive endpoint inhibition of reproduction (0.8 μg/L dissolved silver).

Reliable data for Hyallela azteca were available from Diamond et al. (1990), Rodgers et al. (1997) and Kusi and Maier (2022). As the studies by Diamond et al. (1990) and Rodgers et al. (1997) were both performed at lower hardness than the study by Kusi and Maier (2022), the geometric mean of the two NOEC values for mortality from the former two studies (0.59 and 4 µg/L dissolved silver) was used in the SSD (1.54 μg/L dissolved silver).

 

Key data for three insect species representing two insect orders were available. A NOEC of 0.16 μg/L dissolved silver was calculated for effects on molting in Isonychia bicolor (a mayfly) in a 14-day semi-static exposure test (Diamond et al. 1990). Exposure of Stenonema modestum, a second species of mayfly, for 14 days resulted in a comparable NOEC of 1.0 μg/L measured dissolved silver, also for effects on molting (Diamond et al. 1992). Both of these studies were assigned Klimisch scores of 2 as they were conducted using non-standard species and measured a non-standard endpoint. However, both of these results are considered to be valid and sufficiently robust for PNEC derivation. Key data on Chironomus tentans (a mosquito) were available from a 10-day flow-through water-only exposure conducted by Call et al. (1999). An EC10 of 12.54 μg/L was calculated from the growth data presented in the paper. Rodgers et al. (1997) also conducted exposures with C. tentans and reported a 10-day NOEC of 125 μg/L dissolved silver (measured); these data were used as supporting data.

 

In a 48 hour key study with the rotifer Brachionus calyciflorus exposed to silver nitrate, the EC10 for the most sensitive endpoint (population size) was 0.31 µg/L dissolved silver (Arijs et al. 2021).

 

Key data from two mollusc species are available. Diamond et al. (1990) report a 21-day EC10 of 0.84 μg/L dissolved silver for growth inhibition of Corbicula fluminea derived from a semi static test. From Cremazy et al. (2018), a reliable EC10 of 1.48 μg/L dissolved silver for growth inhibition of Lymnaea stagnalis could be extracted.

 

It is noted that only studies using a soluble silver salt as test substance and reporting filtered (<0.45 μm) silver concentrations have been selected as key studies (because monitoring data are also reported as 0.45 µm–filtered concentrations). As such, several studies did not meet these criteria and were included as supporting studies, for example, Elnabarawy et al. (1986), Nebeker et al. (1983) and Kolkmeier and Brooks (2013).

 

The lowest reliable long-term result for marine crustaceans is a 28-day developmental NOEC of 19 µg/L dissolved silver for Americamysis bahia (Ward et al. 2006). The authors conducted tests at three salinity levels: 10, 20 and 30 ‰. Whilst increases in salinity generally require higher concentrations of dissolved silver to cause a chronic effect, this was not seen in this study which produced NOECs of 6, 34 and 19 µg/L dissolved silver at 10, 20 and 30 ‰ salinity, respectively.

 

The lowest reliable long-term result for marine molluscs is a 24-month reproduction NOEC of 5 μg/L dissolved silver for Crepidula fornicata (Nelson et al. 1983). Larval releases were significantly reduced at 10 μg/L in the parental generation and whilst the number of larvae released by the second generation was reduced at 5 μg/L it was not statistically significant. This test was carried out at 25±2 ‰ salinity.

 

The lowest long-term result for echinoderms is a 30-day NOEC of 8.6 µg/L dissolved silver for Arbacia punctulata for sublethal effects, which included loss of spines or spine turgor and an inability to adhere to surfaces (Ward et al. 2006). This study was initiated with adult sea urchins and at study termination an unsuccessful attempt was made to collect gametes from each surviving adult, indicating that the sea urchins were not conditioned to spawn.

 

The normal salinity range for seawater is 30 - 35 ‰ and therefore the PNEC should be based on the most sensitive result from a test carried out within this range. A long-term saltwater PNEC for silver should therefore be based on the NOEC for effects on the sea urchin, Arbacia punctulata (8.6 µg/L).