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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
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:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
pH buffer MOPS and EDTA were omitted from test media; EDTA was replaced by 1 mg/L DOC (from a natural origin: Suwannee river standard NOM; ID: 2R101N); exposure vessels not placed on orbital shaker; start density of inoculum culture was increased
GLP compliance:
no
Remarks:
Publication, GLP compliance not followed
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) were taken at test initiation (before inoculation with A. flos-aquae) and after 1, 24, 48, and 72 h in both the algae exposures and the chemistry controls.
Vehicle:
no
Details on test solutions:
The test was conducted in modified OECD medium: the pH buffer MOPS was omitted, and EDTA was replaced with 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 polypropylene beakers. 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 chamber until use.
Test organisms (species):
Anabaena flos-aquae
Details on test organisms:
The A. flos-aquae strain UTEX 1444 was purchased from the Culture Collection of Algae at the University of Texas (Austin). On arrival, the cyanobacteria culture was transferred to BG11 medium (Culture Collection of Algae at the University of Texas 2009 - https://utex.org/products/bg-11-medium) and incubated at 21 °C under continuous light.
The inoculum culture was prepared by transferring an aliquot of the A. flos-aquae stock culture kept in BG11 medium to the modified OECD medium and incubated under the same conditions as the actual growth inhibition test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
4.7 ± 0.2 mg Ca/L and 2.9 ± 0.1 mg Mg/L (25 mg/L CaCO3)
Test temperature:
22.8 ±0.6 °C
pH:
pH of fresh test media: 7.8 ± 0.1
pH throughout the test: 7.8 ± 0.2
Dissolved oxygen:
9.4 ± 0.5 mg/L
Nominal and measured concentrations:
Nominal concentrations: control, 0.22, 0.46, 1.0, 2.2, 4.6, 10 and 22 µg Ag/L.
Measured concentrations (time- weighted average of dissolved Ag concentrations in fresh solutions at t 0 h and those measured in algae exposures (1, 24, 48, and 72 h)): <0.01, 0.11, 0.16, 0.35, 0.84, 2.0, 4.3 and 6.6 µg Ag/L.

Total Ag concentrations in fresh medium were on average 27 ± 15% lower compared to the targeted nominal concentrations. Dissolved Ag concentrations in fresh solutions were on average 11 ± 8% lower than total concentrations in fresh solutions and did not change considerably 1 h after inoculation of the solutions with cyanobacteria (dissolved Ag concentrations on average 11 ± 7% lower than in the fresh solutions). During the test period, total Ag concentrations in the algae exposures decreased by 56 ± 7% in the 4 lowest Ag treatments and by 33 ± 2% in the higher Ag treatments. At test termination, dissolved Ag concentrations in the algae exposures were on average 24 ± 6% lower than total concentrations at the same sampling occasion. During the test period, dissolved Ag concentrations decreased by 55 ± 13%, with differences between fresh solutions and exposure solutions at test termination being highest in the lowest Ag treatments. In a previous experiment, Ag concentrations measured in the chemistry controls (not inoculated with algae) decreased on average only 40 ± 20% compared to the dissolved concentrations of the fresh solutions. The latter indicates that part of the decrease in dissolved Ag in the algae exposures is due to algal adsorption and/or uptake. Further analysis of some of the Ag measurements indicates that, especially at lower Ag doses, adsorption of Ag to the test container walls contributes significantly.
Details on test conditions:
Each test concentration was assayed in 3 replicates and the control in 6 replicates. The assays were conducted in acid-washed 125-mL polycarbonate Erlenmeyer flasks (Corning) filled with 60 mL of the test solutions.
Before test initiation, the cell density of the inoculum culture was determined using a Sedge-Wick Rafter counting chamber and a light microscope. To enable the calculation of cell density from fluorescence measurements in the growth inhibition test, a calibration curve was established based on the relationship between fluorescence intensity and cell density (details not shown). Fluorescence intensity was measured using a TECAN Infinite M200 multiwell reader. An excitation wavelength of 590 nm and an emission wavelength of 683 nm were selected based on Simis et al. (2012). This excitation wavelength in the orange-red spectrum showed the highest correlation between fluorescence and cyanobacteria cell densities and targets specifically the phycobilipigments which are the most important light-harvesting pigments in cyanobacteria. The “optimal gain function” (optimal gain 191) was used for determination of fluorescence intensity in the A. flos-aquae exposures.
At test initiation, each test vessel was inoculated with 104 cells/mL (=cell density N0 at t0); these were the “algae exposures.” In addition to the algae exposures, one chemistry replicate per test concentration was used, which received the same treatment during the toxicity test (test conditions, manipulation, and sampling) as the algae exposures; but these chemistry replicates were not inoculated with algae. These chemistry replicates allowed monitoring of the Ag chemistry in the absence of algae and served as background conditions during the fluorescence intensity measurements.
All test vessels were incubated at 24 °C under continuous light (24 h light, 45 µmol/m2/s) and were manually shaken 3 times per day. Cell densities were determined daily (N1, N2, and N3 after 24 [t1], 48 [t2], and 72 [t3] h) based on fluorescence intensity measurements. Growth rate (microns per day) was determined in each replicate of each treatment as the slope of the linear regression of the natural logarithm of cell density versus time.
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.41 µg/L
95% CI:
>= 0.29 - <= 0.52
Nominal / measured:
meas. (TWA)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Details on results:
The calibration curve used to determine cell densities in the algae exposures shows a clear linear relationship between cell densities and fluorescence intensity (R² = 0.998).
Reported statistics and error estimates:
Effect concentrations (NOEC, lowest-observed-effect concentration [LOEC], and ECx) were calculated based on measured dissolved Ag concentrations. All 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 were calculated based on the 2-parameter Weibull concentration–response model using the “drc” package in R, Ver 3.3.2 (R Development Core Team 2016).
The NOECs and LOECs were calculated with the Williams (1971) test, after evaluation of the data for adherence to the underlying assumptions of normality and homogeneity of variances.

Overview of cell density and growth ratesain the different exposure treatments of the 72h-Anabaena flos-aquae growth inhibition test

Nominal Ag

Dissolved Ag

(µg/L)

Time- weighted average

Cell density

(x104cells/mL)

Growth rate

(d-1)

(µg/L)

t 0h

t 1hb

t 24h

t 48h

t 72h

dissolved Agc

Day 1

Day 2

Day 3

t 0h – t 72h

 

 

 

 

 

 

(µg/L)

 

 

Control

<0.01

<0.01

<0.01

<0.01

<0.01

<0.01

3.91±0.34

10.4±1.6

22.5±4.4

1.03±0.05

0.22

0.16

0.16

0.12

0.10

0.09

0.11

3.66±1.00

10.4±2.4

23.4±4.1

1.05±0.06

0.46

0.28

0.28

0.17

0.12

0.10

0.16

3.52±0.38

9.29±1.36

24.4±4.2

1.05±0.05

1

0.6

0.5

0.41

0.27

0.22

0.35

1.90±0.18

6.11±1.58

18.5±3.9

0.99±0.10

2.2

1.4

1.1

1.1

0.66

0.44

0.84

1.99±0.28

1.27±1.21

0.63±0.4

-0.24±0.18

4.6

2.9

2.4

2.1

2.0

1.8

2.0

1.57±0.29

0.40±0.09

0.15±0.03

-0.71±0.06

10

7.5

6.5

4.1

3.7

4.0

4.3

1.20±0.20

0.42±0.08

0.08±0.06

-0.96±0.31

22

11

10

6.5

5.6

5.6

6.6

1.32±0.17

0.56±0.04

0.16±0.05

-0.66±0.10

aAverage of all replicates ± standard deviation is reported.

bSample taken in the exposure vessels approximately 1h after the inoculation with cyanobacteria.

cTime-weighted average of dissolved silver concentrations in fresh solutions at t 0h and those measured in the algae exposures (1h, 24h, 48h & 72h).

Above table shows that a clear concentration–response behavior was observed with increasing Ag doses. At the 3 lowest silver doses, growth was comparable to the control growth. In the higher Ag doses, a negative growth was observed during the 72-h growth inhibition test (i.e., lower cell densities than the inoculum density at test initiation), suggesting that A. flos-aquae was dying in these Ag treatments. Algal clusters were observed at the control and 3 lowest Ag doses but were absent in the higher Ag doses.

Effect concentrations(expressed as measured dissolved silver concentrations) of ionic silver (Ag) to the aquatic speciesAnabaena flos-aquae.

Endpoint

EC10

(µg Ag/L)

EC20

(µg Ag/L)

EC50

(µg Ag/L)

NOEC

(µg Ag/L)

LOEC

(µg Ag/L)

Growth rate

0.41

(0.29-0.52)

0.46

(0.25-0.67)

0.56

(0.16-0.96)

0.35

(4±10)

0.84

(100±0)

ECxvalues were calculated using a Weibull function with 2 parameters. 95% confidential intervals are reported between parentheses.

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

Validity criteria fulfilled:
yes
Remarks:
Control increased 28-fold over 72 h, i.e. in line with OECD validity criteria (≥16-fold). Average control growth rate was 1.03 ± 0.05 d–1 with CV of 5% and CV among the sectional (day-by-day) growth rates in the control was 33%.
Conclusions:
An EC10 of 0.41 μg dissolved Ag /L for the endpoint growth rate was found after 72h exposure of A. flos-aquae to silver nitrate.
Executive summary:

In a 72 hour study with the cyanobacteria Anabaena flos-aquae exposed to silver nitrate, the EC10 for growth rate was 0.41 µg dissolved Ag/L.

 

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

Endpoint:
toxicity to aquatic algae and cyanobacteria
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 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
The nominal test concentrations were prepared in sterile modified AAP growth medium under sterile conditions. The medium was prepared with reduced EDTA concentrations and compounds including chloride were replaced by nitrate compounds.
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
The nominal test concentrations were prepared in sterile modified AAP growth medium under sterile conditions. The medium was prepared with reduced EDTA concentrations and compounds including chloride were replaced by nitrate compounds.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: At test start samples of the control media and of each test concentration were taken from each test solution preparation just before distributing it to the test replicates per treatment (i.e. after equilibration). After 24 hours, 48 hours and at test end, samples of representative replicates per treatment level were taken. Total silver was measured at test initiation and test termination only, whereas the concentration of conventional and truly dissolved Ag (from test vessels) additionally was measured after 24 hours and 48 hours.
Additionally, samples were taken for the measurement of particle size and zeta potential in test media at the test start (i.e. after equilibration). After 72 hours, the size and zeta potential in the test media was measured in samples taken from the additional vessels (no algae). Total and ‘truly’ and ‘conventional’ dissolved silver concentrations were also measured in samples taken from the additional vessels at test start and 72 hours.
Except for the determination of truly dissolved silver, single samples of 10 mL were taken for metal analysis at test start, after 24 hours, 48 hours and the end of the test. For the determination of truly dissolved silver triplicate samples of 10 mL were taken for metal analysis at test start, after 24 hours, 48 hours and the end of the test.

- Sample storage conditions before analysis: For total silver analysis, the samples were taken directly from the solutions and transferred into disposable polyethylene vials. All samples were acidified with HNO3 (1.25 mL concentrated HNO3) and stored in a refrigerator (about 4 °C) until further analysis.
For the determination of size and zeta potential single 10 mL samples of the test dispersion were taken. Measurements were performed immediately at the day of sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution was prepared by weighing 89.51 mg of silver nitrate and transferring into a plastic vessel, filled up with purified water to 1L followed by stirring for one hour. Directly after the stirring the stock solution was sonicated for 3 minutes in a sonication bath (Frequence: 35 kHz) and afterwards the dispersion was carefully hand-shaken for around 30 seconds. The test concentrations were prepared by adding 5.56, 17.6, 55.6, 176, 556 and 1760 µL of the stock solution into 1 L modified AAP medium. Again, directly after the addition the dispersion was sonicated for 3 minutes in a sonication bath (Frequence: 35 kHz) and carefully hand-shaken for around 30 seconds.
The freshly prepared test solutions were then left to stand for 2 hours to reach equilibrium. Following equilibration, samples were taken for chemical/particle analysis after further careful shaking, and then the test solutions were filled into the test (and additional) vessels after more careful shaking. Test item solutions for all five test concentrations were prepared separately.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: 61.81 SAG
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen
- Age of inoculum (at test initiation): exponentially-growing algae, the culture duration of the pre-cultures was 3 days.
- Method of cultivation: The stock cultures are maintained fulfilling the criteria of the OECD guideline (culture medium recommended by Bringmann und Kühn (1980)).

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): not, the stock cultures are cultivated in the culture medium recommended by Bringmann und Kühn (1980)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Modified AAP medium: 0.1 mmol/L (Ca-hardness)
Test temperature:
21.5 - 22 ᵒC
pH:
Control
Test start: 7.38
Test termination: 8.36
Test cultures
Test start: 7.25 to 7.35
Test termination: 7.27 to 8.47
Dissolved oxygen:
0.375 mg O2/L
Salinity:
N/A
Conductivity:
94.5 µS/cm
Nominal and measured concentrations:
Nominal: 0.316, 1.00, 3.16, 10.0, 31.6 µg Ag/L
Mean measured concentrations of total Ag: 0.113, 0.475, 2.35, 11.2 and 28.0 µg Ag/L
Mean measured conventional dissolved Ag concentrations: 0.039, 0.133, 0.742, 9.20 and 25.7 µg Ag/L.
Mean measured concentrations of truly dissolved Ag: 0.003, 0.299, 8.03 and 24.0 µg Ag/L
Details on test conditions:
TEST SYSTEM
- Test vessel: conical polycarbonate flasks (about 200 mL) covered with silicone-sponge caps, initially 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 test vessels were filled with 100 mL of test medium
- Initial cells density: 10,000 cells/mL
- Control end cells density: 170,900 cells/mL (St. dev. = 12.1)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8

GROWTH MEDIUM
- Standard medium used: yes, culture medium recommended by Bringmann und Kühn (1980)*
*Bringmann, G. and Kühn, R. (1980). Comparison of the toxicity thresholds of water pollutants to bacteria, algae, and protozoa in the cell multiplication inhibition test. Water Research 14(3), 231-241.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: modified AAP growth medium; the medium was prepared with reduced EDTA concentrations (to 50%) and compounds including chloride were replaced by nitrate compounds.
- Culture medium different from test medium: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes, the test concentrations were prepared in sterile modified AAP growth medium under sterile conditions.
- Light intensity and quality: 'daylight' type light, 101 to 108 µE m-2 s-1 in incubation chamber 1 and from 99.3 to 104 µE m-2 s-1 in incubation chamber 2
- Other: The cultures were oscillated by continuously stirring on a laboratory shaker with 150 rpm.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The cell concentrations were determined in the inoculum culture prior to the addition to the test vessels at test start and after 24, 48 and 72 h in the test cultures.
- Determination of cell concentrations: electronic particle counter (CASY® TT, Innovatis, Germany)
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: yes
- Test concentrations: 0.1, 1.0 and 10 µg Ag/L
- Results used to determine the conditions for the definitive study: yes, the concentrations to be tested in the definite test were selected on the basis of the results from the range-finding test.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol is tested as primary standard following internal SOPs in a non-GLP test twice a year (the latest test: August 2016).
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.46 µg/L
95% CI:
>= 0.31 - <= 0.62
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (total fraction)
Basis for effect:
growth rate
Remarks on result:
other: based on mean measured total Ag
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.52 µg/L
95% CI:
>= 2.13 - <= 2.98
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (total fraction)
Basis for effect:
growth rate
Remarks on result:
other: based on mean measured total Ag
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
2.35 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (total fraction)
Basis for effect:
growth rate
Remarks on result:
other: based on mean measured total Ag
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.47 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (total fraction)
Basis for effect:
growth rate
Remarks on result:
other: based on mean measured total Ag
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.36 µg/L
95% CI:
>= 0.29 - <= 0.42
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (total fraction)
Basis for effect:
other: yield
Remarks on result:
other: based on mean measured total Ag
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.82 µg/L
95% CI:
>= 0.73 - <= 0.92
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (total fraction)
Basis for effect:
other: yield
Remarks on result:
other: based on mean measured total Ag
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.47 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (total fraction)
Basis for effect:
other: yield
Remarks on result:
other: based on mean measured total Ag
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.11 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (total fraction)
Basis for effect:
other: yield
Remarks on result:
other: based on mean measured total Ag
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.1 µg/L
95% CI:
>= 0.05 - <= 0.16
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: based on mean measured conventional dissolved Ag
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.96 µg/L
95% CI:
>= 0.72 - <= 1.32
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: based on mean measured conventional dissolved Ag
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.74 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: based on mean measured conventional dissolved Ag
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.13 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: based on mean measured conventional dissolved Ag
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.1 µg/L
95% CI:
>= 0.08 - <= 0.12
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
other: yield
Remarks on result:
other: based on mean measured conventional dissolved Ag
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.24 µg/L
95% CI:
>= 0.21 - <= 0.27
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
other: yield
Remarks on result:
other: based on mean measured conventional dissolved Ag
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.13 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
other: yield
Remarks on result:
other: based on mean measured conventional dissolved Ag
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.04 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
other: yield
Remarks on result:
other: based on mean measured conventional dissolved Ag
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.005 µg/L
95% CI:
>= 0.003 - <= 0.008
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: based on mean measured truly dissolved Ag
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.285 µg/L
95% CI:
>= 0.219 - <= 0.365
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: based on mean measured truly dissolved Ag
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.299 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: based on mean measured truly dissolved Ag
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.003 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: based on mean measured truly dissolved Ag
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.001 µg/L
95% CI:
>= 0.001 - <= 0.002
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
other: yield
Remarks on result:
other: based on mean measured truly dissolved Ag
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.016 µg/L
95% CI:
>= 0.014 - <= 0.018
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
other: yield
Remarks on result:
other: based on mean measured truly dissolved Ag
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
<= 0.003 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
other: yield
Remarks on result:
other: based on mean measured truly dissolved Ag
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.003 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
other: yield
Remarks on result:
other: based on mean measured truly dissolved Ag
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): At the end of the test (day 3), the cells were normal and there was almost no cell debris in the control and at test concentration of 0.316 µg Ag/L. Slightly less intact cells but normal appearance, almost no cell debris were observed at ttest concentrations of 1.00 µg Ag/L. At 3.16 and 10.0 and 316 µg Ag/L there were clearly less intact cells and cells debris observed. At the highest test concentration of 31.6 µg Ag/L cell debris and barely intact cells were noted.
- Other: 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):
- Results with reference substance valid? Yes, results were in good agreement with the results of an international ring test with an ErC50 of 3.38 ± 1.30 mg/L
- EC50: ErC50 value of 3.35 mg/L (95 % confidence limits: 3.27 - 3.44 mg/L)
Reported statistics and error estimates:
For the growth test, mean average growth rates were calculated (entire exposure period of 0 - 3 d) and calculation of the percent inhibition compared to controls of growth rate or sectional growth rate [r], and yield [y] for the exposure period were performed.
The test results of the growth inhibition test were statistically analysed to determine an EC50, EC20 and EC10 value together with 95 % confidence intervals using a non-linear regression model (3-parametric cumulative distribution function according to Bruce and Versteeg (1992) for yield and linear regression (probit) for growth rate. Individual replicate responses were used for the regression analysis.
The NOEC values were determined using appropriate statistical methods (Williams` Multiple Sequential t-Test. The computer program ToxRat was used for statistical evaluations.

Effective concentrations based on mean measured total Ag concentrations

Table 1: Percent inhibition of growth rate and yield compared to controls after 72 hours.

Mean measured
total Ag
[µg Ag/L]

% Inhibition of yield

% Inhibition of growth rate

Control

0

0

0.11

0.26 (-)

0.01 (-)

0.47

19.2 (+)

4.08 (+)

2.35

94.0 (+)

53.0 (+)

11.2

99.4 (+)

85.9 (+)

28.0

99.5 (+)

88.1 (+)

(+) statistically significant difference between controls / (-) no significant difference between controls and treatments. Williams t-test (growth rate) and Welsh t-test with Bonferroni Adjustment (yield), significance level 0.05, one-sided smaller.

Table 2: Effective concentrations based on mean measured total Ag concentrations for the exposure ofR. subcapitatafor 72 hours.

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

Parametera

 

EC10

EC20

EC50

LOEC

NOEC

Growth rate (r)

Value

0.46

0.82

2.52

2.35

0.47b

 

95 %-cl lower

0.31

0.61

2.13

 

 

 

95 %-cl upper

0.62

1.03

2.98

 

 

Yield (y)

Value

0.36

0.48

0.82

0.47

0.11

 

95 %-cl lower

0.29

0.41

0.73

 

 

 

95 %-cl upper

0.42

0.55

0.92

 

 

a: ECx values for growth rate were calculated using linear regression and for yield a non-linear regression model was used.

b: The NOEC for growth rate was calculated to be at 0.11 µg Ag/L based on geometric mean measured concentrations of total Ag. However, due to the low inhibition of 4.08% at a concentration of 0.47 µg Ag/L, the NOEC was set to 0.47 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.

Effective concentrations based on conventional dissolved Ag

Table 3: Percent inhibition of growth rate and yield compared to controls after 72 hours.

Mean measured
conventional dissolved Ag
[µg Ag/L]

% Inhibition of yield

% Inhibition of growth rate

Control

0

0

0.04

0.26 (-)

0.01 (-)

0.13

19.2 (+)

4.08 (+)

0.74

94.0 (+)

53.0 (+)

9.20

99.4 (+)

85.9 (+)

25.7

99.5 (+)

88.1 (+)

(+) statistically significant difference between controls / (-) no significant difference between controls and treatments. Williams t-test (growth rate) and Welsh t-test with Bonferroni Adjustment (yield), significance level 0.05, one-sided smaller.

Table 4: Effective concentrations based on mean measured concentrations of conventional dissolved Ag for the exposure ofR. subcapitatafor 72 hours.

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

Parametera

 

EC10

EC20

EC50

LOEC

NOEC

Growth rate (r)

Value

0.10

0.22

0.96

0.74

0.13b

 

95 %-cl lower

0.05

0.14

0.72

 

 

 

95 %-cl upper

0.16

0.31

1.32

 

 

Yield (y)

Value

0.10

0.14

0.24

0.13

0.04

 

95 %-cl lower

0.08

0.11

0.21

 

 

 

95 %-cl upper

0.12

0.16

0.27

 

 

a: ECx values for growth rate were calculated using linear regression and for yield a non-linear regression model was used.

b: The NOEC for growth rate was calculated to be 0.04 µg Ag/L based on geometric mean measured concentrations of conventional dissolved Ag. However, due to the low inhibition of 4.08% at a concentration of 0.13 µg Ag/L, the NOEC was set to 0.13 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.

 

Effective concentrations based on truly dissolved Ag

Table 5: Percent inhibition of growth rate and yield compared to controls after 72 hours.

Mean measured
truly dissolved Ag
[µg Ag/L]

% Inhibition of yield

% Inhibition of growth rate

Control

0

0

0.003

19.2 (+)

4.08 (+)

0.299

94.0 (+)

53.0 (+)

8.03

99.4 (+)

85.9 (+)

24.0

99.5 (+)

88.1 (+)

(+) statistically significant difference between controls / (-) no significant difference between controls and treatments. Williams t-test (growth rate) and Welsh t-test with Bonferroni Adjustment (yield), significance level 0.05, one-sided smaller.

Table 6: Effective concentrations based on mean measured concentrations of truly dissolved Ag for the exposure ofR. subcapitatafor 72 hours.

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

Parametera

 

EC10

EC20

EC50

LOEC

NOEC

Growth rate (r)

Value

0.005

0.020

0.285

0.299

0.003b

 

95 %-cl lower

0.003

0.012

0.219

 

 

 

95 %-cl upper

0.008

0.030

0.365

 

 

Yield (y)

Value

0.0014

0.0031

0.0157

≤ 0.003

< 0.003

 

95 %-cl lower

0.0012

0.0028

0.0136

 

 

 

95 %-cl upper

0.0016

0.0037

0.0183

 

 

a: ECx values for growth rate and yield were calculated using linear regression.

b: The NOEC for growth rate was calculated to be below 0.003 µg Ag/L based on geometric mean measured concentrations of truly dissolved Ag. However, due to the low inhibition of 4.08% at this concentration, the NOEC was set to 0.003 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.

Note:ECx values were calculated based on the four highest test concentrations. Since a calculation of the geometric mean measured concentration over 72 hours was not possible for the lowest test concentration, the concentrations was excluded from the evaluation.

Validity criteria fulfilled:
yes
Remarks:
The cell number in the control increased by a factor of 170.9 within 72 hour, the mean of the replicate CV % in the section-by-section growth rate of controls: 8.84 %, CV of average specific growth rate at test end in replicate control cultures: 1.4 %
Conclusions:
Based on mean measured total Ag concentrations, the 72 hour ErC50 was 2.52 µg Ag/L and ErC10 was 0.46 µg Ag/L. For yield an EyC50 and EyC10 were 0.82 and 0.36 µg Ag/L, respectively. The NOECs for growth rate and for yield were 0.47 and 0.11 µg Ag/L, respectively.

Based on mean measured conventional dissolved Ag concentrations, the 72 hour ErC50 was 0.96 µg Ag/L and ErC10 was 0.10 µg Ag/L. For yield an EyC50 and EyC10 were 0.24 and 0.10 µg Ag/L, respectively. The NOECs for growth rate and for yield were 0.13 and 0.04 µg Ag/L, respectively.

Based on mean measured truly dissolved Ag concentrations, the 72 hour ErC50 was 0.285 µg Ag/L and ErC10 was 0.005 µg Ag/L. For yield an EyC50 and EyC10 were 0.01579 and 0.0014 µg Ag/L, respectively. The NOECs for growth rate and for yield was 0.003 µg Ag/L.

Executive summary:

The 72 hour toxicity of silver nitrate to the uni-cellular green alga Raphidocelis subcapitata was determined in a static system (OECD 201) exposed to nominal concentrations of 0.316, 1.00, 3.16, 10.0 and 31.6 µg Ag/L.

The nominal test concentrations were prepared in sterile modified AAP growth medium under sterile conditions. The medium was prepared with reduced EDTA concentrations and compounds including chloride were replaced by nitrate compounds. The concentrations of the test item in the test media were determined by chemical analysis of silver in the aqueous phase of all treatment levels by ICP-MS at test initiation, after 24 h, 48 h and at the test termination of the growth test (LOQ = 0.001 µg/L). Three different types of measurements were conducted: Total Ag, Conventional dissolved Ag after filtration of a subsample through 0.45 µm PSE filters and Truly dissolved Ag after filtration with centrifugal filters at 3000 x g. The particle size and the zeta potential were measured from samples of an extra analytical vessel without algae to characterise the test item in test media at test initiation and test termination. The evaluation of the results was based on the geometric mean measured concentrations of total Ag, conventional dissolved Ag and truly dissolved Ag.

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

For PNEC derivation, reliable EC10 for Pseudokirchneriella subcapitata on the endpoints yield and growth rate of 0.10 μg/L could be extracted from this study (expressed as conventional dissolved Ag).

Endpoint:
effects on growth of green algae
Type of information:
experimental study
Adequacy of study:
key study
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:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Sampling method:
Metal concentrations at the beginning, prior to each water renewal and at the end of the toxicity test were measured in triplicates using an inductively coupled plasma- optical emission spectrometer (ICP-OES; ICP Optima 8300, Perkin Elmer, USA).
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
All stock solutions were prepared by dissolving the respective chemicals in distilled water, and stored in non-transparent glass bottles to minimize degradation during the test periods. The stock solutions were further diluted using sterile pre-dechlorinated tap water or a culture Bold's Bristol medium (BBM) with slight adjustments (i.e., without-/with-0.3 g L-1 EDTA) to obtain working solutions at designated nominal concentrations of Ag for toxicity tests.
Test organisms (species):
Chlorella vulgaris
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): collected from cultures at the University of Hong Kong.
- Age of inoculum (at test initiation): Fresh culture (<72 h)
- Initial cell density / individuals: 50 000

ACCLIMATION
- Acclimation period: 30d
- Culturing media and conditions (same as test or not): same = Bold's Bristol medium (with- or without-EDTA)
- Any deformed or abnormal cells observed: no
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
not applicable
Hardness:
- Medium without EDTA: 477-1140 mg/L
- Medium with EDTA: 85-110 mg/L
Test temperature:
25°C
pH:
6.6
Dissolved oxygen:
6.4-6.5 mg/L
Conductivity:
control: 132-133 microS/cm
test concentrations: 718-745 microS/cm
Nominal and measured concentrations:
Nominal: 0, 0.25, 0.5. 1, 2, 4, 8 microgram/L

Measured concentrations (microgram/L): Medium without EDTA:
0.01 ± 0.01
0.25 ± 0.01
0.50 ± 0.02
0.99 ± 0.02
1.99 ± 0.09
3.98 ± 0.14
7.95 ± 0.33

Measured concentrations (microgram/L): Medium with EDTA:
0.02 ± 0.01
0.25 ± 0.01
0.47 ± 0.04
0.98 ± 0.02
1.97 ± 0.09
3.93 ± 0.17
7.94 ± 0.27
Details on test conditions:
TEST SYSTEM
- Test vessel: test vial
- Test solution volume: 0.02 L
- Renewal rate of test solution: During the test period, 40% of test solutions in each treatment were renewed once every 24 h.
- Initial cells density: 5.0 × 104 cells mL-1
- Control end cells density: 16.3 ± 0.5 (± standard derivation; without EDTA) – 16.7± 0.4 (with EDTA) fold of cell growth in control

GROWTH MEDIUM
- Standard medium used: yes: culture Bold's Bristol medium (BBM) with slight adjustments (i.e., without-/with-0.3 g L-1 EDTA)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: distilled water
- Culture medium same as test medium:

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: 16h light: 8h dark
- Light intensity and quality: 100 foot-candles of continuous cool-white fluorescent light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- growth

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 0, 0.25, 0.5, 1, 2, 4, 8 microgram/L (nominal)
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.9 µg/L
95% CI:
> 0.7 - < 1
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: With EDTA
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.9 µg/L
95% CI:
> 0.8 - < 1
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: without EDTA
Details on results:
- Exponential growth in the control (for algal test): yes
- Concentration-response:
y = 100/(1+10^((3.36 – x) ×2.28)) (without EDTA)
y = 100/(1+10^((3.41 – x) ×2.05)) (with EDTA)
Reported statistics and error estimates:
Wilcoxon’s test was performed for each algal species against Ag to examine whether the overall dissolved metal concentrations differed between the test solutions without- and with-EDTA. One-way analysis of variances (ANOVA) was conducted to test whether there are variances of Ag concentrations during the test durations. All statistical computations were performed using SPSS (version 20, Chicago, IL, USA; significant level α = 0.05).
Since there were no differences of the Ag concentrations at the beginning, prior to each water renewal (for animals) and at the end of the toxicity test (ANOVA, p > 0.05), average of the measured Ag concentrations were used for EC10/EC50 and LC10/LC50 calculations. After constructing a concentration-response relationship, toxicity endpoint values (i.e., EC10/EC50 or LC10/LC50) and their respective 95% CIs were calculated using sigmoidal dose response (variable slope) non-linear regression based on Hill’s model if the relationship was a non-linear one. If the relationship followed a linear concentration-response relationship, a simple linear regression was employed for determining the toxicity endpoints (GraphPad PrismTM, version 5.00, San Diego, CA, USA).
Validity criteria fulfilled:
yes
Conclusions:
The 72 h EC10 (growth) of AgNO3 to Chlorella vulgaris was determined to be 0.9 μg Ag/L
Executive summary:

In a non-GLP, OECD 201, test the 72 h EC10 (growth) of AgNO3 to Chlorella vulgaris was determined to be 0.9 μg Ag/L.

Description of key information

Data for three algae and cyanobacteria species were used to derive the freshwater PNEC by statistical extrapolation. These were an EC10 of 0.10 μg Ag/L for Pseudokirchneriella subcapitata (Mertens et al. 2019), an EC10 of 0.41 µg Ag/L for Anabaena flos-aquae (Arijs et al 2021) and an EC10 of 0.9 µg/L for Chlorella vulgaris (Wang et al. 2019). The lowest reliable chronic value is an EC10 of 0.10 μg Ag/L for Pseudokirchneriella subcapitata. This has been selected as the chronic ERV for classification purposes.

A NOEC of 1.2 µg Ag/L for Champia parvula (Steele et al. 1983) was used as supporting data for the derivation of the marine PNEC.

Key value for chemical safety assessment

Additional information

Key long-term data for two algae and cyanobacteria species are available.

 

Data for Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) are reported by Mertens et al. (2019). The Mertens et al. (2019) study was conducted according to OECD guideline 201 and the principles of GLP. The test media used was modified synthetic AAP growth media, in which EDTA concentrations were reduced, and media components containing chloride were replaced by nitrate compounds. Total and dissolved silver (both <0.45 µm and <3kDa) concentrations were measured in all exposures, The EC10 and EC50 values for inhibition of algal growth rate were 0.1 and 0.96 µg/L <0.45 µm dissolved silver (measured), respectively. The EC10 and EC50 values for inhibition of algal growth rate were 0.005 and 0.285 µg/L <3kDa µm dissolved silver (measured), respectively. The corresponding EC10 and EC50 values for algal yield were 0.1 and 0.24 µg/L <0.45 µm dissolved silver (measured), respectively, and 0.0014 and 0.0157 µg/L <3kDa µm dissolved silver (measured), respectively. For PNEC derivation, a reliable EC10 on the endpoints yield and growth rate of 0.10 μg/L was extracted from this study (expressed as conventional dissolved Ag).

Wang et al. (2019) also exposed Pseudokirchneriella subcapitata to silver nitrate and determined the 72 h EC10 for growth to be 0.6 μg Ag/L; these data were used as supporting data.

 

In a 72 hour study with the cyanobacteria Anabaena flos-aquae exposed to silver nitrate, the EC10 for growth rate was 0.41 µg dissolved Ag/L (Arijs et al. 2021).

 

In a 72 hour study with Chlorella vulgaris exposed to silver nitrate, the EC10 for growth was determined to be 0.9 μg Ag/L (Wang et al. 2019).

The lowest long-term result for marine algae is a 14-day reproductive NOEC of 1.2 µg/L total silver for Champia parvula (Steele and Thursby 1983). Day 0 analytical values were used to derive the toxicity values as the lowest three concentrations were below the reported limit of detection (1.0 µg/L). The Day 0 data for undetectable concentrations were based on the dilution of the volume of standard added to the flasks. EDTA was present at 100 µg/L in the exposure medium and it is likely that some of the silver would have complexed with this and potentially been unavailable to the test organisms. The loss of silver over the 7-day period in the concentrations was approximately 50% (most likely as a result of sorption to algal cells and the test vessel). Similar results are reported by Thursby and Steele (1986) using the same methodology, but without the addition of EDTA. However, no analytical confirmation of the test concentrations was carried out and there is limited reporting of the method and results.

 

These data, together, are considered sufficiently robust to be used as supporting data for determination of the AF when deriving the PNEC, but not directly for PNEC derivation.