Silver chloride

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

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 221 (Lemna sp. Growth Inhibition Test)

Deviations:

yes

Remarks:

a modified Swedish Standard (SIS) Lemna growth medium was used: 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)

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) in fresh media (samples taken before medium renewal) were taken on days 0, 3, and 5 and in old media (samples taken after medium renewal) on days 3, 5, and 7. Samples for analysis of Dissolved Organic Carbon (DOC) of fresh media (samples taken before medium renewal) were taken at test initiation and of old media (samples taken after medium renewal) on day 3. Samples for measurement of major anions for every test concentration were taken at test initiation and test termination.

Vehicle:

no

Test organisms (species):

Lemna minor

Details on test organisms:

The aquatic freshwater plant L. minor originated from an in-house culture which was initiated in 2015 from plants purchased at Blades Biological. Plants were acclimated for 2 wk prior to test initiation in the modified growth medium at 24 °C.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

10.4 ± 0.5 mg Ca/L and 3.8 ± 0.3 mg Mg/L (42 mg/L CaCO3)

Test temperature:

24.7± 0.9 °C

pH:

pH fresh test medium: 7.1 ± 0.1
pH throughout test: 7.4 ± 0.25

Dissolved oxygen:

85 ± 4%

Nominal and measured concentrations:

Nominal concentrations: control medium, 0.32, 1, 3.2, 10, 32, 100, 320, and 1000 µg Ag/L.
Measured concentrations (geometric mean dissolved Ag): <0.035, 0.13, 0.42, 1.30, 4.31, 18.3, 60.4, 243 and 336 µg/L.

Total Ag concentrations in fresh medium were 0 to 30% lower than nominal concentrations. Losses of Ag in the fresh solutions are most likely due to adsorption of Ag to storage vessels despite the use of polypropylene vessels . At the highest Ag dose level (1000 µg nominal Ag/L solution), only 54% of nominal Ag was recovered in the total fraction, suggesting that Ag solubility may have been exceeded in this solution and that Ag precipitation may have occurred. This hypothesis is consistent with the yellowish color at this dose level compared to uncolored solutions in the other treatments and with speciation modeling using Visual MINTEQ, suggesting that a significant fraction of Ag is precipitating as AgCl. Therefore, this treatment was not further considered in any of the concentration–response fittings. Dissolved concentrations in fresh solutions of the 0.32 to 320 µg nominal Ag/L treatments were on average 17 ± 7% lower than total concentrations in fresh solutions. Total concentrations decreased during the exposure to 55 ± 8% (average concentrations in old solutions ± standard deviation) of the total Ag concentrations in fresh solutions. Dissolved Ag concentrations in old solutions were on average 15 ± 7% lower than the total Ag concentrations in old solutions and 44 ± 7% lower than dissolved Ag concentrations in fresh solutions. Hence, as recommended in the OECD protocol, concentration–response analysis was based on the geometric mean of the measured dissolved Ag concentrations in fresh and old solutions. Geometric mean dissolved Ag concentrations were on average 51 ± 14% lower than nominal concentrations.

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 food-grade polypropylene vessels of 300 mL filled with 100 mL of the test medium and covered with transparent punctured polypropylene covers.
At test initiation, each test unit received 4 plants, each with 3 fronds (i.e., 12 fronds per vessel). The toxicity test was incubated in a 24 °C growth chamber under continuous light (99–112 µmol/m2/s).
The test was semistatic, with complete test medium renewals on days 3 and 5.
Growth of L. minor was monitored during the exposure period as number of fronds and total frond area. Total frond area was determined based on image analysis using a Nikon D5300 camera under strong back-lighting and linking the pixel density of L. minor leaves to the actual frond area using the photo software Image-J. At test termination, the root length was measured using a slide caliper (VWR) on 10 randomly selected plants per test vessel, and the dry weight (after 3 d drying at 55 °C) of all plant colonies (fronds and roots) was measured using a Mettler Toledo balance (AX 105). Growth rate was expressed on the basis of the total frond numbers and total frond area. Growth rate was calculated based on the slope of the relation between ln(Xti) and ti, where Xti is the number of fronds or total frond area (in square millimeters) at time ti and ti is the time since the test initiation.

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

14 µg/L

95% CI:

>= 7 - <= 29

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (dissolved fraction)

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

5.2 µg/L

95% CI:

>= 3.2 - <= 8.5

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (dissolved fraction)

Basis for effect:

frond area

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

1.4 µg/L

95% CI:

>= 0.4 - <= 4.2

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (dissolved fraction)

Basis for effect:

total root length

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

19 µg/L

95% CI:

>= 3.9 - <= 91.9

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (dissolved fraction)

Basis for effect:

dry weight

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.
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 average^a growth rate in the different exposure treatments of the 7d-Lemna minor tests for the growth rate endpoints (r_n& r_a), root length and dry weight.

Nominal Ag	Dissolved Ag in fresh solutions^b (µg/L)	Dissolved Ag in old solutions^b (µg/L)	Geometric mean dissolved Ag	Growth rate (frond number) rn	Growth rate (frond area) ra	Root length	Dry weight
(µg/L)			(µg/L)	(d^-1)	(d^-1)	(mm)	(mg)
0	<0.035	<0.035	<0.035	0.40±0.02	0.30±0.02	9.8±1.8	5.2±1.0
0.32	0.18±0.01	0.09±0.01^c	0.13	0.41±0.01	0.31±0.01	10.8±0.9	6.4±0.2
1	0.59±0.04	0.30±0.04	0.42	0.40±0.003	0.30±0.004	10.2±1.0	6.4±0.6
3.2	1.8±0.2	0.97±0.07	1.30	0.41±0.02	0.30±0.01	9.2±0.6	6.3±0.8
10	6.0±1.6	3.4±0.5	4.31	0.36±0.02	0.28±0.02	8.2±0.7	5.4±0.8
32	28±4	14±5	18.3	0.36±0.002	0.24±0.02	5.2±0.5	4.8±0.3
100	84±6	48±13	60.4	0.31±0.01	0.20±0.01	3.8±0.4	3.3±0.3
320	292±17	206±27	243	0.27±0.02	0.14±0.02	3.7±0.7	2.3±0.3
(1000)d^c	351±9	333±55	336	(0.29±0.01)	(0.17±0.004)	(3.4±0.6)	(3.1±0.3)

^aAverage of all replicates ± standard deviation is reported.

^bAverage of three samples± standard deviation is reported.

^cBelow limit of quantification of ICP-MS (Limit of quantification 0.12 µg/L)

^dThe responses of the 1000 µg nominal Ag/L treatment were not taken into account for concentration response fitting, as measurements of actual silver concentrations indicates that silver precipitation likely occurred in the exposure solution.

Above table shows that all endpoints showed a clear concentration–response behavior with increasing Ag dosing levels.

The concentration response of the dry weight endpoint showed a significant hormesis effect. This effect was not observed for any of the other endpoints. The corresponding effect concentrations are reported in the table below.

Effect concentrations (expressed as the geometric mean of the measured dissolved Ag concentrations in fresh and old solutions) of ionic silver (Ag) to the aquatic species Lemna minor.

Endpoint	EC10 (µg Ag/L)	EC20 (µg Ag/L)	EC50 (µg Ag/L)	NOEC (µg Ag/L)	LOEC (µg Ag/L)
Growth rate (frond number)	14 (7-29)	62 (42-92)	769* (381-1550)	1.3 (-1.8±5.4)	4.3 (9.9±5.0)
Growth rate (frond area)	5.2 (3.2-8.5)	18 (13-25)	159 (124-205)	1.3 (0.4±3.5)	4.3 (9.5±5.3)
Root length	1.4 (0.4-4.2)	4.8 (2.2-10.5)	42 (25.1-68.9)	4.31 (16.1±7.5)	18.3 (47.5±5.4)
Dry weight	19.0 (3.9-91.9)	41.8 (14.5-120.4)	162 (78-336)	18.3 (6.6±4.9)	60.4 (35.8±4.9)

EC_xvalues were calculated using a log-logistic concentration response model with 2 parameters.

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.

^*Extrapolated outside the tested concentration range (geometric mean of the highest silver treatment used for concentration response fitting was 243 µg/L).

Validity criteria fulfilled:

yes

Remarks:

Average doubling time in the control treatments: 1.75 ± 0.10 d. Average control growth rate: 0.40 ± 0.02 d–1 (rn) and 0.30 ± 0.02 d–1 (ra). Average end root length in control: 9.8 ± 1.8 mm, average control dry weight : 5.2 ± 1.0 mg.

Conclusions:

Based on the ECx values, root length was the most sensitive endpoint after 7d exposure of L. minor to silver nitrate.. The EC10 and EC50 values for root length were 1.4 and 41.6 µg dissolved Ag/L, respectively. The growth rate (rn and ra) had the lowest NOEC and LOECs: 1.3 (NOEC) and 4.3 (LOEC) µg dissolved Ag/L. The intratreatment variation was generally higher for the root length and dry weight endpoints.

Executive summary:

In a 7 day study with the aquatic plant Lemna minor exposed to silver nitrate, the EC10 for the most sensitive endpoint (root length) was 1.4  µg dissolved Ag/L.

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

Description of key information

Key long-term data are available for the aquatic plant species Lemna minor: an EC10 of 1.4 µg/L Ag (Arijs et al. 2021).

Additional information

Key long-term data for one species of macrophyte are available. In a 7 day growth inhibition study (performed according to OECD Guideline 221) with the aquatic plant Lemna minor exposed to silver nitrate, the EC10 for the most sensitive endpoint (root length) was 1.4  µg dissolved Ag/L (Arijs et al. 2021).