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Ecotoxicological information

Toxicity to aquatic plants other than algae

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Reference
Endpoint:
toxicity to aquatic plants other than algae
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:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Vehicle:
yes
Test organisms (species):
Lemna minor
Details on test organisms:
Source: Exponential growing plant monoculture of Lemna minor (Umweltbundesamt, FGIII 2.5, Überwachungsverfahren Abwasserentsorgung, Schichauweg 58, D-12307 Berlin).
Culture: All-glass vessel containing sterile Swedish Standard (SIS) medium (Table 2) and the exponentially growing plant monoculture. Healthy Lemna minor plants consist of colonies comprising 2-5 fronds.
Cultivation: At least seven days before testing, sufficient colonies were transferred aseptically into fresh sterile medium and cultured for 7-10 days under the conditions of the test.
Illumination: Continuous (6500–10000 lux) from Osram Fluora L18W77 (Osram AG, Winterthur, Switzerland) and CH Lighting F18T8/6500K EUP (CH Lighting CO., Ltd., China)
Temperature: 24 ± 2 °C
Control of sensitivity: Yearly, with 3,5-dichlorophenol
Test type:
static
Water media type:
freshwater
Total exposure duration:
7 d
Test temperature:
24 ± 2 °C
pH:
6.5 at the beginning of the test.
Nominal and measured concentrations:
Prior to the definitive test a non-GLP range finding test with the nominal concentrations of 10, 100 and 500 mg/l of Acid Red 062’s active ingredient was performed.
Based on the results of this non-GLP range finding test, the test concentrations for the definitive test were 624, 279, 125, 55.9 and 25.0 mg/l nominal concentration of the test item, corresponding to 500, 223.5*, 100, 44.7 and 20.0 mg/l of the active ingredient (constant spacing factor of 2.235).
Details on test conditions:
Test vessel: 400 ml beakers, all-glass, with 200 ml of test medium. The beakers were covered with black paper up until the 200 ml mark to ensure that illumination comes only from above and not from the sides.
Test medium: Recommended Swedish Standard (SIS) medium (Table 2), prepared with ultra-pure water
Pre-culture: Exponentially growing plant monoculture of Lemna minor (the culture was visibly free from contamination by other organisms such as algae and protozoa).
Illumination: Intensity: continuous (6500–10000 lux) from Osram Fluora L18W77 (Osram AG, Winterthur, Switzerland) and CH Lighting F18T8/6500K EUP (CH Lighting CO., Ltd., China)
Homogeneity: ±15% (target; but see Deviations in section 1.10.2)
Incubation: Beakers were incubated on a black non-reflecting surface.
Reference substance (positive control):
no
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
392 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
357 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: dry weight
Details on results:
Effects on frond numbers
With respect to growth rate inhibition, the following effects as compared to the untreated controls were observed: 74% at 500 mg/l. No significant effects were observed at the concentrations nominal concentrations of 224, 100, 44.7 and 20.0 mg/l (Table 4, Figures 1 and 2).
Based on these effects and the measured concentrations of the active ingredient, the median effect concentration with respect to the frond number’s growth rate (frond number ErC50) of Acid Red 062 to Lemna minor was calculated to be 392 mg/l (95% confidence limits: 390–393 mg/l).  The ErC10 was 237 mg/l (95% confidence limits: 235–240 mg/l); while the NOErC was determined to be 205 mg/l.

Effects on dry weight
With respect to growth rate inhibition, the following effects as compared to the untreated controls were observed: 77% at 500 mg/l, 17% at 224 mg/l and 9% at 44.7 mg/l. No significant effects were observed at the nominal concentrations of 100 and 20.0 mg/l (Table 6, Figure 4).
Based on these effects and the measured concentrations of the active ingredient, the median effect concentration with respect to the dry weight’s growth rate (dry weight r ErC50) of Acid Red 062 to Lemna minor was calculated to be 357 mg/l (95% confidence limits: 240–451 mg/l). The ErC10 was 150 mg/l (95% confidence limits: 32–228 mg/l); while the NOErC was estimated to be 94.7 mg/l (expert judgement based on the fact that the significant 9% effects observed at 44.7 mg/l are solely due to one data point (replicate A) showing much higher effects than all other replicates of 20.0, 44.7 and 100 mg/l.

Appearance of the plants at the end of the test

In the blank controls and the 20.0, 44.7 and 100 mg/l test concentrations, the plants all looked healthy and no clear difference differences could be noted.

At 224 mg/l, the plants looked a bit paler.

At 500 mg/l, the plants did not grow and showed red leaves (signs of chlorosis). This appearance was already noted after 2 days.

Validity criteria fulfilled:
yes
Conclusions:
EC50 = 392 mg/l with respect of frond number
Executive summary:

The toxicity to the aquatic plant Lemna Minor were determined following OECD 221.

The results don't show any toxic effects to Lemna minor under the test conditions.

Description of key information

EC50 = 392 mg/L

Key value for chemical safety assessment

EC50 for freshwater plants:
392 mg/L

Additional information

A test was performed on the tested substance following OECD 221.

The results show that the tested item is not toxic to Lemna minor with EC50 =392 mg/L.

Lemna minor is an aquatic plant that develops his leaves on the surface of the water, while nourishing substances are taken from the water solution. With this tests the observed effect is only related to the potential toxicity of the substance and not to the potential shading effect of a classical Alga study. A deviation to the protocol has been applied to the test recommended for dyes (Michael Cleuvers a, Hans-Toni Ratte, Phytotoxicity of coloured substances: is Lemna Duckweed an alternative to the algal growth inhibition test? Chemosphere 49 (2002) 9–15): "Beakers will be incubated on a black non-reflecting surface. Additionally, the walls of the incubation chambers will also be covered with black fabric in order to avoid reflection".