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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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
February from 10 to 17, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
The read across approach is detailed into the document attached into the "information panel" of the Aquatic Toxicity endpoint summary.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
adopted 23th March 2006
Deviations:
yes
Remarks:
temperature ranged between 18.4 and 26.4°C, which is out of the required range of 24 ± 2 °C; the deviation did not impact the test results
Qualifier:
according to guideline
Guideline:
other: EU Method C.26 (Lemna sp. Growth Inhibition Test)
Version / remarks:
7 December 2015
Deviations:
yes
Remarks:
temperature ranged between 18.4 and 26.4°C, which is out of the required range of 24 ± 2 °C; the deviation did not impact the test results
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Analysis of the test concentrations were conducted at the beginning of the test, as well as after 3, 5 and 7 days of exposure.
Details on test solutions:
Since the test item resulted to be soluble, the test solutions were prepared by respective dilutions of a stock solution (test item dissolved in SIS Medium with SIS Medium.
Test organisms (species):
Lemna minor
Details on test organisms:
TEST ORGANISM
- Common name: duckweed.
- Source: exponential growing plant monoculture of Lemna minor, from Umweltbundesamt, FGIII 2.5, Überwachungsverfahren Abwasserentsorgung, Schichauweg 58, D-12307 Berlin.

CULTIVATION
- Cultivation: all-glass vessel containing sterile Swedish standard-Medium
- 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
Limit test:
no
Total exposure duration:
7 d
Test temperature:
The temperature ranged between 18.4 and 26.4 °C (i.e. 6.0 °C variation), which is out of the required range of 24 ± 2 °C. Since the test vessels were randomly re-placed, since all test vessels were exposed to the same temperature, since the toxicity is calculated based on the blank control, and since the growth criterion in the control was fulfilled without a too large variation, this deviation is not expected to have a significant impact on the outcome of the study.
The test item did not show any signs of precipitation, at any of the test concentrations.
pH:
The pH value in the control drifted by 0.6units during the whole test period, which is therefore in the range allowed by the guideline (required: not more than 1.5).
Nominal and measured concentrations:
611, 193, 61.1, 19.3 and 6.11 mg/l nominal concentration of the test item, corresponding to 500, 158.1, 50.0, 15.8 and 5.00 mg/l of the active ingredient
Details on test conditions:
TEST SYSTEM
- Test vessel: 400 ml beakers, all-glass, with 200 ml of test medium.
-- Type of cover: 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.
- Incubation: beakers were incubated on a black non-reflecting surface.
- No. of frond per plant: 2-4 fronds per plant.
- No. of fronds per replicate: 9-12 fronds per replicate.
- No. of vessels per concentration: three replicates.
- No. of vessels per control : six replicates.

GROWTH MEDIUM
- Standard medium used: yes; Swedish standard-Medium.

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: Swedish standard-Medium, prepared with ultra-pure water.
- Adjustment of pH: the pH of the SIS Medium was adjusted before the test to pH 6.5 ± 0.2.

OTHER TEST CONDITIONS
- Photoperiod: continuous light.
- Light intensity and quality: the light intensity was 7500-8250 lux (mean 7939 lux; max. variation ± 6 %) at the start of the test and 6950-8340 lux (mean 7663 lux, max. variation ± 9 %) at the end of the exposure period.
- Homogeneity: the light homogeneity was in the required ±15 % range.

EFFECT PARAMETERS MEASURED
- Observations: the number of fronds was counted on day 0, 3, 5 and 7. Changes in plant development, e.g. in frond size, appearance, indication of necrosis, chlorosis or gibbosity, colony break-up, loss of buoyancy, root length and appearance were noted. Significant changes in the test medium (e.g.precipitation, growth of algae in the test vessel) were also noted.
- Dry weight: determined from duckweed at day 0 in six additional blank replicates (similar to those used in the test), and at day 7 in all blanks and all test vessels, after drying at 60 °C at least overnight.

CONDITIONS MEASUREMENT
- pH: Determined in the combined replicate test solutions at the beginning and at the end of the test.
- Light intensity: Measured at the beginning and at the end of the test over the whole test area at points the same distance from the light source as the Lemna fronds.
- Temperature: measured in an additional glass vessel containing test medium and Lemna, at least daily.

RANGE-FINDING STUDY
Prior to the definitive test a non-GLP range finding test was performed.
- Test concentrations: 10, 100 and 500 mg/l of test item active ingredient (nominal). The photometry determinations indicate that the test item concentrations were stable. Static conditions were therefore applied in the definitive test.
- Results used to determine the conditions for the definitive study: the percentages of inhibition, based on frond number were
at 10 mg/l: 3.4 % growth rate inhibition, 8.1 % yield inhibition
at 100 mg/l: 35 % growth rate inhibition, 61 % yield inhibition
at 10 mg/l: 59 % growth rate inhibition, 82 % yield inhibition
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
615 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
889 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
197 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
178 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
EFFECTS ON FROND NUMBER
With respect to growth rate inhibition, the following effects as compared to the untreated controls were observed: 39 % at 500 mg/l and 23 % at 158 mg/l. No significant effects were observed at the concentrations 50.0, 15.8 and 5.00 mg/l.
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 test item to Lemna minor was calculated to be 615 mg/l (95 % confidence limits: 439–1115 mg/l). The ErC10 was 62.0 mg/l (29.6–92.3 mg/l); while the NOErC was determined to be 49.1 mg/l.

With respect to yield inhibition, the following effects as compared to the untreated controls were observed: 66 % at 500 mg/l and 46 % at 158 mg/l. No significant effects were observed at the concentrations 50.0, 15.8 and 5.00 mg/l.
Based on these effects and the measured concentrations of the active ingredient, the median effect concentration with respect to the frond number’s yield (frond number EyC50) of test item to Lemna minor was calculated to be 197 mg/l (95 % confidence limits: 141–300 mg/l). The EyC10 was 29.2 mg/l (9.26–50.6 mg/l); while the NOEyC was determined to be 49.1 mg/l.

EFFECTS ON DRY WEIGHT
With respect to growth rate inhibition, the following effects as compared to the untreated controls were observed: 34 % at 500 mg/l and 23 % at 158 mg/l. No significant effects were observed at the concentrations 50.0, 15.8 and 5.00 mg/l.
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 ErC50) of test item to Lemna minor was calculated to be 889 mg/l (95 % confidence limits: 546–2429 mg/l). The ErC10 was 57.5 mg/l (22.5–91.2 mg/l); while the NOErC was determined to be 49.1 mg/l.

With respect to yield inhibition, the following effects as compared to the untreated controls were observed: 65 % at 500 mg/l and 52 % at 158 mg/l. No significant effects were observed at the concentrations 50.0, 15.8 and 5.00 mg/l.
Based on these effects and the measured concentrations of the active ingredient, the median effect concentration with respect to the dry weight’s yield (dry weight EyC50) of test item to Lemna minor was calculated to be 178 mg/l (95 % confidence limits: 124–280 mg/l). The EyC10 was 20.8 mg/l (6.01–38.0 mg/l); while the NOEyC was determined to be 49.1 mg/l.

APPEARANCE OF THE PLANTS AT THE END OF THE TEST
In the blank control the plants were healthy with dark green fronds and roots reaching to the bottom of the test vessel.
At 5.00 mg/l, the plants looked like the blanks, with dark green fronds and roots reaching to the bottom of the test vessel.
At 15.8 and 50.0 mg/l, the plants were healthy with dark green fronds and roots reaching to the bottom of the test vessel; the plants were slightly smaller compared to the blank control.
At 158 mg/l, the fronds were dark green and somehow misshapen; the size of the roots were only two thirds of the blank control and the plants were significantly smaller as compared to the blank controls.
At 500 mg/l, the plants werevery small, with plants and fronds partly blue discolored; the size of the roots were only two thirds of the blank control.

TEST CONCENTRATIONS
The determination of test cioncentrations showed that the test item decreased over the whole 7-day test period. The measured concentrations of the active ingredient at the beginning of the test were 514, 163, 49.3, 16.0 and 4.93 mg/l and 345, 113, 50.6, 14.3 and 4.24 mg/l (i.e. 67, 69, 103, 90 and 86 %, respectively, of the initial value) after 7 days.
Therefore, the effective concentrations (ErC50 and EyC50) were assessed based on the geometric mean (GM) of the measured concentrations of the active ingredient.
Validity criteria fulfilled:
yes
Remarks:
doubling time of frond number in the control was be less than 2.5 days (60 h), corresponding to approximately a seven-fold increase in seven days and an average specific growth rate of 0.275 d-1
Conclusions:
ErC50 (7d): 615 mg/l (meas. geom mean), based on frond number
EyC50 (7d): 197 mg/l (meas. geom mean), based on frond number
Executive summary:

The inhibitory effects of test item to the duckweed Lemna minor were investigated over a period of 7 days, based on the frond number and biomass (dry weight), following the guideline OECD 221. The test solutions were prepared by respective dilutions of a stock solution in Swedish standard-Medium (SIS Medium).

The test was performed at 611, 193, 61.1, 19.3 and 6.11 mg/l nominal concentration of the test item, corresponding to 500, 158.1, 50.0, 15.8 and 5.00 mg/l of the active ingredient.

Three parallel test vessels were used for each test concentration of the test item and six vessels for the blank controls.

The test concentrations of test item during the 7-day test period were determined by photometry at the beginning of the test, as well as after 3, 5 and 7 days of exposure. These analyses confirmed the right dosage of the test item, and showed that the concentrations of the loadings decreased over the whole 7-day test period after 7 days and did not remain within 80-120 % range of the initial value (67, 69, 103, 90 and 86 %, respectively, of the initial value). Therefore, the effective concentrations (ErC50 and EyC50) were assessed based on the geometric mean (GM) of the measured concentrations of the active ingredient.

The two endpoints frond number and biomass (dry weight) were investigated at days 3, 5 and 7, and each of them were assessed as growth rate and yield.

The results of the toxicity assessment of to the duckweed Lemna minor are summarized below, showing ECx values based on the measured concentrations of active ingredient:

ErC50 (7d): 615 mg/l (meas. geom mean), based on frond number

ErC50 (7d): 889 mg/l (meas. geom mean), based on dry weight

EyC50 (7d): 197 mg/l (meas. geom mean), based on frond number

EyC50 (7d): 178 mg/l (meas. geom mean), based on fdry weight

The No Observed Effect Concentration was 49.1 mg/l in all the cases (determined by Dunnett's test).

Conclusion

ErC50 (7d): 615 mg/l (meas. geom mean), based on frond number

EyC50 (7d): 197 mg/l (meas. geom mean), based on frond number

Description of key information

ErC50 (7d): 615 mg/l (meas. geom mean), based on frond number

EyC50 (7d): 197 mg/l (meas. geom mean), based on frond number

Key value for chemical safety assessment

EC50 for freshwater plants:
615 mg/L

Additional information

There is no information about the potential toxicity of Acid Violet 048 to acquatic plants, thus the available information on the structural analogous Similar Substance 01 has been taken into consideration; the read across approach can be considered as appropriate and suitable to assess the property under investigation (details about the approach are reported into the document attached into the "information panel" of the Aquatic Toxicity endpoint summary).

The inhibitory effects of test item to the duckweed Lemna minor were investigated over a period of 7 days, based on the frond number and biomass (dry weight), following the guideline OECD 221. The test concentrations of test item during the 7-day test period were determined by photometry; the analyses showed that the concentrations of the loadings decreased over the whole 7-day test period and did not remain within 80-120 % range of the initial value (67, 69, 103, 90 and 86 %, respectively, of the initial value). Therefore, the effective concentrations (ErC50 and EyC50) were assessed based on the geometric mean (GM) of the measured concentrations of the active ingredient.

The results of the toxicity assessment of to the duckweed Lemna minor are summarized below, showing ECx values based on the measured concentrations of active ingredient:

ErC50 (7d): 615 mg/l (meas. geom mean), based on frond number

ErC50 (7d): 889 mg/l (meas. geom mean), based on dry weight

EyC50 (7d): 197 mg/l (meas. geom mean), based on frond number

EyC50 (7d): 178 mg/l (meas. geom mean), based on fdry weight

The No Observed Effect Concentration was 49.1 mg/l in all the cases (determined by Dunnett's test).

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