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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
Remarks:
HPLC-MS/MS
Details on sampling:
Analytical samples were taken at 0 hours (initial value) from fresh test solutions and after 7 days from aged test solutions from all test concentrations and control. For each sampling also a retain sample was taken.
Vehicle:
no
Details on test solutions:
Application
The necessary amount of test item for preparing a stock solution was weighed on a weighing scoop and transferred to a volumetric flask. Test medium (See Appendix B) was added up to the bench mark and the solution was homogenised by shaking. Afterwards the solution was clear and blue. Lower test solutions were prepared by dilution of appropriate solutions with test medium

Sample Work-Up Procedure
After the receipt in the analytical laboratory, the test medium samples (10 mL) were stored deep-frozen (≤ - 18 °C) until analysis. At the day of analysis, the samples were thawed to ambient temperature and 10 mL of acetonitrile were added and the samples were shaken well using a Vortex-Mixer. If necessary, the samples were further diluted with acetonitrile/test medium (1:1, v/v) prior to analysis by HPLC-MS/MS.
Test organisms (species):
Lemna gibba
Details on test organisms:
The test organism was the aquatic plant duckweed, Lemna gibba G 3 (Alismatales: Araceae). The plants used in this study were taken as aseptic clones from the cultivation of the testing facility. They were primarily cultured by Dr. Janet Slovin, Horticulture Crops Quality Laboratory, U.S. Department of Agriculture. BARC-West, Bldg. 050 HH-4, Beltsville, MD 20705, U.S.A.
The plants were cultured in appropriate glass beakers under continuous illumination with OSRAM light tubes (L30/32-930 and L30/W72-965), at 6500 - 10000 lux. The temperature was 24 ± 2 °C. Before the test was started, plants were cultured under the same illumination and temperature conditions as those to be used for the toxicity test. Young, light-green plants of similar size and comprising 2 - 4 fronds were transferred onto fresh medium and cultured for 7-10 days prior to testing, with two further transfers onto fresh medium before initiating the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
23.8 - 24.5 °C
pH:
fresh solution: 7.49 - 7.53
aged solution: 8.70 - 9.07
Nominal and measured concentrations:
Nominal: 57.9, 11.6, 2.31, 0.463, 0.0926, 0.0185 mg/L and control. Considering the purity of the test item of 86.4 % w/w this corresponds to 50.0, 10.0, 2.00, 0.400, 0.0800 and 0.0160 mg pure substance/L.
Measured concentrations (geometric mean) [mg/L]: 59.6, 11.4, 1.94, 0.273, 0.0463, 0.00611
Details on test conditions:
Test Conditions
- Test procedure: Static test
- Duration: 7 days
- Temperature: 23.8 – 24.5 °C
- pH-value of fresh solution: 7.49 – 7.53
- Light intensity (mean): 7760 lux
- Light tubes: Light cartridge with T8- luminescent tubes (daylight, 900 mm), LT30W/865 daylight
- Exposure to light: Continuously
- Number of fronds: 12 per test vessel

Test design
Colonies consisting of 2 - 4 fronds were transferred from the inoculum culture into the test vessels containing a total of 12 fronds, each. The size of plants and fronds were similar in appearance in each test vessel. A randomised location of the test vessels was used to minimise the influence of spatial differences in light intensity or temperature in the incubation chamber. A 7-day static test design was employed. Three replicates were used for each test item concentration, six replicates for the control.

Test Medium
The medium used for the test was 20xAAP Medium for Lemna gibba (according to OECD 221, composition see Appendix B). The pH was adjusted to 7.5 ± 0.1, if necessary.

Test Units
Test vessels were 250 mL glass beakers each containing ~150 mL test solution to allow the plants to grow without touching the sides of the glass vessels.

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
0.069 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
frond number
Remarks:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 59.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
frond number
Remarks:
growth rate
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
frond number
Remarks:
growth rate
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
0.139 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks:
growth rate (dry weight)
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 59.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks:
growth rate (dry weight)
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.046 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks:
growth rate (dry weight)
Details on results:
The measured content of special Brilliant Blue FFR was between 65 % and 98 % of nominal in the fresh samples with a mean initial concentration of 81 % of nominal. In the aged samples the measured content was between < LOQ and 108 % of nominal with a mean measured concentration of 66 % of nominal.
Results with reference substance (positive control):
3,5-Dichlorophenol is tested as the toxic reference item in a separate study twice a year to confirm the sensitivity of the test organism against compounds with known effects under the test conditions. The last test was perfomed in May 2017.
The EC50 for growth rate of frond numbers was determined as 6.78 mg/L and for growth rate of dry weight as 4.63 mg/L.
Reported statistics and error estimates:
A test for normality of the data was performed by calculating the Shapiro-Wilk’s statistic, a test for homogeneity of the data was performed according to Levene. The NOEC and LOEC were determined by using a multiple comparison method (Dunnetts-t-test, left sided, yield of dry weight and growth rate of dry weight, Jonckheere Terpstra test, left sided, for yield of frond numbers and growth rate of frond numbers). The EC10, 20, 50-values were determined by probit analysis following normal distribution. The calculation of the EC50 for growth rate of frond numbers and growth rate of dry weight was not indicated since the inhibition was below 50 % at the highest test item concentration. The evaluation of data was performed by SAS® (2002-2010).
Validity criteria fulfilled:
yes
Remarks:
doubling time of frond numbers in the control was less than 2.5 days
Conclusions:
For Special Brilliant Blue FFR the following values were determined after 7 d towards Lemna gibba under exposure conditions. The EC50-value for growth rate of frond numbers and growth rate of dry weight was determined to be > 59.4 mg/L for both. The NOEC for growth rate of frond numbers was set at 0.00611 mg/L and for growth rate of dry weight at 0.0463 mg/L.
Executive summary:

A study was conducted to assess the toxicity of Special Brilliant Blue FFR against Lemna gibba G3 according to OECD 221 (23 March 2006) 'Lemna, Growth Inhibition Test' to investigate the aquatic phytotoxic effect of Special Brilliant Blue FFR. To achieve this, a series of test item concentrations in aqueous solutions was prepared (measured based on active ingredient (geometric mean) 59.6, 11.4, 1.94, 0.273, 0.0463, 0.00611 mg/L), and the plants were allowed to grow in these concentrations, as well as in controls without the test item, for a test period of 7 days under controlled conditions.

Colonies consisting of 2 - 4 fronds were transferred from the inoculum culture into the test vessels containing a total of 12 fronds, each. Test solutions were prepared with 3 replicates, negative control was prepared with 6 replicates. The test volume was 150 mL.

Frond numbers and the appearance of the colonies were checked on t = 0, 3, 5 and 7 days as well as any change in plant development, frond size, necrosis and additional observations of test media or other abnormalities. The dry weight of the fronds was determined at the end of the test. The NOEC and LOEC were determined by using a multiple comparison method (Dunnetts-t-test, left sided, yield of dry weight and growth rate of dry weight, Jonckheere Terpstra test, left sided, for yield of frond numbers and growth rate of frond numbers). The EC10, 20, 50-values were determined by probit analysis following normal distribution. The calculation of the EC50 for growth rate of frond numbers and growth rate of dry weight was not indicated since the inhibition was below 50 % at the highest test item concentration. The evaluation of data was performed by SAS® (2002-2010).

After 7 d an EC 50 > 59.6 mg/L was determined for frond number and dry weight (both growth rate) of Lemna gibba G3 under exposure conditions. The lowest NOEC and lowest EC10 were determined for frond number (growth rate) to be 0.00611 mg/L and 0.0685 mg/L, respectively.

It is stated in OECD 221 that for Lemna "toxicity estimates should be based on frond number and one additional measurement variable (total frond area, dry weight or fresh weight), because some substances may affect other measurement variables much more than the frond number". Thus the frond number (growth rate) was taken as the main endpoint of this study, as this is the most sensitive endpoint.

Description of key information

A study was conducted to assess the toxicity of Special Brilliant Blue FFR against Lemna gibba G3 according to OECD 221 (23 March 2006) 'Lemna, Growth Inhibition Test' to investigate the aquatic phytotoxic effect of Special Brilliant Blue FFR. To achieve this, a series of test item concentrations in aqueous solutions was prepared (measured based on active ingredient (geometric mean) 59.6, 11.4, 1.94, 0.273, 0.0463, 0.00611 mg/L), and the plants were allowed to grow in these concentrations, as well as in controls without the test item, for a test period of 7 days under controlled conditions.

Colonies consisting of 2 - 4 fronds were transferred from the inoculum culture into the test vessels containing a total of 12 fronds, each. Test solutions were prepared with 3 replicates, negative control was prepared with 6 replicates. The test volume was 150 mL.

After 7 d an EC 50 > 59.6 mg/L was determined for frond number and dry weight (both growth rate) of Lemna gibba G3 under exposure conditions. The lowest NOEC and lowest EC10 were determined for frond number (growth rate) to be 0.00611 mg/L and 0.0685 mg/L, respectively.

It is stated in OECD 221 that for Lemna "toxicity estimates should be based on frond number and one additional measurement variable (total frond area, dry weight or fresh weight), because some substances may affect other measurement variables much more than the frond number". Thus the frond number (growth rate) was taken as the main endpoint of this study, as this is the most sensitive endpoint.

Key value for chemical safety assessment

EC50 for freshwater plants:
59.6 mg/L
EC10 or NOEC for freshwater plants:
0.006 mg/L

Additional information

Should read: EC50 > 59.6 mg/L, NOEC >= 0.00611 mg/L