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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:
From February 8th to 15th, 2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
source study has reliability 1
Justification for type of information:
Justification for Read Across is detailed in the IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
For determination of test item concentrations, samples were taken from each concentration level and from the control at the start and at the end of each renewal periods (renewals on day 3 and 5).
Concentrations were analysed by HPLC method with UV/VIS detection.
Vehicle:
no
Details on test solutions:
Test item stock solutions were first prepared by mechanical dispersion (at the start of each renewal period) of 0.1320 g of test item in 1000 ml test medium in the first renewal period, 0.1323 g of test item in 1002 ml medium in the second renewal period and 0.1325 g test item in 1003 ml medium in the third renewal period (OECD medium), to obtain the nominal concentration of 132.0 mg test item/l.
The stock solutions were placed into an ultrasonic bath for approximately 10 minutes. The further test solutions were prepared by appropriate dilution of these stock solutions.
Dilution water (OECD medium) was used as untreated control solution.
The test solutions were freshly prepared in the testing laboratory just before introduction of plants (at the start of each renewal period).
Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Strain: l emna gibba (G3)
- Source (laboratory, culture collection): Friedrich Schiller Universität, Institut für Allgemeine Botanik und Pflanzenphysiologie, Jena, Germany
- Justification of strain: lemna gibba is a fast-growing species. The small size, the simple structure, vegetative reproduction and the short generation time makes it suitable and convenient for culturing and testing.

ACCLIMATION
- Preculture: 7 days before testing,
- Culturing media and conditions: sufficient colonies are transferred from the stock culture aseptically into fresh sterile medium (OECD medium) and cultured under the conditions of the test prior to beginning the test.

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
The cultures were maintained at a temperature in the range of 24 ± 2 °C which was checked at the beginning of the study and every 24 hours (in a surrogate flask filled with water in the climate chamber).
In addition, the temperature was continuously measured (with a min/max thermometer) within the climate chamber during the experimental period.
T: 22.3 - 25 °C in climate chamber; 23 - 23.4 °C in test vessels.
pH:
The pH was checked at the start and at the end of each renewal period in the test concentrations and the control. The pH of the control medium did not increase by more than 1.5 units during the test.
pH: 8.68 - 8.12
Nominal and measured concentrations:
Nominal concentrations: 0.4, 1.3, 4, 12.9, 41.3, 132 mg/l
Geometric mean of measured concentrations: 0.04, 0.10, 1.95, 8.17, 31.73 and 125.72 mg/l
Details on test conditions:
TEST SYSTEM
- Incubation chamber used: climate chamber
- Test vessel: all-glass beakers with 400 ml total capacity
- Fill volume: 160 ml
- Type of cover: glass petri dish
- Agitation: no
- Renewal rate of test solution: on day 3 and 5
- Control end cells density:
- No. of colonies per vessel: 2 colonies with 4 fronds and 1 colony with 3 fronds per vessel
- No. of fronds per colony: 11
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM (same as test medium)
- Standard medium used: OECD medium (OECD 201)
- Details: the reason of the medium choice is that the test item dissolved in 20XAAP medium precipitates from the solution after approximately 24 hours (also after filtration through a 0.45 μm membrane filter); however, the test item dissolves properly in OECD medium and did not precipitate. Although the use of OECD medium is a deviation from the guideline, it is considered to have not any presumed effect on the study and that the study can be considered scientifically valid.
Reconstituted growth medium (OECD medium, according to OECD 201) was used as dilution water in the experiment.
Separate stock solutions were first prepared in deionised water. The growth medium was prepared by adding an appropriate volume of:
1- stock solution 1 (macronutrients)
2- stock solution 2 (iron)
3- stock solution 3 (trace elements)
4- stock solution 4 (bicarbonate)

WATER PARAMETERS
Substance Final concentration in the prepared growth medium
NH4Cl 15.0 mg/l
MgCl2 5.6 mg/l*
CaCl2 × 2 H2O 18.0 mg/l
MgSO4 × 7 H2O 15.0 mg/l
KH2PO4 1.6 mg/l
FeCl3 × 6 H2O 64.0 µg/l
Na2EDTA × 2H2O 100.0 µg/l
H3BO3 185.0 µg/l
MnCl2 × 4 H2O 415.0 µg/l
ZnCl2 3.0 µg/l
CoCl2 × 6 H2O 1.5 µg/l
CuCl2 × 2 H2O 0.01 µg/l
Na2MoO4 × 2 H2O 7.0 µg/l
NaHCO3 50.0 mg/l

OTHER TEST CONDITIONS
- Photoperiod: continous
- Light intensity and quality: 6500 - 10000 lux using fluorescent light tubes (spectral range 400-700 nm); differences in light intensity between measurements points did not exceed ± 15 %, thus providing equal conditions for each culture.

EFFECT PARAMETERS MEASURED
- Determination of frond number: yes
- Determination of dry weight: test item was adsorbed to the roots and to the underside of the leaves in each concentration level during the experiment, thus dry weight results were unusable for statistical evaluation (calculation of ECx values, determination of NOEC and LOEC values).

RANGE-FINDING STUDY
First test
It was a static test. The used test solutions were prepared by mechanical dispersion. 0.0611 g of test item was dissolved in 611 ml dilution water (20X AAP medium according to OECD 221) in order to obtain the concentration of 100 mg/l. This stock solution was placed into an ultrasonic bath for 10 minutes thereafter the non-dissolved test material was removed by filtration through a 0.45 µm membrane filter. The further test solutions were prepared by appropriate dilution of this stock solution.
Two replicates per test item treated groups (containing 11 fronds in total per test vessel) were exposed to each concentration for a period of 7 days. A concurrent control was run parallel (with three replicates).
Nominal test concentration: 0.1, 1, 10, 100 mg/l. Test item was adsorbed on the roots of the plants. At 100 mg/l, test item precipitated by the end of the test.

nominal con. mg/l control 0.1(2) 1(2) 10(2) 100(1,2)
average frond no. (day 7) 41.33 44 41 39 40
growth rates µ 0.188 0.198 0.188 0.180 0.184
% inhibition µ - -5.14 0.17 4.16 2.11
1: test item precipitaed on the bottom by the end of the test
2: test item was adsorbed on the roots of the plants

Second test
It was a semi-static test with two renewal periods on day 3 and on day 5. The used test solutions were prepared by mechanical dispersion. 0.4004 g of test item was dissolved in 800.8 ml dilution water in the first and in the second renewal period (OECD medium) in order to obtain the concentration of 500 mg/l 0.4001 g of test item was dissolved in 800.2 ml dilution water in third renewal period (OECD medium) in order to obtain the concentration of 500 mg/l. The stock solutions were placed into an ultrasonic bath for 10 minutes. The further test solutions were prepared by appropriate dilution of this stock solution (at the start of each renewal period).
Two replicates per test item treated group (containing 11 fronds in total per test vessel) were exposed to each concentration for a period of 7 days. A concurrent control was run parallel (with three replicates).
Nominal test concentration: 1, 10, 100, 500 mg/l. Test item was adsorbed on the roots of the plants at all concentrations; starting for 10 mg/l, test item was adsorbed on the underside of leaves.
nominal test concentration % at start % after 24 h
1 49 5
10 85 50
100 99 92
500 103 101

nominal con. mg/l control 1(1) 10(2) 100(2) 500(2)
average frond no. (day 7) 41 30 19.5 19.5 10
growth rates µ 0.188 0.143 0.082 0.082 -0.014
% inhibition µ - 23.83 56.46 56.46 107.63
1: test item was adsorbed on the roots of the plants
2: test item was adsorbed to the roots and to the underside of the leaves
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
25.513 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.:
0.149 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
< 0.04 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
0.04 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Details on results:
Biological results
All biological results are based on the geometric mean of the measured concentrations.

Dry weight
Test item was adsorbed to the roots and to the underside of the leaves in each concentration level during the experiment, thus dry weight results were unusable for statistical evaluation (calculation of ECx values, determination of NOEC and LOEC values).

Frond number
The frond number in each test vessel was determined at the start and at the 3rd, 5th, 7th days. The final frond numbers in each treatment at 7th day minus the number of introduced fronds were used for the statistical calculations.

Growth Rate
The average specific growth rates were calculated based on frond numbers and dry weights for each treatment and each parallel.
The average specific growth rate (based on frond number) was statistically significantly different from the control group at each concentrations level (Dunnett t-Test, 2-sided (α=0.05)).
Accordingly the NOEC related to growth rate (based on frond number) was determined to be < 0.04 mg/L and the LOEC was determined to be 0.04 mg/l.
The 168-h ErfnC50 was determined to be 25.513 mg/l (95 % confidence limits: 13.920 – 54.570 mg/l).

Growth rates (µ) and percentage inhibition of µ based on frond number
Meas. conc. [mg/l] Growth rate (r) and % inhibition of r 0-168 h (based on frond number)
r % Ir
Control 0.281 0.00
0.04 0.206* 26.55
0.10 0.199* 29.17
1.95 0.194* 31.14
8.17 0.168* 40.20
31.73 0.137* 51.33
125.72 0.103* 63.29
*: statistically significantly different compared to the control values (Dunnett t-Test, 2-sided (α=0.05))

Growth rates (µ) and percentage inhibition of µ based on dry weight**
Meas. conc. [mg/l] Growth rate (r) and % inhibition of r 0–168 h (based on dry weights)
r % Ir
Control 0.17446 0.00
0.04 0.17839 -2.26
0.10 0.14205 18.58
1.95 0.12553 28.05
8.17 0.15238 12.65
31.73 0.15581 10.69
125.72 0.18834 -7.95
Remark: negative inhibition means growth stimulation.
**: statistical evaluation was not performed

Yield
The yield was calculated based on frond numbers and dry weights for each treatment and each parallel.
Yield (based on frond number) was statistically significantly different from the control group at each concentration level (Dunnett t-Test, 2-sided (α=0.05)).
Accordingly the NOEC related to yield (based on frond number) was determined to be < 0.04 mg/l and the LOEC was determined to be 0.04 mg/l.
The 168-h EyfnC50 was determined to be 0.149 mg/l (95 % confidence limits: 0.065 – 0.284 mg/l).

Yield (y) and percentage inhibition of yield based on frond number
Meas. conc. [mg/l] Yield (y) and % inhibition of yield 0–168 h (based on frond number)
y % Iy
Control 67.67 0.0
0.04 35.67* 47.29
0.10 33.33* 50.74
1.95 31.67* 53.20
8.17 24.67* 63.55
31.73 17.67* 73.89
125.72 11.67* 82.76
*: statistically significantly different compared to the control values (Dunnett t-Test, 2-sided (=0.05))

Yield (y) and percentage inhibition of yield based on dry weight**
Meas. conc. [mg/l] Yield (y) and % inhibition of yield 0–168 h (based on dry weight)
y % Iy
Control 0.00119 0.00
0.04 0.00124 -3.63
0.10 0.00085 29.05
1.95 0.00070 41.34
8.17 0.00095 20.67
31.73 0.00098 17.60
125.72 0.00136 -13.97
Remark: negative inhibition means growth stimulation.
**: statistical evaluation was not performed


Observations during the test
Symptoms, changes of Lemna gibba plants observed during the test
Meas. conc. [mg/l] 3rd day of exp. 5th day of exp. 7th day of exp.
Symptoms Degree of change Symptoms Degree of change Symptoms Degree of change
Control* – – – – – –
0.04(1) – – – – – –
0.10(2,3) – – Chlorosis * Chlorosis *
1.95(4) – – Chlorosis * Chlorosis **
Root length **
Frond size **

8.17(4,5) Frond size * Frond size * Chlorosis **
Chlorosis * Root length **
Frond size **
31.73(4, 5) Frond size * Frond size * Chlorosis **
Root length * Chlorosis * Root length **
Frond size * Root length * Frond size ***
125.72(4,5) Frond size * Frond size ** Chlorosis **
Root length * Chlorosis * Root length **
Root length * Frond size ***

* Some morphological changes were observed in case of the roots and leaves (shorter roots were observed and the leaves were located in small knots).
1: test item was adsorbed to the roots after 7 days.
2: test item was adsorbed to the roots after 3 and 5 days.
3: test item was adsorbed to the roots and to the underside of the leaves after 7 days.
4: test item was adsorbed to the roots and to the underside of the leaves after 3, 5 and 7 days.
5: test item was precipitated on the surface of the test solution and was adsorbed to the wall of the test vessels after 7 days.

Legend:
"–": the plants were healthy, there were not any symptoms observed
*: slight, **: medium, ***: strong change

Abbreviations:
Root length: decrease of the root length
Frond size: decrease of frond size
Chlorosis: chlorosis of fronds
Results with reference substance (positive control):
EyfnC50 (7 day, yield based on frond numbers): 6.177 mg/l
ErfnC50 (7 day, growth rate based on frond numbers): 8.617 mg/l
EydwC50 (7 day, yield based on dry weight): 5.668 mg/l
ErdwC50 (7 day, growth rate based on dry weight): 7.144 mg/l

Measured concentrations

nominal conc. mg/l 1st renewal 2nd renewal 3rd renewal geometric mean mg/l
start end start end start end
132 132 121 130 122 131 119 125.72
41.3 39.2 19.5 38.4 32.2 38.3 28.2 31.73
12.9 11 4.53 11.3 6.92 11.6 6.6 8.17
4 2.82 0.812 2.93 1.56 3 1.75 1.95
1.3 0.155 0.025(1) 0.184 0.069 0.264 0.087 0.1
0.4 0.063 0.025(2) 0.064 0.025(2) 0.104 0.025(1) 0.04
control not detected not detected not detected not detected not detected not detected -

LOQ= 0.05 mg/l, LOD=0.025 mg/l

1: LOQ/2 (the measured value was below the LOQ)

2: LOD (the measured value was below the LOD)

Validity criteria fulfilled:
yes
Conclusions:
Growth rate of frond number: EC50 = 25.513 mg/l (95 % conf. limits 13.920 - 54.570)
Yield of frond number: EC50 = 0.149 mg/l (95 % conf. limits 0.065 - 0.284)
Executive summary:

Method

Test on Duckweed (Lemna gibba); preculture was prepared 7 days before test; healthy colonies with 3-4 fronds. OECD medium was used as dilution water.

Based on the results of the preliminary experiments, nominal concentrations of 0.4, 1.3, 4.0, 12.9, 41.3 and 132.0 mg/l were used in the main study. As measured concentrations deviated more than 20 % from the nominal during the experiment, geometric mean of the measured concentrations were used: 0.04, 0.10, 1.95, 8.17, 31.73 and 125.72 mg/l. Biological results are based on the geometric mean of the measured test item concentrations.

Test design: 7-day exposure in testing solutions; 6 concentrations with 3 replicates and control group with 6 replicates; stock solution was diluted with OECD medium to reach the concentration range; 400 ml Petri dishes-covered glass beakers were filled with 160 ml testing solutions, two Lemna gibba colonies with four, and one colony with three fronds were added to each vessel; assessment of growth and morphological changes on days 3, 5 and 7.

Test type was semi-static, with renewals on day 3 and 5.

Analytics: for determination of test item concentrations, samples were taken from each concentration level and from the control at the start and at the end of each renewal periods. Concentrations were analysed by HPLC method with UV/VIS detection.

Results

NOEC and LOEC, for both response variables (i.e. growth rate and yield) based on frond number were calculated. Calculations based on the dry weights results were not possible because of the large amount precipitation of test item on plants.

For the determination of the LOEC and NOEC, Dunnett t-Test (a= 0.05) was used by SPSS PC+ software.                              

For determination of EC50 values with 95 % confidence limits (based on frond number), Probit analysis was performed using SPSS PC+software.

EC50 = 25.513 mg/l (13.920 - 54.570 mg/l), growth rate

EC50 = 0.149 (0.065 - 0.284 mg/l), yield

NOEC < 0.04 mg/l

LOEC = 0.04 mg/l

All validity criteria were met, thus the study can be considered as valid.

Description of key information

Frond number, growth rate: EC50 (7d) = 25.513 mg/l

NOEC (7d) < 0.04 mg/l, LOEC (7d) = 0.04 mg/l

Key value for chemical safety assessment

EC50 for freshwater plants:
25.513 mg/L

Additional information

There are no information about the potential toxicity of Basic Yellow 094 to aquatic plants, thus the available information on the structural analogue Similar Substance 03 have 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 IUCLID section 13).

Similar Substance 03 was tested according to OECD guideline 221, performing a semi-static test with renewal of solution at day 3 and 5. Biological results were expressed as geometric means of measured concentrations. Analytical monitoring was done by HPLC method with UV/VIS detection.

Based on the preliminary experiment, nominal concentrations were 0.4, 1.3, 4, 12.9, 41.3, 132 mg/l, corresponding to geometric means of 0.04, 0.1, 1.95, 8.17, 31.73 and 125.72 mg/l.

Test results were only as frond numbers (growth rate and yield); dry weight results were not used for statistical evaluation, as test item adsorbed to the roots and to underside of leaves at each concentration level.