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Ecotoxicological information

Toxicity to aquatic plants other than algae

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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:
8 Feb 2017 to 17 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
2006
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Basic Red 14 sulfate
- Appearance: violet to sparkling dark blue solid
- Batch No.: 4030158820
- Date of manufacture: 31 March 2014
- Expiry date: March 2018
- BASF test substance number: 16/0122-1
- Purity: 100 weight% main component
- Water solubility: 74.1 g/L ± 0.4 g/L at 20.0°C ± 0.5°C
- Storage condition of test material: at ambient temperature without exposure to light in a tightly sealed container
Analytical monitoring:
yes
Details on sampling:
Samples of all test item concentrations and the control at exposure initiation and at exposure termination were chemically analyzed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item in the amount of 120.0 mg was weighed in a glass beaker and quantitatively transferred into flask by multiple washing with the 20X AAP medium and filled up to 2000 mL. The test item concentration of 60 mg/L was visually homogeneous. Lower test item concentrations were prepared by sequential dilutions with the 20X AAP medium.
- Controls: 20X AAP medium
Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name: duckweed
- Strain: CPCC 310
- Source: The duckweed Lemna gibba (Linné) CPCC 310 is cultivated in a standard laboratory culture at the Institute of Industrial Organic Chemistry, Branch Pszczyna, Department of Ecotoxicology, Laboratory of Aquatic Toxicology, according to the OECD Guideline No. 221 (2006) recommendations. The culture of Lemna gibba CPCC 310 (stock G3) on agar bevels was received from Canadian Phycological Culture Centre (CPCC), Department of Biology, University of Waterloo, Ontario, Canada.
- Culturing conditions: Duckweed Lemna gibba was transferred from agar bevels to the fresh 20X AAP medium in glass beakers with a capacity of 600 mL with transparent lids and incubated at room temperature with constant illumination (pre-culturing). The duckweed culture was inoculated to fresh medium once a week. A pre-culture was started seven days before exposure.
- Culture medium: The 20X AAP medium was prepared on the basis of deionised water by adding stock solutions of reagent-grade chemicals. The medium was sterilised by autoclaving before addition of the stock solution of sodium hydrogen carbonate – the buffering ingredient. After 24 h of aeration, the pH value of test medium was measured and adjusted with 0.1 M HCl, if necessary. The stock solutions were renewed on regular basis for Lemna gibba culturing and stored in a refrigerator. The alkalinity determined for 20X AAP medium was 292 mg/L as NaHCO3, the hardness was 299 mg/L as CaCO3.
- Health:Only organisms in good physiological condition without any discoloration were used for the inoculation of the test item concentrations and the control.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Hardness:
299 mg/L as CaCO3
Test temperature:
24.4 – 25.1 °C
pH:
- Start: 7.33 – 7.43
- End: 8.33 – 8.97
Conductivity:
1589 µS/cm
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 0.06, 0.19, 0.6, 1.9, 19 and 60 mg/L (based on range-finding test)
- Measured concentrations test start: - Measured concentrations test termination:
Details on test conditions:
TEST SYSTEM
- Incubation chamber used: yes, thermostatic chamber (incubator with shaker, ILW400STD Pol-Eko-Aparatura, Poland)
- Test vessel: glass beakers with a capacity of 600 mL
- Fill volume: 400 mL of each treatment
- Type of cover: transparent lids
- Incubation: test vessels were arranged at random and repositioned twice during exposure
- No. of colonies per vessel: 3
- No. of fronds per colony: 3
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, 20X AAP medium

TEST MEDIUM/ WATER PARAMETERS
- Test medium: same as culture medium, see 'Details on test organisms'
- Intervals of water quality measurement: The pH values were measured in all test item concentrations and the control before division into replicates at exposure initiation as well as in all test item concentrations and the control at exposure termination in pooled replicates.

OTHER TEST CONDITIONS
- Photoperiod: constant illumination,
- Light intensity and quality: 7633 - 8298 lux, fluorescent light source (cool white light)

EFFECT PARAMETERS MEASURED:
- Determination of frond number: the total number of fronds in each test vessel (as well as changes in plant development) was counted on days 2, 5, and after 7 days of exposure. Only visibly distinct fronds were counted.
- Determination of biomass: The dry weight of the representative sample of the duckweed culture used as the inoculum was measured at exposure initiation. The dry weight of all plants from each test vessel was measured after exposure termination. Colonies were dried at approximately 60 °C in a laboratory oven.
- Additional: frond size, shape and appearance (necrosis, chlorosis, gibbosity or bending of fronds), colony breakup or loss of buoyancy, root length and appearance
- Other parameters measured: temperature, measured continuously; light intensity, measured at exposure initiation, twice during exposure and at exposure termination; pH: at test start (before division into replicates) and at test termination.

RANGE-FINDING STUDY
- Test concentrations: 0 (control), 0.01, 0.1, 1.0, 10 and 50 mg/L
- Results used to determine the conditions for the definitive study: Effects on growth rate inhibition ranged from 1.8% to 63.8% (frond number) and from 2.8% to 71.1% (dry weight); effects on yield inhibition ranged from 5.4% to 89.6% (frond number) and from 9.5% to 93.8% (dry weight).
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
33.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.I.: 29.56 - 38.70
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
1.65 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.I.: 1.25 - 2.09
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
6.69 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% C.I.: 5.64 - 7.96
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
0.47 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% C.I.: 0.32 - 0.66
Details on results:
Inhibition of the growth rate based on frond number started at a concentration of 0.6 mg/L (5.3% inhibition) and increased in a dose-dependent manner up to 58.9% inhibition at 60 mg/L. For (additional) determined effect values and information on growth inhibition see 'Any other information on results incl. tables'.

VISUAL SIGNS OF PHYTOTOXICITY
At exposure termination, in the test item concentrations of 0.19 and 0.06 mg/L, no distinctive changes from the development of plants in the control were observed. In the test item concentration of 0.6 mg/L, slightly bending down of fronds were observed. In the test item concentration of 1.9 mg/L, slightly bending down of fronds, and colored roots and fronds were observed. In the test item concentrations of 19 and 6 mg/L, overlapping colonies, bending down of fronds, shortened roots, and colored roots and fronds were observed. In the test item concentration of 60 mg/L, breaking up of colonies, spots of necrosis, shortened roots, and colored roots and fronds were observed.
Results with reference substance (positive control):
7-d ErC50, based on frond number = 9.94 mg/L (95% C.I.: 8.14 - 12.12). The results are within the range given in the literature reference.
Reported statistics and error estimates:
Probit method calculations and analysis by Shapiro-Wilk’s Test on Normal Distribution, Levene’s Test on Variance Homogeneity (with Residuals), Williams Multiple Sequential t-test Procedure, Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm.

Table: Growth rate and yield evaluation after 7 days

Nominal test

item

concentration

[mg/L]

Based on frond number

Based on dry weight

% inhibition at

exposure

termination (day 7)

(growth rate)

% inhibition at

exposure

termination (day 7)

(yield)

% inhibition at

exposure

termination (day 7)

(growth rate)

% inhibition at

exposure

termination (day 7)

(yield)

Control

0

0

0

0

0.06

2.7

7.3

2.9

8.4

0.19

1.2

3.7

-1.2*

-2.7*

0.6

5.3

13.8

3.3

9.7

1.9

9.1

22.9

16.7

38.7

6

23.1

48.6

33.8

63.6

19

42

71.6

51.8

80.2

60

58.9

84.4

67.6

89.5

* calculated inhibition values are lower than 0%: the measured dry weight after exposure termination was higher than the measured dry weight in the control.

Table: Endpoint values of growth rate based on nominal test item concentrations

Endpoint value [mg/L]

Frond number

Dry weight

0-2 d

0-5 d

0-7 d

0-7 d

ErC10

3.31 (2.23 – 4.45)

1.58 (1.20 – 1.98)

1.65 (1.25 – 2.09)

0.94 (0.82 – 1.07)

ErC20

6.79 (5.16 – 8.41)

4.13 (3.44 – 4.84)

4.65 (3.87 – 5.45)

2.6 (2.36 – 2.84)

ErC50

26.83 (22.99 – 31.74)

26.03 (23.12 – 29.57)

33.6 (29.56 – 38.70)

18.22 (17.11 – 19.44)

LOEC

1.9

1.9

0.6

1.9

NOEC

0.6

0.6

0.19

0.6

Table: Endpoint values of yield based on nominal test item concentrations

Endpoint value [mg/L]

Frond number

Dry weight

 

0-2 d

0-5 d

0-7 d

0-7 d

EyC10

2.16 (1.34 – 3.06)

0.59 (0.40 – 0.81)

0.47 (0.32 – 0.66)

0.38 (0.29 – 0.47)

EyC20

4.37 (3.10 – 5.66)

1.38 (1.04 – 1.75)

1.18 (0.87 – 1.50)

0.82 (0.68 – 0.97)

EyC50

16.81 (13.95 – 20.43)

7.04 (5.98 – 8.31)

6.69 (5.64 – 7.96)

3.72 (3.32 – 4.16)

LOEC

1.9

1.9

0.6

1.9

NOEC

0.6

0.6

0.19

0.6

Validity criteria fulfilled:
yes
Remarks:
see 'Any other information on materials and methods incl. tables'

Description of key information

In duckweed (L. gibba), the 7-d ErC50 (growth rate, based on frond number) and 7-d EyC50 (yield, based on frond number) are 33.60 mg/L and 6.69 mg/L, respectively. The 7-d ErC10 and EyC10 values are 1.65 mg/L and 0.47mg/L, respectively.

Key value for chemical safety assessment

Additional information

The toxicity to aquatic plants other than algae was determined in a study according to OECD TG 221 and in compliance with GLP criteria. In this study 3 replicates (6 replicates for control) of 3 uniform, healthy-looking duckweed (L. gibba), with 3 fronds each, were introduced in test vessels containing test substance concentrations of 0 (control), 0.06, 0.19, 0.6, 1.9, 6, 19 and 60 mg/L. Plants were exposed for 7 days under static conditions. Analytical confirmation of nominal test concentrations showed that all test concentrations remained well within ±20% of nominal concentrations throughout the test. Therefore, effect concentrations are expressed as nominal. Observations of frond number, size, shape and appearance (necrosis, chlorosis, gibbosity or bending of fronds), colony breakup or loss of buoyancy, root length and appearance were made on day 2 and 5 and at the end of the exposure. At the end of the test period, dry weight was determined. Throughout the test a decrease in frond numbers was visible with increasing test item concentrations. Effects on plant development were visible from a test item concentration of 0.6 mg/L. The EC50-values for inhibition of the specific growth rate of fronds (ErC50) and dry weight (ErdwC50) were 33.60 mg/L and 18.22 mg/L, respectively. The EC50-values for inhibition of yield (EyC50) and dry weight inhibition of yield (EydwC50) were 6.69 mg/L and 3.72 mg/L, respectively. The ErC10 and EyC10 values, based on frond numbers, are 1.65 mg/L and 0.47 mg/L, respectively.