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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
Study period:
22 January - 03 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
23rd March, 2006
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 761/2009, Annex VI, C.26: „Lemna sp. Growth Inhibition Test“. Dated 24 August 2009
Version / remarks:
24 August 2009
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series on Testing and Assessment No. 23, Paris September 2000
Version / remarks:
September 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Date of production:22 February 2016
Expiry date: 22 February 2021
Analytical monitoring:
yes
Details on sampling:
Based on the results of the preliminary experiment, the nominal concentrations of 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L were investigated in the main study. The measured concentrations deviated more than 20 % from the nominal at the start and at the end of the study, therefore the geometric mean of the measured concentrations were calculated to determine exposure concentration. The corresponding calculated concentrations were 0.10, 0.30, 0.55, 0.91 and 1.69 mg/L, equivalent to the nominal 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L, respectively (based on total product). Biological results and endpoints are based on the measured and calculated concentrations.
Vehicle:
no
Details on test solutions:
As the test item is poorly soluble in deionized water as well in the test medium, preparation of test solutions was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23). The appropriate test item amounts were suspended in the appropriate amounts of the dilution water (20X AAP Medium; see 5.4) in order to give the loading rates of 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L. The test suspensions were handled in ultrasonic bath for 10 minutes thereafter stirred rigorously for a period of 24 hours to achieve equilibrated test concentrations. These test suspensions were then filtrated through a membrane filter* (0.45 µm) to separate the possible non-dissolved test material. The test solutions were freshly prepared in the testing laboratory just before introduction of the test organisms at the start of the test.
Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name (Strain): Lemna gibba (G3)
- Source: Friedrich Schiller Universität, Institut für Allgemeine Botanik und Pflanzenphysiologie,Jena, Germany
- Preculture: 7 days before testing, sufficient colonies were transferred from the stock culture aseptically into fresh sterile medium and cultured under the conditions of the test prior to beginning the test.
- Initial frond number: The initial frond number in the test cultures was 11. The number of colonies and fronds was identical in each test vessel.
- Replicates and controls: The test included three replicates at each test concentration and six replicates for the control group
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Remarks on exposure duration:
The study was performed over an exposure period of 7 days in 20X AAP Medium.
Post exposure observation period:
none
Hardness:
not measured
Test temperature:
The cultures were maintained at a temperature in the range of 24 +/- 2 ºC (22.6 – 25.8°C in the climate chamber and 23.0 – 23.9 °C in the test vessels), 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.
pH:
The pH was checked at the start and at the end of the test in the test concentrations and in the control. The pH of the control medium did not increase by more than 1.5 units. The pH values ranged between 7.86 - 8.88 during the test.
Dissolved oxygen:
not measured
Salinity:
not applicable
Conductivity:
not applicable
Nominal and measured concentrations:
The corresponding measured concentrations were 0.10, 0.30, 0.55, 0.91 and 1.69 mg/L, equivalent to the nominal 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L, respectively (based on total product).
Details on test conditions:
The test containers were kept during the test in a climate chamber with controlled environmental conditions.

Test type: Static test. Based on the results obtained during analytical method validation (Study number: 805-100-2578) the test item is stable for the duration of 7 days in 20X AAP Medium. Therefore no renewal of the test and control solutions was necessary during the test.

Temperature: The cultures were maintained at a temperature in the range of 24 +/- 2 ºC (22.6 – 25.8°C in the climate chamber and 23.0 – 23.9 °C in the test vessels), 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.
pH: The pH was checked at the start and at the end of the test in the test concentrations and in the control. The pH of the control medium did not increase by more than 1.5 units. The pH values ranged between 7.86 - 8.88 during the test.

Light intensity: The test vessels were placed randomly on a tray, illuminated continuously at a light intensity between 6500-10000 lux (measured 7945 - 8025 lux, SD: 25 lux) using fluorescent light tubes (with a spectral range of 400-700 nm). The light intensity was checked and recorded at the start of the test at the position occupied by test containers. The differences in light intensity between the measurement points (i.e. position of fronds) did not exceed +/- 15 % and therefore provided equal conditions for each test culture.

Test units: All-glass beakers (total capacity of 400 mL) were used and were covered by glass petri dishes. The volume of the test liquid in the vessels was 160 mL. Each test unit was uniquely identified with study code, test item concentration (and control) and replicate.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol is tested at least twice a year.
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
> 1.69 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Growth Rate (based on frond number and dry weight)
Remarks on result:
other: EC10 is higher than the nominal concentration of 100 mg/L of the test item in the test medium, which corresponds to the mean measured concentration of 1.69 mg/L
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 1.69 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Growth Rate (based on frond number and dry weight)
Remarks on result:
other: the EC50 is higher than the nominal concentration of 100 mg/L of the test item in the test medium, which corresponds to the mean measured concentration of 1.69 mg/L
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
1.69 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Growth Rate (based on frond number and dry weight)
Remarks on result:
other: the test item had no toxic effect at aquatic saturation on Lemna gibba
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
> 1.69 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Growth Rate (based on frond number and dry weight)
Remarks on result:
other: the overall LOEC is higher than the nominal concentration of 100 mg/L of the test item in the test medium, which corresponds to the mean measured concentration of 1.69 mg/L
Details on results:
Biological Results

All biological results are based on the calculated mean concentrations (based on total product).

Growth Rate

The average specific growth rate was not statistically significantly different from the control group at any observed test item concentration based on frond number and dry weight (Dunnett t-test, one-sided, α=0.05).
The 168-h NOEC was determined to be 1.69 mg/L, while the 168-h LOEC was determined to be greater than 1.69 mg/L (solubility level of the highest loading rate of 100 mg/L in the test medium).

Yield

The yield was not statistically significantly different from the control group at any observed test item concentration based on frond number and dry weight (Dunnett t-test, one-sided, α=0.05).
The 168-h NOEC was determined to be 1.69 mg/L, while the 168-h LOEC was determined to be greater than 1.69 mg/L (solubility level of the highest loading rate of 100 mg/L in the test medium).
Results with reference substance (positive control):
For the evaluation of the quality of the Lemna gibba cultures and the experimental conditions, 3,5-dichlorophenol is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study number: 392-221-3267) with reference item 3,5-dichlorophenol was: 08 – 15 September 2017.
Endpoints of this study were: EyfnC50 (7 day, yield based on frond numbers): 3.980 mg/L, ErfnC50 (7 day, growth rate based on frond numbers): 6.053 mg/L, EydwC50 (7 day, yield based on dry weight): 2.906 mg/L, ErdwC50 (7 day, growth rate based on dry weight): 6.206 mg/L.
Reported statistics and error estimates:
The doubling time of frond number in the control was 2.24 days (less than 2.5 days). The validity criterion was within acceptable limit and therefore the study can be considered as valid.

Results of the Preliminary Range-finding Test

Nominal concentrations
[mg/L]

Control

0.1

1

10

50

100

Average number of fronds
(day 7)

80.0

75.5

89.0

78.5

63.0

61.0

Growth Rates (µ)
[0-7d]

0.283

0.275

0.297

0.281

0.249

0.245

% Inhibition of µ
[0-7d]

-

2.89

-4.78*

0.92

12.17

13.67

* Negative value means growth increase

Calculation of Exposure Concentrations

Nominal
concentration
(mg/L)

Measuredconcentration(mg/L)

Mean measuredconcentration
(mg/L)

Start of the Study

End of the Study

Control

-

-

-

6.25 (WAF*)

<LOD**

<LOD**

0.10

12.5 (WAF*)

0.28

0.32

0.30

25 (WAF*)

0.55

0.56

0.55

50 (WAF*)

0.94

0.89

0.91

100 (WAF*)

1.82

1.57

1.69

-      not detected

*   water accommodated fraction (OECD No. 23.)

**test concentration measured < LOD (Limit of detection) was calculated as LOD(OECD No. 23.)

Growth rates (µ) and Percentage Inhibition ofµbased on Frond Number

Concentration
(mg/L)

Growth rate (µ) and % inhibition ofµ

Nominal

Measured

0–168 h(based on frond number)

µ

% Iµ

Control

-

0.310

-

6.25 (WAF*)

0.10

0.314

-1.35**

12.5 (WAF*)

0.30

0.307

0.69

25 (WAF*)

0.55

0.314

-1.52**

50 (WAF*)

0.91

0.310

-0.11**

100 (WAF*)

1.69

0.308

0.54

*   water accommodated fraction (OECD No. 23.)

** negative value indicates increase in comparison to the control

 

Growth rates (µ) and Percentage Inhibition ofµbased on Dry Weight

Concentration
(mg/L)

Growth rate (µ) and % inhibition ofµ

Nominal

Measured

0–168 h(based on dry weight)

µ

% Iµ

Control

-

0.3309

-

6.25 (WAF*)

0.10

0.3337

-0.86**

12.5 (WAF*)

0.30

0.3276

0.98

25 (WAF*)

0.55

0.3368

-1.79**

50 (WAF*)

0.91

0.3239

2.10

100 (WAF*)

1.69

0.3307

0.04

*   water accommodated fraction (OECD No. 23.)

**negative value indicates increase in comparison to the control

Yield (y) and Percentage Inhibition of Yield based on Frond Number

Concentration
(mg/L)

Yield (y) and % inhibition of yield

Nominal

Measured

0–168 h(based on frond number)

y

% Iy

Control

-

85.17

-

6.25 (WAF*)

0.10

88.00

-3.33**

12.5 (WAF*)

0.30

83.67

1.76

25 (WAF*)

0.55

88.33

-3.72**

50 (WAF*)

0.91

85.33

-0.20**

100 (WAF*)

1.69

84.00

1.37

*   water accommodated fraction (OECD No. 23.)

** negative value indicates increase in comparison to the control

 

Yield (y) and Percentage Inhibition of Yield based on Dry Weight

Concentration
(mg/L)

Yield (y) and % inhibition of yield

Nominal

Measured

0–168 h(based on dry weight)

y

% Iy

Control

-

0.0062

-

6.25 (WAF*)

0.10

0.0064

-2.27**

12.5 (WAF*)

0.30

0.0061

2.54

25 (WAF*)

0.55

0.0065

-4.67**

50 (WAF*)

0.91

0.0059

5.15

100 (WAF*)

1.69

0.0062

0.08

*   water accommodated fraction (OECD No. 23.)

** negative value indicates increase in comparison to the control

 

Symptoms, Changes ofLemna gibbaPlants Observed during the Test

Concentration
(mg/L)

3rdday of
the experiment

5thday of
the experiment

7thday of
the experiment

Nominal

Measured

Symptoms

Degree of change

Symptoms

Degree of change

Symptoms

Degree of change

Control

-

n

-

n

-

n

-

6.25 (WAF*)

0.10

n

-

n

-

n

-

12.5 (WAF*)

0.30

n

-

n

-

n

-

25 (WAF*)

0.55

n

-

n

-

n

-

50 (WAF*)

0.91

n

-

n

-

n

-

100 (WAF*)

1.69

n

-

n

-

n

-

*   water accommodated fraction (OECD No. 23.)

Validity criteria fulfilled:
yes
Conclusions:
The test item Leuco Sulfur Blue 15 had no toxic effect at aquatic saturation on Lemna gibba; the EC10, EC50 and overall LOEC is higher than the nominal concentration of 100 mg/L of the test item in the test medium, which corresponds to the mean measured concentration of 1.69 mg/L, based on water accommodated fraction of the total product.
Executive summary:

The influence of the test item on the growth of the freshwater aquatic plant Lemna gibba (duckweed) was investigated in a 7‑day static test, based on the OECD Guideline No. 221 “Lemna sp. Growth Inhibition Test” (2006) with nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg product/L.

Based on the results the EC50 and NOEC for the test item were determined to be > 100 mg/L and 100 mg/L (nominal concentration, corresponding to the mean measured concentration of 1.69 mg/L), respectively.

All validity criteria were met and therefore the study can be considered as valid. All biological results are based on the mean measured test item concentrations of the total product.

Description of key information

The test item Leuco Sulfur Blue 15 had no toxic effect at aquatic saturation on Lemna gibba; the EC10, EC50 and overall LOEC is higher than the nominal concentration of 100 mg/L of the test item in the test medium, which corresponds to the mean measured concentration of 1.69 mg/L, based on water accommodated fraction of the total product.

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:
1.69 mg/L

Additional information

The influence of the test item on the growth of the freshwater aquatic plant Lemna gibba (duckweed) was investigated in a 7‑day static test, based on the OECD Guideline No. 221 “Lemna sp. Growth Inhibition Test” (2006) with nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg product/L.

Based on the results the EC50 and NOEC for the test item were determinedto be > 100 mg/L and 100 mg/L,respectively.

All validity criteria were met and therefore the study can be considered as valid. All biological results are based on the mean measured test item concentrations of the total product.