Registration Dossier

Ecotoxicological information

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:
toxicity to aquatic plants other than algae
Remarks:
Toxicity to the Aquatic Plant Lemna gibba
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 761/2009, C.26.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solution of the substance in water

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples were taken at the end of the test from all test media and the control of the actual test by pouring together the contents of each treatment.
The samples were diluted by a factor of 1.25 with acetonitrile.
Additional samples of the control blank and the dilution solvent were taken at test start and test end without any sample treatment.

Test solutions

Vehicle:
no
Details on test solutions:
Test Concentrations: 145.1, 45.9, 14.5, 4.6 and 1.5 mg test item/L;
corresponding to 100.0, 31.6, 10.0, 3.17 and 1.03 mg active ingredient/L and a control.
Control: In the control, test medium was used without addition of the test item.
Dosage of Test Item: The test medium of the highest test concentration The test media were prepared just before introduction of the Lemna (= start of the test).
Appearance of the Test Item in Test Medium:
The test media showed an intense dark blue coloration in the highest test concentration. The intensity of the colouration decreased with decreasing test concentration. The test item was observed to lie at the bottom in all test concentrations after 3, 5, and 7 days.

Test organisms

Test organisms (species):
Lemna gibba
Details on test organisms:
Species: Lemna gibba G 3
Origin: The fronds introduced in the test were taken from ibacon's in-house laboratory culture.
Breeding Conditions: The plants are cultured in the laboratories of ibacon under standardised conditions according to the test guidelines.
Pre-Culture: For at least 7 days under test conditions with weekly media exchange.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d

Test conditions

Test temperature:
23 to 25 °C
pH:
pH values at test start 7.5 to 7.6, at the end of the test 8.5 to 8.7
Nominal and measured concentrations:
145.1, 45.9, 14.5, 4.6 and 1.5 mg test item/L;
corresponding to 100.0, 31.6, 10.0, 3.17 and 1.03 mg active ingredient/L and a control.
Details on test conditions:
Test Environment: Controlled environment room
Water Temperature: The temperature was measured daily in a test vessel filled with water and incubated under the same conditions as the test vessels.
23 to 25 °C
pH-Values: The pH-value of each treatment was measured at test start and at test end.
7.5 to 7.6 at test start;
8.5 to 8.7 at test end
Light Regime: Continuous illumination
Light Intensity: 7270 Lux (mean value) with a range of 7080 to 7520 lux.
The illumination was achieved by fluorescent tubes, installed above the test vessels. Measurements were performed once during the test at 6 places distributed over the experimental area at the surface of the test media. Differences in light intensity over the test area did not exceed ± 15 %.
Reference substance (positive control):
yes
Remarks:
For the evaluation of the quality of the Lemna gibba and the experimental conditions the substance 3,5-dichlorophenol is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrationsopen allclose all
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
frond number
Remarks:
yield
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks:
yield
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks:
growth rate
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
12.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
frond number
Remarks:
yield
Remarks on result:
other: 95 % CL
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
71.8 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks:
yield
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks:
growth rate
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
< 1.03 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
frond number
Remarks:
yield
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
< 1.03 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks:
yield
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
2.88 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
9.84 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks:
growth rate
Remarks on result:
other: 95 % CL
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
1.03 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
frond number
Remarks:
yield
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks:
yield
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks:
growth rate
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
3.17 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
frond number
Remarks:
yield
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks:
yield
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks:
growth rate
Details on results:
Biological Results
Growth Inhibition: The 7-day EyC50 was determined to be > 100 mg active ingredient/L for frond number and dry weight.
The 7-day ErC50 was determined to be > 100 mg active ingredient/L for frond number and dry weight.
The 7-day EyC10 was calculated to be < 1.03 and < 1.03 mg active ingredient/L for frond number and dry weight, respectively.
The 7-day ErC10 was calculated to be 2.88 and 9.84 mg active ingredient/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 1.03 and 3.17 mg active ingredient/L for frond number and 31.6 and 100.0 mg active ingredient/L for dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be 10.0 and 31.6 mg active ingredient/L for frond number and 31.6 and 100.0 mg active ingredient/L for dry weight, respectively
Around each test beaker a light tight ring was placed and the bottom of each beaker was covered with black foil to illuminate all test organisms from one direction (top) only and reduce the influence of the strongly coloured test media in the higher test concentrations to a minimum. An influence of the colouration cannot be excluded and inhibition effects in the lower concentrations might be explained by the colouration and the reduced light availability of the plants than by toxic effects.

Reported statistics and error estimates:
Statistical Analysis:
The ErC50 and the EyC50 (see Definitions), the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by probit analysis using linear weighted regression (frond number) and probit analysis (biomass).
For the determination of the 7-day LOEyC, NOEyC, LOErC and NOErC values significant differences at the test concentrations compared to the control values were tested by the Williams t-test.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Doubling Time of Frond Number in Control: 2.1 days, validity criterion was met.
Conclusions:
The influence of Sanolin Lave Blue A VP 5453 on the growth of the freshwater plant Lemna gibba was assessed in a static concentration-response test.
The 7-day EyC50 was determined to be > 100 mg active ingredient/L for frond number and dry weight.
The 7-day ErC50 was determined to be > 100 mg active ingredient/L for frond number and dry weight.
Executive summary:

Title:

Sanolin Lave Blue A VP 5453: Toxicity to the Aquatic Plant Lemna gibba in a Static Growth Inhibition Test

Purpose:

The purpose of this test was to determine the inhibitory effect of the test item Sanolin Lave Blue A VP 5453 on the growth of the freshwater aquatic plantLemna gibba. Cultures ofLemna gibbawere exposed in a static test to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 7 days.

The test method of application and the test system are recommended by the test guidelines andLemna gibbais one of the recommended test species.

The purpose of the analytical part of this study was to verify the concentration of the test item in the test medium.

Guidelines/Recommendations:

-     Commission Regulation (EC) No 761/2009, Annex, Part C, C.26.: "Lemnasp. Growth Inhibition Test", Official Journal of the European Union (EN), dated August 24, 2009

-     OECD Guideline 221: "Lemnasp. Growth Inhibition Test", adopted March 23, 2006

-     SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

 

 

Material and Methods

 

Test Item:

Sanolin Lave Blue A VP 5453; batch no.:CHM8 000373; purity:68.9 %, according to certificate of analysis.

Test Species:

Lemna gibba G 3

Test Design:

This study encompassed6treatment groups (5dose rates of the test item and a control) with threereplicates per test concentration and control.

At test start 12 fronds were introduced in each replicate and incubated for 7 days under static conditions. The frond numbers were determined on day 3, 5 and 7. The dry weight of each replicate was determined at test termination.

Endpoints:

Yield and growth rate based on frond number and dry weight.

Test Concentrations:

145.1, 45.9, 14.5, 4.6 and 1.5 mg test item/L;

corresponding to 100.0, 31.6, 10.0, 3.17 and 1.03 mg active ingredient/L and a control.

Test Conditions:

Water temperature:23 to 25°C; pH values at test start7.5 to 7.6, at the end of the test8.5 to 8.7; continuous illumination; mean light intensity:7270lux (7080 to 7520lux).

 

 

Table1. Summary of Biological Results

Parameter

Yield
(frond number)
[mg active ingredient/L]

Growth rate
(frond number)
[mg active ingredient/L]

Yield
(dry weight)
[mg active ingredient/L]

Growth rate
(dry weight)
[mg active ingredient/L]

EC50(7-day)

>100

>100

>100

>100

95 % conf. limits

n.d.

n.d.

n.d.

n.d.

 

 

 

 

 

EC20(7-day)

12.2

>100

71.8

>100

95 % conf. limits

5.02 - 29.6

n.d.

n.d

n.d.

 

 

 

 

 

EC10(7-day)

< 1.03

2.88

< 1.03

9.84

95 % conf. limits

n.d.

< 1.03 -7.35

n.d.

1.03 - 93.3

 

 

 

 

 

7-day NOEC

1.03

10.0

31.6

31.6

7-day LOEC

3.17

31.6

100.0

100.0

n.d.: not determinable
Values refer to nominal
active ingredient

 

Analytical Results:

The quantification of the test item Sanolin Lave Blue A VP 5453 in the test samples was performed using liquid chromatography with UV detection.

At the start of the test 102% of the nominal test concentrations were found (average of all test concentrations). After 7 days test duration, 100% of the nominal value was determined (average of all test concentrations). During the test the Lemna were exposed to a mean of 101% of nominal. Therefore, all reported results refer to nominal concentrations.

 

 

Conclusion:

The influence of Sanolin Lave Blue A VP 5453 on the growth of the freshwater plant Lemna gibba was assessed in a static concentration-response test.

The 7-day EyC50was determined to be > 100 mg active ingredient/Lfor frond number and dry weight.

The 7-day ErC50was determined to be>100 mg active ingredient/Lfor frond number and dry weight.

The 7-day NOEyC and the LOEyC were determined to be1.03 and3.17 mg active ingredient/Lfor frond number and31.6 and100 mg active ingredient/Lfor dry weight, respectively.

The 7-day NOErC and the LOErC were determined to be10.0 and31.6 mg active ingredient/Lfor frond number and31.6 and100 mg active ingredient/Lfor dry weight, respectively.

This study is classified acceptable and satisfies the guideline requirements for aquatic plant studies.