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Diss Factsheets

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:
2017-04-14 to 2017-06-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD, EU guideline, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 761/2009, C.26.
Qualifier:
according to guideline
Guideline:
other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 4,8-diamino-1,5-dihydroxy-9,10-dioxoanthracene-2,6-disulphonate
EC Number:
220-678-3
EC Name:
Disodium 4,8-diamino-1,5-dihydroxy-9,10-dioxoanthracene-2,6-disulphonate
Cas Number:
2861-02-1
Molecular formula:
C14H10N2O10S2.2Na
IUPAC Name:
disodium 4,8-diamino-1,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2,6-disulfonate

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Duplicate samples were taken at the start and at two test medium renewals from the freshly prepared test media of all test concentrations and from the control.
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 renewal periods, duplicate samples of the aged test media and control were sampled corresponding to the fresh samples and at the end of the test.
Additional samples of the control blank were taken at each sampling date without any sample treatment.

Test solutions

Vehicle:
no
Details on test solutions:
Test Concentrations: 100, 32, 10, 3.2, 1.0 and 0.32 mg test item/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 of nominal 100 mg test item/L was prepared by dissolving 100.4, 101.0, 101.8, 100.5, 100.0, 101.0 and 101.2 mg test item into 1004, 1010, 1018, 1005, 1000, 1010 and 1012 mL test water by intense stirring for 15 minutes, respectively. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
The test media were prepared just before introduction of the Lemna (= start of the test) and each test medium renewal.
Appearance of the Test Item in Test Medium:The test medium was coloured by the test item ranging from light blue (0.32 to 1 mg test item/L) to blue (3.2, 10 and 32 mg test item/L) to dark blue (100 mg test item/L).

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

Test conditions

Test temperature:
23 to 25 °C
pH:
7.4 to 7.6 in the freshly prepared media;
8.0 to 8.3 in the aged test media
Nominal and measured concentrations:
100, 32, 10, 3.2, 1.0 and 0.32 mg test item/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-values were measured in all freshly prepared and aged test media.
7.4 to 7.6 in the freshly prepared test media;
8.0 to 8.3 in the aged test media
Light Regime: Continuous illumination
Light Intensity: 8583 Lux (mean value) with a range of 8200 to 8810 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.:
17.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
24.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
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:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
3.38 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
3.59 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
11.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
19.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
1.41 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
1.31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
3.56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
5.13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
The 7-day EyC50 was calculated to be 17.9 and 24.5 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50 was calculated to be > 100 mg test item/L for frond number and dry weight, respectively.
The 7-day EyC10 was calculated to be 1.41 and 1.31 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC10 was calculated to be 3.56 and 5.13 mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 1.0 and 3.2 mg test item/L for frond number and 3.2 and 10 mg test item/L for dry weight, respectively. The 7-day NOErC and the LOErC were determined to be 1.0 and 3.2 mg test item/L for frond number and 3.2 and 10 mg test item/L for dry weight, respectively
Shape of Fronds: The shape of fronds and colonies after the test period of 7 days was not different to those in the control up to and including the nominal test concentration of 10 mg test item/L.
At the higher test item concentrations the fronds showed deviations from the control replicates after 7 days; i.e. slight chlorosis and larger fronds (32 mg test item/L), slightly gibbous growth and blue coloured roots (100 mg test item/L) and shortened roots and slightly separated fronds (32 and 100 mg test item/L).

Reported statistics and error estimates:
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.
For the determination of the 7-day LOEC and NOEC 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: 1.8 days
Conclusions:
The influence of the test item on the growth of the freshwater plant Lemna gibba was assessed in a semi-static concentration-response test.
The 7-day ErC50 was calculated to be > 100 mg test item/L for frond number and dry weight, respectively.
During the test the test organism were exposed to a mean of 96 % of nominal. Therefore, all reported results refer to nominal concentrations.
Executive summary:

Title: Toxicity to the Aquatic Plant Lemna gibba in a Semi-Static Growth Inhibition Test

Purpose: The purpose of this study was to determine the inhibitory effect of the test item on the growth of the freshwater aquatic plant Lemna gibba. For this purpose, cultures of Lemna gibba were exposed in a semi-static test with daily water renewal to various concentrations 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 and Lemna gibba is the recommended test species. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

Guidelines/Recommendations:

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

OECD Guideline 221: "Lemna sp. Growth Inhibition Test", adopted March 23, 2006

OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000

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: batch no.: 19.05.2016g

Test Species: Lemna gibba G 3

Test Design: This study encompassed 7 treatment groups (6 dose rates of the test item and a control) with three replicates per test concentration and control.

At test start 12 fronds were introduced in each replicate and incubated for 7 days under semi-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: 100, 32, 10, 3.2, 1.0 and 0.32 mg test item/L and a control.

Test Conditions: Water temperature: 23 to 25 °C; pH values at test start in the freshly prepared test media 7.4 to 7.6, at the end of the test in the aged test media 8.0 to 8.3; continuous illumination; mean light intensity: 8583 lux (8200 to 8810 lux).

Conclusion: The influence of the test item on the growth of the freshwater plant Lemna gibba was assessed in a semi-static concentration-response test.

The 7-day EyC50 was calculated to be 17.9 and 24.5 mg test item/L for frond number and dry weight, respectively. The 7-day ErC50 was calculated to be > 100 mg test item/L for frond number and dry weight, respectively. The 7-day NOEyC and the LOEyC were determined to be 1.0 and 3.2 mg test item/L for frond number and 3.2 and 10 mg test item/L for dry weight, respectively. The 7-day NOErC and the LOErC were determined to be 1.0 and 3.2 mg test item/L for frond number and 3.2 and 10 mg test item/L for dry weight, respectively The quantification of the test item in the test samples was performed using liquid chromatography with UV detection. In the freshly prepared test media at the start of the test and at the renewal of the test media 104% of the nominal test concentrations were found (average of all test concentrations). In the aged test media 89% of the nominal value was determined (average of all test concentrations). During the test the test organism were exposed to a mean of 96% of nominal. Therefore, all reported results refer to nominal concentrations.

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