Registration Dossier

Ecotoxicological information

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:
toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
23 March 2006
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Trisodium 8-hydroxypyrene-1,3,6-trisulphonate
EC Number:
228-783-6
EC Name:
Trisodium 8-hydroxypyrene-1,3,6-trisulphonate
Cas Number:
6358-69-6
Molecular formula:
C16H10O10S3.3Na
IUPAC Name:
trisodium 8-hydroxypyrene-1,3,6-trisulfonate
1
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
Dihydrogen oxide
impurity 1
Chemical structure
Reference substance name:
Sodium chloride
EC Number:
231-598-3
EC Name:
Sodium chloride
Cas Number:
7647-14-5
Molecular formula:
ClNa
IUPAC Name:
sodium chloride
impurity 2
Reference substance name:
Sodium sulphate
EC Number:
231-820-9
EC Name:
Sodium sulphate
Cas Number:
7757-82-6
Molecular formula:
Na2SO4
IUPAC Name:
Sodium Sulphate
impurity 3
Reference substance name:
1,3,6-pyrene trisulfonic acid
Molecular formula:
C16H10O9S3
IUPAC Name:
1,3,6-pyrene trisulfonic acid
Test material form:
solid: particulate/powder
Details on test material:
- Batch number: CHT91021
- Trade name: Pyranin 120%

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Photometric analysis of the test item concentration in the test solutions was performed to determine exposure concentration and stability of trisodium 8-hydroxypyrene-1,3,6-trisulphonate during exposure.

Test solutions

Vehicle:
no
Details on test solutions:
According to the results obtained in a range-finding pre-test the study was performed as a static test in five test concentrations with a spacing factor of 2.

Based on the analytical report the purity of the test item was determined to be 77.4%. Therefore a stock solution with 129.6 mg/L test item was prepared to achieve a test concentration of 100 mg/L Pyranin in the highest treatment. The stock solution was prepared by adding 129.6 mg test item to 1000 mL test medium (Steinberg medium) and stirring for 10 min. using a magnetic stirrer at room temperature. Five nominal test item concentrations of 129.6 mg/L, 64.8 mg/L, 32.4 mg/L, 16.2 mg/L and 8.1 mg/L (spacing factor of 2) was prepared by diluting the stock solution with test medium. This corresponds to an active ingredient concentration of 100 mg/L, 50 mg/L, 25 mg/L, 12.5 mg/L and 6.25 mg/L.

Test organisms

Test organisms (species):
Lemna minor
Details on test organisms:
I. Origin:
Tests were carried out with the duckweed Lemna minor obtained from the German Federal Environment Agency (UBA), Berlin-Marienfelde.

II. Culturing conditions:
- The sterile permanent stock culture was grown on agar medium prepared according to Jungnickel & Augsten in 100 mL Erlenmeyer flasks sealed with cellulose stoppers at room temperature
- After a maximum period of 2 months the plants were transferred sterile into freshly prepared medium
- The stock culture used in the test had been cultivated in the labs of Hydrotox since March 2014
- 21 days before the start of the test plant colonies were transferred from the sterile agar stock culture into 150 mL beakers containing 100 mL Steinberg Medium
- After 7 and 14 days they are transferred into fresh medium and grown until using for the test
- This pre-culture was cultivated under test conditions (24°C ± 2°C, 85 – 135 μE m-2 s-1)
- At the earliest after a 7 days adaption period the plants were used in the test
- At least six beakers were prepared to gain enough plant material for the test
- Colonies consisting of 2 to 4 visible fronds were transferred from the inoculum culture and randomly assigned to the test vessels under aseptic conditions

Study design

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

Test conditions

Hardness:
No data
Test temperature:
25.1 – 25.7°C
pH:
5.6-6.9
Dissolved oxygen:
No data
Salinity:
n.a.
Conductivity:
No data
Nominal and measured concentrations:
Nominal test item concentrations of 129.6 mg/L, 64.8 mg/L, 32.4 mg/L, 16.2 mg/L and 8.1 mg/L (spacing factor of 2) correspond to an active ingredient concentration of 100 mg/L, 50 mg/L, 25 mg/L, 12.5 mg/L and 6.25 mg/L.
Details on test conditions:
I. EXPOSURE CONDITIONS:
- 3-4 Lemna plant colonies consisting of 9 fronds were transferred from the pre-culture into the test and control vessels at the start of the test
- Test vessels were covered with watch glasses to minimize evaporation from the test vessels
- The test vessels were placed in an illuminated shelf which is located in a temperature controlled laboratory
- Four fluorescent tubes (Osram L36W/840, CoolWhite) were placed with a distance of 50 cm above the test vessels to obtain a light intensity of 100 μE/m² s +/- 6.6 % (PAR)
- Illumination took place permanently
- Temperature was in a range of 25.1 to 25.7°C during exposure time
- Two times during the seven-day exposure period locations of the test vessels were randomised to minimise the influence of spatial differences in light intensity or temperature

II. MEASUREMENT OF FROND AREA AND FROND NUMBER:
- Frond number and frond area were determined in each test and control vessel at the start of the experiment, at day 2, at day 5 and at the end (day 7)
- For this the digital image evaluation system Scanalyzer from LemnaTec was applied. Image recognition was manually checked for every picture on the screen and if necessary misreads were corrected manually
- Frond number and frond area were documented in a report file automatically by the software

III. DATA AND REPORTING:
- For each single vessel of controls and the treatments the specific growth rates were calculated section by section for each measurement (day 0-2, day 2-5 and day 5-7) and over the entire test duration (7 days)
- The mean value and the variation coefficient for the control replicates were calculated

IV STATISTICS:
- Statistical differences between the growth rate of the test and the control vessels were checked with the software Toxrat (ToxRat Professional, version 2.10, ToxRat Solutions GmbH, Alsdorf).
Reference substance (positive control):
yes
Remarks:
dichlorophenol (Sigma-Aldrich, Steinheim, Germany, Lot No. MKBS0425V, from September 2015)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
7 d
Dose descriptor:
EC10
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:
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
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The measured test item concentrations in the test item treatments were 85.4 –106.2 % of the nominal concentrations. Therefore the EC values are given as nominal concentrations.
Results with reference substance (positive control):
The EC50(growth rate, frond number) for the reference substance 3,5-Dichlorophenol was 3.3 mg/L (2.7–3.9 mg/L 95%-CI) which is in-line with the requirements of the ISO 20079 (2005) [Lemna, Growth Inhibition Test] of 2.2–3.8 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Doubling time of the frond number in the controls must be less than 2.5 days (within a period of 60 hours); This corresponds approx. to a 7-fold increase within the7-day test duration and an average specific growth rate of 0.275 per day
Conclusions:
Under the conditions of the present study trisodium 8-hydroxypyrene-1,3,6-trisulphonate did not induce growth inhibition of Lemna minor at a nominal concentration of higher than 100 mg/L after 7 days of exposure. The measured test item concentrations in the test item treatments were 85.4–106.2 % of the nominal test item concentrations. Therefore the EC values are given as nominal concentrations.
Executive summary:

A growth inhibition test with the freshwater aquatic plant Lemna minor was conducted according to OECD 221 (23 March 2006) in order to investigate the aquatic phytotoxic effect of trisodium 8-hydroxypyrene-1,3,6-trisulphonate. Duckweed is a model organism for aquatic fresh water plants. The purpose of this test is to determine the effects of a test item on the growth of Lemna minor during a test period of 7 days. The test endpoint was inhibition of growth, expressed as logarithmic increase in measurement variable (average specific growth rate of frond number and frond area) during the exposure period. From the inhibition of the average specific growth rates recorded in a series of dilutions of the test solution compared to the control, dose-response curves and the corresponding ECxvalues were determined. At the start of the test and at day 2, day 5 and at the end of the test (day 7) the frond number and the frond area were determined using the digital image evaluation system Scanalyzer from LemnaTec (Würselen). Tests were carried out with the duckweed Lemna minor obtained from the German Federal Environment Agency (UBA), Berlin-Marienfelde. The sterile permanent stock culture is grown on agar medium prepared according to Jungnickel & Augsten (Details see Annex I) in 100 mL Erlenmeyer flasks sealed with cellulose stoppers at room temperature. After a maximum period of 2 months the plants are transferred sterile into freshly prepared medium. According to the results obtained in a range-finding pre-test the study was performed as a static test in five test concentrations (129.6 mg/L, 64.8 mg/L, 32.4 mg/L, 16.2 mg/L and 8.1 mg/L) with a spacing factor of 2 which corresponds to an active ingredient concentration of 100 mg/L, 50 mg/L, 25 mg/L, 12.5 mg/L and 6.25 mg/L. For the control only test medium was used. The test solutions (A-E) and negative control (NC) was prepared in one batch and distributed of the replicates. Test solutions were prepared with 3 replicates, negative control was prepared with 6 replicates. The test volume was 100 mL. Two additional replicates of NC, A, C and E were prepared without test organism for the chemical analysis. 3-4 Lemna plant colonies consisting of 9 fronds were transferred from the pre-culture into the test and control vessels at the start of the test. Test vessels were covered with watch glasses to minimize evaporation from the test vessels. Frond number and frond area were determined in each test and control vessel at the start of the experiment, at day 2, at day 5 and at the end. For each single vessel of controls and the treatments the specific growth rates were calculated section by section for each measurement (day 0-2, day 2-5 and day 5-7) and over the entire test duration (7 days). The mean value and the variation coefficient for the control replicates were calculated. All Validity criteria were met. Under the conditions of the present study trisodium 8-hydroxypyrene-1,3,6-trisulphonate did not induce growth inhibition of Lemna minor at a nominal concentration of higher than 100 mg/L after 7 days of exposure. The measured test item concentrations in the test item treatments were 85.4–106.2 % of the nominal test item concentrations. Therefore the EC values are given as nominal concentrations. This toxicity study is classified as acceptable and satisfies the guideline requirements for the growth inhibition test with Lemna minor.