Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: All validity criteria are fulfilled.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: solid, powder
- Analytical purity: > 99%
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date:
- Lot/batch No.: 06060902
- Expiration date of the lot/batch: June 2009
- Storage condition of test material: room temperature
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Water solubility (under test conditions): < 1 mg/l

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples in 400 mL test vessels were prepared for analytical determinations by the test site AQura GmbH, Marl, Germany. 200 ml of the concentrations 6.25; 25; 100 mg/L culture medium and culture medium (control) were filled each into the test vessels and stored under the same conditions as the other test vessels. After 0 h, 24 h, 48 hand 72 h an amount of 30 mL of each sample was transferred to a 50 mL glass flask, which was closed with screw cap and stored in a freezer (approx. -20°C). At the end of the algae test the samples were sent to the test site for analysis. For transport the samples were shipped on ice and held frozen until analytical determination was performed.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Six 2 L beakers were prepared. One was filled with 1.5 L of culture medium for the control.
The following test substance concentrations of Linkwell TBzTD in 1 L culture medium each were used for test substance preparations:
6.25 mg + 1000 mL culture medium
12.5 mg + 1000 mL culture medium
25 mg + 1000 mL culture medium
50 mg + 1000 mL culture medium
100 mg + 1000 mL culture medium
The volumes of culture medium were weighed into the five 2L beakers each, stirred with magnetic stirrer (300 rpm) and the corresponding amounts of the test substance were added to the stirring medium. Preparations were stirred for 24 h.
The test substance preparations were then filtered through a filter paper circle (diameter 320 mm) (Schleicher &Schuell, Dassel, Germany). The control was treated the same way.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: 86.81
- Source (laboratory, culture collection): "Sammlung von Algenkulturen", Göttingen (SAG),
(Collection of Algae Cultures, Institute for Plant physiology), University of Göttingen, Germany
- Age of inoculum (at test initiation):
- Method of cultivation: A slant agar tube with Desmodesmus subspicatus was stored in the refrigerator. Two 300 mL conical flasks, filled with 100 mL of sterile culture medium were inoculated once with algae from the slant agar.
Twice a week (on Monday and Thursday) the algae stock culture was transferred: One·mL of the stock culture was diluted in two 300 mL conical flasks with 100 mL of fresh sterile culture medium. After two weeks, these stock cultures were discarded and new cultures started as described above.
The culture flasks were stored in an incubator (type: KBP 6395LL Tritec, Hannover, Germany). The temperature was controlled at 25 ± 1 QC and the lighting supplied by fluorescent tubes Osram Dulux L, "Cool White" was 0.72 x 1020 photons per m2s = 8000 ± 1000 Lux, measured with a spherical collector.
To facilitate transfer of carbon dioxide, the algal medium was continuously stirred (magnetic stirrer, Variomag Telemodul 40 S, H + P Labortechnik, Munich, Germany).
To ensure that stock cultures were not contaminated, they were spread on a Caso agar plate with an inoculation loop once a week (on Thursday). The agar plate was incubated like the submerged algae cultures and was evaluated the following Monday. Contaminated stock
cultures were discarded.
Temperature and lighting were measured once a week and the measurements were archived.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
of culture medium 0.182 mmol/l = 1.02 °dH
(calculated from the concentrations of magnesium sulfate and calcium chloride)
Test temperature:
24.8 to 25.2 °C
pH:
at start of exposure time: 7.77.to 7.99
at end of exposure time: 9.81 to 10.44
see Table 1
Nominal and measured concentrations:
see Table 2
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL conical flasks with cotton-wool plugs
- Material, size, headspace, fill volume: 150 ml


- Initial cells density: 5000 cells per ml
- No. of vessels per concentration (replicates): 3 + 1 for analytical determinations
- No. of vessels per control (replicates): 6 + 1 blank


GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: double distilled water
- Ca/Mg ratio: 2,07 (mol/mol; calculated)

OTHER TEST CONDITIONS
- Sterile test conditions: yes (All solutions were autoclaved for 15 min at 121 °C)
- Adjustment of pH: The pH value of the stock solutions was adjusted to 8.1 with 1N NaOH.
- Photoperiod: 72 h
- Light intensity: 8000 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
during test photometry : wavelength 578 nm, cuvette width 5 cm, spectrophotometer UV-1602 (Shimadzu, Duisburg, Germany)
during precultivation: electronic particle counter Coulter Z2 (Coulter Electronic, Krefeld, Germany)


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 6.25; 12.5; 25; 50; 100 mg/l
Based on a NON-GLP pre-test the test substance Linkwell TBzTD was poorly soluble in water and the test was performed with the test substance preparations of 6.25; 12.5; 25; 50; 100 mg test substance/L culture medium.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
63.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
181.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: extrapolation by statistical program ToxRat®
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
50
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
25
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
520.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: extrapolation by statistical program ToxRat®
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Unusual observations were not made.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test substance was poorly soluble in water. After 24h of stirring the test substance preparations were filtered to eliminate substance deposits on the surface of the liquid and on
the bottom of the beaker.

For the parameters yield and growth rate the EC50 values could not be determined by the statistic program ToxRat® due to mathematical
reasons; therefore the EyC50 and ErC50 values were >100 mg/L.
For the growth rate the EC50 value could not be determined by the statistic program ToxRat® due to mathematical reasons.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- ErC50: 0.8 mg/l
- Other:

Any other information on results incl. tables

Table 3: Inhibition in Yield and Growth rate after 72 h

 mg/l 0 (control)  6.25  12.5  25  50  100 
Yield * 10^4 (mean cell number)  85.821  83.334  84.512  82.968  77.213  74.352 
Inhibition (%)  0.0  2.9  1.5  3.3  10.0  13.4 
Growth rate  1.458  1.448  1.453  1.447  1.423  1.410 
Inhibition (%)  0.0  0.7  0.3  0.8  2.4  3.3 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The validation criteria were fulfilled as algal growth in the control between 0 and 72h exceeded the factor of 16 as requested by the underlying OECD guideline. With the current test it was found to be 79.4.
The mean coefficient of variation of the section-by-section (0 - 24; 24 - 48 and 48 - 72 h) growth rate in the control cultures must not exceed 35 % and it was 23.2 % in the current test.
The coefficient of variation of the average specific growth rate in the control cultures, measured during the exposure period (0-72 h) must not exceed 7 % and it was 0.9 % in the current test.
Temperature during the exposure period (O-72h) was in the required range with values between 24.8 and 25.2°C as well as illumination with values of 8000 Lux.
The pH values were in the required range, as reported in Table 1.
Executive summary:

The test substance Linkwell TBzTD-Tetrakis(phenylmethyl)thioperoxydi(carbothioamid) was tested in the Alga, Growth Inhibition Test according to the OECD Guideline for the Testing of Chemicals No. 201 (23 March 2006) using the single cell green algae Desmodesmus subspicatus as test organism.

• The experiment was carried out as a static test, Le. the test substance preparations were not exchanged for the whole exposure period of 72 hours.

• Based on a NON-GLP pre-test the test substance Linkwell TBzTD is poorly soluble in water and the test was performed with the test substance preparations of 6.25; 12.5; 25; 50; 100 mg test substance/L culture medium.

• After 24h of stirring the test substance preparations were filtered to eliminate substance deposits on the surface of the liquid and the bottom of the beaker. After filtration test substance preparations were present as clear solutions.

• During this study the test substance concentrations of nominal 6.25; 25; 100 mg/L culture medium and the control (culture medium) were determined by the test site AQura GmbH,Marl, Germany by HPLC analysis~ This phase of the study is described in detail in the Contributor Report.

• The test solutions and the control solutions were inoculated with 5 x 103 algae per mL.

Validation experiments and the pre-cultivation ensured that the algae were of appropriate viability and sensitivity. After 0, 24, 48 and 72h exposure period the extinction of an aliquot of the cultures was measured photometrically for determination of algae growth. Yield and growth rate were calculated.

• The results of this study show that the test substance Linkwell TBzTD induced inhibition of algal growth after 72 hours of exposure. For the parameters yield and growth rate the ECso values could not be determined by the statistic program ToxRat® due to mathematical reasons; therefore the EyCso and ErCso values were >100 mg/L.

The LOEC and NOEC values were 50 mg/L and 25 mg/L, respectively, after 72 h of exposure, based on nominal concentrations.

Based on analytically determined concentrations (see Table 2) no results could be stated because the test substance concentrations were below the limit of quantification.