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
Study period:
2017-07-25 - 2017-07-28
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:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Physical state/ Appearance:liquid/yellowish, clear
Specific details on test material used for the study:
Name of test substance: Dispex Ultra FA 4404
Chemical name: Phosphonic acid, P,P'-(1-hydroxyethylidene)bis-, compd. with 2-aminoethanol (1:?)
Test substance No.: 16/0276-1
Batch identification: 0013537161
CAS No.: 42220-47-3
Composition: component 1: ethanolamine: 19.7 g/100 g (1H-NMR)
component 2: (1-hydroxy-1-phosphono-ethyl)phosphonic acid: 28.3 g/100 g (1H-NMR) / 28.1 g/100 g (31-PNMR)
side component 3: H3PO3: 0.9 g/100 g (1H-NMR) /
1.0 g/100 g (31-PNMR)
side component 4: H3PO4: 0.2 g/100 g (31-PNMR)
Expiry date: 30 Dec 2017
Date of production: 14 Apr 2015
Physical state/ Appearance:
liquid/yellowish, clear
Water solubility: soluble in any ratio
Storage conditions: The test substance was stored at ambient temperature.
Water: 49.9 g/100 g

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Sampling was performed at the start of the exposure (0 h) from the inoculated replicate 7 of each concentration > 2.0 mg/L and the control. The 3 lower concentrations were not analyzed, since they were expected to be clearly below the LoQ of the analytical method.

At the end of the exposure (72 h) samples were taken from the combined inoculated (+ algae) replicates of the three highest test concentrations and of the combined inoculated control group replicates. Additionally, samples were collected from the uninoculated (-algae) replicate 0 of the 3 highest test concentrations and the control at the end of the exposure (72 h).

Test solutions

Vehicle:
no
Details on test solutions:
The test substance item is an UVCB mixture of components, which are soluble in the test medium in the concentration range selected for this study. The test solutions were prepared separately for the three highest test concentrations and by dilution of a stock solution with 2.0 mg/L for the three lowest test concentrations. The stock test solutions were prepared by directly adding of test substance to test medium and stirring for approximately 20 min until visibly dissolved. The pH-value was adjusted in the 2 highest test concentrations to the pH-value of the control, since it deviated by more than 0.5 units from the control. For each test concentration stock test solutions of 111% of the nominal concentration were prepared to compensate for the dilution by the later addition of algal inoculum. Inoculum culture was added at a ratio of 1:10. After this, the inoculated solutions were distributed to the replicates of each test concentration.
All stock solutions and test solutions were visibly clear and completely dissolved following preparation.

The test concentrations were prepared according to the following scheme:

Nominal concentration [mg/L] 111% stock test solution (before inoculum addition) [mg/L] Weighed amount of test substance [mg] Final Volume test stock solution [mL]
0 (control) - - 1000
2.0 2.22 4.45 2000
6.39 7.09 7.11 1000
20 22.2 22.24 1000

To ensure the precise amount of test substance in the test solutions the lower test concentrations were prepared by dilution of the test stock solution 2.0 mg/L according to the following dilution scheme.

Dilution scheme:

Nominal concentration [mg/L] 111% stock test solution (before inoculum addition) [mg/L] Volume of stock test solution 2.0 mg/L [mL] Final Volume test solution [mL]
0.064 0.071 32 1000
0.20 0.222 100 1000
0.639 0.709 320 1000

The pH-value was adjusted in the 2 highest test concentrations to the pH-value of the control, since it deviated by more than 0.5 units from the control (7.5 and 7.8 in the highest and next highest concentration versus 8.1 in the control). Before the dilution was performed, the stock solutions were checked for complete dissolution of the test substance. All stock test solutions appeared colorless and clear.

For each test concentration stock test solutions of 111% of the nominal concentration were prepared to compensate for the dilution by the later addition of algal inoculum.

Inoculum culture was added to the stock test solutions at a ratio of 1:10. After this, the inoculated solutions were distributed to the replicates of each test concentration.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Supplier:
The algae were obtained at regular intervals from SAG (Collection of algal cultures in Göttingen) and were kept in liquid culture in the Laboratory of Ecotoxicology of the Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany.

Study design

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

Test conditions

Test temperature:
22.4 - 22.5 °C
Nominal and measured concentrations:
Test groups: 0 (control), 0.064, 0.20, 0.64, 2.0, 6.39 and 20 mg/L as nominal concentrations based on test substance item mass without correction for purity or composition, which is equivalent to
0 (control), 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L based on the content of all components of the test substance item other than water (50.1%,)
Details on test conditions:
Test conditions
Test duration: 72 hours
Test vessels: Erlenmeyer flasks (nominal volume 250 mL) plugged with gas permeable silicone sponge caps
Test media: OECD media
Test volume: 100 mL
Initial cell density: 0.5 x 104 cells/mL
Test chamber: Vötsch Industrietechnik GmbH Bioline (VB1014) controlled climate cabinet.
Test temperature: 22.4 - 22.5 °C
Illumination: Artificial light, type universal white (OSRAM L 25), permanent illumination. To minimize the potential effect of slight variations in illumination, the test vessels were rearranged daily.
Light intensity: Average 6588 – 7211 lux (within ± 15% variability) at a wave length of 400 - 700 nm
Shaking rate: Continuous (approx. 85 rpm)
Test parameter: Algal growth measured as in vivo chlorophyll-a fluorescence (pulsed excitation with light flashes having a wavelength of 430 nm)
Route of exposure: Static exposure via the test medium.

The cell density of the inoculum culture in exponential growth phase was determined (Section 3.3.1) and then adjusted to 5 x 104 cells/mL to obtain the correct initial cell density in the test replicates of 0.5 x 104 cells/mL and the correct nominal concentrations of the test solutions, inoculum culture was added to each 111% stock test solution at a ratio of 1:10.
Reference substance (positive control):
yes

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.99 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4.14 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.241 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.17 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
EC10: 0.99 mg/l nominal corresponds to 0.497 mg/l a.i.
EC50: 4.14 mg/l nominal corresponds to 2.078 mg/l a.i
Results with reference substance (positive control):
Reference substance toxicity testing
In order to verify that the algal cultures are responding normally to toxic stress, tests with a reference substance (potassium dichromate) are conducted. Reference substance tests are conducted according to OECD 201 guidelines and in accordance with GLP, but without a GLP status. The results from the reference substance test are compared to potassium dichromate EC50 values published in ISO test guideline 8692, which represent the typical response range for the algal species tested.

According to the test ISO guideline 8692 the EC50 values of the reference substance potassium dichromate should be in the range: ErC50 = 0.92 – 1.46 mg/L after 72 hours for Pseudokirchneriella subcapitata.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In a 72-hour algal growth study, cultures of Pseudokirchneriella subcapitata were exposed to Dispex Ultra FA 4404 at nominal concentrations of 0 (control), 0.064, 0.20, 0.64, 2.0, 6.39 and 20 mg/L under static conditions for in accordance with the OECD 201 guideline. The water pH, temperature and lighting intensity were all maintained within acceptable guideline specifications. The following statistical effect concentrations were obtained after 72 hours of exposure:
Growth rate: EC10: 0.99 mg/l and EC50: 4.14 mg/l nominal

Since the measured concentrations of the test substance in the test solutions above the LoQ did sufficiently confirm the nominal concentrations, the effect concentration can be expressed relative to the nominal concentration for the evaluation of the test substance.
Executive summary:

In a 72-hour algal growth study, cultures of Pseudokirchneriella subcapitata were exposed to test item Phosphonic acid, P,P'-(1-hydroxyethylidene)bis-, compd. with 2-aminoethanol (1:?) at nominal concentrations of 0 (control), 0.064, 0.20, 0.64, 2.0, 6.39 and 20 mg/L under static conditions for in accordance with the OECD 201 guideline. The water pH, temperature and lighting intensity were all maintained within acceptable guideline specifications. The following statistical effect concentrations were obtained after 72 hours of exposure:

Growth rate:  EC10: 0.99 mg/l and EC50: 4.14 mg/l  nominal

Since the measured concentrations of the test substance in the test solutions above the LoQ did sufficiently confirm the nominal concentrations, the effect concentration can be expressed relative to the nominal concentration for the evaluation of the test