Spiro[piperidine-1,1'-pyrrolidinium] bifluoride

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

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:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2008

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Stability of test concentration/s during exposure: Examined by chemical analysis (DOC) at 0, 24, 48 and 72 hours.

Analytical monitoring:

yes

Details on sampling:

Stability of test concentration/s during exposure: Examined by chemical analysis (DOC) at 0, 24, 48 and 72 hours.

Vehicle:

no

Details on test solutions:

PRE-TREATMENT OF TEST ITEM AND PREPARATION OF TEST ITEM CONCENTRATIONS
To produce the only test item concentration 200.1 mg of the test item were added to 1 litre of dilution water. The pH was measured to be 5.4 and was adjusted to pH 7.8.
100 mL of the solution were taken and 0.802 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL. For each test item concentration and the control 6 replicates were prepared. All flasks were sealed with cotton stoppers.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Name: Desmodesmus subspicatus (formerly Scene-desmus subspicatus) Strain No. 86.81 SAG
Source: Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
Maintenance and Acclimatisation : Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21 - 24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 μE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a cell counter (Z2, Beckman Coulter).
Preparation of pre cultures: Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium.
Test cultures : The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

21 - 24 °C

pH:

7.9 - 8.0

Nominal and measured concentrations:

nominal: 100 mg/L

Details on test conditions:

EXPOSURE CONDITIONS
Test vessels: 300 mL Erlenmeyer flasks with cotton stoppers test volume: 100 mL
Culturing apparatus: Shaking incubator in which a temperature in the range 21 °C to 24 °C was maintained at +/- 2 °C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm. Temperature was measured and recorded daily.
Light intensity: A light intensity ranging from 60 to 120 μE x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was measured. The light intensity was checked before the start of the study.
Cell density measurements: Cell densities were measured in a cell counter (Z2, Beckman Coulter) by taking aliquots from each test flask, which were not replaced.
Experimental design: 1 test concentration plus 1 control Initial cell density in the test cultures approximately 5000 cells per millilitre. Additionally highest test concentration without algae.
Test item concentration/s: 100 mg/L
Method of administration: direct weighing
Duration of exposure: 72 hours
Criteria of effects: The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r] of the algal population.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Yield

Details on results:

No toxic effects against algae were observed at a limit test concentration of 100 mg/L (nominal) under exposure conditions.
The results are expressed in terms of nominal concentrations. The substance has not a definite or unique structure and consists of several components. The content of the test item during the exposure period was verified by DOC determination.

Reported statistics and error estimates:

All calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10). For the calculations all algae counts were divided by a factor of 10000.

Analysis

Test item concentration	DOC values [mg/L]
[mg/L]	0 h	24 h	48 h	72 h
Control	--	--	--	2.7973
100	58.544	52.343	49.595	55.092
100 without algae	58.885	50.452	54.971	55.499

 

Validity criteria fulfilled:

yes

Conclusions:

No toxic effects of RFR 6634 against algae were observed at a limit test concentration of 100 mg/L (nominal) under exposure conditions (EC50 > 100 mg/L, NOEC >= 100).

Executive summary:

A study was performed to assess the adverse effects of RFR 6634 on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations.

The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to a limit test concentration of nominally 100 mg/L of RFR 6634 dissolved in dilution water.

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration (NOEC) and a Lowest Observed Effect Concentration (LOEC) according to STUDENT-t test for Homogeneous Variances. The following values were determined:

ErC 50* (0-72 h): > 100

ErC 10* (0-72 h): > 100

NOEC [r]* (tα 0.05): ≥ 100

LOEC [r]* (tα 0.05): > 100

* Reduction of growth rate (ErCx, NOEC [r]) is the preferred endpoint according to OECD 201 and for regulatory purposes in the EU. Results relating to yield (EyCx, NOEC [y]) were calculated to fulfil regulatory requirements in some countries (but not in the EU) and are given in the results section of this report.

No toxic effects against algae were observed at a limit test concentration of 100 mg/L (nominal) under exposure conditions.

The results are expressed in terms of nominal concentrations. The substance has not a definite or unique structure and consists of several components. The content of the test item during the exposure period was verified by DOC determination.

Description of key information

A study was performed to assess the adverse effects of RFR 6634 on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations.

The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to a limit test concentration of nominally 100 mg/L of RFR 6634 dissolved in dilution water.

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration (NOEC) and a Lowest Observed Effect Concentration (LOEC) according to STUDENT-t test for Homogeneous Variances. The following values were determined:

ErC 50* (0-72 h): > 100

ErC 10* (0-72 h): > 100

NOEC [r]* (tα 0.05): ≥ 100

LOEC [r]* (tα 0.05): > 100

* Reduction of growth rate (ErCx, NOEC [r]) is the preferred endpoint according to OECD 201 and for regulatory purposes in the EU. Results relating to yield (EyCx, NOEC [y]) were calculated to fulfil regulatory requirements in some countries (but not in the EU) and are given in the results section of this report.

No toxic effects against algae were observed at a limit test concentration of 100 mg/L (nominal) under exposure conditions.

The results are expressed in terms of nominal concentrations. The substance has not a definite or unique structure and consists of several components. The content of the test item during the exposure period was verified by DOC determination.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Results should read "EC 50 (0 -72 h): > 100 mg/L and NOEC ≥ 100"