4,4'-[(1,10-dioxodecane-1,10-diyl)bis(oxy)]bis(2,2,6,6-tetramethylpiperidine-1-oxidanyl)

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Adopted December 1992

Deviations:

not specified

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,10-bis(2,2,6,6-tetramethyl-1-piperidinyloxy)-1,10-dioxodecane

EC Number:

417-440-8

EC Name:

1,10-bis(2,2,6,6-tetramethyl-1-piperidinyloxy)-1,10-dioxodecane

Cas Number:

2516-92-9

Molecular formula:

C28 H50 N2 O6

IUPAC Name:

bis(1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl) decanedioate

Details on test material:

Identification code (lab) = TKA 40075

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: During the final test samples for analysis were taken from the control, 10, 32 and 100 %{v/v) of the filtrate
- Sampling method:
Frequency: at t=0h and t=72h
Volume: 10 ml
- Sample storage conditions before analysis:
Samples taken at t=0 h were stored in a deep-freeze until analysis.
Sample taken at t=72 h were analyzed on the day of sampling.

Test solutions

Vehicle:

no

Details on test solutions:

The test solutions were prepared using a stock solution in ISO-medium.
For this purpose the test substance was added quantitatively to obtain a supersaturated solution (nominal 100 mg/l). The stock solution was stirred for ca. 92h and passed through a rough paper filter. From this filtrate the required volumes for preparation of the test concentrations were added to ISO-medium. Subsequently, a volume of 0.36 ml of the algal suspension was added. The final volume of the test solutions was 50 ml per vessel. At the start of the test all test solutions were clear and colorless.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): CCAP

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

pH:

7,9-8,2

Nominal and measured concentrations:

nominal: 10, 18, 32, 56 and 100% (v/v) of a filtrate of nominal 100 mg/L with an actual test substance concentration of ca. 0,62 mg/L yielding an actual range of 0,06, 0,11, 0,20, 0,35 and 0,65 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all glass
- Initial cells density: 1 * 10^4 cells/ml
- Control end cells density: 56,7 * 10^4 cells/ml
- No. of organisms per vessel: 1*10^4 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, ISO-medium.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: 7000-8000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber; spectrophotometer
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations:
According to data of the sponsor, water solubility of TKA 40075 (GU 10-415) was < 0.5 mg/l at 20 °C. A supersaturated stock solution of 100 mg/l medium was stirred for ca. 94 hours to reach the maximum attainable concentration. At the end of the stirring period, the stock solution was turbid with undissolved test substance particles. This suspension was then filtered through a rough paper filter to remove the larger undissolved test substance particles. Algal suspensions were exposed to solutions containing 0.1, 1, 10 and 100% (v/v) of the filtrate.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: above maximum water solubility (<0.5 mg/L)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: above maximum solubility (<0.5 mg/L)

Details on results:

At the start of the test an average of 0.62 mg/l was found in the filtered solution of nominal 100 mg/l.
Note that this actual concentration was lower than the maximum attainable concentration in the acute toxicity test in Daphnia magna (NOTOX project 141075, ca. 16 mg/l) and in the 96-hour acute toxicity study in carp (NOTOX project 141086, ca. 20 mg/l). This was due to the fact that, in these studies Cremophor was used as disperging agent, whereas Cremophor cannot be used in an legal test.
The test substance concentrations were below the detection limit of 0.19 mg/l in the samples taken from the 10 and 32 % (^/y) solution of the filtrate nominal 100 mg/l.
At the end of the 72-hour period measured concentrations had decreased to levels below the detection limit (<0.19 mg/l). No difference was observed between samples taken from the highest test concentration with or without algae.

No significant differences were recorded between the values for cell growth or growth rate in any of the test solutions. Note that relatively low extinction values were recorded in two of the six control-vessels. These
lower values reduced the average cell density to such an extent that it was substantially lower than the average cell densities of most treated groups. This was the major reason for the negative values for both growth inhibition and growth rate reduction at the lower test concentrations.


In the control the cell density increased by an average factor of 57 within three days.
Analysis of samples taken from the solution with and without algae failed to show that the actual exposure concentration remained above 80 % relative to the initial concentration. However, it should be noted that the initial concentration of 0.6 mg/l was above the maximum solubility of TKA 40075 (GU 10-415) in water (< 0.5 mg/l at 20°C).
Further, all test conditions remained within the ranges prescribed by the protocol.

Results with reference substance (positive control):

The 72-hour EC50 for growth inhibition (EBC50:0-72h) was 0.69 mg/l. The 72-hour EC50 for growth rate reduction (ERC50:0-72h) was 1.32 mg/l.
These results were in agreement with what was expected on the basis of historical data:
The historical ranges of the 72h EC50 for growth inhibition and the 72h EC50 for growth rate reduction lie between 0.32 and 1.8 mg/l, with < 50 % response at 0.32 mg/l and > 50 % response at 1.8 mg/l.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Scenedesmus capricornutum. TKA 40075 (GU 10-415) did not reach concentrations in water, that inhibited cell growth or reduced growth rate of this fresh water algae species.
Hence, the EC50 for both cell growth and growth rate was > 0.62 mg/l based on intial concentrations, i.e. far above the maximum water solubility. The NOEC was 0.62 mg/l for effects on both cell growth and growth rate.

Executive summary:

Fresh Water Algal Growth Inhibition Test,with TKA 40075 (GU 10-415).

The study procedures described in this report were based on the EEC Directive 92/69, Publication No. L383 Part C-3 adopted December, 1992; OECD guideline No. 201, Adopted June 7, 1984; and ISO Standard 8692, First edition, 15 November 1989.

According to data of the sponsor, water solubility of TKA 40075 (GU 10- 415) was < 0.5 mg/l at 20 °C.
After a range-finding test, a final test was performed exposing exponentially growing algal cultures to a range of dilutions of a rough filtrate prepared at 100 mg/l after stirring for ca. 92 hours.

The average actual concentration of the active substance in the filtrate was found to be 0.62 mg/l based on analysis taken at the start of the test. Hence, based on this value, the test solutions contained TKA 40075

(GU 10-415) concentrations of 0.06, 0.11, 0.20, 0.35 and 0.62 mg/l.
At the end of the 72-hour period, measured concentrations had decreased to levels below the lowest detection level of 0.19 mg/l.

Under the conditions of the present study with Scenedesmus capricornutum. TKA 40075 (GU 10-415) did not reach concentrations in water, that inhibited cell growth or reduced growth rate of this fresh water algae species.

Hence, the ECSO for both cell growth and growth rate was > 0.62 mg/l based on intial concentrations, i.e. far above the maximum water solubility. The NOEC was 0.62 mg/l for effects on both cell growth and growth rate.