N,N'-(decane-1,10-diyldi-1(4H)-pyridyl-4-ylidene)bis(octylammonium) dichloride

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

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 104663
- Purity: 98.9%

OTHER SPECIFICS:
White powder
Test substance is not volatile

Analytical monitoring:

yes

Details on sampling:

- Sampling method: initially and after 72 hours

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: 86.81
- Source: Sammlung für Algenkulturen, Pflanzenphysiologisches Institut der Unversität Göttingen, Germany

ACCLIMATION
- Acclimation period: 3 days of preculture prior to the test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 - 22 °C

pH:

7.18 – 7.40 (initial)
7.17 – 7.48 (after 72 h)

Nominal and measured concentrations:

Initial concentrations: Control, 20, 36, 65, 117, 210 µg/L
Actual concentrations: see below

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks
- Initial cells density: 1 x 10E4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes. Fully artificial medium as recommended by OECD guideline 201

OTHER TEST CONDITIONS
- Light intensity and quality: 8000 lux +/- 20 % in a wavelength range between 400 and 700 nm

EFFECT PARAMETERS MEASURED: cell multiplication inhibition (microscopic counting chamber); calculation of growth rate and biomass production; sampled at 24, 48, 72 hours

Reference substance (positive control):

not required

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.036 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.02 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.:

0.055 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

Test not performed. However, sensitivity of the algal strain used is regularly tested by the testing facility. In the period of the Octenidine dihydrochloride test EbC50 and ErC50 for potassium dichromate as reference substance were 610 µg/L and 740 µg/L, respectively. Both values are well within the range required by the OECD 201 guideline.

Reported statistics and error estimates:

ECx calculation by probit analysis,
NOEC derivation by analysis of variance (Dunnett’s test)

Cell concentration data

Test substance Concentration (nominal) [mg/l] Cell concentrations (mean values) [cells x 10^-4/ml] measured 0 h 24 h 48 h 72 h Control 1.0 3.1 7.1 27.5 0.020 1.0 2.6 4.6 23.9 0.036 1.0 2.5 5.6 9.2 0.065 1.0 2.2 3.2 3.4 0.117 1.0 2.5 2.6 1.8 0.210 1.0 1.7 2.3 1.9

Validity criteria fulfilled:

yes

Conclusions:

Octenidine dihydrochloride was found to be acutely very toxic towards algae.

Executive summary:

The green alga Desmodesmus subspicatus (formerly Scenedesmus subspicatus CHODAT) was used as the test organism in toxicity tests of octenidine dihydrochloride towards algae. The test was performed according to the guidelines OECD 201 and 92/69/EWG, C.3. Algae were exposed to five concentrations of the test substance, ranging nominally from 0.02 to 0.21 mg/L in a geometric series and a control group. Initial cell densities were 1 x 10⁴cells/mL. The test was performed at 21 - 22 °C in a fully artificial medium according to OECD 201. Cell densities were assessed at 24, 48 and 72 hours using a microscopic counter chamber. Physico-chemical water parameters and octenidine dihydrochloride concentrations were measured at 0 and 72 hours. Concentrations were analysed by an appropriate HPLC method with UV detection at 280 nm.

Octenidine dihydrochloride was found to be toxic towardsD. subspicatusin the low microgram-per-litre range.

All validity criteria according to the test guideline OECD 201 are met within the study.

A clear very steep concentration-response relationship can be established from the study results.

Analytical monitoring of the actual test concentrations in vessels with algae gave inconsistent and unsatisfactory results due to unknown reasons. Analysis of concentrations in a blank vessel without algae revealed time-dependent depletion to 47% of the initial value after 96 h. This percentage is in accordance with the results from a fully reliable analytical procedure. Subsequently, the geometric mean of the measured initial (0 h) and final percent values (72 h) is used as an average value of octenidine dihydrochloride exposition over the test duration of 72 h:

(98·42)^½=64.2

Mean average concentration = 64.2% of nominal concentration

The decreasing concentrations cannot be ascribed to volatility or hydrolysis, as octenidine dihydrochloride is non-volatile and stable in aequeous solution. Another possibility is adsorption to glass container walls and algae, therefore the results from the blank vessel without algae can reliably be used as an estimate for non-biological adsorption.

NOErC	0.013 mg/L (measured average) (calculation 0.02 mg/L * 0.642 = 0.013 mg/L)
ErC50	0.034 mg/L (measured average) (calculation 0.055 mg/L * 0.642 = 0.034 mg/L)
EbC50	0.023 mg/L (measured average) (calculation 0.036 mg/L * 0.642 = 0.023 mg/L)

Description of key information

Algae were found to be slightly less sensitive with EC₅₀s of 0.023 mg/L and 0.034 mg/L for biomass production and growth rate, respectively, and a (long-term) NOEC of 0.013 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.034 mg/L

EC10 or NOEC for freshwater algae:

0.013 mg/L

Additional information

Analytical monitoring of the test substance was performed during this study. However, for unknown reasons results from vessels with organisms delivered inconsistent and unreliable results. Therefore, measured concentrations refer to the results from blank vessels without organisms, which were set up in parallel to the biotests.