N,N'-hexamethylenebis(cinnamylideneamine)

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:

From October 04 to 15, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on test solutions:

The following test flasks were set up:
• Test solution (Tn); containing test medium, test item and algae (three replicates per concentration)
• Blank control (Bn); containing test medium and algae (six replicates)
Because the test item is pure with a limited solubility (44 mg/l), the test solutions were prepared by respective dilutions of a saturated stock solution. The respective quantity of test item was added to a sterile empty glass vessels, sterile-filtered algal medium was added and this mixture was moderately stirred overnight. After this incubation time, the test suspension was filtered and the resulting stock solution was diluted (so that the concentrations were spaced by a factor 3.16) with sterile Algal medium into sterile test flasks (glass fiber filter, 1-2 µm). The pH was measured. Finally, each flask (containing the resulting sterile test medium) was inoculated with an exponentially growing preculture of algae.
Pure Algal medium, also sterile filtered into sterile test flasks and inoculated with the exponentially growing preculture of algae mentioned above, served as blank controls.
For the incubation, the test flasks were then placed on an orbital shaker at a nominal shaking speed of about 130 revolutions per min.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Test organism: Axenic slope culture of Desmodesmus subspicatus 86.81 SAG (Collection of Algal Cultures, Institute of Freshwater Ecology, University of Göttingen, D-37073 Göttingen, Germany
Culture: 250 ml flask containing 100 ml of sterile OECD medium (Table 2) inoculated with cell material from axenic slope culture
Illumination: Continuous (2000–3000 lux) from Osram Fluora L18W77 and Osram Daywhite L18W840 (Osram AG, Winterthur, Switzerland)
Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
Control of sensitivity: None – the culture is re-purchased annually

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 to 24°C, maintained at ± 2°C in a thermo-controlled room

pH:

Initially adjusted to 8 ± 0.2; the pH of the control medium should not increase by more than 1.5 units during the test

Nominal and measured concentrations:

100, 31.6, 10.0, 3.16 and 1.00 mg/l loading rate(these loadings refer to the corresponding dilutions (spacing factor 3.16) of a 100 mg/l saturated stock solution).

Details on test conditions:

Test vessel 250 ml flasks, all-glass, with 100 ml of test medium, shaken (130 rpm)
Test medium Recommended OECD medium
Preculture Exponentially growing culture of Desmodesmus subspicatus
Illumination Source: continuous from Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland)
Light intensity : the light intensity amounts to about 3000 lux which corresponds to about 40 μE•m-2•s-2
Homogeneity: the light intensity is maintained within ±15% from the average light intensity over the incubation area. The test vessels are repositioned every day within the incubation area to minimize variation.
Starting cell density 0.5–0.85 µg/ml with respect to dry weight corresponding to about 2–5•103 cells/ml (i.e. OD680 of about 0.005 units)

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 0.627 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 5.31 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.23 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 0.326 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.698 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.23 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Test concentrations

The concentrations of Intercure 3 in the test media were measured by HPLC analysis at the beginning and after 24, 48 and 72 h of exposure. These analyses confirmed the limited solubility of the test item (13% of the 100 mg/l loading rate of the saturated stock solution) and showed that the concentrations decreased over the 72 h test period (24, 9, 3, 1 and 46 % of the initially measured concentration in the 1.00, 3.16, 10.0, 3.16 and 100 mg/l test levels respectively).

Therefore, the effective concentrations E_rC_x/E_yC_xwere assessed based on the geometric mean of the measured concentrations of the test item over the whole 72 h test period.

Determination of the effect concentrations based on growth rate

The dose-response curve encompassed the range from no observed effects to 63 % inhibition, as compared to the blank controls.

The median effect concentration with respect to growth rate (E_rC₅₀/0–3 days) of Intercure 3 to Desmodesmus subspicatus was calculated to be 5.31mg/l (95 % confidence limits: 4.61–6.10 mg/l).

The low effect concentration with respect to growth rate E_rC₁₀was calculated to be 0.627 mg/l (95 % confidence limits: 0.436–0.836 mg/l).

The no observed effect concentration (NOE_rC) with respect to growth rate was determined to be 0.230 mg/l.

Determination of the effect concentrations based on yield (biomass production)

The dose-response curve encompassed the range from no observed effects to 95% inhibition, as compared to the blank controls.

The median effect concentration with respect to yield (E_yC₅₀/0–3 days) of Intercure 3 to Desmodesmus subspicatus was calculated to be 0.698 mg/l (95 % confidence limits: 0.647–0.766 mg/l).

The low effect concentration with respect to yield E_yC₁₀was calculated to be 0.326 mg/l (95 % confidence limits: 0.272-0.369 mg/l).

The no observed effect concentration (NOE_yC) with respect to yield was determined to be 0.230mg/l.

Validity criteria fulfilled:

yes

Conclusions:

ErC50 (72h) = 5.31 mg/l
EyC50 (72h) = 0.698 mg/l

Executive summary:

Method

The effective concentrations (E_rC₁₀, E_rC₅₀and E_yC₁₀, E_yC₅₀) as well as the no observed effect concentrations (NOE_rC and NOE_yC), of Intercure® 3 (CAS No.140-73-8) to the green alga Desmodesmus subspicatus based on the average specific growth rate and the yield (algal biomass production) were investigated over a period of 72 h, according to the OECD guideline 201.

The test solutions were prepared by respective dilutions of a saturated stock solution.

The test concentrations were100, 31.6, 10.0, 3.16 and 1.00 mg/l loading rate. These loadings refer to the corresponding dilutions (spacing factor 3.16) of a 100 mg/l saturated stock solution.

Three parallel test vessels were used for each test concentration of the test item and six vessels for the blank controls.

Results

The 72 h E_rC₅₀and E_yC₅₀values of Intercure® 3 on the green alga Desmodesmus subspicatus were 5.31 mg/l and0.698 mg/l, respectively. These values are based on the geometric mean of the measured concentrations.

Description of key information

ErC50 (72h) = 5.31 mg/l

EyC50 (72h) = 0.698 mg/l

Key value for chemical safety assessment

EC50 for freshwater algae:

5.31 mg/L

EC10 or NOEC for freshwater algae:

0.23 mg/L

Additional information

The effective concentrations (E_rC₁₀, E_rC₅₀and E_yC₁₀, E_yC₅₀) as well as the no observed effect concentrations (NOE_rC and NOE_yC), of Intercure® 3 (CAS No.140-73-8) to the green alga Desmodesmus subspicatus based on the average specific growth rate and the yield (algal biomass production) were investigated over a period of 72 h, according to the OECD guideline 201.

The test solutions were prepared by respective dilutions of a saturated stock solution.

The test concentrations were100, 31.6, 10.0, 3.16 and 1.00 mg/l loading rate. These loadings refer to the corresponding dilutions (spacing factor 3.16) of a 100 mg/l saturated stock solution.

Three parallel test vessels were used for each test concentration of the test item and six vessels for the blank controls.