N,N-bis(2-hydroxypropyl)benzamide

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 July 13th to august 30th, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For the analysis of the actual test item concentrations the following samples were taken:
Just before test start: - duplicate samples from each test medium (without algae)
- duplicate samples from the control (without algae)
After 72 hours (stability samples): - duplicate samples from each test medium (without algae)
- duplicate samples from the control (without algae)
For the 72-hour stability samples additional flasks with adequate volumes of the freshly prepared test media of all test concentrations and the control were incubated under the same conditions as in the actual test but without algae.
All samples were deep-frozen (at about -20 °C) immediately after sampling. Based on pre-experiments for investigation of the storage stability (non-GLP) the test item is sufficiently stable in the test water under these storage conditions.
The concentrations of test item were analyzed in the duplicate test media samples from the test concentrations of nominal 46 and 100 mg/L from both sampling times (0 and 72 hours). The samples from other test concentrations were not analyzed, since they were below the determined 72-hour NOEC. From the control samples only one of the duplicate samples was analyzed from the corresponding sampling times.

Vehicle:

no

Details on test solutions:

The test was started by inoculation of 10000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up three days prior to the test at the same conditions as in the test.
The test design included three replicates per test concentration and six replicates of the control. Volumes of 15 mL algal suspension for each replicate were continously stirred by magnetic stirrers in 50 mL Erlenmeyer flasks.

Test organisms (species):

other: Scenedesmus subspicatus

Details on test organisms:

The test organism used for the study was Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Sammlung von algenkulturen Gottingen. The algae had been grown in the RCC laboratories under standardized conditions according to test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not specified

Hardness:

0.24 mmol/L as CaCO3

Test temperature:

22 °C

pH:

7.7-7.9 at the start.
8.9 at the end of the experiment

Dissolved oxygen:

Not specified

Salinity:

Analytical grade salts were dissolved in purified water to obtain the following nominal concentrations:
CaCl2 * 2H2O: 18.0 mg/L
MgSO4 * 7H2O: 15.0 mg/L
NaHCO3: 50.0 mg/L
NH4Cl: 15.0 mg/L
MgCl2 * 6H2O: 12.0 mg/L
KH2PO4: 1.6 mg/L

Conductivity:

Not specified

Nominal and measured concentrations:

NOMINAL
4.6, 10, 22, 46 and 100 mg/L, plus control (0mg/L)
The analytically determined test item concentrations in the analyzed test media varied in the range from 101 to 103% of the nominal values, therefore nominal values were used for conclusion.

Details on test conditions:

The flasks were covered with glass dishes. They were incubated in a temperature controlled water bath and continusly illuminated at a measured light intensity of about 8800 Lux (mean value). This illumination was achieved by fluorescent tubes installed above the test flasks.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

89 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

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.:

46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The analytically determined test item concentrations in the analyzed test media varied in the range from 101 to 103% of the nominal values. Consequently, test item was stable during the test period and the results were reported using nominal concentrations.
The microscopic examination of the algal cells after 72 hours test period showed no difference between the algal growing in test media at 46 mg/L and in the control. The shape and size of algal cells were clearly not affected by test item.
Cell density in the control increased by about 60 times after 72 hours with respect to the start. Thus, algal growth in the control was sufficiently high to fulfil the validity criterion of a 16-factor growth.
Test item affected algal biomass at a concentration of 100 mg/L. Therefore the NOEC of the substance on algae was established at 46 mg/L.
From the regression curve of toxicity, EC10 was calculated to be about 89 mg/L (87-91 mg/L), while EC50 was higher than 100 mg/L.
No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.
Test item had a statistically significant inhibitory effect also on the growth rate at 100 mg/L, according to the Dunnet test. However, the inhibition of the growth rate was <10% at 100 mg/L. The significance of the Dunnett test was attributed to the very low variability of the algal growth in the replicates of each test concentration.

Results with reference substance (positive control):

Not performed

Validity criteria fulfilled:

yes

Conclusions:

Test item affected algal biomass at a concentration of 100 mg/L. Therefore the NOEC of the substance on algae was established at 46 mg/L.
From the regression curve of toxicity, EC10 was calculated to be about 89 mg/L (87-91 mg/L), while EC50 was higher than 100 mg/L.

Executive summary:

A GLP study according to OECD TG 201 was performed to assess growth inhibition of test item on algae Scenedesmus subspicatus. Nominal concentrations tested were 4.6, 10, 22, 46 and 100 mg/L, plus control (0mg/L). The analytically determined test item concentrations in the analyzed test media varied in the range from 101 to 103% of the nominal values. Consequently, test item was stable during the test period and the results were reported using nominal concentrations. Test item affected algal biomass at a concentration of 100 mg/L after 72 hours. Therefore the NOEC of the substance on algae was established at 46 mg/L. From the regression curve of toxicity, EC10 was calculated to be about 89 mg/L (87-91 mg/L), while EC50 was higher than 100 mg/L. Test item had a statistically significant inhibitory effect also on the growth rate at 100 mg/L, according to the Dunnet test. However, the inhibition of the growth rate was <10% at 100 mg/L. The significance of the Dunnett test was attributed to the very low variability of the algal growth in the replicates of each test concentration.

Description of key information

In a study according to OECD TG 201, the substance affected algal biomass growth at a concentration of 100 mg/L.

EC10 was determined to be 89 mg/L, while EC50 was higher than 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

89 mg/L