2-[(4-aminophenyl)azo]-1,3-dimethyl-1H-imidazolium chloride

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

Study period:

16Oct1998 to 03Dec1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

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)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-[(4-aminophenyl)azo]-1,3-dimethyl-1H-imidazolium chloride

EC Number:

306-764-4

EC Name:

2-[(4-aminophenyl)azo]-1,3-dimethyl-1H-imidazolium chloride

Cas Number:

97404-02-9

Molecular formula:

C11H14N5.Cl

IUPAC Name:

2-[(1E)-2-(4-aminophenyl)diazen-1-yl]-1,3-dimethyl-2,3-dihydro-1H-imidazol-1-ium chloride

Test material form:

solid: particulate/powder

Details on test material:

See individual study reports for purity information and CofA where applicable

Specific details on test material used for the study:

Batch no.: CGF-FO 20088/0010
Expiration date: April 14, 2000
Purity: 98%
Stability in water: 2 days

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

For the analytical measurements of the test item concentrations duplicate samples were taken at the start of the test from the freshly prepared test media (without algae) of all test concentrations and from the control.

For the determination of the stability of the test item under the test conditions, and for the maintenance of the test item concentrations during the test period respectively, sufficient 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), and were sampled in duplicate at the end of the test (after the 72 hours test period).

All samples were deep-frozen (at about -20 °C) immediately after sampling.

The concentrations of the test item MIP ORANGE 3100 were measured in the duplicate test media samples from the test concentrations of nominal 4.6 - 22 mg/l from both sampling times (0 and 72 hours). From the control samples only one of the duplicate samples was analysed from each of both sampling times (0 and 72 hours). The samples from the three lowest test concentration of nominal 0.46 - 2.2 mg/l were not analysed, since this concentrations were below the determined 72-hour NOEC. The analytical procedure and results are described in the attached analytical report.

All remaining samples are kept stored at about -20 °C to enable additional analyses. After delivery of the final report all samples will be discarded.

Test solutions

Vehicle:

no

Remarks:

Test item prepared in the test water (synthetic test water)

Details on test solutions:

Previously to the start of the test, the test medium of the highest test concentration of nominal 22 mg/l was prepared by dissolving 22 mg of the test Item completely in 1000 ml test water by stirring for 15 minutes. Adequate volumes of this intensively mixed test medium were added to test water to prepare the test media of the lower nominal test concentrations of 10, 4.6, 2.2, 1.0 and 1.46 mg/l. Additionally, a control was tested in parallel (test water without addition of the test item).

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Study design

Test type:

static

Water media type:

other: synthetic test water, prepared according to OECD 201

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

N/A

Test conditions

Hardness:

Calculated water hardness of the test water: 0.24 mmol/l (= 24 mg/l) as CaCO3

Test temperature:

23°C

pH:

8.0 to 9.0 in control. 8.0 to 7.9 at highest concentration of test item.

Dissolved oxygen:

Not Measured

Salinity:

Not Measured

Conductivity:

Not Measured

Nominal and measured concentrations:

0.46 mg/L nominal, actual not measured (3 of 6 concentrations measured)
1.0 mg/L nominal, actual not measured (3 of 6 concentrations measured)
2.2 mg/L nominal, actual not measured (3 of 6 concentrations measured)
4.6 mg/L nominal, 4.5 mg/L actual
10 mg/L nominal, 9.6 mg/L actual
22 mg/L nominal, 20 mg/L actual

Details on test conditions:

The test was started (0 hours) by inoculation of a biomass of 10,000 algal cells per ml test medium. These cells were taken from an exponentially growing pre-culture, which was set up 3 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 in the control. Volumes of 15 ml algal suspension per replicate were continuously stirred by magnetic stirrers in 50 ml Erlenmeyer flasks.

The flasks were covered with glass dishes. They were incubated in a temperature controlled water bath at 23 °C, and continuously illuminated at the measured light intensity of about 7400 Lux (mean value), range: 7200 to 7900 Lux (minimum and maximum value of measurements at 6 places distributed over the experimental area at the surface of the test media). This illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed above the water bath in a distance of about 35 cm from the test flasks. The test vessels were labelled with the RCC project number and all necessary additional information to assure unmistakable identification.

The test duration was 72 hours.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 8.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

ca. 18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 6.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

ca. 45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

At the microscopic examination of the shape of the algal cells after 72 hours test period no difference was observed between the algae growing in the test concentration of nominal 10 mg/l and the algal cells in the control. Thus, the shape of the algal cells growing at least up to this test concentration was obviously not affected.

In the control the cell density has increased from nominal N = 1 x 10^4 cells/ml at the start of the test (0 hours) to N = 192 x 10^4 cells/ml (mean value) after 72 hours by a factor of approximately 190 (Table 1). Thus, the algal growth in the control meets the validity criteria (increase by a factor of at least 16).

At the start of the test, the pH values in the test media was 8.0, at the end of the test pH values were measured between 7.9 and 9.0 (Table 6). This increase of the pH during the test was obviously caused by the C02-consumption of the algae due to their rapid growth respectively their high densities (although the test media have been intensively stirred).

Results with reference substance (positive control):

N/A

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The influence of the test item on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the Commission Directive 92/69/EEC, Part C.3, 1992, and the OECD Guideline No. 201, 1984.

The nominal test concentrations were 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/l, and in parallel a control.

The mean analytically determined test item concentrations in the analysed test media varied in the range from 92% to 98% of the nominal values (see Table 2 in the attached analytical report). In the test media, incubated under the test conditions, the test item MIP ORANGE 3100 was sufficiently stable during the test period of 72 hours. Therefore, all biological results are related to the nominal concentrations of the test item.

The test item had a statistically significant inhibitory effect on both, the mean biomass b and the mean growth rate \i of Scenedesmus subspicatus after the exposure period of 72 hours first at the nominal concentration of 10 mg/l. Thus, this test concentration was determined as the 72-hour LOEC (lowest concentration tested with toxic effects).

The 72-hour NOEC (highest concentration tested without toxic effects after a test period of 2 hours) was determined at the nominal concentration of 4.6 mg/l, since up to and including this test concentration both, the mean biomass b and the mean growth rates p of the algae were statistically not significantly lower than in the control.

EC values by biomass in mg/L were as follows:
EC50 = 8.5, EC10 = 4.0, EC90 = 18

EC values by growth rate in mg/L were as follows:
EC50 = 17, EC10 = 6.5, EC90 = 45

Executive summary:

The influence of the test item MIP ORANGE 3100 on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the Commission Directive 92/69/EEC, Part C.3, 1992, and the OECD Guideline No. 201, 1984.

 

The nominal test concentrations were 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/l, and in parallel a control.

 

The mean analytically determined test item concentrations in the analysed test media varied in the range from 92% to 98% of the nominal values (see Table 2 in the attached analytical report). In the test media, incubated under the test conditions, the test item was sufficiently stable during the test period of 72 hours. Therefore, all biological results are related to the nominal concentrations of the test item.

 

The test item had a statistically significant inhibitory effect on both, the mean biomass b and the mean growth rate \i of Scenedesmus subspicatus after the exposure period of 72 hours first at the nominal concentration of 10 mg/l. Thus, this test concentration was determined as the 72-hour LOEC (lowest concentration tested with toxic effects).

 

The 72-hour NOEC (highest concentration tested without toxic effects after a test period of 2 hours) was determined at the nominal concentration of 4.6 mg/l, since up to and including this test concentration both, the mean biomass b and the mean growth rates p of the algae were statistically not significantly lower than in the control.

 

EC values by biomass in mg/L were as follows:

EC50 = 8.5, EC10 = 4.0, EC90 = 18

 

EC values by growth rate in mg/L were as follows:

EC50 = 17, EC10 = 6.5, EC90 = 45