2-[[4-(dimethylamino)phenyl]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:

07Aug2018 to 01Feb2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

278-601-4

EC Name:

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

Cas Number:

77061-58-6

Molecular formula:

C13H18N5.Cl

IUPAC Name:

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

Test material form:

solid: particulate/powder

Details on test material:

Batch 1054934

Specific details on test material used for the study:

Identification: Basic red 51
Batch (Lot) Number: 1054934
Expiry date: 29 November 2020 (expiry date)
Physical Description: Violet powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. The method of analysis is described in the appended Analytical Report (Appendix 6).
Frequency at t=0 h and t=72 h.
Volume 3.0 mL from the approximate centre of the test vessels.
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at nominally 0.32 mg/L but without algae and samples for analysis were taken at the start and at the end of the test period.
Additionally, reserve samples of 3.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of Basic red 51 tested was a violet powder with a purity of 99.9% which was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item.

Preparation of test solutions started with the highest concentration of 100 mg/L. No other treatment than vigorous shaking was needed to completely dissolve the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colored at the end of the preparation procedure. Coloration of test solutions increased with increasing concentrations of the test item from light pink to red.

Test organisms

Test organisms (species):

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

Details on test organisms:

Species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), strain: NIVA CHL 1
Source In-house laboratory culture.
Reason for selection This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Study design

Test type:

static

Water media type:

other: M2 Test Medium

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

24 hours was used in the initial experiment which was a combined limit/ range finding test , 72 hours for full test.

Post exposure observation period:

N/A

Test conditions

Hardness:

Hardness (Ca+Mg) 0.24 mmol/L; (24mg CaCO3/L)

Test temperature:

21 24°C

pH:

8.1 ± 0.2

Dissolved oxygen:

The suspensions were continuously aerated

Salinity:

N/A

Conductivity:

N/A

Nominal and measured concentrations:

All values quoted in mg/L
0 hours
Nominal (Measured)
0.1 (0.106)
0.18 (0.18)
0.32 (0.313)
0.32 (0.33) - without algae for comparative purposes
0.56 (0.558)
1.0 (1.01)

72 hours
Nominal (Measured)
0 (0.002) Extrapolated
0.1 (0.043)
0.18 (0.0776)
0.32 (0.178)
0.32 (0.276) - without algae for comparative purposes
0.56 (0.456)
1.0 (0.865)

Details on test conditions:

Combined Limit/Range-Finding Test
The project started with a combined limit/range-finding test. Six replicates of exponentially
growing algae were exposed to a control and a nominal concentration of 100 mg/L, in the
limit test. Test procedure and conditions were similar to those applied in the final test with the
following exceptions:
• Three replicates per concentration were exposed to nominally 0.10, 1.0 and 10 mg/L, in the combined range-finding test.
• Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Lower concentrations were measured only at the end of the exposure period.
• pH was only measured in the control and the highest test concentration, at the start and at the end of the test.
• At the end of the test algae were not observed under a microscope to verify a normal and healthy appearance.

Test Concentrations
• Basic red 51 0.10, 0.18, 0.32, 0.56 and 1.0 mg/L.
• Control Test medium without test item or other additives.
• Replicates 3 replicates of each test concentration,
• 6 replicates of the control,
• 1 or 2 replicates of each test concentration without algae.

Test duration: 72 hours
Test type: Static
Test vessels: 250 mL, all-plastic culture flasks, containing 30 mL of test solution
Test Medium M2
Cell density: An initial cell density of 1 x 104 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 117 to 119 μE.m-2.s-1.
Incubation Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.


Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. The method of analysis is described in the appended Analytical Report (Appendix 6).

Frequency at t=0 h and t=72 h.
Volume 3.0 mL from the approximate centre of the test vessels.
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates with algae were pooled at each concentration
before sampling.

Compliance with the quality criteria regarding maintenance of actual concentrations was
checked by running a test vessel at nominally 0.32 mg/L but without algae and samples for
analysis were taken at the start and at the end of the test period.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

EC50 for growth rate inhibition (ERC50)

Effect conc.:

>= 0.33 - <= 0.34 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

EC50 for yield inhibition (EYC50)

Effect conc.:

>= 0.24 - <= 0.26 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: EC50 for yield inhibition (EYC50)

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

growth rate inhibition

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

yield inhibition

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Details on results:

A concentration-related increase of inhibition of both growth rate and yield was observed during the test. Statistically significant inhibition of growth rate and yield was found at average exposure concentrations of 0.24 mg/L.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to an average exposure concentration of 0.24 mg/L when compared to the control.

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), Basic red 51 inhibited growth rate and yield of this fresh water algae species significantly at average exposure concentrations of 0.24 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was 0.33 mg/L with a 95% confidence interval ranging from 0.33 to 0.34 mg/L.
The 72h-EC50 for yield inhibition (EYC50) was 0.25 mg/L with a 95% confidence interval ranging from 0.24 to 0.26 mg/L.
The 72h-NOEC for growth rate inhibition was 0.12 mg/L.
The 72h-NOEC for yield inhibition was 0.12 mg/L.

Reported statistics and error estimates:

The 72h-EC50 for growth rate inhibition (ERC50) was 0.33 mg/L with a 95% confidence interval ranging from 0.33 to 0.34 mg/L.
The 72h-EC50 for yield inhibition (EYC50) was 0.25 mg/L with a 95% confidence interval ranging from 0.24 to 0.26 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), Basic red 51 inhibited growth rate and yield of this fresh water algae species significantly at average exposure concentrations of 0.24 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was 0.33 mg/L with a 95% confidence interval ranging from 0.33 to 0.34 mg/L.
The 72h-EC50 for yield inhibition (EYC50) was 0.25 mg/L with a 95% confidence interval ranging from 0.24 to 0.26 mg/L.
The 72h-NOEC for growth rate inhibition was 0.12 mg/L.
The 72h-NOEC for yield inhibition was 0.12 mg/L.

Executive summary:

The objective of the study was to evaluate Basic red 51 for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEC, EC₁₀, EC₂₀and EC₅₀for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of Basic red 51 tested was a violet powder with a purity of 99.9% which was completely soluble in test medium at the concentrations tested.All test solutions were clear and colored at the end of the preparation procedure. Coloration of test solutions increased with increasing concentrations of the test item from light pink to red.

A final test was performed based on the results of a preceding combined limit/range-finding test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominally 0.10, 0.18, 0.32, 0.56 and 1.0 mg/L. The initial algal cell density was 10⁴cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were at the level of nominal concentrations (98 – 106%). These concentrations decreased to 40 – 86% of initially measured at the end of the test.. Based on these results, average exposure concentrations were calculated to be 0.067, 0.12, 0.24, 0.50 and 0.93 mg/L, respectively.

A concentration-related increase of inhibition of both growth rate and yield was observed during the test. Statistically significant inhibition of growth rate and yield was found at average exposure concentrations of 0.24 mg/L. 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters (based on average exposure concentrations) obtained in this study are summarized in the table below.

Parameter (mg/L)		NOEC	EC10	EC20	EC50
Growth rate	Value	0.12	0.24	0.27	0.33
	lower 95%-cl		0.23	0.26	0.33
	upper 95%-cl		0.24	0.27	0.34
Yield	Value	0.12	0.16	0.19	0.25
	lower 95%-cl		0.14	0.17	0.24
	upper 95%-cl		0.18	0.20	0.26

cl – confidence limit