1,1,2-trimethyl-3-(4-sulphonatobutyl)-1H-benz[e]indolium

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:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,1,2-trimethyl-3-(4-sulphonatobutyl)-1H-benz[e]indolium

EC Number:

263-961-7

EC Name:

1,1,2-trimethyl-3-(4-sulphonatobutyl)-1H-benz[e]indolium

Cas Number:

63149-24-6

Molecular formula:

C19H23NO3S

IUPAC Name:

1,1,2-trimethyl-3-(4-sulfobutyl)-1H-benzo[e]indol-3-ium

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sampling method:
Frequency at t=0 h and t=72 h
Volume 1.8 mL
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at 10% of the SS but without algae and samples for analysis were taken at the start and at the end of the test period.
Additionally, reserve samples of 1.8 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 V173671 tested was a light blue powder with a purity of >99 mol% and 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 applying a 30 minute period of magnetic stirring to accelerate dissolution of 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 colorless at the end of the preparation procedure except for the highest test concentration which was clear and very light blue.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

between 23 and 24°C

pH:

7.9 - 8.5

Nominal and measured concentrations:

0.32, 1.0, 3.2, 10, 32 and 100 mg/L.
Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 0.13, 0.66, 2.4, 7.9, 29 and 93 mg/L, respectively. During the exposure period, the concentrations changed to 123, 100, 103, 92, 66 and 36% of initial, respectively, at the end of the test.
Based on these results, the Time Weighted Average (TWA) concentrations were calculated and used to express effect parameters.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Static
- aeration: continuous
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 115.5 x 10^4 cells/mL
- No. of organisms per vessel: 1 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 1 x 10^4 cells/mL
- No. of vessels per control (replicates): 1 x 10^4 cells/mL
- No. of vessels per vehicle control (replicates): 1 x 10^4 cells/mL

GROWTH MEDIUM
- Standard medium used: yes
M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:

TEST MEDIUM / WATER PARAMETERS
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous (24h)
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 79 to 81 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
pH At the beginning and at the end of the test.
Temperature of medium Continuously in a temperature control vessel.
Appearance of the cells At the end of the final test, microscopic observations were performed on the highest test concentration to observe for any abnormal appearance of the algae.
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: A final test was performed based on the results of a preceding combined limit/range-finding test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

24 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

55 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 56 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 56 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

24 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

29 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

38 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 56 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 0.13, 0.66, 2.4, 7.9, 29 and 93 mg/L, respectively. During the exposure period, the concentrations changed to 123, 100, 103, 92, 66 and 36% of initial, respectively, at the end of the test.
Based on these results, the Time Weighted Average (TWA) concentrations were calculated and used to express effect parameters.
The concentrations measured in the samples taken from solutions with algae were comparable with the concentrations measured in the corresponding samples without algae. Hence, the presence of algae did not affect the concentration of the test item in test medium throughout the test.
It should be noted that the two highest test concentrations showed a change in colour from colourless or very light blue at the start of the test to slightly purple to purple at the end of the test (both in the vessels with algae and without algae). Lower test concentrations did not visually change colour. Furthermore, an additional peak was observed in the chromatograms of the highest test concentration, which also increased over time, suggesting degradation (see figure 1 of the appended Analytical Report). However, effect parameters are based on the measured peak of the test item concentration and therefore this extra peak does not influence the study results or conclusions.

Statistically significant inhibition of growth rates and yield with V173671 was only found at the highest test concentration of 56 mg/L. The NOEC based on statistical significance was thus determined to be 24 mg/L for both effect parameters.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a TWA concentration of 56 mg/L when compared to the control.

Results with reference substance (positive control):

Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was 1.11 mg/L with a 95% confidence interval ranging from 1.10 to 1.13 mg/L.
The 72h-ERC50 was within the expected range of 0.92 and 1.46 mg/L as specified in ISO International Standard 8692, February 2012, and the historical range of 0.86 and 2.3 mg/L, which is based on reference tests performed at the Test Facility during the last ten years.
In conclusion, the sensitivity of this culture of Raphidocelis subcapitata was in agreement with ISO International Standard 8692, February 2012 and the historical data collected at the Test Facility.

Reported statistics and error estimates:

See table below

Any other information on results incl. tables

Effect Parameters

Parameter (mg/L)		NOEC	EC10	EC20	EC50
Growth rate	Value	24	55	>56	>56
	lower 95%-cl		51
	upper 95%-cl		58
Yield	Value	24	29	38	>56
	lower 95%-cl		23	32
	upper 95%-cl		37	44

cl – confidence limit.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, V173671 inhibited growth rate and yield of this freshwater algae species significantly at a TWA concentration of 56 mg/L.
The 72h-EC10 for growth rate inhibition (ERC10) was 55 mg/L (95% C.I.: 51 - 58 mg/L). The 72h-EC50 for growth rate inhibition (ERC50) was beyond the range tested, i.e. exceeded a TWA concentration of 56 mg/L.
The 72h-EC10 for yield inhibition (EYC10) was 29 mg/L (95% C.I.: 23 - 37 mg/L). The 72h-EC50 for yield inhibition (EYC50) was beyond the range tested, i.e. exceeded a TWA concentration of 56 mg/L.
The 72h-NOEC for growth rate inhibition was 24 mg/L based on statistical significance.
The 72h-NOEC for yield inhibition was 24 mg/L based on statistical significance.

Executive summary:

The objectiveofthe study was to evaluate V173671 for its ability to generate toxic effects in Raphidocelis 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.

The batch of V173671 tested was a light blue powder with a purity of >99 mol% and completely soluble in test medium at the concentrations tested.

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 0.32, 1.0, 3.2, 10, 32 and 100 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 concentrationsat the start of the testwere 0.13, 0.66, 2.4, 7.9, 29 and 93 mg/L, respectively. During the exposure period, the concentrations changed to 123, 100, 103, 92, 66 and 36% of initial, respectively, at the end of the test. Based on these results, the Time Weighted Average (TWA) concentrations were calculated and used to express effect parameters.

Statistically significant inhibition of growth rates and yield with V173671 was only found at the highest test concentration of 56 mg/L (nominal: 100 mg/L). The NOEC based on statistical significance was thus determined to be 24 mg/L for both effect parameters.

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

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg/L)		NOEC	EC10	EC20	EC50
Growth rate	Value	24	55	>56	>56
	lower 95%-cl		51
	upper 95%-cl		58
Yield	Value	24	29	38	>56
	lower 95%-cl		23	32
	upper 95%-cl		37	44

cl – confidence limit.