Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatesilicate

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:

16 June 2017 - 15 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatesilicate

EC Number:

278-270-6

EC Name:

Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatesilicate

Cas Number:

75627-12-2

Molecular formula:

C54H58N4O6.MoO4 + C54H58N4O6.SiO3

IUPAC Name:

3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-10λ⁴-xanthen-10-ylium dioxomolybdenumbis(olate) silicate

Test material form:

solid: particulate/powder

Details on test material:

Physical appearance: pink powder
Storage conditions: at room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0, t=24 and t=72
Volume: 2.0 mL
Storage: Samples were stored in a freezer (≤ 15°C) until analysis.

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 the limit concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: Preparation of test solutions started with loading rates individually prepared at 1.0, 10 and 100 mg/L in the initial combined limit/range-finding and range-finding tests, and a loading rate of 100 mg/L in the subsequent limit test. An overnight period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as test concentrations.
- Evidence of undissolved material: The two highest WAFs were clear and slightly pink, while all remaining test solutions were clear and colorless at the end of the preparation procedure. No Tyndall effect was observed in any of the test solutions when they were observed with a laser pen.
At the end of the range-finding test, a floating layer and undissolved material were observed in the solution of the highest test concentration. In the limit test, a pink colored coating of the test vessels was observed from 48 hours of exposure onwards. Additionally, small pink-colored bubbles were observed floating on the surface of respective test solutions.
- Controls: Test medium without test item or other additives.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture

CULTIVATION:
- Method: 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.
- Light intensity: 60 to 120 µE/m^2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm
- Medium different from test medium: yes, M1

ACCLIMATION
- Acclimation period: 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.
- Acclimation medium different from test medium: no, M2 (according to OECD 201)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3 mg/L

Test temperature:

23 - 24°C

pH:

At test start: 7.9 - 8.0
At test end: 8.1 - 8.3

Nominal and measured concentrations:

Nominal test concentrations: WAF prepared at a limit concentration of 100 mg/L
Measured test concentrations (TWA): 0.35 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, open, fill volume: 50 mL
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 232.2 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- Other: 2 extra replicates of the limit concentration without algae

TEST MEDIUM / WATER PARAMETERS
- Standard test medium used: yes, M2
- Source of dilution water: Milli-RO water
- Culture medium different from test medium: yes, M1

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: continuous illumination using TLD-lamps with a light intensity within the range of 82 to 83 µE/m^2/s

EFFECT PARAMETERS MEASURED: growth rate at 72 hours.
- Additional measurements: pH at the beginning and at the end of the test. Temperature: continuously in a temperature control vessel; Appearance of the cells: at the end of the final test in the highest concentration by microscopic observations.
- Determination of cell concentrations: 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 (pathlength =20 mm). Algal medium was used as blank. Two extra test vessel per concentration without algae were used as background for the determination of the algal cell density.

COMBINED LIMIT/ RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: no, growth rate inhibition and yield inhibition were found to decrease with increasing test concentration, whereby the inhibition observed at the lowest test concentration was comparable to the inhibition observed in the extra control without the calcium and magnesium salts. Therefore, it was decided to repeat thetest as a range-finding test to investigate the reproducibility of the outcomes.

REPEATED RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L
- Results used to determined the conditions for the definitive study: yes, the results of the first range-finding study were confirmed and based on the results of both preceding tests it was decided to perform a limit test as the final study using a limit concentration of 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (June 2017)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.35 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.35 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

- In two preliminary tests, algal growth was observed to be less affected at higher test concentrations. This indicated that the inhibition observed in the combined limit/range-finding test had more likely been related to the reduced levels of nutrient salts rather than a toxic effect due to exposure to the test item. The results suggest that the test item had a compensating effect on growth inhibition considering that the effects observed in the control group without calcium and magnesium salts were almost identical to the dose level of 1.0 mg/L in the combined limit/range-finding test.
- Results of analysis showed that the test item was decreased to 20-47% of initial after 24 hours and to 11- 91% after 72 hours in the first range-finding study. In the final test, the test concentration was decreased to 71-75% of initial after 24 hours and to 29-49% of initial after 72 hours. Based on these results, the TWA for the limit dose was calculated to be 0.35 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 72h-ErC50: 0.99 mg/L (95% CI: 0.97-1.0 mg/L)
- Other: the result fell within the historical data range.

Reported statistics and error estimates:

An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Two-sample t-test Procedure, α=0.05, one-sided, smaller).
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 1 Growth rate and percentage inhibition for the total test period

C.I. Pigment Red 81:2 TWA conc. (mg/L)	Mean	Std. Dev.	n	% Inhibition
Control	1.815	0.0291	6
0.35	1.699	0.0177	6	6.38*^#

* effect was statistically significant;^#effect biologically not relevant (<10%)

Table 2 Growth rate and percentage inhibition at different time intervals

C.I. Pigment Red 81:2 TWA conc. (mg t.i./L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.699		1.989		1.756
0.35	6	1.774	-4.40	1.608	19.16	1.715	2.35

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see 'Any other information on results incl. tables'

Conclusions:

The 72h-EC50 for growth rate was beyond the range tested, i.e. exceeded an average concentration of 0.35 mg/L being considered the maximum soluble concentration in a filtrate prepared at a loading rate of 100 mg/L. The 72h-NOEC for growth rate inhibition was 0.35 mg/L.

Executive summary:

A study was performed to assess the effect of C.I. Pigment Red 81:2 on the growth rate of fresh water algae (Pseudokirchneriella subcapitata) after 72 hours of exposure. The study was conducted in accordance with OECD 201 and GLP.

 

A combined limit/range-finding study was performed with test solutions prepared individually at loading rates of 1.0, 10 and 100 mg/L by applying an overnight of magnetic stirring to reach maximum dissolution of the test item in the test medium. The resulting aqueous mixtures were filtered through 0.45 µm membrane filters, whereafter the Water Accommodated Fractions (WAFs) were used for testing. The same preparation of test solutions was used for the final test.

 

In the final test, six replicates of exponentially growing algal cultures were exposed to a WAF prepared at a limit concentration of 100 mg/L. The initial algal cell density was 10^4cells/mL. Samples were taken from the 0 mg/L and the 100 mg/L test solution and analysed for confirmation of actual exposure concentrations at the start and after 24 and 72 hours of exposure. Test concentrations were 71 -75% of initial after 24 hours and 29 -49% of initial after 72 hours. Based on these results, the time weighted average (TWA) exposure concentration was calculated to determine the effect concentrations. The TWA was calculated to be 0.35 mg/L.

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, no significant inhibition of growth rate was recorded at a TWA concentration of 0.35 mg/L being considered the maximum soluble concentration in a filtrate prepared at a loading rate of 100 mg/L. The 72h-EC50 for inhibition of growth rate (ErC50) of C.I. Pigment Red 81:2 was >0.35 mg/L. The 72h-NOEC for growth rate inhibition of C.I. Pigment Red 81:2 was 0.35 mg/L.

All acceptability criteria were met and therefore the study was considered to be valid.