Alkali salt of substituted aryl sulfonyl triazinyl amino hydroxy sulfonyl aryl diazo naphthalene sulfonate

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:

10 October 2011 to 13 October 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD, EU and US test guidance in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

Name: Reactive Red F08-0146

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Analytical measurements were performed at the control and at the applied test concentration levels at the beginning and end of the test. On each occasion six replicate samples were taken from the test solution and one sample was taken from the control solution.
The nominal concentrations of Reactive Red F08-0146 used in the main experiment were: 12.5; 25; 50; 100 and 200 mg/L.

Test solutions

Vehicle:

no

Details on test solutions:

An amount of 200 mg test substance was diluted in 1000 mL OECD medium in order to give the 200 mg/L test concentration. The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae.

Test organisms

Test organisms (species):

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

Details on test organisms:

EXPERIMENTAL ORGANISMS

Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Initial cell number: The initial cell number in the test cultures was 104 cells/mL.
Pre-culturing: The pre-culture was intended to give an amount of alga suspension suitable for the inoculation of test cultures. The pre-culture was incubated under the conditions of the study in an aerated Algal Growth Medium and used when still exponentially growing (after an incubation period of 3 days). The cell count of above culture was determined by microscopic method and this cell suspension was diluted with Algal Growth Medium to 107 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure period

Test conditions

Hardness:

Not measured.

Test temperature:

Culture temperature was checked at the beginning of the study and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 23.1 – 23.5 °C measured in the flask and between 22.9 and 23.9 °C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the study, in the control and each concentration (in the ‘treated group’). The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.78 – 7.82 at the start and 7.67 – 9.20 at the end of the study.

Dissolved oxygen:

Not measured.

Salinity:

Not applicable.

Nominal and measured concentrations:

The nominal concentrations of Reactive Red F08-0146 used in the main experiment were: 12.5; 25; 50; 100 and 200 mg/L. The corresponding calculated test item concentrations were: 12.8; 26.3; 53.3; 105.0 and 213.5 mg/L.

Details on test conditions:

Other test conditions:
Light Intensity
The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 109 μE/m2/s, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm) and it is checked periodically.

DESCRIPTION OF THE TEST PROCEDURE
The test item is a highly water soluble deep-coloured dye. A spectral analysis of each concentration was made to show the absorption of light at the wavelengths required by chlorophyll. As the amount of light for photosynthesis was significantly affected by the shading effect of the coloured test solution, a modified test was performed in order to separate physical effect of the coloured material from its true toxic effects.
The purpose of this test method is to compare the algae growth of algal cells which are in contact with the test item solution, and the growth of algal cells with the test item solution as only a light filter in front of the algae suspension.
To assure the appropriate position of the test solutions, two superposed flat flasks (which are open for the light only from above) were used per replicate for this experiment.
The following three types of treatment were tested: control, treated and light filter groups.
The following shows the position of the test solutions in the pairs of flasks in the test groups. In each case the algal cells are in the lower flask, where the only light available passes through the upper flat flask (the sides of the flask will be covered). The depth of the liquid in the upper flask was 50% of that in the lower flask (on the basis that the ‘average’ algal cell in the lower flask will be suspended at the point of 50% of the depth).

Control group Treated group Light filter group
OECD medium OECD medium Test Item + OECD medium
Algae + OECD medium Algae + Test Item + OECD medium Algae + OECD medium

The test was performed with six replicates in the control group and three replicates per treated and light filter group in each concentration level.
The flasks were capped with air-permeable stoppers and continuously shaken by a laboratory orbital shaker during the exposure in order to keep the alga cells in suspension. Volume of algal suspension in the lower flask was 20 mL per replicate.
The exposure time was 72 hours. The test was started (0 hours) by inoculation of a biomass of approximately 104 algal cells per mL test medium.

The test was performed with six replicates in the control group and three replicates per treated and light filter group in each concentration level.
The flasks were capped with air-permeable stoppers and continuously shaken by a laboratory orbital shaker during the exposure in order to keep the alga cells in suspension. Volume of algal suspension in the lower flask was 20 mL per replicate.
The exposure time was 72 hours. The test was started (0 hours) by inoculation of a biomass of approximately 104 algal cells per mL test medium.

Preliminary Range Finding Test
A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations can be selected for use in the definitive test. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test group (light filter and treated group) and three for the control group.

OBSERVATIONS
The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
Microscopic observation of the algal cells in each concentration (in each group) and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

ANALYTICAL MEASUREMENTS
Analytical re-validation was performed because of a new method; therefore, analytical measurements were deviated from the validated analytical method according to the following:
− Detection wavelength was changed from 235 nm to 510 nm.
− Calibration series were prepared in alga test medium instead of acetonitrile : water (6:4).
− Evaluation of the chromatograms was modified, because not only the peak of the main component, Reactive Red F08-0146 was evaluated, but also the peak of it’s transformation product. For the calculations the sum of the two peaks were used.

LIGHT ABSORBTION MEASUREMENTS
The light absorptions of the control and the test item solutions were measured photometrically to show the absorption of light at the wavelengths required by chlorophyll. The light adsorption measurements were performed in the range of 350-700 nm because the absorption maximum of the Chlorophyll A is about 430 and 660 nm and the absorption maximum of Chlorophyll B is about 450 and 640 nm.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Corrected result

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Corrected result

Details on results:

VALIDITY
The cell density in the control cultures increased by a factor of 65.83 within three days.
The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 11.28 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.53 %.
All validity criteria were met, therefore the study can be considered as valid.

CONCENTRATIONS OF THE TEST ITEM
The nominal concentrations of test item were 12.5, 25, 50, 100 and 200 mg/L.
The corresponding calculated test item concentrations were: 12.8; 26.3; 53.3; 105.0 and 213.5 mg/L.
As the measured concentrations deviated not more than 20 per cent from the nominal in most cases, biological results are based on the nominal concentrations.

LIGHT ABSORPTION MEASUREMENTS
The light absorptions of the control and the test item solutions were measured photometrically in the range of 350-700 nm. The absorption maximum of the Chlorophyll A is about 430 and 660 nm and the absorption maximum of Chlorophyll B is about 450 and 640 nm.
The maximum light absorption of the test item was at approximately at 510 nm in the test solutions. However there was a little significant absorption at wavelengths required by chlorophyll in the test solutions as well.

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS
Thin cells were observed in the light filter groups at the concentration of 50 mg/L 48 and 72 hours after the treatment, furthermore it could be also observed at the two highest concentrations of 100 and 200.0 mg/L in both the treated and filter groups 24; 48 and 72 hours after the start of the test.

The average specific growth rates and the yields are detailed in the tables below.

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate (Batch Number: 0769128) is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 11/168-022AL) with the reference item Potassium dichromate is: 11 - 14 July 2011.
The 72h ErC 50 : 0.97 mg/L, (95 % confidence limits: 0.88 – 1.07 mg/L)
The 72h EyC 50 : 0.51 mg/L, (95 % confidence limits: 0.47 – 0.56 mg/L

Reported statistics and error estimates:

Statistical comparisons of average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (a= 0.05) by TOXSTAT software. The ErC50 and EyC50 values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software.

Any other information on results incl. tables

AVERAGE GROWTH RATES

Table 1: Growth Rates (u) during the Test Period

Nominal Concentration [mg/L]		Growth rate (u)
		0-24 h	0-48 h	0-72 h
		u	u	u	significance	difference	significance
Control		0.0533	0.0622	0.0582	-	-	-
12.5	treated	0.0529	0.0609	0.0572	n.s.	0.0009+	n.s.
	filter	0.0538	0.0617	0.0577	n.s.
25	treated	0.0529	0.0573	0.0566	n.s.	0.0008	n.s.
	filter	0.0569	0.0613	0.0574+	n.s.
50	treated	0.0441	0.0577	0.0488	*	0.0015	n.s.
	filter	0.0498	0.0573	0.0501	*
100	treated	0.0401	0.0492	0.0454	*	0.0038	*
	filter	0.0458	0.0577	0.0492+	*
200	treated	0.0345	0.0320	0.0309+	*	0.0121	*
	filter	0.0345	0.0479	0.0431	*

n.s.: statistically not significantly different compared to the control values (Bonferroni t-Test; a = 0.05)

* : statistically significantly different compared to the control values (Bonferroni t-Test; a = 0.05) : at these values the rounding of the EXCEL and TOXSTAT software was different. The table contains the values calculated with EXCEL.

Table 2: Percentage inhibition of 72h Growth Rates

Nominal Concentration [mg/L]		% inhibition of u (0-72h)
		% inhibition	corrected % inhibition
Control		-	-
12.5	treated	1.7	0.9
	filter	0.8
25	treated	2.6	1.4
	filter	1.2
50	treated	16.0	2.2
	filter	13.9
100	treated	21.9	6.6
	filter	15.3
200	treated	46.8	20.9
	filter	25.9

Table 3: Yield (Y) during the Test Period

Nominal Concentration [mg/L]		Yield (Y) 0-72h
		Y	significance	difference	significance
Control		64.8	-	-	-
12.5	treated	60.3	*	4.3	n.s.
	filter	62.7	n.s.
25	treated	58.0	*	3.7	n.s.
	filter	61.7	n.s.
50	treated	32.7	*	4.0	n.s.
	filter	36.0	*
100	treated	25.3	*	8.3	*
	filter	33.7	*
200	treated	8.3	*	13.0	*
	filter	21.3	*

 n.s.: statistically not significantly different compared to the control values (Bonferroni t-Test; a = 0.05)

* : statistically significantly different compared to the control values (Bonferroni t-Test; a = 0.05)

Table 4: Percentage Inhibition of Yield

Nominal Concentration [mg/L]		% inhibition of Yield (0-72h)
		% inhibition	corrected % inhibition
Control		-	-
12.5	treated	6.9	3.6
	filter	3.3
25	treated	10.5	5.7
	filter	4.9
50	treated	49.6	5.1
	filter	44.5
100	treated	60.9	12.9
	filter	48.1
200	treated	87.1	20.1
	filter	67.1

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of Reactive Red F08-0146 test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata over an exposure period of 72 hours in a modified test system.

The results of this experiment showed that the majority of the observed inhibition effect was also related to the light absorption by the test item. However, a slight inhibition was seen at 200 and 100 mg/L, when the Test Item Reactive Red F08-0146 was in contact with the algae cells for 72 hours. This indicates that there was also a slight toxic effect on the growth of the alga (Pseudokirchneriella subcapitata).

Executive summary:

The effect of Reactive Red F08-0146 test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

Five test concentrations in a geometric series (factor 2.0) and one untreated control were tested in the main experiment. The test design included three replicates at each test group and six replicates for the untreated control.

The test item is a highly water soluble deep-coloured dye and the amount of light for photosynthesis is likely to be significantly affected by the shading effect of the coloured test solution. Therefore, a modified test was performed in order to separate physical effect of the coloured material from its true toxic effects.

Modified test results and results without taking into account the modification are reported separately (according to OECD TG No. 201).

The test item concentration was analytically determined at the start and at the end of the test.

The nominal concentrations of Reactive Red F08-0146 used in the main experiment were: 12.5; 25; 50; 100 and 200 mg/L. The corresponding calculated test item concentrations were: 12.8; 26.3; 53.3; 105.0 and 213.5 mg/L.

As the measured concentrations deviated not more than 20 per cent from the nominal in most cases, biological results are based on the nominal concentrations.

Statistical comparisons of average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.

The calculated endpoints for the effect of the test item were the following:

Parameter (0-72 h)	CORRECTED RESULTS		NON-CORRECTED RESULTS
	Growth rate (r) [mg/L]	Yield (y)  [mg/L]	Growth rate (r) [mg/L]	Yield (y)  [mg/L]
	results are based on the nominal concentrations
EC50 95 % conf. limits	> 200 [722.0 calculated] 335.2 - 1555.4	> 200 [2607.4 calculated] 433.8 - 15674.1	> 200 [235.8 calculated] 173.0 - 321.5	64 .9   56.8 - 74.2
NOEC  LOEC	50 100	50 100	25 50	not determinable 12.5

The results of this experiment showed that the majority of the observed inhibition effect was related to the light absorption by the test item. However, a slight inhibition was seen at 200 and 100 mg/L, when the Test Item Reactive Red F08-0146 was in contact with the algae cells for 72 hours. This indicates that there was also a slight toxic effect on the growth of the alga (Pseudokirchneriella subcapitata).