Reactive Red F03-0318

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:

24 May 2012 to 27 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to EU and OECD 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:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal 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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name: Reactive Red F03-0318

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 three replicate samples were taken from the test solution and one sample was taken from the control solution.
The samples were analysed using HPLC system.

Test solutions

Vehicle:

no

Details on test solutions:

Separate stock solutions were first prepared in deionised water. The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations.

Untreated Control: Algal growth medium was inoculated with algal cells (without test item) and was examined in parallel to the test item concentrations.
Reference Control: Potassium dichromate

Test organisms

Test organisms (species):

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

Details on test 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:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period

Test conditions

Hardness:

No data

Test temperature:

Culture temperature was checked at the beginning of the experiment 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 22.0 – 22.7°C measured in the flask and between 21.8 and 23.1°C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the experiment, 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 7.17 – 7.28 at the start and 8.02 – 8.60 at the end of the study.

Dissolved oxygen:

No data

Salinity:

Not applicable

Nominal and measured concentrations:

The nominal concentrations of Reactive Red F03-0318 used in the main experiment were: 12.5; 25; 50; 100 and 200 mg/L.

Details on test conditions:

The composition of the growth medium is detailed below under any other information.

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 illustration below under background information 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).

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.
The concentration levels used and results (72 h) of the preliminary range-finding test are summarised in a table under any other information below.

Concentration Levels Investigated in the Main Test

Five test concentrations in a geometric series with a separation factor of 2.0 and one control were used in the main test.
The following nominal concentrations were tested: 12.5, 25, 50, 100 and 200 mg/L.
The corresponding geometric mean of the measured test item concentrations were: 12.2; 24.4; 48.9; 103.4 and 209.5 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 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



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.

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: 298.7 – 1088.2

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: 171.7 – 380.8

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

VALIDITY
The cell density in the control cultures increased by a factor of 67.00 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 10.25 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 1.20 %.
All validity criteria were met, therefore the study can be considered as valid.

TEST CONDITIONS

Temperature

The temperature was in the range of 22.0 – 22.7°C measured in the flask and between 21.8 and 23.1°C measured within the climate chamber.

pH

The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.17 – 7.28 at the start and 8.02 – 8.60 at the end of the study.

Light Intensity

The light intensity at the position occupied by algal culture flasks during the test was about 111 uE/m2/s.

CONCENTRATIONS OF THE TEST ITEM

The nominal concentrations of test item were 12.5, 25, 50, 100 and 200 mg/L.
The corresponding geometric mean of the measured test item concentrations were: 12.2; 24.4; 48.9; 103.4 and 209.5 mg/L.
As the measured concentrations deviated not more than 20 per cent from the nominal in all cases, biological results are based on the nominal concentrations.

LIGHT ABSORPTION MEASUREMENTS

The maximum light absorption of the test item was at approximately at 541 nm in the test solutions. However there was significant absorption at wavelengths required by chlorophyll in the test solutions as well.

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS

Thin cells were observed in both the treated and filter groups at the two highest concentration levels of 100 and 200 mg/L 24, 48 and 72 hours after the treatment.

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

The date of the last study (Study Code: 11/357-022AL) with the reference item Potassium dichromate is (Batch Number: 0769128): 16 - 19 January 2012.
The 72h ErC 50 : 0.98 mg/L, (95 % confidence limits: 0.91 – 1.05 mg/L)
The 72h EyC 50 : 0.68 mg/L, (95 % confidence limits: 0.62 – 0.73 mg/L)

Reported statistics and error estimates:

See above for 95% confidence limits

Any other information on results incl. tables

Average Specific Growth Rates

Growth Rates (u) during the Test Period:

Nominal Concentration [mg/L]		Growth rate (m)
		0–24 h	0–48 h	0–72 h
		m	m	m	significance	difference	significance
Control		0.0589	0.0600	0.0584	–	–	–
12.5	treated	0.0600	0.0601	0.0582	n.s.	0.0005	n.s.
	filter	0.0609	0.0606	0.0584	n.s.
25	treated	0.0498	0.0598	0.0579	n.s.	0.0011	n.s.
	filter	0.0569	0.0602	0.0584	n.s.
50	treated	0.0498	0.0563	0.0530	*	0.0055	*
	filter	0.0578	0.0602	0.0585	*
100	treated	0.0401	0.0528	0.0519	*	0.0040	*
	filter	0.0538	0.0573	0.0559	*
200	treated	0.0401	0.0404	0.0377^+	*	0.0147	*
	filter	0.0498	0.0555	0.0524	*

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

Percentage inhibition of 72h Growth Rates:

Nominal Concentration [mg/L]		% inhibition ofm (0-72h)
		% inhibition	corrected % inhibition
Control		–	–
12.5	treated	0.3	0.4
	filter	0.0
25	treated	0.8	0.8
	filter	0.0
50	treated	9.3	9.4
	filter	-0.1
100	treated	11.1	6.9
	filter	4.3
200	treated	35.4	25.2
	filter	10.2

 

Yield (Y) during the Test Period:

Nominal Concentration [mg/L]		Yield (Y) 0–72h
		Y	significance	difference	significance
Control		66.0	–	–	–
12.5	treated	65.0	n.s.	2.3	n.s.
	filter	66.0	n.s.
25	treated	63.7	n.s.	5.0	n.s.
	filter	66.0	n.s.
50	treated	44.3	*	22.0	*
	filter	66.3	n.s.
100	treated	41.0	*	14.0	*
	filter	55.0	*
200	treated	14.3	*	28.3	*
	filter	42.7	*

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)

 Percentage Inhibition of Yield:

Nominal Concentration [mg/L]		% inhibition of Yield (0-72h)
		% inhibition	corrected % inhibition
Control		–	–
12.5	treated	1.5	1.5
	filter	0.0
25	treated	3.5	3.5
	filter	0.0
50	treated	32.8	33.3
	filter	-0.5
100	treated	37.9	21.2
	filter	16.7
200	treated	78.3	42.9
	filter	35.4

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

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. At 50 and 100 mg/mL, a statistically significant effect was calculated for growth rates, however, this effect (inhibition: µ = 9.4% and 6.9%, respectively) is not biologically significant, but is due to the biological variability of the test system. A slight albeit not dose-dependent inhibition of yield was seen at 50, 100 and 200 mg/L, when the Test Item Reactive Red F03-0318 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 F03-0318 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 per treated and light filter group at each concentration level 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 F03-0318 used in the main experiment were:12.5; 25; 50; 100 and 200 mg/L.The corresponding geometric mean of the measured test item concentrations were: 12.2; 24.4; 48.9; 103.4 and 209.5 mg/L. As the measured concentrations deviated not more than 20 per cent from the nominal in all 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 (a= 0.05) by TOXSTAT software.

The E_rC₅₀and E_yC₅₀values of the test item and their confidence limits were calculated using Probit analysis 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	> 200 [570.2 calculated]	> 200 [255.7 calculated]		> 200 [332.7 calculated]	103.9
95 % conf. limits	298.7 – 1088.2	171.7 – 380.8		227.6 – 486.5	89.9 – 120.1
NOEC	100	100		25	25
LOEC	200	200		50	50

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. At 50 and 100 mg/mL, a statistically significant effect was calculated for growth rates, however, this effect (inhibition: µ = 9.4% and 6.9%, respectively) is not biologically significant, but is due to the biological variability of the test system. A slight albeit not dose-dependent inhibition of yield was seen at 50, 100 and 200 mg/L, when the Test Item Reactive Red F03-0318 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).