Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2011-11-30 to 2012-03-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
Version / remarks:
1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
The following nominal concentrations were tested: 6.25; 12.5, 25, 50 and 100 mg/L.
Concentration of Reactive Yellow F01-0555 in the test solutions was determined at the beginning and at the end of the study.
Each occasion three replicate samples were taken from the test solutions and one sample was taken from the control solution.

Vehicle:
yes
Details on test solutions:
Dilution and Preparation of Testing Solutions:
Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests. An amount of 100 mg test substance was diluted in 1000 mL OECD medium in order to give the 100 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.

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: 6.25; 12.5, 25, 50 and 100 mg/L.

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
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.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
No post exposure observation period.
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 22.6 – 22.9 °C measured in the flask and between 22.2 and 23.2 °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 range of the pH was 7.75 – 7.88 at the start and 8.09 – 9.16 at the end of the study.
Dissolved oxygen:
Not measured.
Salinity:
Not applicable
Nominal and measured concentrations:
The following nominal concentrations were tested: 6.25; 12.5, 25, 50 and 100 mg/L.
The corresponding geometric mean of the measured test item concentrations were: 6.1; 11.9; 24.8; 49.7 and 101.3 mg/L.
As the measured concentrations deviated not more than 20 per cent from the nominal, biological results are based on the nominal concentrations.
Details on 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 111 uE/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 illustration shows the position of the test solutions in the pairs of flasks in the test groups. In each case the algal cells were in the lower flask, where the only light available passes through the upper flat flask (the sides of the flask were 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 was 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.

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 the table under any other information.

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.

LIGHT ABSORPTION 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
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
VALIDITY
The cell density in the control cultures increased by a factor of 65.33 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 8.58 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 1.22 %.
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 6.25; 12.5, 25, 50 and 100 mg/L.
The corresponding geometric mean of the measured test item concentrations were: 6.1; 11.9; 24.8; 49.7 and 101.3 mg/L.
As the measured concentrations deviated not more than 20 per cent from the nominal, biological results are based on the nominal concentrations.


LIGHT ABSORPTION MEASUREMENTS
The maximum light absorption of the test item was at approximately at 413 nm in the test solutions. However there was significant absorption at wavelengths required by chlorophyll in the test solutions as well.

See tables under any other information for details of average specific growth rates and yields.
Results with reference substance (positive control):
Reference 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 ErC50:0.97 mg/L, (95 % confidence limits: 0.88 – 1.07 mg/L)
The 72h EyC50:0.51 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.

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

Nominal Concentration
[mg/L]

Growth rate (µ)

0–24 h

0–48 h

0–72 h

u

u

u

significance

difference

significance

Control

0.0553

0.0607

0.0580

6.25

treated

0.0529

0.0593

0.0580

n.s.

0.0004

n.s.

filter

0.0569

0.0606

0.0584

n.s.

12.5

treated

0.0498

0.0590

0.0576

n.s.

0.0004

n.s.

filter

0.0498

0.0598

0.0580

n.s.

25

treated

0.0441

0.0545

0.0571

n.s.

0.0004

n.s.

filter

0.0498

0.0572

0.0575

n.s.

50

treated

0.0401

0.0549

0.0547

*

0.0012

*

filter

0.0458

0.0567

0.0559

*

100

treated

0.0345

0.0529

0.0542

*

0.0012

*

filter

0.0401

0.0559

0.0555

*

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 2: Percentage inhibition of 72h Growth Rates

Nominal Concentration
[mg/L]

% inhibition of µ
(0-72h)

% inhibition

corrected % inhibition

Control

6.25

treated

0.1

0.7

filter

-0.6

12.5

treated

0.7

0.7

filter

0.0

25

treated

1.6

0.6

filter

1.0

50

treated

5.8

2.1

filter

3.7

100

treated

6.5

2.1

filter

4.4

Table 3: Yield (Y) during the Test Period

Nominal Concentration
[mg/L]

Yield (Y)
0–72h

Y

significance

difference

significance

Control

64.3

6.25

treated

64.0

n.s.

2.0

n.s.

filter

66.0

n.s.

12.5

treated

62.3

n.s.

2.0

n.s.

filter

64.3

n.s.

25

treated

60.0

n.s.

1.7

n.s.

filter

61.7

n.s.

50

treated

50.3

*

4.7

*

filter

55.0

*

100

treated

48.7

*

4.7

*

filter

53.3

*

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

6.25

treated

0.5

3.1

filter

-2.6

12.5

treated

3.1

3.1

filter

0.0

25

treated

6.7

2.6

filter

4.1

50

treated

21.8

7.3

filter

14.5

100

treated

24.4

7.3

filter

17.1

Validity criteria fulfilled:
yes
Conclusions:
The effect of Reactive Yellow F01-0555 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 there was no toxic effect on the growth of the alga (Pseudokirchneriella subcapitata). At 50 and 100 mg/mL, a statistically significant effect was calculated, however, this effect (inhibition: µ = 2.1%, Y = 7.3%) is not biologically significant, but is due to the biological variability of the test system.

The EC50 value (growth rate) was > 100 mg/L and the EC50 value (biomass) was > 100 mg/L
Executive summary:

The effect of Reactive Yellow F01-0555 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 (light filter and treated 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 performedin order to separate physical effects of the coloured material from its true toxic effects.

Modified test results and results without taking into account 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 Yellow F01-0555 used in the main experimentwere: 6.25;12.5; 25; 50 and 100 mg/L. The corresponding geometric mean of the measured test item concentrations were: 6.1; 11.9; 24.8; 49.7 and 101.3 mg/L.

As the measured concentrations deviated not more than 20 per cent from the nominal, 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. TheErC50and EyC50values of the test item and their confidence limits were calculated using Probit analysisby TOXSTAT software.

The calculated endpoints for the effect ofthe 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

> 100

[not calculated]

> 100

> 100

[1832.2 calculated]

> 100

[267.0 calculated]

95 % conf. limits

-

12.78 – not calculated

186.92 – 17959.10

140.66 – 506.93

NOEC

100

100

100

100

LOEC

> 100

> 100

> 100

> 100

The results of this experiment showed that there was no toxic effect on the growth of the alga(Pseudokirchneriella subcapitata). At 50 and 100 mg/mL, a statistically significant effect was calculated, however, this effect (inhibition: µ = 2.1%, Y = 7.3%) is not biologically significant, but is due to the biological variability of the test system.

Description of key information

The EC50 value (growth rate) was > 100 mg/L and the EC50 value (biomass) was > 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information