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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From August 19, 2006 to September 18, 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No methodological modification for coloured substances; algistatic (shading) and algicidal effects cannot be distinguished
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations (nominal): 1.6, 3.1, 6.3, 12.5, 25, 50 and 100 mg/L
- Sampling method: HPLC analysis with UV/Vis detection system
- Sample storage conditions before analysis: Routinely, the samples were analysed immediately. Only in exceptional cases, they were stored overnight deeply frozen and protected from light.
Vehicle:
no
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: Non-axenic strain
- Source (laboratory, culture collection): 'The collection of algal cultures' of the Institute of Plant Physiology at the University of Gottingen (Germany)
- Age of inoculum (at test initiation): Pre-cultures were set up three days before the start of a test
- Method of cultivation: The algal cultures for a test were taken from an exponentially-growing pre-culture and were mixed with the nutrient medium to make up to a final cell density of about 5,000 cells/mL in the test medium.
- Maintenance of stock cultures: Exponentially-growing stock cultures were maintained in the test facility under following conditions -
Temperature: 23+/-2°C
Light intensity: 60-120 µE x mE-2 x sE-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter)
Nutrient medium (according to Bringmann & Kuhn (1977)): Renewed once a week
Cell density measurements: Carried out using a microcell counter

ACCLIMATION
- Acclimation period: 3 d
- Culturing media and conditions (same as test or not): same as test


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21°C to 25°C (maintained at +/- 2°C)
pH:
7.7-8
Nominal and measured concentrations:
- A stock solution was prepared to give the desired series of test concentrations. To achieve this 124.9 mg of the test item was added to 1 L of dilution water.
- Nominal concentration: 1.6, 3.1, 6.3, 12.5, 25, 50 and 100 mg/L
- Measured concentration: Measured concentrations ranged from 98-104% of nominal values at 0 h, and from 80.3-103.3% of nominal values at 72 h
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks with cotton stoppers
- Culturing apparatus: Light chamber in which a temperature in the range 21°C to 25°C and continuous uniform illumination was provided in the spectral range 400 to 700 nm. Magnetic stirrers were used to keep the algae in suspension.
- Light intensity: At the average of the test solutions, a light intensity in the range 80-120 µE x mE-2 x sE-1, or an equivalent range of 7333-8000 lx, was used
-Experimental design: 7 test concentrations with algae, 1 control (without test substance) with algae and 1 control (with highest test substance concentration) without algae
- Initial cells density: 5,000 cells/mL
- Test concentrations (nominal): 1.6, 3.1, 6.3, 12.5, 25, 50 and 100 mg/L
- Method of administration: Stock solution
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Duration of exposure: 72 h
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 3.2-12.6 mg/L (95% confidence limits)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
76.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 56.0-122 mg/L (95% confidence limits)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 1.9-4.8 mg/L (95% confidence limits)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
20.3
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 16.4-25.4 mg/L (95% confidence limits)
Details on results:
The mean cell density during the test, mean growth (b) and mean growth rate (r) are given in Tables 1, 2 and 3, under "Any other information on results including tables" section.
Reported statistics and error estimates:
Analysis of the growth and growth rate were performed by probit analysis, and NOEC were determined by Dunnett's multiple t-Test.

Table 1: Determination of mean cell density

 

Nominal test substance concentration

[mg/L]

Cell density (cells/mL)

(initial cell density = 5000 cells/mL)

24h

48h

72h

Control

18,333

67,222

413,889

1.6

17,778

60,000

432,222

3.1

16,667

53,333

401,111

6.3

15,556

56,667

307,778

12.5

15,556

62,222

247,778

25.0

17,778

53,333

94,444

50.0

16,667

43,333

70,000

100.0

13,333

20,000

40,000

 

Table 2: Determination of mean growth (b)

 

Nominal test substance concentration

[mg/L]

Area under growth curve

Inhibition [%]

Control

267,500

0.0

1.6

268,889

-0.5

3.1

245,556

8.2

6.3

201,111

24.8

12.5

176,667

34.0

25.0

93,333

65.1

50.0

70,000

73.8

100.0

28,333

89.4

 

Table 3: Determination of mean growth rate (r)

  

Nominal test substance concentration

[mg/L]

Growth rate

Inhibition [%]

Control

1.47

0.0

1.6

1.49

-1.0

3.1

1.46

0.7

6.3

1.37

6.7

12.5

1.30

11.6

25..0

0.98

33.5

50.0

0.88

40.2

100.0

0.69

52.9

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 72 h EbC50 and ErC50 for Desmodesmus subspicatus were 20.3 and 76.3 mg/L. Further, the NOECs for biomass and growth rate were determined to be 3.1 and 6.3 mg/L, respectively.
Executive summary:

A study was conducted to determine the acute toxicity of the test substance to the algae Desmodesmus subspicatus according to EU Method C.3 (which is in most parts equivalent to OECD Guideline 201), in compliance with GLP.

Triplicate inocula of the algae were exposed to nominal concentrations of 1.6, 3.1, 6.3, 12.5, 25, 50 and 100 mg/L of the test substance for 72 h under static conditions. Quantification of the applied concentrations was carried out using HPLC/UV-Vis detection system. Recovery rates ranged from 98 - 104% of the nominal values at 0 h, and from 80.3 - 103.3% of the nominal values at 72 h. Biomass and growth rate were determined after 24, 48 and 72 h.

Under the conditions of the study, the 72 h EbC50 and ErC50 for Desmodesmus subspicatus were 20.3 and 76.3 mg/L. Further, the NOECs for biomass and growth rate were determined to be 3.1 and 6.3 mg/L, respectively.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
11 April 2011 - 14 April 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
modified for coloured substances
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Concentration of the test item 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 solution and one sample was taken from the control solution.
The samples were analysed by HPLC using UV detection.
Vehicle:
no
Details on test solutions:
An amount of 50 mg test substance was diluted in 500 mL OECD medium. The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae.

Dilution and Preparation of Testing Solutions

Preparation of test solutions from stock solution
Nominal concentration
[mg/L] Amount of stock solution (mL) Amount of OECD Medium (mL)
100 500 –
31.3 94 q.s. ad 300
9.8 29 q.s. ad 300
3.1 9 q.s. ad 300
1.0 3 q.s. ad 300
q.s. ad = quantum sufficiat ad (a sufficient quantity to make)
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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, Nikolausberger Weg 18, D-37073 Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of LAB Research 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.
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 22.0 – 22.8 °C measured in the flask and between 21.6 and 22.9 °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 7.70 – 7.90 at the start and 7.77 – 8.91 at the end of the study.
Dissolved oxygen:
Not measured.
Salinity:
Not applicable.
Nominal and measured concentrations:
The following nominal concentrations were tested: 1.0, 3.1, 9.8, 31.3 and 100 mg/L
The measured geometric mean test item concentrations were: 0.5; 0.8; 2.9; 11.2 and 47.1 mg/L
Details on test conditions:
TEST CONDITIONS

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 22.0 – 22.8 °C measured in the flask and between 21.6 and 22.9 °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 7.70 – 7.90 at the start and 7.77 – 8.91 at the end of the study.

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 E/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, therefore 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. In each case the algal cells are in a lower flask, where the only light available passes through the upper flat flask (the sides of the flask are 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.

Concentration Levels Investigated in the Main Test

Five test concentrations in a geometric series (factor 3.2) and one control were used in the main test.
The following nominal concentrations were tested: 1.0, 3.1, 9.8, 31.3 and 100 mg/L.
The analysed concentrations deviated more than 20 percent from the nominal during the test therefore the biologically results are based on the measured geometric mean concentration.
The measured mean test item concentrations were: 0.46; 0.78; 2.85; 11.15 and 47.08 mg/L.

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 and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

Calculation of Average Specific Growth Rate

Concentration-effect relationship is calculated by comparing growth rates in control and test cultures in the following way.
The average specific growth rate (μ) for individual cultures is calculated on the basis of the logarithmic increase of biomass during the test period, expressed per day.

Calculation of Yield

Yield is calculated as the biomass at the end of the test minus the starting biomass for each single vessel of controls and treatments. For each test concentration and control, mean yield values are calculated.

Average specific growth rate and yield were calculated for each test flask. Then the mean growth rate and mean yield were determined as arithmetic mean value over all test flasks per treatment.

In order to determine the ‘true’ toxic inhibition for average specific growth rate and yield, the percent inhibition calculated for “treated group” was corrected with the percent inhibition value calculated for “light filter group”.

Statistical Analysis

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.

The inhibition of alga growth was determined from the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).

The ErC50 and EyC50 values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software (based on the calculated geometric mean concentrations).

Statistical comparisons of average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test by TOXSTAT software.
For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.
For determination of ‘true’ NOEC and LOEC, absolute value of difference of growth rate and yield between “treated group” and “light filter group” was calculated and tested on significant differences to the control values by Bonferroni t-Test. Control values were taken as zero (i.e. 10E-09 = ~0) in that case.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. limits - 114.6 – 1681.2 mg/l
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % conf. limits - 48.5 – 280.4 mg/l
Details on results:
VALIDITY

The cell density in the control cultures increased by a factor of 73.17 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 10.41 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.84 %.
All validity criteria were met, therefore the study can be considered as valid.

CONCENTRATIONS OF THE TEST ITEM

For determination of the concentrations, samples were taken from each concentration level and from the control at the start and at the end of the test.
The nominal concentrations of test item were 1.0, 3.1, 9.8, 31.3 and 100 mg/L.

The analysed concentrations were 1.08; 3.05; 9.89; 31.9 and 103.1 mg/L, at the start and <0.4 (LOQ); <0.4 (LOQ); 0.82; 3.9 and 21.5 mg/L at the end of the test.

The measured concentration of the two lowest concentration (1.0 and 3.1 mg/L nominal) were below the limit of quantification (LOQ = 0.4 mg/L) at the end of the test. In order to calculate the mean exposure concentration at this concentration level, the final concentration was taken as the half of the limit of quantification.

The measured geometric mean test item concentrations were: 0.5; 0.8; 2.9; 11.2 and 47.1 mg/L.
Since the analysed concentrations deviated from the nominal concentrations more than ± 20 % in each concentration level at the end of the experiment, the results are based on the measured geometric mean test item 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 maximum light absorption of the test item was at approximately 420 nm in the test solutions. There was significant absorption at 430 and 450 nm (at wavelengths required by chlorophyll).

AVERAGE SPECIFIC GROWTH RATES
Please refer to tables 3 and 4 below.
The results of the statistical evaluation (based on Bonferroni t-Test; alpha=0.05) show that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value at the concentrations of 11.2 and 47.1 mg/L (measured), accordingly the No Observed Effect Concentration (NOEC) was determined as 2.9 mg/L.
The corrected 72 h ErC50 value was determined as higher than 100 mg/L [calculated by Probit analysis (TOXSTAT software) as 439.0 mg/L (95 % confidence limits: 114.6 – 1681.2 mg/L)]).

YIELD

Please refer to Table 5 below
The results of the statistical evaluation (based on Bonferroni t-Test; alpha=0.05) show that the 0-72 h yield was statistically significantly different from the untreated control value at the concentrations of 11.2 and 47.1 mg/L (measured), accordingly the No Observed Effect Concentration (NOEC) was determined as 2.9 mg/L.
The corrected 72 h ErC50 value was determined as higher than 100 mg/L [calculated by Probit analysis (TOXSTAT software) as 116.6 mg/L (95 % confidence limits: 48.5 – 280.4 mg/L)]).
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: 10/332-022AL) with the reference item Potassium dichromate is: 07 - 10 December 2010.
The 72h ErC 50 : 1.40 mg/L, (95 % confidence limits: 1.29 – 1.53 mg/L)
The 72h EyC 50 : 0.92 mg/L, (95 % confidence limits: 0.85 – 1.00 mg/L)
Reported statistics and error estimates:
The cell density in the control cultures increased by a factor of 73.17 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 10.41 %. The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.84 %.
All validity criteria were met, therefore the study can be considered as valid.

Average Specific Growth Rates

 

Table 3:Growth Rates(m)during the Test Period

Measured Concentration
[mg/L]

Growth rate (m)

0–24 h

0–48 h

0–72 h

m

difference

m

difference

m

difference

Control

0.0533

0.0583

0.0596

0.5

treated

0.0569

0.0111

0.0581

0.0021

0.0587

0.0009

filter

0.0538

0.0594

0.0596

0.8

treated

0.0498

0.0031

0.0577

0.0013

0.0585

0.0009

filter

0.0529

0.0573

0.0595

2.9

treated

0.0401

0.0113

0.0564

0.0026

0.0591

0.0010

filter

0.0401

0.0590

0.0596

11.2

treated

0.0289*

0.0113

0.0523*

0.0062*

0.0485*

0.0074*

filter

0.0401

0.0586

0.0560*

47.1

treated

0.0345

0.0113

0.0415*

0.0049*

0.0393*

0.0128*

filter

0.0345

0.0464*

0.0521*

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

 

Table 4:         Percentage inhibition of 72h Growth Rates

Measured Concentration
[mg/L]

% inhibition ofm
(0-72h)

% inhibition

corrected % inhibition

Control

0.5

treated

1.6

1.5

filter

0.1

0.8

treated

1.8

1.6

filter

0.3

2.9

treated

0.9

0.9

filter

0.1

11.2

treated

18.6

12.5

filter

6.1

47.1

treated

34.0

21.4

filter

12.6

 

 

YIELD

 

Table 5:Yield (Y) and Percentage Inhibition of Y during the Test Period

Measured Concentration
[mg/L]

Yield (Y)
0–72h

% inhibition of Y
0–72h

Y

difference

% inhibition

corrected % inhibition

Control

72.2

0.5

treated

67.3

4.7

6.7

6.5

filter

72.0

0.2

0.8

treated

66.7

4.7

7.6

6.5

filter

71.3

1.2

2.9

treated

69.3

5.3*+

3.9

3.7

filter

72.0

0.2

11.2

treated

32.0*

23.3*

55.7

32.3

filter

55.3*

23.3

47.1

treated

16.0*

23.3*

77.8

35.6

filter

41.7*

42.3

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

+:since yield of treated group showed no statistically significantly different at this concentration, thereforethis statistical significance is not considered as an effect at determination of NOEC.

Validity criteria fulfilled:
yes
Remarks:
see above
Conclusions:
Five test concentrations in a geometric series (factor 3.2) 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 nominal concentrations of the test item used in the main experiment were: 1.0; 3.1; 9.8; 31.3 and 100 mg/L. The corresponding measured mean test item concentrations were: 0.5; 0.8; 2.9; 11.2 and 47.1 mg/L.The calculated endpoints for the effect of Reactive Orange F08-0314 were the following:
ErC50 > 100 mg/l (95 % conf. limits - 114.6 – 1681.2); NOErC = 2.9 mg/L
EyC50 > 100 mg/l (95 % conf. limits - 48.5 – 280.4); NOEyC = 2.9 mg/L

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 greater inhibition was seen at 72 hours when the test item was in contact with the algae cells. This indicates that there was also a slightl< toxic effect on the growth of the alga (Pseudokirchneriella subcapitata).

The substance is not considered harmful to algae on the basis of the results observed, and no classification according to CLP is applicable.
Executive summary:

The effect of the 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 3.2) 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.

 

As 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 effect of the coloured material from its true toxic effects.

Modified test results and results without modifying are reported separately (according to OECD TG No. 201).

 

The analysed concentrations deviated more than 20 percent from the nominal during the test therefore the biologically results are based on the measured geometric mean concentration.

The nominal concentrations of the test item used in the main experimentwere:1.0; 3.1; 9.8; 31.3 and 100 mg/L.The corresponding measured mean test item concentrations were: 0.5; 0.8; 2.9; 11.2 and 47.1 mg/L.

 

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.

TheErC50 and EyC50values of the test item and their confidence limits were calculated using Probit analysisby TOXSTATsoftware.

 

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

 

ErC50 > 100 mg/l (95 % conf. limits - 114.6 – 1681.2); NOErC = 2.9 mg/L

EyC50 > 100 mg/l (95 % conf. limits - 48.5 – 280.4); NOEyL = 2.9 mg/L

 

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 greater inhibition was seen at 72 hours when the test item was in contact with the algae cells. This indicates that there was also a slightly toxic effect on the growth of the alga (Pseudokirchneriella subcapitata).

 

The substance is not considered harmful to algae on the basis of the results observed, and no classification according to CLP is applicable.

Description of key information

The 72 h EbC50 and ErC50 for Desmodesmus subspicatus were determined 20.3 and 76.3 mg/L in an OECD 201 test. Further, the NOECs for biomass and growth rate were determined to be 3.1 and 6.3 mg/L, respectively.

However, the test was not conducted in a modified version for coloured test items. Therefore, no conclusion can be reached on the percentage of algistatic effects (photosynthesis block by shading) and of algicidal (true toxic) effects. The results with the structural test item with a modified version of OECD 201 clearly show that a very little minority of the effects observed are related to algicidal effects. The calculated endpoints for the effect of the structural analogue substance were the following:

ErC50  > 100 mg/l (95 % conf. limits - 114.6 – 1681.2); NOErC = 2.9 mg/L

EyC50  > 100 mg/l (95 % conf. limits - 48.5 – 280.4); NOEyC = 2.9 mg/L

The results of this experiment showed that the majority of the observed inhibition effect was related to the light absorption by the structural analogue substance. Based on the results, the target substance is not considered acutely or chronically harmful to freshwater green algae either. The 72 -h ErC50 for the target substance is 100 mg/L and and the 72 -h NOErC 2.9 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
2.9 mg/L

Additional information