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Ecotoxicological information

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 Feb. 10, 1998 to Mar. 27, 1998
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
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Analysis of the test concentrations 12.5, 25, 50, 100 mg/L plus a control; additional analysis of the highest (100 mg/L) and the lowest analytically detected test concentration (12.5 mg/L) without algal inoculum, at test start and after 72 h exposure.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: From stock solution (125 mg/L)

-Control: Inoculum in nutrient medium and water for dilution
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: CHODAT
- Source (laboratory, culture collection): Institute of Plant Physiology, University of Gottingen (Germany)
- Method of cultivation: In a light chamber at 23 ± 2 °C and with a quantum flux Which equals 120 µE/m2.s.

ACCLIMATION
- Any deformed or abnormal cells observed: No

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
None
Post exposure observation period:
No
Hardness:
No data
Test temperature:
No data
pH:
7.5-8.0 (at 0 h)
9.8-10.3(at 72 h)
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
Nominal: 12.5, 25, 50 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks with stoppers
- Type (delete if not applicable): Closed
- Material, size, headspace, fill volume: 300 mL Erlenmeyer flasks, 100 mL of test medium
- Aeration: Yes
- Algal suspension taken from an exponentially growing preculture which serves to adjust an initial cell density of 10,000 cells per mL.
-Control: Inoculum in nutrient medium and water for dilution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionized water

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Counting chamber (Microcellcounter Sysmex F-300 Digitana)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 76.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: -
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 40.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: -
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 76.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: -
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
13.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
cell number
Remarks:
(cell density)
Remarks on result:
other: -
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
21.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
cell number
Remarks:
(cell density)
Remarks on result:
other: -
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 40.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: -
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No

Please refer to 'Attached background material' for the results on the analysis
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Dunnett test: NOEC, LOEC
Probit analysis

RESULTS - ANALYSIS

Nominal concentration test substance [mg/L]

0 h

72 h

Control

<2

< 2

w. algae 12.5

4

3

12.5

4

5

25

7

8

50

14

14

100

27

26

w. algae100

26

27

w.algae = without the addition of algae

All values refer to TOC-determinations. According to the relevant product informations, 1 mg/L TOC equals to 2.9 mg/L of the test substance

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions, both the 72 h EbC50 and ErC50 of the test substance were determined to be >76.9 mg/L measured geometric mean. > 100 mg/L nominal. The NOEC and LOEC of the test substance were determined to be 13.1 and 21.8 mg/L respectively.
Executive summary:

A study was performed to assess the adverse effects of test substance on the growth and growth rate of green algae species Scenedesmus subspicatus CHODAT over several generations. The study was conducted in accordance with EU Method C.3. in compliance with GLP.


Exponentially growing algal cells were exposed for a period of 72 h to the nominal test concentrations of 12.5, 25, 50 and 100 mg/L of test substance dissolved in water.


Analysis of the test concentrations 12.5, 25, 50, 100 mg/L plus a control; additional analysis of the highest (100 mg/L) and the lowest analytically detected test concentration (12.5 mg/L) without algal inoculum, at the test start and after 72 h exposure.


 


The cell densities were measured at 24 h intervals. Inhibition of the algal population was measured as reduction in growth and growth rate, relative to control.


 


Under the test conditions, both the 72 h EbC50 and ErC50 of the test substance were determined to be >76.9 mg/L. The NOEC and LOEC of the test substance were determined to be 13.1 and 21.8 mg/L respectively.


 

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
May 09, 2008 - June 19, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
other: 92/69/EEC, C3
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours.

Samples were taken from the control (replicates R1-R6 pooled) and each test group (replicates R1-R3 pooled) at 0 and 72 hours and stored at approximately -20°C prior to quantitative analysis.
Vehicle:
no
Details on test solutions:
For the purpose of the definitive study, the test material was dissolved directly in the culture medium.
For the composition of the culture medium, see: Any other information on materials and methods incl. tables.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (as written in study report)
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine
Laboratory, Oban, Argyll, Scotland.
- Method of cultivation: The master cultures were maintained in the laboratory
under constant aeration and constant illumination at 21 ± 1°C.

ACCLIMATION
Prior to the start of the test sufficient master culture was added to approximately 100mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 104 -105 cells/mL.

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
24 ± 1℃
The temperature within the incubator was recorded daily.
pH:
The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure.

The pH values of the control cultures were observed to increase from pH 7.6 -7.7 at 0 hours to pH 7.7 – 7.8 at 72 hours. The pH derivation in the control cultures was less than 1.5 pH units after 72 hours and thus within the recommended limits given in the Test Guideline.
Nominal and measured concentrations:
Based on the results of a range-finding test the following test concentrations were assigned to the definitive test: 10, 20, 40, 80 and 160 mg/L.
For information on the range-finding test, see: Any other information on materials and methods incl. tables.
Details on test conditions:
CULTURE MEDIUM
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

PREPARATION OF THE TEST SOLUTIONS
An amount of test material (160 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 320 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 160, 80, 40 and 20 mg/L. An aliquot (125 mL) of each of the stock solutions was separately mixed with algal suspension (125 mL) to give the required test concentrations of 10, 20, 40, 80 and 160 mg/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

EXPOSURE CONDITIONS
As as in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 25 mL of test preparation were used for the control and three flasks each containing 25 mL were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test material.

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 3.24 x 105 cells per mL. This suspension was diluted to a cell density of 9.36 x 103 cells per mL prior to use. At initiation of the test the culture contained a nominal cell density of 4 x 103 cells per mL.

The flasks were plugged with polyurethane foam bungs and incubated at 24 ± 1°C under continuous illumination (intensity approximately 10000 lux) provided by warm white lighting (380-730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Samples were taken at 0, 24, 47 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
40 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
40 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
40 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
For detailed data see: Any other information on results incl. tables.
Results with reference substance (positive control):
- EC50: 1.2 mg/L (base on grown rate)
0.69 mg/L (base on yield)
0.73 mg/L (base on biomass integral)
- Other: LOEC : 1.0 mg/L (base on grown rate)
0.5 mg/L (base on yield)
0.5 mg/L (base on biomass integral)
NOEC : 0.50 mg/L (base on grown rate)
0.25 mg/L (base on yield)
0.25 mg/L (base on biomass integral)
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999-2001).

RESULTS OF RANGE-FINDING TEST:


The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test material during the range-finding test are given in the following table. The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10 mg/l. However, growth was observed to be reduced at 100 mg/l. Based on this information test concentrations of 10, 20, 40, 80 and 160 mg/L were selected for the definitive test.


























































































































Nominal Concentration
(mg/1)



Cell Densities* (cells per mL)



Inhibition Values (%)



0 Hours



72 Hours



Growth Rate



Yield/Biomass
Integral



Control                                                R1



4.05E+03



7.24E+05



 



 



                                                            R2



4.13E+03



7.68E+05



-



‑



Mean



4.09E+03



7.46E+05



 



 



0.10                                                    R1



4.14E+03



8.26E+05



 



 



                                                            R2



4.37E+03



9.45E+05



[3]



[19]



Mean



4.25E+03



8.85E+05



 



 



1.0                                                      R1



4.20E+03



9.17E+05



 



 



                                                            R2



4.08E+03



9.22E+05



 [4]



[23]



Mean



4.14E+03



9.20E+05



 



 



10                                                       R1



4.15E+03



1.07E+06



 



 



                                                            R2



4.19E+03



1.03E+06



[7]



[41]



Mean



4.17E+03



1.05E+06



 



 



100                                                     R1



4.12E+03



5.44E+05



 



 



                                                            R2



4.12E+03



5.70E+05



6



26



Mean



4.12E+03



5.57E+05



 



 



* Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.


R1 and R2 = Replicates 1 and 2


[Increase in growth compared to controls]


 


VALIDITY OF THE TEST:





























Criterion Value requiredValue obtained fulfilled
Cell Density Increase in control Culturesat least by factor 16 within 72 hoursincreased by a factor of 209 after 72 hours.yes
Coeffficient of Variation of Sectional (Daily) Growth Rates in Control Cultures< 35%11%yes
Coefficient of Variation of Average Growth between Control Replicates< 7%3 %yes

RESULTS (Verification of Test Concentrations)























Sample*Nominal Concentration
(mg/L)		Concentration Found (mg/L)Expressed as a Percent of the Nominal Concentration (%)
0 hoursControl
10
20
40
80
160		<LOQ
9.06
19.2
39.1
77.1
149		-
91
96
98
96
93
72 hoursControl
10
20
40
80
160		<LOQ
8.64
19.9
38.3
79.3
159		-
86
100
96
99
99

* All samples stored frozen prior to analysis
LOQ = Limit of quantitation


DISCUSSION


Validity of the Test


All validity criteria of the applied test guideline were met.


Verification of Test concentrations


The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test material in test medium. A method of analysis was validated and proven to be suitable for use.


Cell Densities and pH Values in the Definitive Test:







































































































































































































































































Nominal Concentration
(mg/1)



pH



Cell Densities* (cells per ml)



pH



0 h



0 h



24 h



47 h



72 h



72 h



Control                        R1



7.7



4.42E+03



2.97E+04



1.48E+05



1.03E+06



7.8



                                    R2



7.7



3.95E+03



2.85E+04



1.35E+05



9.17E+05



7.8



                                    R3



7.7



4.10E+03



2.35E+04



1.07E+05



7.66E+05



7.8



                                    R4



7.6



3.75E+03



2.76E+04



1.22E+05



7.24E+05



7.8



                                    R5



7.6



4.02E+03



2.71E+04



1.14E+05



7.23E+05



7.8



                                    R6



7.6



4.00E+03



2.38E+04



1.02E+05



9.06E+05



7.7



Mean



 



4.04E+03



2.67E+04



1.21E+05



8.44E+05



 



10                                R1



7.5



3.93E+03



2.95E+04



1.22E+05



8.78E+05



7.7



                                    R2



7.5



4.18E+03



3.05E+04



1.28E+05



7.87E+05



7.7



                                    R3



7.5



4.14E+03



2.65E+04



1.16E+05



7.36E+05



7.7



Mean



 



4.08E+03



2.88E+04



1.22E+05



8.00E+05



 



20                                R1



7.3



3.93E+03



2.50E+04



1.10E+05



6.09E+05



7.6



                                    R2



7.3



3.75E+03



2.48E+04



1.20E+05



7.70E+05



7.6



                                    R3



7.3



4.15E+03



2.50E+04



1.15E+05



6.98E+05



7.6



Mean



 



3.94E+03



2.49E+04



1.15E+05



6.92E+05



 



40                                R1



7.2



3.95E+03



2.59E+04



1.15E+05



5.93E+05



7.6



                                    R2



7.2



4.09E+03



2.50E+04



1.12E+05



6.09E+05



7.5



                                    R3



7.2



3.87E+03



2.70E+04



1.05E+05



6.82E+05



7.5



Mean



 



3.97E+03



2.60E+04



1.11E+05



6.28E+05



 



80                                R1



7.0



4.20E+03



2.36E+04



1.18E+05



6.71E+05



7.4



                                    R2



7.0



3.97E+03



2.16E+04



8.99E+04



5.73E+05



7.4



                                    R3



7.0



3.97E+03



2.44E+04



1.14E+05



6.66E+05



7.4



Mean



 



4.05E+03



2.32E+04



1.07E+05



6.37E+05



 



160                              R1



6.8



4.25E+03



2.25E+04



9.18E+04



4.78E+05



7.2



                                    R2



6.8



4.36E+03



2.43E+04



1.01E+05



5.35E+05



7.2



                                    R3



6.8



3.91E+03



2.60E+04



1.07E+05



5.04E+05



7.2



Mean


 

4.18E+03



2.43E+04



1.00E+05



5.05E+05


 

* Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for eachf the replicate flasks.


R1-R6 = Replicates 1 to 6


Daily Specific Growth Rates for the Control Cultures in the Definitive Test

























































 



Daily Specific Growth Rate (cells/ml/hour)



Day 0 - 1



Day 1 - 2



Day 2 - 3



Control                                                                  R1



0.084



0.070



0.077



R2



0.082



0.068



0.076



R3



0.074



0.066



0.079



R4



0.080



0.064



0.071



R5



0.080



0.063



0.074



R6



0.074



0.063



0.087



Mean



0.079



0.066



0.077



R1-R6 = Replicates 1 to 6


 


Inhibition of Growth Rate, Yield and Biomass Integral in the Definitive Test





























































































































































































































































































































Nominal Concentration
(mg/1)



Growth Rate
(cells/ml/hour)



Yield
(cells/nil)



Biomass Integral



0 - 72 h



% Inhibition



0 - 72 h



% Inhibition*



0 - 72 h



% Inhibition



Control                                                  R1



0.077



 



1.02E+06



 



1.69E+07



 



  R2



0.075



 



9.13E+05



 



1.51E+07



 



R3



0.073



 



7.62E+05



 



1.25E+07



 



R4



0.072



-



7.21E+05



-



1.24E+07



‑



R5



0.072



 



7.19E+05



 



1.22E+07



 



R6



0.075



 



9.02E+05



 



1.41E+07



 



Mean



0.074



 



8.40E+05



 



1.38E+07



 



SD



0.002



 



1.24E+05



 



1.88E+06



 



10                                                                           R1



0.075



[1]



8.74E+05



 



1.43E+07



[4]



R2



0.073



1



7.83E+05



 



1.34E+07



3



R3



0.072



3



7.32E+05



 



1.24E+07



11



Mean



0.073



1



7.96E+05



5



1.34E+07



3



SD



0.002



 



7.19E+04



 



9.88E+05



 



20                                                                           R1



0.070



5



6.05E+05



 



1.06E+07



23



R2



0.073



1



7.66E+05



 



1.28E+07



7



R3



0.072



3



6.94E+05



 



1.18E+07



15



Mean



0.072



3



6.88E+05



18



1.18E+07



15



SD



0.002



 



8.03E+04



 



1.12E+06



 



40                                                                           R1



0.069



7



5.89E+05



 



1.06E+07



24



R2



0.070



5



6.05E+05



 



1.06E+07



23



R3



0.071



4



6.78E+05



 



1.14E+07



17



Mean



0.070



5



6.24E+05



26



1.09E+07



21



SD



0.001



 



4.75E+04



 



4.84E+05



 



80                                                                           R1



0.071



4



6.67E+05



 



1.15E+07



17



R2



0.069



7



5.69E+05



 



9.58E+06



31



R3



0.071



4



6.62E+05



 



1.14E+07



18



Mean



0.070



5



6.33E+05



25



1.08E+07



22



SD



0.001



 



5.54E+04



 



1.09E+06



 



160                                                                           R1



0.066



11



4.73E+05



 



8.46E+06



39



R2



0.068



8



5.30E+05



 



9.44E+06



32



R3



0.067



9



5.00E+05



 



9.24E+06



33



Mean



0.067



9



5.01E+05



40



9.05E+06



35



SD



0.001



 



2.85E+04



 



5.17E+05



 



 * In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated


R1 - R6 = Replicates 1 to 6


SD = Standard Deviation ,


[Increase in growth as compared to controls]

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the growth of Pseudokirchnerella subcapitata has been investigated over a 72-Hour period and gave an ErC50 (0 - 72 h) of greater than 160 mg/l*, an EyC50 (0 - 72 h) of greater than 160 mg/l*, and an EbC50 (0 - 72 h) of greater than 160 mg/l*. The Lowest Observed Effect Concentration dased on growth rate, yield and biomass integral was 40 mg/l, and the No Observed Effect Concentration was 20 mg/l.

* It was not possible to calculate EC50 values as no concentration tested resulted in greater than 50% inhibition of growth.
Executive summary:

A study was performed to assess the effect of the test material (EC 404-600-7) on the growth of the green alga Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum). The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC) and at the request of the Sponsor further modified with regards to coloured test substances (reduced test volume and increased light intensity) to be in line with OECD GD 23.


Following a preliminary range-finding test, Pseudokirchneriella subcapitata (as written in the study report) was exposed to an aqueous solution of the test material at concentrations of 10, 20, 40, 80 and 160 mg/1 (three replicate flasks per concentration, six contols) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.


Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.


The study was conducted under static conditions with an initial nominal cell density of 4 x 103 cells per mL. At the start of the test all control cultures were observed to be clear colourless solutions. The test cultures were observed to range from yellow coloured solutions at 10 mg/L through to dark orange coloured solutions at 160 mg/L. After the 72-Hour test period all control cultures were observed to be pale green dispersions whilst the test cultures were observed to range from yellow coloured solutions at 10 mg/L through to dark orange coloured solutions at 160 mg/L.


The effect of the test material on the growth of Pseudokirchnerella subcapitata (name as written in the study report) has been investigated over a 72-Hour period and gave an ErC50 (0 - 72 h) of greater than 160 mg/l*, an EyC50 (0 - 72 h) of greater than 160 mg/l*, and an EbC50 (0 - 72 h) of greater than 160 mg/l*. The Lowest Observed Effect Concentration dased on growth rate, yield and biomass integral was 40 mg/l, and the No Observed Effect Concentration was 20 mg/l.


* It was not possible to calculate EC50 values as no concentration tested resulted in greater than 50% inhibition of growth.


 

Description of key information

The effect of the test material on the growth of Pseudokirchnerella subcapitata (name as written in the study report, Everlight 2008) has been investigated over a 72-Hour period and gave an ErC50 (0 - 72 h) of greater than 160 mg/l*, an EyC50 (0 - 72 h) of greater than 160 mg/l, and an EbC50 (0 - 72 h) of greater than 160 mg/l. The Lowest Observed Effect Concentration dased on growth rate, yield and biomass integral was 40 mg/l, and the No Observed Effect Concentration was 20 mg/l.


An algal study (Caspers, 1998) with Scenedesmus subspicatus CHODAT resulted in a nominal EC50 of > 100 mg/L. The NOEC and LOEC of the test substance were determined to be 13.1 and 21.8 mg/L respectively. The high pH of >10 in all samples (control and replicates) assumes rapid degradation of the substance through hydrolysis during the study, but this is a realistic scenario with regard to the real conditions in the dyeing process for which the test substance is used (dyeing with these kind of reactive dyes takes place at pH 10 to 11).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
20 mg/L

Additional information

A study (Everlight, 2008) was performed to assess the effect of the test material (EC 404-600-7) on the growth of the green alga Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum). The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC) and at the request of the Sponsor further modified with regards to coloured test substances (reduced test volume and increased light intensity) to be in line with OECD GD 23.


Following a preliminary range-finding test, Pseudokirchneriella subcapitata (as written in the study report) was exposed to an aqueous solution of the test material at concentrations of 10, 20, 40, 80 and 160 mg/1 (three replicate flasks per concentration, six contols) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.


Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter. The was conducted under static conditions with an initial nominal cell density of 4 x 103 cells per mL. At the start of the test all control cultures were observed to be clear colourless solutions. The test cultures were observed to range from yellow coloured solutions at 10 mg/L through to dark orange coloured solutions at 160 mg/L. After the 72-Hour test period all control cultures were observed to be pale green dispersions whilst the test cultures were observed to range from yellow coloured solutions at 10 mg/L through to dark orange coloured solutions at 160 mg/L.


The effect of the test material on the growth of Pseudokirchnerella subcapitata (name as written in the study report) has been investigated over a 72-Hour period and gave an ErC50 (0 - 72 h) of greater than 160 mg/l*, an EyC50 (0 - 72 h) of greater than 160 mg/l*, and an EbC50 (0 - 72 h) of greater than 160 mg/l*. The Lowest Observed Effect Concentration dased on growth rate, yield and biomass integral was 40 mg/l, and the No Observed Effect Concentration was 20 mg/l.


* It was not possible to calculate EC50 values as no concentration tested resulted in greater than 50% inhibition of growth.


 


Another study was performed (DyStar, 1998) to assess the adverse effects of test substance on the growth and growth rate of green algae species Scenedesmus subspicatus CHODAT over several generations. The study was conducted in accordance with EU Method C.3.


Exponentially growing algal cells were exposed for a period of 72 h to the nominal test concentrations of 12.5, 25, 50 and 100 mg/L of test substance dissolved in water.


Analysis of the test concentrations 12.5, 25, 50, 100 mg/L plus a control; additional analysis of the highest (100 mg/L) and the lowest analytically detected test concentration (12.5 mg/L) without algal inoculum, at the test start and after 72 h exposure.


The high pH of >10 in all samples (control and replicates) assumes rapid degradation of the substance through hydrolysis during the study, but this is a realistic scenario with regard to the real conditions in the dyeing process for which the test substance is used (dyeing with these kind of reactive dyes takes place at pH 10 to 11).


The cell densities were measured at 24 h intervals. Inhibition of the algal population was measured as reduction in growth and growth rate, relative to control.


Both the 72 h EbC50 and ErC50 of the test substance were determined to be >76.9 mg/L as measured geometric mean. Nominally, the EC50 is > 100 mg/L. The NOEC and LOEC of the test substance were determined to be 13.1 and 21.8 mg/L respectively (Caspers, 1998).