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Toxicity to aquatic algae and cyanobacteria

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toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
according to guideline
other: Real Decreto 363/1995 (BOE núm. 133 de Junio de 1995).
GLP compliance:
not specified
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: Selenastrum capricornutum
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection Algae and Protozoo (Cumbria, United Kingdom)
- Method of cultivation:
Temperature: 22 ± 2 ° C
Intensity and quality of illumination: 2500 Lux (The surface where the study is done receives uniform and continuous fluorescent lighting (white-cold type)
Continuous orbital agitation
The reference substance used to verify the response of test organisms is potassium dichromate (K2Cr2O7). The result of periodic verification of the response of the test organisms is IC50 72h = 1.1 mg / L.

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Test temperature:
22 ± 2ºC
Nominal and measured concentrations:
Theoretical tested concentrations: 10 mg/L, 32 mg/L, 100 mg/L, 320 mg/L, 1000 mg/L, 3200 mg/L, 10000 mg/L
Actual concentration of the mother solution tested (active material): 2866,04 mg/L (10.460 mg/L of test material).
Actual tested concentrations (active material): 2,87 mg/L, 9,17 mg/L, 28,66 mg/L, 91,71 mg/L, 286,6 mg/L, 917,1 mg/L, 2866 mg/L
Details on test conditions:
- Test vessel: Test tubes (40 mL)
- Volume of culture used: 10 mL
- Type: closed with a material permeable to air
- Type of flow-through (e.g. peristaltic or proportional diluter): continuous agitation
- Initial cells density: 1,09 10E4 cells/mL
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

- Standard medium used: yes, culture medium defined in AFNOR T90-304, which has been validated by the laboratory since it gives quantitative results comparable to those described in the guideline
- Detailed composition if non-standard medium was used:

Solution 1: Ca(NO3)2·4H2O: 0.4 g / 100 ml H2O
Solution 2: KNO3: 10 g / 100 ml H2O
Solution 3: MgSO4·7H2O: 3 g / 100 ml H2O
Solution 4: K2HPO4: 4 g / 100 ml H2O
Solution 5:
CuSO4· 5H2O: 30 mg / l
(NH4 6Mo7O24·4H2O: 60 mg / l
ZnSO4·7H2O: 60 mg / l
CoCl2 6H2O: 60 mg / l
Mn (NO3)2·4H2O: 60 mg / l
C6H8O·7H2O: 60 mg / l
H3BO3: 60 mg / l
Solution 6:
C6H5FeO7 5H2O: 1.62 g / l
FeSO4·7H2O: 0.62 g / l
FeCl 3·6H2O: 0.62 g / l

Add to 1000 mL of deionized water
Solution 1: 1 mL
Solution 2:1 mL
Solution 3: 1 mL
Solution 4: 1 mL
Solution 5 : 0.5 mL
Solution 6: 0.5 mL

- Sterile test conditions: no
- Adjustment of pH: not specified
- Growth apparatus: Incubation chamber with orbital agitation (Ivymen system)
- Light intensity and quality: 2500 Lux (The surface where the study is done receives uniform and continuous fluorescent lighting (white-cold type)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber Neubauer

- Spacing factor for test concentrations: 3.2
- Test concentrations: 7+control
- Preparation of stock and test solutions: A stock solution has been prepared with a concentration of 10 g / L of the test material. Therefore, 10 g of the material have been dissolved in 100 ml of growth medium by shaking in a volumetric flask, Class A of 100 mL, so that a stock solution of 10000 mg / L has been obtained. Because the study material contains 27.4% of active material, the stock solution has a concentration 2740 mg / L of active material. The different solutions used in the test have been prepared from this stock solution of 1000 mg / L of active substance.
Reference substance (positive control):
Key result
72 h
Dose descriptor:
Effect conc.:
115 mg/L
Nominal / measured:
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Results with reference substance (positive control):
- Results with reference substance valid
- IC50: 1.1 mg/L

The validation criterion considered for this test is:
- the cell density of the control solutions must have been multiplied by a factor greater than 16 in 72h
- the pH of the control solutions must not have varied more than 1.5 units during the test
- The IC50 value 72h of potassium dichromate is in the range of 0.9 - 1.5 mg / l

The results of the cell concentration are shown in the table 1.
Table 1: Calculated cell concentration

 replicates nº of cells/ml     control                    concentration (mg/L)
   0h  72h  2.87  9.17  28.66  91.71  286.6  917.7  2866
 1 1.09E4   2.46E5   2.56E5   2.21E5   1.96E5   1.41E5   9.34E4   7.01E4   5.93E4
 2  1.09E4   2.63E5   2.43E5   2.23E5   1.93E5   1.38E5   9.59E4   6.84E4   6.13E4
 average  1.09E4   2.55E5   2.50E5   2.22E5   1.95E5   1.40E5   9.47E4   6.97E4   6.03E4
 sd  0  5303  8839  1768   1768   1768   1768  1768   74
 CV  0  2  4  1  1  1  2  3  1

Inhibition of algal growth during the test: In the table 2, the percentage of growth inhibition is shown, defined as the biomass at the end of the exposure period (72h) minus the biomass at the beginning of the exposure period (0h).

Table 2: % inhibition algal growth

 Conc. (mg/L)

 % inhibition algal growth

















Validity criteria fulfilled:
The result obtained in this study indicate that the substance has an IC50 value (72h) equal to 115 mg / L active substance

Executive summary:

For the determination of inhibition of algal growth, the used algae has been the unicellular freshwater Pseudokirchneriella subcapitata (formerly Selenastrum Capricornutum). The guideline followed was the OECD 201 (1984) together with the Spanish Royal Decree 363/1995 (BOE No. 133 of June 1995).
The fresh water algae Selenastrum capricornutum has been exposed to the test substance at test concentrations: 10 mg / L, 32 mg / L, 100 mg / L, 320 mg / L, 1000 mg / L, 3200 mg / L and 10000 mg / L. The total duration of the trial has been 72 hours.
The result obtained in this test indicates that the test substance has an IC50 value (72h) of 115 mg /L based on active material.

Description of key information

The short-term toxicity to algae is determined by means of one test conducted on the substance Direct Red 254 TEA salt (CAS 64683-40 -5),

according to the OECD Guideline 201 (Alga, Growth Inhibition Test) that results on one EC50>100 mg/L. At the maximum concentration tested no deaths were observed. Considering the existing information, the substance is classified as not harmful to algae and cyanobacteria.

There is one study available to determine the short-term toxicity of the substance Direct Red 254 sodium salt (CAS 6300 -50 -1) to Daphnia according to OECD Guideline 202.At the maximum concentration tested (EC50(daphnia)>200 mg/L act. ingr.) and considering the existing information, the substance is not harmful to aquatic invertebrates.

There are two additional studies conducted on the structural analogue substance Direct Red 254 TEA salt (CAS 64683-40 -5). TheEC50 (48h) values are 155 mg/L active ingr. and 175 mg/L active ingr. respectively, show that the tested substance is not harmful to aquatic invertebrates.

The tested substances have identical structure in respect to the anionic constituents.

The results of all the studies support the effect of the substance to aquatic invertebrates and the read-across approach between both structures.

Key value for chemical safety assessment

EC50 for freshwater algae:
115 mg/L

Additional information