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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Experimental test result performed according to the OECD test guideline.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The stock solution was prepared by dissolving 1 gm of test chemical in 1000 mL of OECD media to get the final concentration of 1000 mg/L. Futher exposure concentration of 100 mg/l was prepared from the stock solution in OECD media.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Source (laboratory, culture collection): Sterile, unicellular, suspension cultures of algae were obtained from the laboratory for Biological Research in Aquatic Pollution (LABRAP) at the university of
Ghent in Belgium.
- Age of inoculum (at test initiation): exponentially growing phase

ACCLIMATION
- Acclimation period: 2 days prior to initiation
- Culturing media and conditions (same as test or not): The growth medium used for the culturing of Pseudokirchneriella subcapitata is OECD Medium; which was prepared in ultra-pure, autoclaved water prior to any use.
- Any deformed or abnormal cells observed: not observed
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
22 °C±2°C
Nominal and measured concentrations:
Nominal concentration - 100 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flasks
- Material, size, headspace, fill volume: 100 ml conical flasks filled with 60 ml was used for the study.
- Initial cells density: 10000cells/ml
- No. of organisms per vessel: 10000cells/ml
- No. of vessels per concentration (replicates): Two replicates for each test concentration
- No. of vessels per control (replicates): Three replicates for Control

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination(1500Lux)


TEST CONCENTRATIONS
- Test concentrations: 100 mg/l (Nominal concentration)
- Results used to determine the conditions for the definitive study: Growth rate of test organism was noted.

Other:
Incubation :
1. The temperature of the orbital shaking incubator was kept constant throughout the period of exposure of the experiment. The temperature was maintained at 22 ° C±2°C.
2. The test vessels were incubated with a continuous, uniform fluorescent illumination (1500Lux).
3. The pH of the control cultures needs to be noted during the study and the pH of the control medium should not increase by more than 1.5 units during the test.
4. The orbital shaking incubator was set at a speed of 120 revolutions per minute throughout the study period. This is to provide constant shaking to the algal cells to keep them in suspension and to ensure that they do not settle down on the bottom of the test vessel.
5. Study duration : The experimental phase of the study was lasted for a period of 72 hours.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7) was used as a reference substance.
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
Remarks on result:
other: calculated using Student's t-test
Details on results:
The microscopic observations were also noted in each of the experimental flasks. All the cells appeared healthy, sickle shaped and green throughout the test duration in the control and in the experimental flask also no significant changes were observed.
Results with reference substance (positive control):
- Results with reference substance valid?
- EC50: 0.809 mg/l
Reported statistics and error estimates:
Significance difference between control and test concentration (100 mg/l) was calculated using Student's t-test.

Table: Cell count and percent inhibition

Experimental Flasks

and Test

Concentration(mg/L)

0 Hr

Cell

Count

24 Hr

Cell

Count

48 Hr

Cell Count

72 Hr

Cell Count

Avg Specific

Growth Rate

(μ)

Mean Avg

Specific Growth

Rate (μ)

Percent

Inhibition(%)

control

10000

35000

95000

155000

0.91

0.92

 

control

10000

40000

85000

160000

0.92

control

10000

35000

95000

165000

0.93

100 (R1)

10000

20000

60000

155000

0.91

0.92

0

100 (R2)

10000

20000

65000

160000

0.92

Table: pH and Temperature

Test

Concentration(mg/L)

 

 

Experimental

Flasks

pH

 

 

Temperature °C

0 Hours

72 hours

0 Hours

72 hours

control

R1

7.68

6.96

25

25

control

R2

7.71

6.94

25

25

control

R3

7.69

6.96

25

25

100

R1

8.87

7.09

25

25

100

R2

8.88

7.42

25

25

Validity criteria fulfilled:
yes
Conclusions:
Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the 72 hr median effect concentration (ErC50) was determined to be > 100 (nominal concentration).
Executive summary:

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The stock solution was prepared by dissolving 1 gm of test chemical in 1000 mL of OECD media to get the final concentration of 1000 mg/L. Further exposure concentration of 100 mg/l was prepared from the stock solution in OECD media. Green algae were exposed to nominal concentration of test chemical (100 mg/l) in 100 ml conical flasks. Thus, limit test was performed at 100 mg/l test chemical concentration. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.809 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. No significance difference was noted between control and test concentration (100 mg/l) (p<0.05) calculated by using Student's t-test. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be > 100 mg/l. On the basis of this value, test chemical was considered as non-toxic to aquatic algae and hence, considered to be 'not classified' as per the CLP classification criteria.

Description of key information

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata (Experimental study report, 2019). The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The stock solution was prepared by dissolving 1 gm of test chemical in 1000 mL of OECD media to get the final concentration of 1000 mg/L. Further exposure concentration of 100 mg/l was prepared from the stock solution in OECD media. Green algae were exposed to nominal concentration of test chemical (100 mg/l) in 100 ml conical flasks. Thus, limit test was performed at 100 mg/l test chemical concentration. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.809 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. No significance difference was noted between control and test concentration (100 mg/l) (p<0.05) calculated by using Student's t-test. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be > 100 mg/l. On the basis of this value, test chemical was considered as non-toxic to aquatic algae and hence, considered to be 'not classified' as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L

Additional information

Experimental study of the test chemical and supporting weight of evidence study for its functionally similar read across chemical were reviewed for toxicity to aquatic algae and cyanobacteria end point which are summarized as below:

 

In an experimental study from study report (2019), a freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The stock solution was prepared by dissolving 1 gm of test chemical in 1000 mL of OECD media to get the final concentration of 1000 mg/L. Further exposure concentration of 100 mg/l was prepared from the stock solution in OECD media. Green algae were exposed to nominal concentration of test chemical (100 mg/l) in 100 ml conical flasks. Thus, limit test was performed at 100 mg/l test chemical concentration. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.809 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. No significance difference was noted between control and test concentration (100 mg/l) (p<0.05) calculated by using Student's t-test. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be > 100 mg/l. On the basis of this value, test chemical was considered as non-toxic to aquatic algae and hence, considered to be 'not classified' as per the CLP classification criteria.

For the test chemical, an acute test was conducted for 48 hrs for assessing the effect of test chemical (Experimental study report, 2016). The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test). Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) of strain 86.81 SAG obtained from Institute of botany of the ASCR with an initial biomass conc. 5x10(3) cells /ml was used as a test organism for the study. The solution 100 mg/l was prepared by dissolving test chemical in OECD growth medium. Nominal test chemical conc. used for the study was 100 mg/l, i.e, limit test was conducted. Study was performed using Desmodesmus subspicatus as a test organism in a static system. Desmodesmus subspicatus were exposed to test chemical in 50 ml glass vessel in a volume of 15 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 23±2°C with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Cell counting was carried out using electronic particle counter. The differences in means of control and sample were estimated by the t-test for independent groups at a 95 % confidence level, all individual replicates were used (STATISTICA CZ – data analysis software system, version 9.0, StatSoft, Inc.). Statistically significant differences are for p < 0.05. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72hr Inhibition percentage (I%) value was determined to be 4.8% at 100.0 mg/l of test chemical concentration. Thus, EC50 can be considered to be > 100 mg/l, indicating that the test chemical is non-toxic to aquatic algae and therefore considered to be 'not classified' as per the CLP classification criteria.

 

On the basis of the above results, it can be concluded that the test chemicalwas considered as non-toxic to aquatic algae and hence, considered to be ‘not classified’ as per the CLP classification criteria.