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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Experimental test result performed using standard 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 100 mg/l was prepared by dissolving test chemical in OECD growth medium. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Institute of botany of the ASCR
- Initial biomass concentration: 5000 cells /ml
- Method of cultivation: No data available
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23±2°C
pH:
without adjustment
the sample at concentration: 80 mg/l: pH 7.8 changed to pH = 7.7 during the test
control: pH = 8.0 changed to pH = 7.8 during the test
Nominal and measured concentrations:
Nominal test chemical conc. used for the study were 0, 2.5, 5.0, 10, 20, 40 and 80 mg/l, respectively.
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 ml Glass vessel
- Type (delete if not applicable): closed (with air permeable stopper)
- Sample volume: 15 ml
- Initial cells density: 5000 cells/ml
- No. of vessels per concentration (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 6000-8000 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: microscope with counting chamber Cyrus I or electronic particle counter.
- Other: ErC50 was calculated using non-linear regression by the software Prism 4.0
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
20.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 10.7 to 39.3 mg/l
Results with reference substance (positive control):
- Results with reference substance valid
- EC50: 0.77 mg/L (24 hours)
Reported statistics and error estimates:
EC50 was calculated using non linear regression by the software Prism 4.0.

In the control test vessel containing OECD growth medium without test chemical, the coefficient of variation of average growth rate in replicates during the whole test period was 4.5%.

Result of the definitive test:

Sample no.

Sample Information

Conc. (mg/l)

µ (d-1)

I%

EC50 (based on growth rate)

mg/l

95% C. I. (mg/l)

Control

OECD medium

0

1.77

-

 

 

14676

Test chemical

2.5

5.0

10.0

20.0

40.0

80.0

1.61

1.32

1.12

0.98

0.83

0.09

9.0

25.4

36.7

44.6

53.1

94.9

20.5

10.7 to 39.3

Validity criteria fulfilled:
yes
Conclusions:
Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of test chemical, the 72hr median effective concentration (ErC50) value was determined to be 20.5 mg/l (95% CL: 10.7 to 39.3 mg/l).
Executive summary:

Toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae. 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. 5000 cells /ml was used as a test organism. The stock solution 100 mg/l was prepared by dissolving test chemical in OECD growth medium. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Test chemical concentrations were not verified analytically. Nominal test chemical conc. used for the study were 0, 2.5, 5.0, 10, 20, 40 and 80 mg/l, respectively. Study was performed in a static system for 72 hrs. 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. Control solution vessel containing OECD medium without the test chemical was also setup during the study. 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. The 24 hr EC50 value of the reference substance was determined to be 0.77 mg/l. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. In the control test vessel containing OECD growth medium without test chemical, the coefficient of variation of average growth rate in replicates during the whole test period was 4.5% and the specific growth rate in the control was 1.77 per day, indicating that the biomass in the control cultures have increased exponentially by a factor of more than 16 within the 72 hr exposure duration. Thus, the validity criterion of the test has been fulfilled. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr median effect concentration (ErC50) value was determined to be 20.5 mg/l (95% CL: 10.7 to 39.3 mg/l). Thus, based on the EC50 value, test chemical can be considered to be toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 3' 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 (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. Sterile, unicellular, suspension cultures of algae Pseudokirchneriella subcapitata of length 8 – 14 μm and width 2 - 3 μm was used as a test organism. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 300 mg of test chemical in 300 ml of OECD medium to get the final concentration of 1000 mg/L. Test chemical concentrations were verified analytically by UV-VIS spectrophometer. Green algae were exposed to six different nominal concentrations of test chemical (0, 5 mg/L, 10 mg/L, 20 mg/L, 40 mg/L, 80 mg/L and 160 mg/l) in 100 ml conical flasks. 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. Control containing medium without test chemical was used for the study. The cultures were counted and observed daily with the help of an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test chemical). Apart from this, the cell count of each test vessel was also noted with the help of a microscope. Cell densities were recorded in section by section growth rate at 24 hr intervals, which was calculated as specific growth rate. The 72 hr ErC10 (based on growth rate [r]), was calculated or read from the dose/concentration/percentage response curve. Potassium dichromate (K2Cr2O7) was used as a reference substance for the study. Since the concentration of the test chemical being tested has been satisfactorily maintained within ±20% of the nominal concentration throughout the test, all concentrations will be reported as nominal concentration. As per the OECD guideline No. 201 – Alga growth inhibition test, the biomass in the control cultures have increase exponentially by a factor of at least 17 within the 72 hr test period, the mean coefficient of variation by section specific growth rate in the control cultures not exceeded 35% and the coefficient of variation of average specific growth rate during the whole test period in replicate control cultures was not exceeded 10%, thus, fulfilling the validity of the criteria. The 72 hr EC50 value of reference substance was determined to be 0.809 mg/l. On the basis of growth rate of the test organism Pseudokirchneriella subcapitata, the 72 hrs effect concentration (EC10) value was determined to be 0.45 mg/l (nominal concentration) and median effect concentration (ErC50) was determined to be ≥ 160 (nominal concentration) & ≥ 184.46 mg/l (initial measured concentration). On the basis of the EC50 value, chemical was considered to be non-toxic to aquatic algae at environmental relevant concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:
184.46 mg/L

Additional information

Experimental studies of the test chemical and supporting 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. 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. Sterile, unicellular, suspension cultures of algae Pseudokirchneriella subcapitata of length 8 – 14 μm and width 2 - 3 μm was used as a test organism. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 300 mg of test chemical in 300 ml of OECD medium to get the final concentration of 1000 mg/L. Test chemical concentrations were verified analytically by UV-VIS spectrophometer. Green algae were exposed to six different nominal concentrations of test chemical (0, 5 mg/L, 10 mg/L, 20 mg/L, 40 mg/L, 80 mg/L and 160 mg/l) in 100 ml conical flasks. 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. Control containing medium without test chemical was used for the study. The cultures were counted and observed daily with the help of an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test chemical). Apart from this, the cell count of each test vessel was also noted with the help of a microscope. Cell densities were recorded in section by section growth rate at 24 hr intervals, which was calculated as specific growth rate. The 72 hr ErC10 (based on growth rate [r]), was calculated or read from the dose/concentration/percentage response curve. Potassium dichromate (K2Cr2O7) was used as a reference substance for the study. Since the concentration of the test chemical being tested has been satisfactorily maintained within ±20% of the nominal concentration throughout the test, all concentrations will be reported as nominal concentration. As per the OECD guideline No. 201 – Alga growth inhibition test, the biomass in the control cultures have increase exponentially by a factor of at least 17 within the 72 hr test period, the mean coefficient of variation by section specific growth rate in the control cultures not exceeded 35% and the coefficient of variation of average specific growth rate during the whole test period in replicate control cultures was not exceeded 10%, thus, fulfilling the validity of the criteria. The 72 hr EC50 value of reference substance was determined to be 0.809 mg/l. On the basis of growth rate of the test organism Pseudokirchneriella subcapitata, the 72 hrs effect concentration (EC10) value was determined to be 0.45 mg/l (nominal concentration) and median effect concentration (ErC50) was determined to be ≥ 160 (nominal concentration) & ≥ 184.46 mg/l (initial measured concentration). On the basis of the EC50 value, chemical was considered to be non-toxic to aquatic algae at environmental relevant concentrations.

 

Another toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae (Experimental study report. 2018). 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. 5000 cells /ml was used as a test organism. The stock solution 100 mg/l was prepared by dissolving test chemical in OECD growth medium. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Test chemical concentrations were not verified analytically. Nominal test chemical conc. used for the study were 0, 2.5, 5.0, 10, 20, 40 and 80 mg/l, respectively. Study was performed in a static system for 72 hrs. 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. Control solution vessel containing OECD medium without the test chemical was also setup during the study. 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. The 24 hr EC50 value of the reference substance was determined to be 0.77 mg/l. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. In the control test vessel containing OECD growth medium without test chemical, the coefficient of variation of average growth rate in replicates during the whole test period was 4.5% and the specific growth rate in the control was 1.77 per day, indicating that the biomass in the control cultures have increased exponentially by a factor of more than 16 within the 72 hr exposure duration. Thus, the validity criterion of the test has been fulfilled. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr median effect concentration (ErC50) value was determined to be 20.5 mg/l (95% CL: 10.7 to 39.3 mg/l). Thus, based on the EC50 value, test chemical can be considered to be toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

 

For the test chemical, an acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on green algae. The test was performed in accordance with the Scenedesmus cell proliferation inhibition test, DIN 38412 part 9.Test chemical concentrations were not verified analytically. Test chemical conc. used for the study were 0 (control), 7.8, 15.6, 31.3, 62.5, 125, 250 and 500 mg/l (nominal conc.). Study was performed using Scenedesmus subspicatus as a test organism in a static system in quadruplicates (for test conc.). Initial cell concentration used for the study was 10000 cells/ml. Test organisms were exposed to test chemical in 250 mL Erlenmeyer flask. The test vessels were placed at a temperature of 19.85°C under continuous illumination with a light intensity of 6.2 miS/cm. Cell growth was measured fluorometrically in all flasks at 24, 48, 72 and 96 hours of incubation period. On the basis of effect on growth rate of the test organism Scenedesmus subspicatus, the 96 hr EC20, ErC50 and EC90 was determined to be 20, 79 and > 500 mg/l (nominal conc.), respectively. Thus, based on this, chemical was considered as toxic to aquatic algae and hence, considered to be classified in ‘aquatic chronic category 3’ as per the CLP classification criteria. 

 

On the basis of the above results, it can be concluded that the test chemical can be considered as toxic to aquatic algae and considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

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