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Ecotoxicological information

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:
2 (reliable with restrictions)
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:
yes
Details on sampling:
- Concentrations: 0, 1.5, 3.3, 7.26, 15.97 and 35.13 mg/L
- Sampling method:
- Sample storage conditions before analysis: samples were immediatly analysed
Vehicle:
no
Details on test solutions:
The test solution was prepared by dissolving 100mg of test chemical in 100ml of OECD medium to get the final concentration of 1000 mg/L. Stock solution was verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 1.5, 3.3, 7.26, 15.97 and 35.13 mg/L respectively was from the stock solution.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Length: 8 – 14 μm
- Weight: 2 - 3 μm
- Source: 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 and maintained in Laboratory.
- Method of cultivation: OECD medium

ACCLIMATION
- Culturing media and conditions (same as test or not): The medium to be used for the growth of algae was OECD medium. It is a medium composed of macronutrients, micronutrients, alkaline EDTA solution and Iron solution. Stock solution of each of these was prepared separately and then a complete medium was prepared and sterilized. De-ionized water was used to prepare the BBM
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21 to 24 ± 2°C
pH:
7.10-7.84
Nominal and measured concentrations:
Test chemical concentrations used for the study were 0, 1.5, 3.3, 7.26, 15.97 and 35.13 mg/L, respectively.
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, OECD medium was used as a test medium in the study.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination(3000 - 4000 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell counts were measured using microscope.
- Chlorophyll measurement: No data
- Other: The cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to
observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer.

TEST CONCENTRATIONS
- Test concentrations: Six test concentration were: 0, 0.15, 0.19, 0.24, 0.31, 0.40 and 0.52 mg/L
- Results used to determine the conditions for the definitive study: Mortality of test organisms

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 21 to 24 ± 2°C.
2. The test vessels were incubated with a continuous, uniform fluorescent illumination (3000 - 4000 Lux).
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.:
20.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 CI 17.75-24-37 mg/L
Results with reference substance (positive control):
- Results with reference substance valid?
- EC50: 0.868 mg/l
Reported statistics and error estimates:
To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) was determined.

 

Table: Cell count and percent inhibition

Experimental Flasks

and Test

Conc (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

40000

135000

300000

1.13

1.11

-

Control

10000

40000

140000

220000

1.03

Control

10000

45000

145000

320000

1.16

1.5 (R1)

10000

30000

110000

255000

1.08

1.07

3.69

1.5 (R2)

10000

35000

135000

255000

1.08

1.5 (R3)

10000

30000

145000

225000

1.04

3.3 (R1)

10000

25000

95000

165000

0.93

0.94

14.94

3.3 (R2)

10000

20000

105000

170000

0.94

3.3 (R3)

10000

20000

90000

170000

0.94

7.26 (R1)

10000

20000

30000

115000

0.81

0.80

27.31

7.26 (R2)

10000

20000

35000

110000

0.80

7.26 (R3)

10000

20000

35000

110000

0.80

15.97 (R1)

10000

10000

30000

60000

0.60

0.58

47.85

15.97 (R2)

10000

20000

30000

60000

0.60

15.97 (R3)

10000

15000

25000

50000

0.54

35.13 (R1)

10000

10000

25000

45000

0.50

0.46

58.39

35.13 (R2)

10000

10000

20000

35000

0.42

35.13 (R3)

10000

10000

20000

40000

0.46

Table: pH and temperature

Experimental Flasks

and Test

Conc (mg/L)

pH

Temperature °C

0 Hr

72 Hr

0 Hr

72 Hr

Control

7.74

7.84

23.1

23.1

Control

7.64

7.55

23.1

23.1

Control

7.77

7.60

23.1

23.1

1.5 (R1)

7.52

7.26

23.1

23.1

1.5 (R2)

7.52

7.32

23.1

23.1

1.5 (R3)

7.26

7.27

23.1

23.1

3.3 (R1)

7.26

7.31

23.1

23.1

3.3 (R2)

7.22

7.17

23.1

23.1

3.3 (R3)

7.21

7.27

23.1

23.1

7.26 (R1)

7.14

7.10

23.1

23.1

7.26 (R2)

7.12

7.25

23.1

23.1

7.26 (R3)

7.11

7.22

23.1

23.1

15.97 (R1)

7.10

727

23.1

23.1

15.97 (R2)

7.12

7.36

23.1

23.1

15.97 (R3)

7.15

7.38

23.1

23.1

35.13 (R1)

7.10

7.16

23.1

23.1

35.13 (R2)

7.2

7.22

23.1

23.1

35.13 (R3)

7.15

7.25

23.1

23.1

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 (EC50) was determined to be 20.8 mg/l.
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 test solution was prepared by dissolving 100 mg of test chemical in 100 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined by UV-VIS spectrophotometer. Green algae were exposed to nominal concentration of test chemical in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 23.1°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 0.868 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. As the concentration of the test chemical being tested has not been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on measured concentration. On the basis of growth rate of the test organism, the 72 hr EC50 and median effect concentration (EC50) was determined to be 20.8 respectively.

Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic and hence, considered to be 'not classifed' 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. 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 test solution was prepared by dissolving 100 mg of test chemical in 100 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined by UV-VIS spectrophotometer. Green algae were exposed to nominal concentration of test chemical in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 23.1°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 0.868 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. As the concentration of the test chemical being tested has not been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on measured concentration. On the basis of growth rate of the test organism, the 72 hr EC50 and median effect concentration (EC50) was determined to be 20.8 respectively. Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic and hence, considered to be 'not classifed' as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:
50.26 mg/L

Additional information

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

 

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 test solution was prepared by dissolving 100 mg of test chemical in 100 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined by UV-VIS spectrophotometer. Green algae were exposed to nominal concentration of test chemical in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 23.1°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 0.868 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. As the concentration of the test chemical being tested has not been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on measured concentration. On the basis of growth rate of the test organism, the 72 hr EC50 and median effect concentration (EC50) was determined to be 20.8 respectively.

Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic and hence, considered to be 'not classifed' as per the CLP classification criteria.

In an experimental study from study report (2014),a freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Chlorella vulgaris. 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. BBM medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test substance solution was prepared by dissolving 25.767 µl of test substance in 250 ml of BBM to get the final concentration of 103.07 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 1500 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. The microscopic observations were also noted in each of the experimental flasks. All the cells appeared healthy, round and green throughout the test duration in the control and in the experimental flask following changes were observed with increase in test substance concentration like decrease in cell count and discoloration of algal cells. On the basis of the effect of test chemical on growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 50.26 mg/l and 50.12 (calculated from equation and graphically through probit analysis). On the basis of this value, chemical was considered as toxic to aquatic algae. Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic and hence, considered to be 'not classified' as per the CLP classification criteria.

 

Another toxicity to aquatic algae study was conducted for 96 hrs for assessing the effect of test chemical (A.M. Api, et.al, 2016). The study was performed following the german standard guideline DIN 38412 part 9 under static system. On the basis of the effect of test chemical on growth rate and biomass of the test organism Desmodesmus subspicatus, the 72 hr EC50 value was determined to be 22.9 and 20.9 mg/l and the 96 hr EC50 value was determined to be 21.9 and 12.2 mg/l, respectively. Thus, test chemical can be considered as toxic to aquatic algae. Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic and hence, considered to be non classified as per the CLP classification criteria.

 

For the test chemical from peer reviewed journal (Jihai Shaoa, et.al, 2011), toxicity of test chemical was accessed on the growth of green algae Microcystis aeruginosa. Experiments were carried out in 250mL conical flasks containing 94.9mL CT liquid medium, and 100L of test solutions in dimethyl sulfoxide (DMSO). Prior to these experiments, 0.2% (v/v) DMSO had no obvious effect on the growth and photosynthetic processes of M. aeruginosa NIES-843. Five milliliters of exponential-phase cultures of M. aeruginosa NIES-843 (in CT medium) were added to the conical flasks in order to reach a final volume of 100 mL. The final test concentrations were set as 0, 6.67, 10, 15, 22.5, 33.75 mg/L, respectively, and the initial cellular concentrations of M. aeruginosa NIES-843 were 6.5×100000 cells/mL. Each treatment was replicated three times. All treatments were cultured under the same conditions as mentioned above. The increase in M. aeruginosa cell number was calculated after 48 h using a hemocytometer. Chl a and carotenoid content were determined. Cultures were sampled 48 h after inoculation to measure content of photosynthetic pigments. Results based on transcript expression of genes, polyphasic Chl a fluorescence transients and ultrastructural examinations through TEM indicated that the reaction centre of PS II and electron transport at the acceptor side of PS II are the targets responsible for the toxicity of test chemical on the PS II of M. aeruginosa NIES-843. On the basis of the effect of test chemical on growth rate of the test organism, the 48 hr EC50 value was determined to be 21.23±1.87 mg/l.

 

On the basis of the overall results, it can be concluded that the test chemical was considered as toxic to aquatic algae. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic algae at environmental concentrations and hence, considered to be'not classified' as per CLP classification criteria.