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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Experimental test result performed using standard 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:
PREPARATION AND APPLICATION OF TEST SOLUTION
The stock solution 100 mg/L was prepared 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.

- Controls: OECD medium as specified in OECD 201, used as a control and for dilution of the stock solution.
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
- Age of inoculum (at test initiation): 5x103 cells/ml
- Method of cultivation: No data

ACCLIMATION
- Acclimation period: No data
- Culturing media and conditions (same as test or not): No data
- Any deformed or abnormal cells observed: No data
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
±1 hour
Test temperature:
23°C ± 2°C
pH:
The sample at concentration 80.0 mg/L : pH = 7.7 did not change during the test,
Control : pH = 8.2 changed to pH = 7.7 during the test
Nominal and measured concentrations:
Nominal concentrations: 0, 5, 10, 20, 40 and 80 mg/L, respectively
Details on test conditions:
TEST SYSTEM
- Test vessel: 50ml glass vessel
- Type : closed (with air-permeable stopper)
- Material, size, headspace, fill volume: 7.5 ml
- Aeration: No data
- Type of flow-through (e.g. peristaltic or proportional diluter): No data
- Renewal rate of test solution (frequency/flow rate): No data
- Initial cells density: 5x103 cells/mL
- Control end cells density: No data
- No. of organisms per vessel: No data
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): No data
- No. of vessels per vehicle control (replicates): No data

GROWTH MEDIUM
- Standard medium used: Yes

OTHER TEST CONDITIONS
- Adjustment of pH: Without adjustment
- Photoperiod: Continuous
- Light intensity and quality: 6000 lx - 8000 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell counting: Microscope with counting chamber Cyrus I or electronic particle counter.
- Chlorophyll measurement: No
- Other: ErC50 was calculated using non-linear regression by the software Prism 4.0
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
Key result
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
90.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 82.6 – 99.7 mg/L
Results with reference substance (positive control):
- Results with reference substance valid
- ErC50: 0.69 mg/L K2Cr2O7

Control Solution: OECD growth medium without the sample, the coefficient of variation of average growth rate in replicates during the whole test period was 0.7 % .

Validity criteria fulfilled:
yes
Conclusions:
The median effective concentration (ErC50) of the test substance on a freshwater algae Desmodesmus subspicatus was determined to be 90.8 mg/L on the basis of effects on growth rate in a 72 hour study.
Executive summary:

Freshwater algal growth inhibition test was carried out on green algae. Test was conducted according to the OECD guideline 201. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was used as test organism. The stock solution 100 mg/L was prepared 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 conducted under the static system and tested at the concentrations 0, 5, 10, 20, 40 and 80 mg/L respectively. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determined after an exposure period of 72 hrs. Effects on the growth rate of the test organism was studied. The median effective concentration (ErC50) of the test substance on a freshwater algae Desmodesmus subspicatus was determined to be 90.8 mg/L on the basis of effects on growth rate in a 72 hour study. Thus based on the ErC50 value, chemical consider to be toxic but as the chemical was readily biodegradable in water, thus chemical consider to be nontoxic and not classified as per the CLP classification criteria.

Description of key information

Freshwater algal growth inhibition test was carried out on green algae. Test was conducted according to the OECD guideline 201. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was used as test organism. The stock solution 100 mg/L was prepared 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 conducted under the static system and tested at the concentrations 0, 5, 10, 20, 40 and 80 mg/L respectively. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determined after an exposure period of 72 hrs. Effects on the growth rate of the test organism was studied. The median effective concentration (ErC50) of the test substance on a freshwater algae Desmodesmus subspicatus was determined to be 90.8 mg/L on the basis of effects on growth rate in a 72 hour study. Thus, based on the ErC50 value, chemical consider to be toxic but as the chemical was readily biodegradable in water, thus chemical consider to be nontoxic and not classified as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:
90.8 mg/L

Additional information

Following different studies includes experimental study for the test chemical and read-across analogues which is extracted by using mechanistic approach and functionally and structurally like the target chemical to observe the toxicity of test chemical to aquatic algae.

 

Freshwater algal growth inhibition test was carried out on green algae. Test was conducted according to the OECD guideline 201. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was used as test organism. The stock solution 100 mg/L was prepared 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 conducted under the static system and tested at the concentrations 0, 5, 10, 20, 40 and 80 mg/L respectively. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determined after an exposure period of 72 hrs. Effects on the growth rate of the test organism was studied. The median effective concentration (ErC50) of the test substance on a freshwater algae Desmodesmus subspicatus was determined to be 90.8 mg/L on the basis of effects on growth rate in a 72 hour study. Thus, based on the ErC50 value, chemical consider to be toxic but as the chemical was readily biodegradable in water, thus chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

Similarly, the principle of second weight of evidence study was to determine the effect of test substance on the growth of fresh water green algae. The study was conducted following OECD guideline 201- Alga growth inhibition test. Chlorella vulgaris was used as test organism. The test substance was prepared by adding 100 mg of test substance in 200 ml of BBM to get the final concentration of 281.985 mg/L. The sock solution was ultrasonically agitated for 30 minutes to obtain a homogenous solution for the experiment. The remaining test solutions were prepared by dilution from the above stock solution, under aseptic condition. The test concentrations selected for the study were 6.25 mg/L, 12.5 mg/L, 25 mg/L, 50mg/L, 100 mg/L and 200mg/L, respectively, prepared using stock solution of the test substance using de-ionized water and the study conducted under the static system. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. 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) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations 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. The test is considered to be valid as met all the standard parameters. The green algae were exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of test substance. EC50 calculated through probit analysis was determine to be > 200 mg/l. Thus, based on the effects observed on the growth rate of algae, chemical consider to be nontoxic and not classified as per the CLP classification criteria. 

 

 

Above experimental data further supported by the third weight of evidence study. Principle of this study was to evaluate the nature of test chemical when comes in contact with the test organism. Test was conducted according to the OECD guideline 201. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was used as test organism. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. 0, 10,16, 26, 41, 66 mg/l, respectively concentration was used. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determined after an exposure period of 72 hrs. The median effective concentration (EC50) of the test substance on algae was determined to be 23.6 mg/L with 95% CI of 28.9 mg/l to 33.7 mg/l on the basis of growth rate inhibition effects in a 72-hour study. Based on the ErC50 value, the substance chemical was considered likely to be hazardous to aquatic algae and can be considered to be classified in aquatic chronic 3 category. But as the test chemical was readily biodegradable in water, thus on that basis, chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

Similar study was conducted to evaluate the nature of test chemical when comes in contact with the test organism. Test was conducted according to the OECD guideline 201. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was used as test organism. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. 0, 10,16, 26, 41, 66 mg/l, respectively concentration was used. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determined after an exposure period of 72 hrs. The median effective concentration (EC50) of the test substance on algae was determined to be 23.6 mg/L with 95% CI of 28.9 mg/l to 33.7 mg/l on the basis of growth rate inhibition effects in a 72 hour study. Based on the ErC50 value, the substance chemical was considered likely to be hazardous to aquatic algae and can be considered to be classified in aquatic chronic 3 category. But as the test chemical was readily biodegradable in water, thus on that basis, chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

Thus, based on the above experimental studies from various sources, test chemical considers to be nontoxic and not classified as per the CLP classification criteria.

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