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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:
key study
Study period:
17-06-2016 to 20-06-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Experimental test result performed using standard test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Principles of method if other than guideline:
Objective of this study was to observe the action of test chemical when it exposed with the Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) for 72 hrs. Test conducted in accordance with OECD Guideline 201 (Alga, Growth Inhibition Test).
GLP compliance:
no
Analytical monitoring:
not specified
Vehicle:
not specified
Details on test solutions:
The solution of white crystalline powder 100 mg/l was prepared by dissolving the substance in OECD growth medium.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Institute of botany of the ASCR
- Initial biomass concentration: 5x10(3) cells /ml
- Method of cultivation: No data available

ACCLIMATION - No data available
- Acclimation period:
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed:
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
23±2°C
pH:
Test at highest concentration: 8.1 did not change during test
Control: 8.3 changes to 9.1 during test
Nominal and measured concentrations:
100 mg/l concentration were used in the study
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml Glass vessel
- Type (delete if not applicable): closed (with air permeable stopper)
- Sample volume: 30 ml
- Initial cells density: 5x10(3) cells/ml
- No. of vessels per concentration (replicates): 6

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)
Key result
Duration:
72 h
Dose descriptor:
EC0
Effect conc.:
100 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: No effects were observed at 100 mg/l
Results with reference substance (positive control):
- Results with reference substance valid
- EC50: 0.65 mg/L (24 hours)
Reported statistics and error estimates:
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.
Validity criteria fulfilled:
yes
Conclusions:
Based on the no growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical the EC0 was determine to be 100 mg/l after the exposure period of 72 hrs.
Executive summary:

Aim of this study was to evaluate the nature of chemical test chemical when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201. The solution of white crystalline powder 100 mg/l was prepared by dissolving the substance in OECD growth medium. Test conducted at limit concentration of 100 mg/l. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, 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. Based on the no growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical the EC0 was determine to be 100 mg/l after the exposure period of 72 hrs. Based on the EC0 value, substance is likely to be non-hazardous to aquatic algae and cannot be classified as per the CLP classification criteria.

Description of key information

Aim of this study was to evaluate the nature of chemical test chemical when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201. The solution of white crystalline powder 100 mg/l was prepared by dissolving the substance in OECD growth medium. Test conducted at limit concentration of 100 mg/l. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, 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. Based on the no growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical the EC0 was determine to be 100 mg/l after the exposure period of 72 hrs. Based on the EC0 value, substance is likely to be non-hazardous to aquatic algae and cannot be classified as per the CLP classification criteria.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

Various short term studies available for the test chemical were reviewed to determine the toxic nature of test chemical on the growth of aquatic algae and cyanobacteria. The studies are as mentioned below:

 

Aim of this study was to evaluate the nature of chemical test chemical when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201. The solution of white crystalline powder 100 mg/l was prepared by dissolving the substance in OECD growth medium. Test conducted at limit concentration of 100 mg/l. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, 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. Based on the no growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical the EC0 was determine to be 100 mg/l after the exposure period of 72 hrs. Based on the EC0 value, substance is likely to be non-hazardous to aquatic algae and cannot be classified as per the CLP classification criteria.

 

Above study was supported by the second experimental supporting study. The study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test solution was prepared in aseptic condition. The test item was prepared by adding 50 mg of test item in 250ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring for 30 minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item. 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 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. 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. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate. According to the test validity criteria the following criteria are met in this test: Firstly As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 1.501 per day. Secondaly The mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 13.025%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 1.995 %. Hence, the test is considered valid as per OECD guideline, 201. Based on the growth rate inhibition of green alga Chlorella vulgaris by the test chemical, EC50 was observed experimentally was >200 mg/l and the EC30 was determine to be 265.984 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

 

Thus based on the above data and results from experimental studies, chemical consider to be nontoxic to the aquatic algae and cyanobacteria and thus not classified as per the CLP classification criteria.