N-(2-chloroethyl)pyrrolidine hydrochloride

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21.04.2017 to 24.04.2017

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

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Principles of method if other than guideline:

Short term toxicity of test chemical to aquatic algae was performed according to the 201 OECD guideline in a static system.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The stock solution 100 mg/l was prepared by dissolving white powder 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
- Common name:
- 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:

no

Total exposure duration:

72 h

Test temperature:

23±2°C

pH:

Test at highest concentration: 7.8 (change to 7.7 during tests)
Control: 8.1 (changed to 8.2 during test)

Nominal and measured concentrations:

0, 1, 4, 9 and 20 mg/l

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: 5x10(3) 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)

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

4.29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 3.57-5.17

Results with reference substance (positive control):

- Results with reference substance valid
- EC50: 0.75 mg/L (24 hours)

Reported statistics and error estimates:

EC50 was calculated using non linear regression by the software Prism 4.0.

Validity criteria fulfilled:

yes

Conclusions:

Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical the EC50 was determine to be 4.29 mg/l.

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 stock solution 100 mg/l was prepared by dissolving white powder 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.

 

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 determine after an exposure period of 72 hrs.

 

The median effective concentration (ErC50) for the test substance in algae was determined to be 4.29 mg/L on the basis of growth rate inhibition effects in a 72 hour study. Based on the EC50 value, which indicates that the substance is likely to be hazardous to aquatic algae and can be classified as aquatic chronic 2 category as per the CLP classification criteria.

Description of key information

Various experimental data for the target compound were reviewed for to assess the effects of test chemical on the growth of fresh water green alga along with the predicted data which are summarized as 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 stock solution 100 mg/l was prepared by dissolving white powder 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. 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 determine after an exposure period of 72 hrs. The median effective concentration (ErC50) for the test substance in algae was determined to be 4.29 mg/L on the basis of growth rate inhibition effects in a 72 hour study.

 

The above result was supported by another study 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). The test solution was prepared in aseptic condition. The test item N-(2-chloroethyl)pyrrolidine hydrochloride was prepared by adding 54.67 mg of test item in 250 ml of BBM to get the final concentration of 218.7 mg/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 concentrations chosen were set up to the water solubility limit. 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 104cells/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.

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 0.264 per day.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 20.24%.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 9.31%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item N-(2-chloroethyl)pyrrolidine hydrochloride(CAS No. 7250- 67-1) to various nominal test concentrations, EC50 was found to be 11.548 mg/l graphically and through probit analysis.

 

Based on the EC50 values, which indicates that the substance is likely to be hazardous to aquatic algae and can be classified as aquatic chronic 2 category as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

4.29 mg/L

Additional information

Various experimental data for the target compound were reviewed for to assess the effects of test chemical on the growth of fresh water green alga along with the predicted data which are summarized as 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 stock solution 100 mg/l was prepared by dissolving white powder 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. 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 determine after an exposure period of 72 hrs. The median effective concentration (ErC50) for the test substance in algae was determined to be 4.29 mg/L on the basis of growth rate inhibition effects in a 72 hour study.

 

The above result was supported by another study 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). The test solution was prepared in aseptic condition. The test item N-(2-chloroethyl)pyrrolidine hydrochloride was prepared by adding 54.67 mg of test item in 250 ml of BBM to get the final concentration of 218.7 mg/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 concentrations chosen were set up to the water solubility limit. 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 104cells/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.

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 0.264 per day.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 20.24%.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 9.31%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item N-(2-chloroethyl)pyrrolidine hydrochloride(CAS No. 7250- 67-1) to various nominal test concentrations, EC50 was found to be 11.548 mg/l graphically and through probit analysis.

 

Based on the EC50 values, which indicates that the substance is likely to be hazardous to aquatic algae and can be classified as aquatic chronic 2 category as per the CLP classification criteria.