N-potassium phthalimide

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

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 guidelines

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Principles of method if other than guideline:

This study was designed to assess the effect of the test item Potassium phthalimide (CAS No. 1074-82-4) on the growth of green alga Chlorella vulgaris. The study was conducted in accordance with “OECD guideline for testing of chemicals No. 201 – Alga, growth inhibition test”.

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Potassium phthalimide
- Molecular formula (if other than submission substance): C8H5NO2.K
- Molecular weight (if other than submission substance): 185.223 g/mol
- Smiles notation (if other than submission substance): c12c(C(=O)[NH-]C1=O)cccc2.[K+]
- InChI: 1S/C8H5NO2.K/c10-7-5-3-1-2-4-6(5)8(11)9-7;/h1-4H,(H,9,10,11);/q;+1/p-1
- Substance type: Organic
- Physical state: solid
- Storage condition of test material: Ambient Temp (23 to 27 °C) (Keep container tightly closed in a dry & well-ventilated place. Store in cool place. Do not expose to air, light & moisture.)
- Purity: 99.73%
- Other: Stable in recommended storage conditions
- Solubility in water: 1154.942 mg/l

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

The test solution was prepared in aseptic condition. The test item N-potassium phthalimide was prepared by adding 25 mg of test item in 250 ml of BBM to get the final concentration of 100 mg/L. This stock solution was kept for stirring for 1 hour 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 of the culture was kept 1 X 104cells/ml. Care was taken to have a homogeneous solution for the experiment.

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

TEST ORGANISM
- Common name:green alga
- Strain:Chlorella vulgaris
- Source (laboratory, culture collection): The fresh water green alga Chlorella vulgaris, was used as the test system (organism). Sterile, unicellular, suspension cultures of algae were obtained from National Environmental Engineering Research Institute (NEERI), Nagpur and maintained at Unique Ecotox Research Laboratory, Nagpur. The culture was examined under the microscope to confirm that it was unicellular, healthy and not contaminated.
- Method of cultivation: Bold’s Basal Medium(BBM)

ACCLIMATION
- Culturing media and conditions (same as test or not):The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). 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

Total exposure duration:

72 h

Hardness:

No data available

Test temperature:

22 °C ±2°C

pH:

6.8 ±0.3

Dissolved oxygen:

No data available

Salinity:

No data available

Conductivity:

No data available

Nominal and measured concentrations:

Six test concentration were: 3.125mg/l, 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l,100mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks
- Type (delete if not applicable): No data available
- Material, size, headspace, fill volume: Conical flasks of 100 ml size was used for the study.
- Aeration: No data available
- Type of flow-through (e.g. peristaltic or proportional diluter): No data available
- Renewal rate of test solution (frequency/flow rate): No data available
- Initial cells density: 24.73 x104 cells/mL
- Control end cells density: No data available
- 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
- No. of vessels per vehicle control (replicates): No data available

GROWTH MEDIUM
- Standard medium used: No data available
- Detailed composition if non-standard medium was used: The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). 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.


OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No data available
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination(1500Lux)
- Salinity (for marine algae): No data available

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: 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.
- Chlorophyll measurement: No data available
- Other: 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: All the six concentrations were in geometric series spaced by a factor of 2.
- Justification for using less concentrations than requested by guideline: No data available
- Range finding study: No data available
- Test concentrations: Six test concentration were: 3.125mg/l, 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l,100mg/l (Nominal concentrations)
- Results used to determine the conditions for the definitive study: No data available

6.1. Test solution: The test solution was prepared in aseptic condition. The test item N-potassium phthalimide was prepared by adding 25 mg of test item in 250 ml of BBM to get the final concentration of 100 mg/L. This stock solution was kept for stirring for 1 hour 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 of the culture was kept 1 X 104cells/ml. Care was taken to have a homogeneous solution for the experiment.

6.2. Test vessels: All the tests were 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 60ml so that a sufficient amount of head space was left.

6.3. Replicates: For the assessment of algal growth, the study was conducted in replicates. The control vessel was maintained in triplicates as recommended in the OECD guideline and the test concentrations were selected in geometric series which were maintained in duplicates.

6.4. Incubation:
i) The temperature of the orbital shaking incubator was kept constant throughout the period of exposure of the experiment. The temperature was maintained at 22°C ± 2°C.
ii) The test vessels were incubated with a continuous, uniform fluorescent illumination(1500Lux).
iii) 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.
iv) 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.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

133.35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Control vessels: The microscopic observations were noted down in each of the control vessel. All the cells appeared healthy, round and green throughout the study duration in the control. Also, the drift in pH in the control vessels did not increase by >1.5 units when observed on 72 hours as compared to 0 hours. The average pH drift observed in the control vessels was 0.26 units.

Test vessels: The effect of the test item on the green algae Chlorella vulgaris culture was observed at nominal test concentration of >3.125 mg/L, 6.25 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L,
100 mg/L. All the six concentrations were in geometric series spaced by a factor of 02. The microscopic observations were also noted in each of the test vessel. All the cells appeared
healthy, round and green throughout the study duration and no significant changes were observed up to the concentration of 100 mg/l . EC50 was found to be 133.35 mg/l graphically through probit analysis.

Reported statistics and error estimates:

To obtain a quantitative concentration-response relationship by regression analysis, a line arizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) was determined.

Table 1: Showing the average cell count using Haemocytometer of the test vessels at an equal interval of 24hrs, 48hrs and 72hrs

Test vessels and test concentration	24 Hours	48 Hours	72 Hours
Control
Replicate 1	24000	34000	51200
Replicate 2	26000	40400	52000
Replicate 3	27600	40800	47600
CAS No. 1074 -82 -4
3.125 mg/l
Replicate 1	26800	34000	36400
Replicate 2	25200	32400	36000
6.25 mg/l
Replicate 1	23600	31600	34800
Replicate 2	26000	30000	34000
12.5 mg/l
Replicate 1	21200	28400	32400
Replicate 2	21200	27600	30800
25 mg/l
Replicate 1	19600	26000	28800
Replicate 2	18000	24400	27600
50 mg/l
Replicate 1	18400	22400	25600
Replicate 2	15600	23200	24400
100 mg/l
Replicate 1	16000	21200	24000
Replicate 2	17200	19600	23600

 

 

Table 2: Showing the values of average specific growth rate and percentage inhibition after an interval of 72 hours

	CONTROL		3.125mg/l		6.25mg/l		12.5mg/l		25mg/l		50mg/l		100mg/l
Average Specific Growth rate (µ )	R1	0.544	R1	0.431	R1	0.416	R1	0.392	R1	0.353	R1	0.313	R1	0.292
	R2	0.550	R2	0.427	R2	0.408	R2	0.375	R2	0.338	R2	0.297	R2	0.286
	R3	0.520
Mean of Avg. Specific growth rate	0.538		0.429		0.412		0.383		0.346		0.305		0.289
Percentage Inhibition (%I)	_		20.295		23.458		28.734		35.781		43.247		46.279

 

 

 Table 3: Depicting pH values at 0 Hours and after 72 Hours of test item exposure to algae

Test vessels and test concentration	0 Hours	72 Hours
CONTROL
Replicate 1	7.34	6.83
Replicate 2	7.36	6.86
Replicate 3	7.34	6.85
Average	7.36	6.85
CAS No. 1074 -82 -4
3.125 mg/l
Replicate 1	7.58	6.98
Replicate 2	7.43	6.92
6.25 mg/l
Replicate 1	7.41	7.12
Replicate 2	7.35	7.11
12.5 mg/l
Replicate 1	7.27	7.06
Replicate 2	7.28	7.13
25 mg/l
Replicate 1	7.20	7.18
Replicate 2	7.19	7.10
50 mg/l
Replicate 1	7.14	7.11
Replicate 2	7.13	7.06
100 mg/l
Replicate 1	7.09	7.08
Replicate 2	7.07	7.05

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

The effect of test item N-potassium phthalimide, CAS No. 1074-82-4 was studied on the growth of fresh water green alga Chlorella vulgaris. After 72 hours of exposure to test item N-potassium phthalimide (CAS No. 1074-82-4) at various nominal test concentrations, EC50 was determined to be 133.35 mg/l graphically and through probit analysis.

Executive summary:

The effect of test item N-potassium phthalimide, CAS No. 1074-82-4 was studied on the growth of fresh water green alga Chlorella vulgaris. The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 3.125 mg/L,6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. EC50 calculated graphically through probit analysis was observed to be 133.35 mg/L. Thus based on this value, it can be concluded that the substance can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP classification criteria.

Description of key information

The effect of test item N-potassium phthalimide, CAS No. 1074-82-4 was studied on the growth of fresh water green alga Chlorella vulgaris (UERL Study report, Sustainability Support Services (Europe) AB, Report no. 1074 -82 -4/01/2017/AT, 2017). The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 3.125 mg/L,6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. EC50 calculated graphically through probit analysis was observed to be 133.35 mg/L. Thus based on this value, it can be concluded that the substance can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

133.35 mg/L

Additional information

For toxicity to aquatic algae and cyanobacteria endpoint, 1 experimental study which is supported by the 1 predicted data of target chemical Potassium phthalimide (CAS no. 1074 -82 -4), alongwith the study of its closest read across chemicals using log kow as the primary descriptor, the studies were reviewed to summarize the following information:

 

In an experimental key study report for target chemical N-potassium phthalimide, (CAS No. 1074-82-4), the effect of test item N-potassium phthalimide, CAS No. 1074-82-4 was studied on the growth of fresh water green alga Chlorella vulgaris (UERL Study report, Sustainability Support Services (Europe) AB, Report no. 1074 -82 -4/01/2017/AT, 2017). The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 3.125 mg/L,6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. EC50 calculated graphically through probit analysis was observed to be 133.35 mg/L. Thus based on this value, it can be concluded that the substance can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP classification criteria.

 

Short term toxicity on aquatic algae and cyanobacteria of target chemical Potassium phthalimide (CAS no. 1074 -82 -4), is predicted using the EPI Suite ECOSAR version 1.10 (2017). The 96 hours EC50 was estimated to be 413.131 mg/l on green algae for substance Potassium phthalimide (CAS no. 1074 -82 -4) with growth inhibition effects. Thus, based on this value, it can be concluded that the test chemical Potassium phthalimide can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.

In a supporting study of read across chemical Phthalimide (CAS no. 85-41-6), short term toxicity to algae study was carried out for 72 hrs (J-CHECK, 2016). The study was based on the effects of the read across compound Phthalimide (CAS no. 85-41-6) on green algae in a static fresh water system. On the basis of effect on growth rate of the test organism green algae, the 72 hr EC50 and NOEC value was determined to be > 150 and 1.1 mg/l and on the basis of areas under the growth curves, the 72 hr EC50 and NOEC value was determined to be 62 and 6.5 mg/l, respectively. Thus, based on the EC50 value (> 150 mg/l), it can be concluded that the read across substance Phthalimide can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.

 

For read across chemical Phthalimide (CAS no. 85-41-6), short term toxicity to Selenastrum capricornutum study was carried out for 72 hrs (OECD SIDS, 2005). The study was performed according to OECD Guideline 201 (Alga, Growth Inhibition Test). The study was based on the effects of the read across compound Phthalimide (CAS no. 85-41-6) on Selenastrum capricornutum in a static fresh water system at an average temperature of 22.1°C. Test chemical conc. used for the study were0, 10.6, 19.1, 34.3, 61.7, 111.1 and 200.0 mg/l, respectively. 300 mL net triangle conical glass flask was used as a test vessel. The flasks were incubated in a shaking water bath at 22.1 degrees C with continuous illumination at 4000 - 5000 lux. Based on the effect on growth rate of the test organism Selenastrum capricornutum, the 72 hrs ExC50 and NOEC value was determined to be 161 and 10.7 mg/l, respectively. Thus, based on EC50 value, it can be concluded that the read across substance Phthalimide can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.

 

In a supporting study of another read across chemical N-(Cyclohexylthio)phthalimide (CAS no. 17796-82-6), short term toxicity to Selenastrum capricornutum (green algae) study was carried out for 96 hrs (HSDB, 2016). The study was based on the effects of the read across compound N-(Cyclohexylthio)phthalimide (CAS no. 17796-82-6) on Selenastrum capricornutum (green algae) in a static fresh water system.The test algae were obtained from the US EPA Environmental Research Laboratory in Corvallis, Oregon. Test chemical conc. used for the study were 0, 6, 10, 32, 56 and 100 mg/l, respectively. Initial cell density of algae was 2 × 104cells/ml. All tests were carried out in triplicates. Statistical analysis involved converting each test concentration to a logarithm, and the corresponding percentage decrease of in vivo chlorophyll a or cell numbers was converted to a probit. The EC50 and 95% confidence limits were then calculated by linear regression. The toxicity of the test substance to algae was similar throughout the 96 hr exposure. There was no significant difference between growth of the control and solvent control cultures after 96 hr of exposure by either measured parameter. Based on effect on biomass and growth of the test organism Selenastrum capricornutum (green algae), the 96 hr EC50 value was determined to be 21 mg/l and on the basis of chlorophyll measurement, the 96 hr EC50 value was determined to be 22 mg/l and NOEC value was 6 mg/l, respectively. Thus, based on the EC50 value, it can be concluded that the read across substance N-(Cyclohexylthio)phthalimide can be considered as toxic to aquatic organisms and thus can be classified as aquatic chronic category 3 as per the CLP criteria.Since the chemical is readily biodegradable in nature, chemical N-(Cyclohexylthio)phthalimide can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.

 

Based on the overall reported results of experimental data and various studies for target chemical and of its read across substance (from authoritative database and secondary source), it can be concluded that the test substance Potassium phthalimide can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.