Potassium nitrite

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:

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

Vehicle:

no

Details on test solutions:

The test solution was prepared by dissolving 500 mg of test chemical in 500 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined. The final solubility value obtained after analytical detection is 123.55 mg/L. To get the maximum dissolution, stock solution was stirred for To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/ml. Care was taken to have a homogeneous solution for the experiment.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Size: 8 – 14 μm length, 2 - 3 μm width
- 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 at Unique Ecotox Research Laboratory, Nagpur.
- 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, which was prepared in ultra-pure, autoclaved water prior to any use.
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 °C±2°C

Nominal and measured concentrations:

Nominal concentrations used for the study were 0, 0.25, 0.5, 1, 2, 4 and 8 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
- Detailed composition if non-standard medium was used: The medium to be used for the growth of algae was OECD medium.

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: Spectrophotometer - The absorbance values of each test vessel and control vessel was noted at 680nm.The OECD medium 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
- 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: Test concentration were: 0, 0.25, 0.5, 1, 2, 4 and 8 mg/l (Nominal concentrations).
- 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 22 ° C±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)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.971 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI – 0.513 to 1.839 mg/l

Results with reference substance (positive control):

Results with reference substance valid
- EC50: 0.75 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 Concentration(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	75000	215000	1.02	1.03
control	10000	35000	80000	220000	1.03
control	10000	45000	75000	220000	1.03
0.25 (R1)	10000	40000	50000	130000	0.85	0.85	17.48
0.25 (R2)	10000	35000	55000	125000	0.84
0.5 (R1)	10000	25000	25000	75000	0.67	0.67	34.95
0.5 (R2)	10000	30000	25000	75000	0.67
1 (R1)	10000	20000	15000	35000	0.42	0.39	62.14
1 (R2)	10000	10000	10000	35000	0.37
2 (R1)	10000	10000	30000	25000	0.31	0.31	69.90
2 (R2)	10000	10000	20000	30000	0.31
4 (R1)	10000	10000	15000	20000	0.23	0.23	77.66
4 (R2)	10000	10000	15000	25000	0.23
8 (R1)	10000	10000	10000	15000	0.14	0.18	82.52
8 (R2)	10000	10000	10000	20000	0.23

 

EC50-- 0.971 (95% CI – 0.513-1.839)

 

Table: pH and temperature

Test Concentration(mg/L)	Experimental Flasks	pH		Temperature °C
		0 Hours	72 Hours	0 Hours	72 Hours
control	R1	7.5	7.28	23	23
control	R2	7.5	7.3	23	23
control	R3	7.5	7.4	23	23
0.25	R1	7.5	7.79	23	23
0.25	R2	7.5	7.88	23	23
0.5	R1	7.6	7.70	23	23
0.5	R2	7.7	7.65	23	23
1	R1	7.7	7.64	23	23
1	R2	7.6	7.59	23	23
2	R1	7.5	7.50	23	23
2	R2	7.6	7.59	23	23
4	R1	7.7	7.51	23	23
4	R2	7.6	7.56	23	23
8	R1	7.6	7.52	23	23
8	R2	7.6	7.55	23	23

Validity criteria fulfilled:

yes

Conclusions:

Based on the growth inhibition of green algae by the test chemical, the EC50 was determined to be 0.971 mg/l (95% CI – 0.513 to 1.839 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. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Pseudokirchneriella subcapitata (green algae) was used as a test organism for the study. Initial cell density of the culture was kept at 1 х 10000 cells/ml. OECD medium was used as a growth medium for the study. The test solution was prepared by dissolving 500 mg of test chemical in 500 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined. The final solubility value obtained after analytical detection is 123.55 mg/L. To get the maximum dissolution, stock solution was stirred for To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/ml. Care was taken to have a homogeneous solution for the experiment. Nominal test chemical concentration used for the study were 0, 0.25, 0.5, 1, 2, 4 and 8 mg/L. Green algae were exposed to various 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 22±2°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. Control (containing only the test medium) was also included in the test.One positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. The cultures were observed daily with the helpof an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae. Apart from this, the cell count of each test vessel was also noted with the help of an automated cell counter. In the control vessel, all cells appeared healthy,round and green throughout the study duration. The microscopic observations were also noted in each of the test vessel. EC50 value of the reference substance was determined to be 0.75 mg/l. Based on the growth inhibition of green algae by the test chemical, the EC50 was determined to be 0.971 mg/l (95% CI – 0.513 to 1.839 mg/l). Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic organisms at environmental relevant concentrations and hence, considered to be classified in ‘’aquatic acute category 1’’ 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. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Pseudokirchneriella subcapitata (green algae) was used as a test organism for the study. Initial cell density of the culture was kept at 1 х 10000 cells/ml. OECD medium was used as a growth medium for the study. The test solution was prepared by dissolving 500 mg of test chemical in 500 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined. The final solubility value obtained after analytical detection is 123.55 mg/L. To get the maximum dissolution, stock solution was stirred for To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/ml. Care was taken to have a homogeneous solution for the experiment. Nominal test chemical concentration used for the study were 0, 0.25, 0.5, 1, 2, 4 and 8 mg/L. Green algae were exposed to various 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 22±2°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. Control (containing only the test medium) was also included in the test.One positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. The cultures were observed daily with the helpof an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae. Apart from this, the cell count of each test vessel was also noted with the help of an automated cell counter. In the control vessel, all cells appeared healthy,round and green throughout the study duration. The microscopic observations were also noted in each of the test vessel. EC50 value of the reference substance was determined to be 0.75 mg/l. Based on the growth inhibition of green algae by the test chemical, the EC50 was determined to be 0.971 mg/l (95% CI – 0.513 to 1.839 mg/l). Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic organisms at environmental relevant concentrations and hence, considered to be classified in ‘’aquatic acute category 1’’ as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.971 mg/L

Additional information

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Pseudokirchneriella subcapitata (green algae) was used as a test organism for the study. Initial cell density of the culture was kept at 1 х 10000 cells/ml. OECD medium was used as a growth medium for the study. The test solution was prepared by dissolving 500 mg of test chemical in 500 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined. The final solubility value obtained after analytical detection is 123.55 mg/L. To get the maximum dissolution, stock solution was stirred for To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/ml. Care was taken to have a homogeneous solution for the experiment. Nominal test chemical concentration used for the study were 0, 0.25, 0.5, 1, 2, 4 and 8 mg/L. Green algae were exposed to various 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 22±2°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. Control (containing only the test medium) was also included in the test.One positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. The cultures were observed daily with the helpof an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae. Apart from this, the cell count of each test vessel was also noted with the help of an automated cell counter. In the control vessel, all cells appeared healthy,round and green throughout the study duration. The microscopic observations were also noted in each of the test vessel. EC50 value of the reference substance was determined to be 0.75 mg/l. Based on the growth inhibition of green algae by the test chemical, the EC50 was determined to be 0.971 mg/l (95% CI – 0.513 to 1.839 mg/l). Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic organisms at environmental relevant concentrations and hence, considered to be classified in ‘’aquatic acute category 1’’ as per the CLP classification criteria.