1-dodecylpyridinium chloride

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

Details on sampling:

- Concentrations: 0, 3, 4.8, 7.68, 12.29 and 19.66 mg/L, respectively.
- Sample storage conditions before analysis: Test samples were analysed immediately after sampling

Vehicle:

no

Details on test solutions:

The test solution was prepared by dissolving 250 mg of test chemical in 250 ml of OECD medium to get the final concentration of 100 mg/L. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/mL.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Length: 8 – 14 μm
- Weight: 2 - 3 μm
- 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 in Laboratory.
- 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.
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 to 24 ± 2°C

Nominal and measured concentrations:

Test chemical concentrations used for the study were 0, 3, 4.8, 7.68, 12.29 and 19.66 mg/L (factor of 1.6), 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): Three replicates for each test concentration
- No. of vessels per control (replicates): Three replicates for Control

GROWTH MEDIUM
- Standard medium used: yes, OECD medium was used as a test medium in the study.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination(6000-10000Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell counts were measured using microscope.
- 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
- Spacing factor for test concentrations: All the six concentrations were in geometric series spaced by a factor of 1.6.
- Test concentrations: test concentration were: 0, 3, 4.8, 7.68, 12.29 and 19.66 mg/L (Nominal concentrations)
- Results used to determine the conditions for the definitive study: Growth rate 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 (1500Lux).
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) was used as a reference substance.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.94 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The microscopic observations were also noted in each of the experimental flasks. All the cells appeared healthy, sickle shaped and green throughout the test duration in the control.

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50: 1.57 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: Assessment of Dose Range concentrations

Sr. no.		Concentrations (mg/l)		Wavelength		Absorbance (mean)		Temperature (°C)
1		Blank		258		0.0001		25°C
2		2.356		258		0.0295		25°C
3		7.068		258		0.0907		25°C
4		14.136		258		0.1907		25°C
5		21.204		258		0.2832		25°C
6		28.272		258		0.3798		25°C
7		35.340		258		0.4722		25°C

 

The absorbance and concentrations were recorded at 258 nm.

Table: Concentration After Analytical Determination

				0 hours		0 hours		72 hours		72 hours
Sr. no.		Concentrations (mg/l)		Absorbance (mean)		Analytical concentrations		Absorbance (mean)		Analytical concentrations
1		Control		0.00		0.01		0.00		0.05
2		3		0.03		2.58		0.04		2.93
3		4.8		0.06		4.82		0.06		4.73
4		7.68		0.10		7.12		0.12		8.99
5		12.29		0.15		11.39		0.17		12.4
6		19.66		0.23		17.04		0.25		18.56

 

Conclusion: The test concentrations were measured and found to remain within 80 – 120 % of nominal. Hence, the ErC 50 was expressed based on nominal concentrations.

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	130000	310000	1.14	1.14	-
control	10000	35000	115000	295000	1.13
control	10000	35000	115000	310000	1.14
3	10000	20000	65000	1 250000	0.84	0.83	27.30
3	10000	25000	60000	115000	0.81
3	10000	30000	60000	120000	0.83
4.8	10000	20000	40000	70000	0.65	0.65	43.10
4.8	10000	20000	40000	70000	0.65
4.8	10000	20000	35000	70000	0.65
7.68	10000	10000	20000	45 000	0.50	0.48	58.30
7.68	10000	20000	20000	40000	0.46
7.68	10000	20000	20000	40000	0.46
12.29	10000	20000	20000	30000	0.37	0.24	78.60
12.29	10000	15000	15000	20000	0.23
12.29	10000	15000	15000	15000	0.14
19.66	10000	15000	15000	20000	0.23	0.23	79.70
19.66	10000	15000	15000	20000	0.23
19.66	10000	15000	15000	20000	0.23

 

Table: pH and Temperature

Test concentration (mg/l)	Experimental flasks	pH		Temperature (°C)
		0 hours	72 hours	0 hours	72 hours
control	R1	7.89	8.41	21.3	21.3
control	R2	7.82	8.61	21.3	21.3
control	R3	7.80	8.69	21.3	21.3
3	R1	7.87	8.47	21.3	21.3
3	R2	7.83	8.39	21.3	21.3
3	R3	7.91	7.99	21.3	21.3
4.8	R1	7.85	7.98	21.3	21.3
4.8	R2	7.80	7.87	21.3	21.3
4.8	R3	7.79	7.80	21.3	21.3
7.68	R1	7.84	8.09	21.3	21.3
7.68	R2	7.92	7.90	21.3	21.3
7.68	R3	7.95	8.03	21.3	21.3
12.29	R1	8.03	8.03	21.3	21.3
12.29	R2	7.98	8.13	21.3	21.3
12.29	R3	8.01	8.00	21.3	21.3
19.66	R1	7.99	8.04	21.3	21.3
19.66	R2	8.05	8.01	21.3	21.3
19.66	R3	8.02	7.98	21.3	21.3

 

 

Validity criteria fulfilled:

yes

Remarks:

* The average increase in cell count in control group is >16 fold. * The coefficent of variation between replicates was <7 % (i.e., reported to be 0.838%) * coefficent of variation in the control % CV of Average specific <35% (i.e., reported to be 4.33%).

Conclusions:

Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the 72 hr median effect concentration (ErC50) value was determined to be 5.94 mg/l (nominal conc.).

Executive summary:

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 250 mg of test chemical in 250 ml of OECD medium to get the final concentration of 100 mg/L. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/mL. Further, exposure concentrations of 0, 3, 4.8, 7.68, 12.29 and 19.66 mg/L mg/l (factor of 1.6), respectively was from the stock test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 21 to 24 ± 2°C and with a continuous uniform illumination of 6000 -10000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 1.57 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16, the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% (i.e., reported to be 0.838%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 4.33%), respectively thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (ErC50) value was determined to be 5.94 mg/l (nominal conc.). Thus, test chemical was considered as toxic to aquatic algae at environmental relevant concentrations and hence, considered to be classified in 'aquatic chronic category 2' 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 Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 250 mg of test chemical in 250 ml of OECD medium to get the final concentration of 100 mg/L. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/mL. Further, exposure concentrations of 0, 3, 4.8, 7.68, 12.29 and 19.66 mg/L mg/l (factor of 1.6), respectively was from the stock test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 21 to 24 ± 2°C and with a continuous uniform illumination of 6000 -10000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 1.57 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16, the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% (i.e., reported to be 0.838%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 4.33%), respectively thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (ErC50) value was determined to be 5.94 mg/l (nominal conc.). Thus, test chemical was considered as toxic to aquatic algae at environmental relevant concentrations and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

5.94 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 Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 250 mg of test chemical in 250 ml of OECD medium to get the final concentration of 100 mg/L. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/mL. Further, exposure concentrations of 0, 3, 4.8, 7.68, 12.29 and 19.66 mg/L mg/l (factor of 1.6), respectively was from the stock test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 21 to 24 ± 2°C and with a continuous uniform illumination of 6000 -10000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 1.57 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16, the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% (i.e., reported to be 0.838%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 4.33%), respectively thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (ErC50) value was determined to be 5.94 mg/l (nominal conc.). Thus, test chemical was considered as toxic to aquatic algae at environmental relevant concentrations and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.