N-tert-butylacrylamide

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)

Deviations:

no

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control) and 100 mg/L
- 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 200 mg of test chemical in 200 ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final stock solution obtained was 1000 mg/L, verified analytically by UV-Vis Spectrophotometer. The final solubility value obtained after analytical detection is 603.94 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 Denmark Technical University, Denmark and is 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.
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

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 (control) and 100 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): Six replicates for each test concentration
- No. of vessels per control (replicates): Six 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(7000-8000Lux)

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
- Test concentrations: test concentration were: 0 (control) and 100 mg/l.
- 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 (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.:

> 100 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: 0.868 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 test concentrations

Sr. No	Concentrations mg/L	Wavelength (nm)	Absorbance	Temperature (°C)
1	blank	200	0.00	25°C
2	1	200	0.16	25°C
3	2	200	0.32	25°C
4	3	200	0.47	25°C
5	4	200	0.64	25°C
6	5	200	0.77	25°C
7	6	200	0.92	25°C
8	7	200	1.11	25°C
9	8	200	1.17	25°C
10	9	200	1.36	25°C
11	10	200	1.54	25°C

 

The absorbance and concentrations were recorded at 200 nm.

Table: Concentration after analytical Determination

SR. No	concentrations (mg/L)	Absorbance (mean)	Analytical concentrations	Absorbance (mean)	Analytical concentrations
1	control	0.00	0.00	0.00	0.00
2	100	0.14*	92 mg/l	1.00*	98.5 mg/l
Note:Dilutions were made in case of*marked absorbance. Concentration values are recorded accordingly.

The test concentrations were measured and were maintained within 80 – 120 % of nominal. Hence, the EC50 (>100 mg/L) was expressed based on nominal concentrations. 

Table: pH and Temperature

Test Concentration(mg/L)	Experimental Flasks	pH		Temperature °C
		0 hours	72 hours	0 hours	72 hours
Control	R1	7.80	7.87	25	25
Control	R2	7.82	7.86	25	25
Control	R3	7.90	7.79	25	25
Control	R4	7.95	7.86	25	25
Control	R5	7.98	7.97	25	25
Control	R6	7.94	7.99	25	25
100 (R1)	R1	8.05	8.10	25	25
100 (R2)	R2	8.02	8.19	25	25
100 (R3)	R3	8.04	8.20	25	25
100 (R4)	R4	8.07	8.24	25	25
100 (R5)	R5	8.09	8.28	25	25
100 (R6)	R6	8.08	8.30	25	25

 

The pH was measured at the beginning of the test and after 72 hr of exposure. The pH of the control medium did not increase by more than 1.5 units during the test.

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	30000	10000	220000	1.03	1.04	-
Control	10000	35000	80000	200000	1.00
Control	10000	25000	95000	225000	1.04
Control	10000	40000	105000	225000	1.04
Control	10000	30000	90000	240000	1.06
Control	10000	30000	95000	255000	1.08
100 (R1)	10000	30000	80000	215000	1.02	1.04	0.16
100 (R2)	10000	30000	75000	225000	1.04
100 (R3)	10000	35000	70000	235000	1.05
100 (R4)	10000	25000	90000	220000	1.03
100 (R5)	10000	25000	85000	230000	1.05
100 (R6)	10000	30000	95000	230000	1.05

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 2.63%) * coefficent of variation in the control % CV of Average specific <35% (i.e., reported to be 13.75%).

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 >100 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 200 mg of test chemical in 200 ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final stock solution obtained was 1000 mg/L, verified analytically by UV-Vis Spectrophotometer. 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 and 100 mg/l was from the stock test concentrations. Thus, limit test was performed. 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 7000 -8000 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. All control and test vessel conc. were performed in six replicates. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 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 2.63%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 13.75%), 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 >100 mg/l (nominal conc.).Thus, test chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be 'not classified' 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 200 mg of test chemical in 200 ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final stock solution obtained was 1000 mg/L, verified analytically by UV-Vis Spectrophotometer. 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 and 100 mg/l was from the stock test concentrations. Thus, limit test was performed. 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 7000 -8000 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. All control and test vessel conc. were performed in six replicates. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 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 2.63%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 13.75%), 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 >100 mg/l (nominal conc.).Thus, test chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 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 200 mg of test chemical in 200 ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final stock solution obtained was 1000 mg/L, verified analytically by UV-Vis Spectrophotometer. 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 and 100 mg/l was from the stock test concentrations. Thus, limit test was performed. 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 7000 -8000 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. All control and test vessel conc. were performed in six replicates. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 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 2.63%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 13.75%), 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 >100 mg/l (nominal conc.).Thus, test chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.