Oxacycloheptadec-10-en-2-one

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: - Concentrations: 0 (control) and 8 mg/L, 13.6 mg/L, 23.12 mg/L, 39.304 mg/L and 66.817 mg/L (test concentrations)

- Sample storage conditions before analysis: Test samples were analysed immediately after sampling

Vehicle:

yes

Remarks:

ethanol

Details on test solutions:

The test solution was prepared by dissolving 100 mg of test chemical in 10 mL of solvent ethanol giving a concentration of 10000 mg/L. 1 mL of this solution was then diluted to 100 mL with OECD medium to get the final concentration of 100 mg/L. This followed the amount of solvent to be used as given in the OECD guideline 201. To have a better growth and visibility of cells, the initial cell count of the culture was kept 5000 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 (laboratory, culture collection): Sterile, unicellular, suspension cultures of algae were obtained from Microbiotest, Belgium and it is maintained with utmost care in the ESSEM facility at Nagpur.
- Method of cultivation: OECD medium

-Maintenance: The growth medium used for the culturing of test organism is OECD Medium; which was prepared in ultra-pure, autoclaved water prior to any use. The temperatures were maintained at 21 to 24°C, controlled at ± 2°C. The Light intensity provided to the culture, it is measured each time and should be 4440  - 8880  LUX at the surface and a continuous light phase is maintained for the test duration.

-Pre-culture: Pre-culture was set up two days prior to initiation of the test. It is grown under identical exposure conditions.

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

pH:

Control: 7.67 - 8.30
Solvent control: 7.68 - 8.45
Test concentrations: 7.58 - 8.44

Nominal and measured concentrations:

Test chemical concentrations used for the study were 0 (control) and 8 mg/L, 13.6 mg/L, 23.12 mg/L, 39.304 mg/L and 66.817 mg/l (spacing factor = 1.7).

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: 5000 cells/mL
- No. of organisms per vessel: 5000 cells/mL
- No. of vessels per concentration (replicates): Three replicates for each test concentration
- No. of vessels per control (replicates): Three replicates control
- No. of vessels per vehicle control (replicates): Three replicates for vehicle 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 hr light – 8 hr dark
- Light intensity and quality: 4440 - 8880 LUX


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: Five test concentrations (number of replicates 03) were selected, which were arranged in geometric series with the factor of 1.7.

- Range finding study: A range finding study was conducted prior to main study with the test concentrations of 0.1, 1, 10, and 100 mg/L of test item along with control and solvent control groups containing OECD medium and test system. The percent inhibition of 11.111, 20.261, 72.549, and 92.81 % were observed at 72 h in the test concentrations of 0.1, 1, 10, and 100 mg/L respectively.
- Test concentrations: Test chemical concentrations used for the study were 0 (control) and 8 mg/L, 13.6 mg/L, 23.12 mg/L, 39.304 mg/L and 66.817 mg/L.


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 21 to 24 ± 2°C.
2. The test vessels were incubated with a continuous, uniform light of 4440 - 8880 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 ±10 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.:

16.604 mg/L

95% CI:

> 14.43 - < 18.82

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Percent yield was found to be 18.75, 46.875, 64.063, 82.813, and 87.5 % for test concentration 8, 13.6, 23.12, 39.304, and 66.817 % respectively.

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50: 2.8 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 (ErC) were determined.

Table 1: Assessment of Dose Range concentrations

Sr. No	Concentrations mg/L	Wavelength (nm)	Absorbance	Temperature (°C)
1	blank	197.5	0.0001	25°C
2	10	197.5	0.3837	25°C
3	20	197.5	0.6276	25°C
4	30	197.5	0.9089	25°C
5	40	197.5	1.2062	25°C
6	50	197.5	1.4830	25°C
7	60	197.5	1.7446	25°C

 

The absorbance and concentrations were recorded at 197.5 nm.

 

Table 2: 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	Blank	0.001	0.046	0.002	0.076
2	8	0.259	8.705	0.245	8.235
3	13.6	0.408	13.702	0.373	12.522
4	23.12	0.672	22.544	0.608	20.419
5	39.304	1.205	40.452	0.972	32.639
6	66.817	0.41*	68.81	0.385*	64.57

 

Note: Note: Dilutions made wherever marked *. Concentration was calculated manually.

 

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 No 3: 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	5000	15000	45000	15000	1.1337	1.1220	-
control	5000	15000	40000	135000	1.0986
control	5000	20000	50000	15000	1.1337
Solvent control	5000	20000	45000	110000	1.0303	1.0353	-
Solvent control	5000	15000	45000	115000	1.0452
Solvent control	5000	10000	40000	110000	1.0303
8	5000	15000	35000	100000	0.9986	0.9688	6.400
8	5000	15000	35000	90000	0.9635
8	5000	10000	30000	85000	0.9444
13.6	5000	10000	30000	60000	0.8283	0.8372	19.100
13.6	5000	15000	25000	65000	0.8550
13.6	5000	10000	30000	60000	0.8283
23.12	5000	15000	25000	40000	0.6931	0.7193	30.500
23.12	5000	10000	25000	45000	0.7324
23.12	5000	10000	20000	45000	0.7324
39.304	5000	10000	15000	25000	0.5365	0.5117	50.600
39.304	5000	10000	20000	25000	0.5364
39.304	5000	10000	15000	20000	0.4621
66.817	5000	10000	15000	20000	0.4621	0.4301	58.500
66.817	5000	10000	15000	15000	0.3662
66.817	5000	10000	10000	20000	0.4621

 

Table No 4: pH AND TEMPERATURE

Test Concentration(mg/L)	Experimental Flasks	pH
		0 Hours	72 Hours
control	R1	7.69	8.21
control	R2	7.67	8.26
control	R3	7.72	8.30
Solvent control	R1	7.69	8.36
Solvent control	R2	7.70	8.45
Solvent control	R3	7.68	8.41
8	R1	7.60	7.64
8	R2	7.58	7.74
8	R3	7.63	7.78
13.6	R1	7.70	8.13
13.6	R2	7.68	7.91
13.6	R3	7.65	7.95
23.12	R1	7.91	8.08
23.12	R2	7.98	8.02
23.12	R3	8.01	7.98
39.304	R1	8.10	8.44
39.304	R2	8.13	8.41
39.304	R3	8.10	8.39
66.817	R1	8.09	7.78
66.817	R2	8.12	8.01
66.817	R3	8.14	8.19

 

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.

Validity criteria fulfilled:

yes

Remarks:

* The biomass of the control cultures have increased by factor 16. * The coefficent of variation between replicates was <7 % (reported to be 0.830%) * coefficent of variation in the control % CV of Average specific <35% (i.e., reported to be 21.783 %).

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 > 16.6041 mg/l (nominal conc.) with 95% CI of 14.4327 to 18.8284 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 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 5000 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 100 mg of test chemical in 10 mL of solvent ethanol giving a concentration of 10000 mg/L. 1 mL of this solution was then diluted to 100 mL with OECD medium to get the final concentration of 100 mg/L. This followed the amount of solvent to be used as given in the OECD guideline 201. To have a better growth and visibility of cells, the initial cell count of the culture was kept 5000 cells/mL. Potassium dichromate (K2Cr2O7) was used as a reference substance. Test organisms were in length of 8 – 14 μm and weight of 2 - 3 μm. The cultures of algae were obtained from Microbiotest, Belgium and it is maintained with utmost care in the ESSEM facility at Nagpur. The growth medium used for the culturing of test organism is OECD Medium; which was prepared in ultra-pure, autoclaved water prior to any use. The temperatures were maintained at 21 to 24°C, controlled at ± 2°C. The Light intensity provided to the culture, it is measured each time and should be 4440  - 8880  LUX at the surface and a continuous light phase is maintained for the test duration. Pre-culture was set up two days prior to initiation of the test. It is grown under identical exposure conditions. A range finding study was conducted prior to main study with the test concentrations of 0.1, 1, 10, and 100 mg/L of test item along with control and solvent control groups containing OECD medium and test system. The percent inhibition of 11.111, 20.261, 72.549, and 92.81 % were observed at 72 h in the test concentrations of 0.1, 1, 10, and 100 mg/L respectively. Based on the result of range finding test, confirmatory test had was performed. Five test concentrations (number of replicates 03) were selected, which were arranged in geometric series with the factor of 1.7. Test chemical concentrations used for the study were 0 (control) and 8 mg/L, 13.6 mg/L, 23.12 mg/L, 39.304 mg/L and 66.817 mg/L. 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 4440 - 8880 lux. The speed of the orbital shaking incubator was set at a 120 ±10 rpm throughout the study period. The cultures were counted and observed daily with the help of a microscope. All control, solvent control and  test vessel conc. were performed in three replicates. The pH value in control vessels was determined to be in range of  7.67 to 8.30, in solvent control it was 7.68 to 8.45. And in test concentrations vessels the pH was determined to be in range of  7.58 to 8.44. The 72 hr EC50 value of the reference substance (K2Cr2O7) was determined to be 2.8063 mg/l. Percent yield was found to be 18.75, 46.875, 64.063, 82.813, and 87.5 % for test concentration 8, 13.6, 23.12, 39.304, and 66.817 % respectively. The biomass of the control cultures have increased exponentially by a factor of 16 (specific growth rate of 0.92 day-1) within the 72 hr test periods. 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 0.830%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 21.783 %), 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. 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 > 16.6041 mg/l (nominal conc.) ) with 95% CI of 14.4327 to 18.8284 mg/L. Since, the test chemical is readily biodegradable in water, chemical can be considered as non-toxic to aquatic algae and thus can be 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 5000 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 100 mg of test chemical in 10 mL of solvent ethanol giving a concentration of 10000 mg/L. 1 mL of this solution was then diluted to 100 mL with OECD medium to get the final concentration of 100 mg/L. This followed the amount of solvent to be used as given in the OECD guideline 201. To have a better growth and visibility of cells, the initial cell count of the culture was kept 5000 cells/mL. Potassium dichromate (K2Cr2O7) was used as a reference substance. Test organisms were in length of 8 – 14 μm and weight of 2 - 3 μm. The cultures of algae were obtained from Microbiotest, Belgium and it is maintained with utmost care in the ESSEM facility at Nagpur. The growth medium used for the culturing of test organism is OECD Medium; which was prepared in ultra-pure, autoclaved water prior to any use. The temperatures were maintained at 21 to 24°C, controlled at ± 2°C. The Light intensity provided to the culture, it is measured each time and should be 4440  - 8880  LUX at the surface and a continuous light phase is maintained for the test duration. Pre-culture was set up two days prior to initiation of the test. It is grown under identical exposure conditions. A range finding study was conducted prior to main study with the test concentrations of 0.1, 1, 10, and 100 mg/L of test item along with control and solvent control groups containing OECD medium and test system. The percent inhibition of 11.111, 20.261, 72.549, and 92.81 % were observed at 72 h in the test concentrations of 0.1, 1, 10, and 100 mg/L respectively. Based on the result of range finding test, confirmatory test had was performed. Five test concentrations (number of replicates 03) were selected, which were arranged in geometric series with the factor of 1.7. Test chemical concentrations used for the study were 0 (control) and 8 mg/L, 13.6 mg/L, 23.12 mg/L, 39.304 mg/L and 66.817 mg/L. 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 4440 - 8880 lux. The speed of the orbital shaking incubator was set at a 120 ±10 rpm throughout the study period. The cultures were counted and observed daily with the help of a microscope. All control, solvent control and  test vessel conc. were performed in three replicates. The pH value in control vessels was determined to be in range of  7.67 to 8.30, in solvent control it was 7.68 to 8.45. And in test concentrations vessels the pH was determined to be in range of  7.58 to 8.44. The 72 hr EC50 value of the reference substance (K2Cr2O7) was determined to be 2.8063 mg/l. Percent yield was found to be 18.75, 46.875, 64.063, 82.813, and 87.5 % for test concentration 8, 13.6, 23.12, 39.304, and 66.817 % respectively. The biomass of the control cultures have increased exponentially by a factor of 16 (specific growth rate of 0.92 day-1) within the 72 hr test periods. 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 0.830%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 21.783 %), 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. 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 > 16.6041 mg/l (nominal conc.) ) with 95% CI of 14.4327 to 18.8284 mg/L. Since, the test chemical is readily biodegradable in water, chemical can be considered as non-toxic to aquatic algae and thus can be considered to be "not classified' as per the CLP classification criteria. 

Key value for chemical safety assessment

EC50 for freshwater algae:

16.604 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 5000 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 100 mg of test chemical in 10 mL of solvent ethanol giving a concentration of 10000 mg/L. 1 mL of this solution was then diluted to 100 mL with OECD medium to get the final concentration of 100 mg/L. This followed the amount of solvent to be used as given in the OECD guideline 201. To have a better growth and visibility of cells, the initial cell count of the culture was kept 5000 cells/mL. Potassium dichromate (K2Cr2O7) was used as a reference substance. Test organisms were in length of 8 – 14 μm and weight of 2 - 3 μm. The cultures of algae were obtained from Microbiotest, Belgium and it is maintained with utmost care in the ESSEM facility at Nagpur. The growth medium used for the culturing of test organism is OECD Medium; which was prepared in ultra-pure, autoclaved water prior to any use. The temperatures were maintained at 21 to 24°C, controlled at ± 2°C. The Light intensity provided to the culture, it is measured each time and should be 4440  - 8880  LUX at the surface and a continuous light phase is maintained for the test duration. Pre-culture was set up two days prior to initiation of the test. It is grown under identical exposure conditions. A range finding study was conducted prior to main study with the test concentrations of 0.1, 1, 10, and 100 mg/L of test item along with control and solvent control groups containing OECD medium and test system. The percent inhibition of 11.111, 20.261, 72.549, and 92.81 % were observed at 72 h in the test concentrations of 0.1, 1, 10, and 100 mg/L respectively. Based on the result of range finding test, confirmatory test had was performed. Five test concentrations (number of replicates 03) were selected, which were arranged in geometric series with the factor of 1.7. Test chemical concentrations used for the study were 0 (control) and 8 mg/L, 13.6 mg/L, 23.12 mg/L, 39.304 mg/L and 66.817 mg/L. 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 4440 - 8880 lux. The speed of the orbital shaking incubator was set at a 120 ±10 rpm throughout the study period. The cultures were counted and observed daily with the help of a microscope. All control, solvent control and  test vessel conc. were performed in three replicates. The pH value in control vessels was determined to be in range of  7.67 to 8.30, in solvent control it was 7.68 to 8.45. And in test concentrations vessels the pH was determined to be in range of  7.58 to 8.44. The 72 hr EC50 value of the reference substance (K2Cr2O7) was determined to be 2.8063 mg/l. Percent yield was found to be 18.75, 46.875, 64.063, 82.813, and 87.5 % for test concentration 8, 13.6, 23.12, 39.304, and 66.817 % respectively. The biomass of the control cultures have increased exponentially by a factor of 16 (specific growth rate of 0.92 day-1) within the 72 hr test periods. 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 0.830%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 21.783 %), 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. 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 > 16.6041 mg/l (nominal conc.) ) with 95% CI of 14.4327 to 18.8284 mg/L. Since, the test chemical is readily biodegradable in water, chemical can be considered as non-toxic to aquatic algae and thus can be considered to be "not classified' as per the CLP classification criteria.