Ferrate(4-), hexakis(cyano-C)-, methylated 4-[(4-aminophenyl)(4-imino-2,5-cyclohexadien-1-ylidene)methyl]benzenamine copper(2+) salts

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 according to the guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The test item was prepared by dissolving 1 gm of test chemical in 1000 ml of BBM to get the final concentration of 5.5358 mg/L. This stock solution was kept for stirring for 72 hr to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test chemical. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 10000cells/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
- Source (laboratory, culture collection): laboratory for Biological Research in Aquatic Pollution (LABRAP) at the university of Ghent in Belgium
- 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

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 °C±2°C

Nominal and measured concentrations:

0, 0.3, 0.39, 0.507, 0.659, 0.856 and 1.112 mg/l. All the six concentration were in geometric series spaced by a factor of 1.3

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 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: Yes
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination(1500Lux)

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
- 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 1.3.
- Test concentrations: Six test concentration were: 0, 0.3, 0.39, 0.507, 0.659, 0.856 and 1.112 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 (3000-4000Lux).
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):

no

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.988 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: calculated graphically through probit analysis

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.988 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, round and green throughout the test duration in the control and in the experimental flask also no significant changes were observed.

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

Test vessels and	24 Hours	48 Hours	72 Hours
test concentration
Control
Replicate1	52670	113940	165665
Replicate2	53370	105550	164945
Replicate3	51390	110550	169650
Test chemical
0.3mg/l
Replicate1	47375	114000	164000
Replicate2	46300	106300	159710
0.39mg/l
Replicate1	42805	106300	162500
Replicate2	44510	101745	161500
0.507mg/l
Replicate1	42415	97725	145600
Replicate2	41055	96650	144550
0.659mg/l
Replicate1	33315	95520	130270
Replicate2	30495	93475	133830
0.856mg/l
Replicate1	28225	90060	133300
Replicate2	24840	86220	126500
1.112mg/l
Replicate1	27485	84050	126500
Replicate2	25545	81330	125600

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

	CONTROL		0.3 MG/L		0.39 MG/L		0.507 MG/L		0.659 MG/L		0.856 MG/L		1.112 MG/L
Average Specific Growth rate (μ )	R1	0.94	R1	0.93	R1	0.93	R1	0.89	R1	0.86	R1	0.86	R1	0.84
	R2	0.93	R2	0.92	R2	0.93	R2	0.89	R2	0.86	R2	0.85	R2	0.84
	R3	0.94
Mean of Avg. Specific growth rate	0.94		0.93		0.93		0.86		0.86		0.85		0.84
Percentage Inhibition (%I)	-		1.06		1.03		4.95		8.29		8.89		10.43

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

Test vessels and	0 Hours	72 Hours
test concentration
CONTROL
Replicate1	7.72	8.18
Replicate2	7.08	8.90
Replicate3	7.80	8.10
Average	7.53	8.39
Test chemical
0.3mg/l
Replicate1	8.11	8.62
Replicate2	8.01	6.73
0.39mg/l
Replicate1	7.53	8.37
Replicate2	7.62	8.14
0.507mg/l
Replicate1	8.21	8.03
Replicate2	8.02	7.54
0.659mg/l
Replicate1	7.94	8.08
Replicate2	8.01	7.45
0.856mg/l
Replicate1	7.12	6.65
Replicate2	7.20	6.15
1.112mg/l
Replicate1	6.91	6.46
Replicate2	6.50	7.01

Validity criteria fulfilled:

yes

Conclusions:

Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the EC10 and EC50 value was determined to be 0.9876 and > 0.9876 mg/l.

Executive summary:

This study was designed to assess the toxic effects of the test compound on the freshwater green alga Pseudokirchneriella subcapitata. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). To have a better growth and visibility of cells, the initial cell density of the culture was kept 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. 0.3mg/l, 0.39mg/l, 0.507mg/l, 0.659mg/l, 0.856mg/l, 1.112mg/l concentrations were prepared. All the six concentration were in geometric series spaced by a factor of 1.3. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. 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) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For 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. By 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. The test was considered valid as conducted according to the OECD guideline, 201. Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the EC10 and EC50 value was determined to be 0.9876 and > 0.9876 mg/l through graphically probit analysis. Thus, test chemical can be considered as toxic to aquatic algae at environmental relevant concentrations.

Description of key information

This study was designed to assess the toxic effects of the test compound on the freshwater green alga Pseudokirchneriella subcapitata (Experimental study report, 2018). Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). To have a better growth and visibility of cells, the initial cell density of the culture was kept 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. 0.3mg/l, 0.39mg/l, 0.507mg/l, 0.659mg/l, 0.856mg/l, 1.112mg/l concentrations were prepared. All the six concentration were in geometric series spaced by a factor of 1.3. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. 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) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For 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. By 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. The test was considered valid as conducted according to the OECD guideline, 201. Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the EC10 and EC50 value was determined to be 0.9876 and > 0.9876 mg/l through graphically probit analysis. Thus, test chemical can be considered as toxic to aquatic algae at environmental relevant concentrations.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.988 mg/L

Additional information

This study was designed to assess the toxic effects of the test compound on the freshwater green alga Pseudokirchneriella subcapitata (Experimental study report, 2018). Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). To have a better growth and visibility of cells, the initial cell density of the culture was kept 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. 0.3mg/l, 0.39mg/l, 0.507mg/l, 0.659mg/l, 0.856mg/l, 1.112mg/l concentrations were prepared. All the six concentration were in geometric series spaced by a factor of 1.3. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. 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) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For 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. By 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. The test was considered valid as conducted according to the OECD guideline, 201. Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the EC10 and EC50 value was determined to be 0.9876 and > 0.9876 mg/l through graphically probit analysis. Thus, test chemical can be considered as toxic to aquatic algae at environmental relevant concentrations.