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

Endpoint summary

Description of key information

Short term toxicity to fish

An acute toxicity to fish study was carried out for 96 hr for evaluating the effect of test chemical (Experimental study report, 1989). The study was conducted as per EEC-Guideline L251 Vol. 27, 84/449/EEC: C. Zebra-Fish (Brachydanio rerio) of length 23 mm (19-28 mm), weight of 0.13 g (0.07-0.23 g) was used as a test organism. Test organism was acclimated for a period of 33 days. Feeding was not done during the study and fishes were also not fed with food 24 hours prior to exposure. Fishes (total 10 fish/conc.) were exposed to different concentrations (3.2, 5.8, 10, 18, 32, 58 (mg/l) of test chemical in test aquaria. Study was performed under static conditions at a temperature of 22±1°C, hardness of 160 mg/l CaC03 for 96 hrs under fluorescent light, 16 hours daily. The LC-50 values were calculated according to BERKSON, JASA 48 <1953), LC-values were graphically determined on gausso-logarithmic probability paper. No mortalities were observed in the control vessel. On the basis of effect of test chemical on mortality of the test organism, the 96 hr LC0 and LC100 value was determined to be 5.8 and > 58 mg/l, respectively. On the other hand, 96 hr median lethal concentration (LC50) was determined to be 12 mg/l determined graphically and 14 mg/l with 95% C. I. - 7.1-21 mg/l, respectively. Thus, test chemical was considered as toxic and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Long term toxicity to fish

Based on the prediction done using ECOSAR version 1.1, the long term toxicity on fish was predicted for test substance. On the basis of effects observed in a freshwater system, the NOEC value for the test substance is estimated to be 3 mg/l for fish for 28 days of exposure. Based on the NOEC value it can be concluded that the test chemical was considered as non toxic to fish.

Short term toxicity to aquatic invertebrates

An acute immobilisation test was carried out for assessing the effect of test chemical on aquatic invertebrates (Experimental study report. 2002). The study was performed following the principles of the EEC Directive 93/32/EEC (December 1992) method. Daphnia magna Straus (Water flea) was used as a test organism. The test substance was stirred in M4-medium for about 20 hours. Undisolved test substance was recoved by centrifugation (approx. 20 min about 17700 G). The result is a clear solution. The study was performed with an aqueous extract (eluate) of the test substance. Daphnids were exposed to different test chemical conc. (0, 0.01, 0.1, 1, 10 and 100 mg/l) for 48 hrs. Swimming ability of the daphina after 24 and 48 h; O2-content and pH-values at the begin and end of the exposure period were measured. Based on the effect of test chemical on mobility of the test organism, the 48 hr EC0, EC50 and EC100 value was determined to be 1, 6.5 and 100 mg/l, respectively. Thus, test chemical was considered as toxic and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.

Long term toxicity to aquatic invertebrates

Based on the prediction done using ECOSAR, the long term toxicity on aquatic invertebrate Daphnia magna was predicted for test substance. Study conducted under the flow-through system. The NOEC value for the test substance is estimated to be1.9 mg/l for aquatic invertebrate for 21 days of exposure. Based on the NOEC value it can be concluded that the test chemical was considered as non toxic to aquatic invertebrate at environmentally relevant concentrations.

Toxicity to aquatic algae and cyanobacteria

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.

Toxicity to microorganisms

Toxicity of test chemical was studied on microorganisms (Experimental study report, 1989). The study was performed according to EEC method. Activated sludge collected from the sewage treatment plant of CH-4106 Therwil on 16/10/89 was used as a test bacteria. The preparation was carried out according to the method described in the guideline. The sludge was separated from the aqueous layer by settling instead of centrifugation. The sludge concentration in the test bottles was 1.6 g/L. 21.74, 7.21, 2.93, 1 and 0.25 mg of the test substance were weighed and added to the test medium. The volume was adjusted to 200 ml with water and aerated for 3 hours. Study was carried out in a 250 ml BOD flasks with gas inlet at a temperature of 20 ± 2°C for 3 hrs. 3,5-Dichlorophenol was used as a reference substance. The effects were observed on the respiration of aerobic waste water bacteria for 3 hours. Graphically determined IC50 value for the reference substance was evaluated to be 14 mg/l. Inhibitory concentrations for respiration of the bacteria were graphically determined based on nominal concentrations. Thus, the IC20, IC50 and IC80 value was determined to be 5, 87 and > 100 mg/l, respectively.

Additional information

Short term toxicity to fish

An acute toxicity to fish study was carried out for 96 hr for evaluating the effect of test chemical (Experimental study report, 1989). The study was conducted as per EEC-Guideline L251 Vol. 27, 84/449/EEC: C. Zebra-Fish (Brachydanio rerio) of length 23 mm (19-28 mm), weight of 0.13 g (0.07-0.23 g) was used as a test organism. Test organism was acclimated for a period of 33 days. Feeding was not done during the study and fishes were also not fed with food 24 hours prior to exposure. Fishes (total 10 fish/conc.) were exposed to different concentrations (3.2, 5.8, 10, 18, 32, 58 (mg/l) of test chemical in test aquaria. Study was performed under static conditions at a temperature of 22±1°C, hardness of 160 mg/l CaC03 for 96 hrs under fluorescent light, 16 hours daily. The LC-50 values were calculated according to BERKSON, JASA 48 <1953), LC-values were graphically determined on gausso-logarithmic probability paper. No mortalities were observed in the control vessel. On the basis of effect of test chemical on mortality of the test organism, the 96 hr LC0 and LC100 value was determined to be 5.8 and > 58 mg/l, respectively. On the other hand, 96 hr median lethal concentration (LC50) was determined to be 12 mg/l determined graphically and 14 mg/l with 95% C. I. - 7.1-21 mg/l, respectively. Thus, test chemical was considered as toxic and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Long term toxicity to fish

Based on the prediction done using ECOSAR version 1.1, the long term toxicity on fish was predicted for test substance. On the basis of effects observed in a freshwater system, the NOEC value for the test substance is estimated to be 3 mg/l for fish for 28 days of exposure. Based on the NOEC value it can be concluded that the test chemical was considered as non toxic to fish.

Short term toxicity to aquatic invertebrates

Various experimental studies of the test chemical were reviewed for short term toxicity to aquatic invertebrate end point which are summarized as below:

 

In an experimental study from study report (2002), an acute immobilisation test was carried out for assessing the effect of test chemical on aquatic invertebrates. The study was performed following the principles of the EEC Directive 93/32/EEC (December 1992) method. Daphnia magna Straus (Water flea) was used as a test organism. The test substance was stirred in M4-medium for about 20 hours. Undisolved test substance was removed by centrifugation (approx. 20 min about 17700 G). The result is a clear solution. The study was performed with an aqueous extract (eluate) of the test substance. Daphnids were exposed to different test chemical conc. (0, 0.01, 0.1, 1, 10 and 100 mg/l) for 48 hrs. Swimming ability of the daphina after 24 and 48 h; O2-content and pH-values at the beginning and end of the exposure period were measured. Based on the effect of test chemical on mobility of the test organism, the 48 hr EC0, EC50 and EC100 value was determined to be 1, 6.5 and 100 mg/l, respectively. Thus, test chemical was considered as toxic and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.

 

Another short term toxicity to aquatic invertebrate study was carried out for assessing the effect of test chemical (2002). The study was performed following the principles of the EEC Directive 93/32/EEC (December 1992) method. Daphnia magna Straus (Water flea) was used as a test organism. The test substance was stirred in M4-medium for about 20 hours. Undisolved test substance was removed by centrifugation (approx. 20 min about 17700 G). The result is a clear solution. The study was performed with an aqueous extract (eluate) of the test substance. Daphnids were exposed to different test chemical conc. (0, 0.01, 0.1, 1, 10 and 100 mg/l) for 48 hrs. Swimming ability of the daphina after 24 and 48 h; O2-content and pH-values at the beginning and end of the exposure period were measured. Based on the effect of test chemical on mobility of the test organism, the 48 hr EC0, EC50 and EC100 value was determined to be 1, 10.8 and 100 mg/l, respectively. Thus, test chemical was considered as toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

 

In a supporting study, short term toxicity of test chemical was studied on invertebrates. No stock solution was prepared (Experimental study report, 1989). The test substance was added directly to the water in the tanks. Test conducted on Daphnia magna Straus 1820 obtained from CIBA-GEIGY Ltd., Facilities, Basel. Cultures of daphnia are maintained in glass vessels containing approx. 2.5 L of reconstituted water at 20 ± 1°C (water is renewed partially thrice weekly). At each renewal the daphnia are fed a suspension of green algae (Scenedesmus subspicatus) supplemented by a suspension of TETRAMIN-extract in such quantities that the food is consumed after 24h. ≤24h before the start of the test, reproductive daphnia are separated from the young by sieving all individuals through a 800 um seive. This operation is repeated 6h before the start of the test and the young (6-≤24h of age) are retained for the test. Total 20 daphnia per concentration and control 4 replicates of 5 daphnia each were exposed with the test chemical conc. (0.0010, 0.0018, 0.0032, 0.0058, 0.010 0.018, 0.032, 0.058, 0.010 mg/l) in a test beakers covered with watch glasses. The study was performed under static conditions at hardness of 240 mg CaC03/L., temperature of 20 ± 1°C under fluorescent light of approx. 1500 lux, 16 hours daily for 24 hrs. The EC-50 values were calculated according to the maximum likelihood method, probit model (Mc Cullagh, P., Nelder, J.A., 1983: Generalized linear models, Chapman & Hall, London) EC-values were graphically determined on gausso-logarithmic probability paper. The 24 hour EC50 value of test chemical in Daphnia magna based on the effect of immobilisation was determined graphically as 0.088 mg/l and calculated as 0.15 mg/l (95% confidence limits 0.085-0.52 mg/l). The 24 hr EC0 and EC100 was evaluated to be 0.018 mg/l and >0.10 mg/l, respectively.

 

On the basis of the above results, it can be concluded that the test chemical was considered as toxic to aquatic invertebratesat environmental relevant concentrations and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.

Long term toxicity to aquatic invertebrates

Based on the prediction done using ECOSAR, the long term toxicity on aquatic invertebrate Daphnia magna was predicted for test substance. Study conducted under the flow-through system. The NOEC value for the test substance is estimated to be1.9 mg/l for aquatic invertebrate for 21 days of exposure. Based on the NOEC value it can be concluded that the test chemical was considered as non toxic to aquatic invertebrate at environmentally relevant concentrations.

Toxicity to aquatic algae and cyanobacteria

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.

Toxicity to microorganisms

Toxicity of test chemical was studied on microorganisms (Experimental study report, 1989). The study was performed according to EEC method. Activated sludge collected from the sewage treatment plant of CH-4106 Therwil on 16/10/89 was used as a test bacteria. The preparation was carried out according to the method described in the guideline. The sludge was separated from the aqueous layer by settling instead of centrifugation. The sludge concentration in the test bottles was 1.6 g/L. 21.74, 7.21, 2.93, 1 and 0.25 mg of the test substance were weighed and added to the test medium. The volume was adjusted to 200 ml with water and aerated for 3 hours. Study was carried out in a 250 ml BOD flasks with gas inlet at a temperature of 20 ± 2°C for 3 hrs. 3,5-Dichlorophenol was used as a reference substance. The effects were observed on the respiration of aerobic waste water bacteria for 3 hours. Graphically determined IC50 value for the reference substance was evaluated to be 14 mg/l. Inhibitory concentrations for respiration of the bacteria were graphically determined based on nominal concentrations. Thus, the IC20, IC50 and IC80 value was determined to be 5, 87 and > 100 mg/l, respectively.

On the basis of the available information of aquatic toxicity studies, it can be concluded that the test chemical was considered as toxic to aquatic organisms at environmental relevant concentrations andhence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.