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Ecotoxicological information

Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
Ethanedinitrile, hydrogen cyanide (HCN), potassium cyanide and sodium cyanide can be considered as a chemical category, along with and acetone cyanohydrin (ACH, also known as 2-hydroxy-2-methylpropanenitrile), based on structural similarity, common breakdown/metabolic products in physical and biological systems, and similar physico-chemical properties. Particular attention is paid to the dissociation constant of HCN. Ethanedinitrile breaks down in aqueous solution into cyanide ion (CN-) and cyanate ion (OCN-) (Cotton and Wilkinson 1980). Ethanedinitrile due to its low log Kow (0.07) and relatively high solubility in water (2.34 g/L) needs to get dissolved in aqueous solutions in lungs to enter the body. The rate of hydrolysis of ethanedinitrile is very fast (Ajwa 2015). Also, in the vast majority of environmental and physiologic conditions, the cyanide salts will dissolve in water to form hydrogen cyanide. The physico-chemical hazards and toxicity therefore result from the activity of HCN. An ECETOC Task Force, in the 2007 ECETOC Joint Assessment of Commodity Chemicals (JACC) Report No. 53, “Cyanides of Hydrogen, Sodium and Potassium, and Acetone Cyanohydrin (CAS No. 74-90-8, 143-33-9, 151-50-8 and 75-86-5)” supports the development of the chemical category inclusive hydrogen cyanide, sodium and potassium cyanides. Hydrogen cyanide (Index No.006-006-00-X) and salts of hydrogen cyanides (Index No.006-007-00-5) are both listed in Annex VI, Table 3.1 of Regulation (EC) No. 1272/2008, entry 006-007-00-5, and are restricted in comparable ways taking into account physical characteristics. Thus, the assignment of ethanedinitrile to a chemical category does not result in a less protective regulatory status.
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 203 Fish, Acute toxicity test
Deviations:
yes
Remarks:
Resembling the guideline with broadened scope of research by inclusion of juvenile, fry and egg stages
GLP compliance:
no
Remarks:
Not reported, but performed (1978) by a prestigious US leading public associations using standardized methods
Test organisms (species):
other: see below
Details on test organisms:
1. Fathead minnow, Pimephales promelas (Rafinesque)
2. Bluegill, Lepomis macrochirus (Rafinesque)
3. Rainbow trout, Oncorhynchus mykiss, formerly Salmo gairdneri (Richardson).
Test type:
flow-through
Water media type:
freshwater
Total exposure duration:
96 h
Test temperature:
4.0 - 30.0 °C
pH:
6.0 - 8.0
Dissolved oxygen:
3.36 - 9.26 mg/l
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.121 - < 0.352 mg/L
Basis for effect:
mortality
Remarks:
Fathead minnow eggs
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.081 - < 0.122 mg/L
Basis for effect:
mortality
Remarks:
Fathead minnow swim-up fry
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 0.082 - < 0.137 mg/L
Basis for effect:
mortality
Remarks:
Fathead minnow juveniles
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.58 - < 0.935 mg/L
Basis for effect:
mortality
Remarks:
Bluegill eggs
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.232 - < 0.371 mg/L
Basis for effect:
mortality
Remarks:
Bluegill swim-up fry
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.075 - < 0.125 mg/L
Basis for effect:
mortality
Remarks:
Bluegill juveniles
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.057 mg/L
Basis for effect:
mortality
Remarks:
Oncorhynchus mykiss
Details on results:
95% confidence limit was 0.0557 – 0.0587 mg. L-1

 










Acute toxicity varied from 0.057 mg. L-1for juvenile rainbow trout to 0.191 mg. L-1for field stocks of juvenile fathead minnows. Juvenile fish were more sensitive at lower temperatures and at oxygen levels below 5 mg. L-1. The difference in median lethal concentration between field stock fathead minnows, the most resistant species tested, and rainbow trout juveniles was approximately threefold. Eggs of all tested species were the most resistant life stage. Slope of the log-probit toxicity curves is smallest for egg tests and increases to that for juveniles.




 










Table8.2.2.1 – 3. Acute toxicity of HCN to fathead minnow eggs expressed as 96-hour LC50and median lethal concentrations at hatching














































































°C



DO mg.L-1



pH



96-hour LC50


mg.L-1



95%


Confidence limits



Hatch


LC50


mg.L-1



99%


Confidence limits



15.2



6.36



7.86



0.352



274-453



0.126



90.9-174



20.0



6.13



7.88



0.273



162-463



0.118



97.3-142



24.9



3.51



7.72



0.202



130-314



0.116



86.6-157



24.8



4.46



7.95



0.121



77.3-190



0.113



83.0-154



25.0



5.52



7.90



0.184



115-293



0.180



122-266



25.0



6.34



8.00



0.196



140-274



0.162



135-193



24.9



7.25



7.99



0.202



-



0.187



-



 


Table8.2.2.1 – 4.: Acute toxicity of HCN to fathead minnow swim-up fry expressed as 96-hour LC50and median lethal threshold concentrations




























































°C



DO mg.L-1



pH



96-hour LC50


mg. L-1



95%


Confidence limits



LTC


mg. L-1



95%


Confidence limits



15.0



6.38



7.86



0.122



104-143



0.102



92.8-113



20.0



6.14



7.89



0.0991



88.9-111



0.0961



83.6-110



24.6



3.77



7.84



0.0816



71.2-93.6



0.0816



71.2-93.6



24.7



5.14



7.96



0.108



90.3-130



0.108



90.3-130



24.9



6.17



8.02



0.113



96.5-133



0.113



96.5-133



 


Table8.2.2.1 – 5.: Acute toxicity of HCN to fathead minnow juveniles expressed as 96-hour LC50and median lethal threshold concentrations









































































































°C



DO mg.L-1



pH



96-hour LC50


mg. L-1



95%


Confidence limits



LTC


mg. L-1



95%


Confidence limits



15.0



6.07



7.86



0.121



116-125



0.119



115-123



20.0



3.58



7.70



0.128



109-149



0.123



105-143



19.8



4.68



7.80



0.0824



76.4-88.9



0.0824



76.4-88.9



20.0



5.20



7.78



0.125



117-133



0.123



116-132



20.0



6.07



7.91



0.137



122-153



0.137



122-153



20.0



7.13



7.90



0.131



124-138



0.131



124-138



24.8



3.58



7.75



0.106



87.9-129



0.106



87.9-129



25.0



5.08



7.83



0.119



111-129



0.119



111-129



25.1



6.13



7.98



0.129



124-133



0.129



124-133



25.2



7.04



7.96



0.120



113-128



0.120



113-138



 


Table8.2.2.1 – 6.: Acute toxicity of HCN to bluegill eggs and swim-up fry expressed as 96-hour LC50and median lethal threshold of hatching concentrations


















































































































°C



DO mg.L-1



pH



96-hour LC50


mg. L-1



95%


Confidence limits



LTC


mg. L-1



95%


Confidence limits



Eggs



 



 



 



 



 



 



25.2



3.39



7.70



-



-



0.690



461-1033



25.0



4.99



7.79



-



-



0.535



240-1192



25.1



6.09



7.92



-



-



0.693



572-841



25.0



6.90



7.90



-



-



0.580



343-980



Fry



 



 



 



 



 



 



20.0



5.99



7.89



0.365



188-709



0.205



156-270



24.9



3.59



7.72



0.232



147-366



0.109



99.9-120



24.9



5.08



7.80



0.232



147-366



0.149



117-189



24.9



6.01



7.93



0.276



241-316



0.218



193-247



24.8



6.81



7.90



0.271



200-368



0.194



110-340



 


Table 8.2.2.1 –7.: Acute toxicity of HCN to bluegill juveniles expressed as 96-hour LC50and median lethal threshold of hatching concentrations









































































































°C



DO mg.L-1



pH



96-hour LC50


mg. L-1



95%


Confidence limits



LTC


mg. L-1



95%


Confidence limits



8.4



6.08



7.80



0.083



-



0.0617



59.3-64.2



9.7



8.35



7.94



<0.092



-



-



-



15.0



6.07



7.83



0.087



81.0-93.8



0.0871



81.0-93.8



15.1



7.03



7.92



0.075



66.0-85.2



 



 



17.8



7.97



8.12



0.099



-



 



 



20.0



6.06



7.86



0.108



103-112



0.108



103-112



25.1



3.48



7.71



0.0997



86.0-116



0.0997



86.0-116



25.0



5.05



7.78



0.113



55.8-227



0.113



55.8-227



24.9



6.17



7.92



0.120



109-133



0.120



109-133



24.9



6.90



7.86



0.125



115-135



0.125



115-135



 


 


 


Table 8.2.2.1 –8.: Acute toxicity of HCN to Oncorhynchus mykiss juveniles expressed as 96-hour LC50and median lethal threshold concentrations
























°C



DO mg.L-1



pH



96-hour LC50


mg. L-1



95%


Confidence limits



LTC


mg. L-1



95%


Confidence limits



10



8.80



7.80



0.0572



55.7-58.7



-



-



 


Table 8.2.2.1 –9.: Effect data














Parameter



96 hours



LC50[mg.L-1]*



Fathead minnow eggs : from 0.121 to 0.352 mg. L-1


Fathead minnow swim-up fry: from 0.081 to 0.122 mg. L-1


Fathead minnow juveniles: from 0.082 to 0.137 mg. L-1


Bluegill eggs: from 0.580 to 0.935 mg. L-1(expresed as LTC)


Bluegill swim-up fry: from 0.232 to 0.371 mg. L-1


Bluegill juveniles: from 0.075 to 0.125 mg. L-1


Oncorhynchus mykiss: 57.2 μg. L-1(95% confidence limit was 0.0557 – 0.0587 mg. L-1)













*Based on the mean measured concentrations.



Conclusions:
For most species, juveniles were most sensitive and eggs most resistant to HCN. Temperature has a significant effect on acute toxicity of HCN with juvenile fish in general becoming more sensitive at lower temperatures. Oxygen below 5 mg.l-L results in increased sensitivity for all tested juveniles.
Long-term and chronic toxicity to fish:
Exposure of aquatic organisms is not expected as the fumigant is used for treatment of wood in enclosed spaces. The only emission of the active substance to the air will occur during ventilation of the fumigated wood. The substance will be dispatched in the air and will get quickly diluted due to its high volatility. The substance will stay in the air based on its physical and chemical properties and will not transfer to other environmental compartments such as soil and water as confirmed by models in the environmental fate part of dossier (10.3 Fate and behaviour in air).
Fish early life stage toxicity test:
Exposure of aquatic organisms is not expected as the fumigant is used for treatment of wood in enclosed spaces. The only emission of the active substance to the air will occur during ventilation of the fumigated wood. The substance will be dispatched in the air and will get quickly diluted due to its high volatility. The substance will stay in the air based on its physical and chemical properties and will not transfer to other environmental compartments such as soil and water as confirmed by models in the environmental fate part of dossier (10.3 Fate and behaviour in air).
Executive summary:

Materials and methods


Different stages used for acute tests were eggs, fry and juveniles. Eggs and fry were randomly placed in test chambers with 20 litres of test solution; eggs and fry were tested on screen bottomed acrylic cylinders covered with a bakelite lid and held in a 20-liter chamber. Sodium cyanide from the stock solution was delivered to test chambers from the interminent-flow diluters. NaCN hydrolyses to form free cyanide – CN-ion and molecular HCN. At pH 6.0 – 8.0 in most natural water the molecular (un-ionized) HCN predominates with less than 6 % free cyanide occurring in the ionic form below pH 8 at 25 °C. Eggs and fry were tested immediately on introduction to the test chambers; juveniles were held in test chambers for three days prior to HCN expose. Free cyanide concentrations in each chamber were determined daily.


Observations on mortality were made daily. Acute toxicity of hydrogen cyanide was determined at temperatures from 4 °C to 30 °C and oxygen concentrations from 3.36 to 9.26 mg. L-1on different species life stages, with single water source and using uniform test procedures. 337 acute toxicity tests were designed to determine the 96-hour median lethal HCN concentration (LC50) and the median lethal threshold concentration (LTC).


Free cyanide concentration in each chamber was determined daily by the spectrophotometric method according to Epstein with calculated HCN concentrations based on corresponding pH and temperature measurements and using the dissociation constants of molecular HCN. This method was used as standard method for examination of water by the renowned US leading public associations.


 


Table 8.2.2.1 – 1.: Test organisms






















Criteria



Details



Species/strain



1. Fathead minnow,Pimephales promelas(Rafinesque)


2. Bluegill,Lepomis macrochirus(Rafinesque)


3. Rainbow trout,Oncorhynchus mykiss, formerlySalmo gairdneri (Richardson).



Source



1. Fathead minnow were cultured in test laboratory from brood stock originally obtained from the U.S. EPA´s ERL, Duluth. Juvenile wild-stock fatheads were collected from Como Lake in St. Paul.


2. Bluegill was obtained from wild stock with eggs spawned and fry hatched in the laboratory.


Juvenile bluegills were collected from local waters.


3.Oncorhynchus mykisswere obtained as newly hardened eggs or as 24-hour fry from state hatcheries.



Age/size



1. Sac fry, length 5-; Swim-up, length 5-, Juveniles, length 26-


2. Sac fry, length; Swim-up, length, Juveniles, length 13-


3. Juveniles, length 40 -68 mm


 



 














Pre-treatment



Juveniles held at test conditions for 7 days before being placed in the test chambers. Juveniles from the field were given prophylactic treatment with neomycin and tetracycline at 20 mg. L-1for 4-hour periods on 3 consecutive days.



Feeding of animals during test



No



 


Table 8.2.2.1 – 2.: Test system



























Criteria



Details



Test type



Flow-through



Volume of test vessels



Test chambers for juveniles and trout swim-up were glass aquaria 50x24x20 cc high filled with 20 litres of test solution.


Eggs and fry were tested in screen bottomed acrylic cylinders each covered with a bakelite lid and held in a 20-liter chamber - the portion of water from each cycle flowed upward through the screen to the outlet.



Number of animals/vessel



1. 25-50 eggs; 25 sac fry; 25 swim-up fry; 10 juveniles


2. 25-50 eggs; 10-50 sac fry; 10-50 swim-up fry; 10-20 juveniles


3. 10 juveniles



Number of vessels/ concentration



1




Results and discussion:


Acute toxicity varied from 0.057 mg. L-1for juvenile rainbow trout to 0.191 mg. L-1for field stocks of juvenile fathead minnows. Juvenile fish were more sensitive at lower temperatures and at oxygen levels below 5 mg. L-1. The difference in median lethal concentration between field stock fathead minnows, the most resistant species tested, and rainbow trout juveniles was approximately threefold. Eggs of all tested species were the most resistant life stage. Slope of the log-probit toxicity curves is smallest for egg tests and increases to that for juveniles.


LC50       


Fathead minnow eggs : from 0.121 to 0.352 mg.L-1


Fathead minnow swim-up fry: from 0.081 to 0.122 mg. L-1


Fathead minnow juveniles: from 0.082 to 0.137 mg. L-1


Bluegill eggs: from 0.580 to 0.935 mg. L-1(expresed as LTC)


Bluegill swim-up fry: from 0.232 to 0.371 mg. L-1


Bluegill juveniles: from 0.075 to 0.125 mg. L-1


Oncorhynchus mykiss: 0.0572 mg. L-1


(95% confidence limit was 0.0557 – 0.0587 mg. L-1).


Table 8.2.2.1 – 3. Acute toxicity of HCN to fathead minnow eggs expressed as 96-hour LC50and median lethal concentrations at hatching














































































°C



DO mg.L-1



pH



96-hour LC50


mg.L-1



95%


Confidence limits



Hatch


LC50


mg.L-1



99%


Confidence limits



15.2



6.36



7.86



0.352



274-453



0.126



90.9-174



20.0



6.13



7.88



0.273



162-463



0.118



97.3-142



24.9



3.51



7.72



0.202



130-314



0.116



86.6-157



24.8



4.46



7.95



0.121



77.3-190



0.113



83.0-154



25.0



5.52



7.90



0.184



115-293



0.180



122-266



25.0



6.34



8.00



0.196



140-274



0.162



135-193



24.9



7.25



7.99



0.202



-



0.187



-



Table 8.2.2.1 – 4.: Acute toxicity of HCN to fathead minnow swim-up fry expressed as 96-hour LC50and median lethal threshold concentrations




























































°C



DO mg.L-1



pH



96-hour LC50


mg. L-1



95%


Confidence limits



LTC


mg. L-1



95%


Confidence limits



15.0



6.38



7.86



0.122



104-143



0.102



92.8-113



20.0



6.14



7.89



0.0991



88.9-111



0.0961



83.6-110



24.6



3.77



7.84



0.0816



71.2-93.6



0.0816



71.2-93.6



24.7



5.14



7.96



0.108



90.3-130



0.108



90.3-130



24.9



6.17



8.02



0.113



96.5-133



0.113



96.5-133



Table 8.2.2.1 – 5.: Acute toxicity of HCN to fathead minnow juveniles expressed as 96-hour LC50and median lethal threshold concentrations









































































































°C



DO mg.L-1



pH



96-hour LC50


mg. L-1



95%


Confidence limits



LTC


mg. L-1



95%


Confidence limits



15.0



6.07



7.86



0.121



116-125



0.119



115-123



20.0



3.58



7.70



0.128



109-149



0.123



105-143



19.8



4.68



7.80



0.0824



76.4-88.9



0.0824



76.4-88.9



20.0



5.20



7.78



0.125



117-133



0.123



116-132



20.0



6.07



7.91



0.137



122-153



0.137



122-153



20.0



7.13



7.90



0.131



124-138



0.131



124-138



24.8



3.58



7.75



0.106



87.9-129



0.106



87.9-129



25.0



5.08



7.83



0.119



111-129



0.119



111-129



25.1



6.13



7.98



0.129



124-133



0.129



124-133



25.2



7.04



7.96



0.120



113-128



0.120



113-138



Table 8.2.2.1 – 6.: Acute toxicity of HCN to bluegill eggs and swim-up fry expressed as 96-hour LC50and median lethal threshold of hatching concentrations


















































































































°C



DO mg.L-1



pH



96-hour LC50


mg. L-1



95%


Confidence limits



LTC


mg. L-1



95%


Confidence limits



Eggs



 



 



 



 



 



 



25.2



3.39



7.70



-



-



0.690



461-1033



25.0



4.99



7.79



-



-



0.535



240-1192



25.1



6.09



7.92



-



-



0.693



572-841



25.0



6.90



7.90



-



-



0.580



343-980



Fry



 



 



 



 



 



 



20.0



5.99



7.89



0.365



188-709



0.205



156-270



24.9



3.59



7.72



0.232



147-366



0.109



99.9-120



24.9



5.08



7.80



0.232



147-366



0.149



117-189



24.9



6.01



7.93



0.276



241-316



0.218



193-247



24.8



6.81



7.90



0.271



200-368



0.194



110-340



Table 8.2.2.1 – 7.: Acute toxicity of HCN to bluegill juveniles expressed as 96-hour LC50and median lethal threshold of hatching concentrations



















































°C



DO mg.L-1



pH



96-hour LC50


mg. L-1



95%


Confidence limits



LTC


mg. L-1



95%


Confidence limits



8.4



6.08



7.80



0.083



-



0.0617



59.3-64.2



9.7



8.35



7.94



<0.092



-



-



-



15.0



6.07



7.83



0.087



81.0-93.8



0.0871



81.0-93.8



15.1



7.03



7.92



0.075



66.0-85.2



 



 



 


Table 8.2.2.1 – 6.: Acute toxicity of HCN to bluegill eggs and swim-up fry expressed as 96-hour LC50and median lethal threshold of hatching concentrations


















































































































°C



DO mg.L-1



pH



96-hour LC50


mg. L-1



95%


Confidence limits



LTC


mg. L-1



95%


Confidence limits



Eggs



 



 



 



 



 



 



25.2



3.39



7.70



-



-



0.690



461-1033



25.0



4.99



7.79



-



-



0.535



240-1192



25.1



6.09



7.92



-



-



0.693



572-841



25.0



6.90



7.90



-



-



0.580



343-980



Fry



 



 



 



 



 



 



20.0



5.99



7.89



0.365



188-709



0.205



156-270



24.9



3.59



7.72



0.232



147-366



0.109



99.9-120



24.9



5.08



7.80



0.232



147-366



0.149



117-189



24.9



6.01



7.93



0.276



241-316



0.218



193-247



24.8



6.81



7.90



0.271



200-368



0.194



110-340



 Table 8.2.2.1 – 7.: Acute toxicity of HCN to bluegill juveniles expressed as 96-hour LC50and median lethal threshold of hatching concentrations



















































°C



DO mg.L-1



pH



96-hour LC50


mg. L-1



95%


Confidence limits



LTC


mg. L-1



95%


Confidence limits



8.4



6.08



7.80



0.083



-



0.0617



59.3-64.2



9.7



8.35



7.94



<0.092



-



-



-



15.0



6.07



7.83



0.087



81.0-93.8



0.0871



81.0-93.8



15.1



7.03



7.92



0.075



66.0-85.2



 



 



*Based on the mean measured concentrations.


Conclusion        


For most species, juveniles were most sensitive and eggs most resistant to HCN. Temperature has a significant effect on acute toxicity of HCN with juvenile fish in general becoming more sensitive at lower temperatures. Oxygen below 5 mg.l-L results in increased sensitivity for all tested juveniles.


 


Fish full life cycle test


Exposure of aquatic organisms is not expected. The substance will be dispatched in the air and will get quickly diluted due to its high volatility. The substance will stay in the air based on its physical and chemical properties and will not transfer to other environmental compartments such as soil and water as confirmed by models in the environmental fate part of dossier, CA 7.3.2.


Bioconcentration in fish


Exposure of aquatic organisms is not expected. The substance will be dispatched in the air and will get quickly diluted due to its high volatility. The substance will stay in the air based on its physical and chemical properties and will not transfer to other environmental compartments such as soil and water as confirmed by models in the environmental fate part of dossier, CA 7.3.2.


Ethanedinitrile has log Pow=0.07 (CA 2.7) therefore bioaccumulation is not probable.

Description of key information

Acute toxicity varied from 0.057 mg. L-1for juvenile rainbow trout to 0.191 mg. L-1for field stocks of juvenile fathead minnows. Juvenile fish were more sensitive at lower temperatures and at oxygen levels below 5 mg. L-1. The difference in median lethal concentration between field stock fathead minnows, the most resistant species tested, and rainbow trout juveniles was approximately threefold. Eggs of all tested species were the most resistant life stage. Slope of the log-probit toxicity curves is smallest for egg tests and increases to that for juveniles.

Key value for chemical safety assessment

Additional information