Oxalonitrile

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

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.

Guideline:

OECD Guideline 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.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

>= 0.057 - <= 0.057 mg/L

95% CI:

>= 55.7 - <= 58.7

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

>= 0.075 - <= 0.125 mg/L

95% CI:

>= 55.8 - <= 227

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

>= 0.232 - <= 0.365 mg/L

95% CI:

>= 147 - 709

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

other: LTC

Effect conc.:

>= 0.535 - <= 0.693 mg/L

95% CI:

>= 240 - <= 1 192

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

>= 0.082 - <= 0.137 mg/L

95% CI:

>= 76.4 - <= 153

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

>= 0.081 - <= 0.122 mg/L

95% CI:

>= 71.2 - 143

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

>= 0.121 - <= 0.352 mg/L

95% CI:

>= 77.3 - <= 463

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Conclusions:

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).

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 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-
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

The concentration – percent mortality data were analysed with a logarithmic – probability (log-probit) program. LC₅₀and medial lethal threshold concentrations (TLC) were calculated.

 

 

Table 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(expressed 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 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 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 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 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 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.: 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 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(expressed 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.

 

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.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

read-across based on grouping of substances (category approach)

Remarks:

Kovacs 1982

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

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:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

Similar to guideline with broadened scope of research.

Principles of method if other than guideline:

performed by a prestigious leading public associatious using standardized methods

GLP compliance:

not specified

Remarks:

performed by a prestigious leading public associatious using standardized methods

Details on sampling:

Rainbow trout (Oncorhynchus mykiss, formerly known as Salmo gairdneri) purchased form La Pisciculture, Mt. Sutton, Sutton (Qué.), were held in 200-liter fiberglass oval-shaped tanks at a temperature of 12 ± 1°C. Chemical characteristics of the dechlorinated Montreal city water used during the experiments were as follows; alkalinity 87 mg.L-1 as CaCO3; hardness 127 mg.L-1 as CaCO3; CO2 0.47 mg.L-1. See Table 8.2.1 – 3. for oxygen and pH levels.
After at least 2-week acclimation in the 200-liter holding tanks at 12 °C, the fish to be tested at 6 and 18 °C were segregated and acclimated to the desired water temperatures by gradual adjustments of about 1 °C.d-1. The fish were then held for 7 days at these temperatures, prior to transfer into the white polyethylene (68 x 57.5 x 42 cm) test tanks, where they were held for further 2 weeks at the test temperatures (i.e. acclimated for 3 weeks at test temperatures). The tanks were connected to a flow-through system provided with thermally controlled water, at a flow of 1 L.min.-1 in each tank, providing 99% replacement in 6 h. The entire 24-tank assembly was illuminated evenly by fluorescent light, controlled by a time switch to provide a 12-hour photoperiod (8:30-20:30). During this period the fish were fed daily but no food was given for 48 h before the tests.

Vehicle:

no

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Water media type:

freshwater

Total exposure duration:

96 h

Test temperature:

6 °C, 12 °C, 18 °C

Nominal and measured concentrations:

0.018 - 0.056 mg/HCN/l.

Details on test conditions:

Acute toxicity of HCN to rainbow trout at 6, 12, and 18°C. At hour zero a calculated amount of cyanide stock solution was mixed into each tank to immediately produce the predicted concentrations which were then maintained by Mariotte bottles. Cyanide concentration was monitored in the test tanks twice per day. The measured concentrations never differed by more than 1% from the predicted values.
The concentrations tested (mg HCN.L-1) ranged from 0.018 to 0.056 at 6°C, 0.032 to 0.087 at 12°C, and 0.042 to 0.087 at 18°C. These ranges were selected from preliminary tests and included concentrations causing zero and 100% mortality. Control groups were maintained at each temperature under identical conditions.
The acute toxicity tests were carried out to determine the 96-hour LC50 of HCN to rainbow trout at three temperatures, 6, 12 and 18 °C.
The bioassays were carried out in accordance with standard methods. The fish weighed 9-18 g with the mean weight of 12 g, they were randomly chosen, acclimated as described above, and segregated for each test concentration and control group.
On the day the bioassays began, the tanks were cleaned and a calculated amount of cyanide stock solution was mixed into each tank to immediately produce the predicted concentration. This was determined to be zero hour, and the desired concentrations were then maintained by metering cyanide stock solutions from Mariotte bottles.
Cyanide was monitored in the test tanks twice per day and the cyanide flows were adjusted when necessary. The desired concentrations never varied by more than one percent of the predicted values.
The concentrations tested (mg HCN.l-1) ranged from 0.018 to 0.056 at 6 °C, 0.032 to 0.087 at 12 °C, and 0.042 to 0.087 at 18 °C. These ranges were selected empirically such that the approximate 96-hour LC50 values, determined by preliminary screening bioassays would be close to the median of the eight concentrations tested and would include concentrations causing zero and 100% mortality.
Control groups were maintained at each temperature under identical conditions.
Observations on mortality were taken regularly. The criterion of death was the absence of respiratory movements and lack of response to probing with a glass rod upon which the fish were removed, weighed and their fork length measured.

Key result

Duration:

96 h

Dose descriptor:

LC50

Remarks:

6 °C

Effect conc.:

0.02 - 0.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Remarks:

12 °C

Effect conc.:

0.04 - 0.05 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Remarks:

18 °C

Effect conc.:

0.06 - 0.07 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Sublethal observations / clinical signs:

Acute toxicity of hydrogen cyanide to rainbow trout acclimated and tested at different temperatures in flow-through system for 96 hours:

	6 °C	12 °C	18 °C
96-hour LC50mg.L^-1HCN (95% confidence interval)	0.028 (0.024-0.035)	0.042 (0.038-0.046)	0.068 (0.064-0.072)
Slope function	1.30	1.30	1.12
Highest cyanide conc. with no mortality in 96 hours. mg HCN.L^-1	0.018	0.032	0.060
Lowest cyanide conc. with 100% mortality in 96 hours. mg HCN.L^-1	0.037	0.053	0.087
Average weight of fish (range). g.	11.79 (9.05-15.98)	12.41 (8.29-16.02)	13.19 (9.63-17.21)
Average fork length of fish (range). mm	102 (95-110)	102 (90-111)	106 (95-119)
Mean pH (range)	8.06 (7.9-8.1)	8.10 (8.06-8.11)	7.82 (7.78-7.90)
Mean oxygen saturation (range) %	89.2 (88.2-90.1)	97.1 (95.2-98.5)	86.8 (85.7-88.3)

Conclusions:

Temperature and HCN concentration effect on acute toxicity is documented. LC50 values from the study are:
LC50 = 0.028 ± 0.004 mg.L-1 at 6 °C
LC50 = 0.042 ± 0.004 mg.L-1 at 12 °C
LC50 = 0.068 ± 0.004 mg.L-1 at 18 °C
Warm acclimated rainbow trout survived longer in lethal concentrations of cyanide. Toxicity curves clearly showed that the temperature effect on the acute toxicity of cyanide is concentration dependent. At slow lethal concentrations, cyanide is more toxic at lower temperatures, whereas at rapid lethal levels the reverse occurs; the reversal takes place at 0.10 mg HCN.L-1.

Executive summary:

Ethanedinitrile hydrolyses into HCN in water; one molecule of ethanedinitrile produces one molecule of HCN. HCN is the molecule carrying the cyanide-like toxicity of ethanedinitrile as a parental compound. Molecular weight of ethanedinitrile is 2.1 times higher than that of HCN. Therefore, to estimate ethanedinitrile toxicology aquatic data from HCN aquatic toxicology data, it is possible to multiply the toxicity concentrations such as LC50of HCN by 2.1 to estimate the toxicity of some concentrations of ethanedinitrile in water. Alternatively, ethanedinitrile concentration in water can be divided by 2.1 to derive the maximum HCN concentration which arises from hydrolysis of ethanedinitrile. This is a highly conservative value since a significant amount of ethanedinitrile escapes from water due to its high volatility. Only one acute toxicity study to fish was chosen to be assessed in more detail due to its overall good performance and the chosen test species rainbow trout (Oncorhynchus mykiss,formerly known asSalmo gairdneri)is the same species recommended in Regulation (EU) No 283/2011. Nevertheless, there are extensive data on acute toxicity to fish which do not vary extensively. The scientific articles generally contain more replicates in the experimental setup and therefore their outcomes are statistically more powerful than OECD tests where the number of test animals is limited. OECD test are also performed only once and thus a potential error during the testing cannot be corrected by a repeated independent experiment. Due to extensive acute toxicity testing on fish in the past, we expect that a new acute toxicity study according to OECD 203 would be redundant and would not bring new or scientifically more correct information in regards to toxicity to fish. It would also lead to excessive vertebrate testing which is against the principles of the 3 R (reduction, refinement, and replacement). The chosen study resulted in the most conservative (lowest) LC50out of 119 LC50values from fish acute studies reported in the review of in Gensemer et al. (2005) from 17 fish species. This is mostly due to the fact that the study was performed on the most sensitive species and was also enlarged to assess LC50below standard temperature recommended by the relevant OECD guideline. It is therefore obvious that standard OECD test cannot achieve such low LC50and that this study should be used for risk assessment due to its highly conservative outcomes in respect to EFSA (2015). From the methodological point of view this study is superior to the standard OECD study, for instance by improvements designed to evaluate specific toxicity of HCN e.g. by inclusion of lower temperature for the test itself.

In this study the resistance of juvenile rainbow trout to cyanide was markedly affected by temperature (table below) and each 96-hour LC₅₀value differed significantly from those obtained at other temperatures.

Acute toxicity of hydrogen cyanide to rainbow trout acclimated and tested at different temperatures in flow-through system for 96 hours:

	6 °C	12 °C	18 °C
96-hour LC50mg.L^-1HCN (95% confidence interval)	0.028 (0.024-0.035)	0.042 (0.038-0.046)	0.068 (0.064-0.072)
Slope function	1.30	1.30	1.12
Highest cyanide conc. with no mortality in 96 hours. mg HCN.L^-1	0.018	0.032	0.060
Lowest cyanide conc. with 100% mortality in 96 hours. mg HCN.L^-1	0.037	0.053	0.087
Average weight of fish (range). g.	11.79 (9.05-15.98)	12.41 (8.29-16.02)	13.19 (9.63-17.21)
Average fork length of fish (range). mm	102 (95-110)	102 (90-111)	106 (95-119)
Mean pH (range)	8.06 (7.9-8.1)	8.10 (8.06-8.11)	7.82 (7.78-7.90)
Mean oxygen saturation (range) %	89.2 (88.2-90.1)	97.1 (95.2-98.5)	86.8 (85.7-88.3)

The cyanide level required to kill 50 % of the trout in 96 hours was 2.4 times lower at 6 than at 18 °C.

LC50 values from the study are:

LC50 = 0.028 ± 0.004 mg.L-1 at 6 °C

LC50 = 0.042 ± 0.004 mg.L-1 at 12 °C

LC50 = 0.068 ± 0.004 mg.L-1 at 18 °C.

Warm acclimated rainbow trout survived longer in lethal concentrations of cyanide. Toxicity curves clearly showed that the temperature effect on the acute toxicity of cyanide is concentration dependent. At slow lethal concentrations, cyanide is more toxic at lower temperatures, whereas at rapid lethal levels the reverse occurs; the reversal takes place at 0.10 mg HCN.L-1.

The median survival time (MST) for each test concentration was estimated.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Test organisms (species):

Poecilia reticulata

Details on test organisms:

Fish size: 2.0 +/- 1.0 cm; number of organisms: 3 pcs per concentration in preliminary test, 7 pcs per concentration in basic test

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

96 h

Remarks on exposure duration:

16 hours /day

Test temperature:

22.1 °C - 22.4 °C

pH:

6.71 - 7.65

Dissolved oxygen:

without aeration

Details on test conditions:

The LC50 of ethanedinitrile towards Poecilia reticulata was determined in a 96-hour study according to OECD 203. Semi-static conditions were chosen for the test with complete water renewal every 24 hours. Number of concentrations during preliminary test: 5 + 1 dilution-water control series (0.1, 0.5, 1.0, 2.0, 4.0 mg/L). Number of concentrations during basic test: 6 + 1 dilution-water control series (0.6, 1.2, 1.5, 1.8, 2.4, 4.0 mg/L). During the testing period the fish were not fed, the medium wasnt aerated.

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

It is obvious from the data that the dose response curve is very steep. There was no effect at 1.2 mg/L, thus this is the NOEC concentration in this study.

Sublethal observations / clinical signs:

Table 8.2.1 – 1. Preliminary test –Poecilia reticulata Concentration [mg/L]  C            0.1         0.5         1.0         2.0         4.0 Mortality [%]        0        0            0            0            100        100

Concentrations for basic test were selected according to the results of preliminary test. These concentrations and percentage of mortality can be found inTable 8.2.1 – 2.

Table 8.2.1 – 2. - Basic test –Poecilia reticulata Concentration [mg/L]   C            0.6         1.2         1.5         1.8         2.4         4.0 Mortality [%]         0         0            0            57.1      100        100        100

96h LC50 = 1.40 mg/L It is obvious from the data that the dose response curve is very steep. There was no effect at 1.2 mg/L, thus this is the NOEC concentration in this study.

Conclusions:

The calculated 96h LC50 was 1.40 mg/L. The estimated NOEC is 1.2 mg/L. The test conditions (such as dissolved oxygen, no mortality in control, temperature, and pH) were in line with the OECD requirements. However, it was not possible to estimate the concentration of ethanedinitrile after 24 hours in the used media. This study clearly demonstrates that significant exposure of water environment by ethanedinitrile in natural conditions is not possible due to the instability of ethanedinitrile and its volatility.

Executive summary:

The LC50 of ethanedinitrile towardsPoecilia reticulatawas determined in a 96-hour study according to OECD 203. Semi-static conditions were chosen for the test with complete water renewal every 24 hours. Number of concentrations during preliminary test: 5 + 1 dilution-water control series (0.1, 0.5, 1.0, 2.0, 4.0 mg/L). Number of concentrations during basic test: 6 + 1 dilution-water control series (0.6, 1.2, 1.5, 1.8, 2.4, 4.0 mg/L).

Conditions:

• Test system: Poecilia reticulata, family: Poeciliidae
• Test temperature: from 22.1°C to 22.4°C
• pH: 6.71 – 7.65
• Time of exposure: 96 hours
• Test procedure used: semi-static (testing medium changed every 24 hours)
• Light conditions: 16 hours/day
• Medium volume: 150 ml per 1 test organism Fish size:                             
• Fish size: 2.0 +/- 1.0 cm Other conditions: 
• without feeding, without aeration
• Other conditions: without feeding, without aeration
• Number of organism: 3 pcs per concentration in preliminary test, 7 pcs per concentration in basic test.

Table 8.2.1 – 1. Preliminary test –Poecilia reticulata Concentration [mg/L]  C            0.1         0.5         1.0         2.0         4.0 Mortality [%]        0        0            0            0            100        100

Concentrations for basic test were selected according to the results of preliminary test. These concentrations and percentage of mortality can be found inTable 8.2.1 – 2.

Table 8.2.1 – 2. - Basic test –Poecilia reticulata Concentration [mg/L]   C            0.6         1.2         1.5         1.8         2.4         4.0 Mortality [%]         0         0            0            57.1      100        100        100

The calculated 96h LC50 was 1.40 mg/L. The estimated NOEC is 1.2 mg/L. The test conditions (such as dissolved oxygen, no mortality in control, temperature, and pH) were in line with the OECD requirements. However, it was not possible to estimate the concentration of ethanedinitrile after 24 hours in the used media. This study clearly demonstrates that significant exposure of water environment by ethanedinitrile in natural conditions is not possible due to the instability of ethanedinitrile and its volatility.

Description of key information

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.

 

Ethanedinitrile: The calculated 96h LC50 was 1.40 mg/L. The estimated NOEC is 1.2 mg/L. The test conditions (such as dissolved oxygen, no mortality in control, temperature, and pH) were in line with the OECD requirements. However, it was not possible to estimate the concentration of ethanedinitrile after 24 hours in the used media. This study clearly demonstrates that significant exposure of water environment by ethanedinitrile in natural conditions is not possible due to the instability of ethanedinitrile and its volatility.

 

Hydrogen cyanide: Temperature and HCN concentration effect on acute toxicity is documented. LC50 values from the study are: LC50 = 0.028 ± 0.004 mg.L-1 at 6 °C LC50 = 0.042 ± 0.004 mg.L-1 at 12 °C LC50 = 0.068 ± 0.004 mg.L-1 at 18 °C Warm acclimated rainbow trout survived longer in lethal concentrations of cyanide. Toxicity curves clearly showed that the temperature effect on the acute toxicity of cyanide is concentration dependent. At slow lethal concentrations, cyanide is more toxic at lower temperatures, whereas at rapid lethal levels the reverse occurs; the reversal takes place at 0.10 mg HCN.L-1.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

ca. 0.028 mg/L