Copper cyanide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-03-17 to 2008-05-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental study performed according to the OECD 203 guideline and under GLP compliance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

OECD, 1993. OECD Guidelines for Testing of Chemicals No. 203 “Fish, Acute Toxicity Test (Adopted: July 17, 1992)”.

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Test concentrations were verified by chemical analysis. Water samples were taken from the controls and each exposure level at the start (0-hour), at the middle (48-hour), and at the end (96-hour) of the test for analysis.

Vehicle:

no

Details on test solutions:

Preparation of the test solutions
After 1106 mg of the test substance was added in a 11,000 mL glass tank and dilution water was added up to the mark of the tank to give the nominal concentration of 100 mg/L, this solution was then stirred for about 48 hours. Serial dilutions of this test solution were prepared with the dilution water to give desired series of exposure levels which were nominally 50, 25, 12.5, 6.3, and 3.1 mg/L. For the control group, only the dilution water was used.
No remarkable phenomenon was observed in the test solution.

Test organisms (species):

Oryzias latipes

Details on test organisms:

SOURCE
Fish culture-room (Room No. 324 of Building No. 7) in the Division of Non-clinical Studies, Korea Institute of Toxicology (KIT), Korea Research Institute of Chemical Technology (KRICT), Daejeon, Korea.

CULTURING METHODS
Approximately three month old fish, which was produced through natural spawning in the culturing room of KIT, was used for the toxicity test. The fish were cultured at water temperature of 22-26°C, and photoperiod as following: 16 hr of light and 8 hr of dark, including 30 minute dawn and dusk transition periods. The fish were fed daily with brine shrimp nauplii (MyunSun Co., Ltd., Korea) in the morning and Tetramin flake (Tetra®, Germany) in the afternoon.

ACCLIMATATION AND FOOD SUPPLY
The stock of fish, 54 fish in a 40 L glass container, was acclimated to test conditions 7 days before the definitive test. Food was not given during the 24 hour period prior to exposure. The batch of fish was observed to be in good condition prior to use in the test. The test fish were maintained with water temperature of 23.0-24.7°C, and DO of 7.2-7.6
Control fish were measured at the end of the exposure period. The mean total length and mean body weight were 2.4±0.1 cm (Mean±SD) and 0.11±0.01 g (Mean±SD), respectively.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

The water in this study had a residual chlorine and total hardness (as CaCOs) of <0.01 mg/L and 42.5 mg/L (Date of analysis: December 4, 2007), respectively

Test temperature:

23.5±0.2°C

pH:

7.41-7.82

Dissolved oxygen:

6.0-8.9 mg/L (71-104% of ASV)

Nominal and measured concentrations:

- Nominal concentration, mg/L: Control, 3.1, 6.3, 12.5, 25.0, 50.0, 100.0
- Mean measured concentration, mg/L: Control, 0.07, 0.15, 0.29, 1.24, 2.38

Details on test conditions:

TEST MEDIUM / WATER PARAMETERS
- ulturing and dilution water: Tap water was passed through a membrane filter (1 pm) to remove particulate matter and then a high-grade activated carbon filter to remove any organic contaminants. The water was analyzed at the Korea Testing & Research Institute (KTR), Seoul, Korea to monitor water quality at 4-month intervals based on the water quality standards of U.S. EPA (Environmental Protection Agency) for fish toxicity test. The results of water analysis showed that the water quality met the water quality standard of U.S. EPA. The water in this study had a residual chlorine and total hardness (as CaCOs) of <0.01 mg/L and 42.5 mg/L (Date of analysis: December 4, 2007), respectively.
- Medium renewal: This test substance was stable in the test solution for 96 hours (see “Analytical Report”). Therefore, the definitive test was carried out without renewal of the test solutions (static system).
- Environmental conditions: Dissolved oxygen and pH were recorded daily for all treatments. However, water temperature was recorded daily at control. The water temperature, dissolved oxygen (DO) and pH during the exposure period were maintained as follows:
. Temperature: 23.5±0.2°C
. Dissolved oxygen: 6.0-8.9 mg/L (71-104% of ASV).
. pH: 7.41-7.82
The photoperiod in this study was 16 hours of light and 8 hours of dark, including 30 minute dawn and dusk transition periods. The light intensity was measured prior to the test and at the end of the test. The intensity ranged from 1055 to 1072 lux.
No food and air was provided during the test period.
- Analysis of the test substance in the test solution: Test concentrations were verified by chemical analysis. Water samples were taken from the controls and each exposure level at the start (0-hour), at the middle (48-hour), and at the end (96-hour) of the test for analysis.

STATISTICAL ANALYSIS AND EXPRESSION OF THE RESULTS
The LC50 values and 95% confidence limits were calculated using the Probit Method (EPA/600/4-85/013, 1985) since partial lethal concentrations were observed at more than two concentrations between 0% and 100% death concentration.
The "no-observed effect concentration" is the highest concentration tested at which no immobilization or significant symptoms of intoxication to exposure were observed.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The fish were examined daily for symptoms of intoxication and mortality. The criteria of death employed in this study were (i) absence of respiratory movement and (ii) absence of response to external stimulation.

TEST CONCENTRATIONS
- Range-finding: The range-finding test of this test substance was conducted at the nominal concentrations of 0.1, 1, 10, and 100 mg/L under static system. 251 mg of the test substance was added in a 2500 mL glass tank and dilution water was added up to the mark of the tank to give 100 mg/L of nominal concentration. This solution was stirred during 48 hours and then filtered to remove undissolved test substance using 0.45 pm membrane (Advantec, Toyo Roshi Kaisha Ltd., Japan). This filtered solution was diluted with dilution water serially to prepare 0.1, 1.0, and 10 mg/L (nominal test concentrations) of test solutions.
Five fish were exposed to the each test vessel without replication and the number of dead fish was observed at each test for 48 hours.
- Test concentrations: nominal test concentration (mg/L): control, 3.1, 6.3, 12.5, 25, 50, 100; mean measured test concentration (mg/L): control, 0.07, 0.15, 0.29, 0.59, 1.24, 2.38
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test, 100% mortality at the nominal concentration of 100 mg/L and no mortality were observed at the nominal concentration of 10 mg/L. Therefore, the definitive test was conducted in the concentrations between 1-100 mg/L of nominal concentrations without replication.
Each vessel contained 5 L of test solution and seven fish. There wasn’t any replicate in this test.

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

0.87 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% Confidence limit: 0.49 - 1.88 mg/L

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% Confidence Limit: 0.38 - 1.03 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- OBSERVATION AND ACUTE TOXICITY
There were no mortalities or adverse effects observed in any of the fish exposed to control during the test. However, all seven fish died in the test solutions of 100 mg/L (mean measured concentration: 2.38 mg/L). Some fish exposed to the test substance were seen losing equilibrium and swimming mainly at the bottom.
All results were expressed in terms of the mean measured concentration.

- DISCUSSION
The measured concentration of the test substance (2.38 mg/L) in the test solution for the definitive test was not same as the saturated concentration (3.42 mg/L) in water obtained from the preliminary solubility test. The difference was considered to be caused by the slight difference between the used methods preparing the test solutions in the two tests. About 100 mg of test substance was added to the 1 L of dilution water and stirred for 48 hours for preliminary solubility test and 1106 mg of test substance was added to 11 L of dilution water for definitive test.
Although the nominal concentration of was 100 mg/L and this nominal test concentration was the same with above nominal test concentration, the test condition was not same because of the difference of the amount of dilution water and test substance. In addition, it was judged that the difference of the solubility of test substance was observed because of the difference of physical force such as stirring and ultrasonication during preparation of test solution. Because of the difference of solubility, however, 96 hr- LCso value did not show the difference at above of the level of statistical significance.
Because the statistical methods that are used to calculate LC50 values require only partial mortality data including 0% and 100% mortality data. As shown in the results of definitive test, 100% mortality was observed at the highest test concentration (2.38 mg/L). Partial mortality and 0% mortality were observed at below the highest test concentrations. Although 100% mortality was observed at above the highest concentrations, therefore, these data were not used to calculate LC50.

RESULTS OF THE CHEMICAL ANALYSIS
- Method validation
Specificity
There was no interference of other elements since the emission line of 324.754 nm was selected for copper.
Linearity
The linear regression equation was y = -209.24x - 6.0798 (r2 = 1.0000) and there was a linear relationship (Figure 1).
Precision
The coefficients of variance (CV,%) for 1, 5, and 30 mg/L were presented in Table 1. CV (%) values of intra-batch analysis were 0.71-1.04%, 0.41-0.75%, 0.59-1.38% and CV (%) values of inter-batch analysis were 1.58%, 0.84% and 1.98%. The values were within the precision criteria of 15%.
Accuracy
The relative error (RE, %) for 1, 5, and 30 mg/L were presented in Table 1. RE (%) values of intra-batch analysis were 12.50-15.70%, 3.66-5.06%, 0.78-4.89% and RE (%) values of inter-batch analysis were 13.60%, 4.24% and 2.7%. The values were within the accuracy criteria of 15%.
Limit of Detection
The limit of detection of copper was provided by instrument company was 5.4 ug/L at 324.754 nm.
Limit of Quantitation
The limit of quantitation was 0.05 mg/L for this analysis.

- Stability test of test substance
The measured concentration of 100 mg/L (nominal concentration) of test solution was 3.17 mg/L (3.17% of nominal concentration) at 0 hour. The concentrations were 2.86-2.99 mg/L during 96 hours and they were 90.2-94.3% of initial concentrations (Table 2).

- Analysis of test substance
At 0 hour (the start of the test), the mean measured concentration was 0.07, 0.15, 0.30, 0.60, 1.25, and 2.45 mg/L (2.26-2.50% of nominal concentration) and 0.07, 0.14, 0.29, 0.59, 1.22, and 2.32 mg/L (2.22-2.44% of nominal concentration) at 48 hours (in the middle of the test). The mean concentration measured at 96 hours (the end of the test) was 0.07, 0.15, 0.29, 0.59, and 1.26 mg/L (2.26-2.52% of nominal concentration).

- Conclusion
The concentrations of the test substance measured were 2.22-2.52 mg/L and the geometric mean measured concentration was as follows.
-Nominal concentration, mg/L: Control, 3.1, 6.3, 12.5, 25.0, 50.0, 100.0
- Mean measured concentration, mg/L: Control, 0.07, 0.15, 0.29, 1.24, 2.38

Results with reference substance (positive control):

No data

Sublethal observations / clinical signs:

Please, see the attached study report

Validity criteria fulfilled:

not specified

Conclusions:

The 96h-LC50 value (and 95% confidence limits) and the 96hr NOEC (No-observed effect concentration) expressed in terms of the measured concentration of Copper cyanide to ricefish (Oryzias latipes} were determined as 0.62 mg/L (0.38-1.03 mg/L), and 0.30 mg/L, respectively.

Executive summary:

The study was conducted to assess the acute toxicity of Copper cyanide to ricefish, Oryzzzxslatipesunder static system.

The study was conducted in accordance with the National Institute of Environmental Research(NIER),Notification No. 2007-29 (December 28, 2007), [Annex 5] Chapter 2-3 "Fish, Acute Toxicity Test” and the OECD Guidelines for Testing of Chemicals No. 203 “Fish, Acute Toxicity Test (Adopted: July 17, 1992)”.

The toxicity test was performed in control and nominal concentrations of 3.1, 6.3, 12.5, 25, 50 and 100 mg/L, with mean measured concentrations of 0.07, 0.15, 0.29, 0.59, 1.24, and 2.38 mg/L, respectively. Seven fish were exposed to each treatment concentration and control. Observations were made on the number of dead fish and the incidence of sub-lethal effects after 24, 48, 72, and 96 horns of exposure.

The following values, expressed in terms of the mean measured concentrations, were obtained from the test.

(Unit: mg/L, mean measured concentration)

Exposure time	24-hour	48-hour	72-hour	96-hour
LC50	NA	NA	0.87	0.62
95% confidence limits	NA	NA	0.49-1.88	0.38-1.03
No observed effect concentration	NA	NA	0.29	0.29

 

All results were expressed in terms of the mean measured concentration (geometric mean). At 0-hour (the start of the test), the mean measured concentrations were 0.07, 0.15, 0.30, 0.60, 1.25, and 2.45 mg/L (2.26-2.50% of the nominal concentration). The mean measured concentrations were 0.07, 0.14, 0.29, 0.59, 1.22, and 2.32 mg/L (2.22-2.44% of the nominal concentration) at 48-hour (the middle of the test) and the mean concentrations measured at 96-hour (the end of the test) were 0.07, 0.15, 0.29, 0.59, and 1.26 mg/L (2.26-2.52% of the nominal concentration).

Description of key information

The 96h-LC50 value (and 95% confidence limits) and the 96hr NOEC (No-observed effect concentration) expressed in terms of the measured concentration of Copper cyanide to ricefish (Oryzias latipes} were determined as 0.62 mg/L (0.38-1.03 mg/L), and 0.30 mg/L, respectively.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.62 mg/L