Copper cyanide

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

Study period:

2008-03-17 to 2008-04-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental study on the registered substance performed according to the OECD 201 guideline and under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

March 23, 2006

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

See test material information

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 h) and at te end (72 h) of the test for analysis.
10 mL (x 3 replicates) of test solution was taken from the each test vessel prepared at the beginning of the test. At the end of the test,
test solutions for each test concentration were and then pooled solutions were centrifuged slightly (5 000 rpm, 10 min) to remove growth algae. 10 mL (10 mL x 3 replicates) of solutions were taken from upper phase and put into the 20 mL of vials. 0.3 mL of nitric acid solution (61%) was addedinto the samples and then the samples were sent to the chemical analysis laboratory at Korea Research Institute of Chemical Technology for analysis of copper.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
After 101 mg of the test substance was added in 1 000 mL beaker and dilution water (OECD nutrient medium) was added up to the mark of the beaker to produce the nominal concentration of 100 mg/L, this solution was then stirred for about 48h and then filtered to remove undissolved test substance using 0.45 um membrane (Advantec, Toyo Roshi Kaisha Ltd, Japan). Serial dilutions of this test solution were prepared with the dilution water to give desired series of exposure levels which were nominally 0.1, 0.26, 0.64, 1.6, 4.0, and 10.0 mg/L. For the control group, only the dilution water was used.

No remarkable phenomenon was observed in the test solution.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: No UTEX 1648
- Source (laboratory, culture collection): Division

PRE-CULTURE
Two to four days before the test, sterile OECD nutrient medium (OECD, 2006) was inoculated about 5.0x10^3 cells/ml from a stock culture and incubated in a shaking incubator under continuous illumination (shaking rate: about 100 times/min, and light intensity: 60-120 uE/m2/s) at 21-24°C to give an algal suspension in log phase growth.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

No remark

Post exposure observation period:

No data

Hardness:

No data

Test temperature:

23.6 +/- 0.1°C

pH:

7.94 - 9.52

Dissolved oxygen:

No data

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

Nominal: 0.1, 0.26, 0.64, 1.6, 4.0 and 10.0 mg/L
Mean measured concentrations (geo mean): 8.5, 18.1, 42.4, 107.3, 251.9 and 109.6 ug/L.

Details on test conditions:

MEDIUM
The algae were cultivated and tested in sterile OECD nutrient medium, prepared according to the OECD test guideline (2006).

MEDIUM RENEWAL
Not applicable for static tests

ENVIRONMENTAL CONDITIONS
During the test period, the temperature was recorded daily and continuously using a recorder. At the start o the test, the pH of the test solutions was measured in additional flasks specifically fo pH measurement. At the end of the test, the pH was measured from Replicate 1 of each test concentrations. At the start and end of the test, light intensity was measured using a light meter (LI-250A light meter, quantum sensor) at the surface level of the test solutions.
Simple for measurements of temperature, pH and light intensity were taken at 0, 24, 48 and 72 hours and the cell densities were determined by direct counts using a BECKMAN COULTER tm Multisizer 3 particle analyzer.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The inhibition percentage of the growth rate for each treatment was calculated based on both average growth rate and yield.

EXPERIMENTAL DESIGN
The test was started by inoculating 5.1x10^3 celles/ml in 250 ml conical flasks each containing 100 ml of test solution under sterile conditions. These cells were taken from an exponentially growing pre-culture under the same conditions as for the test.

CULTURE CONDITIONS
The inoculated flasks were then placed in a shaking incubator (DAIHAN Scientific, KOREA). The cultures were incubated, without media renewal, for 72h nder continous illumination. The gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker oscillating at 100 cycles per minute.

EVALUATION OF DATA
Plots of growth curves were produced using the measured cell concentrations in test cultures and controls together with the concentrations of the test substance and the times of measurement.

RANGE-FINDING STUDY
- Nominal concentrations: 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study:
Average growth inhibition %: -2.0, 61.1, 76.4 and 76.4% at 0.1, 1, 10 and 100 mg/L respectively
Yield: -11.6, 97.0, 99.0 and 99.0% at 0.1, 1, 10 and 100 mg/L respectively

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

35 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 21.1 - 55.6 ug/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

20.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 0.00 - 27.5 ug/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

50.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 27.0 - 64.1 ug/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

25.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 16.2 - 35.9 ug/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

89.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 78.9 - 99.7 ug/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

40.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 26.8 - 72.5 ug/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

18.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: average growth rate and yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

42.4 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: average growth rate and yield

Details on results:

Concentration of the test substance in the test solution: At 0 hour (start of the test), the mean measured concentrations were 6.8, 18.2, 44.3, 110.8, 266.1 and 622.7 ug/L (6.2 - 7.0% of the nominal concentrations). The mean concentrations measured at 72h (end of the test) were 10.7, 18.0, 40.6, 104.0, 238.5 and 592.5 ug/L (5.9 - 10.7% of the nominal concentrations).
The mean concentrations of the test substance measured at the nominal concentrations of 0.1, 0.26, 0.64, 1.6, 4.0 and 10.0 mg/L during the definitive test were 8.5, 18.1, 42.4, 107.3, 251.9 and 109.6 ug/L, i.e., 6.1 - 8.5% of the nominal concentrations.

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

No data

See the attached study report

Validity criteria

- Increase of biomass in the control cultures within the 72-hour test period: 207.2-fold (criteria: > or = 16-fold)

- Mean coefficient of variation for section-by-section specific growth rate in the control cultures: 21.2% (criteria: < or = 35%)

- Coefficient of variation of average specific growth rates in replicate control cultures during the whole test period: 2.6% (criteria: < or = 7%)

- Change of pH in control medium during the test period: 0.36 (criteria: < or = 1.5)

Therefore, all validity criteria of the study were met.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the 72hr-EC50 values expressed in terms of the mean measured concentration of copper cyanide to the algae Pseudokirchneriella subcapitata were determined as 89.1 ug/L (95% confidence limits 78.9 - 99.7 ug/L) for average growth rate, and 40.9 ug/L (26.8 - 72.5 ug/L) for yield.

Executive summary:

This study was performed to assess the inhibitory effect of copper cyanide on the growth of the unicellular green alga, Pseudokirchneriella subcapitata (Strain No UTEX 1648). This study was conducted in accordance with the OECD 201 guideline and under GLP conditions.

The toxicity test was performed with control and nominal concentrations of 0.1, 0.26, 0.64, 1.6, 4.0 and 10.0 mg/L each in triplicates, with mean measured concentrations of 8.5, 18.1, 42.4, 107.3, 251.9 and 109.6 ug/L. Cultures were incubated on a shaking incubator under continous illumination at 23.6 +/- 0.1°C for 72 hours. Growth was monitored daily by determining the cell density of each culture by direct counts using a BECKMAN COULTERtm Multisizer 3 particle analyzer.

Under the test conditions, the 72hr-EC50 values expressed in terms of the mean measured concentration of copper cyanide to the algae Pseudokirchneriella subcapitata were determined as 89.1 ug/L (95% confidence limits 78.9 - 99.7 ug/L) for average growth rate, and 40.9 ug/L (26.8 - 72.5 ug/L) for yield. The 72h-NOEC was determined to be 18.1 ug/L.

All results were expressed in terms of the mean measured concentrations (geo mean). At 0 hour (start of the test), the mean measured concentrations were 6.8, 18.2, 44.3, 110.8, 266.1 and 622.7 ug/L (6.2 - 7.0% of the nominal concentrations). The mean concentrations measured at 72h (end of the test) were 10.7, 18.0, 40.6, 104.0, 238.5 and 592.5 ug/L (5.9 - 10.7% of the nominal concentrations).

All validty criteria of the study were met, as stated in the test guideline. Therefore, this study can be considered acceptable for that endpoint.

Description of key information

Under the test conditions, the 72hr-EC50 values expressed in terms of the mean measured concentration of copper cyanide to the algae Pseudokirchneriella subcapitata were determined as 89.1 ug/L (95% confidence limits 78.9 - 99.7 ug/L) for average growth rate, and 40.9 ug/L (26.8 - 72.5 ug/L) for yield. The 72h-NOEC was determined to be 18.1 ug/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

89.1 µg/L

EC10 or NOEC for freshwater algae:

18.1 µg/L