Copper hydroxide nitrate (Cu2(OH)3(NO3))

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Substance taken as reference was nano-sized rendering the results very conservative.

Data source

Reference

Reference 1

Reference Type:

secondary source

Title:

Unnamed

Year:

2009

Reference 2

Reference Type:

publication

Title:

Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata

Author:

Aruoja, V., Dubourguier, H.C., Kasemets, K and Kahru A.

Year:

2009

Bibliographic source:

Science of the Total Environment 407, 1461-1468 (2009)

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Copper oxide

EC Number:

215-269-1

EC Name:

Copper oxide

Cas Number:

1317-38-0

Molecular formula:

CuO

IUPAC Name:

copper (II) oxide

Constituent 2

Reference substance name:

CuO

IUPAC Name:

CuO

Details on test material:

Formula: CuO
Copper oxide, nanosized: particle size around 30 nm compared with bulk CuO

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Details on test solutions:

CuO nanosized was purchased from Sigma-Aldrich and bulk CuO from Alfa Aesar. Sock suspensions were prepared in algae medium immediately before each experiment (100 mg/L CuO). Before use, they were ultrasonicated for 30 minutes.
The appearance of nano and bulk CuO suspensions did not change: nano CuO suspension remaine more homogeneous and muc darker than the bulk formulation.

Test organisms

Test organisms (species):

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

Study design

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Yes

Test conditions

Test temperature:

24°C

Details on test conditions:

The substances under investigation were incubated with P. subcapitata at 24 °C±1 °C for at least 72 h in 20-ml glass incubation vials containing 5 ml of algal growth medium (OECD, 1984). The vials were shaken on a transparent table and constantly illuminated from below with Philips TL-D 38W aquarelle fluorescent tubes (enhanced irradiation between 400 and 500 nm). All assays were run in three replicates with initial algal cell count of 10000 cells/ml and algal biomass was measured by chlorophyll fluorescence. Briefly, 50 μl culture samples were transferred to a 96-well plate, 200 μl of ethanol was added to each sample and the plate was shaken for 3 h in the dark. Thereafter the fluorescence was measured with a microplate fluorometer (excitation 440 nm, emission 670 nm; Fluoroscan Ascent, Thermo Labsystems, Finland). The metal oxide suspensions did not fluoresce under these conditions and also their light absorbances at these excitation and emission wavelengths were below detection limit (Multiskan Spectrum, Thermo Electron Corp., Finland). During the exponential growth phase, the chlorophyll fluorescence (RFU) correlated with cell density
determined by counting in Neubauer hemocytometer. Phase contrast as well as fluorescence micrographs were taken with Olympus CX41 microscope equipped with DP71 camera.
In order to evaluate the shading effects of particle suspensions, special double layer glass vials were constructed by sealing a smaller vessel (cut from a similar vial) under the 20 ml test culture vessel. The lower vessel was filled with 3 ml of algal growth medium but not inoculated with algae. For shading controls the same amount of metal oxide particles than in the respective test vessel was added to the lower vessel.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

421 µg/L

Nominal / measured:

estimated

Conc. based on:

element

Remarks:

Copper (nanosized CuO)

Basis for effect:

growth rate

Remarks on result:

other: 373-852

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

710 µg/L

Nominal / measured:

estimated

Conc. based on:

element

Remarks:

Copper (nanosized CuO)

Basis for effect:

growth rate

Remarks on result:

other: 556-1092

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

504 µg/L

Nominal / measured:

estimated

Conc. based on:

element

Remarks:

Copper (nanosized CuO)

Basis for effect:

growth rate

Remarks on result:

other: 439-931

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8.03 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Remarks:

Copper (bulk CuO)

Basis for effect:

growth rate

Remarks on result:

other: 6.58-14.9

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11.55 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Remarks:

Copper (bulk CuO)

Basis for effect:

growth rate

Remarks on result:

other: 8.64-18.9

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

9.1 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Remarks:

Copper (bulk CuO)

Basis for effect:

growth rate

Remarks on result:

other: 7.28-16.1

Details on results:

Free ionic copper is considered the active ingredient in inorganic copper salts, and is believed to be repsonsible for adverse effects. The bioavailablity of the copper ion at target sites determines the severity of effects. Both copper oxide and and copper hydroxide nitrate are considered sparingly soluble inorganic copper salts and would be expected to dissociate similarly within the organism. As test material is in nano form, results are considered to be very conservative.
As aqueous suspensions of nanoparticles are opaque and thus may inhibit the growth of algae also by absorption of light, the special constructed vials for incubation of algae allowed to differentiate the true inhibitory effect of metal oxide from the light shading effect.
In control experiments, there was no significant effect on the 72 h growth of algae regardless of the concentrations of CuO.
In the experiments, CuO nanoparticles were clearly more toxic towards algae than the bulk form: the 72 h EC50 values being 0.71 and 11.55 mg Cu/l, respectively. There were no visible aggregates present in the growth medium. Of the concentrations tested, complete inhibition of growth occurred at 6.4 mg Cu/l with nanoparticles but at 25.6 mg Cu/l with bulk CuO. When the incubation was extended several days beyond the standard 72 h, growth was observed in case of some initially toxic nano CuO concentrations (1.6 mg Cu/l of nano CuO).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, the NOEL for nano-sized Copper oxide, using a green algae, was 421 µg Cu/L. Based on a content of 53% Copper in Basic Copper Nitrate, the fish NOEL for this substance is estimated to be 794 µg/L.