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Administrative data

bioaccumulation in aquatic species, other
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Well performed study. However not conducted according to a guideline and adsorption was measured instead of bioaccumulation

Data source

Reference Type:
Accumulation of zirconium by microalgae and cyanobacteria
Geoffrey W. Garnham, Geoffrey A. Codd, Geoffrey M. Gadd
Bibliographic source:
Appl Microbiol Biotechnol (1993) 39:666-672

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
In this study the accumulation (adsorption / desorption) of Zr as the soluble species [Zr4(OH)s(H20)16)8+] by several species of aquatic microalgae and cyanobacteria has been investigated.
GLP compliance:
not specified

Test material

Details on test material:
- Analytical grade
- supplied by BDH (Dagenham, Essex, UK).

Sampling and analysis

Details on sampling:
For Zr uptake experiments:
Three replicate samples (1 ml) were taken from the cell suspension at time intervals after the addition of Zr and centrifuged in 1.5 ml Eppendorf tubes using an Eppendorf 5412 microfuge (1 min, 8000g), after which the supematant was removed and stored for Zr concentration analysis

For Zr desorption experiments:
Three replicate samples (1 ml) were then taken from the cell suspensions and centrifuged in 1.5-ml Eppendorf tubes using an Eppendorf 5412 microcentrifuge (1 min, 8000g) after which the supernatant was removed and the Zr concentration measured.

Test solutions

Details on preparation of test solutions, spiked fish food or sediment:
For Zr uptake experiments:
For Zr uptake experiments, 10-ml cell suspensions were incubated in 25-ml acid-washed plastic vials, which were shaken in
the light (300 µE m^-2 s^-1) at 23°C unless stated otherwise. Aliquots from 10 mM and 100 mM zirconyl chloride aqueous solutions were added to give final Zr concentrations in the range 0.5- 50 mM.

Test organisms

Test organisms (species):
other: Synechococcus PCC6301, Synechocystis PCC 6803, Plectonema boryanum, Chlorella emersonii, Scenedesmus obliquus, C. reinhardtii
Details on test organisms:
Axenic cultures of Chlamydomonas reinhardtii 1132a, Scenedesmus obliquus 2763a (obtained from Dr. J. C. Collins, Department of Evolutionary and Environmental Biology, University of Liverpool, P.O. Box 147, Liverpool, L69 3BX), Chlorella emersonii 2118b, Synechocystis PCC (Pasteur Culture Collection) 6803, Synechococcus PCC 6301 and Plectonema boryanum UTEX (University of Texas) 594 were grown at 23°C in 100 ml of BG11 medium, which comprised: 1.5 g NaNO3, 0.04 g K2HPO4, 0.075 g MgSO4x7H2O, 0.036 g CaCl2 x 2 H2O, 0.006 g citric acid, 0.006 g ferric ammonium citrate, 0.001 g disodiurn ethelynediaminetetraacetate (Na2EDTA), 0.02 g NaCO3, 1 ml trace metal mix A5 (2.86 g H3BO3, 1.81 g MnCl2 x 4 H2O, 0.222 g ZnSO4 x 7 H2O, 0.039 g Na2MoO2 x 2 H2O, 0.079 g CuSO4 x 5 H2O, 0.0494 g Co (NO3)2 x 6 H2O in 1 l distilled water) in 1 l distilled water (Allen 1968). The medium was autoclaved (120°C, 15 min) before being inoculated with Scenedesmus obliquus, C. emersonii, C. reinhardtii (106 cells ml^-1), Synechocystis PCC 6803, Synechococcus PCC 6301 (4 x 10^6 cells ml^-1) or 2 ml of a P. boryanum culture of optical density at 680 nm (09680) approx. 10, since this is a filamentous organism. Cultures were incubated in 250-ml conical flasks with rotary incubation at 150 cycles min^-1, at 23°C and with a photon fluence irradiance rate on the surface of the flask of 120 µE m^-2 s^-1 provided by white fluorescent tubes.

Study design

Route of exposure:
Test type:
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
4 h
Total depuration duration:
24 h

Test conditions

Test temperature:
23 °C
Details on test conditions:
- Material, size, headspace, fill volume: 25 mL acid-washed plastic vials
- Biomass loading rate:cell densities of 5 x 10^8 ml^-1 (Synechocysts PCC 6803 and Synechoeoccus PCC 6301), 5 x 10^6 ml^-1 (C.emersonii), 5 x 10^7 ml^-1 (Scenedesmus obliquus), 1 x 10^7 ml^-1 (C. reinhardtii) and to an OD680 of approx. 6 for P. boryanum (all approximately equivalent to 4 mg dry weight (wt) of biomass ml^-1)
- Replicates: 3

- Growth medium: See "Details on test organisms"

- Adjustment of pH: 10 mM sodium acetate buffer , pH 5.0
- Light intensity: 300 µE m-2 s-1
- Test concentrations: Aliquots from 10 mM and 100 mM zirconium dichloride oxide aqueous solution were added to give a final concentration in the range of 0.5-50 mM
Nominal and measured concentrations:
Nominal concentrations : 0.5 to 50 mM of Zr
Details on estimation of bioconcentration:
The amount of Zr taken up by cells was calculated from the reduction of Zr in the buffer, after taking into account any binding to the plastic vial (determined in control experiments without cells).

Cell suspensions were prepared in 10 mM sodium acetate buffer, pH 5.0, with 100 µM zirconium dichloride oxide. Cell suspensions were then incubated for at least 24 h in the light. Three replicate samples (1 mL) were taken from the cell suspensions and centrifuged after which the supernatant was removed and the Zr concentration measured by polarography. Aliquots (5 ml) of the remaining cells were then separated from the buffer by filtration through a 0.45-µm cellulose nitrate membrane filter, diameter 47 mm (Whatman International, Maidstone, Kent, UK), washed quickly with 10 mM
sorbitol to remove any intercellular Zr and resuspended in 5 ml of one of the following desorption agents: 10 mM sodium acetate buffer, pH 5.0; 10 mM acetic acid; 10 mM HC1; 10 mM CaCl2 x2 H20 and 10 mM NaCl. Triplicate samples (250 µl) were taken at time intervals, centrifuged (1 min, 8000g) and the Zr concentration in supernatants measured by polarography

Results and discussion

Details on results:
Zirconium uptake by experimental organisms
Uptake by all the microbial species in 10 mM sodium acetate buffer, pH 5.0, consisted of a single phase that was independent of light or the presence of the metabolic inhibitor CCCP (Table 1). This phase was complete within 5 min with no further uptake over a further 4-h incubation, as illustrated by the accumulation of Zr by C. emersonli. The order of the amount of Zr uptake from a 1 mM concentration for the different species was C. emersonii> S. obliquus > Synechococcus PCC 6301 > P. boryanum > C. reinhardtii > Synechocystis PCC 6803 (Table 1).
However, Accumulation of Zr, as the [Zr4(OH)s(H20)16] 8+ species, by all the microalgal and cyanobacterial species examined was due to a single phase of metabolismindependent "biosorption" with no apparent intracellular.uptake being observed

Desorption of Zr
Desorption of Zr from both microalgae and cyanobacteria was rapid with 28 to 87% of the Zr accumulated being removed within 5 rain depending on the desorption agent used. The order of desorption efficiency for the microalgae and cyanobacteria was 10 mM NaCI > 10 mM acetic acid > 10 mM HCI > 10 mM CaCl2 > 10 mM sodium acetate buffer, pH 5.0 except for C. reinhardtii where the order was 10 mM NaC1 > 10 mM CaCl2 > 10 mM HCl > 10 mM acetic acid > 10 mM sodium acetate buffer, pH 5.0.

Any other information on results incl. tables

Table 1. Accumulation of zirconium (Zr) by microalgae and cyanobacteria

Organism Zr accumulation (µmol g dry wt-1)
After 5 min

 After 4 h

In the light  In the light In the dark  In the light with CCCP
Synechococcus PCC6301  19.9 +/- 0.1  18.2 +/- 0.8  18.7 +/- 0.5  18.5 +/- 0.2
Synechocystis PCC 6803 1.85 +/- 0.2 2.1 +/- 0.5 1.80 +/- 0.5 2.20 +/- 0.5
Plectonema boryanum 16.8 +/- 0.1 16.0 +/- 0.1 16.1 +/-0.2 17.0 +/- 0.5
Chlorella emersonii  25.0 +/- 1.0  25.6 +/- 0.7 23.0 +/- 0.5 24.0 +/- 0.2
Scenedesmus obliquus 21.3 +/- 0.5 22.0 +/- 0.6 21.0 +/- 0.3 21.0 +/-0.5
C. reinhardtii  11.2 +/- 0.6  7.50 +/- 0.5 7.8 +/- 0.5 7.6 +/- 0.6

Applicant's summary and conclusion

Accumulation of Zr was tested using several microalgae and cyanobacteria. Only low values of accumulation could be detected for all test organisms. However, the accumulation of Zr was due to a single phase of metabolism independent "biosorption" with no apparent intracellular uptake being observed. Additionally, the desorption experiment with a rapid removement of Zr show that the "accumulation" is more an adsorption giving worst-case accumulation values. Nevertheless, since the worst-case values are low, a bioaccumulation potential is not expected.