Lutetium trinitrate

Toxicity to microorganisms

Administrative data

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Adequacy of study:

other information

Study period:

2004

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

unsuitable test system

Remarks:

A Klimisch score of 3 was attributed for the following reasons: (1) The species of Lu introduced as the test item is not perfectly clear: we consider it is lutetium trinitrate on the basis that the stock solution corresponds to 10 g/L of 99.999% Lu2O3 in 4% HNO3, (2) V. fischeri is considered of low relevance for STP micro-organisms considered under REACH.

Data source

Reference

Reference Type:

publication

Title:

Lutetium Speciation and Toxicity in a Microbial Bioassay: Testing the Free-Ion Model for Lanthanides

Author:

Weltje L., Verhoof L. R. C. W., Verweij W., Hamers T.

Year:

2004

Bibliographic source:

Environmental Science & Technology 38: 6597-6604

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The bioluminescence response of the bacterium Vibrio fischeri was studied at different lutetium concentrations in the presence and absence of natural and synthetic organic ligands [citrate, malate, oxalate, acetate, ethylenediaminetetraacetate (EDTA), and nitrilotriacetate (NTA)]. Tests performed in the presence of ligands aim at assessing the modulation of toxicity by those ligands that were chosen on the basis of their differences in complexing strength for lutetium and their different charges in fully dissociated state. It should be noted that citrate, malate, oxalate, and acetate are naturally occurring ligands, while EDTA and NTA in the environment are of anthropogenic origin.

GLP compliance:

not specified

Test material

Test material information

Test material form:

not specified

Details on test material:

- Source of test material: CPI International, Santa Rosa, CA.
- Description of test material: ICP stock solution at 10 000 ppm of 99.999% Lu2O3 in 4% HNO3. On this basis, it was concluded that bacteria were exposed to lutetium trinitrate.
No other information are available.

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: All solutions were prepared in Milli-Q water (Millipore Waters, Milford, MA). Using this medium, two lutetium series were prepared:
(1) a logarithmically ordered range of lutetium concentrations with no added ligands (10 concentrations between 0.15 µM and 2.56 mM).
(2) two lutetium concentrations (5 and 50 µM) each associated with the six following organic ligands: EDTA (Sigma Chemical Co., ca. 99%), NTA (Sigma Chemical Co., ca. 99.5%), citrate (J. T. Baker, 99.8%), malate (Acros, 99%), oxalate (Merck, 99.5%), and acetate (J. T. Baker, 99.5%). All six ligands were also tested as pure substances (i.e., with no added lutetium) to ensure that ligands were not toxic over the range of concentrations utilized in the experiments.
- Controls: Each series had its own control, consisting of a 0.355 M NaNO3 solution at pH 5.50.

Test organisms

Test organisms (species):

Vibrio fisheri

Details on inoculum:

- Test organisms: Lyophilized bacteria (V. fischeri, NRRL B-11177).
- Source: Microtox Acute Reagent, Azur Environmental, Carlsbad, CA).
- Preparation for exposure: Bacteria were prepared for the experiments as described by Hamers et al. (2001). First, the condition of the bacteria was tested by observing luminescence in the control over a period of 5 min. If the bacterial condition was satisfactory (a small increase of light emission followed by a slow decrease), 75 µL of the bacterial suspension (in 0.355 M NaNO3, pH 5.50) was injected into each well of the plate.
No further data.

Study design

Test type:

static

Water media type:

other: V. fischeri is a marine bacterium usually studied in NaCl. It can also be exposed in a 0.355 M NaNO3 solution. While the 0.355 M ionic strength is much larger than that of freshwater, high ionic strength and Na+ are needed for undisturbed performance.

Remarks:

This NaNO3 solution was chosen because nitrate has a weak complexing affinity for lanthanides and thus has a limited influence on free-ion concentrations.

Limit test:

no

Total exposure duration:

30 min

Test conditions

Hardness:

No data

Test temperature:

20 +/- 3 °C

pH:

Measurements of pH revealed that it shifted upward during the experiments from the initial 5.50 +/- 0.10 to an average value of 6.30 +/- 0.39. This is apparently due to the activity of the bacteria, because the bacterial suspension, which was also prepared with a NaNO3 solution of pH 5.5, had an average pH of 6.95 +/- 0.12 at the end of the experiment.

Dissolved oxygen:

No data

Salinity:

0.355 M ionic strength in the bacterial suspension, no value for the final test medium.

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentrations:
- Experiment without ligands: 10 Lu concentrations between 0.15 µM and 2.56 mM (between 0.026 and 447.92 mg/L).
- Experiment with ligands: 2 Lu concentrations of 5 and 50 µM (0.875 and 8.75 mg/L).

Measured concentrations: not available (no analytical monitoring).

Details on test conditions:

TEST SYSTEM
- Test vessel: white 96-well plates (Luminoscan LB 96P WMP 25).
- No. of vessels per concentration (replicates): 3 replicates.
- No. of vessels per control (replicates): 3 replicates.
- Bacterial suspension volume per well: 75 µL.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: All test solutions were prepared in Milli-Q water (Millipore Waters, Milford, MA).

EFFECT PARAMETERS MEASURED:
- Measured parameter: After 7.5, 15, 22.5 and 30 min, the luminescence of each well was measured and expressed in relative light units (RLU). The response data, that is RLU, were converted to percentages of the control response, of which the average was set to 100%.
- Measuring apparatus: Luminometer was equipped with an automatic injector (Labsystems Luminoscan RS).

No further data.

Reference substance (positive control):

yes

Remarks:

Cu solutions (Johnson Matthey, AAS standard solution, Specpure, 1000 ppm in 5% HNO3) was used as positive control.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

30 min

Dose descriptor:

EC50

Effect conc.:

0.24 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Free Lu3+

Basis for effect:

other: Bioluminescence

Remarks on result:

other: (equivalent to: 30min-EC50 = 1.37 µM, conversion using molecular weight)

Remarks:

Experiment without ligand

Effect concentrations 2

Duration:

30 min

Dose descriptor:

EC50

Effect conc.:

0.25 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Total dissolved Lu

Basis for effect:

other: Bioluminescence

Remarks on result:

other: (equivalent to: 30min-EC50 = 1.43 µM, conversion using molecular weight)

Remarks:

Experiment without ligand

Effect concentrations 3

Duration:

30 min

Dose descriptor:

EC50

Effect conc.:

>= 0.13 - <= 0.38 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Free Lu3+

Basis for effect:

other: Bioluminescence

Remarks on result:

other: (equivalent to: 30min-EC50 >= 0.74 µM to <= 2.15 µm, conversion using molecular weight)

Remarks:

Experiment with ligands

Effect concentrations 4

Duration:

30 min

Dose descriptor:

EC50

Effect conc.:

>= 0.17 - <= 7.56 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Total dissolved Lu

Basis for effect:

other: Bioluminescence

Remarks on result:

other: (equivalent to: 30min-EC50 >= 0.96 µM to <= 43.2 µm, conversion using molecular weight)

Remarks:

Experiment with ligands

Details on results:

For the experiment with ligands, the ranges in the above table cover the EC50 values obtained for the different ligands. Detailed values ligand per ligand were reported below:
- Exposure to Lu without ligand: 30min-EC50 = 0.24 mg/L (expressed as free Lu3+) and 0.25 mg/L (expressed as total dissolved Lu).
- Expoure to Lu in the presence of EDTA: 30min-EC50 = 0.14 mg/L (expressed as free Lu3+) and 0.17 mg/L (expressed as total dissolved Lu).
- Expoure to Lu in the presence of NTA: 30min-EC50 = 0.13 mg/L (expressed as free Lu3+) and 0.79 mg/L (expressed as total dissolved Lu).
- Expoure to Lu in the presence of citrate: 30min-EC50 = 0.34 mg/L (expressed as free Lu3+) and 7.56 mg/L (expressed as total dissolved Lu).
- Expoure to Lu in the presence of malate: 30min-EC50 = 0.22 mg/L (expressed as free Lu3+) and 3.41 mg/L (expressed as total dissolved Lu).
- Expoure to Lu in the presence of oxalate: 30min-EC50 = 0.38 mg/L (expressed as free Lu3+) and 5.56 mg/L (expressed as total dissolved Lu).
N.B. In the publication, EC50 were expressed in µM and they were converted in mg/L by dividing them by the molecular weight of lutetium (174.967 g/mol).

Experiments with the pure ligands (without lutetium) showed no toxicity in the concentration ranges tested, except for acetate. At concentrations above 1 mM, acetate has toxic effects on V. fischeri. However, these high acetate concentrations would be needed to study Lu complexation. Consequently, acetate was excluded from the Lu ligand experiments, because its complexing intensity is too low at nontoxic concentrations.

Results with reference substance (positive control):

The 15min-EC50 for free Cu2+ was 1.04 µM. The outcome of this positive control experiment implies a good physiological quality of the bacteria and, more importantly, attests to the reproducibility of this biotest.

Reported statistics and error estimates:

All linear and nonlinear least-squares regressions were performed with the software package Prism, release 2.01 (GraphPad Software, San Diego, CA).

Applicant's summary and conclusion

Validity criteria fulfilled:

not specified

Conclusions:

Under the conditions of this study, the following 30min-EC50 values were obtained on Vibrio fisheri:
- Exposure to lutetium without ligand: 30min-EC50 = 0.24 mg/L (expressed as free Lu3+) and 0.25 mg/L (expressed as total dissolved Lu).
- Exposure to lutetium in the presence of organic ligands: 30min-EC50 = 0.13 to 0.38 mg/L (expressed as free Lu3+) and 0.17 to 7.56 mg/L (expressed as total dissolved Lu).

Executive summary:

The bioluminescence response of the bacterium Vibrio fischeri was studied at different lutetium concentrations in the presence and absence of six natural and synthetic organic ligands: citrate, malate, oxalate, acetate, ethylenediaminetetraacetate (EDTA), and nitrilotriacetate (NTA). Tests performed in the presence of ligands aim at assessing the modulation of toxicity by those ligands that were chosen on the basis of their differences in complexing strength for lutetium and their different charges in fully dissociated state. It should be noted that citrate, malate, oxalate, and acetate are naturally occurring ligands, while EDTA and NTA in the environment are of anthropogenic origin. Two sets of experiments were conducted:

(1) test of a logarithmically ordered range of lutetium concentrations with no added ligands (10 concentrations between 0.15 µM and 2.56 mM).

(2) test of two lutetium concentrations (5 and 50 µM) each associated with the six organic ligands.

Bioluminescence was assessed after 7.5, 15, 22.5 and 30 min.

Under the conditions of this study, the following 30min-EC50 values were obtained on Vibrio fisheri:

- Exposure to lutetium without ligand: 30min-EC50 = 0.24 mg/L (expressed as free Lu3+) and 0.25 mg/L (expressed as total dissolved Lu).

- Exposure to lutetium in the presence of organic ligands: 30min-EC50 = 0.13 to 0.38 mg/L (expressed as free Lu3+) and 0.17 to 7.56 mg/L (expressed as total dissolved Lu).