Praseodymium(III,IV) oxide

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:

30 November 2007 - 18 March 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: triplicate samples were taken from each test medium and from the control just before the start of the test and after 24, 48 and 72 hours. The concentrations of praseodymium were analytically determined in two samples of the dilutions 1:4.6, 1:2.1 and of the saturated solution with the loading rate of 100 mg/L and in two control samples from each sampling date. The samples from the lower test concentrations (dilutions 1:10 and 1:21) were not analysed, since these test concentrations were below the 72-hour NOELR and, thus, not relevant for the interpretation of the biological results.
- Sampling method: data not available
- Sample storage conditions before analysis: immediately after sampling, the samples were acidified with 10 % (v/v) nitric acid to stabilise the test material during the storage period. Then, the samples were stored in PE flasks at ambient temperature and protected from light until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
According to the results of a pre-experiment (non-GLP), the test material was not completely soluble at the concentration of 100 mg/L in the test water and no homogeneous dispersion could be prepared.
Therefore, a saturated solution of the test material with the loading rate of 100 mg/L and the dilutions 1:2.1, 1:4.6, 1:10 and 1:21 of the saturated solution were tested. Additionally, a control was tested in parallel (test water without test material).
The test method is based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures.
The dispersion with the loading rate of 100 mg/L was prepared by dispersing 200.1 mg of the test material in 2000 mL of test water. The test material was mixed into the test water as homogeneously as possible using ultrasonic treatment for 15 minutes and intense stirring. No auxiliary solvent or emulsifier was used. The dispersion was stirred on a magnetic stirrer at room temperature in the dark for 24 hours. After this stirring period, the stirrer was switched off to allow the undissolved test material to deposit at the bottom of the stirring vessel for 24 hours.
The equilibrated test medium (saturated solution) was carefully separated from the undissolved test material and used as the highest concentrated test medium. Additionally, adequate volumes of the saturated solution were diluted with test water for the preparation of the test media with lower test material concentrations. The test media were prepared just before the start of the test (= addition of algae). The test concentrations were based on the results of a range-finding test and on the results of pre-experiments to determine the water solubility of the test material (non-GLP). The stirring time of 24 hours was chosen according to the results of a pre-experiment in which the maximum concentration of dissolved praseodymium was determined after the stirring time of 24 hours.
- Eluate: no
- Differential loading: yes
- Controls: blank (test water without test material)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): yes, on the bottom of the stirring vessel, but not in the final test solution.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus subspicatus CHODAT
- Strain: No. 86.81 SAG
- Age of inoculum (at test initiation): the algal cells were taken from an exponentially growing pre-culture, which was set up three days prior to the test under the same conditions as in the test.
- Method of cultivation: cultivated at the laboratory in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water.
- Source: supplied by the Collection of Algal Cultures (SAG, Institut for Plant Physiology, University of Göttingen, 37073 Göttingen, Germany)

ACCLIMATION
- Acclimation period: three days
- Culturing media and conditions: see method of cultivation above
- Any deformed or abnormal cells observed: data not available

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

no

Hardness:

0.24 mmol/L (= 24 mg/L as CaCO3)

Test temperature:

21 - 22 °C

pH:

At the start of the test, the pH of the test media and the control ranged from 8.2 to 8.6. At the end of the test, pH values between 8.0 and 9.4 were measured. The increase of the pH during the test was caused by the uptake of CO2 by the algae due to their growth.

Dissolved oxygen:

not measured

Salinity:

not applicable

Nominal and measured concentrations:

nominal loading rates: dilution 1:21 (loading rate 4.8 mg/L), dilution 1:10 (loading rate 10 mg/L), dilution 1:4.6 (loading rate 22 mg/L), dilution 1:2.1 (loading rate 48 mg/L), saturated solution (loading rate: 100 mg/L)
mean measured concentrations: dilution 1:4.6 (6 μg/L), dilution 1:2.1 (loading rate 17 μg/L), saturated solution (loading rate: 37μg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: Erlenmeyer flasks covered with glass dishes
- Material, size, headspace, fill volume: 50 mL flasks, filled with 15 mL of algal suspension
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): none (static test)
- Renewal rate of test solution (frequency/flow rate): a static, non-renewal exposure system was used.
- Initial cells density: 5000 algal cells per mL of test medium
- Control end cells density: 371341.5 algal cells per mL of test medium
- No. of vessels per concentration (replicates): three replicates
- No. of vessels per control (replicates): six replicates

GROWTH MEDIUM
- Standard medium used: yes, the algae were cultivated in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile purified water
- Total organic carbon, Particulate matter, Metals, Pesticides, Chlorine, Alkalinity, Ca/mg ratio, Conductivity: data not available
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured and recorded in each test concentration and the control at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily. The concentration of phosphate was determined in duplicate in the test media and the control at the start of the test and then daily until the end of the test using a photometric method (Merck Spectroquant phosphate test 1.14848.0001). Prior to the determination, the algal cells were removed by filtration trough glass fibre microfilters (GF/C Whatman). The 24, 48 and 72 hour samples were taken from the separately incubated test media with algae which were also used for analytical purposes.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: the measured light intensity was about 7600 Lux (mean value) and was achieved by fluorescent tubes (Philips TLD 36W/840) installed above the test flasks.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: A small volume of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced. The algal biomass in the samples was determined by fluorescence measurement (BIOTEK Multi-Detection Microplate Reader, Model FLx800). The measurements were performed at least in duplicate. Inhibition of algal growth was determined from: (i) the area under the growth curves (AUC), biomass integral, (ii) the specific growth rates (µ), and (iii) the yield (Y)
- Other: In addition, after 72 hours of exposure, a sample was taken from the control and from the highest test concentration. The shape and size of the algal cells were examined microscopically in these samples.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: yes
- Test concentrations of the range finding study: no data
- Results used to determine the conditions for the definitive study: no data

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL not determined

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

48 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

BIOLOGICAL RESULTS:
- Exponential growth in the control (for algal test): yes (In the control, the biomass increased by a factor of 74 over 72 hours)
- Observation of abnormalities (for algal test): Unusual cell shape, Colour differences, Flocculation, Adherence to test vessels, Aggregation of algal cells: no
- Other: The size and shape of the algal cells was not affected.

APPEARANCE OF THE TEST MEDIUM:
No remarkable observations were made concerning the appearance of test media. All test media were clear solutions throughout the test period.

PHOSPHATE CONCENTRATIONS:
The analysis of the impact of the loading rate on the phosphate amount in the test media showed that the saturated solution had a statistically significant impact on the phosphate concentration in the test medium at all time points (except at 72 hours, where the loss of phosphate over time had a stronger impact on the phosphate concentration than the test material). The dilution 1:2.1 (loading rate of 48 mg/L) had a statistically significant impact on the phosphate concentration at 0 and 48 hours. At the test start, this impact was significant even at the dilution 1:10 (loading rate of 10 mg/L) and above. The loss of phosphate can be explained by the formation of insoluble complexes of phosphate with the test material (which is a well-known behaviour of rare earth elements in the environment) during stirring of the dispersion. The depletion of phosphate in the test medium during the test might have been the reason for the inhibition of algal growth determined at these test concentrations. Thus, growth inhibition due to a secondary effect (i.e. the complexation of the essential algal nutrient phosphate by the test material) cannot be excluded.

ANALYTICAL MONITORING:
The concentrations of praseodymium (Pr) were measured in samples taken daily from the dilutions 1:4.6 (loading rate 22 mg/L), 1:2.1 (loading rate 48 mg/L) and from the saturated solution (loading rate 100 mg/L).
During the test, the concentrations of praseodymium measured in the dilution 1:4.6 ranged from 3 to 7 μg/L, in the dilution 1:2.1 from 13 to 16 μg/L, and in the saturated solution from 24 to 37 μg/L, respectively. The solubility limit reached during this test was thus different from that obtained during the water solubility test (see IUCLID section 4.8). Such contrasting results could be explained by the different water media used in the water solubility test and ecotoxicological studies (media containing analytical grade salts).

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72 hour EC50 for the growth rate = 0.64 mg/L (acceptance range: 0.44 - 1.16 mg/L) (potassium dichromate)

Reported statistics and error estimates:

The EL10 and EL50 values (the respective loading rates of the test material corresponding to 10 and 50 % inhibition, respectively, compared to the control) for the different growth parameters and their 95 % confidence intervals were calculated as far as possible by Probit Analysis. The EL90 could not be calculated for AUC and growth rate, as the inhibition of the parameters was below 90 % at all test concentrations.
For the determination of the LOELR and NOELR, the AUC, the growth rate and the yield at the test concentrations were compared to the corresponding control values by multiple Dunnett’s tests (one-sided, alpha = 0.05).

Table 1: Biomass of Algae 

Treatment / Dilution	Rep. no.	Biomass of algae* (relative Fluorescence units)
		24 hours	48 hours	72 hours
Control	1 2 3 4 5 6	2.7 2.7 2.6 3.0 3.1 3.0	15.6 15.1 17.6 16.5 17.8 16.3	56.7 64.7 61.0 69.6 54.0 59.2
	Mean SD	2.8 0.2	16.5 1.1	60.9 5.6
1:21	1 2 3	2.6 2.8 3.1	17.5 17.8 18.6	71.9 54.4 70.1
	Mean SD	2.8 0.3	18.0 0.5	65.5 9.6
1:10	1 2 3	2.8 3.0 3.0	16.8 17.5 15.3	58.5 56.2 55.8
	Mean SD	3.0 0.1	16.5 1.2	56.8 1.5
1:4.6	1 2 3	2.6 3.2 3.1	14.1 18.6 13.5	55.5 63.6 47.5
	Mean SD	3.0 0.3	15.4 2.8	55.6 8.0
1:2.1	1 2 3	2.4 2.3 2.4	13.2 16.0 14.8	51.0 47.9 44.8
	Mean SD	2.4 0.0	14.7 1.4	47.9 3.1
Saturated solution	1 2 3	2.0 2.4 2.6	7.8 9.3 7.4	8.4 8.3 7.8
	Mean SD	2.3 0.3	8.2 1.0	8.2 0.3

SD: Standard deviation

*: The biomass was determined by fluorescence measurement (duplicate measurements) and is given as relative fluorescence units (x 10³). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 0.82 x 10³ relative fluorescence units).

 

Table 2: Areas under the Growth Curves (AUC)

Treatment / Dilution	Loading rate (mg/L)	Areas under the growth curves AUC (10³*day) And inhibition of AUC (IAUC)
		0 - 24 hours		0 - 48 hours		0 - 72 hours
		AUC	IAUC(%)	AUC	IAUC(%)	AUC	IAUC(%)
Control	-	1.0	0.0	9.9	0.0	47.7	0.0
1:21	4.8	1.0	1.0	10.6	-7.3	51.5	-7.9
1:10	10	1.1	-5.9	10.0	-1.5	45.9	3.9
1:4.6	22	1.1	-5.8	9.4	4.3	44.1	7.6
1:2.1	48	0.8*	22.4	8.5	13.9	38.9*	18.4
Saturated solution	100	0.8*	25.4	5.2*	47.3	12.6*	73.7

*: mean value significantly lower than in the control

(according to Dunnett’s tests, one-sided, alpha = 0.05)

Table 3: Average Growth Rates (µ) 

Treatment / Dilution	Loading rate (mg/L)	Average growth rate μ (day^-1) and inhibition of μ (Ir)
		0 - 24 hours		0 - 48 hours		0 - 72 hours
		µ	Ir (%)	µ	Ir (%)	µ	Ir (%)
Control	-	1.24	0.0	1.50	0.0	1.44	0.0
1:21	4.8	1.24	0.6	1.54	-2.9	1.46	-1.6
1:10	10	1.29	-3.5	1.50	-0.1	1.41	1.5
1:4.6	22	1.28	-3.2	1.46	2.6	1.40	2.2
1:2.1	48	1.07*	13.8	1.44	4.0	1.36*	5.5
Saturated solution	100	1.04*	16.4	1.15*	23.4	0.77*	46.6

*: mean value significantly lower than in the control

(according to Dunnett’s tests, one-sided, alpha = 0.05)

  

Table 4: Yield (Y) 

Treatment / Dilution	Loading rate (mg/L)	Yield Y (x 10³) and inhibition of Y (Iy)
		0- 2 4 hours		0 - 48 hours		0 - 72 hours
		Y	Iy (%)	Y	Iy (%)	Y	Iy (%)
Control	-	2.0	0.0	15.7	0.0	60.0	0.0
1:21	4.8	2.0	1.0	17.2	-9.5	64.6	-7.7
1:10	10	2.1	-5.9	15.7	-0.3	56.0	6.7
1:4.6	22	2.1	-5.8	14.6	6.9	54.7	8.8
1:2.1	48	1.6*	22.4	13.8	11.6	47.1*	21.6
Saturated solution	100	1.5*	25.4	7.4*	53.0	7.3*	87.8

 *: mean value significantly lower than in the control

(according to Dunnett’s tests, one-sided, alpha = 0.05)

Table 5: Section-by-section growth rates

Treatment / Dilution	Loading rate (mg/L)	Section-by-section growth rates (day^-1) and inhibition of the growth rates (Ir)
		0 - 24 hours		24 - 48 hours		48 - 72 hours
		µ	Ir (%)	µ	Ir (%)	µ	Ir (%)
Control	-	1.24	0.0	1.76	0.0	1.30	0.0
1:21	4.8	1.24	0.6	1.85	-5.4	1.29	1.5
1:10	10	1.29	-3.5	1.72	2.3	1.24	5.3
1:4.6	22	1.28	-3.2	1.64	6.6	1.29	1.4
1:2.1	48	1.07	13.8	1.81	-3.0	1.19	9.1
Saturated solution	100	1.04	16.4	1.26	28.4	0.00	99.8

 

Table 6: Phosphate concentrations in the test media and in the control

Treatment / Dilution	Loading rate (mg/L)	Phosphate (mg PO4/L)
		0 hours			24 hours			48 hours			72 hours
		Sample 1 + 2		mean	Sample 1 + 2		mean	Sample 1 + 2		mean	Sample 1 + 2		mean
Control	-	1.24	1.24	1.24	0.99	1.03	1.01	0.80	0.76	0.78	0.07	0.19	0.13
1:21	4.8	1.12	1.13	1.13	1.11	1.07	1.09	0.75	0.82	0.78	0.13	0.06	0.09
1:10	10	1.09	1.01	1.05	0.98	0.99	0.99	0.75	0.76	0.75	< 0.03	< 0.03	< 0.03
1:4.6	22	0.93	0.93	0.93	0.80	0.88	0.84	0.61	0.58	0.59	< 0.03	< 0.03	< 0.03
1:2.1	48	0.60	0.62	0.61	0.59	0.46	0.53	0.25	0.21	0.23	< 0.03	< 0.03	< 0.03
Saturated solution	100	< 0.03	< 0.03	< 0.03	< 0.03	< 0.03	< 0.03	< 0.03	< 0.03	< 0.03	< 0.03	< 0.03	< 0.03

 

Validity criteria fulfilled:

yes

Remarks:

The control biomass is multiplied by 74 over 72 hours (threshold >16), the mean coeff. of variation of the daily growth rates was 20 % (threshold <35 %), and the coeff. of variation of the average specific growth rates was 2.1 % (threshold <7 %)

Conclusions:

The test material had a statistically significant inhibitory effect on the growth (AUC, growth rate and yield) of Scenedesmus subspicatus after the test period of 72 hours at the loading rate of 48 mg/L and above. Thus, this loading rate was determined as the 72 hour LOELR. The 72 hour NOELR was determined to be the loading rate of 22 mg/L. The 72 hour EL50 was > 100 mg/L. However, a significant phosphate depletion was observed at the highest tested concentrations, probably due to a complexation process with the test material. As a consequence, it is possible that the reduction of growth observed was caused by an indirect effect of phosphate lack, rather than a toxic effect of the test material.

Executive summary:

In a 72 hour toxicity study, cultures of the green algal species Scenedesmus subspicatus were exposed to the test material at the loading rates of 4.8, 10, 22, 48 and 100 mg/L under static conditions in accordance with the standardised guidelines OECD 201 and EU Method C.3.

The NOELR, the LOELR and EL50 values based on the growth rate were 22 mg/L, 48 mg/L and > 100 mg/L, respectively.

Additional remark:

The analysis of the impact of the loading rate on the phosphate amount in the test media showed that the saturated solution had a statistically significant impact on the phosphate concentration in the test medium at all time points (except at 72 hours, where the loss of phosphate over time had a stronger impact on the phosphate concentration than the test item). The dilution 1:2.1 (loading rate of 48 mg/L) had a statistically significant impact on the phosphate concentration at 0 and 48 hours. At test start, this impact was significant even at the dilution1:10 (loading rate of 10 mg/L) and above. The depletion of phosphate in the test medium during the test might have been the reason for the inhibition of algal growth determined at these test concentrations. As a result, growth inhibition due to a secondary effect (i.e. the complexation of the essential algal nutrient phosphate by the test item) cannot be excluded.

Description of key information

The NOELR, the LOELR and EL50 values based on the growth rate were 22 mg/L, 48 mg/L and > 100 mg/L, respectively. It is important to note that a significant phosphate depletion was observed at the two highest tested concentrations (saturated solution: loading rate = 100 mg/L, dilution 1:2.1: loading rate = 48 mg/L), probably due to a complexation process with the test item. As a consequence, the reduction of growth here observed, and leading to the LOELR and NOELR above cited, was probably caused by an indirect effect of phosphate lack, rather than a toxic effect of praseodymium oxide.  

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

22 mg/L

Additional information

The key study was conducted in line with GLP and the standardised guidelines OECD 201 and EU Method C.3. It was assigned a reliability score of 1 in accordance with the criteria detailed by Klimisch (1997).

In a 72 hour toxicity study, cultures of the green algal species Scenedesmus subspicatus were exposed to the test material at the loading rates of 4.8, 10, 22, 48 and 100 mg/L under static conditions.

The NOELR, the LOELR and EL50 values based on the growth rate were 22 mg/L, 48 mg/L and > 100 mg/L, respectively.