Yttrium oxide (Y2O3), europium-doped

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:

17 February - 20 February 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study was performed according to OECD Guideline No. 201 and in accordance with GLP principles. The study is considered to be reliable with restrictions due to the absence of KH2PO4 in the test medium in the full test. This approach was chosen because Yttrium Oxide, Europium-doped was found to remove phosphate from aqueous solutions and consequently it was not possible to determine the real toxicity of Yttrium Oxide, Europium-doped to algae in an earlier test.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(2006)

Deviations:

yes

Remarks:

see principles of method

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(2009)

Deviations:

yes

Remarks:

see principles of method

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Version / remarks:

(2004)

Deviations:

yes

Remarks:

see principles of method

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23 (2000)

Principles of method if other than guideline:

As a member of the rare earth oxides family, yttrium oxide may remove phosphate from aqueous solutions.
In order to investigate the nature of the effects of Yttrium Oxide, Europium-doped on algae growth, two parallel tests were performed: a full test in medium without phosphate, and a limit test in medium spiked with phosphate. Effects can be direct, i.e. toxicity of Yttrium Oxide, Europium-doped, or indirect, i.e. depletion of phosphate from the test medium caused by the test substance.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Singular samples for possible analysis were taken from all test concentrations and the controls according to the schedule below. In addition, the filter containing undissolved residue was retained for possible analysis.
- Nominal concentrations (final test): 0 (blank control), 0 (extra control without KH2PO4), 10, 18, 32, 56 and 100% of a 0.45 μm filtered solution prepared at a loading rate of 100 mg test substance/L.
- Sampling method: pipetting specified volume from test vessels. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Frequency: at t=0 h, t=24 h and t=72 h
- Volume: 4.8 ml
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis (≤ -15°C).
- On the day of analysis, the samples (4.8 ml) were defrosted at room temperature. Samples were diluted in a 1:25 (v:v) ratio with HNO3 and analysed by aid of ICP-MS for yttrium. If necessary, the samples were further diluted with 4% (v/v) HNO3 in M2-medium to obtain concentrations within the calibration range.

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTIONS
- Method: Preparation of test solutions started with the highest loading rate of 100 mg test substance/L M2-medium containing no phosphate. A 15-minute period of treatment with ultrasonic waves was applied followed by a two-day period of magnetic stirring to ensure maximum dissolution of the test substance in the test medium. The resulting dispersion was subsequently filtered through a 0.45 µm membrane filter (Whatman, RC55) to remove the undissolved fraction of the test substance. The obtained Water Soluble Fraction (WSF) was divided into two parts.
* One part was used as highest test concentration in a full test. The lower test concentrations were prepared by subsequent dilutions of the filtrate in medium containing no phosphate. The final test solutions were all clear and colourless. The control without phosphate was treated in the same way.
* The second part of the WSF was spiked with 0.1 mL of pre-dissolved phosphate solution (16 g K2HPO4/L M2-medium) and used as the limit concentration in a limit test. The control was spiked with phosphate at the same rate. The final phosphate concentration was similar to the concentration usually applied in standard M2-medium (i.e. 1.6 mg/L).

APPLICATION OF TEST SOLUTIONS
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration, after which 1 mL of an algal suspension was added.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green microalgae
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60-720 µE/m^2/s at 400-700 nm) in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: Three days before the start of the test, cells from the algal stock culture were inoculated in pre-culture medium (M2) at a cell density of 1 x 10^4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Culturing media and conditions (same as test or not): yes for limit test (phosphate replenishing after preparation of test solution); no for the full test (M2 without phosphate)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

22°C (start)
21-22°C (during exposure period)

pH:

8.0-8.3 (limit test)
8.0-8.2 (full test)

Nominal and measured concentrations:

Limit test:
- Nominal concentrations: blank control (phosphate-free M2-medium spiked with KH2PO4), 100% of a 0.45 μm filtered solution prepared at a loading rate of 100 mg test substance/L (WSF) and spiked with KH2PO4.
- Measured concentrations: 0 and 0.17 mg Y/L (0-72 h TWA)

Full test:
- Nominal concentrations: blank control (phosphate-free M2-medium), 10, 18, 32, 56 and 100% of a 0.45 μm filtered solution prepared at a loading rate of 100 mg test substance/L (WSF) in phosphate-free medium.
- Measured concentrations: < 0, 0.023, 0.041, 0.059, 0.12 and 0.23 mg Y/L (0-72 h TWA)

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass, with 100 mL capacity and filled with 50 mL of test solution
- Type (delete if not applicable): closed (capped)
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
Limit test (medium spiked with phosphate)
- Control end cells density (72 h): 190.4 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
Full test (medium without phosphate)
- Control end cells density (72 h): 21.7 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (M2)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap-water purified by reverse osmosis.
- Culture medium different from test medium: no.
- Intervals of water quality measurement:
* pH: at the start and end of test.
* Temperature of medium: Continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: 84 to 87 µE/m^2/s; TLD-lamps

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank and in the full test the extra replicates as background for the treated solutions.
- Appearance of the cells: At the end of the test microscopic observations were performed on the highest test concentrations from both tests (i.e. medium w/o phosphate, and spiked with phosphate) to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 1.8
Limit test
- Test concentrations: 0 mg/L, loading rate 100 mg test substance/L (= 0.17 mg Y/L TWA)
Full test
- Test concentrations: 0 mg/L, loading rates at 10, 18, 32, 56 and 100% of 100 mg test substance/L (= 0.023, 0.041, 0.059, 0.12 and 0.23 mg Y/L TWA, resp.)

Reference substance (positive control):

yes

Remarks:

K2Cr2O7 (Potassium dichromate)

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.17 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

element

Remarks:

Ytrium

Basis for effect:

growth rate

Remarks on result:

other: Limit test

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.17 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

element

Remarks:

Ytrium

Basis for effect:

other: growth rate, yield

Remarks on result:

other: Limit test

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.23 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

element

Remarks:

Ytrium

Basis for effect:

growth rate

Remarks on result:

other: Full test

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.091 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

element

Remarks:

Yttrium

Basis for effect:

growth rate

Remarks on result:

other: Full test

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.059 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

element

Remarks:

Yttrium

Basis for effect:

other: growth rate, yield

Remarks on result:

other: Full test

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Full test; calculated based on MWs of element and test substance

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Full test; calculated based on MWs of element and test substance

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.077 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Full test; calculated based on MWs of element and test substance

Details on results:

1. Limit test
- Exponential growth in the control (for algal test): yes (factor is 58.9 after 48 h)
- Observation of abnormalities (for algal test): no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: yes

The coefficient of variation for section-by-section growth rates in the control was 43.2% over a 72 h period, indicating that growth was not exponential during the whole exposure period. Therefore, it was decided to calculate the effect parameters on the basis of a 48-h exposure period, where the coefficient of the section-by-section growth rates was 39.5%. This value is considered acceptable in light of the fact that the other two validity criteria of the test were fulfilled. No significant differences were recorded between the values for growth rate and yield at the highest test concentration when compared to the control group during the 48-h exposure period.

Acceptability of the test (based on 48-h test period as growth was not exponential for the whole 72-h period):
a) In the control, cell density increased by an average factor of >16 within 2 days.
b) The mean coefficient of variation for section-by-section growth rates in the control cultures exceeded 35% (i.e. 39.5%). However, since other criteria were met, this was considered acceptable.
c) The coefficient of variation of average specific growth rates during the test period in replicate control did not exceed 7% (i.e. 2.5%).

2. Full test
- Exponential growth in the control (for algal test): yes (factor is 21.7 after 72 h)
- Observation of abnormalities (for algal test): no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: yes

Growth rates and yields were in the range of the controls at the three lowest test concentrations during the 72-h period, whereas the growth rate of algae exposed to the two highest concentrations were increasingly reduced. Statistically significant reduction was observed at the two highest concentrations. (see any other information on results incl. tables).

Acceptability of the test (based on 72-h test period):
a) In the control, cell density increased by an average factor of >16 within 2 days.
b) The mean coefficient of variation for section-by-section growth rates in the control cultures exceeded 35% (i.e. 102%). However, it was expected that the growth in the test without phosphate would not be exponential as phosphate is an important nutrient for algal growth. Therefore, this does not invalidate the test.
c) The coefficient of variation of average specific grwoth rates during the test period in replicate control did not exceed 7% (i.e. 1.6%).

Results with reference substance (positive control):

- K2Cr2O7 (Potassium dichromate): The EC50 for growth rate reduction (ErC50: 0-72h) was 1.3 mg/L with a 95% confidence interval ranging from 1.3 to 1.4 mg/L. These values are within the historical ranges obtained for the positive control with the in-house algal laboratory culture.

Reported statistics and error estimates:

- Determination of the NOEC and calculation of the EC50
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used.
* Limit test: the substance proved to be non-toxic (EC50 > maximum soluble concnetration, thus EC50 could not be calculated
* Full test: An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or inhibition of yield (Welch-t-test for Inhomogeneous Variances with Bonferroni-Holm adjustment, alpha = 0.05, one-sided, smaller). Note that the effects were determined by comparing the Yttrium Oxide, Europium-Doped treated groups with blank control. Calculation of the EC50 values was based on probit analysis using linear maximum likelihood regression with the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding time weight average (TWA) exposure concentrations of the test substance. Calculations were performed with ToxRat Professional v, 2.10.05.

Limit test (48 h test period, test medium replenished with phosphate)

 

Percentage inhibition of growth rate

Ytrium TWA concentration (mg Y/L)	Mean (day^-1)	Std. Dev.	n	% Inhibition
Control	2.035	0.0512	6	0.0
0.17	2.133	0.0269	6	-4.8

 

Percentage inhibition of yield

Ytrium TWA concentration (mg Y/L)	Mean	Std. Dev.	n	% Inhibition
Control	57.87	6.019	6	0.0
0.17	70.34	3.957	6	-21.5

 

Full test (72 h test period, phosphate-free test medium)

 

Percentage inhibition of growth rate (total test period)

Ytrium TWA concentration (mg Y/L)	Mean (day^-1)	Std. Dev.	n	% Inhibition
Control	1.026	0.0162	6	0.0
0.023	1.045	0.0214	3	-1.9
0.041	0.977	0.0563	3	4.8
0.059	0.954	0.0417	3	7.0
0.12	0.904	0.0158	3	11.9*
0.23	0.659	0.0139	3	35.7*

* effect was statistically significant

 

Percentage inhibition of yield

Ytrium TWA concentration (mg Y/L)	Mean	Std. Dev.	n	% Inhibition
Control	20.72	1.057	6	0.0
0.023	22.01	1.489	3	-6.2
0.041	17.90	3.036	3	13.6
0.059	16.59	2.148	3	19.9
0.12	14.07	0.722	3	32.1*
0.23	6.235	0.305	3	69.9*

* effect was statistically significant

 

Validity criteria fulfilled:

yes

Conclusions:

In this study with Pseudokirchneriella subcapitata it was shown that:
- Yttrium Oxide, Europium-Doped inhibits the growth rate and yield of this fresh water algae species significantly at a TWA concentration of 0.12 mg Y/L in medium without phosphate (corresponding to 0.16 mg test substance/L) (LOEC).
- In medium containing phosphate, Yttrium Oxide, Europium-Doped does not inhibit growth and yield of Pseudokirchneriella subcapitata at its limit of solubility in medium.

Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201, freshwater algae (Pseudokirchneriella subcapitata) were exposed to Yttrium Oxide, Europium-Doped under static conditions. Two tests were performed: 1. a limit test with phosphate replenished test medium (blank control and 100% of a 0.45 μm filtered solution prepared at a loading rate of 100 mg /L), and 2. a full test in phosphate-free test medium at the following nominal concentrations: control without KH2PO4, 10, 18, 32, 56 and 100% of a 0.45 μm filtered solution prepared at a loading rate of 100 mg /L. The full-test endpoints for growth rate reduction are as follows:

72 h-ErC50: >0.23 mg Y/L based on time weighted average (TWA) exposure concentrations (corresponding to >0.30 mg test substance/L),

72 h-ErC10: 0.091 mg Y/L based on TWA exposure concentrations (corresponding to 0.12 mg test substance/L),

72 h-NOErC: 0.059 mg Y/L, based on TWA exposure concentrations (corresponding to 0.077 mg test substance/L).

The study is considered to be reliable with restrictions due to the absence of KH2PO4 in the test medium in the full test. This approach was chosen because Yttrium Oxide, Europium-doped was found to remove phosphate from aqueous solutions and consequently it was not possible to determine the real toxicity of Yttrium Oxide, Europium-doped to algae in an earlier test with regular test water.

Description of key information

In a 72 h toxicity study conducted according to OECD guideline 201, freshwater algae (Pseudokirchneriella subcapitata) were exposed to Yttrium Oxide, Europium-Doped under static conditions. Endpoints are expressed in terms of time weighted average (TWA) concentrations.
72 h-ErC50: >0.23 mg Y/L, corresponding to >0.30 mg test substance/L (inhibition of growth rate during test period: 35.7%)
72 h-ErC10: 0.091 mg Y/L, corresponding to 0.12 mg test substance/L
72 h-NOErC: 0.059 mg Y/L, corresponding to 0.077 mg test substance/L

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.12 mg/L

Additional information

Phosphorous (provided through phosphate) is one of the critical nutrients for algal growth in aquatic systems. Also, it is known from literature, and practice in waste water treatments, that rare earth oxides and salts are able to remove excess phosphate from aqueous solutions (ex. La^[1],[2]). The process involves adsorption of phosphate onto the surface of the rare earth oxide beads.^[3]-[7] It seems that the adsorption process is selective for phosphate.

 

Two studies were performed according to OECD 201 under static conditions:

1.    First study

Since only little phosphate is present in test water (max 2 mg/L in M2), it can be assumed that all phosphate is captured by/adsorbed onto the yttrium oxide beads during preparation of the test solution at a loading rate of 100 mg/L, leaving the undiluted filtrate without any phosphate (a crucial nutrient for algae). All other test solutions are dilutions of the undiluted filtrate with test medium, i.e. the higher the dilution factor the more phosphate is present in test solutions. Thus, in dilutions of the WSF, the algal growth should not be inhibited significantly, whereas algal growth is expected to be reduced in the undiluted filtrate due to insufficient nutrition (phosphate) for algae, possibly in combination with intrinsic toxicity of the substance. Therefore, controls with and without KH2PO4 were used in order to gain insight on the impact of substance-related phosphate removal from test medium on algae growth.

Nominal test concentrations: blank control, control without KH2PO4, 10, 18, 32, 56 and 100% of a 0.45 μm filtered solution prepared at a loading rate of 100 mg/L, corresponding average concentrations were 0.011, 0.021, 0.041, 0.079 and 0.15 mg Y/L, respectively.

 

Based on the uncertainty of the cause of the effects seen in the test system during the first study, a second algae study was performed.

 

2.    Second study:

A bulk test solution at a loading rate of 100 mg substance/L was prepared in test medium prepared without phosphate. Prior to testing the filtrates (WSF) of this test solution it was split into two portions: one part was used in a limit test, and the other part in a full test with various dilutions of the 100% WSF solution in medium without phosphate. The limit test was performed at 100% of the WSF and the test solution was replenished with phosphate to yield levels commonly used in M2 media, just before addition of test organisms.

Nominal test concentration (prepared in M2-medium without phosphate):

- limit test: blank control (replenished with phosphate), 100% of a 0.45 μm filtered solution prepared at a loading rate of 100 mg/L (replenished with phosphate), corresponding to a time weighted average (TWA) concentration of 0.17 mg Y/L.

- full test: control without KH2PO4, 10, 18, 32, 56 and 100% of a 0.45 μm filtered solution prepared at a loading rate of 100 mg/L, corresponding time weighted average (TWA) concentrations were 0.023, 0.041, 0.059, 0.12 and 0.23 mg Y/L, respectively.

 

Comparison of the test results from both algae studies shows that observed effects (toxicity) in the first test are more severe. However, in view of the phosphate depleting properties of the substance, the toxicity may be ascribed to a combination of effects, i.e. phosphate depletion (indirect) and intrinsic toxicity (direct) of the substance. This finding is supported by the reported growth rate reduction in both tests in undiluted filtrates (100% of WSF prepared at a loading rate of 100 mg/L): 64.8% in study 1, where phosphate was present during preparation of the test solution, and 35.7% in study 2, where phosphate was deliberately excluded from test solutions.

Thus, the full test of study 2 appears to best reflect the actual intrinsic toxicity of the substance. Therefore the results from this study are used as key information for intrinsic properties of the substance: 72-h ErC50 > 0.23 mg Y/L or > 0.30 mg test substance/L, 72-h ErC10 = 0.091 mg Y/L or 0.12 mg test substance/L, 72-h NOEC = 0.059 mg Y/L or 0.077 mg test substance/L.

 

[1] P. Ninget al.; Journal of Environmental Sciences 20 (2008), 670–674

[2] E.V. Kleberet al.; US Patent 3,956,118 (1976)

[3] S. Recillaset al.;Water Science and Technology (2012), 66(3), 503-509.

[4] P.Satapathyet al.; Asian Journal of Chemistry (2011), 23(7), 3055-3058

[5] H. Guoet al.;Rare Metals (Beijing, China) (2011), 30(1), 58-62

[6] L. Liuet al.;Anquan Yu Huanjing Xuebao (2007), 7(2), 66-67

[7] B.M. Shukla and R.S. Tripathi;Proc. Chem. Symp., 1st (1970), 2, 95-101