Zirconium zircon with encapsulated cadmium selenium sulphide

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Series on testing and assessment, Number 29. Guidance document on transformation/dissolution of metals and metal compounds in aqueous media. ENV/JM/MONO (2001)9, 23-July 2001.

Version / remarks:

According to OECD Series on Testing and Assessment # 29 (2001), “For hazard classification purposes the results of the dissolution/transformation protocol are compared with existing ecotoxicity data for metals and metal compounds. However, for purposes such as data validation, there might be cases where it may be appropriate to use the aqueous medium from a completed transformation test directly in an OECD 202 and 203 daphnia and fish ecotoxicity test. If the CaCl2.2H2O and MgSO4.7H2O concentrations of the transformation medium are reduced to one-fifth of the ISO 6341 medium, the completed transformation medium can also be used (upon the addition of micronutrients) in an OECD 201 algae ecotoxicity test.” Therefore, to assess the hazard potential of this pigment containing several different metals, an ecotoxicity validation test was performed applying the dissolved fraction collected after 7-d of transformation/dissolution of the pigment in the respective ecotoxicity medium.

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2011-02-07

Analytical monitoring:

yes

Details on sampling:

Dissolved cadmium, selenium and zirconium concentrations were measured in all test solu-tions at the beginning of the test prior to the addition of the algae and in samples of the algae cultures taken from replicates pooled per treatment at the end of the test.

Duplicate samples of approximately 20 mL were taken. The samples were filtered by means of 0.2 µm polyether sulphone membrane syringe filter at room temperature (20-25°C), transferred into disposable polyethylene vials (Scintillation vials, Sarstedt, Nuembrecht, Germany), acidified with HNO3 (final concentration 1 % HNO3), and stored until chemical analysis. The concentra¬tions of dissolved cadmium, selenium and zirconium were determined by ICP-MS. The results were assured by recovery experiments and measurement of certified reference materials.

Details on test solutions:

The preparation of the applied test media was performed following to OECD 29 ‘Guidance document on transformation / dissolution of metals and metal compounds in aqueous media’, (2001; document ENV/JM/MONO(2001)9) [12]. This guidance was the basis of pre-treatment of the test item for setting up the concentration in the test media.
For the test 0.05, 0.5, 5.0, 50 and 500 mg of IPC-2013-001 (Silicic acid, zirconium salt, cadmium pigment-encapsulated) were transferred into glass flasks and brought to the volume of 500 mL with growth medium to obtain nominal loadings of 0.1, 1.0, 10.0, 100 and 1000 mg/L (Table 6).
All flasks were agitated on laboratory shakers (Gerhardt, Laboshake RO 500, Königswinter, Germany and Sartorius Certomart SII, Sartorius AG, Goettingen, Germany) at 100 rpm at 21.5 + 1.5°C. After 7 days the media were filtrated and applied for the following algal test.

Initial weight of test item into daphnia test media.
Media Nominal loading [mg/L] Loading in mg/0.5 L media
Algae test media 0 0
Algae test media 0.1 0.050
Algae test media 1 0.502
Algae test media 10 5.001
Algae test media 100 50.002
Algae test media 1000 500.0

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata, Chlorophycea, Chlorophyta.
Origin: SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen, Catalog No 61.81 SAG.
Cultivation: The stock cultures are maintained fulfilling the criteria of the OECD 201 guideline [5]. Prior to testing a pre-culture will be established in standard OECD growth medium to obtain exponentially-growing algae for the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

no data

Test temperature:

Temperature was between 21 °C and 22 °C.

pH:

The pH of the controls was 8.0 at the beginning of the test and ranged between 7.95 – 8.30 at test end.

Dissolved oxygen:

not applicable

Salinity:

not applicable

Nominal and measured concentrations:

Measured selenium, cadmium and zirconium concentrations concentrations day 0 – day 3 in filtered test samples

Loading of test Se Cd Zr Se Cd Zr
item, nominal day 0 day 0 day 0 day 3 day 3 day 3
[mg/L] [µg/L] [µg/L] [µg/L] [µg/L] [µg/L] [µg/L]
0 0.82 0.03 0.1 0.21 0.04 1 0.85 0.06 10 0.37 0.85 0.04 0.26 0.81 0.34
100 1.62 1.79 1000 14.0 9.63 0.34 13.7 8.52 0.93
(internal calculation were performed with more digits, values were rounded)

Details on test conditions:

A sterilised synthetic OECD medium [4] was used as growth medium (Table 2). As stated in the guideline OECD 201, the molar ratio of EDTA to iron slightly exceeds unity in this medium. This prevents iron precipitation and at the same time, chelation of heavy metal ions is minimised (molar ratio EDTA / Fe(III+) = 1.135). Considering the molar ratio and the stability constants of EDTA with Fe(III+), Zr(IV+) and Cd(II+) ions (log Ks are 25.7, 27.7 – 29.5 and 17.4, respectively [13], [14], [15], [16], the chelation of zirconium and cadmium remains negligible in comparison with nominal test concentrations.

All stock solutions and the medium were prepared with purified water processed using a water purifier “Pure Lab Ultra”. The pH of the medium in the range of 7.5 to 8.0 was obtained at equilibrium between the carbonate system of the medium and the partial pressure of CO2 in atmospheric air.

Validity of the test
The algae growth inhibition test fulfils the validity criteria of OECD 201 [4]:
• The cell number in the control cultures increased by a factor of 171 within the test period of 72 h (validity criterion: > 16).
• Evaluation of the sectional growth rates of the controls:
The mean of the replicate coefficients of variations in the section-by-section growth rate of controls was 9.1 % (validity criterion  35 %).
• The coefficient of variation of average specific growth rate in replicate control cultures during the whole test period was 2.1 % (validity criterion  7%).

Test conditions
Light intensity, temperature, and pH-values of the cultures were in accordance with the OECD 201 guideline [4]. Light intensity ranged between 7353 - 7503 lux, temperature was between 21 °C and 22 °C. The pH of the controls was 8.0 at the beginning of the test and ranged between 7.95 – 8.30 at test end. In the treatments, the initial pH ranged between 7.98 and 8.13, at test end the pH was between 8.01 and 8.30. Refer to A.2.1 for raw data.

Test item concentrations
Dissolved cadmium, selenium and zirconium concentrations in the test media were assessed analytically by means of ICP-MS. The minimum level of quantification (LOQ) was
Se: 0.06 µg/L
Cd: 0.003 µg/L
Zr: 0.03 µg/L.
The substance was analysed in samples of freshly prepared test solutions and control solutions at test start, and from pooled replicates per test concentration and controls at test end. Se, Cd and Zr measurements of acidified samples were performed in less than three months after sampling. According to DIN EN ISO 5667-3: 2003 dissolved in aqueous samples (waste-, ground- and surface-water) metals are stable for at least six months.

Test vessels
Test vessels were sterile 250 mL conical glass flasks covered with air-permeable silicone-sponge caps. Test vessels were acid-washed with 10% HNO3 and then rinsed six times with purified water prior to use by autoclaving.

Incubation conditions
The culture vessels were continuously illuminated with a light intensity (day light: OSRAM “cool white”) adjusted to 4440 - 8880 lux close to the surface of the liquid (equivalent to 60-120 µE m-² s-²). Light measurements [lux] were made using a cosine (2 π) receptor (LI-189 with radiation sensor, LI-COR) each day. The test culture temperature was maintained at 22.0°C ± 2°C, and was also measured in one test vessel per day. The pH of the solutions was measured in one test vessel per test concentration, including the control, at the start of the test and in every test vessel at the end of the test. The pH of the controls did not vary by more than 1.5 units. The position of each test vessel was randomised within the incubator and rearranged daily. The algae cultures were continuously re-suspended by rotation on an orbital laboratory shaker (150 rpm) (INFORS, Switzerland).

Determination of cell density
Cell concentrations were determined using an electronic particle counter (CASY 1 Model TT, Roche Diagnostics GmbH, Germany). The accuracy of the electronic counts was checked against manual cell counts under the microscope, as per internal standard operating procedures. The cell concentrations were determined after 24, 48 and 72 h in samples taken directly from the test vessels.

Test concentrations
Based on the results of the range finder test , the following WAF loadings of Silicic acid, zirconium salt, cadmium pigment-encapsulated were agreed upon by the monitor: 0.1, 1.0, 10.0, 100 and 1000 mg/L.
The study was performed with four replicates of each test concentration and eight replicates of the control.

Preparation of the test media
As the test item is a multi-component substance, its toxicity was determined using water ac-commodated fractions (WAFs). The WAFs for the various test concentrations were prepared individually in accordance with the OECD guidance document on transformation/ dissolution of metals and metal compounds in aqueous media. No. 29.

Amounts of 0.05, 0.5, 5.0, 50 and 500 mg test item were transferred into glass flasks and brought to the volume of 500 mL with growth medium to obtain nominal loadings of 0.1, 1.0, 10.0, 100 and 1000 mg/L, respectively. One flask (1000 mL) was filled with growth medium only for the control. Flasks were stirred at 100 rpm for 7 days at room temperature (~20°C). For a preparation of test solutions the insoluble parts of each WAF were separated from the aqueous phase by filtration through a 0.22 µm filter (Syringe Filters DIAFIL PS (Polyether sulfone, PES) DIA-Nielsen GmbH & Co. KG, Düren, Germany) under sterile conditions. The filtrated WAFs were directly used for the algal growth test.

Test performance
Eight test vessels were prepared for the control and four replicates for the test concentra¬tions. The test vessels allocated for the test concentration were filled with 100 mL of the filtered WAFs containing the test item. The blank control consisted of growth medium only.

The cell density of the inoculum culture was determined, and then an adequate aliquot of the inoculum culture (628.9 µL) was added into the test solutions to obtain the required cell density of 10,000 cells/mL.

The cell number of each test vessel was recorded after 24, 48 and 72 h in order to determine algal growth.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at a loading of 100 mg/L after 7 days (T/D fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at a loading of 1000 mg/L after 7 days (T/D fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at a loading of 1000 mg/L after 7 days (T/D fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at a loading of 100 mg/L after 7 days (T/D fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at a loading of 1000 mg/L after 7 days (T/D fraction)

Basis for effect:

biomass

Remarks:

(inhibition of yield)

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at a loading of 1000 mg/L after 7 days (T/D fraction)

Basis for effect:

biomass

Remarks:

(inhibition of yield)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at a loading of 100 mg/L after 7 days (T/D fraction)

Basis for effect:

biomass

Remarks:

(inhibition of yield)

Details on results:

The algae growth inhibition test fulfils the validity criteria of OECD 201:
• The cell number in the control cultures increased by a factor of 171 within the test period of 72 h (validity criterion: > 16).
• Evaluation of the sectional growth rates of the controls:
The mean of the replicate coefficients of variations in the section-by-section growth rate of controls was 9.1 % (validity criterion  35 %).
• The coefficient of variation of average specific growth rate in replicate control cultures during the whole test period was 2.1 % (validity criterion  7%).

Measured selenium, cadmium and zirconium concentrations concentrations day 0 – day 3 in filtered test samples

Loading of test Se Cd Zr Se Cd Zr
item, nominal day 0 day 0 day 0 day 3 day 3 day 3
[mg/L] [µg/L] [µg/L] [µg/L] [µg/L] [µg/L] [µg/L]
0 0.82 0.03 0.1 0.21 0.04 1 0.85 0.06 10 0.37 0.85 0.04 0.26 0.81 0.34
100 1.62 1.79 1000 14.0 9.63 0.34 13.7 8.52 0.93
(internal calculation were performed with more digits, values were rounded)

Results with reference substance (positive control):

The sensitivity of the test organism is routinely checked using 3,5-dichlorophenol as primary standard following internal SOPs. The tests are conducted as non-GLP studies according to the OECD guideline 201. The nominal ErC50 value of 2.42 mg/L (95% confidence limits 1.95 – 3.03 mg/L) is in good agreement with the results of an international ring test with ErC50 of 3.38 ± 1.30 mg/L

Recoveries of fortified test medium

Sample	Element	Measured concentration [µg/L]	Calculated level after addition [µg/L]	Recovery [µg/L] / [%]
1000 mg test substance/L A d 3	Se	13.7	19.8	22.3 / 113
1000 mg test substance/L A d 3 aged	Cd	8.5	16.4	17.2 / 105
1000 mg test substance/L A d 0	Zr	0.3	8.6	8.3 / 96.7

 

Method validation summary

Validation parameter	Results	Comment
Selectivity	similar data with different isotopes and gas modes for ICP-MS method	no interferences due to test media observed
Linearity	applied calibration functions were linear	seeTable22correlation coefficient
Limit of detection (LOD)	Se: 0.02 µg/L Cd: 0.001 µg/L Zr: 0.01 µg/L	seeTable22
Limit of quantification (LOQ)	Se: 0.06 µg/L Cd: 0.003 µg/L Zr: 0.03 µg/L	seeTable22
Reagent blanks	Se: < LOD = < 0.02 µg/L (n =7) Cd: < LOD/LOQ = < 0.001 / 0.003 µg/L (n = 6) One reagent blank above LOQ (0.005 µg/L) Zr: < LOD/LOQ = < 0.01 / 0.03 µg/L (n = 4) In three reagent blanks above LOQ (0.04 µg/L; 0.13 µg/L; 0.41 µg/L)	-
Accuracy and precision	Se: mean recovery for TMDA-70 (diluted to 2.58 µg Se/L): 103 ± 4 % (n = 3) Cd: mean recovery for TMDA-70 (diluted to 14.5 µg Cd/L): 97.2 ± 4.6 % (n = 3)	corresponds to concentration range of samples
Accuracy and precision	Se: mean recovery for TM 15.2 (diluted to 7.55 µg Se/L): 114 ± 7.2% (n = 3) Cd: mean recovery for TM 15.2 (diluted to 6.5 µg Cd/L): 106 ± 1.3 % (n = 3)	corresponds to concentration range of samples
Accuracy and precision	mean recovery for recalibration standard CPI (diluted to 10 µg Se/L): 93.8 ± 1.6 (n = 3) mean recovery for recalibration standard CPI (diluted to 10 µg Cd/L): 88.5 ± 6.1 (n = 3)	corresponds to concentration range of samples
Accuracy and precision	mean recovery for Zr recalibration (diluted to 1 µg Zr/L): 95.1 ± 19.3 % (n = 3)	corresponds to concentration range of samples
Accuracy and precision	mean recovery for Zr recalibration (diluted to 5 µg Zr/L): 89.8 ± 6.6 % (n = 3)	corresponds to concentration range of samples
Trueness	recovery from fortification of samples 96.7 – 113 % (n=3)

Validity criteria fulfilled:

yes

Remarks:

see "Details on result"

Conclusions:

In this study the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (nominal concentrations): The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 hours were >1000 mg test item/L and >1000 mg test item/L, respectively. The NOEC-values for the inhibition of specific growth rate and yield after 72 hours were 100 and 100 mg test item/L, respectively. All effect values are given based on nominal concentrations of the test substance.

Executive summary:

The toxic effects of silicic acid, zirconium salt, cadmium pigment encapsulated on the growth rate of algae (Pseudokirchneriella subcapitata) were studied in a state-of-the-art test according to OECD Guideline 201 (Wenzel, 2013). The EC50 value for the 72-h inhibition of growth rate of P. subcapitata is  > 1000 mg/L (dissolved fraction at a loading of 1000 mg/L after 7 days of dissolution).

.

 

The long-term toxic effects of silicic acid, zirconium salt, cadmium pigment encapsulated on the growth rate of algae (Pseudokirchneriella subcapitata) were studied in a state-of-the-art test according to OECD Guideline 201 (Wenzel, 2013). The EC10 value for the 72-h inhibition of growth rate of P. subcapitata is  > 100 mg/L (dissolved fraction at a loading of 100 mg/L after 7 days of dissolution).

Description of key information

The results of a key study with Zirconium zircon with encapsulated cadmium selenium sulphide indicate low potential for toxicity to freshwater algae. The respective 72h EC50 of Zirconium zircon with encapsulated cadmium selenium sulphide on growth rate of freshwater algae (Pseudokirchneriella subcapita), derived from a GLP-conform OECD 201 test, is > 1000 mg/L (dissolved fraction of Zirconium zircon with encapsulated cadmium selenium sulphide at a loading of 1000 mg/L after stirring for 7 days). The respective 72 h EC10 of Zirconium zircon with encapsulated cadmium selenium sulphide on growth rate of freshwater algae (Pseudokirchneriella subcapita) is > 100 mg/L (dissolved fraction of Zirconium zircon with encapsulated cadmium selenium sulphide at a loading of 100 mg/L after stirring for 7 days) (Wenzel, 2013). All effect values are given based on nominal concentrations of the test substance. Nevertheless, read-across to the assessment entity soluble cadmium substances is applied since cadmium cations determine fate and toxicity in the environment.