Dimolybdenum trizinc nonaoxide

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, 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: All concentrations analysed for zinc and molybdenum.
- Sampling method: Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were analyzed immediately, samples were acidified by the chemist before analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (the first approximate 100 mL used to pre condition the filter was discarded) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 0.48, 1.0, 2.2, 4.8 and 10% v/v saturated solution. An aliquot (800 mL) of each of the stock solutions was separately inoculated with 5.5 mL of algal suspension to give the required test concentrations of 0.48, 1.0, 2.2, 4.8 and 10% v/v saturated solution. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

other: Raphidocelis subcapitata (formally Pseudokirchneriella subcapitata)

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata (formally Pseudokirchneriella subcapitata).
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): Not specified
- Method of cultivation: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1°C until the algal cell density was approximately 10^4 to 10^5 cells/mL.

ACCLIMATION
- Acclimation period:Acclimated until the algal cell density reached approximately 10^4 to 10^5 cells/mL.
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: Not specified

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

The water hardness (calculated) of the test water will be 0.15 mmol/L (= 15 mg/L as CaCO3).

Test temperature:

Temperature was maintained at 24 ±1ºC throughout the test.

pH:

7.43 to 8.54 in test vessels and 7.94 to 8.57 in control vessels. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guideline.

Dissolved oxygen:

Not measured

Salinity:

Not applicable

Conductivity:

Not measured

Nominal and measured concentrations:

Nominal: 0.48, 1.0, 2.2, 4.8 and 10 mg/L.
Measured (0 hour) zinc concentrations: 0.0312, 0.0646, 0.138, 0.295 and 0.667 mg/L.
Measured (0 hour) molybdenum concentrations: 0.0446, 0.0912, 0.203, 0.438 and 0.973 mg/L.
Calculated concentration of the test substance in the sample based on the measured (0 hour) zinc component: 0.0846, 0.176, 0.374, 0.800 and 1.81 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass conical flask
- Type (delete if not applicable): open (plugged with polyurethane foam bungs)
- Material, size, headspace, fill volume: 250 mL flasks filled with 100 mL test media.
- Aeration: No
- Initial cells density: 5x10^3 cells/mL
- Control end cells density: 1.58 to 1.88^5 cells/mL.
- No. of organisms per vessel: Cell density varied
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes but with modifications to accound for the potential chelating effect of the test substance on essential ions required for algal growth. This is in accordance with the OECD Guideline 23.
- Detailed composition if non-standard medium was used: The culture medium used for both the second range-finding and definitive tests was the same as that used to maintain the stock culture however it was enhanced with 198.4 mg/L extra calcium chloride dihydrate (CaCl2.2H2O) in order to mitigate potential chelating effects of the test item. The initial range-finding test used culture media without the enhanced calcium chloride dehydrate.

TEST MEDIUM / WATER PARAMETERS
- As per OECD guideline

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 380 to 730 nm (intensity approximately 7000 lux).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 23, 49 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominal inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density. To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Standard as per OECD guidelines
- Range finding study
- Test concentrations: Initial range-finder; 0.10, 1.0 10 and 100 mg/L saturated solution. The same concentrations were used in the second range-finder although the media was fortified to investigate whether toxic effects were due to the test item forming complexes with the test media and thereby reducing the nutrients available for algal growth or if the test item itself was causing toxicity. The test media was enhanced with extra CaCl2.2H2O to overcome possible complexation effects.
- Results used to determine the conditions for the definitive study: Based on the results from the first and second range-finding tests the following test concentrations were assigned to the definitive test: 0.48, 1.0, 2.2, 4.8 and 10 mg/L saturated solution.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.72 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

Test item calculated based on zinc component

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.175 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

Test item calculated based on zinc component

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes

- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.48 and 1.0 mg/L saturated solutions; however, some cell damage was observed at 2.2 mg/L saturated solution and much cell debris and broken down burst cells were observed to be present in the test cultures at 4.8 and 10 mg/L saturated solutions.

- Any stimulation of growth found in any treatment: None noted

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72 Hour test period the control, 0.48 and 1.0 mg/L saturated solutions were observed to be green dispersions. At the test concentration of 2.2 mg/L saturated solution cultures were observed to be very pale green to pale green dispersions. The test concentrations 4.8 and 10 mg/L saturated solutions were observed to be clear colorless solutions.

Validation criteria: Cell concentrations of the control cultures increased by a factor of 199 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours was 5.00 x 10^3 cells per mL and mean cell density of control at 72 hours was 9.95 x 10^5 cells per mL. The mean coefficient of variation for section by section specific growth rate for the control cultures was 4% and hence satisfied the validation criterion given in the OECD Guideline which states that the mean must not exceed 35%. The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hours) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test. The resultant 72 hour ErC50 was 1.5 mg/L (1.3 to 1.7 mg/L). The NOEC was 0.50 mg/L. The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) A Dunnetts t test was carried out on the growth rate and yield data after 72 hours to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Effect values for growth rate and yield.

	Growth Rate				Yield
	EC50	95% Confidence Limits	NOEC	LOEC	EC50	95% Confidence Limits	NOEC	LOEC
Nominal Concentration (mg/L)	4.2	3.6 – 5.0	1.0	2.2	1.8	1.6 – 2.0	1.0	2.2
Zinc Measured Concentrations (mg/L)	0.27	0.23 – 0.31	0.0646	0.138	0.11	0.10 – 0.13	0.0646	0.138
Test Item calculated Concentrations (mg/L)	0.72	0.61 – 0.83	0.175	0.374	0.31	0.28 – 0.35	0.175	0.374

Cell densities and inhibition for growth rate and yield.

Nominal Concentration (mg/L Saturated Solution)	Growth Rate (cells/mL/hour)		Yield (cells/mL)
0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.076	-	1.18E+06	-
	R2	0.072		8.72E+05
	R3	0.073		9.35E+05
	R4	0.075		1.13E+06
	R5	0.074		1.01E+06
	R6	0.071		8.15E+05
	Mean	0.074		9.90E+05
	SD	0.002		1.44E+05
0.48	R1	0.072	3	8.67E+05	15
	R2	0.072	3	8.93E+05
	R3	0.070	5	7.63E+05
	Mean	0.071	4	8.41E+05
	SD	0.001	-	6.86E+04
1.0	R1	0.073	1	9.28E+05	7
	R2	0.072	3	9.03E+05
	R3	0.073	1	9.31E+05
	Mean	0.073	2	9.20E+05
	SD	0.001	-	1.53E+04
2.2	R1	0.057	23	2.89E+05	69
	R2	0.055	26	2.56E+05
	R3	0.061	18	3.88E+05
	Mean	0.058	22	3.11E+05
	SD	0.003	-	6.86E+04
4.8	R1	0.031	58	4.25E+04	95
	R2	0.031	58	4.21E+04
	R3	0.033	55	5.00E+04
	Mean	0.032	57	4.49E+04
	SD	0.001	-	4.44E+03
10	R1	0.011	85	6.41E+03	99
	R2	0.014	81	9.20E+03
	R3	0.013	82	7.88E+03
	Mean	0.013	83	7.83E+03
	SD	0.002	-	1.39E+03

*       =In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R      =Replicate

SD    =Standard Deviation

Validity criteria fulfilled:

yes

Conclusions:

An OECD guideline GLP compliant study with green algae provided a 72 hour ErC50 value of 0.27 mg/L based on the measured zinc concentrations. The respective NOEC value based on growth rate was 0.0646 mg/L. The ErC50 and NOEC values based on the test substance calculated from the measured zinc concentrations was 0.72 and 0.175 mg/L, respectively.

Description of key information

An OECD guideline GLP compliant study with green algae provided a 72 hour ErC50 value of 0.27 mg/L based on the measured zinc concentrations. The respective NOEC value based on growth rate was 0.0646 mg/L. The ErC50 and NOEC values based on the test substance calculated from the measured zinc concentrations was 0.72 and 0.175 mg/L, respectively.

The effect concentration based on the measured zinc concentrations is considered to be the most conservative for chemical safety assessment purposes.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.27 mg/L

EC10 or NOEC for freshwater algae:

0.065 mg/L