Dimolybdenum trizinc nonaoxide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations were analysed
- 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 48 hours for quantitative analysis. All 0 hour samples were acidified and taken for immediate analysis; the 48 hour samples were acidified and stored in the fridge prior to analysis. Duplicate sets of samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.
- Sample storage conditions before analysis: Analysed immediately or stored under refrigeration for 48 hour samples.

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 test water 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). A series of dilutions was made from this saturated solution to give further test concentrations of 10, 18, 32 and 58 mg/L saturated solution. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus 1820
- Source: In-house laboratory cultures originally supplied by the University of Sheffield.
- Age of parental stock (mean and range, SD): Parental daphnia originally from 7th brood.
- Feeding during test: No

ACCLIMATION
- Acclimation period: 4th to 5th brood used on test. Parental daphnia were 7th brood.
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: Mixture of algal suspension (desmodesmus subspicatus) and GM 300 food suspension. The quantity of feed provided was dependent on the age of the daphnia and was between 0.10 and 0.25 mg TOC per daphnia per day.
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): None noted

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

Test temperature:

Temperature was maintained at approximately 20 °C to 21°C throughout the test.

pH:

7.27 to 7.93 in test vessels and 8.07 to 8.16 in controls.

Dissolved oxygen:

8.55 to 8.89 mgO2/L in test vessels and 8.61 to 8.93 in controls.

Salinity:

No applicable

Conductivity:

< 5 µS cm^-1

Nominal and measured concentrations:

Nominal: 10, 18, 32, 58 and 100 mg/L.
Measured zinc (0 hour): 1.66, 2.92, 5.26, 9.66 and 16.8 mg/L
Measured molybdenum (0 hour): 2.70, 4.84, 8.76, 16.2 and 27.8 mg/L
Calculated concentration of the test substance in the test sample (0 hour) based on zinc as potentially the most ecologically relevant component: 4.5, 7.93, 14.3, 26.2, 45.5.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass jar
- Type (delete if not applicable): open (covered to reduce evaporation)
- Material, size, headspace, fill volume: 100 mL of test medium in a 150 mL jar
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Reconstituted water - ISO Medium. The reconstituted water had a pH of 7.8 ±0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value. Media is standard as per OECD guideline. The diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: Throughout the definitive test the light intensity was observed to be in the range 926 to 997 Lux.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: No immobilization was observed at the test concentration of 1.0 mg/L saturated solution; however, a small number of daphnids was observed to be immobilized at 0.1 and 10 mg/L saturated solution and 100% immobilization was observed at 100 mg/L saturated solution. Reduced mobility of the Daphnia were observed in 10 mg/L saturated solution test concentration. As the immobilization observed at 0.1 and 1 mg/L was, at most, 10% the test concentrations of 10, 18, 32, 58 and 100 mg/L saturated solution were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.13 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element (dissolved fraction)

Remarks:

Zinc

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5.79 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

Test item calculated based on zinc component

Basis for effect:

mobility

Details on results:

Analytical method:
Linearity: The data was found to have a linear correlation within the calibration range of 0.020 to 5 mg/L zinc. The R² fit of the calibration curve to the data was 0.9998, and was considered to be acceptable. The data was found to have a linear correlation within the calibration range of 0.020 to 10 mg/L molybdenum. The R² fit of the calibration curve to the data was 0.9998, and was considered to be acceptable.

Accuracy (recovery) and precision: A set of recovery samples accurately fortified at relevant concentrations of zinc and molybdenum was prepared five-fold and analyzed. The method was considered to be sufficiently accurate and precise for the purposes of this test. The test sample results were not corrected for recovery. Procedural recoveries were analyzed alongside each analysis and considered acceptable. The results have been reported with the test samples.

Limit of quantification: The limit of quantification (LOQ) was determined as the lowest usable standard concentration with a precision less than 10% RSD. Using this method the LOQ was determined to be 0.020 mg/L as zinc and molybdenum.

Verification of test concentrations: Analysis of the test preparations at 0 hours showed measured test concentrations to range from 1.66 to 16.8 mg/L as zinc, giving calculated concentrations of test item in the range of 4.50 to 45.5 mg/L. There was no significant change in the measured concentrations of zinc at 48 hours and so the results are based on 0 hour measured concentrations.

Solubility observations: At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. The 48 hour EC50 was determined to be 0.8 mg/L (0.12 to 1.5 mg/L). The results of the inter laboratory tests and a Technical Corrigendum to ISO 6341 give a 24-Hour EC50 for potassium dichromate within the range 0.6 mg/L to 2.1 mg/L. Accordingly the results from the positive control study were within the normal range for this reference item.

Reported statistics and error estimates:

The EC50 values and associated confidence limits at 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. The Lowest Observed Effect Concentration (LOEC) and the NOEC at 24 and 48 hours were calculated using the Step-down Roa-Scott-Cochran-Armitage analysis. The control response of 5% immobilization was compensated using Abbotts formula. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Immobilisation data (definitive):

Nominal concentration (mg/L)	24 hours						48 hours
	Cumulative immobilised Daphnia (initial 5 per replicate)
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4	Total	%
Control	0	0	0	0	0	0	0	0	1	0	1	5
10	0	0	0	0	0	0	0	0	2	2	4	20
18	3	3	4	5	15	75	4	4	5	5	18	90
32	5	5	5	5	20	100	5	5	5	5	20	100
58	5	5	2	0	12	60	5	5	5	5	20	100
100	4	4	4	4	16	80	5	5	5	5	20	100

Effect data:

	48 hours
	EC50	95% confidence limits	LOEC	NOEC
Nominal concentrations (mg/L)	13.0	11.3 – 14.9	18	10
Zinc Measured Concentrations (mg/L)	2.13	1.86 – 2.44	2.92	1.66
Test item calculated concentrations (based on zinc concentrations) (mg/L)	5.79	5.04 – 6.63	7.93	4.50

Validity criteria fulfilled:

yes

Remarks:

No more than 10% of the control daphnids showed immobilization or other signs of disease or stress and the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Conclusions:

An OECD guideline GLP compliant study with Daphnia magna provided a 48 hour EC50 value of 2.13 mg/L based on measured zinc concentrations and 5.79 mg/L based on the test substance calculated from the measured zinc concentrations.

Description of key information

An OECD guideline GLP compliant study with Daphnia magna provided a 48 hour EC50 value of 2.13 mg/L based on measured zinc concentrations and 5.79 mg/L based on the test substance calculated from the measured zinc concentrations.

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

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.13 mg/L