Tris(nitrato-O)nitrosylruthenium

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

Study period:

31 Oct 2019 - 26 June 2020

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)

Version / remarks:

13 April 2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

purity: 10.13%Ru

Analytical monitoring:

yes

Details on sampling:

The ruthenium concentrations in the aqueous solutions were measured during the study. Fresh samples were taken from all five test solutions and the control at the beginning of the test and at medium renewal at 24 hours prior to distribution to the test vessels. Samples of aged solutions were taken from one representative replicate at media renewal and test end. Aged samples were filtered through a 0.22 µm PES filter before measurement.
In addition, for all samples a second set of samples was taken as retain samples.
All samples were acidified and stored at 4 – 8 °C until analysis.

Vehicle:

no

Details on test solutions:

The highest test concentration was prepared by weighing in approx. 100 mg of the test item and transferring it to 1000 mL dilution water, resulting in a concentrations of 100 mg test item/L. The test medium was stirred for 24 hours at room temperature (about 20°C).
Since undissolved test item was observed after 24 hours, the test medium was filtered using a 0.22 µm PES filter to remove undissolved test item.
Afterwards, the test medium was diluted to obtain the lower test concentrations.
The test was performed under semi-static test conditions. Test solutions were renewed after 24 hours. The test solutions were freshly prepared before test start and before media renewal.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test organisms were juvenile Daphnia magna, 4 – 24 hours old.
Origin of the cladocerans is the German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene.
Specimens used in the test were bred in the laboratory of the Fraunhofer IME.

Pre-Treatment:
Adult Daphnia, at least 3 weeks old, were separated from the stock population by sieving.
Batches of 30 to 50 animals were held at room temperature in ca. 1.8 L dilution water for one week. During this week the daphnids were fed daily with an algal suspension (Desmodesmus subspicatus) and ArtemioFluid (JBL). Algae growing in the log-phase were centrifuged and the pellet was re-suspended in a few mL of medium. 5 mL of this suspension was given to 1.8 L Daphnia medium.
The water was changed three times per week.
Newborn D. magna were separated by sieving, the first generation was discarded.
Individuals applied in the test were transferred with a bore Pasteur pipette a few hours after sieving to ensure applying only healthy specimens.

Test type:

semi-static

Water media type:

freshwater

Remarks:

Purified, Cu-reduced drinking water was used as holding- and dilution water.

Limit test:

no

Total exposure duration:

48 h

Hardness:

Hardness of the Cu-reduced water is below the recommended range. Therefore, it is enriched with an appropriate amount of CaCl2 to values of 250 – 350 mg CaCO3/L.

Test temperature:

18.8-19.5°C (19.2 °C mean temperature)

pH:

6.71 – 8.18 at all treatment levels

Dissolved oxygen:

7.05 mg/L and 8.84 mg/L (80.1 – 98.8 % of air saturation)

Conductivity:

250 µS/cm

Nominal and measured concentrations:

Nominal concentrations:
range finding test: 1 -10 - 100 mg test item/L
The concentrations to be tested in the definite test were selected based on results from the range-finding test. The following nominal loadings with a spacing factor of 2.0 will be applied: Control, 6.25, 12.5, 25.0, 50.0 and 100 mg/L (corresponding to 0.633, 1.27, 2.53, 5.07 and 10.1 mg Ru/L).

Measured concentrations:
The concentration of the test item in the aqueous test solution was assessed by analysis of ruthenium at the start of the test, at media renewal after 24 hours, and at test end. An ICP-OES system was used for measurements of ruthenium in the test solutions.
Concentrations of freshly prepared test media showed recovery rates between 90.0 and 98.6 % of nominal concentrations. Concentrations in aged test media showed recovery rates between 90.2 and 99.6 % of nominal concentrations and therefore remained stable with 98.6 to 103 % of initial concentrations.
The evaluation of effects was based on the nominal concentrations of 0.633, 1.27, 2.53, 5.07 and 10.13 mg Ru/L, equivalent to 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L (considering ruthenium content of 10.13 % in the test item).

Details on test conditions:

light intensity: 739 - 866 lx (corresponding to 9.85 – 11.5 µE/(m².s)) throughout the test
alkalinity: 1.6 mmol/L
total hardness: 1.2 mmol/L

Reference substance (positive control):

yes

Remarks:

K2Cr2O7 - the latest 24h-EC50 (March, 2020) was 1.868 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 10.13 mg/L

Nominal / measured:

nominal

Conc. based on:

element (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

No significant signs of disease or stress like discoloration or abnormal behavior were observed in any replicate.

Results with reference substance (positive control):

latest 24h-EC50 (March, 2020) was 1.868 mg/L

Reported statistics and error estimates:

The evaluation of the concentration-effect-relationships and the calculations of effect concentrations was based on the nominal test item concentrations since the measured concentrations varied less than ± 20% of the nominal concentrations throughout the test.

As the results showed inhibition of < 50 % no EC50 could be calculated.
The NOEC and LOEC values were determined using appropriate statistical methods (e.g. Fisher`s Exact Binomial Test for quantal response). As no statistically significant effects were observed, the NOEC is given as ≥ highest test concentration.
The computer program ToxRat was used for statistical evaluations.

Nominal conc. Test item [mg/L]	Nominal conc. Ru [mg/L]	Total Introduced	Mobile	Immobile	% Immobility	% Immobility after compensation *
Control	Control	20	18	2	10.0	0.0
6.25	0.633	20	17	3	15.0	5.0 (-)
12.5	1.27	20	19	1	5.0	0.0 (-)
25.0	2.53	20	15	5	25.0	15.0 (-)
50.0	5.07	20	15	5	25.0	15.0 (-)
100	10.13	20	17	3	15.0	5.0 (-)

* The control response of 10.0% was compensated using Abbott`s formula.(; -) statistically not significant different from the control

Validity criteria fulfilled:

yes

Remarks:

mortality in controls does not exceed 10 %, the dissolved oxygen concentration at the end of the test (48 h) ≥ 3 mg/L in control and test vessels.

Conclusions:

The short-term toxicity to Daphnia magna was determined according to OECD Guideline 202 (GLP compliant). The test item had no statistically significant effect on immobility of the daphnids up to the highest test concentrations. The EC50 for immobility was set to be >100 mg test item/L and the NOEC was determined to be ≥100 mg test item/L (based on nominal concentrations). Considering the ruthenium content of 10.13 % in the test item, the EC50 and NOEC was determined to be >10.13 mg Ru/L and ≥ 10.13 mg Ru/L, respectively.

Executive summary:

A study was performed to investigate the influence of the test item Tris(nitrato-O)nitrosylruthenium on the mobility of Daphnia magna. The test was conducted under semi-static conditions over a period of 48 hours with one media exchange after 24 hours. Effects on immobilization were determined daily. 

The daphnids were placed in water containing the test item in nominal concentrations of 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L. Considering the ruthenium content of 10.13 %, 0.633, 1.27, 2.53, 5.07 and 10.13 mg ruthenium/L were introduced.

Cu-reduced dilution water enriched with CaCl₂was used as test medium. A control with CaCl₂enriched Cu-reduced dilution water only was run in parallel.

The concentrations of the test item in the media were confirmed by measurements of ruthenium (Ru) concentrations at beginning of the test, before and after media renewal at 24 hours and at test end using an ICP-OES system.

Concentrations of freshly prepared test media showed recovery rates between 90.0 and 98.6 % of nominal concentrations. Concentrations in aged test media showed recovery rates between 90.2 and 99.6 % of nominal concentrations. During incubation in the test, test concentrations remained stable with 98.6 to 103 % of initial concentrations.

The measured concentrations were within a range of 80 – 120 % of nominal concentrations and remained stable during the test. Therefore, the evaluation of effects was based on the nominal concentrations.

At test end, the mortality was ≤ 10 % in the control. The dissolved oxygen content was > 3 mg/L in control and test vessels throughout the test. The mean water temperature was in the range of 20 ± 2 °C. Thus, the test was considered valid according to OECD 202.

The test item had no statistically significant effect on the immobilization of the daphnids up to and including the highest test concentrations of 100 mg test item /L, corresponding to 10.13 mg Ru/L. The EC₅₀was determined at >100 mg test item/L (>10.13 mg Ru/L), the NOEC was≥100 mg test item/L (≥10.13 mg Ru/L).

Description of key information

The EC50 for immobility of D. magna was >100 mg test item/L, corresponding to >10.13 mg Ru/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 µg/L

Additional information

The short-term toxicity to Daphnia magna was determined according to OECD Guideline 202 (GLP compliant). Based on the nominal concentrations, the EC₅₀for immobilization of the test organisms was determined at > 100 mg test item/L, equivalent to 10.13 mg Ru/L. The NOEC was≥100 mg test item/L, equivalent to≥10.13 mg Ru/L.