Tris(nitrato-O)nitrosylruthenium

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:

16 Jan 2020 - 29 May 2020

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)

Version / remarks:

28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Ru content: 10.13%

Analytical monitoring:

yes

Details on sampling:

The concentrations of the test item were determined by ICP-OES measurements of ruthenium at test initiation and test termination.

Samples (2 of about 20 mL) were taken from all test solutions and controls at the beginning of the test prior to addition of the algae.
At the end of the growth test, samples were taken directly from representative replicates per test concentration and controls. Aged samples (72 hours) were filtered using a 0.22 μm PES filter. Optional, unfiltered samples were taken additionally.
At test initiation there were two sets of non-filtered samples (6 samples) and at test termination there was one set of filtered samples (6 samples) and one set of samples was not filtered (6 samples). In addition, for each set a set of retain samples was taken.

All samples were stabilised by acidification and stored at 4 ± 3 °C until further analysis, if necessary.

Vehicle:

no

Details on test solutions:

An amount of 15.032 mg of the test item was transferred into a sterile glass flask filled up to 1 L with growth medium (sterile) to obtain the highest test concentration.
The test media was stirred for 24 hours at room temperature (about 20°C). The highest test concentration then was serially diluted with sterile growth medium to obtain the other test concentrations. For the control growth medium without test item was used. All work was conducted on a clean bench using sterile equipment.

Test organisms (species):

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

Details on test organisms:

Species: Raphidocelis subcapitata, Chlorophycea, Chlorophyta.
Origin: SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, 37073 Göttingen.
Strain number: Catalog No 61.81 SAG.
Cultivation: The stock cultures were maintained fulfilling the criteria of the OECD guide-line (culture medium recommended by Bringmann und Kühn (1980) [6]). Prior to testing a pre-culture was established in OECD growth medium (as described below) to obtain exponentially-growing algae for the test. The culture duration of the pre-cultures was 3 days.
cell numbers: cell concentrations were determined in the inoculum culture prior to the addition to the test vessels at test start and after 24, 48 and 72 h in the test cultures.
The cell density was measured using an electronic particle counter (CASY TT, OMNI Life Science GmbH & Co. KG, Bremen, Germany).

Test type:

static

Water media type:

freshwater

Remarks:

sterilised synthetic growth medium (OECD medium) according to OECD 201

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.0 °C during the test

pH:

The pH of the controls increased during the test from 7.28 at test start to a mean value (n = 8) of 7.92 at test end. In the test concentrations, the initial pH in the treatments ranged from 7.00 to 7.12 and the mean pH value was between 7.73 and 7.86 at the end of the test.

Nominal and measured concentrations:

Nominal test concentrations: control, 0.150, 0.475, 1.50, 4.75 and 15.0 mg test item/L, equivalent to control, 0.015, 0.048, 0.152, 0.481 and 1.52 mg Ru/L (factor: 3.16).
At test initiation, the measured concentrations ranged from 96.3% to 98.7% of nominal concentrations and therefore were within a range of 80 to 120% of the nominal concentrations. The measured concentrations did not remain within ± 20% throughout the test period of 72 hours (% of initial: 63.8% to 100%; % of nominal: 61.9 to 98.4%). The evaluation of biological results was therefore based on the geometric mean measured concentrations 0.117, 0.412, 1.40, 4.41 and 14.7 mg test item/L (re-calculated from the measured Ru concentration based on a Ru content of the test item of 10.13%).

Details on test conditions:

light intensity: OSRAM Standard “cool white”, 60 - 120 µE m-2 s-1 (4440 - 8880 lux), light intensity as measured daily using a cosine (2 π) receptor (LI-250A, LI-COR) in µE m-2 s-1 at level of the test media surface and at different positions in the incubation chamber. measured value: 95.22 to 98.83 µE m-2 s-1
During the course of the test all test vessels were kept on a laboratory shaker (Incubation Shaker Multitron®, INFORS Typ HT, Switzerland) shaking continuously at 150 rpm.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% CI 0.01-0.08

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95%CI 1.14-2.91

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.08 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.117 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

The NOEC for growth rate was determined with <0.117 mg test item/L. However, due to an inhibition of 5.8 % at 0.117 mg test item/L, i.e. an effect below 10% the NOEC is not considered to be ecotoxicologically relevant. Generally, it is recommended by OECD and EFSA to apply the EC10 approach in preference to the NOEC approach for the environmental risk assessment. However, the ErC10 value is below the lowest test concentration of 0.117 mg test item/L and therefore extrapolated. Based on an inhibition of 5.70% at 0.117 mg test item/L, the NOEC is considered to be more reliable.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.117 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: yield

Details on results:

A concentration-dependent inhibiting effect on the growth of the freshwater green algae was observed over the range of the tested concentrations.
Microscopic examinations were performed to verify a normal and healthy appearance of the inoculum culture and to detect any abnormal appearance of the algae (as may be caused by the exposure to the test substance) at the end of the test.
The NOEC for growth rate was determined with <0.117 mg test item/L. However, due to an inhibition of 5.8 % at 0.117 mg test item/L, i.e. an effect below 10% the NOEC is not considered to be ecotoxicologically relevant. Generally, it is recommended by OECD and EFSA to apply the EC10 approach in preference to the NOEC approach for the environmental risk assessment [5], [14], [15]. However, the ErC10 value is below the lowest test concentration of 0.117 mg test item/L and therefore extrapolated. Based on an inhibition of 5.70% at 0.117 mg test item/L, the NOEC is considered to be more reliable.

Results with reference substance (positive control):

Sensitivity of the test organism is routinely checked using 3,5-dichlorophenol as primary standard following internal SOPs in a non-GLP test twice a year. The latest (March 2020) ErC50 value of 2.92 mg/L (nominal; 95% confidence limits: 2.64 – 3.24 mg/L) is in good agreement with the results of an international ring test with an ErC50 of 3.38 ± 1.30 mg/L.

Reported statistics and error estimates:

Four replicates of each test concentration and eight replicates for the control were prepared.

Validity criteria fulfilled:

yes

Remarks:

cell number in control cultures increased by a factor of 81.2, mean of replicate CV % in controls was 15 %, coefficient of variation of average specific growth rate at test end in replicate control cultures was 3.2 %

Conclusions:

The 72-hour toxicity of Tris(nitrato-O)nitrosylruthenium to the unicellular green alga Raphidocelis subcapitata was determined in a static system (OECD 201). 72-hour ErC50 for the inhibition of growth rate was determined to be at 1.8 mg test item/L. The ErC10 was determined to be at 0.03 mg test item/L. The NOEC for growth rate was determined with at 0.117 mg test item/L. However, due to an inhibition of 5.8 % at 0.117 mg test item/L, i.e. an effect below 10% the NOEC is not considered to be ecotoxicologically relevant. Generally, it is recommended by OECD and EFSA to apply the EC10 approach in preference to the NOEC approach for the environmental risk assessment. However, the ErC10 value is below the lowest test concentration of 0.117 mg test item/L and therefore extrapolated. Based on an inhibition of 5.70% at 0.117 mg test item/L, the NOEC is considered to be more reliable.

Executive summary:

A study was performed to determine the toxicity of Tris(nitrato-O)nitrosylruthenium on the growth of the unicellular freshwater green alga Raphidocelis subcapitata.

Exponentially-growing cultures of the green alga were exposed to five concentrations of the test item plus a control over several generations under defined conditions for 72 hours according to the OECD guideline 201.

The nominal test concentrations were prepared with sterile growth medium under sterile conditions. For the examination of the inhibition of alga growth, eight replicates of the control (test medium only) and four replicates of each treatment were tested at the following nominal concentrations: 0.150, 0.475, 1.50, 4.75 and 15.0 mg test item/L, equivalent to 0.015, 0.048, 0.152, 0.481 and 1.52 mg Ru/L (factor: 3.16).

The concentrations of the test item in the media were confirmed by ICP-OES measurements of ruthenium at test initiation and test termination. The LOQ was at 0.0027 mg Ru/L.

At test initiation, the measured concentrations ranged from 96.3% to 98.7% of nominal concentrations and therefore were within a range of 80 to 120% of the nominal concentrations. The measured concentrations did not remain within ± 20% throughout the test period of 72 hours (% of initial: 63.8% to 100%; % of nominal: 61.9 to 98.4%). The evaluation of biological results was therefore based on the geometric mean measured concentrations 0.117, 0.412, 1.40, 4.41 and 14.7 mg test item/L (re-calculated from the measured Ru concentration based on a Ru content of the test item of 10.13%).

A concentration-dependent inhibiting effect on the growth of the freshwater green algae was observed over the range of the tested concentrations.

The respective 72-hour E_rC₅₀for the inhibition of growth rate was determined to be at 1.80 mg test item/L. The E_rC₁₀was determined to be at0.03 mg test item/L and the NOEC for growth rate was set to be at 0.117 mg test item/L. The E_rC₁₀value is below the lowest test concentration of 0.117 mg test item/L and therefore extrapolated. Based on an inhibition of 5.70% at 0.117 mg test item/L, the NOEC is considered to be more reliable.

Description of key information

The 72-hour NOEC and EC50 for the inhibition of growth rate was determined to be at 0.117 and 1.8 mg test item/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.8 mg/L

EC10 or NOEC for freshwater algae:

0.117 mg/L

Additional information

The 72-hour toxicity of Tris(nitrato-O)nitrosylruthenium to the unicellular green alga Raphidocelis subcapitata was determined in a static system (OECD 201; GLP compliant). The 72-hour ErC50 for the inhibition of growth rate was determined to be at 1.8 mg test item/L. The NOEC was determined to be at 0.117 mg test item/L