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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Jan 2020 - 7 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:
Rh content: 11.11%
Analytical monitoring:
yes
Details on sampling:
The concentrations of dissolved rhodium were determined by ICP-MS 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. Since the test solutions were filtered already in the process of the test media preparation a further filtration step was not necessary.
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 was one set of 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 with concentrated nitric acid and stored at 4 ± 3 °C until further analysis.
Vehicle:
no
Details on test solutions:
An amount of 100.08 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 72 hours at 30 – 35°C to achieve maximum solubility. To separate insoluble test item from the test medium, the highest test concentration was filtered through a 0.22 μm PES filter (polyether sulphone, DIA Nielsen, Dueren, Germany).
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):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 7.15 at test start to a mean value (n = 8) of 7.76 at test end. In the test concentrations, the initial pH in the treatments ranged from 7.02 to 7.08 and the mean pH value was between 7.82 and 7.83 at the end of the test.
Nominal and measured concentrations:
Based on the results of the range-finder, the following nominal concentrations with a spacing factor of 2 were selected: 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L, equivalent to 0.694, 1.39, 2.78, 5.55 and 11.11 mg Rh/L.

At test initiation, the measured concentrations were 0.016, 0.036, 0.069, 0.150 and 0.290 mg Rh/L (2.4 – 2.7% of nominal) and therefore not within a range of 80 – 120% of the nominal concentrations.
The measured concentrations mainly remained within ± 20% throughout the test period of 72 hours. At test termination, the measured concentrations were 0.014, 0.029, 0.064, 0.133 and 0.283 mg Rh/L (79.2 – 97.6% of initial; 2.1 – 2.5% of nominal).
The evaluation of biological results was based on the geometric mean measured concentrations. After re-calculation of the measured Rh concentrations into test item concentrations, the evaluation was based on geometric mean measured concentrations of 0.14, 0.29, 0.60, 1.27 and 2.58 mg test item/L.
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: 86.95 to 102.10 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
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.82 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 0.61-1.09
Key result
Duration:
72 h
Dose descriptor:
EC50
Remarks:
extrapolated value (highest concentration was 2.58 mg test item/L
Effect conc.:
4.38 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 2.69-6.97; extrapolated value (max 34.7% inhibition of growth rate at 2.58 mg test item/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.32 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.:
1.24 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.
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 55.8, mean of replicate CV % in controls was 17.6 %, coefficient of variation of average specific growth rate at test end in replicate control cultures was 3.5 %
Conclusions:
The 72-hour toxicity of Tris(triphenylphosphine) rhodium (I) chloride to the unicellular green alga Raphidocelis subcapitata was determined in a static system (OECD 201). T2-hour ErC50 for the inhibition of growth rate was determined to be at 4.38 mg test item/L (which is above the highest test concentration (2.58 mg test item/L) and therefore extrapolated). The ErC10 was determined to be at 0.82 mg test item/L
Executive summary:

A study was performed to determine the toxicity of Tris(triphenylphosphine) rhodium (I) chloride 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: 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L, equivalent to 0.69, 1.39, 2.78, 5.55 and 11.1 mg Rh/L (factor: 2).

Due to the very low water solubility of the test item a stock solution was prepared using 100 mg test item/L test medium, which was stirred for 72 hours at 30 – 35°C to achieve maximum solubility. This is in accordance with the OECD series on testing and assessment no. 23.

The concentrations of the test item in the media were confirmed by ICP-MS measurements of rhodium at test initiation and test termination. The LOQ was at 2 x 10-6mg Rh/L.

At test initiation, the measured concentrations ranged from 2.4 to 2.7% of nominal concentrations and therefore were not within a range of 80 to 120% of the nominal concentrations. The measured concentrations mainly remain within ± 20% throughout the test period of 72 hours (% of initial: 79.2 to 97.6%; % of nominal: 2.1 to 2.5%). The evaluation of biological results was based on the geometric mean measured concentrations of 0.14, 0.29, 0.60, 1.27 and 2.58 mg test item/L (re-calculated from the measured Rh concentration based on a Rh content of the test item of 11.11%).

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 ErC50for the inhibition of growth rate was determined to be at 4.38 mg test item/L, which is above the highest test concentration and therefore extrapolated. The ErC50was set to be above the highest test concentration of 2.58 mg test item/L. The ErC10was determined to be at 0.82 mg test item/L and the NOEC for growth rate was at 0.29 mg test item/L.

For yield, an EyC50of 1.24 mg test item/L was determined. The EC10values for yield was at 0.32 mg test item/L and the NOEC was at 0.29 mg test item/L.

Description of key information

The 72-hour EC10 and EC50 for the inhibition of growth rate was determined to be at 4.38 (extrapolated value; >2.58) and 0.82 mg test item/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
4.38 mg/L
EC10 or NOEC for freshwater algae:
0.82 mg/L

Additional information

The 72-hour toxicity of tris(triphenylphosphine)rhodium (I) chloride 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

4.38 (extrapolated value; >2.58) mg test item/L.

The ErC10 was determined to be at 0.82 mg test item/L.