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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:
November 2015 till March 2016
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:
2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
, 2011-02-07
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1 mg/L & 100 mg/L (pre-test 1 and 2 & main test)

- Sampling method: To assess the initially dissolved concentrations of the metal and the organic component, all test solutions were sampled at the beginning of the exposure period prior to addition of the algae. At test termination, samples from representative replicates per treatment level and the additionally prepared vessels without algae were taken for chemical analysis. Dissolved metal concentrations and the concentrations of the organic component (acid) were measured in all test solutions sampled at the beginning of the test prior to addition of the algae. After 72 hours, samples were taken from representative growth test replicates and test vessels without algae of each treatment level. The samples were filtered (0.2 µm polyether-sulphone membrane syringe filter) at room temperature (20 - 25°C). A volume of 20 mL of the solutions was then transferred into disposable polyethylene vials (Sarstedt, Nuembrecht, Germany). The samples for the europium analysis were acidified with HNO3 (final concentration 1 % HNO3), and stored at 4°C until further analysis. The samples for the organic component analysis were frozen until analysis.

- Sample storage conditions before analysis: For analysis of europium 20 mL of aqueous samples were transferred into disposable 50 mL polyethylene vials (Sarstedt, Nuembrecht, Germany), and acidified by addition of 200 µL of concentrated nitric acid for stabilisation. Samples are stable for at least half a year under these conditions. Moreover, to prove stabilisation, acidified samples from day 0 were quantified again during the measurement series for samples of day 3.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

Range-finding test (Pre-test):
Non-GLP pre-tests were performed with nominal test item concentrations ranging between 1 and 100 mg test item/L including chemical analysis of europium at the end of the growth tests.
The test solutions were prepared as described below (main test) with the exception that the stirring time in the pre-tests was about 60 hours.

In the second pre-test, the pH of the 100 mg/L test solution was adjusted to the pH of the controls and the algale growth was compared to that in a not pH adjusted medium. The pH adjustment did not improve the test design.

Main test:
The nominal concentrations to be tested in the definitive test were selected on the basis of results from the range-finding test. Reason for choosing these test item concentrations was to show that the NOEC is ≥ 1 mg/L and the EC50 is > 100 mg/L in a GLP test. Hence, the test design is in accordance with the Regulation (EC) No 1272/2008 and will allow classification of the substance under concern. The pH values of the test solutions were not adjusted.

Concentration: Control, 1.00 and 100 mg pure test item/L (purity > 98 %)

The concentrations were assessed by chemical analysis at start and at test termination after 72 hours.

Preparation:
An amount of 1.996 mg test item was given into 2 L of growth medium and 99.991 mg test item into 1 L growth medium to prepare the test medium and to achieve nominal test concentrations of 1 and 100 mg test item/L. Afterwards the test media was transferred into Erlenmeyer flasks with a volume of 3 L in total and were stirred at 100 rpm using a magnetic stirrer for 7 days at room temperature. For the growth test the solutions were filtered using a 0.22 µm polyether sulfone (PES) filter (Nalgene bottle top filter). Thereby, possibly insoluble parts were separated from the aqueous phase and the solution was sterilised.
All filtration and dilution work was conducted under a clean bench using sterile equipment.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species:
Pseudokirchneriella subcapitata, Chlorophycea, Chlorophyta,
(other name Raphidocelis subcapitata, former name Selenastrum capricornutum).

Origin:
SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen, Catalog No. 61.81 SAG.

Cultivation and pre-culture:
The stock cultures are maintained fulfilling the criteria of the OECD guideline (OECD No. 201). The sterilised synthetic growth medium according to Bringmann and Kühn was used as growth medium for the stock cultures.

Before the onset of a test, a pre-culture is established in the OECD 201 growth medium under test conditions to obtain exponentially-growing algae for the test. The culture duration of the pre-cultures was 3 days.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21.5°C
pH:
The pH of the controls decreased slightly during the test, the pH was 7.69 at test start and 7.73 at test termination (between 7.68 and 7.78). The initial pH values of the test solutions cultures were between 7.19 and 7.68. The mean pH values of the treatment levels varied from 7.78 at a nominal concentration of 1 mg test item/L to 7.28 at a nominal concentration of 100 mg test item/L at the end of the test.
Nominal and measured concentrations:
test substance:
1 mg/L and 100 mg/L (test substance)
0.22 mg/L and 22.0 mg/L Metal concentration
0.782 mg/L and 78.2 mg/L acid concentration

measured:
< 0.43 % metal concentration of nominal (day 0-3)
107-113% acid concentration of nominal (day 0-3)
Details on test conditions:
TEST SYSTEM
The test vessels used were 250 mL conical glass flasks covered with air-permeable silicone-sponge caps. The test vessels and caps were sterilised by autoclaving prior to use.
The culture vessels were incubated at a temperature in a range of at 21 to 24 °C, controlled at ± 2 °C, with a light intensity (day light: OSRAM Standard “cool white”) adjusted to approximately 60 - 120 µE m-2 s-1 (4440 - 8880 lux). Measurements were made of the direct light above the tray of the incubator. The cultures were oscillated by continuously stirring on a laboratory shaker with 150 rpm (Incu-bation Shaker Multitron®, INFORS, Switzerland). The light intensity was measured using an illu-minance meter LI-250A (LI-COR, Lincoln, USA) with a cosine (2 pi) receptor in µE m-2 s-1 at level of the test media surface daily. During the exposure period, the incubation temperature was measured daily with a calibrated thermometer in an additionally prepared control vessel kept under the same conditions. The pH values were measured in the additionally prepared replicate at the beginning of the test and directly in the test vessels at the end of the test.

Cell concentrations were determined using an electronic particle counter (CASY 1 Model TT, Roche Diagnostics GmbH, Germany). The correctness of the electronic counts was checked by comparison with a standardised particle solution following internal standard operation procedures. The cell density of the pre-culture was determined at test initiation and the volume required to achieve the initial cell concentration of 10,000 cells/mL was calculated. During the test, the cell concentrations were determined after 24, 48 and 72 h in samples taken directly from the test vessels.

- Initial cells density: 10,000 cells/mL
- Control end cells density: 203.6 * E4 cells/mL
- No. of vessels per concentration (replicates): 8
- No. of vessels per control (replicates): 8


GROWTH MEDIUM
The sterilised synthetic OECD medium according to OECD 201 was used as growth medium. In this medium, the molar ratio of EDTA to iron slightly exceeds unity (molar ratio Fe(III+) / EDTA = 1 : 1.135). Considering the molar ratio of Fe and EDTA, the high stability constant of EDTA with Fe(III+) ions (log Ks 25) and the presence of other metals (Co, Cu, Mn, Zn, in the test medium, the chelation of europium contained in the test item (log Ks 17.3) was considered to be negligible in comparison with the nominal test concentrations. However, the OECD 201 medium with reduced EDTA was used. In a pre-test, it was demonstrated that the growth of P. subcapitata in the OECD 201 growth medium with 50 % EDTA concentration fulfilled the validity criteria of the OECD 201 guideline while it did not in an EDTA-free OECD 201 medium.

TEST CONCENTRATIONS
The nominal concentrations to be tested in the definitive test were selected on the basis of results from the range-finding test. Reason for choosing these test item concentrations was to show that the NOEC is ≥ 1 mg/L and the EC50 is > 100 mg/L in a GLP test. Hence, the test design is in accordance with the Regulation (EC) No 1272/2008 and allows classification of the test item. The pH values of the test solutions in the main test were not adjusted.
Reference substance (positive control):
yes
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: organic content (acid)
Remarks:
rr within 80-120%
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 mg/L
Nominal / measured:
nominal
Conc. based on:
other: organic content (acid)
Remarks:
rr within 80-120%
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: organic content (acid)
Remarks:
rr within 80-120%
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 mg/L
Nominal / measured:
nominal
Conc. based on:
other: organic content (acid)
Remarks:
rr within 80-120%
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
63.2 mg/L
Nominal / measured:
nominal
Conc. based on:
other: organic content (acid)
Remarks:
rr within 80-120%
Basis for effect:
biomass
Remarks on result:
other: 95 %-cl (5.01 - 74.7 mg/L)
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
6.77 mg/L
Nominal / measured:
nominal
Conc. based on:
other: organic content (acid)
Remarks:
rr within 80-120%
Basis for effect:
biomass
Remarks on result:
other: 95 %-cl (0.90 - 52.8 mg/L)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.11 mg/L
Nominal / measured:
nominal
Conc. based on:
other: organic content (acid)
Remarks:
rr within 80-120%
Basis for effect:
biomass
Remarks on result:
other: 95 %-cl (0.25 - 17.8 mg/L)
Details on results:
- Exponential growth in the control (for algae test): yes
Test start: day 1, day 3: no abnormalities at control and 1 mg/L, high number of cell agglomerates at 100 mg/L

Test termination: no abnormalities at control and 1 mg/L, high portion of cell debris, very few intact cells at 100 mg/L
Results with reference substance (positive control):
The 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 nominal ErC50 value (August 2015) of 2.62 mg/L (95 % confidence limits: 2.52 - 2.72 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:
The test results of the growth inhibition test showed growth rate inhibition around 20 % and yield inhibition of > 50%. The yield data was statistically analysed to determine an EC50, EC20 and EC10 value with their 95% confidence intervals using linear regression procedures. At first, non-linear regression procedures, e.g. a 3-parametric cumulative normal distribution function according to Bruce and Versteeg (1992), were fitted to the data. Since there was a significant fit, this 3-parametric normal cumulative distribution function F(x) = b0*[NormalCDF(b1-log10(x)/b2 + zOpt)] was fitted to the data (b0-b2: parameters; zOpt: adjustment to have the EC10 as parameter b1; x: concentration). Individual replicate responses were used for the regression analysis. Individual replicate responses were used for the regression analysis. More information on the statistical approaches can be found in OECD 54. The NOEC values for growth rate and yield were determined with the Williams Multiple Sequential t-test Procedure. All statistical analyses were conducted by the computer program ToxRat®.

For the determination of the effects on algae growth, eight replicates of the control (test medium only) and of each test concentration were tested. Dissolved metal and organic component (acid) concentrations were measured at test start (0h) and at the end of the test (72h) using ICP-OES and HPLC, respectively.The recovery rates (rr) of metal at test start and at test termination were < 0.43% of nominal. During the test, samples with and without algae were measured after 72 hours, whereas the metal concentration in the samples with algae at a nominal test concentration of 100 mg/L were less than the concentration without algae (rr 0.021 vs. 0.33%). Nevertheless, recovery rates of organic component (acid) ranged between 108 and 113% of the nominal values in all samples at test start and between 107 and 110% at test termination, confirming the nominal test item concentrations. No significant differences in concentration regarding organic component (acid) were measured in samples with and without algae after 3 days of testing. Due to a recovery ranging between 80 and 120 % for organic component (acid), the evaluation of the effect data was based on the nominal test item concentrations.

According to the Pourbaix diagram, the respective precipitates at a pH at and above 8.5 as metal hydroxide that’s may be the reason for the increase of metal concentrations in solution. For the Pourbaix diagram please refer to the "illustration" below.

Validity criteria fulfilled:
yes
Conclusions:
The impact of the test item on the growth of the freshwater green algae species Pseudokirchneriella subcapitata was determined as follows:

NOEC (growth rate) = >= 1 mg/L
72h-ErC50 (growth rate) > 100 mg/L
72h-ErC20 (growth rate) > 100 mg/L

All effect concentrations are based on nominal concentrations of the test item as the recovery rates at day 0 and after 3 days of exposure are within 80-120% of nominal concentration of for the organic component (acid).

Description of key information

The impact of the test item on the growth of the freshwater green algae species Pseudokirchneriella subcapitata was determined as follows:

72h-ErC50 (growth rate) > 100 mg/L

NOEC (growth rate) >= 1 mg/L

All effect concentrations are based on arithmetic mean measured concentrations of the test item.

Key value for chemical safety assessment

Additional information