9-Anthracenecarboxylic acid, (triethoxysilyl)methyl ester

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Data waiving:

other justification

Justification for data waiving:

other:

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2019-09-16 to 2019-09-19

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:

yes

Remarks:

only a dose range finder performed

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

Samples (2 x about 5 mL) were taken from all test solutions and controls at the beginning of the test prior to addition of the algae (2 x 6 samples, analysis and retain samples).
The test item was analysed using GC-MS.

Sample preparation
A sample volume of 50 mL of each sample was transferred into a 60 mL glass vials. 25 μL of the internal standard solution (150 μL of stock II Anthracen in 10 mL of Acetone) were added. Samples were extracted with 5 mL of Hexane and shaking for ca. 30 minutes. 4 mL of the organic phase are taken from the sample and concentrated to 150 μL under a nitrogen stream subsequently followed by GC-MS measurement.

Vehicle:

no

Details on test solutions:

A non-GLP dose range finder was initiated with test item concentrations 100 % (saturated solution) 1:10 and 1:100 dissolution in triplicate.

Preparation of the test media:
An amount of 100.2 mg of the test item was transferred into a sterile glass flask. 1 L of growth medium (sterile) were added to obtain the highest test item concentration. The test media was stirred for about 72 hours at room temperature (about 20°C). The test media was filtered (0.22 μm PES filter) to remove undissolved test item. After that, the filtrate was used as the highest test concentration for testing. The other test concentrations were obtained by dilution (1:10 and 1.100) of the highest test concentration with sterile growth medium, which was treated in the same way as the test media of the highest test concentration. For the control, growth medium without test item was used. The growth medium for the control was treated in the same way as the highest test concentration with regard to stirring and filtration. 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, Selenastraceae, Chlorophycea, Chlorophyta. (therein referenced as Pseudokirchneriella subcapitata).
Origin:
SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Karspüle 2, D-37073 Göttingen, Catalog No 61.81 SAG.
Cultivation:
The stock cultures are maintained fulfilling the criteria of the OECD guideline (culture medium recommended by Bringmann und Kühn (1980)). Prior to testing, a pre-culture will be established in standard OECD growth medium 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 to 24 °C, controlled at ± 2°C

pH:

7.65

Nominal and measured concentrations:

saturated solution (100%), 1:10 and 1:100 dissolution and control in triplicate
No test item could be detected in test item solution at test start and after 72 h.

Details on test conditions:

The cultures were incubated in a laboratory shaker (Incubation Shaker Multitron®, INFORS Typ HT, Switzerland) and kept moving continuously by shaking on an orbital laboratory shaker (150 rpm) (INFORS, Switzerland).
The culture vessels were incubated at a temperature in the range of 21 to 24 °C, controlled at ± 2°C, with a light intensity (OSRAM Standard “cool white”) adjusted to 60 - 120 μE m-2 s-1 (4440 - 8880 lux) close to the surface of the liquid. The light intensity was 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 shaker. The solution pH was measured in one test vessel per test concentration including the control. At the end of the test, the solution pH values were measured directly in the test vessels.

The 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). For the growth test, three replicates of each test concentration and three replicates of the control were prepared. The algal pre-culture (668 μL at a cell density of 1.498 x 106 cells/mL) was added to the test vessels to achieve the initial cell concentration of 10,000 cells/mL. At the beginning of the test, the initial cell concentration was calculated based on the cell number of the pre-culture. During the test, the cell concentrations were determined after 24, 48 and 72 hours. All test preparations were performed under sterile conditions.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

72 h

Dose descriptor:

EC50

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

ErC(50) > solubility of test material in test medium

Duration:

72 h

Dose descriptor:

NOEC

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

NOErC >= solubility of test material in test medium

Details on results:

Since no test item could be measured after filtration of the stock solution (equal to highest test concentration) through a 0.22 μm PES filter, other filter were purchased and tried to determine the effect of the filter material on the recovery of the test item. Therefore, test solution was prepared as described above with slight modifications. Due to the very low water solubility (< 2 μg/L at 20.3 °C and pH 6.5) a log Pow of 5.6 and a potentially photosensitivity of the test item, the highest test concentration was prepared using 100 mg test item per liter test medium in a 1 L amber glass vessel and stirred (300 – 500 rpm) over 24 hours to coat the vessel with the test substance before testing, to avoid adsorption of the test item to the glass vessel during testing. After 24 hours of stirring the solution was discarded. A new stock solution was prepared using 100 mg test item/L test medium in a 1L amber glass vessel and stirred over 72 hours to achieve the maximum water solubility. This is in accordance with the OECD series on testing and assessment no. 23. The test substance did not change in terms of the colour (slightly yellowish) and of its behaviour over 72h of stirring. Afterwards, the test solution was filtered through a PTFE filter with a pore size of 0.45 μm and a cellulose acetate filter with a pore size of 0.22 μm, respectively. For the PTFE filter with a larger pore size, it was observed that even the pore size is larger, no filtrate could be gathered due to a quick clogging of the filters. For the cellulose acetate filters and the modifications in test solution preparation the same results as for the PES filter was achieved and the determined concentrations of the test item were below the lowest calibration level. The filter cake with test substance was appeared to be still yellowish.

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 (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.

Inhibition of growth rate and yield compared to controls after 72 hours.

Nominal conc. [mg test item/L]	Growth rate		Yield
	Growht rate [1/d]	% inhibition	Yield [cells x 104/mL]	% Inhibition
1.00	1.559	-0.9	107	-3.6
10.0	1.565	-1.2	110	-6.6
100	1.544	0.1	102	0.9

(+) significantly/ (-) not significantly different from controls, Williams Multiple Sequential t-test Procedure,

significance level 0.05, one-sided smaller.

- negative value indicates increase of the observed parameter compared to the control

Validity of the test:

• The cell number in the control cultures increased by a factor of 32.7 within the 72-hour test period (validity criterion: > 16).

• Evaluation of the sectional growth rates of the controls: The mean of the replicate coefficients of variation (CV %) for section-by-section growth rate of controls was 19.8% during the test period (validity criterion ≤ 35%).

• The coefficient of variation of average specific growth rates in replicate control cultures at test end was 0.5 % (validity criterion ≤ 7%).

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) >= water solubility of the test item in test medium
72h-ErC50 (growth rate) > water solubility of the test item in test medium

The test item was not detectable (lowest calibration limit 0.05 µg test item/L) in the test item solutions neither at test start nor at the end after 72 h. However, there were no concentration-dependent inhibiting effects on the growth of the freshwater green algae over the range of the tested concentrations. Based on the highly insolubility of the test item a full test was not performed in accordance with Regulation (EC) 1907/2006 Annex VII column 2.

Executive summary:

The 72-hour toxicity of the test item to the unicellular green alga Pseudokirchneriella subcapitata was determined in a non-GLP dose range finding static freshwater system according to OECD 201 (2011). Due to the low water solubility of the test item (< 2 µg/L (20°C/pH 6.5)), a stock solution was prepared using 100 mg test item/L test medium and stirred over 72 hours to achieve the maximum water solubility (in accordance with the OECD series on testing and assessment no. 23). Based on a serial dilution by factor 10, nominal concentrations of 100 mg test item/L (saturated solution), 1:10, 1:100 and 0 (control) were tested. The dissolved test item concentrations in all test solutions were measured by GC-MS (lowest calibration level 0.05 µg test item/L) before start (0 h) and at test end (72 h). In all test solutions the test item was not detectable neither at test start nor at the end after 72 h. Concentration-dependent inhibiting effects by the test item on the growth of the freshwater green algae over the range of the tested concentrations were not observed. The 72h ErC50 and EyC50 were determined to be > solubility of the test item in test solution (equivalent to a nominal loading of 100 mg/L). The NOEC (growth rate and yield) was determined to be ≥ solubility of the test item in test solution (equivalent to a nominal loading of 100 mg/L). All validity criteria of the test were met.

Description of key information

The effect of the test substance on Raphidocelis subcapitata was assessed in a 72 h toxicity test conducted in freshwater following OECD 201 (2011). 3 concentrations (between 100% saturated solution and 1%) and blank were tested in a non-GLP dose range-finding test. There were no concentration-dependent inhibiting effects on the growth of the freshwater green algae over the range of the tested concentrations. Hence, the EC50 (72h) is > solubility of the test item in test medium (dissolved test item). A main test was not performed since the test item concentration was below the lowest calibration level of 0.05 µg test item/L.

Based on the above and in accordance with Regulation (EC) 1907/2006 Annex VII column 2, a study does not need to be conducted if there are mitigating factors indicating that aquatic toxicity is unlikely to occur for instance if the substance is highly insoluble in water which is the case for that substance.