5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-[O-(2-cyanoethyl)-N,N′-diisopropylphosphoramidite]

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:

2018-10-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted March 2006, corrected July 28, 2011, equivalent to Commission Regulation (EC) No 761/2009, C.3., 2009

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

solubility in water: < 0.51 mg/L (20°C, pH 7.2) according to study R 014/2017; DMT-dT Phosphoramidite: Water solubility in distilled water (column elution method); WeylChem InnoTec GmbH (see also chapter 4.8)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Filtrate of 100 mg test item/L representing the highest test concentration and dilutions of 1:3, 1:9, 1:27 and 1:81 of this filtrate, and a control were tested
- Sampling method: The samples were taken from the biological phase of the study. Collecting, storage and handing over of the samples were the Study Director’s responsibility. The information concerning the samples was provided by the Study Director. Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment. Additional samples of the control and of the dilution solvent were taken at each sampling without any sample treatment.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. Afterwards the samples were again stored deep frozen (≤ -20 °C) and will be kept stored up to the date of the final report.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was not well soluble in test water. In this study, only the inhibitory effects of dissolved test item were tested and thus no concentrations above the solubility limit of the test item in test water were tested. The preparation of the test solution was conducted following the recommendations of the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 23). Therefore, a supersaturated stock solution of nominal 100 mg test item/L was prepared by suspending 100.6 mg test item into 1006 mL test water by intense stirring for 5 minutes. The stock suspension was shaken overhead for 48 hours at room temperature in the dark to achieve a maximum dissolved concentration. Then, non-dissolved fractions of the test item were separated from the test medium by membrane filtration (0.45 µm cellulose acetate filter). After adjusting the pH to 8.0 using 1 M NaOH the solution with dissolved test item was used as the test medium of the highest test concentration and to prepare the desired 1:3, 1:9, 1:27 and 1:81 dilutions. The test media were prepared just before introduction of the algae (= start of the test).
- Eluate: N/A
- Differential loading: N/A
- Controls: In the control, test water was used without addition of the test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): No vehicle used
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Non-dissolved fractions are reported. The non-dissolved fractions of the test item were separated from the test medium by membrane filtration (0.45 µm cellulose acetate filter). There were no remarkable observations in the test medium.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (KORSHIKOV) formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
- Strain: Strain No. 61.81 SAG
- Source (laboratory, culture collection): The algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen (SAG)", 37073 Göttingen, Germany.
- Method of cultivation: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.
Reference Item: For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
ACCLIMATION
- Acclimation period: N/A
- Culturing media and conditions (same as test or not): same as test.
- Any deformed or abnormal cells observed: No.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
Water temperature was: 21.6 to 23.3 °C

pH:

The pH was measured in all test item concentrations and the control at the start and the end of the test. pH was:
7.9 to 8.0 at test start and 9.8 to 10.0 at test end

Nominal and measured concentrations:

The water solubility of test item was indicated to be low (< 0.51 mg/L) and when preparing the fresh media particles were floating on the surface or were lying on the bottom of the flask. Therefore, a filtrate of 100 mg test item/L representing the highest test concentration and dilutions of 1:3, 1:9, 1:27 and 1:81 of this filtrate, and a control were tested. All tested measured values were below the detection limit (i.e. < 0.01 mg/L).

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Erlenmeyer flasks of 50 mL volume with approximately 50 mL of test medium
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): No, static
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: 5000 algal cells per mL test medium; These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test start under the same conditions as in the test.
- Control end cells density: 1 134 170 000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates):6
- No. of vessels per vehicle control (replicates): N/A
The test was performed with three replicates per test concentration and six replicates in the control. Volumes of 50 mL of algal suspension per replicate were continuously stirred with magnetic stirrers in 50 mL Erlenmeyer flasks. The flasks were covered with air permeable glass dishes and incubated in a water bath. The flasks were placed in a random order and were repositioned each day to minimize differences in test conditions.
Additionally, one replicate of each test concentration and of the control was prepared without algae to provide a "blank" for the spectrophotometric measurements. The additional replicates were incubated under the same conditions as described above. The blank values were subtracted from the absorption measured in the samples containing algae in order to eliminate absorption caused by the test item.
GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water, Analytical grade salts were added to achieve OECD medium
- Culture medium different from test medium: No
- Intervals of water quality measurement: Start and end; Test conditions were recorded with suitable instruments and documented.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
Mean light intensity: 5708 lux (range: 5270 to 6360 lux)
- Salinity (for marine algae): N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae). Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.
- Chlorophyll measurement: No

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Justification for using less concentrations than requested by guideline: N/A

Range finding study
- Test concentrations: filtrate of 100 mg/L and dilutions thereof of 1:10, 1:100 and 1:1000 and a control
- Results used to determine the conditions for the definitive study: Yes
Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions. The test item was shaken overhead 48 hours and the undissolved test item was separated by filtration (0.45 µm cellulose acetate filter). Approx. 10 000 algal cells per mL test medium were exposed to the filtrate of 100 mg/L and dilutions thereof of 1:10, 1:100 and 1:1000 and a control. Two replicates were tested per treatment group. No effect on the algae was observed. The pre-experiments were not performed in compliance with the GLP-Regulations and are excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the study number of the present study.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Filtrate of nominal 100 mg test item/L

Basis for effect:

growth rate

Remarks on result:

not determinable because of methodological limitations

Remarks:

Concentration must be below LOD of 0.01 mg/L though a supersatured solution of the test item was prepared.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Filtrate of nominal 100 mg test item/L

Basis for effect:

growth rate

Remarks on result:

not determinable because of methodological limitations

Remarks:

Concentration must be below LOD of 0.01 mg/L though a supersatured solution of the test item was prepared.

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Filtrate of nominal 100 mg test item/L

Basis for effect:

growth rate

Remarks on result:

not determinable because of methodological limitations

Remarks:

Concentration must be below LOD of 0.01 mg/L though a supersatured solution of the test item was prepared.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Filtrate of nominal 100 mg test item/L

Basis for effect:

other: Yield

Remarks on result:

not determinable because of methodological limitations

Remarks:

Concentration must be below LOD of 0.01 mg/L though a supersatured solution of the test item was prepared.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Filtrate of nominal 100 mg test item/L

Basis for effect:

other: Yield

Remarks on result:

not determinable because of methodological limitations

Remarks:

Concentration must be below LOD of 0.01 mg/L though a supersatured solution of the test item was prepared.

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Filtrate of nominal 100 mg test item/L

Basis for effect:

other: Yield

Remarks on result:

not determinable because of methodological limitations

Remarks:

Concentration must be below LOD of 0.01 mg/L though a supersatured solution of the test item was prepared.

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No. The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a filtrate of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to the highest concentration tested.
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The water solubility of test item was indicated to be low (< 0.51 mg/L) and when preparing the fresh media, particles were floating on the surface or were lying on the bottom of the flask. Therefore, a filtrate of 100 mg test item/L representing the highest test concentration and dilutions of 1:3, 1:9, 1:27 and 1:81 of this filtrate, and a control were tested. The preparation of the test solution was conducted according to the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 23).

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50: 0.963 mg test item/L (95 % C.I.: 0.927 - 1.001 mg test item/L)

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hours ErC50 and the 72 hours EyC50 could not be calculated by a statistical analysis due to the lack of effects and were therefore determined directly from the raw data. For the determination of the 72 hours LOEC and the 72 hours NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Dunnett's t- test.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat® Solutions GmbH.

Biological results:

The EC50, EC20 and EC10 for yield and growth rate were estimated to be greater than the highest test concentration of the filtrate of 100 mg test item/L, which, however, could not be quantified analytically as the content was below the limit of detection. The NOEC was determined to be at least the filtrate of 100 mg test item/L. The LOEC was estimated to be greater than the filtrate of 100 mg test item/L. Therefore, no toxicity towards the test organism up to the limit of solubility was detected for the test item.

Analytical results:

The quantification of the test item 5′-O-[bis(4-methoxyphenyl)phenylmethyl]-2′-deoxythymidine, 3′-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite] in the test samples was performed using liquid chromatography with UV detection. The chosen analytical method was the most sensitive method applicable. The concentrations of the test item were analysed in the duplicate test media samples from all test concentrations (filtrate of the test item stock solution and its dilutions) and the duplicate control samples from all sampling times. No substance was detected in any sample (see Table 1), concluding that the maximum soluble amount of the test item must be below the Limit of Detection (LOD) of the method (0.01 mg test item/L).

 

Validity Criteria of the Study
Cell Density Increase in Control Cultures:	226.8-fold increase within 72 hours and thus, the validity criterion was met.
Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures:	14.7 % and thus, the validity criterion was met.
Coefficient of Variation of Average Growth between Control Replicates:	1.2 % and thus, the validity criterion was met.

 

Table 1. Summary of Analytical Results   Sample Description % of RSD [mg test item/L] nominal^1 [%] n Control n.a. n.a. 4 Dilution 1:81 <LOD n.a. 4 Dilution 1:27 <LOD n.a. 4 Dilution 1:9 <LOD n.a. 4 Dilution 1:3 <LOD n.a. 4 Filtrate of 100 <LOD n.a. 4           ^1mean value of all measured samples per treatment group RSD: relative standard deviation per treatment group n: number of analysed samples n.a.: not applicable LOD: 0.01 mg/L

Validity criteria fulfilled:

yes

Conclusions:

No effect on the growth or yield of Pseudokirchneriella subcapitata was observed up to the limit of water solubility of the test substance.
The analytical verification could not determine a measured concentration as the concentration of the test item was too low. Although a saturated solution was prepared by stirring for 48 h. The substance is hardly soluble and the limit of detection was 0.01 mg/L.

Executive summary:

In a 72-hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG were exposed to 5′-O-[bis(4-methoxyphenyl)phenylmethyl]-2′-deoxythymidine, 3′-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite] at nominal concentrations of 100, 33, 11, 3.7 and 1.2 mg test item/L under static conditions in accordance with the OECD guideline 201.  No effect was observed up to the limit of water solubility of the test substance. The EC50, EC20 and EC10 for yield and growth rate were estimated to be greater than the highest test concentration of the filtrate of 100 mg test item/L, which, however, could not be quantified analytically as the content was below the limit of detection. The NOEC was determined to be at least the filtrate of 100 mg test item/L. The LOEC was estimated to be greater than the filtrate of 100 mg test item/L. Therefore, no toxicity towards the test organism up to the limit of solubility was detected for the test item. The analytical verification of the concentrations was below the LOD of 0.01 mg test item/L.

 

This toxicity study is classified as acceptable guideline requirements for Freshwater Alga and Cyanobacteria, Growth Inhibition Test with P. subcapitata.

 

Results Synopsis

 

Test Organism: of Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG

Test Type (Flowthrough, Static, Static Renewal): Static

 

Parameter	Yield [mg test item/L]	Growth rate [mg test item/L]
72-hour EC50	>Filtrate of nominal 100 mg/L	>Filtrate of nominal 100 mg/L
95 % conf. interval	n.d.	n.d.
72-hour EC20	>Filtrate of nominal 100 mg/L	>Filtrate of nominal 100 mg/L
95 % conf. interval	n.d.	n.d.
72-hour EC10	>Filtrate of nominal 100 mg/L	>Filtrate of nominal 100 mg/L
95 % conf. interval	n.d.	n.d.
72-hour NOEC	≥Filtrate of nominal 100 mg/L	≥Filtrate of nominal 100 mg/L
72-hour LOEC	>Filtrate of nominal 100 mg/L	>Filtrate of nominal 100 mg/L
n.d. = not determinable

 

Endpoint(s) Effected: Growth rate, Yield

 

Description of key information

No effect on the growth or yield of Pseudokirchneriella subcapitata was observed up to the limit of water solubility of the test substance. The test was conducted according to the OECD guideline 201 and under GLP.

The analytical verification could not determine a measured concentration as the concentration of the test item was too low. Although a saturated solution was prepared by stirring for 48 h. The substance is hardly soluble and the limit of detection was 0.01 mg/L.