5'-O-(p,p'-dimethoxytrityl)thymidine

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:

2017-11-14 to 2018-03-29

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:

23 March 2006, corrected 28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- 4 sampling time points of at each of 6 test concentrations (24 samples). The number of quality controls (QCs) was nine (3 at the beginning, middle, and end of an assay batch, each measured in duplicate)
- The samples were filled into 10 mL glass vials. To each vial, 5 mL acetonitrile was added and samples were stored in the freezer (≤ -18 °C). The sampling was conducted according to the following specification:
• After 0 h, 24 h and 48 h exposure, both additional vessels of NC, A, B, C, D and E were sampled; one sample per replicate: 2 samples of 5 mL per treatment group.
• After 72 h exposure, both additional vessels and 2 replicates from the test vessels of NC, A, B, C, D and E were sampled; one sample per replicate: 4 samples of 5 mL per treatment group.
For each sampled treatment, one of the analytic samples from 0 h, 24 h, 48 h and 72 h was sent to the analytical laboratory at the test site menal GmbH for chemical analysis. The remaining samples were stored as retain samples in the freezer (≤ -18 °C) until finalisation of the study.
- Substance specific analysis was performed at the test site of menal GmbH.
- Concentrations: calibration samples 0.500, 1.00, 2.00, 3.00, 4.00, 6.00, 8.00, 10.0 mg/L (nominal loading rate); quality control samples 0.750, 1.50, 4.00 mg/L (nominal loading rate)
- Sampling method: 5’-O-(4,4’-Dimethoxytrityl)thymidine was quantified by an HPLC-UV method in aqueous samples derived from growth inhibition tests with Raphidocelis subcapitata (algae). Samples were transferred into the HPLC vial and 20 µL are injected into the HPLC-UV system, where 5’-O-(4,4’-Dimethoxytrityl)thymidine is detected at 230 nm.
- Sample storage conditions before analysis: Samples were stored at -20°C ± 10°C until analysis, and least until finalization of the phase report after which they will be destroyed or further stored. On assay day, samples were thawed at room temperatures and directly transferred into an HPLC-vial.
- Shipping: Samples were personally handed over between Hydrotox GmbH and menal GmbH

Vehicle:

no

Details on test solutions:

- Method: The test was performed with OECD TG 201 medium according to OECD 201 (2006). The stock solution was prepared as Water-Soluble Fraction (WSF) by adding 444.0 mg test item (including a factor of 1.11 to consider the dilution caused by addition of the algal inoculum) to 1000 mL test medium and shaking for 48 h using an overhead shaker at 20.3 – 21.0 °C in the dark.
The WSF was filtered through a fibre-glass filter with a retaining range up to 0.6 µm (MN 85/70 BF, Marchery-Nagel, Düren, Germany). The filter was prepared by rinsing with purified water and preconditioning with ca. 100 mL WSF (which was discarded) to reduce adsorption of the test item. This filtered stock solution was used as highest test item loading rate in the test.

- Controls: The negative control (NC; test medium) was treated in the same way as the test item solution.

Into each test vessel, 50 mL test solution (including algal inoculum) was transferred. The negative control and the further test item loading rates were prepared by diluting the stock solution with test medium according to the following scheme (dilution factor: 2):

Treatment group NC A B C D E
Test vesse item loading rate [mg/L] 0 25 50 100 200 400
Stock solution [mL] 0 31.25 62.5 125 250 500
Test medium [mL] 600 468.75 437.5 375 250 0
Test vessels (replicates) 6 3 3 3 3 3
Total volume per test vessel [mL] 50 50 50 50 50 50
Test solution / Test medium [mL] 45 45 45 45 45 45
Algal inoculum suspension [mL] 5 5 5 5 5 5
Additional vessels for chemical analysis 2 2 2 2 2 2
Total volume per test vessel * [mL] 60 60 60 60 60 60
Test solution / Test medium [mL] 54 54 54 54 54 54
Algal inoculum suspension [mL] 6 6 6 6 6 6
The calculated initial algal biomass in the test vessels was 7 × 10^3 cells/mL.
* The volume in the additional vessels for chemical analysis at the start of the test was increased (in relation to the test vessels) to obtain similar volumes as in the test vessels during the exposure period.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata, also known as Raphidocelis subcapitata
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Culture Collection of Algae at the University of Goettingen
- Age of inoculum (at test initiation): 4-day-old pre-culture was used as inoculum in the test
- Method of cultivation: Twice a week, the stock suspension is inoculated into fresh Holm-Hansen medium. Composition: 496 mg/L NaNO3, 39 mg/L K2HPO4, 75 mg/L MgSO4×7H2O, 36 mg/L CaCl2×2H2O, 58 mg/L Na2CO3, 10 mg/L Na2 EDTA×2H2O, 3 mg/L citric acid, 3 mg/L iron citrate, 0.1144 mg/L H3BO3, 0.0724 mg/L MnCl2× 4H2O, 0.0088 mg/L ZnSO4×7H2O, 0.0032 mg/L CuSO4×5H2O, 0.0010 mg/L Na2MoO4×2H2O, 0.0016 mg/L CoCl2×6H2O medium under axenic conditions to keep it in exponential growth.

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): 4 days before test, the same culturing conditions as in the test.
- Any deformed or abnormal cells observed: no obvious abnormality in test nor control

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Measurements also taken at 24 hours and 48 hours

Hardness:

Not reported

Test temperature:

22.8 - 23.0°C

pH:

7.5 - 7.8 (control); 7.6 - 8.0 (test item treatment)

Dissolved oxygen:

Not reported

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal loading rate in the test vessel [mg/L]: 0, 25, 50, 100, 200, 400
Measured concentration in the test vessel (geom. mean) [mg/L]: 0, 0.407, 0.678, 1.30, 1.95, 4.66

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer glass flasks 100 mL
- Type (delete if not applicable): closed, with cellulose stoppers
- Material, size, headspace, fill volume: test vessels are filled to 50 mL
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no, static test
- Initial cells density: 7 x 10E3 cells/mL
- Control end cells density: 11.315 x 10E5 cells/mL
- No. of vessels per concentration (replicates): 3 replicates per concentration
- No. of vessels per control (replicates): 6 replicates per negative control

GROWTH MEDIUM
- Standard medium used: yes, OECD TG 201 medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: solutions to make the 10-fold concentrated test medium were dissolved in deionised water; 1-fold concentrated medium was prepared by diluting 10-fold medium with ultrapure water
- Intervals of water quality measurement: The pH was measured at the start and end of the test; vessels were kept in light incubator (21 - 24 °C, constant within ± 2 °C) and continuously illuminated by lateral fluorescent tubes (58 W each), and mean light intensity was reported.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: Mean light intensity was 71.1 µE m-2 s-1 ± 3.8 %, supplied by lateral fluorescent tubes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): cell density, every 24 h; The inhibition of algal growth was determined from yield and growth rate; calculation according to the OECD guideline 201
- Determination of cell concentrations: To convert the measured surrogate parameter chlorophyll fluorescence into a cell concentration (the measure for biomass), a correlation factor is determined twice a year within the quality check by measuring cell concentrations of different dilutions with the Coulter Counter and the corresponding fluorescence with the microplate fluorescence reader. Chlorophyll fluorescence values are correlated with measured cell concentrations. The slope of the curve gives the conversion factor. By means of the conversion factor, the surrogate parameter chlorophyll fluorescence is converted into a cell concentration
- Chlorophyll measurement: The algal biomass was monitored by measuring the chlorophyll fluorescence after 0 h, 24 h, 48 h and 72 h. From each test vessel, 200 µL test solution was transferred into a 96-well microplate and the fluorescence measured with the fluorescence microplate reader at an excitation wavelength of 465 nm and an emission wavelength of 670 nm. Each measurement was conducted in duplicate. If the variation coefficient was > 10 %, the measurement was repeated. Test medium was measured as blank value and subtracted from the fluorescence values in the test vessels.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes, a preliminary test without GLP was performed before start of this GLP-study
- Test concentrations (in range finding study): Nominal loading rates of 50, 100 and 200 mg/L test item were tested and resulted in inhibition of -10.9, 12.9 and 70.7 % for yield and -4.1 %, 0.9 % and 24.4 % for growth rate (72 h), respectively.
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 4.66 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

5.028 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 3.588 - 6.967

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.975 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 2.256 - 3.924

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.903 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 1.741 - 8.430

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.908 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 1.034 - 3.527

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.312 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 0.697 - 2.470

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.95 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: Yes; the ErC20 and ErC50 values are extrapolated and exceeding the solubility of the substance. The ErC10 value is interpolated and does not exceed the water solubility of the substance. Therefore, the ErC10 (=2.975 mg/L) is set as key value.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

The results for the Inhibition of the growth rate are presented in table 1.       

 

Table1: Inhibition of growth rate after 24 h, 48 h and 72 h exposure.

Nominal test item loading rate [mg/L]	Geometric mean of test item conc. [mg/L]	Inhibition of growth rate [%]
		24 h	48 h	72 h
NC	--	--	--	--
25	0.407*	-3.3	-4.0	-4.0
50	0.678*	4.2	-2.2	-0.9
100	1.30	15.0	-0.3	0.7
200	1.95	11.5	2.4	4.9
400	4.66	20.9	15.5	17.2

* The measured concentration is below the Limit of Quantification but above the Limit of Detection.

 

CHEMICAL ANALYSIS

In the control, the test item was not detected. As the measured test item concentrations are not within ± 20% of the nominal loading rates, according to OECD 201 (2006) and OECD 23 (2000), all results are given in relation to the analytically measured test item concentrations (see table 2). Therefore, the geometric mean of all four measurement times (0, 24, 48, and 72 hours) was calculated.

 

Table2: Analytically measured test item concentrations after 0 h, 24 h, 48 h and 72 h exposure.

Nominal test item loading rate [mg/L]	Sampling time [h]	Measured test item concentration [mg/L]	Geometric mean of test item concentration [mg/L]
NC	0	--	--
	24	--
	48	--
	72	--
25	0	0.470*	0.407
	24	0.340*
	48	0.434*
	72	0.395*
50	0	0.741*	0.678
	24	0.681*
	48	0.622*
	72	0.672*
100	0	1.40	1.30
	24	1.17
	48	1.25
	72	1.40
200	0	1.44	1.95
	24	0.60
	48	0.91
	72	1.16
400	0	1.51	4.66
	24	0.93
	48	0.89
	72	1.47

* The measured concentration is below the Limit of Quantification but above the Limit of Detection.

 

CRITERIA OF VALIDITY

- The biomass (cell concentration) in the control has increased by a factor of 101.6 and therefore ≥ 16 during the test period.

- The mean coefficient of variation for section-by-section growth rate (day 0 - 1, 1 - 2 and 2 - 3) in the control is 14.1 % and therefore ≤ 35 %.

- The coefficient of variation of average specific growth rate during the test period in the control replicates is 2.8 % and therefore ≤ 7 %.

- The test is valid according to OECD Test Guideline 201 (23 March 2006).

Validity criteria fulfilled:

yes

Conclusions:

As the measured test item concentrations are not within ± 20% of the nominal loading rates, results are given in relation to the geometric mean concentrations. The 72-h EC20 based on growth rate of the test item in Pseudokirchneriella subcapitata is is 5.028 mg/L (95% CL 3.588 - 6.967; nominal values). The 72-h EC50 was > 4.66 mg/L. The ErC20 and ErC50 values are extrapolated and exceeding the solubility of the substance. The ErC10 value is interpolated and does not exceed the water solubility of the substance. Therefore, the ErC10 (=2.975 mg/L) is set as key value.

Executive summary:

In a 72 hour acute toxicity study, cultures of Pseudokirchneriella subcapitata, Strain No. 61.81 SAG were exposed to 5’-O-(4,4’-Dimethoxytrityl)thymidine at measured concentrations of 0, 0.407, 0.678, 1.30, 1.95 and 4.66 mg test item/L (nominal: 0, 25, 50, 100, 200 and 400 mg test item/L) under static conditions in accordance with the OECD guideline 201. As the measured test item concentrations were not within ± 20 % of the nominal loading rates, all results are given in relation to the analytically measured test item concentrations. Two of the measured test item concentrations are below the LOQ (i.e. 1 mg/L), the measured concentrations were used for calculation of the effect concentrations. The chemical analysis was conducted using the method with the lowest LOQ possible with the available equipment at the test site. The signal to noise ratio would be too low for a lower LOQ (in the range of or below the Limit of detection). The ErC20 (= 5.028 mg/L) is extrapolated exceeding the solubility of the substance and the ErC50 (> 4.66 mg/L) could not be determined. The ErC10 value is interpolated and does not exceed the water solubility of the substance. Therefore, the ErC10 (=2.975 mg/L) is set as key value. The water solubility of the substance was determined according to the OECD guideline 105 (see section 4.8 in the present dossier) with 3 mg/L.

No abnormalities were observed.

This toxicity study satisfies the guideline requirements for algae growth inhibition study according to OECD guideline 201.

 

Results Synopsis

 

Test Organism:Pseudokirchneriella subcapitata

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

 

Effect levels:

(Lowest/No observed) Effect Concentration (95% Confidence Limits)	Measured test item concentration (mg/L)
	Based on yield (72 hours)	Based on growth rate (72 hours)
EC50	3.903 (1.741 - 8.430)	> 4.66
EC20	1.908 (1.034 - 3.527)	5.028 (3. 588 - 6.967)
EC10	1.312 (0.697 - 2.470)	2.975 (2.256 - 3.924)
LOEC	1.95	1.95
NOEC	1.30	1.30

            

Description of key information

 In a 72-hour algae growth inhibition test cultures of Pseudokirchneriella subcapitata were exposed to 5’-O-(4,4’-Dimethoxytrityl)thymidine at measured concentrations of 0, 0.407, 0.678, 1.30, 1.95 and 4.66 mg test item/L under static conditions in accordance with the OECD 201 (2006) and GLP.

 

The following effect levels - based on the growth rate - are calculated:

- 72 h ErC50 > 4. 66 mg/L

- 72 h ErC10 = 2.975 (95 % C.L. 2.256 – 3.924 mg/L; extrapolated value)

- 72 h NOErC: 1.3 mg/L

- 72 h LOErC: 1.95 mg/L.  

As key value for classification the ErC10 is used. The EC20 nd EC 50 values are extrapolated and exceed the solubility of the test item (i.e. 3mg/L).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

1.3 mg/L